Biorefineries Industrial Processes and Products Status Quo and Future Directions PDF

Biorefineries –
Industrial Processes and

Products
Edited by
Birgit Kamm,
Patrick R. Gruber,
and Michael Kamm
Biorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 1
Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm
Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 3-527-31027-4
Related Titles
Elvers, B. (Ed.)
Handbook of Fuels
Energy Sources for Transportation
2006
ISBN 3-527-30740-0
Olah, G. A., Goeppert, A., Prakash, G. K. S.
Beyond Oil and Gas: The Methanol Economy

2006
ISBN 3-527-31275-7
Shahidi, F. (Ed.)
Bailey’s Industrial Oil and Fat Products

6 Volume Set
2005
ISBN 0-471-38460-7
Ocic, O.
Oil Refineries in the 21st Century

Energy Efficient, Cost Effective, Environmentally Benign
2005
ISBN 3-527-31194-7
Biorefineries –
Industrial Processes and Products
Status Quo and Future Directions
Volume 1
Edited by
Birgit Kamm, Patrick R. Gruber, and Michael Kamm
The Editors n All books published by Wiley-VCH are carefully
produced. Nevertheless, authors, editors, and
Dr. Birgit Kamm publisher do not warrant the information contained
Research Institute in these books, including this book, to be free of
Bioactive Polymer Systems errors. Readers are advised to keep in mind that
biopos e.V. statements, data, illustrations, procedural details or
Kantstr. 55 other items may inadvertently be inaccurate.
14513 Teltow
Germany
Dr. Patrick R. Gruber

President and CEO
Outlast Technologies Inc. Library of Congress Card No.: applied for
5480 Valmont Road British Library Cataloguing-in-Publication Data:
Boulder, CO 80301 A catalogue record for this book is available
USA from the British Library.
Bibliographic information published by
Michael Kamm
Die Deutsche Bibliothek
Biorefinery.de GmbH
Die Deutsche Bibliothek lists this publication in
Stiftstr. 2 the Deutsche Nationalbibliografie; detailed
14471 Potsdam bibliographic data is available in the Internet at
Germany <http://dnb.ddb.de>
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA,

Weinheim, Germany
All rights reserved (including those of translation

into other languages). No part of this book may
be reproduced in any form – by photoprinting,
microfilm, or any other means – nor transmitted
or translated into a machine language without
written permission from the publishers.
Registered names, trademarks, etc. used in this
book, even when not specifically marked as such,
are not to be considered unprotected by law.
Typsetting K+V Fotosatz GmbH, Beerfelden

Printing Betz-Druck GmbH, Darmstadt
Binding Litges & Dopf Buchbinderei GmbH,
Heppenheim
Printed in the Federal Republic of Germany

Printed on acid-free paper
ISBN-13: 978-3-527-31027-2
ISBN-10: 3-527-31027-4
V
Contents
Editors’s Preface XXI
Foreword XXIII
Henning Hopf
Foreword XXV
Paul T. Anastas
List of Contributors XXVII
Volume 1
Part I Background and Outline – Principles and Fundamentals
1 Biorefinery Systems – An Overview 3

Birgit Kamm, Michael Kamm, Patrick R. Gruber, and Stefan Kromus
1.1 Introduction 3
1.2 Historical Outline 4
1.2.1 Historical Technological Outline and Industrial Resources 4
1.2.2 The Beginning – A Digest 5
1.2.2.1 Sugar Production 5
1.2.2.2 Starch Hydrolysis 5
1.2.2.3 Wood Saccharification 5
1.2.2.4 Furfural 6
1.2.2.5 Cellulose and Pulp 6
1.2.2.6 Levulinic Acid 6
1.2.2.7 Lipids 7
1.2.2.8 Vanillin from Lignin 7
1.2.2.9 Lactic Acid 7
1.2.3 The Origins of Integrated Biobased Production 8
1.3 Situation 11
ISBN: 3-527-31027-4
VI Contents
1.3.1 Some Current Aspects of Biorefinery Research

and Development 11
1.3.2 Raw Material Biomass 12
1.3.3 National Vision and Goals and Plan for Biomass Technology
in the United States 14
1.3.4 Vision and Goals and Plan for Biomass Technology in the
European Union and Germany 15
1.4 Principles of Biorefineries 16
1.4.1 Fundamentals 16
1.4.2 Definition of the Term “Biorefinery” 19
1.4.3 The Role of Biotechnology 20
1.4.3.1 Guidelines of Fermentation Section within Glucose-product
Family Tree 21
1.4.4 Building Blocks, Chemicals and Potential Screening 22
1.5 Biorefinery Systems and Design 23
1.5.1 Introduction 23
1.5.2 Lignocellulosic Feedstock Biorefinery 24
1.5.3 Whole-crop Biorefinery 26
1.5.4 Green Biorefinery 29
1.5.5 Two-platform Concept and Syngas 31
1.6 Outlook and Perspectives 32
References 33
2 Biomass Refining Global Impact –

The Biobased Economy of the 21st Century 41
Bruce E. Dale and Seungdo Kim
2.1 Introduction 41
2.2.1 Background and Development of the Fossil Carbon-processing
Industries 42
2.2.2 The Existing Biobased Economy: Renewable Carbon 43
2.2.3 Toward a Much Larger Biobased Economy 44
2.3 Supplying the Biorefinery 45
2.3.1 What Raw Materials do Biorefineries Require and What Products
Can They Make? 45
2.3.2 Comparing Biomass Feedstock Costs With Petroleum Costs 48
2.3.3 How Much Biomass Feedstock Can be Provided at What Cost? 50
2.4 How Will Biorefineries Develop Technologically? 53
2.4.1 Product Yield: The Dominant Technoeconomic Factor 53
2.4.2 Product Diversification: Using the Whole Barrel of Biomass 54
2.4.3 Process Development and a Technical Prerequisite
for Cellulosic Biorefineries 55
2.5 Sustainability of Integrated Biorefining Systems 56
2.5.1 Integrated Biorefining Systems: “All Biomass is Local” 56
Contents VII
2.5.2 Agricultural/Forestry Ecosystem Modeling: New Tools for an Age

of Sustainability 57
2.5.3 Analyzing the Sustainability of Integrated Biorefining Systems:
Some Results 60
2.6 Conclusions 64
Acknowledgements 65
References 65
3 Development of Biorefineries –
Technical and Economic Considerations 67
Bill Dean, Tim Dodge, Fernando Valle, and Gopal Chotani
3.1 Introduction 67
3.2 Overview: The Biorefinery Model 68
3.3 Feedstock and Conversion to Fermentable Sugar 68
3.3.1 Sucrose 70
3.3.2 Starch 70
3.3.3 Cellulose 71
3.4 Technical Challenges 74
3.4.1 Cellulase Enzymes 74
3.4.1.1 Improved Cellulase Production Economics 74
3.4.1.2 Improved Cellulase Enzyme Performance 76
3.4.2 Fermentation Organisms 77
3.4.2.1 Biomass Hydrolyzate as Fermentable Carbon Source 78
3.4.2.2 Production Process as a Whole 79
3.4.2.3 Emerging Solutions 80
3.5 Conclusions 81
Acknowledgments 82
References 82
4 Biorefineries for the Chemical Industry –

A Dutch Point of View 85
Ed de Jong, René van Ree Rea, Robert van Tuil, and Wolter Elbersen
4.1 Introduction 85
4.2 Historical Outline – The Chemical Industry: Current Situation and
Perspectives 86
4.2.1 Overview of Products and Markets 86
4.2.2 Technological Pathways 87
4.2.3 Biomass-based Industrial Products 87
4.2.3.1 Carbohydrates 89
4.2.3.2 Fatty Acids 90
4.2.3.3 Other 91
4.2.4 International Perspectives 92
4.2.4.1 Production 92
4.2.4.2 Integration 92
4.2.4.3 Use and Re-use 93
VIII Contents
4.3 Biomass: Technology and Sustainability 93

4.3.1 Transition to a Bio-based Industry: Sectoral Integration
in the Netherlands 93
4.3.2 Can Sustainability Drive Technology? 96
4.4 The Chemical Industry: Biomass Opportunities – Biorefineries 97
4.4.1 Biomass Opportunities 97
4.4.2 Biorefinery Concept 98
4.4.3 Biomass Availability 100
4.4.4 Primary Refinery 101
4.4.5 Secondary Thermochemical Refinery 102
4.4.6 Secondary Biochemical Refinery – Fermentative Processes 104
4.4.6.1 Feedstocks 105
4.4.6.2 Product Spectrum 105
4.4.6.3 Side Streams and Recycling 106
4.5 Conclusions, Outlook, and Perspectives 106
4.5.1 Biomass – Sustainability 106
4.5.2 Biomass Refining and Pretreatment 107
4.5.3 Conversion Technology 108
4.5.4 Chemicals and Materials Design 108
4.5.5 Dutch Energy Research Strategy (“EOS”) 109
References 109
Part II Biorefinery Systems
Lignocellulose Feedstock Biorefinery
5 The Lignocellulosic Biorefinery –

A Strategy for Returning to a Sustainable Source of Fuels
and Industrial Organic Chemicals 115
L. Davis Clements and Donald L. Van Dyne
5.1 The Situation 115
5.2 The Strategy 115
5.2.1 A Strategy Within a Strategy 116
5.2.2 Environmental Benefits 117
5.2.3 The Business Structure 117
5.2.4 Cost Estimates 118
5.3 Comparison of Petroleum and Biomass Chemistry 118
5.3.1 Petroleum Resources 118
5.3.2 Biomass Resources 119
5.3.3 Saccharides and Polysaccharides 121
5.3.4 Lignin 121
5.3.5 Triacylglycerides (or Triglycerides) 121
5.3.6 Proteins 122
5.4 The Chemistry of the Lignocellulosic Biorefinery 122
5.5 Examples of Integrated Biorefinery Applications 125
Contents IX
5.5.1 Production of Ethanol and Furfural from Lignocellulosic

Feedstocks 125
5.5.2 Management of Municipal Solid Waste 125
5.5.3 Coupling MSW Management, Ethanol, and Biodiesel 126
5.6 Summary 127
References 127
6 Lignocellulosic Feedstock Biorefinery:

History and Plant Development for Biomass Hydrolysis 129
Raphael Katzen and Daniel J. Schell
6.1 Introduction 129
6.2 Hydrolysis of Biomass Materials 129
6.2.1 Acid Conversion 129
6.2.2 Enzymatic Conversion 130
6.3 Acid Hydrolysis Processes 130
6.3.1 Early Efforts to Produce Ethanol 130
6.3.2 Other Products 133
6.4 Enzymatic Hydrolysis Process 134
6.4.1 Early History 134
6.4.2 Enzyme-Based Plant Development 134
6.4.3 Technology Development 135
6.5 Conclusion 136
References 136
7 The Biofine Process – Production of Levulinic Acid, Furfural,

and Formic Acid from Lignocellulosic Feedstocks 139
Daniel J. Hayes, Steve Fitzpatrick, Michael H. B. Hayes,
and Julian R. H. Ross
7.2 Lignocellulosic Fractionation 139
7.2.1 Acid Hydrolysis of Polysaccharides 141
7.2.2 Production of Levulinic Acid, Formic Acid and Furfural 142
7.3 The Biofine Process 144
7.3.1 Yields and Efficiencies of the Biofine Process 145
7.3.2 Advantages over Conventional Lignocellulosic Technology 146
7.3.3 Products of The Biofine Process 147
7.3.3.1 Diphenolic Acid 148
7.3.3.2 Succinic Acid and Derivatives 149
7.3.3.3 Delta-aminolevulinic Acid 149
7.3.3.4 Methyltetrahydrofuran 150
7.3.3.5 Ethyl Levulinate 152
7.3.3.6 Formic Acid 153
7.3.3.7 Furfural 154
7.3.4 Biofine Char 155
7.3.5 Economics of The Biofine Process 158
X Contents
7.4 Conclusion 161

References 162
Whole Crop Biorefinery
8 A Whole Crop Biorefinery System:

A Closed System for the Manufacture of Non-food Products
from Cereals 165
Apostolis A. Koutinas, Rouhang Wang, Grant M. Campbell, and Colin Webb
8.1 Intro 165
8.2 Biorefineries Based on Wheat 167
8.2.1 Wheat Structure and Composition 167
8.2.2 Secondary Processing of Wheat Flour Milling Byproducts 169
8.2.3 Advanced Wheat Separation Processes for Food and Non-food
Applications 173
8.2.3.1 Pearling as an Advanced Cereal Fractionation Technology 173
8.2.3.2 Air Classification 176
8.2.4 Biorefinery Based on Novel Dry Fractionation Processes
of Wheat 176
8.2.4.1 Potential Value-added Byproducts from Wheat Bran-rich
Fractions 178
8.2.4.2 Exploitation of the Pearled Wheat Kernel 180
8.3 A Biorefinery Based on Oats 183
8.3.1 Oat Structure and Composition 183
8.3.2 Layout of a Potential Oat-based Fractionation Process 183
8.3.2.1 Potential Value-added Byproducts from Oat Bran-rich Fractions 185
8.4 Summary 187
References 187
Fuel-oriented Biorefineries
9 Iogen’s Demonstration Process for Producing Ethanol

from Cellulosic Biomass 193
Jeffrey S. Tolan
9.2 Process Overview 193
9.3 Feedstock Selection 194
9.3.1 Feedstock Composition 194
9.3.2 Feedstock Selection 196
9.3.3 Ethanol from Starch or Sucrose 197
9.3.4 Advantages of Making Ethanol from Cellulosic Biomass 197
9.4 Pretreatment 198
9.4.1 Process 198
9.4.2 Chemical Reactions 198
9.4.3 Other Pretreatment Processes 199
Contents XI
9.5 Cellulase Enzyme Production 201

9.5.1 Production of Cellulase Enzymes 201
9.5.2 Enzyme Production on the Ethanol Plant Site 202
9.5.3 Commercial Status of Cellulase 202
9.6 Cellulose Hydrolysis 202
9.6.1 Process Description 202
9.6.2 Kinetics of Cellulose Hydrolysis 203
9.6.3 Improvements in Enzymatic Hydrolysis 205
9.7 Lignin Processing 205
9.7.1 Process Description 205
9.7.2 Alternative Uses for Lignin 206
9.8 Sugar Fermentation and Ethanol Recovery 206
References 207
10 Sugar-based Biorefinery –
Technology for Integrated Production
of Poly(3-hydroxybutyrate), Sugar, and Ethanol 209
Carlos Eduardo Vaz Rossell, Paulo E. Mantelatto, José A. M. Agnelli,
and Jefter Nascimento
10.2 Sugar Cane Agro Industry in Brazil – Historical Outline 209
10.2.1 Sugar and Ethanol Production 209
10.2.2 The Sugar Cane Agroindustry and the Green Cycle 210
10.3 Biodegradable Plastics from Sugar Cane 212
10.3.1 Poly(3-Hydroxybutyric Acid) 212
10.3.1.1 Biodegradable Plastics and the Environment 212
10.3.1.2 General Aspects of Biodegradability 213
10.3.2 Poly(3-Hydroxybutyric Acid) Polymer 214
10.3.2.1 General Characteristics of Poly(3-hydroxybutyric Acid)
and its Copolymer Poly(3-hydroxybutyric Acid-co-3-
hydroxyvaleric Acid) 214
10.3.2.2 Processing of Poly(Hydroxybutyrates) 215
10.4 Poly(3-Hydroxybutyric Acid) Production Process 217
10.4.1 Sugar Fermentation to Poly(3-Hydroxybutyric Acid)
by Ralstonia eutropha 217
10.4.2 Downstream Processing for Recovery and Purification of Intracellular
Poly(3-Hydroxybutyric Acid) 218
10.4.2.1 Processes for Extraction and Purification
of Poly(hydroxyalkanoates) 218
10.4.2.2 Chemical Digestion 218
10.4.2.3 Enzymatic Digestion 219
10.4.2.4 Solvent Extraction 219
10.4.3 Integration of Poly(3-Hydroxybutyric Acid) Production
in a Sugar Mill 221
XII Contents
10.4.4 Investment and Production Cost of Poly(3-Hydroxybutyric Acid)

in a Sugar Mill 222
References 225
Biorefineries Based on Thermochemical Processing
11 Biomass Refineries Based on Hybrid

Thermochemical-Biological Processing – An Overview 227
Robert C. Brown
11.2.1 Origins of Biorefineries Based on Syngas Fermentation 228
11.2.2 Origins of Biorefineries Based on Fermentation of Bio-oils 229
11.3 Gasification-Based Systems 230
11.3.1 Fundamentals of Gasification 230
11.3.2 Fermentation of Syngas 233
11.3.2.1 Production of Organic Acids 234
11.3.2.2 Production of Alcohols 235
11.3.2.3 Production of Polyesters 236
11.3.3 Biorefinery Based on Syngas Fermentation 239
11.3.4 Enabling Technology 240
11.4 Fast Pyrolysis-based Systems 241
11.4.1 Fundamentals of Fast Pyrolysis 241
11.4.2 Fermentation of Bio-oils 244
11.4.3 Biorefineries Based on Fast Pyroylsis 246
11.4.4 Enabling Technologies 248
References 250
Green Biorefineries
12 The Green Biorefiner Concept – Fundamentals and Potential 253

Stefan Kromus, Birgit Kamm, Michael Kamm, Paul Fowler,
and Michael Narodoslawsky
12.2.1 The Inceptions 254
12.2.2 First Production of Leaf Protein Concentrate 254
12.2.3 First Production of Leaf Dyes 257
12.3 Green Biorefinery Raw Materials 258
12.3.1 Raw Materials 258
12.3.2 Availability of Grassland Feedstocks for Large-scale Green
Biorefineries 259
12.3.3 Key Components of Green and Forage Grasses 260
Contents XIII
12.3.3.1 Structural Cell Wall Constituents 260

12.3.3.2 Cell Contents 265
12.4 Green Biorefinery Concept 269
12.4.1 Fundamentals and Status Quo 269
12.4.2 Wet Fractionation and Primary Refinery 271
12.5 Processes and Products 273
12.5.1 The Juice Fraction 273
12.5.1.1 Green Juice 273
12.5.2 GJ Drinks/Alternative Life 275
12.5.2.1 Silage Juice 276
12.5.3 Ingredients and Specialties 277
12.5.3.1 Proteins/Polysacharides 277
12.5.3.2 Cholesterol Mediation 277
12.5.3.3 Antifeedants 277
12.5.3.4 Silica 277
12.5.3.5 Silicon Carbide 278
12.5.3.6 Filter Aids 278
12.5.3.7 Zeolites 278
12.5.4 The Press-Cake (Fiber) Fraction 278
12.5.4.1 Fibers 280
12.5.4.2 Chemicals 282
12.5.4.3 Residue Utilization 283
12.6 Green Biorefinery – Economic and Ecological Aspects 283
Acknowledgment 285
References 285
13 Plant Juice in the Biorefinery –

Use of Plant Juice as Fermentation Medium 295
Margrethe Andersen, Pauli Kiel, and Mette Hedegaard Thomsen
13.3 Biobased Poly(lactic Acid) 296
13.3.1 Fermentation Processes 296
13.3.2 The Green Biorefinery 296
13.3.3 Lactic Acid Fermentation 298
13.3.4 Brown Juice as a Fermentation Medium 298
13.4 Materials and Methods 299
13.4.1 Analytical Methods 299
13.4.1.1 Sugar Analysis 299
13.4.1.2 Analysis of Organic Acids 299
13.4.1.3 Analysis of Minerals 299
13.4.1.4 Analysis of Vitamins 299
13.4.1.5 Analysis of Amino Acids 299
13.4.1.6 Analysis of Protein 299
XIV Contents
13.4.2 Fed Batch Fermentation of Brown Juice with Lb. salivarius

BC 1001 299
13.4.3 Pilot Scale Continuous Fermentation with Lb. salivarius
BC 1001 300
13.4.4 Study of Potato Juice Quality During Aerobic
and Anaerobic Storage 300
13.5 Brown Juice 300
13.5.1 Chemical Composition 300
13.5.2 Seasonal Variations 302
13.5.3 Lactic Acid Fermentation of Brown Juice 305
13.5.4 The Green Crop-drying Industry as a Lactic Acid Producer 306
13.6 Potato Juice 309
13.6.1 Potato Juice as Fermentation Medium 309
13.6.2 The Potato Starch Industry as Lactic Acid Producer 310
13.7 Carbohydrate Source 311
13.8 Purification of Lactic Acid 312
13.9 Conclusion and Outlook 313
Acknowledgments 313
References 313
Part III Biomass Production and Primary Biorefineries
14 Biomass Commercialization and Agriculture Residue Collection 317

James Hettenhaus
14.2.1 Case Study: Harlan, Iowa Corn Stover Collection Project 319
14.2.2 Case Study: Bagasse Storage – Dry or Wet? 321
14.2.2.1 Dry Storage 321
14.2.2.2 Wet Storage 323
14.3 Biomass Value 324
14.3.1 Soil Quality 324
14.3.2 Farmer Value 325
14.3.3 Processor Value 327
14.4 Sustainable Removal 328
14.4.1 Soil Organic Material 328
14.4.2 Soil Erosion Control 329
14.4.3 Cover Crops 331
14.5 Innovative Methods for Collection, Storage and Transport 332
14.5.1 Collection 332
14.5.1.1 Baling 333
14.5.1.2 One-pass Collection 333
14.5.2 Storage 334
14.5.2.1 Density 335
14.5.2.2 Storage Area 335
Contents XV
14.5.2.3 Storage Loss 335

14.5.2.4 Foreign Matter and Solubles 337
14.5.2.5 Storage Investment 337
14.5.3 Transport 337
14.5.3.1 Harvest Transport 338
14.5.3.2 Biorefinery Supply 338
14.6 Establishing Feedstock Supply 339
14.6.1 Infrastructure 340
14.6.1.1 Infrastructure Investment 340
14.6.1.2 Organization Infrastructure 340
14.7 Perspectives and Outlook 341
References 342
15 The Corn Wet Milling and Corn Dry Milling Industry –

A Base for Biorefinery Technology Developments 345
Donald L. Johnson
15.1.1 Corn – Wet and Dry Milling – Existing Biorefineries 345
15.2 The Corn Refinery 346
15.2.1 Wet Mill Refinery 346
15.2.2 Dry Mill Refinery 346
15.2.3 Waste Water Treatment 347
15.3 The Modern Corn Refinery 348
15.3.1 Background and Definition 348
15.3.2 Technologies and Products 348
15.3.3 Refinery Economy 350
15.3.3.1 Refinery Economy of Scale and Location Considerations 350
15.4 Carbohydrate Refining 351
References 352
Part IV Biomass Conversion: Processes and Technologies
16 Enzymes for Biorefineries 357

Sarah A. Teter, Feng Xu, Glenn E. Nedwin, and Joel R. Cherry
16.2 Biomass as a Substrate 359
16.2.1 Composition of Biomass 359
16.2.1.1 Cellulose 359
16.2.1.2 Hemicellulose 360
16.2.1.3 Lignin 360
16.2.1.4 Starch 360
16.2.1.5 Protein 361
16.2.1.6 Lipids and Other Extracts 361
16.2.2 Biomass Pretreatment 361
XVI Contents
16.2.2.1 Dilute Acid Pretreatment 362

16.2.2.2 Ammonia Fiber Explosion 362
16.2.2.3 Hot-wash Pretreatment 362
16.2.2.4 Wet Oxidation 363
16.3 Enzymes Involved in Biomass Biodegradation 363
16.3.1 Glucanases or Cellulases 364
16.3.2 Hemicellulases 364
16.3.3 Nonhydrolytic Biomass-active Enzymes 365
16.3.4 Synergism of Biomass-degrading Enzymes 365
16.4 Cellulase Development for Biomass Conversion 366
16.4.1 Optimization of the CBH-EG-BG System 366
16.4.1.1 BG Supplement 366
16.4.1.2 Novel Cellulases with Better Thermal Properties 367
16.4.1.3 Structure–Function Relationship of EG 370
16.4.2 Other Proteins Potentially Beneficial for Biomass Conversion 371
16.4.2.1 Secretome of Cellulolytic Fungi 371
16.4.2.2 Hydrolases 373
16.4.2.3 Nonhydrolytic proteins 374
16.5 Expression of Cellulases 374
16.6 Range of Biobased Products 375
16.6.1 Fuels 376
16.6.2 Fine/Specialty Chemicals 378
16.6.3 Fuel Cells 378
16.7 Biorefineries: Outlook and Perspectives 380
16.7.1 Potential of Biomass-based Material/Energy Sources 380
16.7.2 Economic Drivers Toward Sustainability 381
References 382
17 Biocatalytic and Catalytic Routes for the Production of Bulk and Fine
Chemicals from Renewable Resources 385
Thomas Willke, Ulf Prüße, and Klaus-Dieter Vorlop
17.1.1 Renewable Resources 385
17.1.2 Products 386
17.1.2.1 Bulk Chemicals and Intermediates 386
17.1.2.2 Fine Chemicals and Specialties 386
17.3 Processes 388
17.3.1 Immobilization 389
17.3.2 Biocatalytic Routes from Renewable Resources to Solvents
or Fuels 390
17.3.2.1 Ethanol Production with Bacteria or Yeasts? 390
17.3.3 Biocatalytic Route from Glycerol to 1,3-Propanediol 393
17.3.3.1 Introduction 393
17.3.3.2 The Process 393
Contents XVII
17.3.4 Biocatalytic Route from Inulin to Difructose Anhydride 397

17.3.4.2 Enzyme Screening 398
17.3.4.3 Genetic Engineering 398
17.3.4.4 Fermentation of the Recombinant E. coli 399
17.3.4.5 Enzyme Immobilization and Scale-up 400
17.3.4.6 Summary 401
17.3.5 Chemical Route from Sugars to Sugar Acids 402
17.3.5.2 Gold Catalysts 403
17.3.5.3 Summary 405
References 405
Subjcet Index 407
Volume 2
Part I Biobased Product Family Trees
Carbohydrate-based Product Lines
1 The Key Sugars of Biomass: Availability, Present Non-Food Uses and

Potential Future Development Lines 3
Frieder W. Lichtenthaler
2 Industrial Starch Platform – Status quo of Production,

Modification and Application 61
Dietmar R. Grüll, Franz Jetzinger, Martin Kozich, Marnik M. Wastyn,
and Robert Wittenberger
3 Lignocellulose-based Chemical Products

and Product Family Trees 97
Birgit Kamm, Michael Kamm, Matthias Schmidt, Thomas Hirth,
and Margit Schulze
Lignin Line and Lignin-based Product Family Trees
4 Lignin Chemistry and its Role in Biomass Conversion 151

Gösta Brunow
5 Industrial Lignin Production and Applications 165

E. Kendall Pye
XVIII Contents
Protein Line and Amino Acid-based Product Family Trees
6 Towards Integration of Biorefinery and Microbial Amino Acid

Production 201
Achim Marx, Volker F. Wendisch, Ralf Kelle, and Stefan Buchholz
7 Protein-based Polymers:
Mechanistic Foundations for Bioproduction and Engineering 217
Dan W. Urry
Biobased Fats (Lipids) and Oils
8 New Syntheses with Oils and Fats as Renewable

Raw Materials for the Chemical Industry 253
Ursula Biermann, Wolfgang Friedt, Siegmund Lang, Wilfried Lühs,
Guido Machmüller, Jürgen O. Metzger, Mark Rüsch gen. Klaas,
Hans J. Schäfer, Manfred P. Schneider
9 Industrial Development and Application of Biobased

Oleochemicals 291
Karlheinz Hill
Special Ingredients and Subsequent Products
10 Phytochemicals, Dyes, and Pigments in the Biorefinery Context 315

George A. Kraus
11 Adding Color to Green Chemistry?

An Overview of the Fundamentals and Potential of Chlorophylls 325
Mathias O. Senge and Julia Richter
Part II Biobased Industrial Products, Materials and Consumer Products
12 Industrial Chemicals from Biomass – Industrial Concepts 347

Johan Thoen and Rainer Busch
13 Succinic Acid – A Model Building Block for Chemical Production

from Renewable Resources 367
Todd Werpy, John Frye, and John Holladay
14 Polylactic Acid from Renewable Resources

Patrick Gruber, David E. Henton, and Jack Starr 381
15 Biobased Consumer Products for Cosmetics 409

Thomas C. Kripp
Contents XIX
Part III Biobased Industry:

Economy, Commercialization and Sustainability
16 Industrial Biotech –
Setting Conditions to Capitalize on the Economic Potential 445
Rolf Bachmann and Jens Riese
Subject Index 463

XXI
Editor’s Preface
In the year 2003 when the idea for this set of books “Biorefineries, Biobased In-
dustrial Processes, and Products” arose, the topic of biorefineries as means of
processing industrial material and efficient utilization of renewable products
had been primarily a side issue beyond the borders of the United States of
America. This situation has changed dramatically over the last two years. Today
in almost every developed and emerging nation much work is being conducted
on biorefinery systems, driven by the rising cost of oil and the desire of to move
away from petrochemical-based systems.
In these books we do not claim to describe and discuss everything that be-
longs or even might belong to the topic of biorefineries – that would be impos-
sible. There are many types of biorefinery, and the state of the technology is
changing very rapidly as new and focused effort is directed toward making bio-
refineries a commercial reality. It is a very exciting time for those interested in
biorefineries – technologies for bio-conversion have advanced to a state in which
they are becoming practical on a large scale, economics are leaning more fa-
vourably to the direction of renewable feedstocks, and chemical process knowl-
edge is being applied to biobased systems.
As the editors of the first comprehensive biorefinery book we saw it as our
duty to provide, first of all, a general framework for the subject – addressing
the main issues associated with biorefineries, the principles and basics of biore-
finery systems, the basic technology, industrial products which fall within the
scope of biorefineries, and, finally, technology and products that will fall within
the scope of biorefineries in the future.
To provide a reliable description of the state of biorefinery research and devel-
opment and of industrial implementations, strategies, and future developments
we asked eighty-five experts from universities, research and development insti-
tutes, and industry and commerce to present their views, their results, their im-
plementations, and their ideas on the topic. The results of their contributions
are thirty-three articles organized into seven sections. Our very special thanks
go to all the authors.
We are especially indebted to Dr. Hubert Pelc from Wiley-VCH publishing,
who worked with us on the concept and then, later, on the development and
implementation of the book. Thanks go also to Dr. Bettina Bems from Wiley-
ISBN: 3-527-31027-4
XXII Editor’s Preface
VCH publishing, who managed with admirable professionalism and very much
patience, and to the three editors and eighty-five authors from three different
continents. We are also indebted to Hans-Jochen Schmitt, also of Wiley-VCH
publishing, who had the not always easy task of arranging the manuscripts in a
form ready for publication.
Maybe in 2030, when a biobased economy utilizing biorefinery technology
has become a fundamental part of national and globally connected economies,
someone will wonder what had been thought and written about the subject of
biorefineries at the beginning of the 21st century. Hopefully this book will be
highly representative. Until then we hope it will contribute to the promotion of
international biorefinery developments.
Teltow-Seehof (Germany) Birgit Kamm

Boulder, CO (USA) Patrick R. Gruber
Potsdam (Germany) Michael Kamm
November 2005
XXIII
Foreword
One-hundred-and-fifty years after the beginning of coal-based chemistry and 50
years after the beginning of petroleum-based chemistry industrial chemistry is
now entering a new era. In the twenty-first century utilization of renewable raw
materials will gain importance in the chemical conversion of substances in in-
dustry. Partial or even complete re-adjustment of whole economies to renewable
raw materials will require completely new approaches in research, development,
and production. Chemical and biological sciences will play a leading role in the
building of future industries. New synergies between biological, physical, chem-
ical, and technical sciences must be elaborated and established and special re-
quirements will be placed on raw material and on product-line efficiency and
sustainability. The necessary change from chemistry based on a fossil raw mate-
rial to biology-based modern science and technology is an intellectual challenge
for both researchers and engineers. Chemists should support this change and
collaborate closely with their colleagues in adjoining disciplines, for example
biotechnology, agriculture, forestry, and the material sciences.
The German Chemical Society will help direct this necessary development by
supporting within its structure new kinds of organization for chemists to work
on this subject in universities, research institutes, and industry.
This two-volume book is based on the approach developed by biorefinery-sys-
tems – transfer of the logic and efficiency of today’s petrochemical product lines
and product family trees into manipulation of biomass. Raw biomass materials
are mechanically separated into substances for chemical conversion into other
products by different methods, which may be biotechnological, thermochemical,
and thermal. Review of biomass processes and products developed in the past
but widely forgotten in the petroleum age will be as important as the presenta-
tion of new methods, processes, and products that still require an enormous
amount of research and development today.
Henning Hopf
President of the German Chemical Society
Frankfurt (Germany)
November 2005
ISBN: 3-527-31027-4
XXV
Foreword
On October 5, 2005, the Nobel Prize Committee made an interesting and im-
portant statement with regard to the prize in chemistry. It said, “This represents
a great step forward for ‘green chemistry’, reducing potentially hazardous waste
through smarter production. [This research] is an example of how important ba-
sic science has been applied for the benefit of man, society and the environ-
ment.” By making this statement, the Nobel committee recognized what a new
generation of scientists has known for quite some time, that by working at the
most fundamental level – the molecular level – we are able to design our prod-
ucts, processes, and systems in ways that are sustainable.
There is general recognition that the current system by which we produce the
goods and services needed by society is not sustainable. This unsustainability
takes many forms. It would be legitimate to note that in our current system of
production we rely largely on finite feedstocks extracted from the Earth that are
being depleted at a rate that cannot be sustained indefinitely. It is equally legiti-
mate to recognize that our current production efficiency results in more than
90% of the material used in the production process ending up as waste, i.e. less
than 10% of the material ends up in the desired product. Yet another condition
of unsustainability is in our current energy use; this not only relies largely on fi-
nite energy sources but also results in degradation of the environment that can-
not be continued as the growing population and demands of the developing
world emerge over the course of the twenty-first century. Finally, the products
and processes we have designed since the industrial revolution have accom-
plished their goals without full consideration of their impact and consequence
on humans and the biosphere, with many examples of toxic and hazardous sub-
stances being distributed throughout the globe and into our bodies.
If we are to change this unsustainable path, it will need the direct and com-
mitted engagement of our best scientists and engineers to design the future dif-
ferently from the past. We will need to proceed with a broader perspective such
that when we design for efficiency, effectiveness, and performance, we now
must recognize that these terms include sustainability – a minimized impact
on humans and the environment.
An essential part of meeting the challenge of designing for sustainability will
be based on the nature of the materials we use as starting materials and feed-
stocks. Any sustainable future must ensure that the materials on which we base
ISBN: 3-527-31027-4
XXVI Foreword
our economic infrastructure are renewable rather than depleting. The rate of re-
newability is also important because certainly one could argue that petroleum is
renewable if you have a few million years to wait. Serious analysis would, how-
ever, necessitate that the rate of renewability is connected to the rate of use.
There are options for how to approach this technological challenge, for example
using waste products from one process as a feedstock for another, that are well
thought through in industrial ecology models. There is, however, recognition
that an essential part of a sustainable future will be based on appropriate and
innovative uses of our biologically-based feedstocks.
This book addresses the essential questions and challenges of moving toward
a sustainable society in which bio-based feedstocks, processes, and products are
fundamental pillars of the economy. The authors discuss not only the important
scientific and technical issues surrounding this transition but also the necessary
topics of economics, infrastructure, and policy. It is only by means of this type
of holistic approach that movement toward genuine sustainability will be able
to occur where the societal, economic, and environmental needs are met for the
current generation while preserving the ability of future generations to meet
their needs.
While it will be clear to the reader that the topics presented in this book are
important, it is at least as important that the reader understand that these topics
– and the transition to a sustainable path that they address – are urgent. At this
point in history it is necessary that all who are capable of advancing the transi-
tion to a more sustainable society, engage in doing so with the level of energy,
innovation, and creativity that is required to meet the challenge.
Paul T. Anastas
Director of the Green Chemistry Institute
Washington, D.C.
November, 2005
XXVII
List of Contributors (Volume 1 and 2)
José A. M. Agnelli Robert C. Brown

Universidade Federal de São Carlos Center for Sustainable Environmental
Departamento de Engenharia Technologies
de Materiais Iowa State University
Rodovia Washington Luis (SP-310) 286 Metals Development Building
São Carlos, São Paulo Ames, IO 50011
Brazil USA
Margrethe Andersen Gösta Brunow

AgroFerm A/S Department of Chemistry
Limfjordsvej 4 University of Helsinki
6715 Esbjerg N A. I. Virtasen aukio 1
Denmark 00014 Helsinki
Finland
Rolf Bachmann
McKinsey and Company Inc Stefan Buchholz
Zurich Office Degussa AG
Alpenstrasse 3 Creavis
8065 Zürich Projecthouse ProFerm
Switzerland Rodenbacher Chaussee 4
63403 Hanau-Wolfgang
Ursula Biermann Germany
Fachbereich Chemie
Carl von Ossietzky Universität Rainer Busch
Oldenburg Dow Deutschland GmbH & Co. OHG
Postfach 2603 Industriestrasse 1
26111 Oldenburg 77836 Rheinmünster
Germany Germany
ISBN: 3-527-31027-4
XXVIII List of Contributors
Grant M. Campbell Tim Dodge

Satake Centre for Grain Process Genencor International
Engineering 925 Page Mill Road
School of Chemical Engineering and Palo Alto, CA 94304
Analytical Science USA
The University of Manchester
Sackville Street Donald L. Van Dyne
Manchester M60 1QD Agricultural Economics
UK University of Missouri – Columbia
214c Mumford Hall
Joel R. Cherry Columbia, MO 65211
Novozymes Biotech Inc USA
1445 Drew Ave
Davis, CA 95616 Wolter Elbersen
USA Agrotechnology and Food Innovations
B. V.
Gopal Chotani P.O. Box 17
Genencor International 6700 AA Wageningen
925 Page Mill Road The Netherlands
Palo Alto, CA 94304
USA Steve Fitzpatrick
Biofine
L. Davis Clements 245 Winter Street
Renewable Products Development Waltham, MA 02154
Laboratories USA
3114 NE 45th Ave.
Portland, OR 97213 Paul Fowler
USA The BioComposites Centre
University of Wales
Bruce E. Dale Bangor
Department of Chemical Engineering Gwynedd LL57 2UW
and Materials Science UK
Michigan State University
East Lansing, MI 48824 Wolfgang Friedt
USA Institut für Pflanzenbau
und Pflanzenzüchtung 1
Bill Dean Justus-Liebig-Universität Giessen
Genencor International Heinrich-Buff-Ring 26–32
925 Page Mill Road 35392 Giessen
Palo Alto, CA 94304 Germany
USA
List of Contributors XXIX
John Frye James R. Hettenhaus

Pacific Northwest National Laboratory CEA Inc
P.O. Box 999/K2-12 3211 Trefoil Drive
Richland, WA 99352 Charlotte, NC 28226
USA USA
Patrick R. Gruber Karlheinz Hill

President and CEO Cognis Deutschland GmbH & Co. KG
Outlast Technologies Incorporated Paul-Thomas-Straße 56
5480 Valmont Road 40599 Düsseldorf
Suite 200 Germany
Boulder, CO 80301
USA Thomas Hirth
Fraunhofer-Institut
Dietmar R. Grüll Chemische Technologie
Südzucker Aktiengesellschaft Joseph-von-Fraunhoferstraße 7
Mannheim/Ochsenfurt 76327 Pfinztal
Wormser Strasse 11 Germany
67283 Obrigheim/Pfalz
Germany John Holladay
Pacific Northwest National Laboratory
Daniel J. Hayes P.O. Box 999/K2-12
Department of Chemical Richland, WA 99352
& Environmental Sciences USA
University of Limerick
Limerick Franz Jetzinger
Ireland Zuckerforschung Tulln
Gesellschaft mbH
Michael H. B. Hayes Josef-Reither-Strasse 21–23
Department of Chemical 3430 Tulln
& Environmental Sciences Austria
Limerick Donald L. Johnson
Ireland Biobased Industrial Products
Consulting
David E. Henton 29 Cape Fear Drive
Nature Works LLC Hertford, NC 27944
(former Cargill Dow LLC) USA
15305 Minnetonka Blvd
Minnetonka, MN 55345 Ed de Jong
USA Agrotechnology and
Food Innovations B.V.
P.O. Box 17
6700 AA Wageningen
The Netherlands
XXX List of Contributors
Birgit Kamm Apostolis A. Koutinas

Research Institute Bioactive Polymer Satake Centre for Grain Process
Systems (biopos e.V.) Engineering
Research Centre Teltow-Seehof School of Chemical Engineering and
Kantstraße 55 Analytical Science
14513 Teltow The University of Manchester
Germany Sackville Street
Manchester M60 1QD
Michael Kamm UK
Biorefinery.de GmbH
Stiftstraße 2 Martin Kozich
14471 Potsdam Zuckerforschung Tulln
Germany Gesellschaft mbH
and Josef-Reither-Strasse 21–23
Laboratories Teltow 3430 Tulln
Kantstraße 55 Austria
14513 Teltow
Germany George A. Kraus
Department of Chemistry
Raphael Katzen Iowa State University
9220 Bonita Beach Road 1605 Gilman Hall
Suite 2000 Ames, IA 50011-3111
Bonita Springs, FL 34135 USA
USA
Thomas C. Kripp
Ralf Kelle Wella AG
Degussa AG Abt. FON
R & D Feed Additives Berliner Allee 65
Kantstrasse 2 64274 Darmstadt
33790 Halle/Westfalen Germany
Germany
Stefan Kromus
Pauli Kiel BioRefSYS-BioRefinery Systems
Biotest Aps Innovationszentrum Ländlicher Raum
Gl. Skolevej 47 Auersbach 130
6731 Tjæreborg 8330 Feldbach
Denmark Austria
Seungdo Kim
Department of Chemical Engineering
and Materials Science
East Lansing, MI 48824
USA
List of Contributors XXXI
Siegmund Lang Achim Marx

Institut für Biochemie Degussa AG
und Biotechnologie Creavis
Technische Universität Projecthouse ProFerm
zu Braunschweig Rodenbacher Chaussee 4
Spielmannstraße 7 63403 Hanau-Wolfgang
38106 Braunschweig Germany
Germany
Jürgen O. Metzger
Frieder W. Lichtenthaler Fachbereich Chemie
Institute of Organic Chemistry Carl von Ossietzky Universität
Darmstadt University of Technology Oldenburg
Petersenstraße 22 Postfach 2603
64287 Darmstadt 26111 Oldenburg
Germany Germany
Wilfried Lühs Michael Narodoslawsky

Institut für Pflanzenbau Graz University of Technology
und Pflanzenzüchtung 1 Institute of Resource Efficient and
Justus-Liebig-Universität Giessen Sustainable Systems (RNS)
Heinrich-Buff-Ring 26–32 Inffeldgasse 21 B
35392 Giessen 8010 Graz
Germany Austria
Guido Machmüller Jefter Nascimento

FB 9 – Organische Chemie PHB Industrial SA
Bergische Universität Fazenda da Pedra
GH Wuppertal s/n – C. Postal 02
Gaußstraße 20 CEP 14150 Servana
42097 Wuppertal São Paulo
Germany Brazil
Paulo E. Mantelatto Glenn E. Nedwin

Centro de Tecnologia Canavieira Novozymes Biotech Inc
(formerly Centro de Tecnologia 1445 Drew Ave
Copersucar) Davis, CA 95616
Fazenda Santo Antonio USA
CP 162
13400-970 Piracicaba
Brazil
XXXII List of Contributors
Ulf Prüße Carlos Eduardo Vaz Rossell

Federal Agricultural Research Centre Centro de Tecnologia Canavieira
(FAL) (formerly Centro de Tecnologia
Institute of Technology and Copersucar)
Biosystems Engineering Fazenda Santo Antonio
Bundesallee 50 CP 162
38116 Braunschweig 13400-970 Piracicaba
Germany Brazil
E. Kendall Pye Mark Rüsch gen. Klaas

Lignol Innovations Corp. Department Technology
3650 Westbrook Mall University of Applied Sciences
Vancouver, BC Neubrandenburg
V6S 2L2 Brodaer Straße 2
Canada 17033 Neubrandenburg
Germany
René van Ree Rea
Energy research Centre Hans J. Schäfer
of the Netherlands (ECN) – Organisch-Chemisches Institut
Biomass Department Universität Münster
P.O. Box 1 Corrensstraße 40
1755 ZG Petten 48149 Münster
The Netherlands Germany
Julia Richter Daniel J. Schell

Institut für Chemie National Bioenergy Center
Universität Potsdam National Renewable Energy
Karl-Liebknecht-Str. 24–25 Laboratory
14476 Golm 1617 Cole Blvd.
Germany Golden, CO 80401-3393
USA
Jens Riese
McKinsey and Company Inc Matthias Schmidt
Munich Office Biorefinery.de GmbH
Prinzregentenstraße 22 Stiftstraße 2
80538 München 14471 Potsdam
Germany Germany
Julian R. H. Ross Manfred P. Schneider

University of Limerick FB 9 – Organische Chemie
Department of Chemical Bergische Universität
& Environmental Sciences GH Wuppertal
Limerick Gaußstraße 20
Ireland 42097 Wuppertal
Germany
List of Contributors XXXIII
Margit Schulze Robert van Tuil

FB Angewandte Naturwissenschaften Agrotechnology and Food Innovations
FH Bonn-Rhein-Sieg B.V.
Grantham-Allee 20 P.O. Box 17
53754 Sankt Augustin 6700 AA Wageningen
Germany The Netherlands
Mathias O. Senge Dan W. Urry

SFI Tetrapyrrole Laboratory BioTechnology Institute
School of Chemistry University of Minnesota
Trinity College Dublin Twin Cities Campus
Dublin 2 1479 Gortner Avenue
Ireland Suite 240
St. Paul, MN 55108-6106
Jack Starr USA
Cargill Dow LLC and
15305 Minnetonka Blvd Bioelastics Inc.
Minnetonka, MN 55345 2423 Vestavia Drive
USA Vestavia Hills, AL 35216-1333
USA
Sarah A. Teter
Novozymes Biotech Inc Fernando Valle
1445 Drew Ave Genencor International
Davis, CA 95616 925 Page Mill Road
USA Palo Alto, CA 94304
USA
Johan Thoen
Dow Europe GmbH Klaus-Dieter Vorlop
Bachtobelstrasse 3 Federal Agricultural Research Centre
8810 Horgen (FAL)
Switzerland Institute of Technology and
Biosystems Engineering
Mette Hedegaard Thomsen Bundesallee 50
Risø National Laboratory 38116 Braunschweig
Biosystems Department Germany
Frederiksbovgvej 399
4000 Roskilde Rouhang Wang
Denmark Satake Centre for Grain Process
Engineering
Jeffrey S. Tolan School of Chemical Engineering and
Iogen Corporation Analytical Science
8 Colonnade Road The University of Manchester
Ottawa Sackville Street
Ontario K2E 7M6 Manchester M60 1QD
Canada UK
XXXIV List of Contributors
Marnik M. Wastyn Thomas Willke

Zuckerforschung Tulln Federal Agricultural Research Centre
Gesellschaft mbH (FAL)
Josef-Reither-Strasse 21–23 Institute of Technology and
3430 Tulln Biosystems Engineering
Austria Bundesallee 50
38116 Braunschweig
Colin Webb Germany
Satake Centre for Grain Process
Engineering Robert Wittenberger
School of Chemical Engineering and Zuckerforschung Tulln
Analytical Science Gesellschaft mbH
The University of Manchester Josef-Reither-Strasse 21–23
Sackville Street 3430 Tulln
Manchester M60 1QD Austria
UK
Feng Xu
Volker F. Wendisch Novozymes Biotech Inc
Institute of Biotechnology 1 1445 Drew Ave
Research Center Juelich Davis, CA 95616
52425 Juelich USA
Germany
Todd Werpy
P.O. Box 999/K2-12
Richland, WA 99352
USA
Biorefineries – Industrial Processes and Products
© 2006 WILEY-VCH Verlag GmbH & Co.
Part I
Background and Outline – Principles and Fundamentals
ISBN: 3-527-31027-4
3
1
Biorefinery Systems – An Overview
1.1
Introduction
The preservation and management of our diverse resources are fundamental po-
litical tasks to foster sustainable development in the 21st century. Sustainable
economic growth requires safe and sustainable resources for industrial produc-
tion, a long-term and confident investment and finance system, ecological
safety, and sustainable life and work perspectives for the public. Fossil resources
are not regarded as sustainable, however, and their availability is more than
questionable in the long-term. Because of the increasing price of fossil re-
sources, moreover, the feasibility of their utilization is declining.
It is, therefore, essential to establish solutions which reduce the rapid con-
sumption of fossil resources, which are not renewable (petroleum, natural gas,
coal, minerals). A forward looking approach is the stepwise conversion of large
parts of the global economy into a sustainable biobased economy with bio-
energy, biofuels, and biobased products as its main pillars (Fig. 1.1).
Fig. 1.1 3-Pillar model of a future biobased economy.
ISBN: 3-527-31027-4
4 1 Biorefinery Systems – An Overview
Whereas for energy production a variety of alternative raw materials (wind,

sun, water, biomass, nuclear fission and fusion) can be established, industry
based on conversion of sustainable material, for example the chemical industry,
industrial biotechnology, and also the fuel generation, depends on biomass, in
particular mainly on plant biomass.
Some change from the today’s production of goods and services from fossil to
biological raw materials will be essential. The rearrangement of whole econo-
mies to implement biological raw materials as a source with increased value re-
quires completely new approaches in research and development. On the one
hand, biological and chemical sciences will play a leading role in the generation
of future industries in the 21st century. On the other hand, new synergies of
biological, physical, chemical, and technical sciences must be elaborated and es-
tablished. This will be combined with new traffic technology, media- and infor-
mation technology, and economic and social sciences. Special requirements will
be placed on both the substantial converting industry and research and develop-
ment with regard to raw material and product line efficiency and sustainability.
The development of substance-converting basic product systems and poly-
product systems, for example biorefineries, will be the “key for the access to an
integrated production of food, feed, chemicals, materials, goods, and fuels of
the future” [1].
1.2
Historical Outline
1.2.1
Historical Technological Outline and Industrial Resources
Today’s biorefinery technologies are based (1) on the utilization of the whole
plant or complex biomass and (2) on integration of traditional and modern pro-
cesses for utilization of biological raw materials. In the 19th and the beginning
of the 20th century large-scale utilization of renewable resources was focused
on pulp and paper production from wood, saccharification of wood, nitration of
cellulose for guncotton and viscose silk, production of soluble cellulose for fi-
bers, fat curing, and the production of furfural for Nylon. Furthermore, the
technology of sugar refining, starch production, and oil milling, the separation
of proteins as feed, and the extraction of chlorophyll for industrial use with al-
falfa as raw material were of great historical importance. But also processes like
wet grinding of crops and biotechnological processes like the production of
ethanol, acetic acid, lactic acid, and citric acid used to be fundamental in the
19th and 20th century.
1.2.2
The Beginning – A Digest
1.2.2.1 Sugar Production

The history of industrial conversion of renewable resources is longer than 200
years. Utilization of sugar cane has been known in Asia since 6000 BC and im-
ports of cane sugar from oversea plantations have been established since the
15th century. The German scientist A. S. Marggraf was a key initiator of the
modern sugar industry. In 1748 he published his research on the isolation of
crystalline sugar from different roots and beet [2, 3]. Marggraf’s student, F. C.
Achard, was the first to establish a sugar refinery based on sugar beet, in Cu-
nern/Schlesien, Poland, in 1801.
1.2.2.2 Starch Hydrolysis

In 1811, the German pharmacist G. S. C. Kirchhoff found that when potato
starch was cooked in dilute acid the starch was converted into “grape sugar”
(i.e. d-glucose or dextrose) [4]. This was not only a very important scientific re-
sult but also the starting point of the starch industry. In 1806 the French emper-
or Napoleon Bonaparte introduced an economic continental blockade which
considerably limited overseas trade in cane sugar. Thus, starch hydrolysis be-
came of interest for the economy. The first starch sugar plant was established
in Weimar, Germany, in 1812, because of a recommendation of J.W. Döbereiner
to grand duke Carl August von Sachsen-Weimar. Successful development of the
sugar beet industry, however, initially obstructed further development of the
starch industry [5]. In 1835, the Swedish Professor J. J. Berzelius developed en-
zymatic hydrolyses of starch into sugar and introduced the term “catalysis”.
1.2.2.3 Wood Saccharification

In 1819 the French plant chemist H. Braconnot discovered that sugar (glucose)
is formed by treatment of wood with concentrated sulfuric acid. 1855, G. F. Mel-
sens reported that this conversion can be carried out with dilute acid also. Acid
hydrolysis can be divided into two general approaches, based on (1) concen-
trated acid hydrolysis at low temperature and (2) dilute acid hydrolysis at high
temperature. Historically, the first commercial processes, named wood saccharif-
ication, were developed in 1901 by A. Classen (Ger. Patent 130 980), employing
sulfuric acid, and in 1909 by M. Ewen and G. Tomlinson (US Patent 938 208),
working with dilute sulfuric acid. Several plants were in operation until the end
of World War I. Yields of these processes were usually low, in the range 75–130
liter per ton wood dry matter only [6, 7]. Technologically viable processes were,
however, developed in the years between World War I and World War II. The
German chemist Friedrich Bergius was one of the developers. The sugar frac-
tions generated by wood hydrolyses have a broad spectrum of application. An
important fermentation product of wood sugar of increasing interest is ethanol.
Ethanol can be used as fuel either blended with traditional hydrocarbon fuel or
as pure ethanol. Ethanol is also an important platform chemical for further pro-
cessing [8].
1.2.2.4 Furfural
Döbereiner was the first to report the formation and separation of furfural by
distillation of bran with diluted sulfuric acid in 1831. In 1845 the English Che-
mist G. Fownes proposed the name “furfurol” (furfur – bran; oleum – oil). Later
the suffix “ol” was changed to “al” because of the aldehyde function [9, 10].
Treatment of hemicellulose-rich raw materials with dry steam in the presence of
hydrogen chloride gave especially good results [11]. Industrial technology for pro-
duction of furfural from pentose is based on a development of an Anglo-American
company named Quaker Oats. The process was been developed in the nineteen-
twenties [E. P. 203 691 (1923), F. P. 570 531 (1923)]. Since 1922 Quaker Oats Cereal
Mill in Cedar Rapids/Iowa, USA, has produced up to 2.5 tons of furfural per day
from oat husks. Since 1934 the process had been established as an industrial fur-
fural plant. Furfural was the cheapest aldehyde, at 16–17 cents per lb (lb = pound;
1 metric ton = 1000 kg = 2204.62442 lb; 1 kg = 0.453592 lb) [12]. Until approxi-
mately 1960 DuPont used furfural as a precursor of Nylon-6.6. Furfural has since
been substituted by fossil based precursors.
1.2.2.5 Cellulose and Pulp

In 1839 the Frenchman A. Payen discovered that after treatment of wood with
nitric acid and subsequent treatment with a sodium hydroxide solution a resi-
due remained which he called “les cellules”, cellulose [13]. In 1854 caustic soda
and steam were used by the Frenchman M. A. C. Mellier to disintegrate cellulose
pulp from straw. In 1863 the American B. C. Tilgham registered the first patent
for production of cellulose by use of calcium bisulfite. Together with his brother,
Tilgham started the first industrial experiments to produce pulp from wood by
treatment with hydrogen sulfite. This was 1866 at the paper mill Harding and
Sons, Manayunk, close to Philadelphia. In 1872 the Swedish Engineer C. D. Ek-
man was the first to produce sulfite cellulose by using magnesium sulfite as
cooking agent [14]. By 1900 approximately 5200 pulp and paper mills existed
worldwide, most in the USA, approximately 1300 in Germany, and 512 in
France.
1.2.2.6 Levulinic Acid

In 1840 the Dutch Professor G. J. Mulder (who also introduced the name “pro-
tein”) synthesized levulinic acid (4-oxopentanoic acid, c-ketovaleric acid) by heat-
ing fructose with hydrochloride for the first time. The former term “levulose”
for fructose gave the levulinic acid its name [15]. Although levulinic acid has
been well known since the 1870s when many of its reactions (e.g. esters) were
established, it has never reached commercial use in any significant volume. In

the 1940s commercial levulinic acid production was begun in an autoclave in
the United States by A. E. Staley, Dectur, Illinois [16]. At the same time utiliza-
tion of hexoses from low-cost cellulose products was examined for the produc-
tion of levulinic acid [17]. As early as 1956 levulinic acid was regarded as a plat-
form chemical with high potential [18].
1.2.2.7 Lipids
From 1850 onward European import of tropical plant fats, for example palm-oil
and coconut oil, started. Together with the soda process, invented by the French
N. Leblanc in 1791, the industrialization of the soap production began and soap
changed from luxury goods into consumer goods. The developing textile indus-
try also demanded fat based products. In 1902 the German chemist W. Nor-
mann discovered that liquid plant oils are converting into tempered fat by aug-
mentation of hydrogen. Using nickel as catalyst Norman produced tempered
stearic acid by catalytic hydration of liquid fatty acids [19]. The so called “fat
hardening” led to the use of European plant oils in the food industry (marga-
rine) and other industries.
1.2.2.8 Vanillin from Lignin

In 1874 the German chemists W. Haarmann and F. Tiemann were the first to
synthesize vanillin from the cambial juice of coniferous wood. In 1875 the com-
pany Haarmann and Reimer was founded. The first precursor for the produc-
tion of vanillin was coniferin, the glucoside of coniferyl alcohol. This precursor
of lignin made from cambial juice of coniferous trees was isolated, oxidized to
glucovanillin, and then cleaved into glucose and vanillin [20]. This patented pro-
cess [21] opened the way to industrial vanillin production. It was also the first
industrial utilization of lignin. Besides the perfume industry, the invention was
of great interest to the upcoming chocolate industry. Later, however, eugenol
(1-allyl-4-hydroxy-3-methoxybenzene), isolated from clove oil, was used to pro-
duce vanillin. Today, vanillin production is based on lignosulfonic acid which
is a side product of wood pulping. The lignosulfonic acid is oxidized with air
under alkaline conditions [22, 23].
1.2.2.9 Lactic Acid

In 1895 industrial lactic acid fermentation has been developed by the pharma-
ceutical entrepreneur A. Boehringer. The Swedish pharmacist C. W. Scheele had
already discovered lactic acid in 1780 and the conversion of carbohydrates into
lactic acid had been known for ages in food preservation (e.g. Sauerkraut) or
agriculture (silage fermentation). Because of the activity of Boehringer the Ger-
man company Boehringer-Ingelheim can be regarded as the pioneer of indus-
trial biotechnology. Both the process and the demand for lactic acid by dyeing
factories, and the leather, textile, and food industries made the company the
leading supplier. In 1932 W. H. Carothers, who was also the inventor of polya-
mide-6.6, developed, together with van Natta, a polyester made from lactic acid,
poly(lactic acid) [24]. In the late 1990s this poly(lactic acid) was commercialized
by the company NatureWorks (Cargill, the former Cargill Dow) [25].
1.2.3
The Origins of Integrated Biobased Production
In the year 1940 the German chemist P. von Walden (noted for his “Inversion
of configuration at substitution reactions”, the so-called “Walden-Reversion”) cal-
culated that in 1940 Germany produced 13 million tons of cellulose leaving 5 to
6 million tons of lignin suitable only as wastage. He then formulated the ques-
tion: How long can national economy tolerate this [26]? Approaches to inte-
grated production during industrial processing of renewable primary products
have a long tradition, starting from the time when industrial cellulose produc-
tion expanded continuously, as also did the related waste-products. Typical ex-
amples of this will be mentioned.
As early as 1878 A. Mitscherlich, a German chemist, started to improve the
sulfite pulp process by fermentation of sugar to ethyl alcohol – it should be
mentioned that sugar is a substance in the waste liquor during sulfite pulp pro-
duction. He also put into practice a procedure to obtain paper glue from the
waste liquor. Both processes were implemented in his plant located in Hof, Ger-
many, in the year 1898 [27].
In 1927 the American Marathon Corporation assigned a group of chemists
and engineers to the task of developing commercial products from the organic
solids in the spent sulfite liquor from the Marathon’s Rothschild pulp and paper
operations close to Wausau, Wisconsin, USA. The first products to show prom-
ise were leather tanning agents. Later, the characteristics of lignin as dispersing
agents became evident. By the mid 1930s, with a considerable amount of basic
research accomplished, Marathon transferred operations from a research pilot
plant to full-scale production [28].
One of the most well known examples is the production of furfural by the Qua-
ker Oats Company since 1922, thus coupling food, i.e. oat flakes, production and
chemical products obtained from the waste [10] (Section 1.2.2). On the basis of fur-
fural a whole section of chemical production developed – furan chemistry.
Agribusiness, especially, strived to achieve combined production from the very
beginning. Modern corn refining started in the middle of the 18th century
when T. Kingsford commenced operation of his corn refining plant in Oswego,
New York [29]. Corn refining is distinguished from corn milling because the re-
fining process separates corn grain into its components, for example starch, fi-
ber, protein and oil, and starch is further processed into a substantial number
of products [30].
The extensive usage of green crops has been aim of industry for decades, be-
cause there are several advantages. Particularly worthy of mention is the work
of Osborn (1920) and Slade and Birkinshaw (1939) on the extraction of proteins
from green crops, for example grass or alfalfa [31].
In 1937 N. W. Pirie developed the technical separation and extraction methods
needed for this use of green crops [32, 33]. By means of sophisticated methods
all the botanical material should have been used, both for production of animal
feed, isolated proteins for human nutrition, and as raw material for further in-
dustrial processes, for example glue production. The residual material, juices
rich in nutrients, had initially been used as fertilizer; later they were used for
generation of fermentation heat based on biogas production [34, 35].
These developments resulted in market-leading technology, for example the
Proxan and Alfaprox procedures, used for generation of protein–xanthophyll
concentrates, including utilization of the by-products, however, predominantly
in agriculture [36].
In the United States commercial production of chlorophyll and carotene by
extraction from alfalfa leaf meal had started in 1930 [37, 38]. For example
Strong, Cobb and Company produced 0.5 ton chlorophyll per day from alfalfa
as early as 1952. The water-soluble chlorophyll, or chlorophyllin, found use as
deodorizing agent in toothpastes, soaps, shampoos, candy, deodorants, and
pharmaceuticals [39].
A historical important step for today’s biorefinery developments was the in-
dustry-politics-approach of “Chemurgy”, founded in 1925 in the US by the Che-
mist W. J. Hale, son-in-law of H. Dow, the founder of Dow Chemical, and C. H.
Herty, a former President of the American Chemical Society. They soon found
prominent support from H. Ford and T. A. Edisons. Chemurgy, an abbreviation
of “chemistry” and “ergon”, the Greek word for work [40], means by analogy
“chemistry from the acre” that is the connection of agriculture with the chemi-
cal industry.
Chemurgy was soon shown to have a serious industrial political philosophy –
the objective of utilizing agricultural resources, nowadays called renewable re-
sources, in industry. There have been common conferences between agriculture,
industry, and science since 1935 with a national council called the “National
Farm Chemurgic Council” [41]. The end of Chemurgy started with the flooding
of the world market with cheap crude oil after World War II; numerous inven-
tions and production processes remained, however, and are again highly news-
worthy. One was a car, introduced by Henry Ford 1941, whose car interior lin-
ing and car body consisted 100% of bio-synthetics; to be specific it had been
made from a cellulose meal, soy meal, formaldehyde resin composite material
in the proportions 70% : 20% : 10%, respectively. The alternative fuel for this car
was pyrolysis methanol produced from cannabis. Throughout the thirties more
than 30 industrial products based on soy bean were created by researchers from
the Ford company; this made it necessary to apply complex conversion methods
[42]. Hale was a Pioneer of ethyl alcohol and hydrocarbon fuel mixture (Power
Alcohol, Gasohol) [43]. This fuel mixture, nowadays called E10-Fuel, consisting
of 10 percent bioethanol and 90 percent hydrocarbon-based fuel, has been the
national standard since the beginning of this millennium in the United States.
Associated with the work of Bergius, 1933 [44], and Scholler, 1923 and 1935
[45, 46], wood saccharification was reanimated at the end of WW II. Beside opti-
mization of the process, use of lignocelluloses was of great interest. The con-
tinuously growing agribusiness left behind millions of tons of unused straw.
Two Americans, Othmer and Katzen, were the main pioneers in the field of
wood saccharification [47]. Between the years 1935 and 1960 several hydrolysis
plants were built in Germany and the United States; in these deal, wood flour,
surplus lumber, and also straw were hydrolyzed [48]. One of the most well
known plants are those of Scholler/Tornesch located in Tornesch, Germany,
with a production rate of 13,000 tons per year, in Dessau, Germany, production
rate 42,000 tons per year, based on wood, in Holzminden, Germany, production
rate of 24,000 tons per year, also based on wood, in Ems, Switzerland, with a
production rate of 35,000 tons per year, also wood based, and the plants in Ma-
dison and Springfield, United States, and the Bergius plants in Rheinau, Ger-
many (Rheinau I, built 1930, with a production rate of 8000 tons per year, based
on surplus lumber; Rheinau II, built 1960 with a production rate of 1200 tons
per year, based on wood) and the plant in Regensburg, Germany, with a produc-
tion rate of 36,000 tons per year [49].
During WW II the plant in Springfield, Oregon, US (using the Scholler-Tor-
nesch process as modified by Katzen) produced 15,000 gallons of ethyl alcohol
per day from 300 tons wood flour and sawdust, i.e. 50 gallons per ton of wood
[50]. The plant in Tornesch, Germany, has been producing approximately 200 li-
ters of ethyl alcohol, purity 100%, per ton wood and approximately 40 kg yeast
per ton of wood. In 1965 there were 14 plants in what was then the Soviet
Union, with a total capacity of 700,000 tons per year and an overall annual
wood consumption of 4 million tons [6].
During the nineteen-sixties wood chemistry had its climax. Projects had been
developed, which made it possible to produce nearly all chemical products on
the basis of wood. Examples are the complex chemical technological approaches
of wood processing from Timell 1961 [51], Stamm 1964 [52], James 1969 [53],
Brink and Pohlmann 1972 [54], and the wood-based chemical product trees by
Oshima 1965 [55]. Although these developments did not make their way into in-
dustrial production, they are an outstanding platform for today’s lignocellulose
conversions, product family trees, and LCF biorefineries (Section 1.5.2).
Most of the above mentioned technologies and products, some of which were
excellent, could not compete with the fossil-based industry and economy; nowa-
days, however, they are prevailing again. The basis for this revival started in the
seventies, when the oil crisis and continuously increasing environmental pollution
resulted in a broad awareness that plants could be more than food and animal
feed. At the same time the disadvantages of intensive agricultural usage, for exam-
ple over-fertilization, soil erosion, and the enormous amounts of waste, were re-
vealed. From this situation developed complex concepts, which have been pub-
lished, in which the aim was, and still is, technological and economical coopera-
tion of agriculture, forestry, the food-production industry, and conventional indus-
try, or at least consideration of integrated utilization of renewable resources.
1.3 Situation 11
Typical examples of this thinking were:

· integrated industrial utilization of wood and straw [56];
· industrial utilization of fast growing wood-grass [57, 58];
· complex utilization of green biomass, for example grass and alfalfa, by agri-
culture and industry [59–61];
· corn wet-grinding procedures with associated biotechnological and chemical
product lines [62];
· modern aspects of thermochemical biomass conversion [63];
· discussion of the concept “organic chemicals from biomass” with main focus
on biotechnological methods and products (white biotechnology) [64–66] and
industrial utilization of biomass [67].
These rich experiences of the industrial utilization of renewable resources, new

agricultural technology, biotechnology, and chemistry, and the changes in ecol-
ogy, economics, and society led inevitably to the topic of complex and integrated
substantial and energy utilization of biomass and, finally, to the biorefinery.
1.3
Situation
1.3.1
Some Current Aspects of Biorefinery Research and Development
Since the beginning of the 1990s the utilization of renewable resources for pro-
duction of non-food products has fostered research and development which has
received increasing attention from industry and politicians [68–70]. Integrated
processes, biomass refinery technology, and biorefinery technology have become
object of research and development. Accordingly, the term “biorefinery” was es-
tablished in the 1990s [1, 71–80]. The respective biorefinery projects are focused
on the fabrication of fuels, solvents, chemicals, plastics, and food for human
beings. In some countries these biorefinery products are made from waste bio-
mass. At first the main processes in the biorefinery involved ethanol fermenta-
tion for fuels (ethanol-oriented biorefineries) [81–85], lactic acid (LA) fermenta-
tion [25, 86], propanediol (PDO) fermentation [87], and the lysine fermentation
[88] especially for polymer production. The biobased polymers poly(lactic acid)
[25], propandiol-derived polymers [89], and polylysine [88] have been completed
by polyhydroxyalkanoates [90] and polymerized oils [91].
Many hybrid technologies were developed from different fields, for example
bioengineering, polymer chemistry, food science and agriculture. Biorefinery sys-
tems based on cereals [92, 93], lignocelluloses [94, 95], and grass and alfalfa [35,
96], and biorefinery optimization tools are currently being developed [97, 98].
The integration of molecular plant genetics to support the raw material supply
is currently being discussed intensely [99, 100].
Broin and Associates has begun the development of a second generation of dry
mill refineries and E. I. du Pont de Nemours has developed an integrated corn-
based biorefinery. In 2001 NatureWorks LCC (to Cargill, former Cargill Dow
LLC) started the industrial production of PLA (PLA-oriented Biorefinery) on the
basis of maize.
Biorefineries are of interest ecologically [101], economically, and to business,
government, and politicians [102–107]. National programs [108, 109], biobased
visions [110], and plans [111] have been developed and the international ex-
change of information is increasing, for example as a result, among others, of
series of international congresses and symposia:
1. BIO World Congress on Industrial Biotechnology and Bioprocessing [112];
2. biomass conferences [113, 114];
3. The Green and Sustainable Chemistry Congress [115]; and
4. the Biorefinica symposia series [116, 117].
Currently, biorefinery systems are in the stage of development world-wide. An

overview of the main aspects, activity, and discussions is the content of this
book. An attempt to systematize the topic “Biorefinery” will be presented below.
1.3.2
Raw Material Biomass
Nature is a permanently renewing production chain for chemicals, materials,

fuels, cosmetics, and pharmaceuticals. Many of the biobased industry products
currently used are results of direct physical or chemical treatment and proces-
sing of biomass, for example cellulose, starch, oil, protein, lignin, and terpenes.
On one hand one must mention that because of the help of biotechnological
processes and methods, feedstock chemicals are produced such as ethanol, buta-
nol, acetone, lactic acid, and itaconic acid, as also are amino acids, e.g. glut-
aminic acid, lysine, tryptophan. On the other hand, only 6 billion tons of the
yearly produced biomass, 1.7–2.0 ´ 1011 tons, are currently used, and only 3 to
3.5% of this amount is used in non-food applications, for example chemistry
[118].
The basic reaction of biomass is photosynthesis according to:
nCO2 nH2 O ! CH2 On nO2
Industrial utilization of raw materials from agriculture, forestry, and agriculture

is only just beginning. There are several definitions of the term “biomass” [118]:
· the complete living, organic matter in our ecological system (volume/non-spe-
cific)
· the plant material constantly produced by photosynthesis with an annual
growth of 170 billion tons (marine plants excluded)
· the cell-mass of plants, animals, and microorganism used as raw materials in
microbiological processes
1.3 Situation 13
Biomass is defined in a recent US program [108, 109]: “The term “biomass”

means any organic matter that is available on a renewable or recurring basis
(excluding old-growth timber), including dedicated energy crops and trees, agri-
cultural food and feed crop residues, aquatic plants, wood and wood residues,
animal wastes, and other waste materials.”
For this reason it is essential to define biomass in the context of the industrial
utilization. A suggestion for a definition of “industrial biomass” [108, 109] is:
“The term “industrial biomass” means any organic matter that is available on a
renewable or recurring basis (excluding old-growth timber), including dedicated
energy crops and trees, agricultural food and feed crop residues, aquatic plants,
wood and wood residues, animal wastes, and other waste materials usable for
industrial purposes (energy, fuels, chemicals, materials) and include wastes and
co-wastes of food and feed processing.”
Most biological raw material is produced in agriculture and forestry and by
microbial systems. Forestry plants are excellent raw materials for the paper and
Fig. 1.2 Products and product classes based on biological raw materials [78].
cardboard, construction, and chemical industries. Field fruits are an pool of or-
ganic chemicals from which fuels, chemicals, chemical products, and biomate-
rials are produced (Fig. 1.2), [69]. Waste biomass and biomass of nature and
agricultural cultivation are valuable organic reservoirs of raw material and must
be used in accordance with their organic composition. During the development
of biorefinery systems the term “waste biomass” will become obsolete in the
medium-term [119].
1.3.3
National Vision and Goals and Plan for Biomass Technology in the United States
Industrial development was pushed by the US President [108] and by the US

congress [109], initially in 2000. In the USA it is intended that by 2020 at least
25% of organic-carbon-based industrial feedstock chemicals and 10% of liquid
fuels (compared with levels in 1994) will be produced by biobased industry. This
would mean that more than 90% of the consumption of organic chemicals in
the US and up to 50% of liquid fuel needs would be biobased products [1].
The Biomass Technical Advisory Committee (BTAC) of the USA in which
leading representatives of industrial companies, for example Dow Chemical,
E. I. du Pont de Nemours, Cargill Dow LLC, and Genencor International, and
Corn growers associations and the Natural Resources Defense Council are in-
volved and which acts as advisor to the US government, has made a detailed
plan with steps toward targets of 2030 with regard to bioenergy, biofuels, and
bioproducts (Table 1.1) [110].
Simultaneously, the plan Biomass Technology in the United States has been pub-
lished [111] in which research, development, and construction of biorefinery
Table 1.1 The US national vision goals for biomass technolo-

gies by the Biomass Technical Advisory Committee [110].
Year Current 2010 2020 2030
BioPower (BioEnergy) 2.8% 4% 5% 5%

Biomass share of electricity (2.7 quad) a) (3.2 quad) (4.0 quad) (5.0 quad)
and heat demand in utilities
and industry
BioFuels 0.5% 4% 10% 20%
Biomass share of demand for (0.15 quad) (1.3 quad) (4.0 quad) (9.5 quad)
transportation fuels
BioProducts 5% 12% 18% 25%
Share of target chemicals that
are biobased
a) 1 quad = 1 quadrillion BTU = 1 German billiarde BTU;

BTU = British thermal unit; 1 BTU = 0.252 kcal,
1 kW = 3413 BTU, 1 kcal = 4.186 kJ
1.3 Situation 15
demonstration plants are determined. Research and development are necessary

to:
1. increase scientific understanding of biomass resources and improve the tailor-
ing of those resources;
2. improve sustainable systems to develop, harvest, and process biomass re-
sources;
3. improve efficiency and performance in conversion and distribution processes
and technologies for development of a host of biobased products and
4. create the regulatory and market environment necessary for increased devel-
opment and use of biobased products.
The Biomass Advisory Committee has established specific research and develop-
ment objectives for feedstock production research. Target crops should include
oil and cellulose-producing crops that can provide optimum energy content and
usable plant components. Currently, however, there is a lack of understanding
of plant biochemistry and inadequate genomic and metabolic information about
many potential crops. Specific research to produce enhanced enzymes and
chemical catalysts could advance biotechnology capabilities.
1.3.4
Vision and Goals and Plan for Biomass Technology
in the European Union and Germany
In Europe there are already regulations about substitution of nonrenewable

resources by biomass in the field of biofuels for transportation [120] and the
“Renewable energy law” of 2000 [121]. According to the EC Directive “On the pro-
motion of the use of biofuels” the following products are regarded as “biofuels”:
(a) “bioethanol”, (b) “biodiesel”, (c) “biogas”, (d) “biomethanol”, (e) “biodimethyl
ether”,
(f) “bio-ETBE (ethyl tertiary-butyl ether)” on the basis of bioethanol,
(g) “bio-MTBE (methyl tertiary butyl ether)” on the basis of biomethanol, and
(h) “synthetic biofuels”, (i) “biohydrogen”, (j) pure vegetable oil
Member States of the EU have been requested to define national guidelines for
a minimum amounts of biofuels and other renewable fuels (with a reference
value of 2% by 2005 and 5.75% by 2010 calculated on the basis of energy con-
tent of all petrol and diesel fuels for transport purposes). Table 1.2 summarizes
this goal of the EU and also those of Germany with regard to establishment of
renewable energy and biofuel [122, 123].
Today there are no guidelines concerning “biobased products” in the Europe-
an Union and in Germany. After passing directives relating to bioenergy and
biofuels, however, such a decision is on the political agenda. The “biofuels” di-
rective already includes ethanol, methanol, dimethyl ether, hydrogen, and bio-
mass pyrolysis which are fundamental product lines of the future biobased
chemical industry.
Table 1.2 Targets of the EU and Germany with regard to the

introduction of technologies based on renewable resources.
Year 2001 2005 2010 2020–2050
Bioenergy 7.5% – 12.5% 26% (2030)

Share of wind power, photo- 58% (2050)
voltaics, biomass and geother-
mal electricity and heat
demand in utilities and indus-
try
Biofuels 1.4% 2.8% 5.75% 20% (2020)
Biomass share of demand in
transportation fuels (petrol
and diesel fuels)
Biobased Products 8% – – –
Share of target chemicals that
are biobased
In the year 2003, an initiative group called “Biobased Industrial Products”

consisting of members from industry, small and middle-class businesses, and
research, and development facilities met and formulated a strategy paper, called
“BioVision 2030” [124]. This strategy paper has been included in the resolution
of the German Government (Deutscher Bundestag) on the topic “Accomplish
basic conditions for the industrial utilization of renewable resources in Ger-
many” [125]. An advisory committee consisting of members of the chemical in-
dustry, related organizations, research facilities, and universities has been estab-
lished to generate a plan concerning the formulation of the objectives for the
third column, bio-products in Europe (Table 1.2) [126].
1.4
Principles of Biorefineries
1.4.1
Fundamentals
Biomass, similar to petroleum, has a complex composition. Its primary separa-

tion into main groups of substances is appropriate. Subsequent treatment and
processing of those substances lead to a whole range of products. Petrochemis-
try is based on the principle of generating simple to handle and well defined
chemically pure products from hydrocarbons in refineries. In efficient product
lines, a system based on family trees has been built, in which basic chemicals,
intermediate products, and sophisticated products are produced. This principle
of petroleum refineries must be transferred to biorefineries. Biomass contains
the synthesis performance of the nature and has different C : H : O : N ratio from
petroleum. Biotechnological conversion will become, with chemical conversion,

a big player in the future (Fig. 1.3).
Thus biomass can already be modified within the process of genesis in such
a way that it is adapted to the purpose of subsequent processing, and particular
target products have already been formed. For those products the term “precur-
sors” is used. Plant biomass always consists of the basic products carbohydrates,
lignin, proteins, and fats, and a variety of substances such as vitamins, dyes, fla-
vors, aromatic essences of very different chemical structure. Biorefineries com-
bine the essential technologies which convert biological raw materials into the
industrial intermediates and final products (Fig. 1.4).
A technically feasible separation operation, which would enable separate use
or subsequent processing of all these basic compounds, is currently in its initial
stages only. Assuming that of the estimated annual production of biomass by
biosynthesis of 170 billion tons 75% is carbohydrates, mainly in the form of cel-
lulose, starch, and saccharose, 20% lignin, and only 5% other natural com-
pounds such as fats (oils), proteins, and other substances [127], the main atten-
tion should first be focused on efficient access to carbohydrates, and their sub-
sequent conversion to chemical bulk products and corresponding final products.
Glucose, accessible by microbial or chemical methods from starch, sugar, or cel-
lulose, is, among other things, predestined for a key position as a basic chemi-
cal, because a broad range of biotechnological or chemical products is accessible
from glucose. For starch the advantage of enzymatic compared with chemical
hydrolysis is already known [128].
For cellulose this is not yet realized. Cellulose-hydrolyzing enzymes can only
act effectively after pretreatment to break up the very stable lignin/cellulose/
Fig. 1.3 Comparison of the basic-principles of the petroleum refinery and the biorefinery.
Fig. 1.4 Providing code-defined basic substances (via fraction-

ation) for development of relevant industrial product family
trees [78, 79].
Fig. 1.5 Possible schematic diagram of biorefinery for

precursor-containing biomass with preference for carbo-
hydrates [78, 79].
hemicellulose composites [129]. These treatments are still mostly thermal, ther-
momechanical, or thermochemical, and require considerable input of energy.
The arsenal for microbial conversion of substances from glucose is large, and
the reactions are energetically profitable. It is necessary to combine degradation
processes via glucose to bulk chemicals with the building processes to their sub-
sequent products and materials (Fig. 1.5).
Among the variety of microbial and chemical products possibly accessible
from glucose, lactic acid, ethanol, acetic acid, and levulinic acid, in particular,
are favorable intermediates for generation of industrially relevant product family
trees. Here, two potential strategies are considered: first, development of new,
possibly biologically degradable products (follow-up products from lactic and le-
vulinic acids) or, second, entry as intermediates into conventional product lines
(acrylic acid, 2,3-pentanedione) of petrochemical refineries [78].
1.4.2
Definition of the Term “Biorefinery”
The young working field “Biorefinery Systems” in combination with “Biobased

Industrial Products” is, in various respects, still an open field of knowledge.
This is also reflected in the search for an appropriate description. A selection is
given below.
The term “Green Biorefinery” was been defined in the year 1997 as: “Green
biorefineries represent complex (to fully integrated) systems of sustainable, en-
vironmentally and resource-friendly technologies for the comprehensive (holis-
tic) material and energetic utilization as well as exploitation of biological raw
materials in form of green and residue biomass from a targeted sustainable re-
gional land utilization” [73]. The original term used in Germany “complex con-
struction and systems” was substituted by “fully integrated systems”. The US
Department of Energy (DOE) uses the following definition [130]: “A biorefinery
is an overall concept of a processing plant where biomass feedstocks are con-
verted and extracted into a spectrum of valuable products. Based on the petrol-
chemical refinery.” The American National Renewable Energy Laboratory
(NREL) published the definition [131]: “A biorefinery is a facility that integrates
biomass conversion processes and equipment to produce fuels, power, and
chemicals from biomass. The biorefinery concept is analogous to today’s petro-
leum refineries, which produce multiple fuels and products from petroleum. In-
dustrial biorefineries have been identified as the most promising route to the
creation of a new domestic biobased industry.”
There is an agreement about the objective, which is briefly defined as: “Devel-
oped biorefineries, so called “phase III-biorefineries” or “generation III-biorefi-
neries”, start with a biomass–feedstock-mix to produce a multiplicity of most
various products by a technologies-mix” [74] (Fig. 1.6).
An example of the type “generation-I biorefinery” is a dry milling ethanol
plant. It uses grain as a feedstock, has a fixed processing capability, and pro-
duces a fixed amount of ethanol, feed co-products, and carbon dioxide. It has al-
most no flexibility in processing. Therefore, this type can be used for compar-
able purposes only.
An example of a type “generation-II biorefinery” is the current wet milling
technology. This technology uses grain feedstock, yet has the capability to pro-
Fig. 1.6 Basic principles of a biorefinery (generation III biorefinery) [78].
duce a variety of end products depending on product demand. Such products

include starch, high-fructose corn syrup, ethanol, corn oil, plus corn gluten
feed, and meal. This type opens numerous possibilities to connect industrial
product lines with existing agricultural production units. “Generation-II biorefi-
neries” are, furthermore, plants like NatureWorks PLA facility [25] (Sections 1.2
and 1.3.1) or ethanol biorefineries, for example Iogen’s wheat straw to ethanol
plant [132].
Third generation (generation-III) and more advanced biorefineries have not
yet been built but will use agricultural or forest biomass to produce multiple
products streams, for example ethanol for fuels, chemicals, and plastics.
1.4.3
The Role of Biotechnology
The application of biotechnological methods will be highly important with the

development of biorefineries for production of basic chemicals, intermediate
chemicals, and polymers [133–135]. The integration of biotechnological methods
must be managed intelligently in respect of physical and chemical conversion
of the biomass. Therefore the biotechnology cannot remain limited to glucose
from sugar plants and starch from starch-producing plants. One main objective
is the economic use of biomass containing lignocellulose and provision of glu-
cose in the family tree system. Glucose is a key chemical for microbial pro-
cesses. The preparation of a large number of family tree-capable basic chemicals
is shown in subsequent sections.
1.4.3.1 Guidelines of Fermentation Section within Glucose-product Family Tree

Among the variety of chemical products, and derivatives of these, accessible
microbially from glucose a product family tree can be developed, for example
(C-1)-chemicals methane, carbon dioxide, methanol; (C-2)-chemicals ethanol,
acetic acid, acetaldehyde, ethylene, (C-3)-chemicals lactic acid, propanediol, propy-
lene, propylene oxide, acetone, acrylic acid, (C-4)-chemicals diethyl ether, acetic
acid anhydride, malic acid, vinyl acetate, n-butanol, crotonaldehyde, butadiene,
2,3-butanediol, (C-5)-chemicals itaconic acid, 2,3-pentane dione, ethyl lactate,
(C-6)-chemicals sorbic acid, parasorbic acid, citric acid, aconitic acid, isoascorbinic
acid, kojic acid, maltol, dilactide, (C-8)-chemicals 2-ethyl hexanol (Fig. 1.7).
Guidelines are currently being developed for the fermentation section of a
biorefinery. The question of efficient arrangement of the technological design
for production of bulk chemicals needs an answer. Considering the manufac-
ture of lactic acid and ethanol, the basic technological operations are very simi-
lar. Selection of biotechnologically based products from biorefineries should be
done in a way such that they can be produced from the substrates glucose or
pentoses. Furthermore the fermentation products should be extracellular. Fer-
menters should have batch, feed batch, or CSTR design. Preliminary product re-
covery should require steps like filtration, distillation, or extraction. Final prod-
uct recovery and purification steps should possibly be product-unique. In addi-
Fig. 1.7 Biotechnological sugar-based product family tree.

tion, biochemical and chemical processing steps should be advantageously con-

nected.
Unresolved questions for the fermentation facility include:
1. whether or not the entire fermentation facility can/should be able to change
from one product to another;
2. whether multiple products can be run in parallel, with shared use of common
unit operations;
3. how to manage scheduling of unit operations; and
4. how to minimize in-plant inventories, while accommodating necessary
change-overs between different products in the same piece of equipment [95].
1.4.4
Building Blocks, Chemicals and Potential Screening
A team from Pacific Northwest National Laboratory (PNNL) and NREL sub-
mitted a list of twelve potential biobased chemicals [98]. A key area of the inves-
tigation as biomass precursors, platforms, building blocks, secondary chemicals,
intermediates, products and uses (Fig. 1.8).
The final selection of 12 building blocks began with a list of more than 300
candidates. A shorter list of 30 potential candidates was selected by using an
iterative review process based on the petrochemical model of building blocks,
chemical data, known market data, properties, performance of the potential can-
didates, and previous industry experience of the team at PNNL and NREL. This
list of 30 was ultimately reduced to 12 by examining the potential markets for
the building blocks and their derivatives and the technical complexity of the
synthetic pathways.
The reported block chemicals can be produced from sugar by biological and
chemical conversions. The building blocks can be subsequently converted to
several high-value biobased chemicals or materials. Building-block chemicals, as
considered for this analysis, are molecules with multiple functional groups with
the potential to be transformed into new families of useful molecules. The
twelve sugar-based building blocks are 1,4-diacids (succinic, fumaric, and malic),
2,5-furandicarboxylic acid, 3-hydroxypropionic acid, aspartic acid, glucaric acid,
glutamic acid, itaconic acid, levulinic acid, 3-hydroxybutyrolactone, glycerol, sor-
bitol, and xylitol/arabinitol [98].
A second-tier group of building blocks was also identified as viable candi-
dates. These include gluconic acid, lactic acid, malonic acid, propionic acid, the
triacids citric and aconitic, xylonic acid, acetoin, furfural, levuglucosan, lysine,
serine, and threonine. Recommendations for moving forward include:
· examining top value products from biomass components, for example aro-
matic compounds, polysaccharides, and oils;
· evaluating technical challenges in more detail in relation to chemical and bio-
logical conversion; and
· increasing the suites of potential pathways to these candidates.
Fig. 1.8 Model of a biobased product flow-chart for biomass feedstock [98].
No products simpler than syngas were selected. For the purposes of this study
hydrogen and methanol comprise the best short-term prospects for biobased
commodity chemical production, because obtaining simple alcohols, aldehydes,
mixed alcohols, and Fischer-Tropsch liquids from biomass is not economically
viable and requires additional development [98].
1.5
Biorefinery Systems and Design
1.5.1
Introduction
Biobased products are prepared for a usable economic use by meaningful com-
bination of different methods and processes (physical, chemical, biological, and
thermal). It is therefore necessary that basic biorefinery technologies are devel-
oped. For this reason profound interdisciplinary cooperation of various disci-
plines in research and development is inevitable. It seems reasonable, therefore,
to refer to the term “biorefinery design”, which means: “bringing together well
founded scientific and technological basics, with similar technologies, products,
and product lines, inside biorefineries”. The basic conversions of each biorefin-
ery can be summarized as follows. In the first step, the precursor-containing
biomass is separated by physical methods. The main products (M1–Mn) and the
by-products (B1–Bn) will subsequently be subjected to microbiological or chemi-
cal methods. The follow-up products (F1–Fn) of the main and by-products can
also be converted or enter the conventional refinery (Fig. 1.6).
Currently four complex biorefinery systems are used in research and develop-
ment:
1. the “lignocellulosic feedstock biorefinery” which use “nature-dry” raw
material, for example cellulose-containing biomass and waste;
2. the “whole crop biorefinery” which uses raw material such as cereals or
maize;
3. the “green biorefineries” which use “nature-wet” biomasses such as green
grass, alfalfa, clover, or immature cereal [78, 79]; and
4. the “biorefinery two platforms concept” includes the sugar platform and the
syngas platform [98].
1.5.2
Lignocellulosic Feedstock Biorefinery
Among the potential large-scale industrial biorefineries the lignocellulose feed-

stock (LCF) biorefinery will most probably be pushed through with the greatest
success. On the one side the raw material situation is optimum (straw, reed,
grass, wood, paper-waste, etc.), on the other side conversion products have a
good position on both the traditional petrochemical and future biobased product
market. An important point for utilization of biomass as chemical raw material
is the cost of raw material. Currently the cost of corn stover or straw is 30 US$/
ton and that of corn is 110 US$/ton (3 US$/ bushel; US bushel corn
=25.4012 kg = 56 lb) [136].
Lignocellulose materials consist of three primary chemical fractions or precur-
sors:
· hemicellulose/polyoses, sugar polymers of, predominantly, pentoses;
· cellulose, a glucose polymer; and
· lignin, a polymer of phenols (Fig. 1.9).
The lignocellulosic biorefinery-regime is distinctly suitable for genealogical com-

pound trees. The main advantages of this method is that the natural structures
and structural elements are preserved, the raw materials are inexpensive, and
large product varieties are possible (Fig. 1.10). Nevertheless there is still a de-
mand for development and optimization of these technologies, e.g. in the field
of separation of cellulose, hemicellulose and lignin, and utilization of the lignin
in the chemical industry.
Fig. 1.9 A possible general equation for conversion at the LCF biorefinery.
Fig. 1.10 Lignocellulosic feedstock biorefinery.
An overview of potential products of an LCF biorefinery is shown in Fig. 1.11. In

particular furfural and hydroxymethylfurfural are interesting products. Furfural is a
starting material for production of Nylon 6,6 and Nylon 6. The original process for
production of Nylon-6,6 was based on furfural (see also Section 1.2.2). The last of
these production plants was closed in 1961 in the USA, for economic reasons (the
artificially low price of petroleum). Nevertheless the market for Nylon 6 is huge.
Fig. 1.11 Products of a lignocellulosic feedstock biorefinery

(LCF-biorefinery, Phase III) [78, 79, 95].
There are, however, still some unsatisfactory aspects of the LCF, for example
the utilization of lignin as fuel, adhesive, or binder. Unsatisfactory because the
lignin scaffold contains substantial amounts of mono-aromatic hydrocarbons,
which, if isolated in an economically efficient way, could add a significant in-
crease in value to the primary processes. It should be noticed there are no natu-
ral enzymes capable of splitting the naturally formed lignin into basic mono-
mers as easily as is possible for natural polymeric carbohydrates or proteins
[137].
An attractive process accompanying the biomass-nylon-process is the already
mentioned hydrolysis of the cellulose to glucose and the production of ethanol.
Some yeasts cause disproportionation of the glucose molecule during their gen-
eration of ethanol from glucose, which shifts almost all its metabolism into
ethanol production, making the compound obtainable in 90% yield (w/w; with
regard to the chemical equation for the process).
On the basis of recent technology a plant has been conceived for production
of the main products furfural and ethanol from LC feedstock from West Central
Missouri (USA). Optimal profitability can be achieved with a daily consumption
of approximately 4360 tons of feedstock. The plant produces 47.5 million gallon
of ethanol and 323,000 tons of furfural annually [74].
Ethanol can be used as a fuel additive. It is also a connecting product to the
petrochemical refinery, because it can be converted into ethene by chemical
methods and it is well-known that ethene is at the start of a series of large-scale
technical chemical syntheses for production of important commodities such as
polyethylene or poly(vinyl acetate). Other petrochemically produced substances,
for example hydrogen, methane, propanol, acetone, butanol, butandiol, itaconic
acid, and succinic acid, can also be manufactured by microbial conversion of
glucose [138, 139].
1.5.3
Whole-crop Biorefinery
Raw materials for the “whole crop biorefinery” are cereals such as rye, wheat,
triticale, and maize. The first step is mechanical separation into corn and straw,
approximately 10 and 90% (w/w), respectively [140]. Straw is a mixture of chaff,
nodes, ears, and leaves. The straw is an LC feedstock and may further be pro-
cessed in a LCF biorefinery.
There is the possibility of separation into cellulose, hemicellulose, and lignin
and their further conversion in separate product lines which are shown in the
LCF biorefinery. The straw is also a starting material for production of syngas
by pyrolysis technology. Syngas is the basic material for synthesis of fuels and
methanol (Fig. 1.13).
The corn may either be converted into starch or used directly after grinding to
meal. Further processing may be conducted by four processes – breaking up, plas-
ticization, chemical modification, or biotechnological conversion via glucose. The
meal can be treated and finished by extrusion into binder, adhesives, and filler.
Fig. 1.12 Whole-crop biorefinery based on dry milling.
Fig. 1.13 Products from a whole-crop biorefinery [78, 79].
Starch can be finished by plasticization (co- and mix-polymerization, compound-

ing with other polymers), chemical modification (etherification into carboxy-
methyl starch; esterification and re-esterification into fatty acid esters via acetic
starch; splitting reductive amination into ethylenediamine, etc., hydrogenative
splitting into sorbitol, ethylene glycol, propylene glycol, and glycerin), and biotech-
nological conversion into poly-3-hydroxybutyric acid [69, 76, 92, 93, 141].
An alternative to traditional dry fractionation of mature cereals into grains

and straw only was been developed by Kockums Construction (Sweden), which
later became Scandinavian Farming. In this crop-harvest system whole imma-
ture cereal plants are harvested. The whole harvested biomass is conserved or
dried for long-term storage. When convenient, it can be processed and fraction-
ated into kernels, straw chips of internodes, and straw meal (leaves, ears, chaff,
and nodes) (see also green biorefinery).
Fractions are suitable as raw materials for the starch polymer industry, the
feed industry, the cellulose industry, and particle board producers, gluten can be
used by the chemical industry and as a solid fuel. Such dry fractionation of the
whole crop to optimize the utilization of all botanical components of the bio-
mass has been described [142, 143]. A biorefinery and its profitability has been
described elsewhere [144].
One expansion of the product lines in grain processing is the “whole crop
wet mill-based biorefinery”. The grain is swelled and the grain germ is pressed,
releasing high-value oils. The advantages of whole-crop biorefinery based on
wet milling are that production of natural structures and structure elements
such as starch, cellulose, oil, and amino acids (proteins) are kept high yet well
known basic technology and processing lines can still be used. High raw mate-
rial costs and, for industrial utilization, the necessary costly source technology
are the disadvantages. Some of the products formed command high prices in,
e.g., the pharmaceutical and cosmetics industries (Figs. 1.14 and 1.15). The ba-
sic biorefinery technology of corn wet mills used 11% of the US corn harvest in
1992, made products worth $7.0 billion, and employed almost 10,000 people
[1 a].
Wet milling of corn yields corn oil, corn fiber, and corn starch. The starch
products of the US corn wet milling industry are fuel alcohol (31%), high-fruc-
tose corn syrup (36%), starch (16%), and dextrose (17%). Corn wet milling also
Fig. 1.14 Whole-crop biorefinery, wet-milling.

Fig. 1.15 Products from a whole-crop wet mill-based biorefinery.
generates other products (e.g. gluten meal, gluten feed, oil) [62]. An overview
about the product range is shown in Fig. 1.15.
1.5.4
Green Biorefinery
Green biorefineries are also multi-product systems and furnish cuts, fractions,
and products in accordance with the physiology of the corresponding plant ma-
terial, which maintains and utilizes the diversity of syntheses achieved by na-
ture. Most green biomass is green crops, for example grass from cultivation of
permanent grass land, closure fields, nature reserves, or green crops, such as lu-
cerne, clover, and immature cereals from extensive land cultivation. Green crops
are a natural chemical factory and food plant and are primarily used as forage
and as a source of leafy vegetables. A process called wet-fractionation of green
biomass, green crop fractionation, can be used for simultaneous manufacture
of both food and non food items [145].
Scientists in several countries, in Europe and elsewhere, have developed green
crop fractionation [146–148]. Green crop fractionation is now studied in approxi-
mately 80 countries [149]. Several hundred temperate and tropical plant species
have been investigated for green crop fractionation [148, 150, 151] and more
than 300,000 higher plants species have still to be investigated. The subject has
been covered by several reviews [73, 146–148, 151–155]. Green biorefineries can,
by fractionation of green plants, process from a few tonnes of green crops per
hour (farm scale process) to more than 100 tonnes per hour (industrial scale
commercial process).
Careful wet fractionation technology is used as a first step (primary refinery)
to isolate the contents of the green crop (or humid organic waste goods) in their
natural form. Thus, they are separated into a fiber-rich press cake (PC) and a
nutrient-rich green juice (GJ).
The advantages of the green biorefinery are high biomass profit per hectare,
good coupling with agricultural production, and low price of the raw materials.
Simple technology can be used and there is good biotechnical and chemical po-
tential for further conversion (Fig. 1.16). Rapid primary processing or use of
preservation methods, for example silage production or drying, are necessary,
both for the raw materials and the primary products, although each method of
preservation changes the content of the materials.
In addition to cellulose and starch, the press cake contains valuable dyes and
pigments, crude drugs and other organic compounds. The green juice contains
proteins, free amino acids, organic acids, dyes, enzymes, hormones, other or-
ganic substances, and minerals. Application of the methods of biotechnology re-
sults in conversion, because the plant water can simultaneously be used for
further treatment. In addition, the lignin–cellulose composite is not as intract-
able as lignocellulose-feedstock materials. Starting from green juice the main fo-
cus is directed toward products such as lactic acid and its derivatives, amino
acids, ethanol, and proteins. The press cake can be used for production of green
feed pellets, as raw material for production of chemicals, for example levulinic
acid, and for conversion to syngas and hydrocarbons (synthetic biofuels). The
residues of substantial conversion are suitable for production of biogas com-
bined with generation of heat and electricity (Fig. 1.17). Reviews have been pub-
lished on the concepts, contents, and goals of the green biorefinery [73, 75,
119].
Fig. 1.16 A “green biorefinery” system.

Fig. 1.17 Products from the green biorefinery. In this illustra-

tion a green biorefinery has been combined with a green
crop-drying plant [78, 79].
1.5.5
Two-platform Concept and Syngas
The “two-platform concept” is one which uses biomass consisting, on average, of

75% carbohydrates which can be standardized as a “intermediate sugar platform”,
as a basis for further conversion, but which can also be converted thermochemi-
cally into synthesis gas and the products made from this. The “sugar platform” is
based on biochemical conversion processes and focuses on fermentation of sugars
extracted from biomass feedstocks. The “syngas platform” is based on thermoche-
mical conversion processes and focuses on the gasification of biomass feedstocks
and by-products from conversion processes [63, 98, 131]. In addition to the gasifi-
cation other thermal and thermochemical biomass conversion methods have also
been described – hydrothermolysis, pyrolysis, thermolysis, and burning. The ap-
plication chosen depends on the water content of biomass [156].
The gasification and other thermochemical conversions concentrate on utiliza-
tion of the precursor carbohydrates and their intrinsic carbon and hydrogen
content. The proteins, lignin, oils and lipids, amino acids, and other nitrogen
and sulfur-containing compounds occurring in all biomass are not taken into
account (Fig. 1.18).
The advantage of this concept is that the production of energy, fuels, and bio-
based products is possible using only slightly complex and low-tech technology,
Fig. 1.18 The sugar platform and the syngas platform [131].
for example saccharification and syngas technology. The sugar platform also en-
ables access to a huge range of family tree-capable chemicals (Figs. 1.7 and 1.8).
In-situ conversion of biomass feedstock into liquid or gas could be one way of
using existing infrastructure (developed pipe network), but with the disadvan-
tages of the need to remove hetero-atoms (O, N, S) and minerals present in the
biomass and the highly endothermic nature of the syngas process [157]. Cur-
rently, production of simple alcohols, aldehydes, mixed alcohols, and Fischer-
Tropsch liquids from biomass is not economically viable and additional develop-
ments are required [98] (Fig. 1.19).
1.6
Outlook and Perspectives
Biorefineries are the production plants in which biomass is economically and

ecologically converted to chemicals, materials, fuels, and energy. For successful
development of “industrial biorefinery technologies” and “biobased products”
several problems must be solved. It will be necessary to increase the production
of substances (cellulose, starch, sugar, oil) from basic biogenic raw materials
and to promote the introduction and establishment of biorefinery demonstra-
tion plants. Ecological transport of biomass must also be developed, for example
utilization of already developed pipe networks.
Another important aspect is committing chemists, biotechnologists, and engi-
neers to the concept of biobased products and biorefinery systems and promot-
ing the combined biotechnological and chemical conversion of substances. Last,
but not least, the development of systematic approaches to new synthesis and
technologies is required to meet the sustainable principles of “ideal synthesis”
and “principles of green chemistry and process engineering” [159–161].
References 33
Fig. 1.19 Syngas-based product family tree [157, 158].
References
1 National Research Council; Biobased In- tres, Anné 1748 [Preußische Akademie,
dustrial Products, Priorities for Research Berlin, 1749]
and Commercialization [National Aca- 3 Marggraf, A. S.; Chym. Schriften (Che-
demic Press, Washington D.C., 2000, mische Schriften) 2 Bd., Berlin 1761 till
ISBN 0-309-05392-7] a) 74 1767
2 Marggraf, A. S.; In: Histoire de l’Acadé- 4 Kirchhoff, G. S. C.; Schweigers Journal für
mie Royale des Sciences et Belles Let- Chemie und Physik, 4 (1812) 108
5 Graebe, C.; History of Organic Chemis- das aromatische Princip der Vanille. Ber.
try. Bd 1 [Verlag Julius Springer, Berlin, dt. chem. Ges., 7 (1874) 608–623
1920, germ.] a) 28, b) 122 f 22 Dignum, M. J. W.; Kerler, J., Verpoorte,
6 Kosaric, N.; Vardar-Sukan, F.; Potential R.; Vanilla Production: Technological,
Sources of Energy and Chemical Prod- Chemical and Biosynthetic Aspects. Food
ucts; In: The Biotechnology of Ethanol Rev. Intern., 17 (2001) 199–219.
[M. Roehr (ed.), Wiley–VCH, Weinheim 23 Vaupel, L., Vanille and Vanillin. Pharm.
et al, 2001 ISBN 3-527-30199-2] 132 Zeit., 38 (2002)
7 Prescott, S. C.; Dunn, C. G.; Industrial 24 Carothers, H.; Dorough, G. L.; and Van
Microbiology. 3rd Ed. [McGraw-Hill, Natta, F. J.; J. Am. Chem. Soc., 54 (1932)
New York, Toronto, London, 1959] 761
8 Osteroth, D.; From Coal to Biomass 25 Gruber, P. R.; O’Brien, M.; Polylactides
[Springer, New York, 1989, ISBN 0-387- “Nature Works” PLA; In: Biopolymers,
50712-4, germ.] 192 ff Polyester III [Y. Doi, A. Steinbüchel
9 McKillip, W. J.; Furan and Derivatives in (eds.), Wiley-VCH, Weinheim, 2002]
Ullmann’s Encyclopedia of Industrial 26 Walden, P., History of Organic Chemis-
Chemistry. 6th Ed., Vol. 15 [Wiley–VCH, try since 1880. Bd. 2 [Graebe, C. (Ed.),
Weinheim, 2003] 187 ff Verlag Julius Springer, Berlin, 1941,
10 www.furan.com germ.] 686
11 Pringsheim, H.; Cellulosechemie, 2 (1921) 27 Pötsch, W. R.; Lexicon of famous Chem-
123 ists. [Bibliographisches Institut, Leipzig,
12 Ullmann, F.; Enzyklopädie der Tech- 1988, ISBN 3-323-00185-0, germ.] a) 305
nischen Chemie. 2. Aufl., 5. Bd. [Urban 28 Borregaard LignoTech; marathon co.;
und Schwarzenberg, Berlin und Wien, http://www.ltus.com.
1930] 442 ff 29 Peckham; B. W.; The First Hundred
13 Payen, A.; Comptes rendus de l’Académie Years of Corn Refining in the United
des Sciences (C. r.), 8 (1839) 51 States. In Corn Annual 2000 [Corn Refi-
14 Gruber, E.; Krause Th., Schurz, J.: Cellu- ners Association, Washington, DC, 2000]
lose; In: Ullmann, Enzyklopädie der 30 Johnson, D. L.; The Corn Wet milling
technischen Chemie, 4. Aufl. Bd. 9 [Ver- and Corn Dry milling industry [in this
lag Chemie, Weinheim, 1975] 184–191 book, 2005]
15 Mulder, G. J.; J. Prakt. Chem., 21 (1840) 31 Slade, R. E.; Birkinshaw, J. H. (ICI); Im-
219 provement in or related to the utilization
16 A. E. Staley, Mfg. Co. A. E. (Decatur Ill.); of grass and other green crops. Brit. Pat.
Levulinic Acid 1942 [C.A. 36, 1612] BP 511,525 (1939)
17 Kitano et al., Levulinic Acid, A New 32 Pirie, N. W.; Chem. Ind., 61 (1942) 45
Chemical Raw Material – Its Chemistry 33 Pirie, N. W.; Nature, 149 (1942) 251
and Use. Chemical Economy and Engi- 34 Heier, W.; Grundlagen der Landtechnik 33
neering Review (1975) 25–29 (1983) 45–55
18 Leonard, R. H.; Levulinic acid as a Basic 35 Kamm, B.; Kamm, M.; The Green Biore-
Chemical Raw Material. Ind. Eng. Chem., finery – Principles, Technologies and
1956, 1331–1341 Products. In: Proceed. 2nd Intern. Symp.
19 Norman, W.; Process for Converting Un- Green Biorefinery, October, 13–14, 1999
saturated Fatty Acids or their Glycerides [SUSTAIN (eds), Feldbach, Austria, 1999]
into Saturated Compounds. BP 1515 , S. 46–69
1903 36 Knuckles, B. E.; Bickoff, E. M.; Kohler,
20 Sandermann, W.; Grundlagen der Che- G. O.; J. Agric. Food Chem., 20 (1972)
mie und chemischen Technologie des 1055
Holzes [Akademische Verlagsge- 37 Schertz, F. M.; Ind. Eng. Chem., 30
sellschaft, Leipzig, 1956] 147 (1938) 1073–1075
21 Tiemann, F., Haarmann, W., Ueber das 38 Shearon, W. H.; Gee, O. F.; Ind. Eng.
Coniferin und seine Umwandlung in Chem., 41 (1949) 218–226
References 35
39 Judah, M. A.; Burdack, E. M.; Caroll, 57 Shen, S. Y.; Wood Grass Production Sys-
R. G.; Ind. Eng. Chem., 46 (1954) 2262– tems for Biomass. Proceed. Midwest For-
2271 est Economist Meeting, Duluth, Minne-
40 Hale, W. J.: The Farm Chemurgic [The sota, April 1982
Stratford Co., Boston, 1934] 58 Shen, S. Y.; Biological Engineering for
41 Borth, Ch.; Pioneers of Plenthy [Bobbs- Sustainable Biomass Production; In: Bio-
Merril Co, Indianapolis, New York, 1939] diversity [E. O. Wilson, Harvard Univer-
42 Lewis, D. L.; The Public Image of Henry sity (ed.), National Academy of Sciences/
Ford [Wayne State University Press, De- Smithsonian Institution, National Aca-
troit, 1976] demic Press, Washington DC, 1988, Ger-
43 Brandt, E. N.; Growth Company – Dow man Edition: “Ende der biologischen
Chemical’s First Century [Michigan State Vielfalt?”, 1992, ISBN 3-89330-661-7]
University press, East Lansing, 1997] 404–416
44 Bergius, F.; Trans. Inst. Chem. Eng. (Lon- 59 Carlsson, R.; Trends for future applica-
don), 11 (1933) 162 tions of green crops. In: Forage Protein
45 Scholler, H.; Dissertation, Technical Uni- Conservation and Utilization. Proceed. of
versity of Munich, Germany 1923 EFC Conf. 1982 [EFC Press, Dublin, Ire-
46 Scholler, H.; French Patent, F.P. 777,824 land, 1982] 57–81
(1935) 60 Carlsson, R.; Green Biomass of Native
47 Katzen, R.; Tsao, G. T.; A View of the Plants and new Cultivated Crops for
History of Biochemical Engineering IN. Multiple Use: Food, Fodder, Fuel, Fibre
Advances in Biochemical Engineering/ for Industry, Photochemical Products
Biotechnology, Vol. 70 [Springer, Berlin, and Medicine. In: New Crops for Food
2000] and Industry [G. E. Wickens, N. Haq,
48 Conrad, T. F.; Holzzuckerbrennerei; In: and P. Day (eds.), Chapman and Hall,
Die Hefen, Vol. II 8 F. Reiff, R. Kautz- London, 1989]
mann, H. Lüers, M. Lindemann (eds.) 61 Dale, B. E.; Biomass refining: protein
[Verlag Hans Carl, Nürnberg, 1962] 437– and ethanol from alfalfa. Ind. Eng. Prod-
444 uct Research and Development, 22 (1983)
49 Prescott, S. C.; Dunn, C. G.; Industrial 446
Microbiology. 3rd Ed. [McGraw-Hill, 62 Hacking, A. J.; The American wet
New York, Toronto, London, 1959] milling industry. In: Economic Aspects
50 Harris, E. E. et al; Madison Wood Sugar of Biotechnology [Cambridge University
Process. Ind. Eng. Chem., 38 (1946) 896– Press, New York, 1986] 214–221
904 63 White, D. H.; Wolf, D.; Research in Ther-
51 Timell, T. E.; Tappi, 44 (1961) 99 mochemical Biomass Conversion [A. V.
52 Stamm, A. J.; Wood and Cellulose Bridgewater, J. L. Kuester (eds.), Elsevier
Science [Ronald Press, New York, 1964] Applied Science, New York, 1988]
53 James, R. L.; In: The pulping of wood, 64 Lipinsky, E. S.; Chemicals from Biomass:
2nd edn., vol. 1 [McGraw-Hill, New York, petrochemical substitution options.
1969] 34 Science, 212 (1981) 1465–1471
54 Brink, D. L.; Pohlmann, A. A.; Tappi, 55 65 Wise, D. L. (ed.); Organic Chemical from
(1972) 381 ff. Biomass [The Benjamin/Cummings
55 Oshima, M.; Wood Chemistry – Process Publishing Co., Inc., Menlo Park, Cali-
Engineering Aspects [Noyes development fornia, 1983]
Corp. New York, 1965, No. 10965] 66 Bailey, J. E.; Ollis, D. F.; Biochemical En-
56 Puls, J.; Dietrichs, H. H.; In: Energy gineering Fundamentals. 2nd edition.
from Biomass. Proceed. of First Europe- [McGraw-Hill, New York, 1986]
an Comm. Inter. Conf. on Biomass, 67 Szmant, H. H.; Industrial Utilization of
Brighton, UK, November 1980 [W. Palz, Renewable Resources [Technomic Pub-
P. Chartier, D.O. Hall (eds), Elsevier, lishing, Lancaster, Pa., 1987]
1981, ISBN: 0-85334-970-3] 348 68 Eggersdorfer, M.; Meyer J.; Eckes, P.;
Use of renewable resources for non-food
materials. FEMS Microbiolol. Rev., 103 vironmental and Economics (E3) Hand-
(1992) 355–364 book [www.bioproducts-bioenergy.gov]
69 Morris D. J.; Ahmed I.; The carbohydrate 81 Lynd L. R.; Cushman J. H.; Nichols R. J.;
Economy: Making Chemicals and Indus- Wyman C. E.; Fuel Ethanol from Cellulo-
trial Materials from Plant Matter [Insti- sic Biomass. Science, 251 (1991) 1318.
tute of Local Self Reliance, Washington 82 Keller, F. A. Integrated bioprocess devel-
D.C. 1992] opment for bioethanol production. In:
70 Bozell, J. J.; Landucci, R.; Alternative handbook ob bioethanol: production and
feedstock program – technical and eco- utilization. [C. E. Wyman (ed), Taylor and
nomic assessment [US Department of Francis, Bristol, Pa. 1996] 351–379
Energy, 1992] 83 Wyman C. E.; Handbook on Bioethanol:
71 Schilling, L. B.; Chemicals from alterna- Production and Utilization. Applied En-
tive feedstock in the United States. ergy Technology Series [Taylor and Fran-
FEMS Microbiol. Rev., 16 (1995) 1001– cis, 1996]
1110 84 Lynd, L.; Overview and evaluation of fuel
72 de la Ross, L. B.; Dale, B. E.; Reshamwa- ethanol from cellulosics biomass: tech-
la, S. T.; Latimer, V. M.; Stuart, E. D.; nology, economics, the environment, and
Shawky, B. T.; An integrated process for policy. Annual Review of Energy and the
protein and ethanol from coastal Bermu- Environment, 21 (1996) 403–465
da grass. Appl. Biochem. Biotechnol., 45/ 85 Galbe, M.; Zacci, G.; A review of the pro-
46 (1994) 483–497 duction of ethanol from softwood. Appl.
73 Soyez, K.; Kamm, B.; Kamm, M. (eds.); Microbiol. Biotechnol., 59 (2002) 618–628
The Green Biorefinery, Proceedings of. 86 Datta, R.; Tasi, S.-P.; Bonsignore, P.;
1st International Green Biorefinery Con- Moon, S. H.; Frank, J. R.; Technological
ference, Neuruppin, Germany, 1997 [Ver- and economics potential of poly (lactic
lag GÖT, Berlin, 1998, ISBN 3-929672- acid) and lactic acid derivatives. FEMS
06-5, German and English] Microbiol. Rev., 16 (1995) 221–231
74 Van Dyne D. L.; Blasé M. G.; Clements 87 Witt, U.; Müller R. J.; Widdecke, H.;
L. D.; A strategy for returning agriculture Deckwer, W.-D.; Synthesis, properties
and rural America to long-term full em- and biodegradability of polyesters based
ployment using biomass refineries. In: on 1,3-propanediol. Macrom. Chem.
Perspectives on new crops and new uses. Phys., 195 (1994) 793–802
[J. Janeck (ed), ASHS Press, Alexandria, 88 Yosida, T.; Nagasawa, T.; e-Poly-L-lysine:
Va., 1999] pp 114–123 microbial production, biodegradation
75 Narodoslawsky, M.; The Green Biorefin- and application potential. Appl. Microbiol.
ery, Proceedings 2nd Intern. Symp. Biotechnol., 62 (2003) 21–26
Green Biorefinery, Feldbach, Austria 89 Potera, C.; Genencor and DuPont create
[SUSTAIN, (ed.), TU Graz, 1999] “green” polyester. Genet. Eng. News, 17
76 Nonato R. V.; Mantellato, P. E.; Rossel (1997) 17
C. E. V.; Integrated production of biodeg- 90 Poirier, Y.; Nawrath, C.; Somerville, C.;
radable plastic, sugar and ethanol. Appl. Production of polyhydroxyalkanoates, a
Microbiol. Biotechnol., 57 (2001) 1–5 family of biodegradable plastics. Bio/tech-
77 Ohara, H. Biorefinery. Appl. Microb. Bio- nology, 13 (1995) 142–150
techn. (AMB), 62 (2003) 474–477 91 Warwel, S.; Brüse, F.; Demes, C.; Kunz,
78 Kamm, B.; Kamm, M.; Principles of M.; Rüsch gen Klaas, M.; Polymers and
Biorefineries. Appl. Microbiol., Biotechnol., Surfactants on the basis of renewable re-
(AMB), 64 (2004) 137–145 sources. Chemosphere, 43 (2001) 39–48
79 Kamm, B.; Kamm, M.; Biorefinery-Sys- 92 Bozell, J. J.; Alternative Feedstocks for
tems. Chem. Biochem. Eng. Q., 18 (2004) Bioprocessing; In: Encyclopedia of Plant
1–6 and Crop Science [R.M. Goodman (ed.),
80 U.S. Department of Energy (DEO); Na- Dekker, New York, 2004, 0-8247-4268-0]
tional Biomass Initiative and Energy, En-
References 37
93 Webb, C.; Koutinas, A. A.; Wang, R.; ergy and Value-Added Products [R. P.
Developing a Sustainable Bioproces- Overend and E. Chornet (eds.), Perga-
sing Strategy Based on a Generic Feed- mon Press, Oxford, UK, 1999] 867–872
stock. Adv. Biochem Eng./Biotechn., 87 104 Bachmann, R.; Bastianelli, E.; Riese, J.;
(2004) 195–268 Schlenzka, W.; Using plants as plants.
94 Gravitis, J.; Suzuki, M.; Biomass Refin- Biotechnology will transform the pro-
ery – A Way to produce Value Added duction of chemicals. The McKinsey
Products and Base for Agricultural Quarterly, 2 (2000) 92–99
Zero Emissions Systems; In: Proc. 99 105 Hettenhaus, J. R.; Wooley, B.; Biomass
Intern. Conference on Agric. Engineer- Commercialization: Prospect in the
ing, Beijing, China 1999 [United Na- Next 2 to 5 Years [NREL, Golden Color-
tions University Press, Tokyo, 1999] ado, 2000, No. NREL/ACO-9-29-039-01]
III-9–III-23 106 Woolsey, J.; Hydrocarbons to Carbohy-
95 Van Dyne, D. L., et al., Estimating the drates, The strategic Dimension; In:
Economic Feasibility of Converting Lig- The Biobased Economy of the 21st
no-Cellulosic Feedstocks to Ethanol Century: Agriculture Expanding into
and Higher Value Chemicals under the Health, Energy, Chemicals, and Materi-
refinery concept: A Phase II Study als. NABC Report 12 [National Agricul-
[University of Missouri, 1999, tural Biotechnology Council, Ithaca,
OR22072-58] New York, 2000, No. 14853]
96 Kurtanjek, Z. (ed.); Chemical and Bio- 107 Eaglesham, A.; Brown, W. F.; Hardy,
chemical Engineering Quarterly, Special R. W. F. (eds.); The Biobased Economy
Issue, 18 (2004) 1–88 of the 21st Century: Agriculture Ex-
97 Marano, J. J.; Jechura, J. L.; Biorefinery panding into Health, Energy, Chemi-
Optimization Tools – Development and cals, and Materials. NABC Report 12
Validation. In: 25th. Symposium on [National Agricultural Biotechnology
Biotechnology for Fuels and Chemi- Council, Ithaca, New York, 2000, No.
cals: Program and Abstracts [National 14853]
Renewable Energy Laboratory, Golden, 108 US President: Developing and Promot-
CO, No. NREL/BK-510-33708, 2003] ing Biobased Products and Bioenergy,
104 Executive Order 13101/13134 [William
98 T. Werpy, G. Petersen (eds.); Top Value J. Clinton, The White House, Washing-
Added Chemicals from biomass (ed) ton D.C. 1999]
[U.S. Department of Energy, Office of 109 US Congress; Biomass Research and
scientific and technical information, Development, Act of 2000 [Washington
2004, No.: DOE/GO-102004-1992, D.C., 2000]
www.osti.gov/bridge] 110 Biomass R&D, Technical Advisory
99 Goddijn, O. J. M.; Plants as bioreactors. Committee; Vision for Bioenergy and
Trends Biotechnol., 13 (1995) 379–387 Biobased Products in the United States
100 Wilke, D.; Chemicals from biotechnol- [Washington D. C. Oct. 2002, www.bio-
ogy: molecular plant genetic will chal- products-bioenergy.gov/pdfs/BioVi-
lenge the chemical and the fermenta- sion_03_Web.pd]
tion industry. Appl. Microbiol. Biotech- 111 Biomass R&D, Technical Advisory
nol., (AMB), 52 (1999) 135–145 Committee; Roadmap for Biomass
101 Anex, R. (ed.).; Journal of Ind. Ecology, Technologies in the United States
Special Issue, 7 (2003) 1–235 [Washington D.C., Dec. 2002, www.bio-
102 Ludgar, R. G.; Woolsey, R. J.; The new products-bioenergy.gov/pdfs/FinalBio-
petroleum. Foreign Affairs, 78 (1999) massRoadmap.pdf ]
88–102 112 Biotechnology Industrial Organisation;
103 Wyman, C. E.; Production of Low Cost World Congress on Industrial biotech-
Sugars from Biomass: Progress, Oppor- nology and Bioprocessing, http://
tunities, and Challenges; In: Biomass: www.bio.org/World%20Congress
A Growth Opportunity in Green En-
113 Biomass Conferences of the Americas; 125 Deutscher Bundestag; Rahmenbedin-

http://www.nrel.gov/bioam/ gungen für die industrielle stoffliche
114 Biomass, a growth opportunity in Nutzung von Nachwachsenden Roh-
green energy and value-added prod- stoffen in Deutschland schaffen, An-
ucts, Proceedings of the 4th Biomass trag 15/4943, Berlin (2005)
Conference of the Americas, Oakland 126 Busch, R.; Hirth, Th.; Kamm, B.;
California, USA, August 29–September Kamm, M.; Thoen, J.; Biomasse-In-
2, 1999 [Elsevier Science Ltd, Overend, dustrie – Wie aus “Bio” Chemie wird.
R.P.; Chornet, E. (ed.)], ISBN: Nachrichten aus der Chemie, 53 (2005)
0080430198, Oxford, UK, 1999] 130–134
115 Green and Sustainable Chemistry Con- 127 Röper, H.; Perspektiven der industriel-
gress; http://www.chemistry.org len Nutzung nachwachsender Roh-
116 Biorefinica – International Symposia stoffe, insbesondere von Stärke und
Biobased Products and Biorefineries; Zucker. Mitteilung der Fachgruppe Um-
www.biorefinica.de weltchemie und Ökotoxikologie der Ge-
117 Kamm, B.; Hempel, M.; Kamm, M. sellschaft Deutscher Chemiker, 7(2) (2001)
(eds.); biorefinica 2004, International 6–12
Symposium Biobased Products and 128 Kamm, B.; Kamm, M.; Richter, K.; Ent-
Biorefineries, Proceedings and Papers, wicklung eines Verfahrens zur Konver-
October, 27 and 28, 2004, [biopos e.V., sion von hexosenhaltigen Rohstoffen
Teltow, 2004, ISBN 3-00-015166-4] zu biogenen Wirk- und Werkstoffen –
118 Zoebelin, H. (ed.); Dictionary of Re- Polylactid aus fermentiertem Roggen-
newable Resources [Wiley-VCH, Wein- schrot über organische Aluminiumlac-
heim, 2001] tate als alternative Kuppler biotech-
119 Kamm B, et al.; Green Biorefinery nischer und chemischer Stoffwandlun-
Brandenburg, Article to development gen. In: Chemie nachwachsender Roh-
of products and of technologies and as- stoffe [P.B. Czedik-Eysenberg (ed.),
sessment. Brandenburgische Umweltbe- Österreichisches Bundesministerium
richte, 8 (2000) 260–269 für Umwelt (BMUJF) Wien, 1997] 83–
120 European parliament and Council; Di- 87
rective 2003/30/EC on the promotion 129 Kamm, B.; Kamm, M.; Schmidt, M.;
of the use of biofuels or other renew- Starke, I.; Kleinpeter, E.; Chemical and
able fuels for transport [Official Journal biochemical generation of carbohy-
of the European Union L123/42, drates from lignocellulose-feedstock
17. 05. 2003, Brussels, 2003] (Lupinus nootkatensis), Quantification of
121 Gesetz für den Vorrang erneuerbarer glucose, Chemosphere (in press)
Energien; Erneuerbare Energiegesetz, 130 US Department of Energy; http://
EEG/EnWGuaÄndG., 29. 03.2000, www.oit.doe.gov/e3handbook
BGBI, 305, (2000) 131 National Renewable Energy Laboratory
122 European parliament and Council; (NREL); http://www.nrel.gov/biomass/
Green Paper “Towards a European biorefinery.html
strategy for the security of energy sup- 132 Tolan, J. S.; Iogen’s Demonstration Pro-
ply” KOM2002/321, 26. 06. 2002, cess for Producing Ethanol from Cellu-
(2002) losic Biomass. In this book, 2005
123 Umweltbundesamt; Klimaschutz durch 133 EuropaBio; White Biotechnology: Gate-
Nutzung erneuerbarer Energien, Re- way to a more sustainable future [Euro-
port 2 [Erich Schmidt Verlag, Berlin, paBio, Lyon, April 2003]
2000] 134 BIO Biotechnology Industry Organisa-
124 BioVision2030-Group: Strategiepapier tion: New Biotech Tools for a cleaner
“Industrielle stoffliche Nutzung von Environment – Industrial Biotechnol-
Nachwachsenden Rohstoffen in ogy for Pollution Prevention, Resource
Deutschland”, Nov. 2003, www.biorefi- Conservation and Cost Reduction,
nica.de/bibliothek
References 39
2004; http://www.bio.org/ind/pubs/ physiological development; In: Hand-

cleaner2004/cleanerReport.pdf book of Plant and Crop Physiology [M.
135 Dti Global Watch Mission Report: Im- Pessarakli (ed.), Marcel Dekker Inc.,
pact of the industrial biotechnology on N.Y., USA, 1994] 941–963.
sustainability of the manufacturing 146 Pirie, N. W.; Leaf Protein – Its agron-
base – the Japanese Perspective, 2004 omy, preparation, quality, and use
136 Dale, B.; Encyclopedia of Physical [Blackwell Scientific Publications, Ox-
Science and Technology, Third Edition, ford/Cambridge, UK, 1971]
Volume 2: (2002) 141–157 147 Pirie, N. W.; Leaf Protein and Its By-
137 Ringpfeil M.; Biobased Industrial Prod- Products in Human and Animal Nutri-
ucts and Biorefinery Systems – In- tion [Cambridge Univ. Press, UK, 1987]
dustrielle Zukunft des 21. Jahrhun- 148 Carlsson, R.; Status quo of the utiliza-
derts? [2001, www.biopract.de] tion of green biomass. In: The Green
138 Zeikus, J. G.; Jain, M. K.; Elankovan, P.; Biorefinery, Proceedings of 1st Interna-
Biotechnology of succinic acid produc- tional Green Biorefinery Conference,
tion and markets for derived industrial Neuruppin, Germany, 1997 [S. Soyez,
products. Appl. Microbiol. Biotechnol., B. Kamm, M. Kamm (eds.), Verlag
51 (1999) 545–552 GÖT, Berlin, 1998, ISBN 3-929672-06-
139 Vorlop, K.-D.; Wilke, Th.; Prüße, U.; 5]
Biocatalytic and catalytic routes for the 149 Carlsson, R.; Food and non-food uses
production of bulk and fine chemicals of immature cereals; In: Cereals – Nov-
from renewable resources: [in this el Uses and Processes [G. M. Campbell,
book], 2005 C. Webb, S. L. McKee (eds), Plenum
140 Wurz, O.; Zellstoff- und Papierherstel- Publ. Corp., New York, USA, 1997],
lung aus Einjahrespflanzen [Eduard pp. 159–167
Roether Verlag, Darmstadt, 1960] 150 Carlsson, R.; Leaf protein concentrate
141 Rossel C. E. V.; Mantellato, P. E.; Agnel- from plant sources in temperate cli-
li, A. M.; Nascimento, J.; Sugar-based mates. In: Leaf Protein Concentrates
Biorefinery – Technology for an inte- [L. Telek, H. D. Graham (eds.), AVI
grated production of Poly(3-hydroxybu- Publ. Co., Inc., Westport, Conn., USA,
tyrate), Sugar and Ethanol, in this 1983] 52–80
book, 2005 151 Telek, L.; Graham, H. D. (eds.); Leaf
142 Rexen, F.; New industrial application Protein Concentrates [AVI Publ., Co.,
possibilities for straw. Documentation Inc., Westport, Conn., USA, 1983]
of Svebio Phytochemistry Group (Dan- 152 Wilkins, R. J. (ed.); Green Crop Frac-
ish) [Fytokemi i Norden, Stockholm, tionation [The British Grassland So-
Sweden, 1986-03-06, 1986] 12 ciety, c/o Grassland Research Institute,
143 Coombs, J.; Hall, K.; The potential of Hurley, Maidenhead, SL6 5LR, UK,
cereals as industrial raw materials: Le- 1977]
gal technical, commercial considera- 153 Tasaki, I. (ed.); Recent Advances in
tions; In: Cereals – Novel Uses And Leaf Protein Research, Proc. 2nd Int.
Processes [G.M. Campbell, C. Webb, Leaf Protein Res. Conf. [Nagoya, Japan,
and S.L. McKee (eds.), Plenum Publ. 1985]
Corp., New York, USA, 1997] 1–12. 154 Fantozzi, P. (ed.); Proc. 3rd Int. Leaf
144 Audsley, E.; Sells, J. E.; Determining Protein Res. Conf., Pisa-Perugia-Viter-
the profitability of a whole crop biore- bo, Italy, 1989
finery; In: Cereals – Novel Uses and 155 Singh, N. (ed.); Green Vegetation Frac-
Processes [G. M. Campbell, C. Webb, tionation Technology [Science Publ.
and S. L. McKee (eds.), Plenum Publ. Inc., Lebanon, NH 03767, USA, 1996]
Corp.; New York, USA; 1997] 191–294 156 Okkerse, C.; van Bekkum, H.; From
145 Carlsson, R.; Sustainable primary pro- fossil to green. Green Chemistry, 4
duction – Green crop fractionation: Ef- (1999) 107–114
fects of species, growth conditions, and
157 Lancaster, M.; The Syngas Economy; 160 Lancaster, M.; The Biorefinery. In:
In: Green Chemistry [The Royal So- Green Chemistry [The Royal Society of
ciety of Chemistry, Cambridge, UK, Chemistry, Cambridge, UK, 2002,
2002, ISBN: 0-85404-620-8] 205 ISBN: 0-85404-620-8] 207
158 Matlack, A. S.; The Use of Synthesis 161 Anastas, P. T.; Warner, J. C.; Green
Gas from Biomass; In: Introduction to Chemistry. Theory and Practice [Oxford
Green Chemistry [Marcel Dekker, New University Press, New York, 1998]
York, 2001, ISBN: 0824704118] 369
159 Clark, J. H.; Green Chemistry. Chal-
lenges and opportunities. Green Chem-
istry, 1 (1999) 1–8
41
2
Biomass Refining Global Impact –
The Biobased Economy of the 21st Century
2.1
Introduction
We are in the early phases of a truly historic transition – from an economy

based largely on petroleum to a more diversified economy in which renewable
plant biomass will become a significant feedstock for both fuel and chemical
production. The development of the petroleum refining industry over the past
century provides many instructive lessons for the future biobased economy and
also many reasons for supposing that the new biobased economy will be differ-
ent from the hydrocarbon economy in crucial ways. This paper explores the
similarities and differences between the petroleum refining and biorefining in-
dustries in a historical context and the implications of these similarities and dif-
ferences for the biobased economy in the long term.
We assume a mature biobased economy – as the petroleum economy is ma-
ture today – and from that assumption we extrapolate likely features of the ma-
ture biobased economy. Among the technical, social, and economic forces that
will drive the mature biobased economy are:
1. yield (using the whole “barrel of biomass”);
2. gradual diversification of biobased products, probably starting with higher-val-
ue chemical products and trending toward fuels over time;
3. the great diversity of biomass resources combined with their considerable
compositional similarity;
4. possible/likely limits on agricultural productivity;
5. integration of biorefining and agricultural ecosystems in a local social and
political context (the “all biomass is local” paradigm); and
6. the sustainability of the mature biobased economy and its most important
underlying resource – productive soils.
ISBN: 3-527-31027-4
42 2 Biomass Refining Global Impact – The Biobased Economy of the 21st Century
2.2
Historical Outline
2.2.1
Background and Development of the Fossil Carbon-processing Industries
Materials that contain carbon, including the primary commercial fuels, virtually
all food and fiber products, and most commodity chemicals, pharmaceuticals,
and nondurable manufactured goods, play an integral role in the world econo-
my [1]. Carbon-rich raw materials originate through the process of photosynthe-
sis in which plants and some bacteria use solar energy to convert atmospheric
carbon dioxide into organic substances including simple sugars, polysacchari-
des, amino acids, proteins, lipids, and aromatic compounds such as lignin.
Some carbon-rich raw materials are derived from fossil sources such as petro-
leum, coal, and natural gas. Fossil sources result from photosynthesis in ancient
times and comprise a large, but nonrenewable, reserve. In contrast, present day
photosynthesis provides a potentially renewable source of carbon.
Renewable agricultural and forestry resources have been used since ancient
times as fuels and raw materials for numerous products. Starting in the late
1700s, at the beginning of the Industrial Revolution, coal began to displace
wood as a fuel. In the mid 1800s the large-scale processing of petroleum to fuel
and chemical products began, taking away some markets from coal and many
more from renewable carbon sources. In the last half of the 20th century, uses
of natural gas began to expand greatly, both as a fuel and as a feedstock for
chemical production [Ref. 1, Chapter 1]. Currently petroleum provides 40% of
the United States’ and 35% of the world’s direct primary energy supply whereas
plant material in all forms provides approximately 10% of the world’s energy
supply.
Although remaining supplies of petroleum, coal, and natural gas are very
large, it is, nonetheless, obvious that the world is using these essentially nonre-
newable resources at a huge and growing rate. Natural processes are simply not
replacing fossil carbon at even a minute fraction of the rate at which we are
using it. For example, some experts believe that the peak rate of production of
conventional oil will occur within this decade whereas others predict this will
occur before mid-century [2, 3]. After that point, conventional, inexpensive oil
production will irreversibly decline. Natural gas production will peak later than
conventional oil, but will still begin permanent decline within the next few de-
cades. Although other sources of petroleum exist (e.g., tar sands, deep-water
oil), they will be more difficult and much more expensive to produce. Whatever
the exact date of peak oil production, we are approaching a major change in the
way we must provide energy and other services to the world’s population.
This paper addresses two related questions:
1. is it realistic to believe that renewable sources of carbon can provide a large
share of the energy and other services currently provided by fossil carbon, par-
ticularly petroleum?
2. what might be the salient characteristics of a mature biobased economy pro-

ducing not only food and fiber but also fuels and chemicals?
We will touch somewhat on question 1 but will treat question 2 more exten-
sively.
An extensive ongoing project called the Role of Biomass in America’s Energy
Future (RBAEF Project) sponsored by the US Department of Energy and the
Energy Foundation is attempting to address the first question with considerable
breadth and depth. The RBAEF Project is led by Professor Lee R. Lynd of Dart-
mouth College (Hanover, New Hampshire, USA), Dr John Sheehan of the Na-
tional Renewable Energy Laboratory (Golden, Colorado, USA), and Mr Natha-
nael Greene of the Natural Resources Defense Council (New York, New York,
USA). We encourage all readers of this article to make themselves aware of the
findings of the RBAEF team as these findings become available. Dr Lynd pro-
vides an outline of some of the expected results in another article in this vol-
ume.
2.2.2
The Existing Biobased Economy: Renewable Carbon
Renewable carbon-based raw materials are produced in agriculture, silviculture

and microbial systems, including managed and unmanaged systems. Although
estimates are necessarily imprecise, the total amount of new carbon-based plant
material fixed yearly by terrestrial plants is of the order of 100 ´ 1012 kg (assum-
ing biomass is on average 50% by weight carbon) [4, 5]. This amount of plant
material has an energy content (via heat of combustion) roughly five times the
energy value of all forms of energy used worldwide and over ten times the en-
ergy content of all petroleum used worldwide [6]. Although this plant resource
is dispersed, has competing uses, and, as a solid, is not in an ideal form for
easy transporting and processing, the size of the renewable carbon resource and
its associated energy content clearly suggest significant potential to provide raw
material and energy services.
The amount of new plant biomass dwarfs the use of fossil carbon to produce
organic chemicals. For example, the United States produces about 100 ´ 109 kg
fine, specialty, intermediate, and commodity organic chemicals each year, or ap-
proximately 0.1% of total world biomass production [7]. Less than 10% of this
total is produced from renewable carbon [1]. The total mass of these organic
chemicals is roughly equal to 40% of US production of corn grain, about twice
the grain that the United States exports each year. In fact, there is pressure on
the US (and the EU) from developing countries to reduce export and other sub-
sidies of their agricultural products. As this occurs, more US grain may rather
quickly find its way into fuel and chemical production. Already approximately 9
billion kg (3 billion gallons) annually of fuel ethanol are produced primarily
from corn in the US, consuming approximately 12% of domestic corn produc-
tion. There is, however, no reasonable expectation that grain will be able to pro-
vide the approximately 750 billion kilograms per year of refined petroleum prod-
ucts used in the United States [8]. That quantity of renewable carbon must
come from crop and forestry residues and energy crops (crops grown specifical-
ly to produce energy products) – not from food/feed grain.
In addition to existing uses of renewable carbon to produce organic chemicals
and fuels, renewable carbon sources provide about 90% of organic materials
such as lumber and paper, natural fibers, and composites, cellulosics, and some
proteins [Ref. 1, Chapter 2]. Finally, renewable carbon provides nearly all of our
food and animal feed. There is no reasonable alternative to using renewable car-
bon to meet food/feed demand, although the efficiency with which this demand
is met is certainly subject to change and innovation. We briefly explore some
possible innovations for food and feed production in this paper – a much more
complete discussion will be available in the RBAEF reports.
In total, the organic chemical, fuels (solid, liquid, and gaseous), carbon-based
materials, food, and feed components of the US domestic economy exceed US
$ 2 trillion per year. Much of this economic activity is already based on renew-
able carbon. The question which naturally arises is: could the fraction of fuels
and chemicals from renewable carbon be significantly increased without inter-
fering with other essential uses of plant material?
Before addressing this issue, we wish to point out two important facts that we
will explore in more detail below. First, there are several largely unexplored alter-
natives for coproducing animal feed and human food with fuels. Doing so would
make the whole enterprise more efficient, and very probably more economically
competitive. Second, both US and European agriculture are currently constrained
by demand, not by ability to produce. Respective national governments have been
paying farmers not to produce to capacity for many years. If large new demands
for renewable carbon were to arise, there is every reason to believe that much more
plant material could be produced on the same or similar acreages.
2.2.3
Toward a Much Larger Biobased Economy
Given the amount of grain available in the US and the EU and the ability to
supply very pure dextrose in large quantities for around US $ 0.20 per kg at
corn wet mills, we suggest that material supply, convenience, and purity consid-
erations favor dextrose derived from corn (and perhaps other grain) as a feed-
stock for organic chemical manufacture. The supply of grain dextrose is more
than adequate to produce all organic chemicals that conceivably can be made
from dextrose. Furthermore, corn yields are tending to increase with time. Giv-
en historical yield increases, each year US agriculture produces an additional
3.5 ´ 109 kg new dextrose equivalents. The biobased products industry will need
to grow very rapidly even to consume the additional dextrose made available
from new corn production. A switch from fossil carbon to renewable carbon for
organic chemicals will occur as conversion technologies improve, conversion
costs decline, and various barriers to entry are overcome.
In most situations it will be difficult for plant biomass to compete economi-

cally with coal as a solid fuel for stand alone electricity generation and we do
not consider this case. However, electricity generation from biomass processing
residues in an integrated processing facility producing liquid fuels and other
products is very attractive. Biomass gasification to produce a natural gas substi-
tute is also a much more attractive possibility than direct combustion, but like-
wise we do not treat it here because of space limitations. Gasification (and even
direct combustion) seem most useful when applied to forest products and resi-
dues. US forest growth has exceeded harvest since the 1940s. Harvested timber,
pulp, and paper have increased from private forestlands at the same time inter-
est in preserving public forests in more or less “unmanaged” conditions has re-
duced the timber, pulp, and paper available from these public lands.
Thus the land and other agricultural resources of the US seem more than
adequate to satisfy current domestic and export demands for food, feed, and fi-
ber and still produce ample raw materials for a much larger biobased economy.
Because the US consumes a disproportionate amount of fuel and chemicals
compared with the rest of the world, there is reason to hope that other coun-
tries can also provide much more of their fuel and chemical needs from renew-
able carbon sources. We offer further evidence below that this is so. The only
situation for which biobased feedstock supply adequacy for materials and chem-
icals does not seem to be the obvious conclusion is a massive increase of liquid
fuel production from renewable carbon. The remainder of this paper deals with
the concept of liquid fuel production from renewable carbon (specifically agri-
cultural crops and crop residues) in mature, integrated processing facilities
called “biorefineries”.
2.3
Supplying the Biorefinery
2.3.1
What Raw Materials do Biorefineries Require and What Products Can They Make?
Although petroleum feedstocks vary somewhat in composition, their composi-

tional variety is much less than for biomass feedstocks. Biomass compositional
variety is both an advantage and disadvantage. Biomass feedstocks consist of
grain, crop residues, oilseeds, sugar crops, forage crops, and a wide variety of woo-
dy crops. Figure 2.1 depicts typical compositions of some of these biomass feed-
stocks. The major components of biomass include carbohydrates (cellulose and
hemicellulose for crop residues, forage crops and woody crops – called lignocellu-
losic materials, starch for grain, and primarily sucrose for sugar crops), lipids
(fats, waxes and oils), proteins of many types, aromatic compounds (primarily lig-
nin), and ash (non carbon minerals such as calcium, phosphorus, potassium, etc.).
An advantage of biomass compositional variety is that biorefineries can make
more classes of products than can petroleum refineries, thus providing addi-
Fig. 2.1 Composition of several biomass species.
tional economic stability and opportunities for new product development. Biore-
fineries can also use a wider range of raw materials than can petroleum refi-
neries. Among the products that might be produced from biomass are liquid
transportation fuels (including both gasoline and diesel substitutes), electricity
and steam, a tremendous variety of chemicals containing carbon, oxygen, hydro-
gen, and nitrogen and combinations of these elements, monomers and poly-
mers, lubricants, adhesives, fertilizers and, significantly, animal feeds and hu-
man foods. Some of these products are summarized in Fig. 2.2 in their life cy-
cle context as possible replacements for petroleum-based or petroleum-depen-
dent products [9].
A disadvantage of biorefineries compared with petroleum refineries is that a
relatively larger range of processing technologies is needed. This is particularly
true for conversion and/or separation of the wider range of components of the
renewable feedstock raw materials, as shown in Fig. 2.1. It is important to note
in Fig. 2.2 that biorefineries will probably operate by first preprocessing (sepa-
rating and reacting) the inlet raw materials to a relatively small range of inter-
mediate products including carbohydrates, protein, syngas (mixtures of carbon
monoxide, hydrogen and carbon dioxide) and a few other products. These inter-
mediate products will then be upgraded by further reaction and separation steps
to a very wide variety of final products. Some of these reaction and separation
steps are well developed, others remain to be developed. In particular, the pro-
cessing technologies to convert lignocellulosic materials economically and in
high yield to carbohydrates and other products are not yet fully available. Once
these and other processing technologies are developed and deployed, however,
they will find use for a much wider variety and much more geographically dis-
persed set of renewable raw materials than is true for petroleum.
Fig. 2.2 Life cycle overview of biobased products [9].
2.3 Supplying the Biorefinery
47
Prototype biorefineries already exist, including corn wet and dry mills, pulp and
paper mills, and other renewable carbon-based processing facilities. Corn wet
mills perhaps best exemplify many of the features that are likely to be found in
mature biorefineries for large-scale fuel production. Wet mills are large, highly
integrated facilities producing a wide range of chemical, biochemical, feed, food,
and fuel products, as outlined in Fig. 2.2. Over 90% of the inlet corn leaves as
value-added products (selling price per kilogram greater than corn feedstock)
[Ref. 1, Chapter 3]. Corn wet mills have continued to add products with time,
particularly higher value chemical/biochemical products. Similarly to petroleum
refineries, wet mills alter their product mix to meet changing market condi-
tions, including seasonal variations in demand.
2.3.2
Comparing Biomass Feedstock Costs with Petroleum Costs
Of course, the widely dispersed and renewable nature of biomass feedstocks is of

little practical importance unless we can reasonably expect to convert these feed-
stocks to products that will compete economically with petroleum-derived prod-
ucts. Fortunately, this is an entirely reasonable and feasible goal. To support this
statement, we note that competitive pressures and continually improving conver-
sion technologies gradually force many high margin new products to eventually
become mature, commodity products with narrow margins. This process has oc-
curred with products as diverse as penicillin, Nylon and, of course, gasoline.
Experience shows that approximately 60–70% of the cost of manufacturing
commodity products depends on the cost of the raw materials from which these
commodities are made [10, 11]. This is true for petroleum-derived commodities
in particular, and explains the large swings in gasoline prices as crude oil prices
change. We envisage a mature biorefining industry producing liquid biofuels to
replace gasoline and diesel fuel. In such a mature biorefining industry the cost
of manufacturing biofuels will also depend very highly on raw material cost.
The question therefore arises: how does the cost of renewable plant biomass
compare with the cost of petroleum? I am indebted to Professors Lee Lynd and
Charles Wyman of Dartmouth College (Hanover, New Hampshire, USA) for
suggesting the approach outlined in Fig. 2.3 below.
Figure 2.3 shows the cost of plant biomass relative to the cost of petroleum
on two different bases – cost per kilogram of material and cost per unit of en-
ergy. Three different horizontal lines are drawn representing different classes of
plant biomass available at three different prices. Crop residues are valued at
$ 20 per ton (US) in Fig. 2.3, hays and forage crops such as low quality alfalfa
are valued at $ 50 per ton, and corn grain at $ 120 per 1000 kg is roughly
equivalent to current US Corn Belt prices of corn at about $ 2.75 per bushel.
Historically, corn grain has been priced at closer to $ 2.00 per bushel in con-
stant dollars, see Fig. 2.4 below.
Figures 2.3 and 2.4 teach several important lessons. First, corn grain at $ 3 per
bushel is roughly equivalent to petroleum at $ 35 per barrel on an energy basis
Fig. 2.3 Relative costs of biomass and petroleum by mass and energy content.
Fig. 2.4 Real and historial corn prices.
(arrow #1 in Fig. 2.3). On a mass basis, however, corn is less than half the cost of
petroleum (arrow #2) – giving corn starch and other corn components real poten-
tial as a feedstock for chemical production to replace petroleum-derived chemicals.
Corn wet millers often use net corn cost to reflect the cost of starch available for
chemical and fuel production after coproduct credits (e.g. for protein and oil) are
subtracted. Typical net corn costs are roughly 70% of the purchase cost of corn,
further reducing the actual cost of corn starch for chemical or fuel production.
Ethanol production from corn grain currently requires various financial incen-
tives to be competitive with gasoline from oil. No such incentives are required
for chemicals from corn starch. Very large efforts are currently in progress to
Table 2.1 Ten required biomass feedstock properties.
1. Economical
2. Stable price and availability
3. Consistent composition
4. Low cost
5. Favorable co-products and by-products
6. Multiple product opportunities
7. Inexpensive
8. Environmentally benign or beneficial
9. Storable
10. Inexpensive
produce chemicals (e.g. lactic acid, 1,3 propanediol, etc.) from corn starch by
either wet milling or dry milling. This is precisely how the petroleum refining
industry developed. Additional valuable products such as plastics were added to
the refinery over a period of decades as these products and processes were in-
vented or improved and economics became attractive for the new products.
Second, lignocellulosic biomass such as crop residues and herbaceous species
(grasses, hays, forage crops) is available at prices that are a fraction (one fifth to
one half) of petroleum costs (at $ 35 per barrel) on an energy basis, and even
less on a mass basis (arrows #3 and #4 in Fig. 2.3). Therefore, given processing
technology that economically and efficiently converts the energy content of lig-
nocellulosic biomass to liquid fuels, we can reasonably expect to derive fuel
products that are available at prices similar to current gasoline and diesel prices.
Sugar fermentation to ethanol is one such process. Over 90% of the energy con-
tent of glucose is captured in the ethanol product from high-yield fermenta-
tions.
Feedstock costs are absolutely critical to commodity chemicals and fuels, and
biomass feedstocks are already much less expensive than petroleum on both a
mass and energy basis. It is impossible to overstate the importance of low cost
renewable carbon feedstocks to the eventual commercial success of large-scale,
integrated biorefineries. Dr Paul Roessler of Dow Chemical (US) makes this
point in a humorous and effective way in his list of ten required biomass feed-
stock properties, given as Table 2.1. In line with the historical development of
other processing industries, as the biomass processing industry develops and
the related technology matures, raw material costs will become dominant in the
cost of manufacture.
2.3.3
How Much Biomass Feedstock Can be Provided at What Cost?
Renewable carbon feedstock prices are critical to the economic success of biore-
fineries. Although we believe data on likely biomass prices are encouraging, the
ultimate possible scale of the industry, and hence its ability to displace petro-
leum, will also be determined by the amount of biomass available at these

prices. We now briefly examine this subject using Table 2.2 to frame our discus-
sion.
Table 2.2 summarizes world production of crop residues. In all, approximately
1.55 ´ 1012 kg residue are produced annually, the equivalent of approximately
600 billion liters of bioethanol at expected ethanol yields for mature technology
of about 400 liters per 1000 kg residue [12]. The figure of 600 billion liters is
close to the volume of gasoline and diesel consumed each year in the US, a very
significant figure indeed.
Rice straw must be removed from fields before planting of the next crop and
is probably available close to collection costs, given the demand for the straw.
Currently most rice straw is field-burned to remove it, a practice becoming less
and less tolerated everywhere. Worldwide, over 700 ´ 109 kg rice straw are pro-
duced annually, mostly in Asia. Almost 200 ´ 109 kg sugar-cane bagasse is col-
lected annually in many locations worldwide. Bagasse is probably available at its
fuel cost or below, because it has only limited value to provide energy for the
sugar mill. Thus nearly one thousand billion kilograms of rice straw and sugar
cane bagasse are probably available at nominal costs.
Considering other residues, approximately 100 billion kg per year corn stover
and wheat straw in the Corn Belt and Great Plains regions of the United States
are probably available at delivered prices of $ 20 per 1000 kg and less [14]. Simi-
lar amounts of residue, mostly wheat straw and barley straw, are available in
Europe, probably at costs comparable with those in the United States. At $ 50
per 1000 kg, crop residue availability increases significantly. Approximately 150
billion kg US crop residues are available at this price compared with 100 bil-
lion kg at $ 20 per 1000 kg [14]. Other countries should also experience in-
creased incentives to collect and utilize crop residues at higher prices. At about
$ 50 per 1000 kg, farmers will also begin to produce hays and grasses specifical-
ly for biorefineries [15], and lignocellulosic biomass supplies will expand greatly.
The extent to which supplies expand at a price of $ 50 per 1000 kg depends
Table 2.2 Worldwide availability of crop residues [13].
Material Africa Asia Europe North Central Oceania South Subtotal

(billion kg) America America America
Corn stover 0.0 33.9 28.6 134 0.0 0.2 7.2 204
Barley straw 0.0 2.0 44.2 9.9 0.2 1.9 0.3 58.5
Oat straw 0.0 0.3 6.8 2.8 0.0 0.5 0.2 10.6
Rice straw 20.9 668 3.9 10.9 2.8 1.7 23.5 731
Wheat straw 5.3 145 133 50.1 2.8 8.6 9.8 354
Sorghum 0.0 0.0 0.4 6.9 1.2 0.3 1.5 10.3
straw
Bagasse 11.7 74.9 0.0 4.6 19.2 6.5 63.8 181
Subtotal 38.0 924 217 219 26.1 19.7 106 1549
largely on the productivity of crop and pasture lands. The RBAEF Project will
treat this subject of productivity in great detail. We simply observe here that the
US has nearly 16 million ha in its Conservation Reserve Program (CRP), land
which is removed from grain crop production but which would be very suitable
for grass and hay production.
In the US, pasture lands (including crop land used as pasture) average about
5600 kg of biomass per ha per year [16]. There is little incentive to increase
production on these lands, because there are no markets for increased hay
production. Biomass production for energy would provide increased incentive
to use these lands efficiently and a severalfold increase in average yield over
time seems entirely likely. For example, some lands supporting dairy cattle in
Michigan are managed intensively for maximum biomass production, including
the use of winter cover crops and corn harvested as silage. Such lands are
currently yielding 20 000 kg dry biomass per ha per year. Double these yields
(40 000 kg ha–1 per year) of other species such as sugarcane and elephant grass
have also been achieved on degraded lands [17]. If all the CRP lands and one
half of crop land used as pasture (15 million ha) achieved 20 000 kg ha–1, an
additional 600 ´ 109 kg biomass would be produced annually in the US.
We believe the large scale conversion of cellulosic biomass to fuel will be
based first on crop residues, given their low cost and availability. There is more
than enough low-cost crop residue in the US and elsewhere to begin such an
industry. As the industry expands, and processing economics improve with re-
search and experience, the “demand pull” for additional biomass will cause the
agricultural research and production sector to learn how to produce much larger
amounts of herbaceous biomass profitably at costs approximating $ 50 per
1000 kg on lands that compete only modestly or not at all with food crop pro-
duction. In addition, biorefineries producing fuels will also produce both pro-
tein and energy feeds for animals, just as corn wet and dry mills producing
ethanol fuel do today. Coproduction of animal feeds with fuel and chemical
products in biorefineries will increase feed supplies and reduce pressure on
cropland.
Before concluding this treatment of biomass feedstock costs, we note that the
wide geographic availability, abundance and variety of biomass will tend to re-
duce the risks of raw material supply availability and reduce price swings. Un-
certain availability and price volatility are major features of the current petro-
leum economy. As an essentially fixed world endowment of easily recoverable
petroleum is gradually consumed, these risks will only grow and an increasing
price paid in terms of national security and stability [2, 18]. Therefore, this eco-
nomic and energy security issue can only grow in importance. Furthermore,
many developed and developing nations which lack petroleum can grow large
quantities of plant biomass, and thereby begin to escape the “development trap”
that petroleum dependence brings in an era of decreasing petroleum supplies
and high and volatile prices [18].
2.4
How Will Biorefineries Develop Technologically?
2.4.1
Product Yield: The Dominant Technoeconomic Factor
Yield (kg salable products per kg purchased raw materials) is usually the domi-
nant factor governing the economics of a given reaction/separation system to
produce commercial products. Because, essentially, all chemical and biological
reactions produce multiple products, the yield of salable products influences the
economics of the system in the following ways.
1. Raw material cost increases per unit of product as yield declines. For exam-
ple, at $ 0.10 per pound of glucose and 90% yield of lactic acid produced by
fermentation of this glucose, the glucose raw material cost is $ 0.11 per
pound of lactic acid. At 50% yield, the raw material cost is $ 0.20 per pound
of lactic acid. Because the cost of manufacturing commodity fuels and chemi-
cals is very dependent on raw material costs, a lower yield significantly in-
creases the cost of manufacture.
2. The cost of the reaction system increases. If a fixed total annual production
rate is required, then at 50% yield almost twice the total reactor volume is
needed compared with 90% yield to provide that amount of product.
3. The cost of the separation system increases even more rapidly than the reac-
tion system. The cost of separation is proportional to the volume of fluid
handled. Under similar reaction conditions, a lower yield means lower con-
centration of product and, therefore, a greater volume of reaction fluid must
be handled for a given production rate. The cost of separation also increases
with the number of components needing separation. Decreasing yield usually
means there are more components that must be separated.
4. The cost of waste treatment increases. Either markets for byproducts must be
found, which is not always possible, or the resulting waste streams must be
treated before disposal, adding to both the capital and operating expense of
the overall system.
Given the dominance of yield in process economics for commodities, several

conclusions regarding biorefinery development for fuels and commodity chemi-
cals arise.
First, fuel production in biorefineries will tend to be performed first in exist-
ing facilities where the yield of products can be improved by adding fuel pro-
duction and where substantial capital investment has already been made, reduc-
ing the risk to innovators. Second, given the mild conditions (moderate pH and
temperature) of biological catalysts, bioprocessing and biotechnology will tend
to be used in biorefineries instead of harsh chemical or thermal processing to
avoid degradation (and thereby the loss of value) of sugars, proteins and other
labile biomolecules. Third, there will be continuing pressure to use all of the
components of the biomass feedstock at their highest possible value. Thus the
number of products will increase over time as will the yield of salable products
per unit of raw material(s) consumed. This is precisely the trend that has oc-
curred historically in the petroleum refining industry.
Because we have pointed out several probable similarities between petroleum
refineries and biorefineries, it is worth pointing out here a very significant dif-
ference between them. Both genetic tools and conventional plant breeding can
be used to develop biomass feedstocks that are particularly designed for process-
ing in the biorefinery. For example, lignin content can be altered to make ligno-
cellulosic biomass easier to process. Also, valuable products such as enzymes
can be produced in plants and recovered in the biorefinery [Ref. 1, Chapter 1].
This capability has no parallel in petroleum refining and is a major advantage
for biorefineries. Continuing advances in the life sciences virtually guarantee
that this advantage will grow with time.
2.4.2
Product Diversification: Using the Whole Barrel of Biomass
The importance of finding valuable uses for all biomass components is illustrat-
ed by the following example based on the composition outlined in Fig. 2.1. As-
sume a corn stover-based biorefinery producing 378 million liters per year of
fuel ethanol at a yield of 420 liters of ethanol per 1000 kg stover. At a 50% re-
moval rate for stover, approximately 200 000 ha corn will be required to provide
the stover. Such a biorefinery will also produce nearly 21 ´ 106 kg minerals (Ca,
K, Mg and P), 20 ´ 106 kg lipids, fats, and waxes, 52 ´ 106 kg protein (equivalent
to the protein produced from nearly 70 000 ha soybeans), electricity from burn-
ing the lignin residue and probably residual sugars for animal feeding. These
conclusions arise directly from the chemical nature of the components of bio-
mass and the realities of chemical and biological processing outlined in Sec-
tion 2.4.1, above. A biorefinery will be in many businesses (fuels, chemicals,
power, feed, etc.) simultaneously. This fact cannot be evaded, but must be faced
and dealt with, hopefully to the benefit of the overall biorefining system.
One significant potential benefit that arises from this analysis is that the net
requirement for agricultural land to provide feed protein decreases by 70 000 ha,
or about 1/3 of the land from which stover is harvested. Put another way, 3 ha
of corn production are now able to replace 1 ha of soybean production, while
still providing all the grain these corn acres produced before and 900 ´ 106 kg
stover for liquid fuel production. If herbaceous biomass species, for example
switchgrass, are grown for energy production, they will probably be higher
yielding and will also contain significantly more protein than corn stover. Thus
such crops should provide even greater net savings of crop land required to
meet protein needs. For example, if switchgrass or another herbaceous species
such as coastal Bermuda grass is produced at dry matter yields of 20 000 kg ha–1
per year (about 9 tons acre–1 year–1) and contains 10% protein of which 80% is
recovered [19, 20], the system will produce 1,600 kg ha–1 protein, over twice the
amount of protein produced per ha of soybeans. Because the United States cur-
rently devotes approximately 30 million ha to soybean production, this emphasis

on using the whole barrel of biomass could lead to substantial coproduction of
energy and protein, but without devoting any new acreage to energy crop pro-
duction.
As described in Section 2.4.1 above, if the various components of biomass are
not used in salable products, they must be disposed of at a cost. If they are not
used in products, they must also be carried along with all the other streams in
the process, adding to the capital and operating costs of all the related process-
ing equipment. Likewise, if these components are not used in products, the re-
maining products must bear a larger portion of the overall costs, particularly
the crucial feedstock costs. Thus many forces converge on a single objective: uti-
lizing the entire “barrel of biomass”. The overall result of this convergence is
quite simple: there is strong and unrelenting pressure to increase the yield and
value of the multiple products of biomass and not to waste even a fraction of
the raw materials.
Because of this driving force, continuous, incremental process improvement
is a key feature of both oil refineries and existing biorefineries. There are, how-
ever, many improvements and innovations that cannot occur until a refinery is
actually operating and producing products and will not result from laboratory
discoveries. We expect this pattern of continuous, incremental improvement in
existing facilities will continue both with existing, starch-based biorefineries. It
will also occur with the next generation of cellulose biorefineries that will come
when key breakthroughs occur. We now briefly discuss breakthroughs required
for cellulose biorefineries.
2.4.3
Process Development and a Technical Prerequisite for Cellulosic Biorefineries
If plant raw material is already inexpensive compared with petroleum and suffi-
ciently abundant to support a large scale biorefining industry, one may legiti-
mately ask: “Why has such an industry not already emerged?” We note that
such industries have arisen: corn wet and dry mills and pulp and paper mills
are examples. For these industries, inexpensive raw materials are coupled with
well developed and efficient processing technology to convert the plant raw ma-
terials to products. For large scale liquid fuel production from cellulosic materi-
als, however, the missing part of the equation is demonstrated, inexpensive pro-
cessing technology to convert inexpensive and abundant cellulosic raw materials
to fuels.
While we believe that the entire barrel of biomass must be used effectively,
use of the carbohydrate component (cellulose and hemicellulose represent ap-
proximately 70% of cellulosic biomass) is the sine qua non for cellulosic biorefi-
neries [Ref. 1, Chapters 4 and 21]. The accessibility (reactivity) of cellulose and
hemicellulose must be increased without significantly degrading these compo-
nents. These carbohydrate polymers must somehow be converted into chemi-
cally reactive intermediate species, as shown in Fig. 2.2. Achieving this objective
requires some sort of pretreatment before enzymatic, biological, or chemical up-

grading of these sugar polymers to more valuable products such as ethanol fuel.
Overcoming the recalcitrance of cellulosic biomass is therefore arguably the sin-
gle most important research/process development obstacle confronting biorefi-
neries processing cellulosic biomass to fuels.
A closely related research objective for cellulose biorefineries is significantly
reducing the cost of enzymes (called “cellulases”) required to convert pretreated
biomass into fermentable sugars. An even more powerful impact on the eco-
nomics of cellulose biorefineries than inexpensive enzymes would occur given
the high yield, one step biological conversion of pretreated biomass to useful
products. This process by which cellulase production, cellulose hydrolysis, and
fermentation of soluble sugars to desired products occur in a single process step
is called “consolidated bioprocessing” [21].
If viable, inexpensive pretreatments to overcome the recalcitrance of cellulosic
biomass are developed, it seems likely they will first be demonstrated in existing
facilities where a cellulose/hemicellulose-containing stream is available for up-
grading to fuels and chemicals. Such existing facilities include pulp and paper
mills, corn wet and dry mills, and perhaps flour mills. When this key piece of
technology is available, the first generation of cellulose biorefineries will be
launched. These pioneer plants will become the loci for further improvements,
both incremental and transformational, in converting cellulosic biomass to a
wide variety of fuels, chemicals, animal feeds, and other products.
2.5
Sustainability of Integrated Biorefining Systems
2.5.1
Integrated Biorefining Systems: “All Biomass is Local”
Because biomass feedstocks are solids and tend to be bulky, there is consider-
able incentive to refine them close to where they are grown. Likewise the waste
streams from biorefineries will tend to be high in organic material and there-
fore biological oxygen demand. But these biorefinery wastes will not be particu-
larly toxic. Many of these waste streams are therefore suitable candidates for re-
turning to agricultural land. Given the capital and operating costs associated
with waste handling in the biorefinery, there is a strong economic incentive to
apply these wastes to land.
Furthermore, agriculture is, by nature, a regional or local activity because of
differences in soil and climate. Therefore crops grown for biorefineries can be
or will be adapted to local conditions. The relatively smaller scale of biorefi-
neries (compared with petroleum refineries) also enhances the likelihood that
the farmers who produce the crops will have some sort of formal or informal
“ownership” of the biorefinery. This pattern of local ownership of the biorefine-
ry, or at least participation in ownership, is observed in many corn dry mills in
the US. Corn wet mills are, however, much larger than dry mills (the largest
wet mills approach the mass throughput rate of petroleum refineries) and are
not farmer-owned. Local ownership of the biorefinery will give farmers an addi-
tional incentive to utilize animal feeds, mineral fertilizers, and organic waste
streams from the biorefinery. We call this framework the “All Biomass is Local”
concept; it is illustrated in Fig. 2.5.
We envisage farmers using locally appropriate agricultural systems growing
biomass specifically for the biorefinery. In the biorefinery, biomass is separated
into its major components. Some of these components (e.g., protein and miner-
als or “ash”) may be salable or usable without further upgrading. The ash is re-
turned as fertilizer to the land. Protein is fed to animals, preferably in a nearby
location to avoid costs of drying and transportation. Animal wastes and organic
waste streams from the biorefinery are used in the agricultural system. The na-
ture of these waste streams makes them particularly appropriate for land appli-
cation to perennial grass systems, where the potential for runoff and leaching
to groundwater is minimized.
Therefore, the convergence of these factors:
· local ownership/participation in biorefineries,
· the widespread geographical distribution of these refineries,
· the fact that biorefineries will be intimately involved with land use practices,
and
· society’s continuing concern with the environment
virtually guarantee that biorefineries will be conceived, designed, built, and op-
erated with an unprecedented emphasis on their local and global environmental
impact. Petroleum refineries largely avoided environmental/social issues when
that industry was in its infancy, but now are being forced to do so at great cost.
Biorefineries that do not adequately address appropriate environmental and so-
cial issues will be at substantial risk of failure.
We believe these environmental and social issues surrounding biorefineries
are best addressed using the concept of sustainability as an organizing frame-
work and life cycle analysis as a powerful analytical methodology [22]. As we il-
lustrate below, we believe that thoughtful, intelligent design and implementa-
tion of integrated agricultural production and biorefining systems can do much
more than simply maintain the environmental status quo. Rather, we believe it
is possible to effect significant improvements in the local, regional, and global
environment by using life cycle analysis of integrated crop production and refin-
ing systems.
2.5.2
Agricultural/Forestry Ecosystem Modeling: New Tools for an Age of Sustainability
Sustainability is a very broad subject. In this paper we do not have the scope to
treat all aspects of sustainability in relation to crop production and biorefining
systems such as those represented in Fig. 2.6. To illustrate an approach that
Fig. 2.5 All biomass is local: integrating agriculture and the biorefinery.
Fig. 2.6 Carbon flows in the CENTURY model [25].

might be taken, however, we consider the dynamics of carbon and nitrogen flow
in agricultural ecosystems. We then analyze how those flows might be manipu-
lated to enhance the sustainability of the agricultural production and biorefining
system when taken as an integrated whole. We also point out what seem to be
some emerging principles for understanding the sustainability of these systems.
Analyzing and modeling carbon and nitrogen flows in agricultural ecosystems
has been an active area of research for over thirty years [23, 24]. Researchers
have studied a variety of biological and nonbiological processes connected with
plant growth. These processes occur in the soil, on the surface and above the
surface of the ground. Researchers have estimated the rates of these processes
based on local factors such as temperature, soil type, rainfall, plant genetic po-
tential and existing pools of carbon such as microbial carbon, standing dead
plant, carbon, etc. Tillage and harvesting practices have also been taken into ac-
count, as have other human inputs such as fertilizers and irrigation. As a result
of such work, flexible, powerful agricultural ecosystem models such as the CEN-
TURY model have been developed [25, 26]. To illustrate the breadth of processes
considered in such ecosystem level studies, a diagram for carbon flow in the
CENTURY model is given below.
Similar agricultural ecosystem models have been developed for nitrogen-con-
taining species and are being developed for phosphorus-containing compounds.
When these ecosystem models are combined with models of the biorefinery
processes, we can use them to evaluate and then improve the sustainability of
integrated biorefining systems. We illustrate this approach below.
2.5.3
Analyzing the Sustainability of Integrated Biorefining Systems: Some Results
We envisaged an integrated biorefining system based on corn grain, soybeans,

and corn stover, i.e. the cellulosic residue remaining after grain is harvested.
We assume that a second harvesting trip through the field is required to cut,
bale, and remove the corn stover. Fuel ethanol is assumed to be produced from
the corn grain by wet milling and additional ethanol from corn stover by acid
hydrolysis and fermentation [27]. Residual fermentation solids are burned to
produce electricity and steam. Steam is used in the plant and excess electricity
is exported to the grid. Soybean oil is converted to biodiesel. To satisfy soil ero-
sion prevention guidelines, a minimum of 1070 kg ha–1 corn stover are left be-
hind. None of the soybean stover is removed. Several different agricultural sce-
narios are investigated to determine the effect of cropping system management
on environmental performance. The cropping system assumptions for sustain-
ability analysis are summarized below in Table 2.3. The acronyms given to each
scenario in Table 2.3 are used in subsequent figures to identify simulation re-
sults.
In all, six different scenarios are modeled and analyzed. Because “all biomass
is local” we choose a particular location, Washington County, Illinois, USA, for
our analysis. Climate and soil data and cropping practices are available for this
Table 2.3 Assumptions for cropping system sustainability analysis.
Basic cropping system

– Corn (plow till) – soybean (no-till): CPSN (grain)
Effect of winter cover crop under no-till corn continuous cultivation
– 0% of corn stover removed: CC (grain) (No winter cover crop)
– Average 56% of corn stover removed: CC (56%) (No winter cover crop)
– Wheat and oat as winter cover crops with 70% corn stover removal: CwCo (70%)
Effect of winter cover crop under no-till corn–soybean rotation
– Wheat and oat as winter cover crops after corn cultivation with 70% corn stover removal:
CwCo (70%)
– Average 54% of corn stover removed: CS (54%) (No winter cover crop)
Cover crop not harvested
location. The agricultural base is conventional midwestern US corn–soybean ro-

tation with only corn grain and soybean harvested, using plow till for corn and
no till cultivation for soybeans (CPSN in Table 2.3). For all other scenarios both
corn and soybeans are grown under no-till cultivation practices, in which soil is
left undisturbed from harvest to planting. A second scenario is the continuous
cultivation of corn (CC), again with only corn grain harvested. A third scenario
includes the harvest and removal of 56% of the corn stover and all the grain un-
der continuous corn cultivation (CC 56%). A fourth scenario is rotation of corn
with soybeans and removal of 54% of the corn stover and all the corn grain and
soybeans (CS 54%).
We explore the use of cover crops in the two final scenarios. In some areas it
is common to plant another crop such as wheat or oats in the late fall after the
corn is harvested. These young plants grow a few centimeters, survive over the
winter and then resume growth in the spring once conditions permit. Cover
crops can be harvested, killed with herbicide or plowed under to increase soil or-
ganic matter. In this analysis we assume that the cover crop is not harvested by
rather is killed with herbicide and the corn or soybean is planted in the dead
cover crop.
Cover crops help eliminate wind and water erosion and are also effective in
removing excess nitrogen fertilizer left behind in the previous cropping cycle.
This excess nitrogen is vulnerable to conversion by various processes to soluble
nitrogen species that leach through soil and are transported to streams, lakes,
rivers and the ocean. This excess nitrogen can also be converted by anaerobic
soil bacteria to nitrous oxide, a potent greenhouse gas. The fifth scenario there-
fore considers continuous corn with 70% removal of stover plus winter wheat
or oats as a cover crop (CwCo 70%). The sixth and last scenario assumes a
corn–soybean rotation from which 70% of the corn stover is harvested and win-
ter cover crops are grown (CwSo 70%).
Soil organic carbon levels were predicted over time using the six scenarios
modeled. Depending on conditions chosen, either static or increasing soil or-
ganic carbon levels are possible as shown in Fig. 2.7, below, as long as cultiva-
tion is no till. The use of winter cover crops enables very substantial removal of
corn stover for industrial uses while still enhancing soil quality over time.
The total amount of protein, lipids, lignin and carbohydrate (including starch,
cellulose and hemicellulose) produced by each of the cropping systems above
was estimated over a 40 year period. Based on data from biorefinery operations
and crop production databases, greenhouse gases were also calculated in kilo-
gram carbon dioxide equivalents per kilogram of each of these species produced.
Figure 2.8 shows greenhouse gas production per kilogram of carbohydrate for
each of the six scenarios.
Once again, the cropping system chosen has a significant effect on the re-
sults. The results range from a net production of 560 g of CO2 kg–1 carbohy-
drate for conventional corn–soybean rotation to 9 g (net carbon sequestration)
kg–1 carbohydrate for continuous corn cultivation under no-till conditions with
winter wheat and 70% stover removal.
A similar life-cycle approach is taken to estimate the greenhouse gas reduction
when ethanol produced using the biorefinery systems outlined above is consumed
in a mid-sized passenger vehicle. Figure 2.9 summarizes these results. All of the
cropping systems result in net greenhouse gas reduction, but there are substantial
differences between cropping systems. Aggressive corn stover removal when
coupled with winter cover crops can result in over 70% reduction in greenhouse
gas formation compared with a gasoline fueled vehicle. But as Fig. 2.7 shows, soil
health can continue to improve even when large amounts of stover are removed.
Finally, even more dramatic reductions in leached nitrogen (inorganic nitro-
gen species escaping the root zone of plants) are possible by judicious choice of
cropping systems and agricultural system management. Using the nitrogen
flow submodel of the CENTURY model to predict the effects of different agri-
cultural practices, it seems possible to reduce inorganic nitrogen losses more
Fig. 2.7 Soil organic carbon trends under different agricultural practices.
Fig. 2.8 Greenhouse gas emissions per kg carbohydrate.
Fig. 2.9 Greenhouse gas reductions under different cropping systems.
than a factor of ten by use of conventional corn production as the base case.
These results are summarized in Fig. 2.10. The results are for a 40 year period
in which these practices are used in a specific geographic location – Washington
County, Illinois, USA.
As expected, the use of winter cover crops greatly reduces nitrogen leaching.
It is also interesting to note that stover removal seems to reduce nitrogen leach-
ing – compare CC with CC (56%). One mechanistic explanation of these results
is that the nitrogen content of the corn stover (approx. 1% by weight) is not
available for conversion to soluble inorganic nitrogen species when the stover is
removed, thereby reducing leaching. Also, the carbohydrates in harvested stover
are not then available to provide metabolic energy for microbial processes that
Fig. 2.10 Inorganic nitrogen losses over 40 years – Washington Country, Illinois.
convert organic nitrogen into inorganic nitrogen. Although stover removal must
be carefully managed to maintain soil fertility, stover removal has powerful envi-
ronmental benefits. These benefits include providing renewable carbon for bior-
efining to fuels and chemicals and reduced inorganic nitrogen leaching.
On the basis of these and other results, we believe that a combination of crea-
tive system design, careful planning and use of powerful ecosystem and biore-
finery modeling tools can help us achieve very significant environmental im-
provements as we develop the biobased economy of the 21st century. We need
not be content with maintaining the environmental status quo or even with
modest improvements. Instead, very significant improvements are possible if
we are both wise and informed.
2.6
Conclusions
The biobased economy will grow rapidly during the 21st century. A combination
of low-cost plant raw materials and gradually improving biorefinery process
technology for converting these raw materials into a variety of fuels, chemicals,
materials, food, and feed will drive the adoption of the biobased economy. The
biological sciences will have a particularly powerful impact on both the raw ma-
terials and the processing technologies underlying the biobased economy. The
biobased economy, and its associated biorefineries, will be shaped by many of
the same forces that shaped the development of the hydrocarbon economy and
its refineries over the past century. These similarities include the importance of
yield (using the whole “barrel of biomass”), continuing diversification of prod-
ucts, and gradual process improvement in functioning biorefineries.
References 65
However, significant differences between the biobased economy and the hy-
drocarbon economy are also apparent. Among these are the great compositional
variety of plant raw material, requiring a greater range of processing technolo-
gies to add value to the basic components, and the much wider geographic dis-
tribution of both raw materials and the associated refineries. This wide geo-
graphic distribution of both raw materials and biorefineries will promote greater
economic/national security and more equitable distribution of wealth. We be-
lieve that supposed limits on agricultural productivity to support the biobased
economy are mostly illusory. There is no “food vs. fuel” conflict. Economic prof-
itability and process efficiency will force the adoption of “food and fuel” scena-
rios. Biorefineries and their associated crop production systems will be highly
integrated. Furthermore, integrated biorefining systems will be designed to
achieve not only economic profitability but also environmental benefits. Truly
transformational environmental benefits can be achieved by creative design of
these integrated biorefining systems.
Acknowledgements
The authors gratefully acknowledge support from Cargill Dow, LLP, from Du-
Pont Biobased Materials, Inc., and from the Center for Plant Products and Tech-
nology at Michigan State University.
References
1 C. J. Arntzen, B. E. Dale (Eds.), Biobased 9 S. Kim, B. E. Dale, J. Ind. Ecol. 2004,

Industrial Products: Research and Commer- 7(3/4), 147–162.
cialization Priorities, National Academy 10 B. E. Dale, TIBTECH. 1987, October
Press, USA, 2000, 26–54. 287–291.
2 C. J. Campbell, J. H. Laherrere, Scientific 11 G. E. Tong, Chem. Eng. Prog. 1978, April
American. 1998, 278 (3), 78–83. 70–83.
3 L. F. Ivanhoe, World Oil 1996, 217(11), 12 C. E. Wyman, Ann. Rev. Energy Environ.
91–94. 1999, 24, 189–226.
4 J. T. Houghton, Ency. Energy Tech. Envi- 13 S. Kim, B. E. Dale, Biomass & Bioenergy
ron. 1995, 1, 491–504. 2004, 26, 361–375.
5 M. Graboski, R. Bain, Biomass Gasifica- 14 P. Gallagher, et al., Biomass from Crop
tion: Principles and Technology, T. B. Reed Residues: Cost and Supply Estimates, US
(Ed.), Noyes Data Corporation, USA, Dept of Agriculture Economic Report
1981, 41–71. 819, USA, 2003.
6 Energy Information Agency, http:// 15 United States Dept. of Agriculture Hay
www.eia.doe.gov/pub/international/iealf/ Reporter http://www.ams.usda.gov/
table18.xls, United States Dept. of En- mnreports/DC_GR310.txt. 2004.
ergy, 2002. 16 United States Dept. of Agriculture Agri-
7 Anon., Chem. Eng. News 2002, 22, 55–63. cultural Statistics Table 6.1 http://
8 Statistical Abstract of the United States, www.usda.gov/nass/pubs/agr04/
Table #958 (http://www.census.gov/prod/ 04_ch6.pdf. 2003.
2001pubs/statab/sec19.pdf), 2000.
17 J. A. Stricker, et al., Production and Man- 24 P. Smith, et al., Quantifying the change in
agement of Biomass/Energy Crops on Phos- greenhouse gas emissions due to natural re-
phatic Clay in Central Florida, Univ. of source conservation practice application in
Florida Cooperative Extension Service Indiana, Final report to the Indiana Con-
Circular 1084, USA, 1993. servation Partnership, Colorado State
18 R. G. Lugar, R.J. Woolsey, Foreign Affairs. University Natural Resource Ecology
1999, 78(1), 88–102. Laboratory and USDA Natural Resources
19 M. E. Ensminger, C. G. Olentine, Jr., Conservation Service, USA, 2002.
Feeds and Nutrition – Complete, The Ens- 25 A. K. Metherall, L. A. Harding, C. V. Cole,
minger Publishing Company, USA, W. J. Parton, CENTURY Soil organic
1978. matter model environment. Technical
20 L. B. de la Rosa, et al., Appl. Biochem. & documentation. Agroecosystem version
Biotech. 1994, 45/46, 483–497. 4.0. Great Plains System Research Unit
21 L. R. Lynd, C. E. Wyman, T. U. Gern- Technical Report No. 4. UDSA-ARS,
gross, Biotech. Prog. 1999, 15, 777–793. USA, 1993.
22 R. Anex (Ed.), J. Ind. Ecol. 2004, 7(3/4). 26 R. H. Kelly, et al., Geoderma 1999, 81,
See articles on pages 75–92, 93–116, 75–90.
117–146 and 147–162. 27 J. Sheehan, et al., Is ethanol from corn
23 L. Kristen, et al., Parameterizing Century stover sustainable?, National Renewable
to model cultivated and noncultivated sites Energy Laboratory Draft Report, USA,
in the loess region of western Iowa, US December 2002.
Geological Survey Report 00-508, US De-
partment of the Interior, USA, 2000.
67
3
Development of Biorefineries –
Technical and Economic Considerations
3.1
Introduction
As the world’s population and economy continue to grow, so does the demand
for goods to sustain that growth. Growth has come at the expense of our non-
renewable resources. It is only a matter of time before the price of petroleum,
upon which the world economy is heavily dependent, will rise to a point where
we will be forced by economic factors to find alternatives. We suspect this time
is not far off in the future and the time is now to explore viable alternatives.
Our nation is fortunate to have abundant agricultural and forest renewable re-
sources and a climate that is amenable to their productive use. If we put our
mind to it, these renewable assets can go a long way to replace our heavy de-
pendence on petroleum and other non-renewables. That process has, in a way,
already begun, as we have been in the business of “bio-refining” in the broader
sense for quite some time. Our forests are harvested to produce a host of prod-
ucts including paper, solvents, building materials, and many more. Our agricul-
ture industry can produce large quantities of grain and other crops, which can
and are being used to produce a host of materials. Starch from grain crops and
sucrose from beet, sugar cane, and other materials are being converted to an in-
creasing number of products and chemicals. Some of the chemicals now being
produced include ethanol, 1,3-propanediol, lactic acid, and ascorbic acid. As the
technology of pathway engineering advances, one can expect many more chemi-
cals (and polymers) to be produced from sugar as a carbon source [1, 2].
Increasing demand for sugars will eventually result in increasing sugar prices
and, ultimately, supply problems. It is logical to assume that as more chemicals
and materials are produced from fermentable sugars as the carbon source, mar-
ket forces will inevitably drive fundamental changes in the agriculture and
chemical sectors.
Genencor International has been active in developing technologies that have
affected the evolution of biorefineries. As part of our efforts in this area, we
have actively explored the concept of using cellulosic biomass to provide the car-
ISBN: 3-527-31027-4
68 3 Development of Biorefineries – Technical and Economic Considerations
bon source for such future refineries. In this pursuit, we have focused on the
conversion of cellulosic biomass into fermentable sugars and on the challenges
of biocatalysis in utilizing those sugar streams [3, 4].
Advances in pathway engineering will result in commercially viable and com-
petitive processes based on renewable feedstocks which in many cases will pro-
vide the low-cost route to production. The cost of carbon to feed biocatalysis is
often more than 50% of the total direct cost of production, and therefore efforts
to reduce this cost play an important role in the overall development of the bio-
refinery concept. It is not surprising that many have focused on cellulosic bio-
mass (e.g., agricultural waste) as the ultimate low cost source of the fermentable
sugar in the biorefinery. Much knowledge has been gained and progress made
on the understanding of cellulosic biomass saccharification to fermentable su-
gars to be used for bioconversion to chemicals.
The challenge before us is to develop the technologies that will be required to
enable this upcoming industry. We will focus here on two of these technologies:
1. enzymes required for conversion of cellulosic biomass to fermentable sugars
within an enabling cost structure; and
2. engineered organisms to produce chemicals competitively.
3.2
Overview: The Biorefinery Model
Biorefineries, often referred to as integrated biorefineries, are processing facil-

ities that use renewable plant materials as feedstocks. Plant materials, compris-
ing carbohydrates and associated oil, protein, lignin, and other components, are
converted in the biorefinery into higher-value chemicals and materials. Our
broad definition of biorefineries includes a initial process that utilizes renewable
carbon feedstocks containing sucrose, starch, and cellulose as shown in Fig. 3.1.
Essential elements of a biorefinery are:
1. multiple feedstock capability and a tolerance of wide variation in those feed-
stocks;
2. feedstock-processing by enzymes to fermentable sugars (and by-product
streams);
3. biocatalyst, which converts sugars to desired product(s), and
4. co-products which are used in the process, recycled through the process, or sold.
3.3
Feedstock and Conversion to Fermentable Sugar
Three basic biorefinery approaches, as portrayed in Fig. 3.2, are evolving on the
basis of the nature of the feedstock, i.e. sucrose, starch, or cellulose.
On a fermentable-carbon-cost basis, the sucrose-based Biorefinery I is cur-
rently the most cost-competitive. In recent years, however, the starch-based Bio-
Fig. 3.1 The overall biorefinery model.
Fig. 3.2 Biorefinery evolution.
refinery II has become more cost competitive as a result of innovations in farm-

ing and milling grain like corn. In principle, the cellulose-based Biorefinery III
will become more competitive as technical and economic challenges are ad-
dressed. As illustrated in Fig. 3.3, fermentable sugars, whether derived from su-
crose, starch, or cellulose, will become cost competitive with petroleum-derived
carbon for production of fuel, chemicals, polymeric materials, and specialty in-
termediates.
Fig. 3.3 Change of cost of fermentable carbon with time for different carbohydrate sources.
3.3.1
Sucrose
The simplest biorefinery system uses sucrose as feedstock (sugar beet, cane,
etc.). Sugar is first extracted from the feedstock. Lignin and cellulosic residuals,
if sugar cane is used, are utilized separately or burned for energy to run the op-
eration. The sucrose-based industry is a significant biorefinery opportunity today
and into the future. Sucrose is second only to cellulose in availability and cur-
rent output far exceeds all other commercial carbohydrates combined. It is esti-
mated that only 1.7% of annual sucrose production goes to non-food uses. It is
possible to imagine a sugar biorefinery as an integrated producer, not only of
sucrose and ethanol, but of important high-value products whose manufacture
could be scaled up or down depending on circumstances, economics, and de-
mand [5]. Not surprisingly, more than 50% of ethanol volume produced in the
world today has sucrose as feedstock.
3.3.2
Starch
The grain wet-milling industry as it exists today is an excellent example of the

biorefinery concept. The grain kernel, e.g. corn, is processed through a series of
steps resulting in a glucose (or multimers of glucose) stream and co-products
such as oil, protein, fiber, and nutrients (e.g. carotenoids and corn steep liquor).
The glucose can be further converted to ethanol, 1,3-propanediol, lactic acid,
etc., by fermentation, and to sweeteners, for example high-fructose syrup, by en-
zymatic conversion. The starch-based biorefinery has been enabled by the devel-
opment of highly efficient and thermostable amylases and the development of
isomerization processes to convert glucose to fructose by immobilized glucose
isomerase enzyme systems.
The corn (and other grain) dry mill is a highly specialized abbreviated version
of the wet mill whereby many of the initial steeping and extraction steps have
been removed and starch is enzymatically converted to glucose and fermented
to ethanol in a process called simultaneous saccharification and fermentation

(SSF). The capital cost of building these less complex bio-refineries is signifi-
cantly lower than for wet mills, but the trade-off is that dry mill co-product
streams (distillers dried grains and solubles, or DDGS, sold into select animal
feed markets) have relatively lower value.
Thermal energy consumption is significant in the milling process. A wet corn
mill is very effective in extracting maximum value, because of the multiplicity
of products and co-products mentioned above. In general, however, a wet grain
mill is capital-intensive, and therefore new ethanol production plants coming
on-line in recent years are mainly of the dry grain mill type. New enzyme tech-
nology developments hold considerable promise for impact here and these refi-
neries will continue to evolve.
3.3.3
Cellulose
Considering the amount of cellulosic biomass available for saccharification to

fermentable sugar, there is a clear opportunity to develop commercial processes
that could generate products that are needed at very high volumes and low sell-
ing price. Most of such products are now being made from non-renewable re-
sources, mainly through oil refineries. These refineries, starting from a complex
mixture (petroleum), use a wide range of unit operations to generate an impres-
sive variety of products that are sold directly or transformed into value-added
products like plastics, fibers, etc. Approximately 17% of the volumes of products
derived from petroleum in the US are classified as chemicals. If these chemi-
cals could be obtained from renewable resources like biomass in a biorefinery,
it would reduce our petroleum dependence and also have a positive environ-
mental impact.
Supplies of starch and sucrose feedstock will probably not be sufficient to
meet the feedstock needs of future biorefineries. As an example, the gasoline
market in the US alone is 150 billion gallons per year. MTBE (methyl tertiary-
butyl ether) replacement at 6% would result in approximately 10 billion gallons
per year of ethanol, which would require about 30% of the US farmland cur-
rently growing corn. Given that ethanol is only one of many compounds that
can flow out of a corn biorefinery, it is likely that other sources of carbon feed-
stock will also be required. Brazil and a few other tropical and sub-tropical
countries use sucrose in biorefineries that produce ethanol. Because it is widely
believed that it is not feasible to produce enough sugarcane (and other sugar
crops) to meet potential fuel ethanol volume requirements, there has been a 50-
plus year effort to develop technologies to convert cellulose from biomass into a
carbon feedstock for the biorefinery of the future.
Cellulosic biomass conversion to fermentable sugars has been explored as the
potentially lowest cost feedstock for the biorefinery of the future. Unlike sugar
energy storage compounds like starch, which can be converted to fermentable
sugar with relative ease, cellulosic biomass is a complicated structure of cellu-
lose (b1–4 glucose linkage), hemicellulose (linked C5 sugars including man-

nose, galactose, xylose, and arabinose), lignin, and numerous minor compo-
nents. This structure has evolved as a support element of plants, and therefore
is not meant to be readily accessible as a carbon source.
Currently, the most promising approach for using this feedstock is enzymatic
hydrolysis of the cellulose content after pretreatment of the fiber to make the
cellulose more accessible to enzymatic attack. The process for conversion of cel-
lulosic biomass to fermentable sugars faces major technical and engineering
challenges that have so far prevented large-scale commercial use of cellulose as
a source of fermentable sugar.
A significant cost component in the overall process to break down cellulosic
biomass has been the cost of cellulase enzymes required to carry out the pro-
cess. As much as 100-fold more native cellulase protein (compared with amylase
protein for breakdown of starch) is required for conversion of pretreated sub-
strate, e.g. corn stover, into fermentable sugars. Given the relatively high cost of
the enzymes and the amount required to produce fermentable sugars, the pro-
cess has not been viable. In addition, the pretreatment of cellulosic biomass,
making cellulose available for the enzymatic hydrolysis, has been a significant
challenge. Many approaches have been explored; however, all suffer from their
capital intensity because of the extreme conditions of the process. The cost im-
pact of processing differences between corn and stover is reflected in Fig. 3.4.
The cost of cellulose-hydrolyzing enzymes, or cellulases, is not included in
this analysis. Through a three-year DOE funded program administered by the
National Renewable Energy Laboratory (NREL), and a one year extension of that
program, major advances have been made toward reducing the cost of enzymes
in ethanol production from pretreated corn stover. The impact of these improve-
ments on cellulase cost, estimated as $ per gallon of ethanol (EtOH) produced
in a bench-scale NREL assay, can be seen in Fig. 3.5.
This improvement has come from reducing the cost of producing the en-
zymes, enhancing the mix of enzymes, and altering, or recruiting, key enzymes
to enable operation at elevated temperatures. However, enzyme requirement re-
mains high, even under the higher-temperature operating conditions engineered
into the multi-enzyme system. It is anticipated that enzymatic hydrolysis cost
will be further reduced by continued improvement in the enzymes and new
processes that use elevated temperatures and more effective pre-treatment pro-
cesses.
One strategy to minimize ethanol production cost is to run simultaneous sac-
charification and fermentation, or SSF, which would use ethanologens engi-
neered to operate in high temperature environments. Also, the fermentation or-
ganism’s ability to utilize C5 sugars derived from the hemicellulose component,
and have acceptable productivity in the presence of numerous byproducts of the
biomass pretreatment process, would lead to lower overall production cost.
Fig. 3.4 Cost components production of fuel ethanol from corn and corn stover [6].
Fig. 3.5 Cellulase enzyme cost estimate based on the NREL assay [6].
3.4
Technical Challenges
3.4.1
Cellulase Enzymes
The challenge of improving cellulase enzymes is significant. Lignocellulose is

not a pure compound on which cellulase activity can be optimized. The compo-
sition and structure of the biomass substrate can vary dramatically depending
on the source of the lignocellulose. Corn stover structure is different from su-
garcane bagasse, which in turn is different from municipal solid waste. In addi-
tion, corn stover from one part of the country can differ significantly from that
from another, because of soil, weather, and other conditions. Finally, different
pretreatment processes affect biomass in unique ways, resulting in even further
differences between structure and composition. Thus the enzyme system re-
quired may need to be optimized for each specific combination of substrate and
pretreatment that would be employed.
In 2000, Genencor International was awarded a three-year US Department of
Energy (DOE) subcontract through the National Renewable Energy Laboratory
(NREL) to reduce the cost of cellulase, on a “per gallon of ethanol produced by
NREL process and assay basis”, by a factor of ten. That goal was achieved within
the three-year timeframe, and the program was extended to further reduce costs
to a factor of twenty. These aggressive goals were approached from two direc-
tions by making significant improvements in cellulase production economics
(reduced cost/gram enzyme) and in cellulase enzyme performance (reduced
grams of enzyme needed).

$ $
Enzyme cost in assay enzyme cost
gallons EtOH g enyzme

g enzyme
enyzme loading 1
gallons EtOH
3.4.1.1 Improved Cellulase Production Economics

An understanding of the components of production cost is a necessary prerequi-
site for effectively reducing the cost. A simplistic, but highly useful, model
would identify fixed and direct costs for each of the three major production pro-
cesses fermentation, enzyme recovery, and formulation:
Total cost Fermentation cost (fixed direct) Recovery cost (fixed direct)
Formulation cost (fixed direct) 2
Most current commercial cellulase products are sold as cell-free, stabilized con-
centrates. Although these formulations meet market needs with regard to appli-
cation and cost, for many enzyme products it is not uncommon for the recovery
and formulation costs to be a major portion of the overall cost. It then follows
that reduced post-fermentation processing would possibly lead to reduced cost,
with no post-fermentation processing being the lowest cost. This is, however,
only practical if the resulting product still meets the needs of the application.
Fermentation broth with no processing was tested in saccharification of pre-
treated corn stover. Performance in the saccharification was indistinguishable
among fresh whole fermentation broth, fresh fully-recovered and formulated
product, and 28-day old fermentation broth. These results suggest that typical re-
covery and formulation costs can be eliminated for use in biorefinery operations,
especially in an integrated plant that both makes and uses the cellulase enzymes.
This leaves the fermentation costs that can be broken down into fixed costs,
for example depreciation and labor, and direct costs, for example utilities and
raw materials.
Fermentation cost Fixed cost (labor depreciation)

Direct cost (utilities other raw materials
carbon/energy source) 3
The single largest cost component for the fermentation process was estimated
to be the carbon/energy source for the culture. For Trichoderma cellulase, the
carbon source has historically been lactose. The cost per unit enzyme produced
is a function of the yield of enzyme on the carbon source and the cost of the
carbon source itself. When performed in a similar manner, the other costs are
proportional to the rate of enzyme production, as measured by volumetric pro-
ductivity. An integrated plan of action was taken to affect both enzyme expres-
sion and the fermentation process. Improvements were made on the production
organism, the production process, and in their interactions.
A conventional mutagenesis and screening approach was applied to the exist-
ing production strain. The work had several objectives. One was to find strains
capable of fast and efficient growth on cellulose. A useful screening method
was adapted from the method of Toyama [7]. In this method, mutated spores
are poured in large numbers with agar in the bottom of a Petri dish. A second
layer is added on top containing cellulose as the sole carbon source. The spores
germinate and grow through the agar eventually emerging on top of the cellu-
lose-agar layer. Those that erupt first are more likely to grow quickly and effi-
ciently on cellulose. Another goal was to disrupt existing regulatory mecha-
nisms that result in catabolite repression and the need for induction of cellulase
expression. Several plate screening methods were developed that used a high
glucose concentration to overcome catabolite repression or glycerol as the car-
bon source to find expression without induction. A third goal was to improve
the secretion of cellulases. Resistance to different chemical agents and selection
for hyper-branched morphological mutants were both employed.
Through successive rounds of mutagenesis and selection with different

screening methods, an improved host strain was developed. Several new traits
were recruited that positively affected fermentation cost. First, the specific
growth rate of the strain was increased by 50%. This reduced the growth time
needed for the fermentation, positively affecting enzyme productivity. The spe-
cific productivity of the strain, enzyme per unit cell mass per unit time, was
also improved. This impacted both key metrics, i.e. yield of enzyme from the
carbon source and volumetric productivity.
The improvement in yield helped reduce cost of the sugar lactose per unit en-
zyme produced. Lactose, a by-product of the dairy industry, typically costs signif-
icantly more than glucose, a product from sucrose, starch, or cellulose process-
ing. Lactose is a mild inducer of cellulase expression with little to no catabolite
repression. Glucose, on the other hand, does not induce cellulase expression
and exhibits high catabolite repression. It would thus seem improbable to re-
place relatively expensive lactose with relatively inexpensive glucose. The disac-
charide sophorose (b-1,2 linkage of two glucose units) is one of the most potent
inducers of cellulase expression known [8]. It was found that treating a concen-
trated glucose solution with whole cellulase at elevated temperatures leads to
the formation of numerous higher sugars, among them sophorose. This treated
solution can then be fed to a cellulase-producing fermentation with perfor-
mance equal to or better than that obtained using lactose. The small amount of
enzyme product that must be recycled to produce sophorose is more than offset
by the reduced cost of glucose relative to lactose and by the improved fermenta-
tion performance. Traditional fermentation optimization was performed on the
improved strain by adjusting the level of salts and other nutrients, temperature,
pH, and glucose/sophorose mixture.
The cumulative effect of all these improvements was very significant, and the
production cost, per unit of enzyme, was reduced by approximately a factor of
seven. These improvements went far beyond what had been deemed probable
for an established and mature product such as whole cellulase. Investigations
were also made to evaluate pretreated corn stover as a carbon feed for cellulose
production; this will be discussed below.
3.4.1.2 Improved Cellulase Enzyme Performance

Reducing enzyme production cost was only part of the solution. As shown in
Eq. (1), improving the performance of the cellulase enzymes, resulting in re-
duced enzyme loading per gallon of ethanol can be equally effective. Several of
different approaches have been used to improve enzyme performance, including
increasing thermal stability, recruitment of novel cellulolytic activity, and in-
creasing specific activity [9–12].
A major focus was on increasing the thermal stability of the cellulase en-
zymes. Cellobiohydrolase I (CBH I) is the major component of whole cellulase.
It also happens to be the component with the lowest thermal stability, as indi-
cated by Tm, the characteristic “melting” temperature. Successful engineering of
CBH I to improve thermal stability also required several strategies. CBH I struc-
ture analysis suggested sites that could affect thermal stability, as did comparing
the structures of CBH I homologues. Random mutagenesis and screening were
also used. An important part of this effort was the development of a small-scale
screening method to quickly and accurately measure the effect of the changes
made. By the incorporation of a large number of specific site changes, Tm of
CBH I was improved significantly. Placing the engineered CBH I into a produc-
tion strain in which the native CBH I gene was deleted did not have a negative
effect on enzyme production levels.
Along with the ability to engineer proteins for increased thermal stability, it is
imperative that the engineered proteins can be expressed at high levels in pro-
duction strains without negatively affecting the other cellulase components.
This has proven to be the case for both T. reesei-engineered CBH I and for
homologues from other fungi. The homologue from Humicola grisea var thermo-
idea was expressed at levels similar to the wild-type CBH I without negatively af-
fecting the production of the other cellulase components.
By analysis of the CBH I structure, several sites were selected that were hy-
pothesized to affect binding of substrate and products in the active site cleft.
Several mutants were made, with significant effects on both Km, the Michaelis-
Menten constant and kcat, the catalytic rate constant.
Many of these improved enzymes have been produced effectively in whole cel-
lulase production strains. As discussed above, improved cellulase production
has been maintained with the improved enzymes incorporated. The resulting
products have been tested in the NREL process scheme and have resulted in an
approximately threefold reduction in enzyme loading.
The work discussed above was performed by Genencor International and col-
laborators as part of a subcontract funded by the DOE administered by NREL.
A similar subcontract was also awarded to Novozymes. The improvements re-
ported to date by both the groups have been quantitatively very similar. The
approaches taken have also been similar. Together, it is clear that the cost of en-
zymes for biomass saccharification has been dramatically reduced from the
baseline in 2000. Enzyme cost can no longer be regarded as the largest barrier
to commercialization of biorefineries.
3.4.2
Fermentation Organisms
A biorefinery would be a processing factory that would separate biomass into

component streams, and transform them into a wide range of products, using
enzymatic and/or fermentation processes. This concept has been around for
some time, but has not been implemented to its full potential. The utilization
of biomass and the development of biorefineries are enormous technological
challenges that no doubt require the solution of multiple problems.
3.4.2.1 Biomass Hydrolyzate as Fermentable Carbon Source

The use of live cell fermentation to convert sugars into commercial products or
intermediates has been exploited successfully for many years. These fermenta-
tion processes are based on the use of relatively clean sugar streams that con-
tain few impurities. However, the full use of all the carbon components in bio-
mass is more complicated. After biomass pretreatment, and enzymatic hydroly-
sis of cellulose (and hemicellulose), a mixture of hexose and pentose sugars and
several degradation by-products, for example furfural, hydroxymethylfurfural,
phenols, and formic, acetic and other acids is obtained. Several of these pre-
treatment and hydrolysis by-products are well known inhibitors of fermentation
processes. The extent of inhibition depends on the fermentation process. Occa-
sionally it is severe and inhibits cell growth completely. To avoid this, the hydro-
lyzates must be diluted or fed during fermentation, complicating the fermenta-
tion processes and reducing the total amount of hydrolyzed biomass that can be
used per unit of time or volume. In other circumstances the impact of the by-
products is less severe, because they do not inhibit growth but instead have a
negative impact on performance of the cells. For example, at certain concentra-
tions, some organic acids, for example formic or acetic, have a negative effect
on cell physiology and increase the amount of energy that cells use to perform
their normal functions (cell maintenance). This increase in energy consumption
is, in turn, reflected in higher consumption of carbon from sugars, with a con-
sequent decrease in the overall yield of product per unit of biomass [13].
Besides the effects of by-products present in hydrolyzed biomass, the highly
concentrated stream of carbohydrates obtained can also be a problem. When ex-
posed to high concentrations of sugars and salts, cells tend to undergo osmotic
stress that, depending on its magnitude, can inhibit cell growth or cause an in-
crease in cell maintenance, leading to problems described above.
Another well-known response of cells to the presence of high concentrations
of carbon sources is that they tend to use the sugars in a particular order and
in an inefficient manner. Cells tend to utilize first the carbon source that is eas-
iest to metabolize and that provides more energy. When this carbon source is
completely consumed or reaches a particular concentration, cellular metabolism
is re-adjusted to utilize another carbon source present in the mixture. This se-
quential use of the carbon sources and the associated metabolic adjustments
complicates fermentation process design because optimum cell-performance is
obtained when some of the growth media components, mainly carbon and ni-
trogen, are present in certain ratios. From the engineering design perspective of
the fermentation process, these ratios are calculated on the basis of total carbon
present in the mixtures. From the cells’ perspective, however, only one or a few
carbon sources at a time are “sensed”, and a carbon-to-nitrogen ratio calculated
on the basis of total carbon content is detected as an unbalanced mixture by the
cells. Very often this leads to poor cell and fermentation process performance.
3.4.2.2 Production Process as a Whole

In addition to addressing problems related to the use of mixtures of complex
sugars and inhibitors, development of commercially viable fermentation pro-
cesses requires proper process integration of the different unit operations to re-
duce costs and disposal of fermentation by-products. Enzymatic hydrolysis of
biomass is currently performed with a mixture of enzymes that work in the 50–
60 8C temperature range and 3–5 pH range. However, most of the production
strains developed so far to utilize biomass hydrolyzates perform better in the
30–40 8C temperature range and the 6–8 pH range. This means temperature
and pH must be adjusted before starting fermentation. pH adjustment is not
only expensive but also produces salts that will have an osmotic effect during
fermentation and must be disposed of properly at the end of the process. Tem-
perature adjustments also increase fermentation costs.
For these reasons it is highly desirable to develop production strains that can
utilize biomass hydrolyzates satisfactorily at 50–60 8C and pH 3–5 [14]. This
would enable relatively inexpensive SSF processes. Furthermore, low pH and
high-temperature fermentation conditions would retard contamination. On the
other hand, it is important to note that at high temperatures the inhibition ef-
fects of toxic compounds in biomass hydrolyzates are also magnified.
An SSF process may not always be the best means of obtaining specific prod-
ucts from biomass hydrolyzates, however. Occasionally the physiochemical prop-
erties of the product(s) must be considered when designing a process. For ex-
ample, a study on production of twelve top value-added chemicals from biomass
identified eight as acids, three of which must be produced in the free-acid form
directly during fermentation. If these are produced in the salt forms first, the
cost impact on product purification and disposal of by-product salts is high [15].
As shown in Table 3.1, pK values of several commercially relevant acids are in
the 3–5 range. For production of these, therefore, the fermentation pH neces-
sary for a product in the free-acid form must be in the 2–4 range, making SSF
an unlikely process choice.
Table 3.1 pK values of some commercially important organic

acids that are or potentially can be produced by fermentation.
Acid pK Value
Pyruvic 2.50
Fumaric 3.03
Malic 3.40
Itaconic 3.84
Lactic 3.86
Aspartic 3.90
Succinic 4.19
Glutamic 4.20
Oxalic 4.21
3.4.2.3 Emerging Solutions

On the basis of the challenges discussed above it is evident that future efforts to
develop commercial processes utilizing biomass hydrolyzates for fermentation
will require production strains different from traditional organisms like yeast,
Bacillus, E. coli, Pseudomonas, Corynebacterium, etc. [14]. Instead, we will need to
find or develop microorganisms capable of performing better at low pH and/or
higher temperatures, and under high concentration of carbohydrates. Further-
more, these new microorganisms would also need to be able to resist the by-
products generated during biomass pretreatment and hydrolysis.
In the last few years there has been an explosion in our knowledge of nontra-
ditional microorganisms. Today, the genomes of hundreds of bacteria and Ar-
chaea are available [16]. In this collection of newly characterized microorgan-
isms we can find almost any physiological trait that we want. These strains can,
furthermore, often be manipulated genetically. Some examples of these are
shown in Table 3.2. From these microorganisms and many others we can learn
the strategies that Nature has selected to deal with high temperatures, high con-
centrations of toxic compounds, sugars, salts, pH, etc., and in some of the ge-
nomes we can also find new enzymes that would enable the use of lignocellulo-
sic materials for fermentation.
On one side, genomics is providing an immense number of physiological so-
lutions. Likewise, advances in other research areas are providing possible solu-
tions to some of the problems mentioned. For example, some mutations have
been designed to eliminate the sequential utilization of carbohydrates present
in complex mixtures [17–19]. Some of these mutations reduce the ability of the
production strains to induce hundreds of genes that could produce undesired
phenotypes during fermentation. Also, use of genomic arrays is helping us un-
Table 3.2 Some examples of the sequenced genomes from

microorganisms with properties relevant for the development
of biorefineries.
Organism name Relevant properties
Thermoplasma acidophilum Optimum growth: 59 8C, pH 2.0

Thermatoga maritima Optimum growth temp. 80 8C. Capable of using starch, cellu-
lose, xylan as carbon sources
Halobacterium sp. NRC-1 Requires 4 m NaCl to grow
Corynebacterium efficiens Grows and produces glutamic acid above 40 8C
Gluconobacter oxydans Extracellular oxidation of a wide range of carbohydrates and
alcohols. The corresponding products (aldehydes, ketones and
organic acids) are secreted almost completely into the
medium
Picrophilus torridus Optimum growth at 60 8C and pH 0.7. Its membrane has very
low proton permeability
Sulfolobus sulfataricus P2 and Grow optimally at 80 8C and pH 2–4
Sulfolobus tokodaii strain 7
3.5 Conclusions 81
derstand the types of physiological response that occur when cells are exposed
to stressful conditions. For example, comparison of E. coli strains capable of
producing ethanol at different levels has shown that increased ethanol tolerance
results from a combination of multiple changes affecting different aspects of
cell physiology [20].
3.5
Conclusions
The importance of the biorefinery concept is growing and the recent increased
demand for fuel ethanol has in large part driven that growth, as shown in
Fig. 3.6.
New products such as the lactic acid and 1,3-propanediol produced from su-
gar in engineered organisms are recent examples of the biorefinery concept pro-
gressing toward reality. Apart from co-product streams resulting from the pro-
cessing of grain or biomass, the resulting sugars are being used as carbon feed
to furnish an ever-growing list of products including fuel and the building
blocks for the synthesis of chemicals and polymers. Commercial production of
1,3-propanediol for Sorona, based on technology developed in a close collabora-
tion between DuPont and Genencor, will mark the emergence of commercial vi-
able application of the biorefinery concept for production of basic chemical
building blocks in competition with petrochemical-derived materials [21].
Many of the biorefinery elements required for financial success seem to be
currently present, for example:
· high-volume, low-cost application (fuel ethanol);
· multiple alternate product streams;
· ability to shift to different products quickly when required; and
· acceptable cost structure (at least for sucrose, starch).
Fig. 3.6 World demand for fuel ethanol over the years.
Several financial requirements will enable the biorefinery concept to become a

reality in the near future:
· parts of the overall biorefinery scheme that can operate in a stand-alone man-
ner from a financial perspective, and the ability to evolve from there;
· the potential for multiple product or co-product streams – note that this may
ultimately drive efforts to engineer crops to maximize their potential;
· the ability to make use of a variety of feedstocks as a hedging strategy and to
take advantage of pricing opportunities;
· manageable capital requirements and favorable investment environment; and
· government enablement (funded demonstration/development pilot plants;
reasonable regulation of biorefinery operations; favorable tax treatment;
strategic market support to give new biorefinery products a head start).
In summary, the concept of the biorefinery is not new. In its simplistic form,
several pieces have been in operation for thousands of years. Biorefineries con-
tinue to evolve depending on market needs, and growth now is being driven by
the rising demand for fuel ethanol. It will continue to evolve in a manner simi-
lar to the petroleum refineries in the 1900s. Ultimately, as technological devel-
opments expand and the range of products grows, biorefineries will utilize a
wide variety of feedstocks, including cellulosic biomass.
Acknowledgments
This work was supported in part by a subcontract from The Office of Biomass
Program, within the DOE Office of Energy Efficiency and Renewable Energy.
The authors thank Colin Mitchinson and Mike Knauf of Genencor International
for their guidance of the work presented.
References
1 G Chotani, T Dodge, A Hsu, M Kumar, 3 Mitchinson, Colin. Improved Cellulases

R LaDuca, D Trimbur, W Weyler, and K for the BioRefinery: A review of Genen-
Sanford. Commercial Production of cor’s progress in the DOE subcontract
Chemicals using Pathway Engineering. for cellulase cost reduction for bioetha-
Biochemica Biophysica Acta, 1543 (2), nol. Stanford GCEP Biomass Energy
434–455, 2000. Workshop, April 2004.
2 Manoj Kumar, Jeff Pucci, Gopal Chotani, 4 Michael Knauf and Mohammed Moni-
and Karl Sanford. Biocatalytic Conver- ruzzaman. Lignocellulosic Biomass Pro-
sion of Renewable Feedstock to Indus- cessing: A Perspective. International Su-
trial Chemicals. In Lignocellulose Bio- gar Journal, 106#1263, 147–150, April
degradation (ACS Symposium Series 2004.
889) Badal C. Saha and Kiyoshi Hayashi, 5 Mary Ann Godshall, Future Directions
editors, Oxford Univ. Press, pp 363–376, For The Sugar Industry, 2003. http://
2004. www.spriinc.org/buton10a.html
References 83
6 Determining the cost of producing etha- for biomass to ethanol commercializa-

nol from corn starch and lignocellulosic tion. http://www.afdc.nrel.gov/pdfs/
feedstocks. NREL/TP-580-28893. Oct 4957.pdf.
2000. 15 Werpy, T. and Petersen G. Eds. Top value
7 Toyama et al. 2002. Applied Biochemis- added chemicals from biomass. Volume
try and Biotechnology, 98–100:257–263. I. Results of screening for potential can-
8 Mary Mandels, Frederick W. Parrish, and didates from sugars and synthesis gas,
Elwyn T. Reese, J. Bacteriol. 1962 Febru- NREL, August 2004. http://
ary; 83(2): 400–408. www.osti.gov/bridge
9 Foreman PK, Brown D, Dankmeyer L, 16 TIGR Microbial Database: a listing of
Dean R, Diener S, Dunn-Coleman NS, microbial genomes and chromosomes in
Goedegebuur F, Houfek TD, England progress. http://www.tigr.org/tigr-scripts/
GJ, Kelley AS, Meerman HJ, Mitchell T, CMR2/CMRGenomes.spl.
Mitchinson C, Olivares HA, Teunissen 17 Hernandez-Montalvo, V., Valle, F., Boli-
PJ, Yao J, Ward M. Transcriptional regu- var F. and Gosset G. Characterization of
lation of biomass degrading enzymes in sugar mixtures utilization by an Escheri-
the filamentous fungus Trichoderma ree- chia coli mutant devoid of the phospho-
sei. J. Biological Chemistry, 278(34): transferase system. Appl. Microbiol. Bio-
31988–31997, 2003. technol. (2001) 57: 186–191.
10 M. Sandgren, P. Gualfetti, A. Shaw, A. 18 Chatterjee, R., Sanville-Millard, C.,
G. Day, L. Gross, T. Alwyn Jones and C. Champion, K., Clark D. and Donnelly
Mitchinson. Comparison of Homologous M. Mutation of the ptsG gene results in
Family 12 Glucosyl hydrolases and Re- increased production of succinate in fer-
cruited Variants Important for Stability. mentation of glucose by Escherichia coli.
Protein Science, 12(4): 848–860, 2003. Appl. Environ. Microbiol. (2001) 67:
11 F. Goedegebuur, J. Phillips, WAH van 148–154.
der Kley, P. van Solingen, L. Dankmeyer, 19 Nichols, N., Dien, B. and Bothast R. Use
S.D. Power, T. Fowler. Cloning and Rela- of catabolite repression mutants for fer-
tional Analysis of 15 Novel Fungal Endo- mentation of sugar mixtures to ethanol.
glucanases from Family 12 Glycosyl Hy- Appl. Microbiol. Biotechnol. (2001) 56:
drolase. Current Genetics, 41:2, 89–98, 120–125.
2002. 20 Gonzalez, R., Tao, H., Purvis J., York, S.,
12 Sandgren M, Shaw A, Ropp T, Wu S, Shanmugam K. and Ingram L. Gene ar-
Bott R, Cameron A, Stahlberg J, Mitchin- ray-based identification of changes that
son C, Jones A. The X-ray crystal struc- contribute to ethanol tolerance in etha-
ture of the Trichoderma reesei family 12 nologenic Escherichia coli: comparison of
endoglucanase 3 (Cel12A) at 1.9 Å J. Mo- KO11 (parent) to LY01 (resistant mu-
lecular Biology, 308(2):295–310, 2001. tant). Biotechnol. Prog. (2003) 19: 612–
13 Zaldivar, J., Nielsen J. and Olsson L. 623.
Fuel ethanol production from lignocellu- 21 C. Nakamura and G. Whited. Metabolic
lose: a challenge for metabolic engineer- engineering for the microbial production
ing and process integration. Appl. Micro- of 1,3 propanediol. Curr. Opin. Biotech-
biol. Biotechnol. (2001) 56: 17–34. nol. 2003 Oct; 14(5): 454–459.
14 Hettenhauss, J. Ethanol fermentation
strains: Present and future requirements
85
4
Biorefineries for the Chemical Industry –
A Dutch Point of View
Ed de Jong, René van Ree, Robert van Tuil, and Wolter Elbersen
4.1
Introduction
As a major policy goal for 2020, the Dutch government has stipulated that 10%
of its energy use should be provided by renewable sources to meet its Kyoto ob-
jectives. Biomass is expected to be a major contributor with an expected share
of more than 50% of the policy goal mentioned. Further, the Ministry of Eco-
nomic Affairs has defined some very ambitious policy targets for biomass in the
longer term (2040), namely 30% fossil fuel substitution in the power and trans-
port sector and 20–45% fossil-based raw material substitution in the industrial
sector. It has been calculated that the energy-substitution policy goal corre-
sponds to a long-term required biomass substitution volume of about 600–
1000 PJth annum–1, in a scenario in which severe energy savings have also been
taken into account (Ministry of Economic Affairs 2003). Adding the very ambi-
tious raw material policy goal an additional biomass substitution volume of sev-
eral hundreds of PJth annum–1 will be required.
Biomass in the Netherlands that is not currently used for food applications is
mainly used as animal feed or as fuel for power (and heat) production. Biomass
is converted mainly by means of direct/indirect cofiring in conventional coal-fired
power plants and also by stand-alone combustion plants (Cuijk, Lelystad). To meet
the longer-term policy ambitions biomass must be applied in additional market
sectors of the Dutch economy, using new thermochemical and (bio)chemical con-
version/production processes, for example advanced gasification and fermentation
technology. A current disadvantage of these processes is that final products will be
produced that are more expensive than their fossil-based alternatives. Prolonged
financial governmental support (e.g. investment subsidies, fiscal measures) neces-
sary to support successful market implementation is currently lacking in the Neth-
erlands. Further, to meet the longer-term policy ambitions, the use of potentially
available relatively cheap organic side- and waste streams will not be sufficient.
The use of dedicated, relatively expensive “energy” crops grown both in and out-
side the Netherlands (imports) is therefore inescapable.
ISBN: 3-527-31027-4
86 4 Biorefineries for the Chemical Industry – A Dutch Point of View
Within this framework it is believed biorefineries will play a major role in the
transition to a more sustainable Dutch economy. Realization of high-efficiency bior-
efining processes at places where biomass can be gathered, grown, and/or imported
and where the “green” products can be sold to a cluster of chemical and material
industries are believed to be key technologies to meet the longer-term policy goals.
The chemical and material industries are founded on innovation. Because of
the emerging interaction between chemistry, biology, and process engineering
the industries of 2020 will be significantly different from those of today. Not
only does technological development encourage changes in the design of pro-
cesses and products, however, pressure from consumers and the general public
also fuels a transition to a more sustainable industry. This stimulates discussion
on if and how the use of renewable resources can add to a future scenario of a
continuing innovative chemical industry taking into account the wishes and
constraints of all current and future stakeholders.
This paper first provides the societal and institutional context for the transi-
tion to sustainability in the evolution of the chemical industry. Second, it reviews
different perspectives on the future of a modified chemical industry, partly result-
ing from emerging technological opportunities. Third, taking into account these
emerging technological opportunities, this paper discusses the potential of the
use of biomass in the chemical industry of today and tomorrow. Special emphasis
will be given to the potential that “biorefineries” offer the chemical industry.
4.2
Historical Outline – The Chemical Industry: Current Situation and Perspectives
The objective of this section is to present an overview of the chemical products

that the current industry produces. It also gives an overview of the technological
pathways involved in the production of these chemical products. This overview
covers the scope of chemical products and chemical intermediates that need to
be produced from biomass and also provides an overview of currently produced
biomass-based chemical products.
4.2.1
Overview of Products and Markets
The oil industry may be divided into several important refinery sectors and
products including: gas for commercial energy supply, heavy gasoline for car
fuels, naphtha for the petrochemical industry, kerosene for aviation fuel, and oil
residues used in bitumen and lubricating oils. This review focuses on the
naphtha fraction in the petrochemical industry and the gamut of chemicals,
products, applications, and markets that can be derived from it.
The current chemical industry’s most important feedstock is naphtha which
can be cracked to obtain a range of olefins, for example ethylene and butanes,
and other small (un)saturated hydrocarbons and aromatic compounds, for ex-
4.2 Historical Outline – The Chemical Industry: Current Situation and Perspectives 87
ample benzene and alkyl benzenes. These simple hydrocarbons form the back-
bone of the possible products that are generated in the chemical industry today.
The scope of different chemicals (and transformations) that can be achieved
from naphtha for the chemical industry is illustrated schematically in Fig. 4.1.
In principal these materials can be transformed to the bulk of chemicals pro-
duced by two initial key pathways:
· they may be directly isolated, used, and transformed by a variety of chemical
techniques to a range of compounds; or
· they may undergo a gasification process to form synthesis gas (CO and H2)
which on recombination enables access to another branch of alternative
chemicals and technologies.
The chemicals described in Fig. 4.1, derived from the small (un)saturated hydro-
carbons and synthesis gas, enables an array of chemicals of technological and
economically important products, applications, and markets to be obtained. For
example:
· vinyl monomers for plastics used in pipe, packaging, and rubber applications;
· monomers for polyester and amide synthesis used in fibers (for textiles), en-
gineering materials, and some container materials;
· solvents for, among others, the paint industry;
· chemicals for the pharmaceutical industry; and
· chemicals for the insecticide and herbicide industry.
4.2.2
Technological Pathways
Closer examination of the chemical transformation steps involved in converting

one substance into another (Fig. 4.1), reveals some generic approaches to the
types of chemistry and technology used in the chemical industry:
· oxidative and reductive techniques are most prolific;
· introduction of nitrogen into chemical structures is most frequently achieved
by initial amination or amidation with ammonia;
· carbonylation reactions are frequently used to make small incremental
changes in chain length and for introduction of new functionality;
· extensive use of gaseous reagents;
· high selectivity of chemical steps by utilization of catalytic materials, therefore
reducing the need for chemical derivatization for transformation; and
· high conversion in chemical steps by use of catalytic materials
4.2.3
Biomass-based Industrial Products
Although most chemicals are of petrochemical origin an important example of

a chemical produced in bulk from a non-petrochemical source is ethanol. Etha-
nol, produced by fermentation of molasses, etc., has been most extensively used
Fig. 4.1 Schematic representation of chemical transformation

steps in the (petro)chemical industry.
Table 4.1 Estimated EU potential of major biomass-based

products (Ehrenberg 2002).
Market sector Total Renewable Potential of Potential share

consumption consumption renewables in 2010 (%)
market (1998) market (1998) in 2010 (kton)
(kton) (kton)
Polymers 33 000 25 500 1.5

Lubricants 4240 100 200 5
Solvents 4000 60 235 12.5
Surfactants 2260 1180 1450 52
in the food and beverage industry, although increasing amounts are also used
in bio-based transportation fuels and to prepare non-food industrial chemical
products. Although ethanol is perhaps the most widely known example of a bio-
based chemical product both in the Netherlands and worldwide, a range of
other bio-based chemical products is produced and used in a variety of indus-
trial applications. These fall into several generic categories:
· naturally occurring carbohydrate polymers;
· fats and oils of plant origin (and, to a lesser extent, of animal origin);
· terpene-based materials;
· chemical products of carbohydrate-containing materials; and
· fermentation products of carbohydrate-containing sources.
A recent study coordinated by the European Renewable Resources and Materials

Association (ERRMA) has evaluated the current situation of biomass use as an
industrial feedstock for chemicals and materials (Ehrenberg 2002). Table 4.1
shows the potential of biomass to replace petrochemical-based products in the
areas of polymers, lubricants, solvents, and surfactants. Many of those applica-
tions, especially lubricants, solvents, and surfactants, can be achieved by direct
extraction of the components from the biomass without additional (bio)conver-
sion steps.
4.2.3.1 Carbohydrates
Today’s bio-based products include commodity and specialty chemicals, fuels,
and materials. Some of these products result from the direct physical or chemi-
cal processing of biomass cellulose, starch, oils, protein, lignin, and terpenes.
Most biomass consists of natural polymers and most biomass is carbohydrate in
nature. This means that most biomass is in the form of carbohydrate polymers
(polysaccharides). These natural polymers can be used both as nature provides
them and as the skeletal framework of other derived polymers.
By far the most abundant of these carbohydrate polymers is cellulose, the
principal component of the cell walls of all higher plants. It is estimated that 75
billion tons of cellulose are biosynthesized and disappear each year, most of the
disappearance being through natural decay. Cellulosic plant materials are used
as fuel, lumber, mechanical pulp, and textiles. Purified cellulose is currently
used to make wood-free paper, cellophane, photographic film, membranes, ex-
plosives, textile fibers, water-soluble gums, and organic-solvent-soluble polymers
used in lacquers and varnishes.
The principal cellulose derivative is cellulose acetate, which is used to make
photographic film, acetate rayon, a variety of thermoplastic products, and lac-
quers. The world’s annual consumption of cellulose acetate is about 750,000
tons. Cellulose acetate products are biodegradable. Commercial production of
lyocell, a cellulosic fiber made from a solvent spinning process, has also started
recently. Lyocell, which, unlike rayon, does not require dry cleaning but is wash-
able and very strong, is the first new textile fiber to be introduced in 30 years.
Cotton is currently the most important non-wood fiber crop. It is mainly used
for weaving and spinning into cloth. Advances in biotechnology and genetic en-
gineering are now enabling development of cotton cultivars with improved pest
resistance, yield, and quality, thereby potentially reducing production costs and
better matching cotton characteristics to specific applications. Natural fibers
other than cotton occupy a variety of niche markets, for example specialty fab-
rics, fiber-reinforced composites, papers, cordage, and horticultural mulches and
mixes.
Heightened environmental concerns are also helping jute, hemp, flax, sisal,
abaca, coir fibers, and products derived from these fibers to find their way into
new markets. The use of natural fibers in fiber-reinforced composites, especially,
has seen tremendous growth in Europe in recent years (van Dam et al. 2004).
4.2.3.2 Fatty Acids

Fatty acids, readily available from plant oils, are used to make soaps, lubricants,
chemical intermediates such as esters, ethoxylates, and amides. These three im-
portant classes of intermediate are used in the manufacture of surfactants, cos-
metics, alkyd resins, polyamides, plasticizers, lubricants and greases, paper, and
pharmaceuticals (Ahmed and Morris 1994). Of the approximately 2.5 million
tons of fatty acids produced in 1991, about 1.0 million tons (40%) were derived
from vegetable and natural oils; the remaining 1.5 million tons were produced
from petrochemical sources. Twenty-five percent of all plant-derived fatty acids
used in the coatings industry comes from tall oil (a byproduct of kraft paper
manufacture). Surfactants are, currently, by far the most important outlet for
fatty acids (Table 4.1). In Europe most raw materials used for surfactant produc-
tion are derived from tropical oils, because of their more suitable chemical
structure. Besides oil-based surfactants there is also a relatively small market
(< 5%) for starch-derived surfactants.
4.2.3.3 Other
Terpenes, derived from woody materials, also give rise to a variety of chemicals
and products. Crude turpentine, isolated from the pulping industry, may be
used to isolate “pine oil” commonly used in cleaning products, alternatively its
components may be isolated and chemically transformed to materials such as
dipentene, which can be polymerized to prepare tacky polymers and used in
chewing gum and food packaging coatings.
Although not widespread in Western Europe and North America, several East-
ern European, Asian, and South American countries use carbohydrate-contain-
ing agricultural residues as a raw material in the chemical industry. For exam-
ple, hydrolysis of starchy materials to glucose with concomitant severe acid-cata-
lyzed degradation can result in oxalic acid, which is used in the leather industry.
Alternatively, pentoses, found in bagasse and corncobs, for example, readily un-
dergo acid-catalyzed dehydration to furfural. Furfural is a flexible chemical raw
material which can be used as a solvent itself in several applications or can be
used to prepare furfuryl alcohol used in resin materials, and tetrahydrofuran, a
common organic solvent. Although many other chemical transformations are
possible, their current commercial status is unclear.
Carbohydrates remain a flexible raw material, and beside “classical” chemical
transformations, biotechnological transformations have also been explored. For
example lactic acid can be used to prepare a biodegradable polymer with inter-
esting properties and has a wide range of potential applications including fibers
and packaging materials (Sreenath et al. 2001). Other biotech products include
citrates to prepare additive chemicals (dyeing, cleaning, and polymer) and fuma-
ric acid in preserving agents and as a component of unsaturated polyesters.
Specialty chemicals can be made using fermentation and enzymatic processes
or directly extracted from plants (or aquatic biomass). It has been shown that
plants can be altered to produce molecules with functionality and properties not
present in existing compounds (e.g. chiral chemicals). Examples of bio-based
specialty chemicals include bioherbicides and biopesticides, bulking and thick-
ening agents for food and pharmaceutical products, flavors and fragrances, nu-
traceuticals (e.g. antioxidants, noncalorific fat replacements, cholesterol-reducing
agents, and salt replacements), chiral chemicals, pharmaceuticals (e.g. Taxol),
plant-growth promoters, essential amino acids, vitamins, industrial biopolymers
such as xanthan gum, and enzymes. Specialty chemical markets currently repre-
sent a wide range of high-value products. These chemicals usually sell for more
than 4 1 kg–1. Although the worldwide market for these chemicals is smaller
than for bulk and intermediate chemicals, the specialty chemicals market now
exceeds $ 3 billion.
It is expected that advances in biotechnologies will have significant impacts
on the growth of the specialty chemicals market.
4.2.4
International Perspectives
In several industrialized countries (i.e. USA, UK, and the Netherlands) govern-
ment, business, society, and science have been engaged in outlining future de-
velopments in the material and chemical industry (American Chemical Society
1996; Molendijk et al. 2004; National Research Council 2000; Okkerse and van
Bekkum 1997; Parris 2004; Sims 2004; UK Foresight program Chemicals Panel
2000; US Department of Energy 1998, 1999; Weaver 2000). Most of these fore-
sight exercises are led by the view that an increased demand for chemicals and
materials will place additional pressure on the use of resources and on the envi-
ronment. Accordingly, the thrust is in finding new technologies and creating
novel materials, processes, and capabilities to bring this growth in line with so-
ciety’s demand for sustainability.
The perspectives indicate a shared interest in shifting from sole dependence on
fossil resources to a chemical and material industry founded on the application of
plant-based resources, derived either from secondary streams (i.e. waste and recy-
cling) or from primary streams (i.e. dedicated production). The discussion sug-
gests that changing the resource base of the chemical and material industries will
induce cleaner processes, safer products and a more effective use of scarce re-
sources. The shift to a bio-based chemical and material industry will alter the tech-
nological basis of the industry quite radically. To substantiate the sustainable cre-
dentials of new products and processes, further research and actual implementa-
tion will indicate what specific technological routes in fact contribute to sustain-
ability. This will be necessary for communication with NGO, the general public,
regulators, and policy makers about, for example, CO2 emissions.
From the perspective of the chemical industry striving for sustainability with
sound economic foundations draws the attention to three key areas.
4.2.4.1 Production
The actual production process has a major environmental impact both on efficient
use of energy and resources and on emission and waste production; this is especially
so in bulk industries. This links the provision of multi-quality biomass and the in-
dustrial production process. Because of the large volumes used, it may have a far-
reaching impact on the environment. Cost reduction, because of cheaper raw ma-
terials or processes with less extreme conditions, will be an important consideration.
4.2.4.2 Integration
Implementing a strategy for sustainability requires coordination between differ-
ent levels of a supply chain, product portfolio, and fine-tuning between distrib-
uted technological capabilities. Key technologies in conversion, extraction, and
separation will lay the foundation for further improvement in bulk production
and the development of products with well defined functionality.
This requires an integrated view on resource use and a strategic view on tech-
nology development. Linking of life sciences, chemistry, energy technology, and
process engineering is required for taking up such a challenge.
4.2.4.3 Use and Re-use

In terms of specific functionality, life cycle and recycling or safety, the actual
performance of end-products importantly defines the shape of a market-oriented
strategy for sustainable resource use. Increasing revenues by generating high-
value products will also be an important consideration. This links production
process with product design and defines new terrain for innovative business en-
terprises. Communicating the sustainability benefits to consumers or users in
the (new) end-use market will enable them to make more informed choices.
To develop a sustainable perspective for the chemical industry a combinatorial
approach integrating functionality provided by new molecules or materials, im-
proved efficiency and safety of production processes, and use and re-use of ma-
terials must be applied. Integrating criteria such as design for functionality or
recycling properties of new materials will encourage the search for new sustain-
able solutions in the chemical industry.
4.3
Biomass: Technology and Sustainability
The chemical industry undergoes rapid and important changes inherent to the
turmoil resulting from transitions at the end of an industry’s current lifecycle.
These changes require new technological, organizational, and commercial an-
swers from the industry. Moreover, the business strategy of the chemical indus-
try will become increasingly dependent on the acceptance or rejection by society
and consumers of its activities and its conduct. This section introduces a per-
spective on a chemical industry that combines technological innovation with a
socially acceptable business strategy.
4.3.1
Transition to a Bio-based Industry: Sectoral Integration in the Netherlands
Products from the chemical, material, and power industries have become an in-
tegral part of our daily lives and demand for these products is projected to in-
crease. A general concern is the intensive use of finite resources, in particular
fossil resources, and, consequently, the industry is in the midst of reconsidering
its current resource use. Two major Dutch companies communicated its transi-
tion to the general public – the chemical company DSM advertised its transition
process to a specialty company while building an image of sustainability and, in
2002, Shell, the energy company, launched a nation-wide advertisement cam-
paign highlighting the future of natural and renewable resources. In this pro-
cess, industrial research and development seems to (re)discover the variation of

quality and specific functionality in renewable resources; this offers the opportu-
nity to meet the demand for healthy and environmentally friendly end-products.
The industry must, however, contend with both established paths of research
and development and its extensive infrastructure in terms of production facil-
ities and equipment. As a result, transition to a bio-based industry requires the
development of new chains and persistent crossing of boundaries between disci-
plines, departments, and sectors.
Innovation in the energy sector is an important driver of technology develop-
ment in the chemical and material industries. Hence, petrochemistry is a con-
stant factor in industrial development, although the use of biomass or renew-
able resources comes to the fore anticipating rises in oil prices or directive mea-
sures related to international agreements, for example EU directive 2003/30/EC
on promotion of the use of biofuels or other renewable fuels for transport.
There are several political drivers for enforcement of such directives. The most
important include:
· the widely accepted threat of global warming as a result of the emission of
greenhouse gases;
· the unwanted dependency on oil-producing countries;
· the recent enlargement of the EU with associated political issues especially re-
lated to agriculture; and
· sustainable development of rural areas and creation of employment.
The question is whether an integrated energy sector and petrochemical industry

is the right or only venue for a transition to a bio-based industry.
Two non-exclusive scenarios can be distinguished:
1. The energy sector and the petrochemical industry integrate further and the
oil price remains the major driving force in business and in innovation.
2. The chemical and materials industry dissociate themselves from the petro-
chemical sector and seeks new potentials of bio-based industrial processes
using renewable resources supplied by the agro-sector, leading to environmen-
tally friendly modes of production and to healthy and sustainable end-prod-
ucts with specific qualities (Coombs 1995).
Most likely vertically integrated companies, including Sabic and Shell, will tend
to stick to the first strategy, whereas companies not involved in exploration, e.g.
Dow, DSM, might tend to follow the second strategy. The selling of the polyole-
fins division of DSM to Sabic and the acquisition of part of Roche can be seen
as an example of this strategy. These scenarios (represented in Fig. 4.2), dis-
cussed on a number of occasions in the Netherlands, draw the attention to the
linking of the agro-sector and the food and feed-processing industry with the
chemicals and materials industry (de Klerk et al. 2002).
Although the Dutch economy still has a strong base in manufacturing, both
of food and non-food products, the agro-sector and the chemicals sector operate
remotely and lack of synergy between these two sectors may hinder progress in
Fig. 4.2 New synthesis between economic clusters.
developing biorefineries with new manufacturing processes and new products.

In the 1980s and 1990s the agro-sector was involved in so-called “agrification”,
i.e. producing industrial crops in rotation with food crops or potatoes, but failed
to create ventures with end-users for its resources in the chemical and manufac-
turing industries (van Roekel and Koster 2000). Pushing the use of renewable
resources without identifying a clear demand, either by industry-based end-
users or consumers, thus seemed to be unproductive and even counterproduc-
tive.
Fine-tuning across sectoral boundaries seems to be an important condition
for changing current resource use. In the following discussion we will identify
the consequences of this condition for the position of the Dutch agrosector and
food industry.
The Dutch agro sector and the food and feed industry are founded not only
in primary production of agricultural crops but equally in processing, distribut-
ing, and transporting primary and secondary flows of agricultural resources.
The combination of intensive agricultural production, the transit of commodi-
ties in Rotterdam harbor and an extensive food processing industry is a specific
quality of the Dutch economy. Import of resources for the food-processing in-
dustry, including starch, sugar, and vegetable oils, results in a high “biomass in-
tensity”. A related factor is the opportunity to direct secondary flows, side, and
waste streams, to other industries; without the absorption of these secondary
flows, mainly by the feed and alcohol producers, the continuity of the process-
ing industry would be in danger (Rabobank International 2001). This sketches a
landscape wherein a new synthesis between sectors is possible. This contrasts,
however, with the current low-value use of renewable resources, mainly in the
feed and power industry. A driver for change in the feed industry might origi-
nate in recent scares about the use of slaughter residues and of potentially haz-
ardous fats and oils in feed, bringing these undesired components in the cycle
for human consumption. The European debate on genetically modified crops
might also result in stricter regulation of the use of these crops in feed and
food. Consequently, the composition of feed is affected by public pressures and
stricter regulation which, in combination with the tendency to reduce the inten-
sive livestock industry in the Netherlands, might lead to the disappearance of
existing markets for secondary flows. This will place high pressure on the food
processing industry, because secondary or waste streams will become a cost
rather than an income generator (Rabobank International 2001).
The short-term question is where the food processing industry and the agro-
sector can market their secondary resource flows and/or plant-based resources;
this is an environmental and economic problem. Therefore, companies, re-
search institutes, and government try to address the issue of creating new
chances for biomass, which includes finding alternative markets for residues
and waste streams. A related question is how to find high-value utilization and
application of these renewable resources, in addition to low-value energy and
bulk products. This will require fine-tuning of the quality, price, and quantity of
renewable resources with demand in the end-user market and functionality re-
quirements of new products. We must, therefore, turn round and move from
demand and functionality to processing and, eventually, to the production or
supply of the raw material.
The next section briefly identifies how different social actors perceive healthy
and sustainable products and processes, to provide a guide for formulating tech-
nology agendas for the chemicals and materials industry.
4.3.2
Can Sustainability Drive Technology?
One of the more important societal driving forces is the drive for sustainability.
Directing innovation and technology development from the perspective of a sus-
tainable, bio-based society is one of the major challenges for the chemicals and
materials industries. In response to this widely conceived public concern, com-
panies try to focus their business strategies both on sustainability, including en-
vironmental concerns, and on consumer demands for safe products. Most com-
panies trying to address the three Ps (planet, people, profit) are, however, well
aware that profit is always the ultimate driving force.
To develop the technology supporting such an ambition still requires a sub-

stantive effort, both in terms of innovative research and in terms of bringing to-
gether different disciplines, such as chemistry, biology, energy technology, and
engineering, and different professional fields, such as design, bulk production
of chemicals, and the supply and storage of renewable or plant-based resources.
Hence, both from a sustainability and business viewpoint an integral approach
to chemical, material, product, and energy outlets must be addressed in biorefi-
neries creating maximum added value from the selected biomass resources.
A sustainable chemical industry must strive for a business strategy that inte-
grates social, safety, health, and environmental objectives with the technological
and economic objectives of its activities. Assembling industry, science, and gov-
ernment is one aspect of developing foresight; consulting a wider range of
stakeholders, i.e. consumers and citizens, about technology strategies is another.
In doing so, interdisciplinary research may be able to contribute to fine-tuning
business strategies and technology strategies. Before formulating the shape of
such a new perspective, a selected number of international perspectives on tech-
nological change in the materials and chemicals industry will be discussed.
4.4
The Chemical Industry: Biomass Opportunities – Biorefineries
The preceding paragraphs have shown the scope of chemical products that are cur-
rently produced and therefore the targeted product portfolio of biomass-based chem-
ical products. It was also shown that drivers for a transition to a bio-based economy
lie not in technological opportunities alone but are a complex combination of so-
cietal, economic, and technological opportunities, challenges, and constraints. This
section will focus in more detail on the main technological opportunities to transfer
biomass, either directly or via chemical intermediates, into chemical products.
4.4.1
Biomass Opportunities
In the realization of a bio-based chemical industry two distinct approaches can

be identified. In the first approach, the value chain approach, value-added com-
pounds in biomass are identified and isolated in different processing and (bio)-
conversion steps. The remaining biomass is then transformed into a universal
substrate from which chemical products can be synthesized. In this approach it
is thought that it is technologically and economically beneficial to extract valu-
able chemicals and polymers from biomass rather than building these com-
pounds from universal building blocks. It can be concluded that the main tech-
nological challenges to aid the economic feasibility of this approach lie in the
area of biomass refining, separation technology, and bioconversion technology.
Far-reaching integration of the food, feed, and chemical industries is, moreover,
required, as is a major investment in infrastructure.
The second approach, the integrated process chain approach, follows the analog
of the petrochemical industry. In this scheme a “universal” substrate is first
transformed into universal building blocks, based on which chemical products
are produced. In this approach it is thought that it is economically and techno-
logically beneficial to build chemicals in highly integrated production facilities.
The main technological challenges for this approach lie in the high-efficiency
transformation of biomass into commonly known building blocks for the petro-
chemical industry (van Tuil 2002).
The main technologies producing chemicals from biomass are:
· biomass refining or pretreatment;
· thermochemical conversion (gasification, pyrolysis, hydrothermal upgrading
(HTU));
· fermentation and bioconversion; and
· product separation and upgrading.
Five main categories of building block can be identified as intermediates in the

production of chemical products from biomass.
· Refined biomass, i.e. biomass in which the valuable components have been
made accessible by physical and/or mild thermochemical treatment. These
components are extracted from the refined biomass. The remaining biomass
then undergoes further transformation.
· BioSyngas. This gas (mainly CO and H2) is a multifunctional intermediate in
the production of materials, chemicals, transportation fuels, power, and/or
heat from biomass; it can easily be used in existing industrial infrastructures
to substitute the conventional fossil-based fuels and raw materials.
· Mixed sugars. These C5 and C6 sugars are further refined substrates for chem-
ical and bioconversion. These substrates mainly originate from side streams in
the food industry and potentially from ligno-cellulosic biomass streams.
· Pyrolysis oil. This oil is produced in fast and flash pyrolysis processes and
can be used for indirect cofiring for power production in conventional power
plants, for direct decentral heating purposes, and potentially as high energy
density intermediate (important for long-distance transportation) bio-based in-
termediate for the final production of chemicals and/or transportation fuels.
· Biocrude. This material is a fossil oil-like mixture of hydrocarbons with low oxy-
gen content. Biocrude results from severe hydrothermal upgrading (HTU) of
(relatively wet) biomass and can, potentially, like its petroleum analog, be used
to produce materials, chemicals, transportation fuels, and power and/or heat.
4.4.2
Biorefinery Concept
A biorefinery is a facility that integrates biomass conversion processes and

equipment to co-produce fuels, power, and chemicals from diverse biomass
sources (Fig. 4.3). The biorefinery concept is analogous to today’s petroleum refi-
neries, which produce multiple fuels and products from petroleum (Fig. 4.4).
Fig. 4.3 Schematic overview general integrated biorefinery process.
Fig. 4.4 Detailed overview integrated biorefinery process.

Industrial biorefineries have been identified as the most promising routes to

the creation of a bio-based economy (Realff and Abbas 2004). Partial biorefi-
neries already exist in some agricultural and forest products facilities (e.g. pulp
mills, corn wet milling, starch and sugar beet refining).
These systems can be improved by better utilization of residues and optimiza-
tion of total added-value creation; new biorefineries can be enhanced by apply-
ing the lessons learned from existing facilities to comparable situations.
By producing multiple products, a biorefinery can take advantage of the natu-
ral complexity and differences between biomass components and intermediates
and therefore maximize the value derived from the biomass feedstock. A biore-
finery might, for example, produce one or several low-volume, but high-value,
chemical products and a low-value, but high-volume, platform chemical and/or
liquid transportation fuel, while generating power and heat for its own use, and
probably enough for sale of electricity. The high-value products enhance profit-
ability, the high-volume chemicals and/or transportation fuels help meet Euro-
pean energy needs and CO2 emission-reduction goals; whereas the power and/
or heat both reduce overall production costs and greenhouse gas emissions.
4.4.3
Biomass Availability
The Netherlands is a small country where land is scarce. Although the options for
primary crop production are limited, the biomass flux of utilized biomass (organic
material) is very high (partially imported). An estimated 42 million dry tons of bio-
mass (13 ton ha–1) (van Dam et al., in preparation) are used and produced in dif-
ferent sectors of the economy (compare with a biomass flux some 5 tonnes for
Germany). As markets change this high turnover of biomass does generate many
opportunities for reallocating streams toward biorefinery feedstocks.
One of the largest biomass streams is produced in the agri-industrial com-
plex. Many of these streams can be regarded as byproducts. Approximately
10 million tonnes (as received) of byproducts are currently mostly (90%) used
for animal feed (Vis 2002; Elbersen et al. 2002). These byproducts vary from
slaughterhouse wastes to discarded frying oil, potato peel, etc. As traditional
markets change (diminish) and markets for biobased energy and products in-
crease, a large part of these streams can become partially available for biorefi-
nery. This change in market demand for feed is already apparent from the de-
cline in livestock (from 4.6 in 1995 to 3.6 million in 2003), and pigs (14.5 mil-
lion in 1995 to 10.7 million in 2003), which leads to a smaller demand for feed.
Another estimate has been made of the potentially available (ligno)cellulose
biomass feedstocks in The Netherlands that could be used for ethanol produc-
tion (de Jong et al. 2003). The total amount of these technically suitable feed-
stocks is approximately 12 million tons (dry weight) per year (about 220 PJth an-
num–1), excluding import and biomass energy crops. The potential feedstocks
are highly variable, however, and include a range of agro-industrial residues,
agricultural wastes, forestry residues, and household organic waste, etc.
As already mentioned, the domestic (primary) biomass energy crop potential

is limited with some 2 million ha of agricultural land of which less than 1 mil-
lion ha is arable land. It is hard to estimate the potential for biomass energy
crops as competing demands for land are uncertain. Estimates range from 0 to
a maximum of 3 million tonnes (300.000 ha at 10 ton dry weight ha–1 annum–1,
18 GJ ton–1 dry weight) equivalent to 50 PJth annum–1 (Minnesma 2003). In the
short term, arable agriculture could be an important source of biorefinery feed-
stocks with more than 1 million tonnes of crop field residues available.
The maximum total Dutch biomass availability (organic residues and crops)
will therefore amount to 300 PJth annum–1 (approx. 15 million tonnes dry
weight). Taking the longer-term (2040) policy ambitions into account, requiring
a biomass substitution volume of about 600–1000 PJth annum–1, it can be con-
cluded that The Netherlands will have to import at least half, if not more, of its
long-term biomass requirements.
The potentially available feedstocks for biorefinery in The Netherlands are
highly variable and most streams are dispersed over the country. Though many
streams currently have other applications the potential is more than 15 million
dry tonnes of biomass. Large-scale biorefinery systems will have to use a variety
of feedstocks to secure feedstock availability (“multi-feedstock plant”). The op-
tion to import biomass over longer distances should also be available.
For medium-term development (< 10 years) a focus on feedstocks which have
the desirable characteristics (for example homogeneous streams low in lignin
and/or low in ash) are desirable. For the longer term (> 10 years), as the scale of
production increases, the feedstock range should be broadened with additional
residues and (woody) energy crops (e.g. willow).
The worldwide average net available biomass potential for non-feed and non-
material purposes is expected to amount 200–700 EJth annum–1 (maximum
1100 EJth annum–1) in 2050 (Lysen 2000). Worldwide, enough biomass will be
available to fulfill the needs. Because other countries will claim the same bio-
mass, the market price will be internationally settled. Timely participation in
the developing international market is a requirement to become an important
global player.
4.4.4
Primary Refinery
Deriving a raw material stream with desired specifications (i.e. amount of ash,
fermentable sugars, lignin) while simultaneously extracting valuable compo-
nents from the heterogeneous biomass streams is one of the major biorefinery
research and development issues. The main research and development areas
which must be addressed before an efficient biomass pretreatment chain can be
established are:
· characterization and standardization of raw materials and products;
· development of a cost-effective infrastructure for production, collection, char-
acterization, storage, identity preservation, pre-processing activities, import
and transportation of feedstocks for bio-based products and bioenergy applica-

tions; and
· development of economically viable pretreatment processes for commercial
use of a range of current and new bio-based feedstocks.
Biorefineries could potentially use complex processing strategies to efficiently

produce a diverse and flexible mixture of conventional products, fuels, electrici-
ty, heat, chemicals, and material products from all available, environmentally ap-
propriate biomass feedstocks. To achieve economically viable biorefineries it is
important that:
· separation and fractionation technologies for high-throughput systems are de-
veloped that produce value-added products and no waste streams; and
· generic solutions are identified that will apply across multiple feedstocks
while simultaneously achieving a zero-waste production system with either di-
rect use or recycling of all components.
4.4.5
Secondary Thermochemical Refinery
Thermochemical-based refinery processes usually consist of the following inter-

connected unit operations: pretreatment (i.e. drying, size reduction), feeding,
conversion (gasification, pyrolysis, HTU), product clean-up and conditioning,
and product end-use. In this subsection only gasification-related aspects are dis-
cussed, because it is expected that gasification will be the key technology in
(secondary) thermochemical refinery processes.
Atmospheric air-blown gasification processes, based on fixed bed, (circulating)
fluidized bed, or indirect dual reactor technology, are commercially available for
product gas (mainly CO, H2, N2, impurities) production. After gas clean-up this
– so called – fuel gas can potentially be used for heat, power, or CHP produc-
tion in a variety of prime movers. At the moment only direct heat production
by coupling of these technologies to conventional (natural gas or diesel fired)
furnaces, and power production by indirect cofiring in coal-fired power plants is
technically and economically feasible.
The market implementation of fully integrated gasification-based systems for
stand-alone power or CHP production is delayed by the high power/CHP pro-
duction costs, mainly caused by the relatively high investment costs of these
new and emerging technologies, and insufficient financial support from the
government.
Oxygen-blown gasification processes, especially applicable for the production
of BioSyngas (mainly CO and H2), based on both bubbling fluidized bed and
entrained-flow technology, are technically not yet available for biomass applica-
tions. Within the framework of the EU Chrisgas-project, TPS et al. are now try-
ing to modify the air-blown pressurized Varnamo gasification plant (Sweden)
for oxygen-blown operation.
Within the sixth Framework Program of the EU, ECN et al. are currently (Oc-
tober 2004) preparing a large STREP proposal concerning the modification of
existing coal-based slagging oxygen-blown entrained-flow-based gasification tech-
nology for 100% biomass use. Main research areas are biomass feeding, gasifi-
cation/slag behavior, product gas cooling, and the commercial applicability of
produced solid waste streams. The final goal is to design, build, and operate
several MWth pilot plants within 4 years, so that large commercial implementa-
tion can become feasible around 2010. The gasification processes mentioned all
require size-reduced and relatively dry (about 15–20% moisture maximum) bio-
mass fuels. “Wet” biomass fuels require rigorous drying before they can be
used.
Alternatively these fuels can potentially be converted by means of sub/super-
critical gasification processes (or fermentation processes, see Section 4.4.6).
Supercritical biomass gasification is performed at conditions above the critical
point of water (374 8C, 221 bar), mostly in the temperature range 500–700 8C.
Under these conditions an H2-rich product gas is produced. Under subcritical
conditions (temperature range approx. 350–400 8C) a methane-rich gas will be
produced. Under these conditions for full carbon conversion very low dry matter
concentrations and a catalyst are required. Although bench/pilot facilities are
available – FZK (D) 100 L h–1 (since 2003), University of Twente (NL) 5–30 L h–1
(since 1998) – the ECN opinion is that some years of laboratory-scale PhD
work will be required before the potential commercial implementation of
this technology. The technology is expected to be developed for “green natural
gas” (SNG) production; for the production of hydrogen, ECN has the opinion
that this technology will not become financially competitive in the longer
term. Some main research items are: feeding, heat exchange and catalyst be-
havior.
Within the framework of the biorefinery concept, two gasification-based path-
ways can be distinguished.
· Application of the biorefinery concept to increase the financial yield of “con-
ventional” gasification processes. By separating highly added-value compo-
nents from raw biomass fuels before conversion, or afterwards from the raw
“products” in product gas clean-up/conditioning, the overall plant economics
could be improved, simplifying market implementation (Fig. 4.5).
· Development of highly efficient advanced gasification-based thermochemical
secondary biorefining processes. By developing advanced catalyst-supported
staged or subcritical gasification processes it is expected that a variety of
“products” could be separated from biomass in such a way that the overall
process will be market competitive, without the need for substantial govern-
mental support.
4.4.6
Secondary Biochemical Refinery – Fermentative Processes
This section focuses on fermentation as the main form of bioconversion. A

large number of chemicals are currently produced by fermentation. These range
from bulk chemicals, for example ethanol (for food and fuel purposes) (Reith et
al. 2002) and lactic acid (as food ingredient or as monomer of polylactic acid)
(Datta et al. 1995), via compounds as amino acids, gluconic acid, and citric acid,
to high-value products such as antibiotics.
The efficient conversion of sugars into ethanol and lactate by fermentation in-
creases interest in fermentation technology as a means of production of bulk che-
micals. Fermentation has several advantages over conventional chemical reactions.
· Fermentation processes are usually one-step syntheses which could reduce in-
vestment costs.
· Microbial biosynthesis offers control over chemical reactions that is unchal-
lenged by state-of-the-art chemical synthesis resulting in highly functional
compounds.
Currently, however, fermentation has several disadvantages which have to be

solved before it will be able to compete with conventional chemical reactions.
Fig. 4.5 Thermochemical biorefinery concept of ECN to in-

crease the financial yield of “conventional” gasification pro-
cesses (Boerrigter et al. 2004).
· The costs of fermentation processes for bulk processes are higher than those
for the corresponding chemical process.
· The natural product spectrum of microorganisms is limited.
· In fermentation processes several side streams are formed which can be coped
with on the small scale but will cause a severe burden on the bulk scale.
4.4.6.1 Feedstocks
Most feedstocks currently used for fermentation processes are based on sugar
beet, sugar cane, and corn. To reduce feedstock costs other substrates must be
used. Several alternative feedstocks are being considered, for example fruit
waste, wood, straw, agricultural waste streams, dung, oils, and fatty acids, etc.
Among these the lignocellulosic materials are the most abundant polysaccha-
ride-containing biomass available in the world and are therefore an extensively
studied feedstock for fermentation processes. For almost all microorganisms
this lignocellulosic material must be hydrolyzed into its component saccharides
by mechanical pretreatment, followed by acid, base, or heat treatment, use of or-
ganic solvents or wet-oxidation to open the matrix, and, usually, enzymatic hy-
drolysis of the cellulose. The costs of the hydrolytic enzymes are the main ex-
pense in feedstock pretreatment and hydrolysis.
Hydrolysates from lignocellulosic materials contain, beside C6 sugars such as
glucose, also C5 sugars, for example xylose, and inhibitors. The relative amount
depends on the type of feedstock and the process used. Bakers’ yeast, used in
the production of ethanol, cannot use these C5 sugars. It has been calculated
that for a competitive process these C5 sugars also must be converted into etha-
nol. This is probably also true for other, future, processes.
4.4.6.2 Product Spectrum

Microorganisms can produce an extremely wide variety of chemical compounds.
Most of these compounds are, however, only intermediates in the overall meta-
bolism and will not be produced in significant amounts. Many of the platform
chemicals used in the chemical industry are, furthermore, not produced by nat-
ural microorganisms. The new fermentation technology must interface directly
with existing processes and installations in classical chemistry to reduce invest-
ment costs, indicating that the biochemical pathways of the microorganisms
must be modified to be able to produce current platform chemicals. Several
physiological aspects (yield, productivity, toxicity of product, use of GMO, by-
product formation, etc.) must be taken into account if this is to be achieved suc-
cessfully. This shows that the production of non-natural compounds by microor-
ganisms can be a complex task. Some successful examples of the modification
of metabolic pathways, however, are the production of 1,3-propanediol for the
production of Sorona by DuPont, the synthesis of muconic acid as a precursor
for adipic acid, and the optimization of succinic acid production (Biebl et al.
1999; Chotani et al. 2000; Zeikus et al. 1999).
4.4.6.3 Side Streams and Recycling

Fermentation processes are usually regarded as “clean” processes. During fer-
mentation, however, some side-streams are created which could be a severe en-
vironmental burden on the bulk scale, for example:
· During ethanol production by bakers’ yeast glycerol and fusel oils are produced.
· Lactic acid is removed from the fermentation broth by complexing with cal-
cium ions resulting in the formation of insoluble calcium lactate. This even-
tually results in the formation of equimolar amounts of gypsum.
· In all fermentations microbial biomass is formed. This can be a considerable
fraction. For example in the experimental production of polyhydroxyalkano-
ates (biopolymers) 20–50% of the product is biomass.
Because gypsum production could also be a problem in the synthesis of other

organic acids, much research has been devoted to in the development of other
down-stream-processing processes, for example membrane electrodialysis. For
the problem of microbial biomass formation another solution must be sought
in which the high-added-value components of the microbial biomass, for exam-
ple pigments, vitamins, and antioxidants are extracted and the remaining mate-
rial could then be recycled as feedstock in the fermentation process.
4.5
Conclusions, Outlook, and Perspectives
The objective of this section is to draw conclusions from previous sections by

presenting an agenda for further activity to facilitate the transition toward the
production of chemicals and chemical products from biomass. We believe that
if these agendas are addressed the sustainable production of chemicals from
biomass will become a realistic option in the future.
4.5.1
Biomass – Sustainability
A transition towards increased use of biomass originates from its possible con-
tribution to sustainable production of energy, fuels, chemicals, and materials.
Several chemicals can, moreover, be more easily or energy-efficiently produced
from biomass than from other feedstock. Many of these products can be ex-
tracted directly from the biomass.
The importance of biomass use towards the sustainability of the production
of chemicals is directly linked to the scale of production. It is, moreover, often
questioned whether the use of biomass for energy, fuels, and chemicals can
form a symbiosis with the use of the same biomass for the production of food,
feed, and materials, for example paper and wood. Also mentioned is the uncer-
tainty of the implication of a change in the main feedstock for energy and fuels
industry on the chemical industry.
4.5 Conclusions, Outlook, and Perspectives 107
What will be the main feedstock for energy and fuels in 50 years time –
water, sunlight, natural gas, or biomass? Are the conversion technologies men-
tioned in this study elegant ways of waste disposal in the food, feed, and cellu-
lose/paper industry? Is biomass mainly of interest for extraction of valuable che-
micals? What will be the impact of a biotechnological revolution on the opportu-
nities and pitfalls of biomass?
Some of these questions have been introduced in this paper. It is, however,
suggested that further insight into the role of biomass in chemicals production
in the Netherlands will be obtained by scenario evaluation. The result of this
evaluation will make it possible to choose feedstock/conversion technology com-
binations that maximize the potential of biomass use for chemicals and chemi-
cal products.
4.5.2
Biomass Refining and Pretreatment
A range of standards is needed to verify performance in the industry and to

help improve marketability. These include standards for environmental quality
of feedstocks and conversion technologies, and accreditation and standards for
energy content and the quality of feedstocks and products. Much of the technol-
ogy and many of the products developed have not been proven under “real
world” conditions and/or face significant certification challenges. These certifica-
tion challenges are often the result of systems that set standards based on the
physical characteristics of petroleum products, rather than on the performance
of the end product.
Improved practices in agriculture, silviculture, and aquaculture can play a sig-
nificant role in increasing yields while reducing required inputs. To achieve
great increases in biomass feedstock availability many issues must be resolved
in harvesting, collection, storage, import, and transport.
Current methods result in low densities of desired components, high trans-
portation costs, and potential storage stability issues. Pre-processing might be
done “on the farm” or even during harvesting or transportation “en route” to
densify, dry, and perhaps initially separate biomass components.
New transportation schemes might include pumping a fluid slurry, torrefica-
tion, pyrolysis, or “pelletizing” biomass locally. All of these advances must be
made while maintaining biodiversity and ensuring the safety and sustainability
of the technologies utilized.
Most biomass is solid, requiring improved material handling systems at the
front end of conversion operations. Breakthroughs in fractionation and separa-
tion technology will be required to produce higher value-added products, to re-
duce processing costs, waste, and environmental impact.
4.5.3
Conversion Technology
Next to already known conventional thermal, chemical, and bioconversion tech-

nologies this report has shown that the potential in conversion technology is en-
ormous, partly as a result of ever-increasing knowledge about thermochemical
and biotechnological pathways for the conversion of biomass into chemical
products. It is suggested that research efforts be focused on the following areas:
· Optimization of thermochemical conversion technologies. This paper has
shown that different technologies have been proposed for thermochemical
conversion of biomass. Further improvements lie in: (1) optimization of effi-
ciency and cost reduction of currently employed conventional technologies,
and (2) development of new advanced technologies, for example catalytic sup-
ported staged or subcritical gasification processes.
· (Bio)catalyst development and bioreactor engineering. Metabolic pathway en-
gineering enables synthesis of very specific catalysts (both whole-cell and en-
zymes) for transformation of refined biomass (mainly mixed sugars, fatty
acids, and syngas) into a variety of chemical intermediates and chemical prod-
ucts. This will lead to the controlled, safe and efficient production of new and
existing chemicals and polymers. Important issues for the efficient use of bio-
catalysts include immobilization, reactor kinetics and design, and separation
of the chemical product from the reaction mixture. As in the pretreatment of
biomass, separation technology also plays an important role in efficient and
cost-effective biocatalytic production processes.
4.5.4
Chemicals and Materials Design
The approaches to the production of chemicals from biomass presented in this

section are resource-based forward-integrated approaches. A design approach
can also be suggested in which a backward-integrated chemical industry designs
chemicals and production methods that fit the application of the chemical prod-
uct. It is thought that whereas the resource-based approach fits current (petro)-
chemical business and production models, the design approach will lead to a
real transition in the production and design of chemicals and materials. This
backward-integrated approach will initially be applicable in specialty chemical
markets but there are also long-term opportunities in bulk chemical markets.
It is thought that with the envisaged development in advanced thermochemi-
cal and bioconversion technologies the role of biomass and a biomimetic
approach to the design of chemicals and materials may add to sustainable pro-
duction, use, and reuse or disposal of chemicals and materials. Further exami-
nation of the role that chemicals and materials design can have on the sustain-
able use of biomass and biomass-based resources is therefore suggested.
References 109
4.5.5
Dutch Energy Research Strategy (“EOS”)
In The Netherlands the Ministry of Economic Affairs has defined a national

subsidy program “Energie OnderzoeksStrategie (EOS)” for co-financing long-
term (> 10 years) technology developments that support the transition process to
a more sustainable society. In this program (2004–2008) an annual subsidy bud-
get of 35 Mio 1 is available for technology development in pre-defined areas.
Biomass-based technology development in direct/indirect cofiring in conven-
tional power plants, gasification, and biorefineries have been selected for co-fi-
nancing. It is expected that for biorefinery technology development about 10 M
1 subsidy will be available for project co-funding for the next four years.
The Energy Research Center of the Netherlands (ECN) – Biomass Depart-
ment, with Agrotechnology and Food Innovations B.V. (A&F), Wageningen Uni-
versity and Research center (WUR), University of Twente (UT), Utrecht Univer-
sity (UU), and Groningen University (RUG), have defined an integral biorefi-
nery-based research and development program for the coming four years. With-
in this program joint projects will be defined on the basis of a common vision
and submitted for co-funding. Research items that will be addressed are: inte-
gral chain analysis and scenario studies to identify platform chemicals and pro-
vide the framework for technological developments; primary refining processes,
including pretreatment; secondary thermochemical and biological refining pro-
cesses, including product separation and upgrading; and some site-specific case
studies to encourage real market implementation.
References
Ahmed, I. and Morris, D. J. (1994) Replac- fuels, and products from biomass, 2nd
ing Petrochemicals with Biochemicals: A World Conference and Technology Exhibi-
Pollution Prevention Strategy for the Great tion on Biomass for Energy, Industry and
Lakes. Minneapolis: Institute for Local Climate Protection, Rome, Italy, 10–14
Self-Reliance. May 2004.
American Chemical Society, American Insti- Chotani, G., Dodge, T., Hsu, A., Kumar, M.,
tute of Chemical Engineers, Chemical LaDuca, R., Trimbur, D., Weyler, W. and
Manufacturers Association, Council for Sanford, K. (2000) The commercial pro-
Chemical Research, Synthetic Organic duction of chemicals using pathway engi-
Chemical Manufacturers Association neering. Biochim Biophys Acta, 1543 (2):
(1996) Technology vision 2020; the U.S. 434–455.
chemical industry (downloaded at http:// Coombs, R. (1995) Firm strategies and
www.acs.org) technological choices. In: Rip et al. (eds)
Biebl, H., Menzel, K., Zeng, A. P., and Managing technology in society: the approach
Deckwer, W. D. (1999) Microbial produc- of constructive technology assessment.
tion of 1,3-propanediol, Appl. Microbiol. Pinter Publishers: London, New York,
Biotechnol., 52, 289–297. 331–345.
Boerrigter H. et al. (2004). Thermal Biore- Dam, J. E. G. van, de Klerk-Engels, B.,
finery; high-efficient integrated production Struik, P. C. and Rabbinge, R. (2004) Sec-
of renewable chemicals, (transportation) uring renewable resources supplies for
changing market demands in a biobased Driving Force Behind a Knowledge Econo-

economy. Industrial Crops and Products, my and Sustainability ... A Vision
in press. Ministry of Economic Affairs (2001) Cataly-
Dam, van J. E. G., C. Boeriu, and J. P. M. sis: key to sustainability (Technology Road-
Sanders – Sustainable plant production map Catalysis initiated by Ministry of Eco-
chains for “green chemicals”. ACS Sympo- nomic Affairs and facilitated by Pricewa-
sium: Feedstocks for the Future: Renew- terhouseCoopers Management Consul-
ables for the Production of Chemicals and tants) (available at http://
Materials, Anaheim, CA, March 28–April www.technologyroadmapping.com).
1, 2004 (in preparation). Minnisma, M. and Hissemoller, M., Bio-
Datta, R., Stai, S.-P., Bonsignore, P., Moon, massa – Een Wenkend Perspectief, IVM,
S.-H., and Frank, J. R. (1995) Technological Februari 2003 (in Dutch).
and economic potential of poly(lactic acid) Molendijk, K. G. P., Venselaar, J., Weterings,
and lactic acid derivatives. FEMS Micro- R. A. P. M. and de Klerk-Engels, B. (2004)
biol. Rev., 16, 221–231. Transitie naar een duurzame chemie.
Diamantidis, N. D., and Koukios E. G. (2000) TNO-rapport R2004/179.
Agricultural crops and residues as feed- National Research Council (2000), Biobased
stock for non-food products in Western industrial products: priorities for research and
Europe. Ind. Crops Products, 11, 97–106. commercialization, Washington D.C.: Na-
de Jong, E., R. R. Bakker, H. W. Elbersen, tional Academic Press.
R. A. Weusthuis, R. H. W. Maas, J. H. Okkerse C. and van Bekkum, H. (1997) To-
Reith, H. den Uil, and R. van Ree (2003) wards a plant-based economy? In: Van Do-
Perspectives for Bioethanol Production in ren H. A. and van Swaaij A. C. (eds),
the Netherlands: feedstock selection and Starch 96 – the book.
pretreatment options, Poster presentation Parris, K. (2004) Agriculture, Biomass, Sus-
at the 25th Symposium on Biotechnology tainability and Policy: an Overview OECD
for Fuels and chemicals, Breckenridge, Workshop on Biomass and Agriculture:
Colorado. Sustainability, Markets and Policies, 10–13
de Klerk, B., Vellema, S. and van Tuil, R. June 2003, Vienna, Austria. OECD Code
(2002) Chemie: de weg naar duurzaam- 512004011P1, pp 27–36.
heid? Chemisch2Weekblad, 16 Feb. 2002, Rabobank International (2001) De Neder-
14–15. landse akkerbouwkolom: het geheel is meer
Ehrenberg, J. (2002) Current situation and dan de som der delen. Rabobank Food and
future prospects of EU industry using re- Agribusiness Research: Utrecht.
newable raw materials, (coordinated by Realff, M. J. and Abbas, C. (2004) Industrial
the European Renewable Resources & Ma- Symbiosis, refining the biorefinery. Jour-
terials Association (ERRMA), Brussels, nal of Industrial Ecology 7:5–9.
February 2002. Reit, J. H., den Uil, H., van Veen, H., de
Elbersen, H. W., F. Kappen and J. Hiddink Laat, W. T. A. M., Niessen, J. J., de Jong, E.,
(2002) Quickscan value-added applications Elbersen, H. W., Weusthuis, R., van Dij-
for byproducts and wastes generated in ken, J. P. and Raamsdonk, L. (2002) Co-
the Food Processing Industry (Hoogwaar- production of bio-ethanol, electricity and
dige Toepassingen voor Rest- en Neven- heat from biomass residues. Proc. of the
stromen uit de Voedings- en Genotmidde- 12th European Conference and Technology
lenindustrie). ATO, Arcadis IMD. For the Exhibition on Biomass for Energy, Indus-
Ministry of Agriculture. try and Climate Protection, June 17–21
Lysen, E. H. (2000) GRAIN: Global Restric- 2002, Amsterdam, The Netherlands.
tions on Biomass Availability for Import Roekel, G. J. van and Koster R. (2000) Succes
to the Netherlands, E2EWAB00.27, – en faalfactoren van de agrificatie in Neder-
Utrecht, The Netherlands, August 2000 land. ATO: Wageningen (report for the
(partly in Dutch). Dutch Ministry of Agriculture).
Ministry of Economic Affairs (2003) The Sims, R. E. H. (2004) Biomass, Bioenergy
Netherlands: Biomass in 2040, The Green and Biomaterials: Future Prospects. OECD
References 111
Workshop on Biomass and Agriculture: for fulfilling a vision to enhance U.S. eco-
10–13 June 2003, Vienna, Austria. OECD nomic security through renewable plant/crop-
Code 512004011P1, pp 37–62. based resource use (available at http://
Sreenath, H. K., Moldes, A. B., Koegel, R. G. www.oit.doe.gov/agriculture/).
and Straub, R. J. (2001) Lactic acid produc- Vis, M. (2002) Beschikbaarheid van reststro-
tion from agriculture residues. Biotechnol. men uit de voedings – en enotmiddelenin-
Lett. 23:179–184. dustrie voor energieproductie. A report for
UK Foresight Programme Chemicals Panel Novem, Utrecht. DEN nr 2020-01-23-03-
(2000) A chemicals renaissance (available at 0003/4700001071 (in Dutch).
(http://www.foresight.gov.uk) Weaver, P., Jansen, L., van Grootveld, G.,
US Department of Energy (1998) Plant/ van Spiegel, E. and Vergragt, Ph. (2000)
Crop-based Renewable Resources 2020: A vi- Sustainable technology development. Green-
sion to enhance U.S. economic security leaf Publishing: Sheffield.
through renewable plant/crop-based resource Zeikus, J. G., Jain, M. K. and Elankovan, P.
use (available at http://www.oit.doe.gov/ (1999) Biotechnology of succinic acid pro-
agriculture/). duction and markets for derived industrial
US Department of Energy (1999) The Tech- products. Appl. Microbiol. Biotechnol. 51,
nology Roadmap for Plant/Crop-based Re- 545–552.
newable Resources 2020: research priorities
Part II
Biorefinery Systems
ISBN: 3-527-31027-4
115
5
The Lignocellulosic Biorefinery –
and Industrial Organic Chemicals
5.1
The Situation
The current, historically high, prices of crude oil are causing economic hardship
for families and businesses worldwide, because of the resulting high energy
prices. The estimated impacts of increasing energy costs for farmers, truckers,
and airlines exceed US $ 13 billion per year for the United States alone. At the
same time, it is clear that increasing demand and a finite supply of petroleum
will sustain the rising prices and increase competition for secure petroleum
supplies.
The strategy outlined here not only addresses these issues, but also provides
major benefits to the environment and to farm incomes. This is all accom-
plished by using known, proven chemical processes (no new research needed)
and at very attractive rates of return on investment (ROI) that will not require
long-term government subsidies when this new industry is established.
5.2
The Strategy
At the most basic level, the strategy is to reduce then, essentially, eliminate de-
pendence on petroleum as the primary source of liquid fuels and industrial or-
ganic chemicals (where “industrial organic chemicals” includes those made
from both petroleum and biological resources). This is accomplished by replac-
ISBN: 3-527-31027-4
116 5 The Lignocellulosic Biorefinery
ing petroleum, the current dominant hydrocarbon source, with hydroxycarbons

derived from plant and animal biomass sources. The biomass carbon resources
are processed in biorefinery complexes that are analogs of present day petro-
chemical complexes. These biorefineries will produce the same, or functionally
equivalent, fuels and industrial chemicals that are currently obtained from pet-
roleum. The difference is that the biomass resources are renewable and, as will
be shown in examples based upon the United States, the biomass resources are
largely derived from materials currently regarded as waste.
The United States annually discards more tonnes of biomass carbon as “trash”
than we consume from petroleum carbon resources. Technologies are, however,
available for converting agricultural and forestry residues and municipal solid
wastes (MSW), which we now discard, into the fuels and chemicals that we con-
sume. Each year the United States consumes approximately 10 million tonnes
of industrial organic chemicals and 325 million tonnes of liquid transportation
fuels.
Currently, the United States converts approximately 15 million tonnes of agri-
cultural products into liquid fuels (ethanol and biodiesel) and discards approxi-
mately 270 million tonnes of agriculturally derived residues in the form of har-
vestable crop residues, animal manure, forest residues, and the organic fraction
of municipal solid wastes.
Since 1933, an average of 15 million hectares of crop land have laid idle an-
nually in the US, representing an additional 75 million tonnes of potential agri-
culturally produced biomass. There is no technical or economic reason why the
US demand for carbon resources could not be met by biomass replacing petro-
leum as the primary carbon source.
The functional equivalent of the petrochemical complex for processing agri-
culturally derived raw and residue materials and MSW processing is a biorefi-
nery. Here the term “biorefinery” is defined as “a production facility that uses
multiple renewable, agriculturally derived raw materials, in combination with
multiple processing methods, to provide the most profitable mix of higher value
chemicals and energy products possible at a given location.”
5.2.1
A Strategy Within a Strategy
An essential component of the structural shift from petroleum to biomass as

the source of carbon is a “two-use” ethic. Everything that grows or is derived
from organic sources (even plastics) should have at least two uses. MSW is col-
lected and recycled to the biorefinery. Other organic materials, for example agri-
cultural residues, used tires and plastics, and human and animal wastes, are
converted into new chemicals or fuels in biorefineries. In this “two-use” ethic,
carbon is recovered and recycled in much the same way that aluminum, iron,
and lead are recycled today.
In contrast with the huge petrochemical complexes of today, biorefineries will
probably be limited in total capacity to 1000 to 2000 tonnes of biomass per day.
5.2 The Strategy 117
This is because the distributed nature of agricultural production, forestry pro-

duction, and municipal solid-waste management strategies limit the amount of
material that can be economically assembled in one area, on a continuing basis,
at an acceptable cost.
This means that biorefineries will be regional, and dispersed. This regionality
not only means greater domestic security through dispersion and redundancy,
but also wider distribution of new jobs and economic activity in rural areas.
5.2.2
Environmental Benefits
The use of biomass as a replacement for petroleum in the production of liquid

fuels and industrial organic chemicals would have immediate and far-reaching
environmental benefits.
· First, biomass resources are renewable. Most are annually renewable. This
means that the carbon exhausted into the atmosphere as carbon dioxide when
liquid fuels are burned would be recycled into new plant growth in the follow-
ing years’ crops. This factor alone will greatly improve air quality worldwide,
and directly address the issue of global warming as a result of greenhouse
gas emissions.
· Second, entire biomass resource needs are available domestically for many
countries. No imports are needed. In the United States we currently have ap-
proximately 360 million tonnes per year of non-fossil carbon raw materials
available to replace some, if not all, of the demand for petroleum for liquid
fuels and industrial organic chemicals.
· Third, the assembly and refining of these resources will create a large number
of jobs, primarily in rural areas.
· Fourth, the production and/or conversion of biomass resources to liquid fuels
and organic chemicals involves processing steps that usually reduce the toxic
burden associated with the petrochemical production of these products.
· Fifth, the required tonnage of carbon-rich biomass can be derived largely
through the implementation of the “two uses” policy, i.e. the recycling of bio-
mass. This provides a very economical input for the biorefinery.
5.2.3
The Business Structure
Because a major source of the biomass needed for this transition is “2nd use”,
this provides a unique opportunity for new business partnerships. Municipal
solid wastes (MSW), agricultural crop residues, and forestry residues comprise
the bulk of the 2nd-use sources.
The biomass input resources are the major component of the operating cost
of production of liquid fuels and industrial organic chemicals for the biorefi-
nery. Because these inputs are now considered to be of low or even negative val-
ue, the owners of these 2nd-use resources can very profitably move these bio-
mass materials into the refining process, but delay taking their share of the
profit until the final products are produced and sold. Thus, the 2nd-use produc-
ers would receive their fair share of the generated profits after the increased val-
ue has been added. This provides a considerably better return than the current
negative, zero, or near zero waste value.
The 2nd-use strategy leads directly to the concept of a business partnership
comprising the biorefinery owner(s) and the biomass production owner(s). For
agricultural wastes and residues, assembly of these inputs could be achieved by
use of already existing cooperatives and/or farmer organizations. Similarly,
MSW inputs could be supplied through existing waste-management companies
or municipal waste-management operations.
It is envisaged that the biomass suppliers would participate equally with the
biorefinery companies in the direction and operation of the partnership. Essen-
tially, the business model is one of vertical integration through a collaborative
partnership or a joint venture business.
5.2.4
Cost Estimates
A biorefinery complex is not cheap. The approximate cost of a 1000 to 2000

tonnes per day multi-product plant is approximately $ 0.5 billion (US). In the
United States, example, the biorefinery will draw raw materials from a radius of
150 to 300 km. This means, allowing for desert and inland waters, that from
300 to 500 biorefineries would eventually be built in the US. This represents an
approximate $ 200 billion investment for the US. This is, however, approxi-
mately the same investment that will be required to replace approximately 200
petroleum refineries and petrochemical complexes that are now more than 30
years old and nearing the end of their useful lives.
5.3
Comparison of Petroleum and Biomass Chemistry
5.3.1
Petroleum Resources
Petroleum is a mixture of hundreds of hydrocarbon compounds. The dominant

chemistry of petroleum is that of linear hydrocarbons, with relatively little un-
saturation or branching. The chemical structure is polymers of the –(CH2)–
mer, with the hydrocarbon chains typically from 4 to 30 units long. Lesser quan-
tities of aromatic compounds (benzene derivatives), naphthenic compounds
(benzene dimers), and anthracenes (benzene trimers) are present. Additional
elements are incorporated into some molecules, for example oxygen, nitrogen,
sulfur, or phosphorus, but the essential elements of petroleum are carbon and
hydrogen.
Utilization of petroleum for the production of liquid fuels and organic chemi-
cals involves both physical separation of the numerous different compounds
and chemical synthesis. Fuels production is primarily a separation process, with
additional synthesis needed for higher-quality products, for example reformu-
lated gasoline, and for removal of sulfur and nitrogen. Crude petroleum is sepa-
rated into different fractions according to molecular size by distillation. Distilla-
tion processes account for approximately three percent of the US total energy
budget.
The largest-volume fuel produced from petroleum is gasoline. Gasoline is a
mixture of smaller (four to eight carbon) straight-chain hydrocarbons recovered
by distillation and synthetically created branched chain hydrocarbons with a
similar number of carbon atoms. Diesel fuel consists of larger (nine to fifteen
carbon) hydrocarbons that are recovered largely by distillation.
Nitrogen, sulfur, and phosphorus are elements that occur naturally in petro-
leum and must be removed from liquid fuels, largely because of the detrimental
environmental effects of their combustion products. The removal processes typi-
cally involve catalytic reactions under extreme operating conditions.
Petrochemical products are, in general, based on chemical addition of organic
functional groups such as hydroxyl, aldehyde, acid, ester, etc., or other elements,
such as oxygen, nitrogen, and halides. Much of the synthetic chemistry used is
based on addition of functional groups to olefin hydrocarbons such as ethylene,
propylene, and butylenes. Ethylene, propylene, and butylene are derived by
high-temperature processing of ethane, propane, and butane recovered from
petroleum crude oil by distillation. Benzene occurs naturally in petroleum, but
most of the benzene family of hydrocarbons is produced synthetically by catalyt-
ic reforming of hexane and/or alkylation reactions. A representation of the pet-
rochemical products families is given in Fig. 5.1.
5.3.2
Biomass Resources
Although a tremendous variety of biomass resources is available, only four basic

chemical structures present in biomass are of significance for production of
fuels and industrial products:
· saccharides and polysaccharides (sugars, starches, cellulose, hemicellulose);
· lignins (polyphenols);
· triacylglycerides or lipids (vegetable oils and animal fats); and
· proteins (vegetable and animal polymers made up of amino acids).
In addition to these basic structural resources, there are hundreds of specific or-
ganic compounds of biomass origin that have commercial uses ranging from
medicinal materials, nutrients and natural products, to industrial products.
Fig. 5.1 Fossil sources of industrial organic chemicals [1].

5.3.3
Saccharides and Polysaccharides
Saccharides and polysaccharides may be characterized as hydroxycarbons be-

cause their basic chemical structure is CH2O. Most hydroxycarbons occur natu-
rally as either five- or six-membered ring structures. This ring structure may in-
clude only one or two connected rings (sugars) or they may be very long poly-
mer chains (cellulose and hemicellulose).
The basic six-sided saccharide structure is exemplified by glucose. Long-chain
polymers of glucose, or other hexoses, may be categorized as either a starch or
a cellulose. The categorization depends on the configuration of the bonds
formed across the oxygen molecule that joins two hexose units.
Starch is an energy-storage compound found in the seeds of many plants and
is readily hydrolyzed enzymatically. Cellulose is found in association with two
other polymers, hemicellulose and lignin, and is much more difficult to hydro-
lyze. Hemicellulose is a polysaccharide that has a large fraction of pentose su-
gars, with some hexoses, and is relatively easy to hydrolyze with acid.
Current industrial uses of starch and hexose sugars include the production of
ethanol and other fermentation products, and the use of starch derivatives for
polymers, absorbents, and adhesives. The pentose sugars from hemicellulose
are a source of furfural and its derivatives and numerous xylose products.
5.3.4
Lignin
Lignin is a network polymer made up of multi-substituted, methoxy, arylpro-

pane, and hydroxyphenol units. The resulting thermosetting polymer serves as
the glue that holds the strands of cellulose and hemicellulose together in plant
fibers to provide structure and strength. Together, the three polymers make up
the largest biologically derived resource on earth – “lignocellulosics”. The “lig-
nocellulose” structure is the basis of the semi-rigid fibers found in all multi-cel-
lular plants. The structural difference between a corn stalk, a tree, a flower
stem, and a piece of waste paper is the difference between the relative amounts
of hemicellulose, cellulose and lignin present and the shape and length of the
fibers formed by the intertwined lignocellulosic chains.
The most significant current industrial uses for lignocellulosics are for construc-
tion materials and for pulp and paper products. The abundance of lignocellulosics is
the basis for regarding them as the key input resources for the biorefinery strategy.
5.3.5
Triacylglycerides (or Triglycerides)
Triacylglycerides (or triglycerides) are the primary component of vegetable oils

and animal fats. Irrespective of origin, these materials have identical chemical
structures and very similar chemical compositions. The basic structure of a tri-
glyceride looks like a comb with three long tines. The backbone of the comb is
a three-carbon hydroxycarbon, dehydrated glycerol, with three medium to long-
chain fatty acids attached. The fatty acid part of the molecule is a 7 to 31-carbon
hydrocarbon chain with an organic acid group at one end. Triglycerides are rela-
tively easily reacted with water or alcohols to form free fatty acids or fatty acid
esters, respectively, with glycerol as co-product.
Vegetable oils and animal fats are an essential part of our diet. As such, they
are available as recyclable materials in the form of recycled cooking oils and as
the “float-grease” fraction recovered in municipal water treatment plants. In the
United States an average of 10 kg of “waste” triglyceride materials is produced
per person annually [2].
Derivatives of fats and oils have extensive use in non-food/feed applications.
Current uses of triglyceride derivatives range from latex paints, high perfor-
mance lubricants and polymers, to biodiesel fuel and personal care products.
The hydrocarbon structure of the fatty acid chain has facilitated the accep-
tance and use of triglycerides and their esters in conjunction with traditional
petrochemical products. In fact, use of fats and oils for the production of indus-
trial chemical products (oleochemical industry) is the largest contribution to the
current industrial chemical industry made by biomass resources today.
5.3.6
Proteins
Proteins are long-chain polyamides based solely upon amino acid units. The
–(NH)– (peptide) bonds characteristic of proteins are the same bonds found in
industrial Nylons. The primary non-food/feed uses for proteins currently are as
leather products, protein glues, and personal-care products. There are opportu-
nities for the creation of “designer proteins”, for example synthetic spider silk
for light-weight, high strength cables or for polymer films and adhesives, but
progress in this area has been somewhat slow.
5.4
The Chemistry of the Lignocellulosic Biorefinery
The production of liquid fuels and industrial chemicals from biomass will rely
most heavily on the utilization of polysaccharide, lignocellulosic, and triacylgly-
ceride feedstocks. These materials are available, easily assembled, and storable
in the large quantities. In addition, these materials may be converted into prod-
ucts that are identical, or functionally equivalent, to current petroleum-based liq-
uid fuels and industrial chemicals.
Petroleum is predominantly a liquid resource, with some gases and a small
fraction of solid materials (waxes and asphalts). As such, every petroleum refi-
nery begins with a massive distillation system to separate the crude oil into a
large number of “cuts” that are further manipulated into the desired products.
5.4 The Chemistry of the Lignocellulosic Biorefinery 123
Chemical manipulation in a petrochemical refinery usually can be characterized

as the synthesis of structure – the conversion of linear hydrocarbons into more
structured and substituted materials, or the creation of very large, synthetic
polymer molecules.
Biomass materials are solids that include a sizable aqueous component. In
general, initial treatment of biomass materials includes steps of drying and
physical size reduction. The biomass materials already have a richly varied
chemical structure. This means that the goal of biomass utilization is, in part,
preservation of the intrinsic functional structures while depolymerizing the orig-
inal material.
The process chemistries used in the depolymerization of biomass materials
and their products are depicted in Fig. 5.2. These processes include:
· Pyrolysis – Treatment of biomass at moderate temperatures (300 to 600 8C) in
the absence of oxygen to cause partial depolymerization of the material. Slow
heating rates tend to favor production of volatile gases (CO, CO2, hydrogen,
methane, ethylene), organic acids and aldehydes, mixed phenols, and char.
High heating rates tend to minimize liquid production and maximize gas
production.
· Gasification – High-temperature (> 700 8C) treatment of biomass in the ab-
sence of oxygen but with addition of steam, and possibly CO2, to maximize
the production of synthesis gas (syngas), a mixture of H2, CO, CO2, and CH4.
Syngas can be used directly as a fuel or as a chemical intermediate in the pro-
duction of ammonia, methanol, and higher alcohols, organic acids and alde-
hydes, synthetic gasoline (using Fischer-Tropsch processing), and isobutene
and isobutane.
· Thermochemical Liquefaction – Pyrolytic processing with addition of H2, CO,
CO2, and selected catalysts to convert the biomass into hydrocarbons, mixed
phenols (from the lignin fraction), and light gases. The key to commercial
utilization of liquefaction processing is the separation and recovery of the
multiple products created during the liquefaction process.
· Hydrolytic Liquefaction – This processing includes the use of acids, alkalis, or
enzymes to depolymerize polysaccharides into their component sugars. These
aqueous-phase reactions are used to provide basic polymers, for example cel-
lulose and fermentable sugars for further processing, or hexose and/or pen-
tose sugars for chemical conversion into other organic compounds.
· Fermentation – Biochemical processing uses microorganisms or/and enzy-
matic reactions to convert a fermentable substrate into recoverable products.
Hexoses, particularly glucose, are the most frequently used fermentation sub-
strates, but pentoses, glycerol, and other hydroxycarbons are also used. Fer-
mentations are most commonly performed in aqueous solution, with the final
products present in modest concentration.
· Chemical Synthesis – Cellulose recovered from a lignocellulosic matrix using hy-
drolytic liquefaction can be treated with several reagents to make materials such
as cellulose acetate, nitrocellulose, and rayon. Xylose from the hydrolytic depo-
lymerization of hemicellulose can be further reacted in the same process to
Fig. 5.2 Biomass-derived industrial organic chemicals.
make furfural. Furfural is the starting point for a large family of derivative
chemical and polymer products. The adipic acid and the hexamethylene dia-
mine used in the original synthesis of Nylon-6,6 were made by acid hydrolytic
depolymerization of hemicellulose from oat hulls, followed by chemical synthe-
sis of the monomers and condensation polymerization to form the Nylon-6,6.
There are several possible ways of using lignocellulosic and other categories of
biomass for production of fuels and chemicals. Because the initial production
steps involve solid materials, preparation and handling of the raw materials is
more complicated and costly than using petroleum liquids. Also, as noted
5.5 Examples of Integrated Biorefinery Applications 125
above, biomass materials are more disperse and less dense than petroleum.
This means that assembly and storage of the raw materials is more complicated
than for petroleum.
Finally, some of the biomass resources, notably crops and crop residues, are
cyclical in production. This means that storage and integration with other re-
sources are necessary in the design and management of a biorefinery complex.
5.5
Examples of Integrated Biorefinery Applications
5.5.1
Production of Ethanol and Furfural from Lignocellulosic Feedstocks
An approach to implementation of a biorefinery using lignocellulosic feedstocks

(LCF) that has been given much attention in the United States, via USDOE-
sponsored programs, is the use of chemical and enzyme treatments to depoly-
merize the LCF to produce fermentable sugars and lignin. The primary goal
has been the production of ethanol. The basic process is:
Lignocellulosics + Water ? Xyloses + Cellulose/Lignin (Acid Process)
Cellulose + Water ? Glucose (Enzyme Process)
Glucose Fermentation ? Ethanol + CO2 + Biomass
Xylose Fermentation ? Ethanol + CO2 + Biomass
Lignin + Biomass ? Heat + Steam
Unfortunately, with ethanol as the sole product, and no subsidies, this process
facility does not make a profit. If, however, the xyloses are diverted from the fer-
mentation process into the production of furfural, a highly versatile intermedi-
ate chemical, the overall operation can be highly profitable. Van Dyne et al. [3]
showed that the optimum capacity for the integrated ethanol/furfural facility is
4360 tonnes per day of LCF. The discounted cash flow rate of return, after
taxes, for this facility is approximately sixteen percent. At capacities below ap-
proximately 750 tonnes per day, the combined ethanol–furfural operation is not
profitable, because the plant capital and operating costs are too large (economy
of scale). On the other hand, the operation becomes increasingly less profitable
for capacities above 4400 tonnes per day because the costs of assembling suffi-
cient feedstock are too high.
5.5.2
Management of Municipal Solid Waste
Municipal solid wastes (MSW) are the largest single source of lignocellulosic
materials available for utilization in modern society. Most localities have sys-
tems for collection and “disposal” of these materials. Too often the “disposal”
consists of burial of the materials. Typically approximately 25% of the waste is
either recyclable materials or inorganic materials such as stones, cement, and

wallboard (gypsum) materials. The remaining 75% is non-recyclable organic sol-
id waste materials (NROSW) that are available for production of a wide range of
fuels and industrial chemicals.
There are numerous locations where the NROSW is burned, either to reduce
the volume or, hopefully, to recover the energy value of the wastes. Using the
biorefinery approach and the “Two-uses” paradigm, the following options should
be considered for the NROSW:
A. Gasification ? Syngas + Ash ? Electrical power + CO2
B. Gasification ? Syngas + Ash ? Chemical synthesis + CO2
C. Gasification ? Syngas + Ash ? Fermentation ? Ethanol
D. Liquefaction ? Mixed organic liquids ? Specialty chemicals and fuels
In many locations the use of the NROSW materials for gasification and the co-
generation of electrical power is not competitive with other power sources, with-
out special incentives. The production of a variety of specialty chemicals from
syngas is commercial technology [4], although the easiest fuel source to use is
often natural gas. Liquefaction processing of coal has long been commercially
used for fuels and chemicals, with South Africa being the prime example.
Liquefaction of biomass is beginning to be used in commercial applications
in some countries, including the US. The liquefaction strategy can be a very
profitable alternative. As an example, a NROSW liquefaction plant handling 140
tonnes per day of MSW and 90 tonnes per day of waste tires costs about US $
30 million. The facility produces a range of liquid fuels and a number of high-
er-value industrial organic chemicals. Using a 10 year project life, the facility
produces a before-tax cumulative net profit of more than US $ 100 million.
5.5.3
Coupling MSW Management, Ethanol, and Biodiesel
Conventional ethanol production uses corn as the source of fermentable sugars

to produce ethanol. The corn typically is dry milled and the entire milled kernel
is added to the saccharification tank before fermentation. The process requires
a sizable amount of process steam and electricity.
Biodiesel production requires modest amounts of energy, but requires an in-
expensive source of fats or oils to be competitive. The oil can be, for example re-
cycled restaurant grease, a virgin vegetable oil, or rendered animal fat. The corn
that goes into the fermentation process contains approximately 3% by weight
corn oil, which passes through the fermentation process as an inert material.
Biodiesel also requires a source of alcohol to make the biodiesel from oil.
MSW, or other lignocellulosics, such as seed hulls, waste paper, etc., can be
burned directly to provide steam and heat, but it is more compatible with the
production of ethanol if the LCF is first converted to syngas to be used as a fuel
gas. Also, the syngas can be converted into methanol – the most commonly
used alcohol for making biodiesel.
References 127
The integrated process scheme for the biorefinery facility is:

Corn ? Milling ? Starch + Germ ? Glucose ? Ethanol
Germ ? Corn oil + Defatted Germ Meal
Gasification + LCF ? Syngas + Ash ? Steam + Electricity
Syngas ? Methanol
Corn Oil + Methanol ? Biodiesel + Glycerol
In this process system, corn and LCF are the inputs and ethanol, biodiesel, de-
fatted corn germ meal, and glycerol are the outputs. Economies of scale in the
biodiesel plant may require additional sources of fats/oils, but this only makes
the processes more profitable. The ethanol product is not used in the biodiesel
because it is more valuable as an intermediate chemical or as a fuel on its own.
5.6
Summary
There is already significant use of renewable biomass resources for industrial

chemicals [5, 6] and recognition of the opportunities for greatly expanded en-
ergy and fuel uses for biomass materials [7–11]. The biorefinery strategy advo-
cated here is combining of the utility of multiple feedstocks with the utility of a
wide array of conversion technologies to create a new, sustainable strategy to
meet the world’s needs for industrial chemicals and liquid fuels.
References
1 Morris, D. and I. Ahmed, The Carbohy- 5 Szmant, H. H., Industrial Utilization of

drate Economy: Making Chemicals and Renewable Resources: An Introduction,
Industrial Materials from Plant Matter, Technomic Publishing Company, Lancas-
The Institute for Local Self Reliance, ter, PA, 1986.
Washington, D.C., 1992. 6 Johnson, R. W. and E. Fritz, Fatty Acids
2 Wiltsee, G., “Urban Waste Grease Re- in Industry, Marcel Dekker, New York,
source Assessment,” NREL/SR-570- 1989.
26141, November, 1998. 7 Donaldson, T. L. and O. L. Culberson,
3 Van Dyne, D. L., M. G. Blaise, and L. D. “Chemicals from Biomass: An Assess-
Clements, “A Strategy for Returning ment of the Potential for Production of
Agriculture and Rural America to Long- Chemical Feedstocks from Renewable
Term Full Employment Using Biomass Resources,” ORNL/TM-8432, June, 1983.
Refineries,” Perspectives on New Crops 8 Wise, D. L., Organic Chemicals from
and New Uses, J. Janick, ed., ASHS Biomass, The Benjamin/Cummings
Press, Alexandria, VA, 1999. Publishing Company, Inc., Cambridge,
4 Spath, P. L. and D. C. Dayton, “Prelimin- MA, 1983.
ary Screening – Technical and Economic 9 Clements, L. D., S. R. Beck and C.
Assessment of Synthesis Gas to Fuels Heintz, “Chemicals from Biomass Feed-
and Chemicals with Emphasis on the stocks,” Chem. Engr. Prog., 79, 59–62
Potential for Biomass-Derived Syngas,” (1983).
NREL/TP-510-34929, December, 2003.
10 Lowenstein, M. Z., Energy Applications New Crops, New Uses, New Markets –
of Biomass, Elsevier Applied Sciences 1992 Yearbook of Agriculture, US De-
Publishers, New York, 1985. partment of Agriculture, Washington,
11 Thames, S., R. Kleiman and L. D. Clem- D.C., 1992.
ents, “How Crops Can Provide Raw Ma-
terials for the Chemical Industry,” in
129
6
Lignocellulosic Feedstock Biorefinery:
History and Plant Development for Biomass Hydrolysis
6.1
Introduction
The current high level of interest in lignocellulosic biomass conversion technol-

ogy is driven by the potential to produce fuels and chemicals to reduce depen-
dence on petroleum, improve air quality, and reduce greenhouse gas emissions.
Research and development efforts and attempts to commercialize biomass hy-
drolysis technology began in the early 1900s. This chapter discusses some of
this early work which set the stage for current efforts to bring to fruition the
lignocellulosic biorefinery. We highlight early efforts to pilot and commercialize
lignocellulosic biomass conversion technology using acid hydrolysis processes
and enzyme-based cellulose hydrolysis.
6.2
Hydrolysis of Biomass Materials
Producing ethanol from lignocellulosic biomass depends on converting the

complex cellulosic and hemicellulosic carbohydrates into simple sugars which
are then fermented to ethanol by a variety of microorganisms. This section
briefly reviews both acid- and enzyme-based methods for hydrolyzing the poly-
meric carbohydrates into their constituent sugars.
6.2.1
Acid Conversion
Hydrolysis of cellulose and hemicellulose (primarily xylan) to sugars can be cat-

alyzed by a variety of acids, including sulfuric, hydrochloric, hydrofluoric, and
nitric acids. The hydrolysis process is represented by the following simple ex-
pressions:
ISBN: 3-527-31027-4
130 6 Lignocellulosic Feedstock Biorefinery: History and Plant Development for Biomass Hydrolysis
cellulose ? glucose ? HMF ? tars

xylan ? xylose ? furfural ? tars
in which HMF is 5-hydroxymethylfurfural. If hydrolysis conditions are severe
(e.g. high temperatures or acid concentrations) a large fraction of the sugars is
degraded to other products, e.g. HMF, furfural, and tars.
Because dilute sulfuric acid is inexpensive, use of this acid is the most stud-
ied in acid conversion processes and these processes are most often used in
plants based on acid conversion technology. Biomass is impregnated with a di-
lute sulfuric acid solution and treated with steam at temperatures ranging from
140–260 8C. At lower temperatures of 140–180 8C, xylan is rapidly hydrolyzed to
xylose with little cellulose degradation. At higher temperatures, cellulose is also
rapidly hydrolyzed to glucose and xylan is quickly converted to furfural and tars.
Concentrated acids are also used to hydrolyze cellulose and hemicellulose to
sugars. Because low temperatures (100–120 8C) are typically used, high yields of
sugars are obtained with little production of degradation products. The econom-
ic viability of this process depends, however, on the successful recovery of acid
at low cost.
6.2.2
Enzymatic Conversion
Sugar yields are limited during acid hydrolysis because sugars are also con-
verted to degradation products. Cellulase, a multi-component enzyme system,
catalyzes cellulose hydrolysis and is 100% selective for conversion of cellulose to
glucose; high yields are, therefore, possible. This enzyme is produced by a vari-
ety of microorganisms, most commonly, the fungus Trichoderma reesei.
Cellulose conversion rates are limited by the ability of the enzyme to access
the cellulosic substrate. To increase accessibility, biomass is subjected to physi-
cal and chemical treatments that disrupt the biomass structure, usually by re-
moving a fraction of the hemicellulose and/or lignin. Effective pretreatment is
necessary to achieve good cellulose-to-glucose conversion yields.
6.3
Acid Hydrolysis Processes
6.3.1
Early Efforts to Produce Ethanol
Before World War II, research was conducted in the United States on hydrolysis
of biomass to produce sugars for production of ethanol with emphasis on the
use of forest and wood-processing wastes; no operation achieved true commer-
cial success, however. Sherrard and Kressman [1] highlight developments in
acid-based hydrolysis technology before World War II. In the early 1900s, a
plant was built in Hattiesburg, MS, USA, to process wood waste using sulfur-
ous acid, but never operated successfully [2]. A commercial operation was also
set up in Georgetown, SC, USA in the 1910s to process wood waste using dilute
sulfuric acid, but eventually failed because of low ethanol yields [3].
Process development was being investigated in Germany at the same time.
One process used concentrated hydrochloric acid [4] to hydrolyze the carbohy-
drate fraction of wood waste. The treated material was then neutralized with
caustic soda to yield a mixture of wood-based sugars and salt (sodium chloride),
used primarily as cattle feed. In another process, a 7300 dry metric ton year–1
plant hydrolyzed wood chips in countercurrent diffusers by contact with 42%
(w/w) HCl to produce a concentrated sugar solution [5]. After World War II the
plant was extensively rebuilt to use a modified process known as the Udic–Rhei-
nau process [6].
Work on sulfuric acid processes was also conducted in Germany at the same
time, leading to the development of the Scholler process [7]. The Scholler pro-
cess used a percolation reactor to hydrolyze wood waste, producing glucose as
the primary product; this was then fermented to ethanol. Many such plants
were built in Germany and Russia before World War II. The extensive use of
water required for this process, however, produced a rather dilute (4%) sugar
stream that was more costly to process.
In the late 1930s, a continuous wood-hydrolysis pilot plant was built in the
United States to produce lignocellulosic plastics, and wood sugars, furfural, and
acetic acid as by-products [3]. The plant was designed to hydrolyze wood slurries
continuously with dilute sulfuric acid by pumping the mixture through heated
hydrolysis tubes and then recovering the solid product. The plant produced
180–260 kg day–1 hydrolyzed product.
At the beginning of World War II molasses was fermented to ethanol as a
raw material for synthesis of butanediol, which in turn was polymerized to pro-
duce synthetic rubber. Because German submarines were sinking transports
from Cuba containing shipments of molasses and ethanol, the US government
made the decision to develop technology for producing ethanol from wood
waste for the synthetic rubber program. The Defense Plant Corporation, an or-
ganization of the US Government, awarded a contract to the Vulcan Copper
and Supply Company (later known as Vulcan Cincinnati) to design and to con-
struct a facility to produce ethanol. The author (Raphael Katzen) was involved
as the senior process engineer and later as the project manager for design and
construction of this facility in Springfield, OR, USA. The plant was designed to
process 270 metric tons (dry basis) day–1 of softwood sawdust trucked from saw-
mills in the area, with the goal of producing 208 L/dry metric ton (50 gal/dry
ton) ethanol.
This plant was based on modifications to the Scholler process, as a result of
work at the Forest Products Laboratory (FPL) of the US Department of Agricul-
ture in Madison, Wisconsin, USA. Based on early work by Ritter [8] and Sher-
rard [9], FPL built a pilot facility that improved upon the Scholler technology,
yielding the Madison-Scholler process [10, 11].
Vulcan Cincinnati’s design for their plant was based on the Madison-Scholler
process and used information and data produced during test runs of the FPL pi-
lot plant. Equipment was designed and fabricated at Vulcan’s shops in Cincinna-
ti, OH, USA, and construction was carried out in Springfield, OR, USA, with
the assistance of contractors. Although construction was stopped at the end of
World War II, the Defense Plant Corporation decided to complete the facility
and test the technology.
Plant construction was completed in 1946 and the plant was started up under
management of the Willamette Valley Wood Chemical Company, a group of in-
dividuals in the local forest products industry. Test runs were then initiated with
Vulcan providing technical management under Raphael Katzen’s supervision.
Despite problems with tar formation and calcium sulfate deposits from neutrali-
zation of sulfuric acid used to hydrolyze the cellulose, the design production
rate was achieved. The process proved too costly to compete with petroleum-de-
rived synthetic ethanol which appeared on the scene at the end of World War
II, however. Efforts were made to utilize the Springfield facility for production
of waxy products, but it was not designed for this operation and the plant was
shut down and dismantled.
With the prevalence of cheap petroleum-derived ethanol and other petrochem-
ical products after World War II, there was little economic incentive to pursue
cellulose hydrolysis technology further. In Germany many of the plants shut
down and most of the Scholler process plants in Russia were converted to sin-
gle-cell yeast production, because it was a more profitable product than ethanol.
Research did continue, however, in various laboratories and pilot plants in dif-
ferent parts of the world. In the 1950s the Tennessee Valley Authority (TVA)
built a dilute-sulfuric-acid-hydrolysis-based pilot plant using percolation reactors
at Muscle Shoals, AL, USA [12]. The New Zealand Forest Products Laboratory
built a similar plant in the 1980s [13]. Several pilot facilities were also built in
the 1980s that performed continuous dilute-sulfuric-acid hydrolysis of cellulose
and hemicellulose and were able to process 1–2 metric dry tons of biomass per
day. Plug-flow reactor systems processing dilute biomass streams were con-
structed by the American Can Company [14] and at the Solar Energy Research
Institute (SERI), now the National Renewable Energy Laboratory (NREL), in
Golden, CO, USA [15]. Systems for processing high-solids biomass streams
were constructed using twin-screw extruders at New York University [16] and in
Canada by Bio-hol/St Lawrence Reactor [17], and TVA installed a new system
using a modified pulp digester [18]. Except for the new reactor at TVA and a
new but similar reactor recently installed at the NREL [19], none of these sys-
tems is currently operational.
In the mid-1980s researchers at the SERI proposed a “progressing batch reac-
tor system” [20] that retained the simplicity of percolation reactors but achieved
countercurrent flow of liquors to reduce sugar losses due to degradation reac-
tions even further compared with percolation reactors. Further testing of the
system, however, did not produce substantial sugar yield improvements [21]. In
the late 1990s, the idea of a countercurrent shrinking bed reactor was also pro-
posed for dilute-acid total cellulose hydrolysis [22], but operational difficulties
limited the effectiveness of this system.
Several economic studies performed by engineering companies in the early
1980s to evaluate dilute-acid total hydrolysis processes for production of ethanol
[23, 24] showed the economics to be favorable. The potential of enzymatic cellu-
lose hydrolysis to achieve better yields was shifting emphasis away from acid
hydrolysis to enzymatic-based processing, however [25].
Although there is no current active effort to build dilute-acid based cellulose
plants, two companies are pursuing concentrated acid hydrolysis processing. Ar-
kenol has examined the ability of using recombinant Z. mobilis to ferment acid
hydrolysates produced by its concentrated-acid hydrolysis process [26] and is
pursuing international opportunities to build a large-scale plant. Masada Re-
sources Group plans to build a waste-handling facility in Middetown, NY, USA,
that will use its patented OxyNol process to recycle or convert municipal solid
waste. Profitable economics for both companies rely on achieving cost-effective
recovery of the acid catalyst.
6.3.2
Other Products
Both glucose and xylose produced by acid-catalyzed hydrolysis of lignocellulosic

biomass will, under the same conditions, further degrade to HMF and furfural,
respectively. Furfural has been produced commercially from biomass but there
has been no commercial interest in producing HMF. The Quaker Oats Company
began production of furfural in the United States in the 1920s [27] and until re-
cently was the major world producer of furfural from agricultural residues [28].
Furfural is produced from agricultural sources including corncobs, oat hulls, rice
hulls, cereal grasses, and sugar cane bagasse. Early plants used batch production
technology, but in 1965, at the recommendation of the Katzen Company [29], Qua-
ker Oats agreed to pilot a continuous process in an existing leased pulping facility
operated by Katzen. Successful results led to the design of the largest furfural
plant in the world by the Katzen Company and the Black Clawson Company of
Middletown, OH, USA. The plant is based on a highly modified Pandia digester
system, originally used to pulp wood and agricultural residues.
The plant was constructed in Belle Glade, FL, USA and began operation in 1966.
The feedstock for the plant was sugar cane bagasse from nearby sugar mills.
When successful operation was demonstrated, the plant capacity was expanded
to process 1800 dry metric tons day–1 bagasse to yield 137 metric tons day–1 furfur-
al. By-product methanol and HMF were burned, with the lignocellulose residue, to
provide steam and electrical energy for the plant. The plant was sited adjacent to a
sugar mill operated by the Sugar Cane Growers Company of Florida, which pro-
vided transport and bulk storage of up to 227 000 metric tons (dry basis) of bagasse
from nearby sugar mills, thereby enabling year-round operation. After 31 years of
continuous operation this plant was shut down in 1997 because of the availability
of lower cost crude furfural from China.
6.4
Enzymatic Hydrolysis Process
6.4.1
Early History
A new and exciting development in biomass conversion technology began when

reports from US troops in the South Pacific during World War II described how
“the green fungus among us” [30, 31] was destroying uniforms and other cotton
gear. Reese, at the Natick Massachusetts Laboratory of the US Army Materials
Command, analyzed samples of cotton items affected by this fungus in the
1950s [32]. He identified the fungus as Trichoderma viride and later named T.
reesei QM 6a as the organism responsible for producing a cellulase complex that
hydrolyzed cellulose to glucose. Serious studies of the use of this enzyme for
biomass conversion began in the early 1970s with the pioneering work of Man-
dels and coworkers [33, 34]; this resulted in the development of improved T. ree-
sei strain QM 9414.
In the late 1970s and early 1980s much research effort was devoted to mutat-
ing the wild T. reesei strains QM 6a or QM 9414 to enhance production of cellu-
lase [34]. One such effort was conducted by researchers at several university la-
boratories in a cooperative program coordinated by Eveleigh at Rutgers Univer-
sity. This cooperative research program resulted in substantial improvement of
the wild-type fungal strain and produced the widely known T. reesei strain RUT
C30 [35]. Many other efforts from around the world have produced modified T.
reesei strains.
6.4.2
Enzyme-Based Plant Development
In the late 1970s several industrial organizations became interested in enzy-

matic conversion of cellulose to sugars and ethanol. One of the first efforts to
develop and pilot this technology was a collaboration between Gulf Oil Chemi-
cals and Nippon Mining in a program carried out by the Bio-Research Corpora-
tion of Japan. This led to the building of a 900 kg day–1 pilot plant in Pittsburg,
KS, USA. In addition to producing cellulase, the plant was the first to use a
novel technology combining cellulase saccharification with glucose fermentation
in the same reactor system, called simultaneous saccharification and fermenta-
tion (SSF) [36, 37]. The plant successfully processed paper mill waste, producing
dilute beer streams containing 30–35 g L–1 ethanol, although problems with
contamination in the SSF fermentors were reported [38, 39].
Although no commercial plants have been built for enzymatic cellulose con-
version, several pilot-scale facilities were built in the 1980s throughout the world
to test a variety of technology and feedstocks. For example, a pilot plant located
in Soustons, France, utilized a Stake process [40] to pretreat biomass, followed
by enzymatic saccharification. Ralph Katzen Associates International, with the
6.4 Enzymatic Hydrolysis Process 135
University of Arkansas and Procter and Gamble, erected a pilot plant at a pulp
mill in Pennsylvania, USA, to process pulp mill waste through disc refiners fol-
lowed by SSF in a 9500-L fermentor [30]. In 1987, a 3000 kg day–1 pilot plant
that processed wheat straw through a batch digester (no catalyst) and used the
washed pretreated solids to produce cellulase that was subsequently used to sac-
charify the remaining washed solids was constructed in the Voest-Alpine Bio-
mass Technology Center [41]. Two pilot plants were also constructed in Japan.
One operated from 1983 to 1987 and processed 500 kg day–1 bagasse or rice
straw. It used mild alkaline pretreatment then enzymatic saccharification of the
pretreated biomass with cellulase produced on Avicel as the carbon source, fol-
lowed by sugar concentration using reverse osmosis and subsequent fermenta-
tion to produce ethanol [42]. The other plant operated from 1986 to 1990 and
processed 1000 kg day–1 cedar wood or white birch chips. It used steam explo-
sion to treat the wood that was then fermented with a strain of Clostridium [43].
In the early 1990s, the US Department of Energy (DOE) and NREL con-
structed a fully integrated 900 kg day–1 pilot plant to produce ethanol from a
variety of lignocellulosic biomass sources [19] that used a modified pulp digester
for pretreatment that has already been discussed. The plant includes unit opera-
tions for feedstock handling, pretreatment in a modified pulp digester, seed cul-
ture production, SSF in 9000-L fermentors, feed tanks for enzyme and nutrient
addition, ethanol stripping in a sieve-tray distillation column, and solid–liquid
separation. The plant has also extensive instrumentation for process control and
data collection. The plant was operated on a corn fiber feedstock during a 15-
day run [44] and was later operated continuously for up to six weeks [45].
The Iogen Company of Ottawa, Canada, recently built a 983,000-L-ethanol-per-
year demonstration-scale plant processing nearly 5 metric tons day–1 agricultural
residue. Because Iogen is a cellulase producer, they can supply the cellulase for
the plant from an adjacent enzyme-production facility. Although little has been
publicly disclosed about this facility and its performance, it has been reported to
produce a 4% alcohol stream from conversion of lignocellulosic biomass [46].
6.4.3
Technology Development
The Biomass Research and Development Technical Advisory Committee, a

group of biomass industry and academic experts, recently issued a “roadmap”
document [47] outlining bioconversion research needs. The recommendations
most relevant to enzyme-based processing are:
1. the need to improve physical and chemical pretreatments before fermentation;
2. the need for cost-effective chemical/enzymatic conversion; and
3. the need to overcome barriers associated with inhibitory substances in hydro-
lysate sugar streams.
Engineering catalyst and microorganisms to improve their tolerance are meth-

ods used to achieve the third goal.
Advances in all three areas will be required to achieve economic viability and
thus success in the marketplace. The Consortium for Advanced Fundamentals
and Innovation (CAFI), a group of independent academic researchers, is colla-
borating in an effort to identify and develop new pretreatment technology. The
most promising technology is uncatalyzed steam explosion, liquid hot water,
pH controlled hot water, flow-through liquid hot water, dilute acid, flow-through
acid, lime, and ammonia-based processing [48].
Development of new and improved enzymes for biomass conversion has been
a focus of the US DOE for the last few years. They have funded cost-shared ef-
forts with the two largest cellulase producers, Genencor International and Novo-
zymes, to produce cost-effective enzymes with a goal of achieving a tenfold cost
reduction that would bring cost down to an estimated $ 0.50 gallon–1 ethanol.
Both companies report success in reaching this goal, but further cost reduction
is required and the DOE’s goal is to achieve an effective enzyme cost of
$ 0.10 gallon–1 ethanol.
Efficient conversion of all sugars derived from biomass to desired products is
required for this technology to become economically viable. Development of ge-
netically modified microorganisms able to utilize other sugars beside glucose
has been an ongoing effort in many laboratories [49, 50]. As also emphasized
by the last recommendation above, the ability to tolerate inhibitory substances
is also a highly desirable characteristic. Currently, however, no microorganisms
can tolerate inhibitory substances in dilute-acid pretreated biomass substrates
without some type of conditioning process that removes inhibitors.
6.5
Conclusion
Early efforts to commercialize acid-based processes for producing products from

cellulose hydrolysis were ultimately unsuccessful, because of competition from
lower-cost petroleum-derived materials. These efforts, and ongoing work on en-
zyme-based processes, are, however, providing valuable knowledge and experi-
ence. The future is the multi-product biorefinery that can utilize all biomass com-
ponents. We need to build upon past efforts and use new advances in technology
to ensure the commercial success of the lignocellulosic feedstock biorefinery.
References
1 E. Sherrard, F. Kressman, Ind. Eng. 4 F. Bergius, Trans. Inst. Chem. Engrs.

Chem. 1945, 37, 5. (London) 1933, 11, 162.
2 Ruttan, J. Soc. Chem. Ind. 1909, 28, 5 F. Bergius, Ind. Eng. Chem. 1937, 29,
1291. 247.
3 R. Katzen, D. Othmer, Ind. Eng. Chem. 6 J. Wright, A. Power, P. Bergeron, Evalua-
1942, 34, 314. tion of Concentrated Halogen Acid Hydro-
lysis Processes for Alcohol Fuel Production,
References 137
Solar Energy Research Institute, SERI/ 27 R. Kottke, Kick-Othmer Encyclopedia of

TR-231-2074, Golden, CO 1984. Chemical Technology, Supplement to 4th
7 H. Scholler, French Patent # 777,824, Ed., John Wiley & Sons, NY, 1998, 155.
1935. 28 J. Levy, K. Yokose, Chemical Economics
8 G. Ritter, Ind. Eng. Chem. Anal. Ed., Handbook, Report 660.5000, SRI Con-
1932, 4, 202. sulting, Menlo Park, CA, USA, 2004.
9 E. Sherrard, Ind. Eng. Chem. 1923, 15, 29 R. Katzen, Personal Communication.
1164. 30 R. Katzen, D. Monceaux, App. Biochem.
10 E. Harris, E. Berlinger, G. Hajny, E. Biotechnol. 1995, 51/52, 585.
Sherrard, Ind. Eng. Chem. 1945, 37, 12. 31 J. Sheehan, M. Himmel, Biotechnol. Prog.
11 E. Harris, E. Berlinger, Ind. Eng. Chem. 1999, 15, 817.
1945, 38, 890. 32 D. Ryu, M. Mandels, Enzyme Microb.
12 N. Gilbert, A. Hobbs, J. Levine, Ind. Eng. Technol. 1980, 2, 91.
Chem. 1952, 44, 1712. 33 M. Mandels, L. Hontz, J. Nystrom, Bio-
13 R. Burton, Proc. of the Royal Soc. of Ca- technol. Bioeng. 1974, 16, 1471.
nada Internat. Symp. on Ethanol from Bio- 34 I. Persson, F. Tjerneld, B. Hahn-Hager-
mass, Royal Society of Canada, Ottawa, dal, Proc. Biochem. 1991, 26, 65.
Canada, 1983, 247. 35 B. Montenecourt, D. Eveleigh, App. Envi-
14 J. Church, D. Wooldridge, Ind. Eng. ron. Microbiol. 1977, 34, 777.
Chem. Res. Dev. 1981, 20, 371. 36 F. Gauss, S. Suzuke, M. Takagi, US Pat-
15 A. Brennen, W. Hoagland, D. Schell, ent 3 990 944, 1976.
Biotechnol. Bioeng. Symp. No. 17, John 37 G. Huff, N. Yata, US Patent 3 990 945,
Wiley & Sons, New York, 1987, 53. 1976.
16 B. Rugg, P. Armstrong, A. Dreiblatt, D. 38 D. Becker, P. Blotkamp, G. Emert, Fuels
Wise, Liquid Fuel Developments, CRC from Biomass and Waste, Ann Arbor
Press, Baco Raton, FL, USA, 1983, 139. Science Publishers, Ann Arbor, MI,
17 R. Lawford, R. Charley, R. Edamura, J. USA, 1981, 375.
Fien, K. Hopkins, D. Potts, B. Zawadzki, 39 K. Bevernitz, S. Gracheck, D. Rivers, D.
H. Lawford, Fifth Canadian Bioenergy Becker, K. Kaupisch, G. Emert, Energy
R&D Seminar, National Research Council from Biomass and Waste XIV, Institute of
of Canada, 1984, 503. Gas Technology, Chicago, 1982, 897.
18 M. Bulls, J. Watson, R. Lambert, J. Bar- 40 J. Heard, W. Schabas, Chem. Eng. 1984,
rier, Energy from Biomass and Waste XIV, 91 (6), 49.
Institute of Gas Technology, Chicago, 41 M. Hayn, W. Steiner, R. Klinger, M. Sin-
1991, 1167. ner, H. Esterbauer, Bioconversion of Forest
19 Q. Nguyen, J. Dickow, B. Duff, J. Farm- and Agricultural Plant Residues, CAB In-
er, D. Glassner, K. Ibsen, M. Ruth, D. ternational, Oxon, U.K. 1993, 33.
Schell, I. Thompson, M. Tucker, Biore- 42 Y. Shirasaka, H. Ishibashi, H. Etoh, H.
source Technol. 1996, 58, 189. Michiki, H. Miyakawa, S. Moriyama, En-
20 J. Wright, P. Bergeron, P. Werdene, Ind. ergy from Biomass and Waste XIII, Insti-
Eng. Chem. Res. 1987, 26, 699. tute of Gas Technology, Chicago, 1989,
21 D. Schell, Personal Communication. 1311.
22 Y. Lee, W. Zhangwen, R. Torget, Biore- 43 S. Matsui, Conference on Alcohol and Bio-
source Technol. 2000, 71, 29. mass Energy Technologies, NEDO-OS-9106,
23 Badger Engineers, Inc., SERI Subcon- New Energy and Industrial Technology
tract Report No. ZX-3-03096-1, 1982. Development Organization, Tokyo, 1991,
24 Stone & Webster Engineering Corp., 27.
SERI Subcontract Report No. ZX-3- 44 D. Schell, C. Riley, N. Dowe, J. Farmer,
03096-1, 1982. K. Ibsen, M. Ruth, S. Toon, R. Lumpkin,
25 J. Wright, Chem. Eng. Prog. 1988, 84, 62. Bioresource Technol. 2004, 179.
26 T. Yamada, M. Fitigati, M. Zhang, App. 45 D. Schell, Personal Communication.
Biochem. Biotechnol. 2002, 98-100, 899. 46 B. Foody, From Presentation at the 27th
Symposium on Biotechnology for Fuels
and Chemicals, Chattanooga, TN, USA, 48 N. Mosier, C. Wyman, B. Dale, R. Elan-

2004. der, Y. Lee, M. Holtzapple, M. Ladisch,
47 Biomass Research and Development Bioresource Technol. 2005, 96, 673.
Technical Advisory Committee, Roadmap 49 R. Bothast, N. Nichols, B. Dien, Biotech-
for Biomass Technologies in the United nol. Prog. 1999, 15, 867.
States, 2002, www.eere.energy.gov/bio- 50 A. Aristidou, M. Penttila, Current
mass/progs/searchdb2.cgi?7219. Opinions in Biotechnol. 2000, 11, 18.
139
7
The Biofine Process – Production of Levulinic Acid, Furfural,
and Formic Acid from Lignocellulosic Feedstocks
Daniel J. Hayes, Steve Fitzpatrick, Michael H. B. Hayes, and Julian R. H. Ross
7.1
Introduction
The energy needs of the developed world are currently over-dependent on the
utilization of finite mineral resources. Although renewable-power technologies,
for example wind and photovoltaics, may, in the future, have major roles in the
production of electricity, provision must still be made for the supply of indus-
trial chemicals and motor fuels that are currently produced predominately from
oil. In fact, of the approximately 170 chemical compounds produced annually in
the US in volumes exceeding 4.5 ´ 106 kg, 98% are derived from oil and natural
gas [1]. The vast majority of modern synthetic products are also derived from
oil. Emerging biorefinery technologies offer a sustainable alternative by utiliza-
tion of carbohydrates, the most abundant organic chemicals on the surface of
the earth. This chapter will focus on the Biofine Process [2, 3], biorefinery tech-
nology that transforms carbohydrate feedstocks into products that include the
platform chemicals levulinic acid, furfural, and formic acid in high yields. The
process involves high-temperature acid-hydrolysis in two reactors and is one of
the most advanced and commercially viable lignocellulosic-fractionating technol-
ogies currently available. The process involves the hydrolysis of polysaccharides
to their monomeric constituents, and these are then in turn continuously con-
verted into valuable platform chemicals.
7.2
Lignocellulosic Fractionation
The major polysaccharides of importance in biomass are the glucans and hemi-
celluloses. Of the glucans (carbohydrate homopolysaccharides consisting of re-
peating d-glucopyranose units), starch and cellulose are the most abundant.
Technologies utilizing starchy feedstocks (e.g. maize) for production of ethanol,
by fermentation of the liberated glucose monomers, are well-established and
ISBN: 3-527-31027-4
140 7 The Biofine Process
Fig. 7.1 Physical structures of cellulose and of starch amylose and amylopectin.
run at relatively high efficiencies. This is because of the comparative ease of

starch hydrolysis, using mainly a-amylase and gluco-amylase enzymes [4]. In
1999 a total of 1.48 billion gallons (ca 5.3 ´ 109 L) of fuel ethanol was blended
with gasoline for use in motor vehicles in the United States. About 94% of this
was produced by fermentation from maize; most of the remainder was from
other grain [5].
Cellulose is much more abundant in nature than is starch, and its annual
production is estimated at 100 ´ 109 tonnes [6]. Furthermore, cellulosic feed-
stocks tend to be more productive and require less energy to produce than
starch crops. Technologies for hydrolysis of the cellulosic feedstocks are cur-
rently not commercially developed at a scale approaching that for starch, how-
ever. This is because cellulose (Fig. 7.1) is of the order of 100 times more diffi-
cult to hydrolyze than starch [7]. The d-anhydroglucopyranose units in cellulose
are linked through b-(1 ? 4)-glycosidic bonds, as opposed to the a-(1 ? 4)-link-
ages in the amylose component of starch and the a-(1 ? 6) amylopectin
branches in starch. The structure of cellulose enables intimate intermolecular
associations that do not occur in starches, and this explains the relative resis-
tance to degradation in cellulose fibrils and microfibrils compared with starch
macromolecules.
7.2.1
Acid Hydrolysis of Polysaccharides
Cellulose is hydrolyzed in pure water by attack by the electrophilic hydrogen

atoms of the H2O molecule on the glycosidic oxygen (Fig. 7.2). This is a very
slow reaction, because of the resistance of the cellulose to hydrolysis. The rate
of the reaction can be increased by use of elevated temperatures and pressures
or can be catalyzed by acids (concentrated or dilute), or by highly selective en-
zymes such as cellulases. The steps involved in the acid-catalyzed hydrolysis of
cellulose are illustrated in Fig. 7.2. The H+ ions equilibrate between the O
atoms in the system, including those of water and the glycoside, with the conse-
quence that there is an equilibrium concentration of protonated glycoside. This
equilibrium tends towards the protonated form of the glycoside with increasing
temperature. The protonated conjugate acid then slowly breaks down to the cy-
clic carbonium ion, which adopts a half chair conformation (while the other
glucopyranose residue retains the OH at C-4). After rapid addition of water, free
sugar is liberated. Because the sugar competes with the water, small amounts
of disaccharides are formed as reversion products.
There is a time/temperature relationship whereby lower acid concentrations
require more extreme conditions and longer times for cellulose degradation.
The use of stronger acid may reduce the costs associated with higher-pressure
vessels, but the costly effects of equipment corrosion and of acid loss may be ex-
cessive. Rates of cellulose hydrolysis may differ according to the degree of crys-
tallinity of the cellulose (i.e. the proportions of crystalline and amorphous cellu-
lose present), a factor which varies between feedstocks.
The mechanism of hydrolysis of hemicellulose polysaccharides is similar to
that illustrated for cellulose in Fig. 7.2 and usually involves protonation of the
glycosidic oxygen. Process conditions do not need to be as severe, however, giv-
en the lower degree of polymerization (formation of the carbonium ion occurs
more rapidly at the end of a polysaccharide chain) and a tendency for the occur-
rence of less intermolecular bonding in most hemicelluloses. The rate of hydro-
lysis of hemicelluloses with a higher uronic acid content may be lower than for
other hemicelluloses, however, as a result of the steric effects of the carboxyl
groups.
Fig. 7.2 Steps involved in the acid hydrolysis of cellulose [8].

The ash content of feedstocks is important because ash tends to lower the
acidity of the mixture – the catalytic hydrogen ion is a function of the concen-
tration of the acidic solution applied and the neutralizing power of the ash [9].
It is therefore useful to measure the titratable alkalinity of feedstocks to ascer-
tain what acid levels may be necessary for their hydrolysis.
7.2.2
Production of Levulinic Acid, Formic Acid and Furfural
The Biofine Process involves the use of dilute sulfuric acid as a catalyst but dif-
fers from other dilute-acid lignocellulosic-fractionating technologies in that free
monomeric sugars are not the product. Instead, the 6-carbon and 5-carbon
monosaccharides undergo multiple acid-catalyzed reactions to give the platform
chemicals levulinic acid (C5H8O3) and furfural (C5H4O2) as the final products.
Hydroxymethylfurfural (HMF) is an intermediate in the production of levulin-
ic acid (4-oxopentanoic acid) from 6-carbon sugars in the Biofine Process. The
series of consecutive reactions involved in its production are illustrated in
Figs 7.3 and 7.4. These reactions have been established by numerous studies
aimed at identification of intermediate products and analyses of pathways for
their further transformation [10]. The enediol (1), obtained by enolization of d-
glucose, d-mannose, or d-fructose, is the key compound in the formation of
HMF. Further dehydration of the enediol (1) yields the product (2); which is
Fig. 7.3 Dehydration of the enediol (1) of D-glucose, D-mannose and D-fructose.
Fig. 7.4 Formation of hydroxymethylfurfural from 3,4-dideoxyglucosulosene-3.
further dehydrated to give 3,4-dideoxyglucosulosene-3 (3). 3,4-dideoxyglucosulo-

sene-3 (3) is readily converted (Fig. 7.4) to the dienediol (4), which eventually re-
sults in the formation of 5-hydroxymethylfurfural (6) via the intermediate cyclic
compound (5). Humic-type compounds can also be produced as side products
in this reaction [11].
If the CH2OH group of the hexoses is, instead, a hydrogen (as with the pen-
toses) a similar procedure occurs, but furfural is now the product.
Furfural
Hydration of HMF, i.e. addition of a water molecule to the C-2–C-3 olefinic

bond of the furan ring, leads to an unstable tricarbonyl intermediate (7) which
decomposes to levulinic acid (LA) (8) and formic acid (HCOOH). A possible re-
action process is shown in Fig. 7.5 [11]. The steps in the brackets in the mecha-
nism below have not been proven and include several assumptions; these inter-
Fig. 7.5 A possible process for formation of LA from HMF [11].

13
mediates were proposed by Horvat et al. [12, 13] based on analysis of C NMR
spectra of the reaction mixture formed in the hydration of HMF.
7.3
The Biofine Process
Feedstock materials for a Biofine plant must be of appropriate particle size (ca
0.5 to 1 cm) to ensure efficient hydrolysis and optimum yields. The feedstock is
therefore initially shredded before the biomass particulates are conveyed by
high-pressure air injection system to a mixing tank. Here the feedstock is mixed
with recycled dilute sulfuric acid (1.5–3%, depending on feedstock and titratable
alkalinity). The Biofine Process then consists of two distinct acid-catalyzed
stages (Fig. 7.6) that are operated to give optimum yields with a minimum of
degradation products and tar formation.
The objective in the first reactor is the dominant, first-order, acid hydrolysis
of the carbohydrate polysaccharides to their soluble intermediates (e.g. HMF).
This reaction is favored by the use of a plug-flow reactor, at a temperature of
210–220 8C and a pressure of 25 bar. The rapid nature of the hydrolysis reaction
means that a residence time of only 12 s is required. Given that the products
are removed continuously, such a small residence time requires that the diame-
ter of the reactor is kept small.
The completely mixed conditions of the second reactor favor the first-order re-
action sequence leading to LA (Fig. 7.5) rather than higher-order tar-forming
condensation reactions. Although the acid concentration remains the same as
Fig. 7.6 Chemical conversion of cellulose to LA (major prod-

uct), formic acid (byproduct), and tars (minor condensation
products) in the two Biofine reactors.
in the first reactor, operating conditions are less severe (190–200 8C, 14 bar).
This reactor is considerably larger than the first, however, because of the need
for a residence time of approximately 20 min. Furfural and other volatile prod-
ucts tend to be removed at this stage while the tarry mixture of LA and residues
are passed to a gravity separator. From here the insoluble mixture goes to a de-
hydration unit where the water and volatiles are boiled off. The heating of the
mixture to boil off the LA is conducted under reduced pressure and results in
the tarry material being “cracked”, to give a bone-dry powdery substance
(“char”). The crude 75% LA product can be purified up to a purity of 98%. The
acid is recovered in the final recycle stage, enabling it to be re-used in the sys-
tem.
In a complete Biofine plant, additional processing may then occur, depending
on the final products required. For example, syngas production from the Biofine
char (a dry, powdery material of calorific value comparable with that of bitumi-
nous coal, and composed of the residual materials in the Biofine Process which
has value as a fuel and as a soil additive) can be conducted in a gasification unit
or the LA can be esterified with ethanol to produce ethyl levulinate. The down-
stream conversions will be discussed further below.
7.3.1
Yields and Efficiencies of the Biofine Process
The maximum theoretical yield of LA from a hexose is 71.6% w/w and formic
acid makes up the remainder [14]. How close to this theoretical yield is achieved
in the conversion process will depend on the degradation reactions involved. In
addition to cellulose and LA there are likely to be many intermediates other
than those presented above. Some authors [12] have estimated there are over
100. These intermediates tend to cross-react and coalesce to form an acid-resis-
tant tar which incorporates many insoluble residues such as humins. Previously
developed technologies that attempted to produce LA from lignocellulosics were
expensive because of low LA yields (approx. 3% by mass) and significant tar for-
mation. The Biofine Process, because of its efficient reactor system and the use
of polymerization inhibitors that reduce excessive char formation [2, 3], achieves
from cellulose LA yields of 70–80% of the theoretical maximum. This translates
to conversion of approximately 50% of the mass of 6-carbon sugars to LA, with
20% being converted to formic acid and 30% to tars. The mass yield of furfural
from 5-carbon sugars is also approximately 70% of the theoretical value of
72.7%, equivalent to 50% of the mass, the remainder being incorporated in the
Biofine char. These claims have been supported by process data from a pilot
plant located in Glens Falls, New York State. This processes one dry tonne of
feedstock per day and has been operational for several test-run periods since
1996. Its construction followed successful laboratory-scale demonstrations of the
viability of the process at the National Renewable Energy Laboratory in Golden,
Colorado. In the latter experiments, paper sludges from nearby paper mills were
initially used as pilot plant feedstocks and gave LA yields ranging from 0.42 to
0.595 kg per kilo of cellulose (between 59 and 83% of the theoretical maximum
yield).
The acid-insoluble ligneous and ash components of the feedstock become in-
corporated in the Biofine char with 100% mass conversion, although the proper-
ties of the resulting materials are likely to be altered under the “cracking” condi-
tions of high-temperature and pressure. For most lignocellulosic feedstocks that
may be processed in a Biofine unit, the dry mass balance of structural polysac-
charides, lignin, and ash is likely to be close to 100%. Some feedstocks may
have a relatively high proportion of extractives (extraneous components that
may be separated from the insoluble cell wall material as a result of their solu-
bility in water or neutral organic solvents). Bark, for example, may contain up
to 25% by mass of extractives (predominately fats, waxes and terpenes) [15]
whereas some grasses may contain a significant proportion (e.g. 20%) of water-
soluble carbohydrates (WSC), depending on the time of year and environmental
conditions. Although these WSC are also potential LA precursors, their fate in
the Biofine process (as with other acid-hydrolysis schemes [16]) is likely to tend
towards tar/residue formation because the process conditions are geared to-
wards the conversion of cellulose and hence may be too strong to give LA as an
end-product from WSC. Other extractive components are also likely to be incor-
porated in the Biofine char. That may be advantageous in instances where the
char is to be combusted, given the relatively high heating values of these impu-
rities [17].
7.3.2
Advantages over Conventional Lignocellulosic Technology
The Biofine Process is entirely chemical and does not rely on the use of any
form of microorganism, as in enzymatic hydrolysis and in conventional dilute/
concentrated acid hydrolysis technologies. The use of biological agents is often
responsible for poor yields and a lower range of feasible feedstocks.
Most dilute acid hydrolysis technologies utilize microorganisms in the fer-
mentation of the fully hydrolyzed monomers (e.g. Saccharomyces cerevisiae [18]).
Some of the more recently developed schemes also utilize microorganisms in
the hydrolysis of cellulose after hemicellulose extraction (simultaneous sac-
charification and fermentation, SSF). Even in the most advanced SSF technol-
ogy the fermentation process takes a substantial time. After pretreatment, the
cellulase enzyme and fermentation organisms require about 7 days to bring
about the conversion to ethanol, compared with approximately 2 days for conver-
sion of starch and approximately 30 min for conversion of cellulose to levulinic
acid in the Biofine Process. Ethanol yields are also reduced as a result of the
formation of sugar degradation products that inhibit the organisms/enzymes
used for fermentation [19].
There are also significant problems associated with the fermentation of non-
glucose sugars, particularly xylose. Although these sugars can be converted to
ethanol by the genetically engineered yeasts that are currently available, for ex-
ample Pachysolen tannophilus [20], ethanol yields are not sufficient to make the
process economically attractive. It also remains to be seen whether the yeasts
can be made “hardy” enough for production of ethanol on a commercial scale
[21]. The inefficient utilization of nonglucose monosaccharide residues is a ma-
jor disadvantage in fermentation schemes because these residues may be a sig-
nificant proportion of the total polysaccharide mass (e.g. xylose makes up ap-
proximately 20% of the total dry mass in much woody and herbaceous bio-
mass). The 50% (by mass) conversion of C5 sugars to furfural in the Biofine
Process looks particularly attractive in such instances.
In avoiding the use of microorganisms, Biofine also enables use of a wider
range of heterogeneous lignocellulosic feedstocks, including those (e.g. cellulo-
sic municipal solid waste, sewage) that contain contaminants that might inhibit
fermentation. The flexibility of the technology for a variety of feedstocks has
been demonstrated over a four-month evaluation period during which the
highly heterogeneous organic fraction of municipal solid waste (from the Bronx
district of New York City) was successfully fractionated [22]. Furthermore, the
lignin content of biomass has no inhibiting effect on the Biofine Process and
this contrasts with enzymatic hydrolysis in which steric hindrance, caused by
lignin–polysaccharide linkages, limits access of fibrolytic enzymes to specific
carbohydrate moieties, this resulting in lower yields or the need for steam-explo-
sion pretreatment [23].
7.3.3
Products of the Biofine Process
LA is a valuable platform chemical because of its particular chemistry – it has

two highly reactive functional groups that enable many synthetic transforma-
tions. LA can react both as a carboxylic acid and as a ketone. The carbon atom
of the carbonyl group is usually more susceptible to nucleophilic attack than
that of the carboxyl group. Because of the spatial relationship of the carboxyl
and keto groups, many of the reactions proceed with cyclization forming hetero-
cyclic molecules (for example methyltetrahydrofuran). LA is readily soluble in
water, alcohols, esters, ketones, and ethers. The worldwide market for pure LA
at a price of $5 kg–1 has been estimated to be about only half a million kilo-
grams. The key to an increased potential marketability for LA is the vast range
of derivatives possible from this platform chemical (e.g. Refs. [24–26]) and its
economical production via the Biofine Process. Figure 7.7 lists some of the sec-
tors that offer markets for the products of the Biofine process. The following
subsections will discuss some of the more promising products which potentially
have the largest markets and hence the greatest potential for significantly re-
placing oil as a source of industrial chemicals and transport fuels.
Fig. 7.7 Possible markets and saleable products from the Biofine process.
7.3.3.1 Diphenolic Acid

Diphenolic acid [4,4-bis-(4'-hydroxyphenyl)pentanoic acid] is prepared by reac-
tion of levulinic acid with two molecules of phenol [27]. It may be a direct re-
placement for bisphenol A (BPA) in polycarbonates, epoxy resins, polyarylates,
and other polymers. The acid also has numerous other uses including applica-
tions in lubricants, adhesives, and paints [28]. It can also copolymerize with
BPA or can replace it in a variety of formulations. It contains a carboxyl group,
absent from BPA, which confers additional functionality useful in polymer syn-
thesis.
Diphenolic Acid Bisphenol A
Diphenolic acid (DPA) was used commercially in various resin formulations be-
fore it was replaced by the petrochemically-derived BPA which could be sup-
plied at a lower price. The reduced cost of LA production made possible with
the Biofine Process may enable DPA to recapture some market share. Extensive
research into near-term applications of DPA, particularly those that displace cur-
rently marketed BPA products, has been conducted at the Rensselaer Polytech-
nic Institute in New York State [29]. In the longer term DPA could be a viable
alternative to oil in the production of plastics.
The cost of LA produced by other technologies is the principle reason for the
high price of DPA (approx. $\phi 6 kg–1). On the basis of Biofine estimates, the
production of DPA from LA from the Biofine Process could result in a market
price of $ 2.40 kg–1. That price, based on Biofine estimates, could result in DPA
capturing 20% of the US market (2.5 ´ 108 kg year–1 for BPA). It may also result
in DPA recapturing some of the 2.5 ´ 106 kg year–1 market it held for its old use
as a coating material.
7.3.3.2
Succinic Acid and Derivatives
Oxidation of levulinic acid can lead to the production of succinic acid (Fig. 7.8).
Currently, succinic acid is produced using a hydrocarbon-based process. A fer-
mentation process using glucose derived from corn syrup can also produce suc-
cinic acid but this is not economically competitive. The most important uses of
succinic acid are in food additives, soldering fluxes, and pharmaceutical prod-
ucts. The US market for succinic acid is approximately 4.50 ´ 108 kg year–1, with
a market price of approximately $ 2.8 kg–1.
Succinic acid can be used to produce tetrahydrofuran (THF), 1,4-butanediol,
and c-butyrolactone (GBL). THF is formed by cyclization of succinic acid to give
succinic anhydride which is then reduced and dehydrated to provide tetrahydro-
furan. THF is a cyclic ether whose major use is as a monomer in the produc-
tion of poly(tetramethylene ether glycol) (PTMEG), a component of, among
other things, polyurethane stretch fibers (Spandex). A smaller amount of THF
is used as a solvent in poly(vinyl chloride) (PVC) cements, pharmaceuticals, and
coatings and as a reaction solvent. The Western European market for tetrahy-
drofuran is estimated to be approximately 7.5 ´ 107 kg, valued at $ 2.6 kg–1. Al-
most 80% of production is used captively, mostly for PTMEG [30].
c-Butyrolactone (C4H6O2) is used as a chemical intermediate in the manufac-
ture of the pyrrolidone solvents. It can be used in the production of pesticides,
herbicides, and plant-growth regulators. Mechanisms for the production of GBL
are currently being refined – catalysts have been identified for the selective re-
duction of succinic acid to GBL in the presence of acetic acid [31]. Although
high GBL yields have been successfully demonstrated, catalyst productivities are
currently still below commercially attractive rates [31]. The market price for 1,4-
butanediol, another possible derivative of succinic acid, is approximately
$2.30 kg–1 [30].
Fig. 7.8 The production of succinic acid in base (e.g. NaOH).
7.3.3.3 Delta-aminolevulinic Acid

d-Aminolevulinic acid (DALA) is a naturally occurring substance present in all
plant and animal cells [32–34]. It is the active ingredient in a range of environ-
mentally benign, highly selective, broad-spectrum herbicides. It has high activity
against dicotyledonous weeds and little activity against monocotyledonous crops
such as corn (maize), wheat, or barley [35]. DALA also has use as an insecticide
[36] and in cancer treatment [37].
DALA
Difficulties experienced in production of DALA from LA involve the selective in-

troduction of an amino group at the C5-position. The most common approach
for activating the C5 position toward amination is bromination of LA in an alco-
hol medium to give mixtures of 5-bromo- and 3-bromoesters that are separated
by distillation [38]. The 5-bromolevulinate is then aminated using a nucleophilic
nitrogen species [39]. These conventional mechanisms give low yields at very
high cost. The National Renewable Energy Laboratory (NREL) process (Fig. 7.9),
significantly improves yields and reduces costs. It also results in the production
of two moles of formic acid per mole of DALA, the resulting DALA being ob-
tained at a purity of greater than 90%. During initial testing in a greenhouse
environment, NREL found that this crude DALA was active as a herbicide.
A significant amount of research is still being conducted on the formation of
DALA from LA. The complexity and low yields of conventional DALA synthesis
techniques mean that it is currently a very expensive product, being used only
for highly selective herbicidal treatment and some cancer therapies. There is a
large potential in the agricultural and horticultural sector for lower-cost Biofine-
derived DALA; however, quantification of this area is not possible at present be-
cause specific commercial formulations must be developed.
7.3.3.4 Methyltetrahydrofuran
The production of fuel additives via renewable feedstocks offers perhaps the
greatest potential for mass-market penetration of LA. Methyltetrahydrofuran
(MTHF) can be added to petroleum in amounts up to 30% by volume with no
adverse effects on performance, and engine modifications are not required.
Some important properties of MTHF are listed in Table 7.1. Although it has a
Fig. 7.9 NREL mechanism for the production of DALA

from LA. Taken from [28].
Table 7.1 Selected properties of MTHF and ethyl levulinate [42–44].
MTHF Ethyl levulinate
Boiling point (102 mmHg), 8C 20 93

Boiling point (Atm.), 8C 80 206.2
Flash point, 8C 11 195
Reid vapor pressure, psig 5.7 < 0.01
Lower heating value, kJ kg–1 32 000 24 300
Specific gravity 0.813 1.016
Octane rating 80
Cetane number – < 10
Lubricity (HFRR micros) – 287
lower heating value than regular petroleum, it has a higher specific gravity and
hence mileage from MTHF blended fuel would be competitive. MTHF substan-
tially reduces the vapor pressure of ethanol when coblended in gasoline. This
has led to the development of “P-Series” fuels, i.e. fuels miscible with petroleum
designed for vehicles with flexible-fuel engines and containing “pentanes-plus”
hydrocarbons from natural gas, ethanol (preferably from biomass), and methyl-
tetrahydrofuran as a co-solvent for ethyl alcohol (high-octane) [40]. P-Series fuels
can be used alone or may be mixed in any proportions with petroleum. Vehicle
tailpipe and evaporative emissions tests have been conducted on three P-Series
formulations by the Environmental Protection Agency [41] and the results have
been compared with those obtained from reformulated gasoline (RFG). It was
found that the formulations had a reduced ozone-forming potential (OFP) and
resulted in reduced emissions of nonmethane hydrocarbons and total hydrocar-
bons – approximately a third of that formed with Phase 2 RFG. It has been esti-
mated that when the MTHF and ethanol are derived from biological materials,
the full fuel-cycle greenhouse gas emissions will be between 45 and 50% below
those of reformulated gasoline [41]. These successful emission and performance
tests have recently resulted in the P-Series formulations being approved by the
US Department of Energy as an alternative gasoline, meeting the requirements
of the Energy Policy Act for automobile fleet usage. It should be noted, however,
that P-Series fuels can only be used in “flexible fuel engines” and have to be
distributed at gasoline stations supplied with pumps specially modified for alco-
hol-based fuels. Their short-term markets may therefore be limited to captive
fleets (e.g. city buses).
Direct conversion of LA to MTHF occurs in low yield, hence indirect routes
are utilized (Fig. 7.10). One possible mechanism involves the catalytic hydroge-
nation of LA to c-valerolactone (GVL) which, on further hydrogenation, yields
1,4-pentanediol and, finally, MTHF [28]. An efficient application of this mecha-
nism was devised by scientists at the Pacific Northwest Laboratory (PNL) in the
US [45]. The process is conducted at elevated temperatures and pressures using
a continuous-flow catalytic reactor. Levulinic acid is pumped into a tube where
it is warmed to approximately 40 8C, then mixed with hydrogen. Both com-
Fig. 7.10 Possible mechanisms for formation of MTHF from LA [28].
pounds are then pumped through a reactor filled with a catalyst in which a se-
ries of chemical reactions occurs at approximately 240 8C and 100 atmospheres
pressure to create MTHF. The procedure requires three moles of hydrogen per
mole LA. Laboratory tests indicated the yield was 83% on a theoretical (molar)
basis [45]. This would be equivalent to a yield of approximately 63 kg (71 L)
MTHF for every 100 kg of LA, or 81 L of MTHF for every 100 L of LA. The yield
of the PNL process is significantly greater than that of other processes. These
usually used mechanisms in which MTHF was a merely a byproduct, with final
yields of approximately only 3%.
The extra costs involved in producing MTHF from LA are minimal – for a
Biofine plant processing 1000 dry tonnes of biomass per day, the estimated ex-
tra capital cost for an MTHF production facility would be $ 10 Mio. The re-
quired 6 kg of hydrogen for every 100 kg of LA could be supplied from the resi-
dual process char (via a syngas production unit, see below) with an estimated
cost of hydrogen production of 5 c kg–1.
In addition to transport, MTHF has value in other markets – it is, for exam-
ple, an excellent general solvent that is, in many regards, superior to tetrahydro-
furan. It should also be noted that catalytic production of MTHF from furfuryl
alcohol is possible, as is the production of dimethyltetrahydrofuran (DMTHF)
from hydroxymethylfurfural (the product of the first Biofine reactor). It is hy-
pothesized that the additional methyl group in DMTHF may afford superior
performance and mileage over MTHF, although vehicle tests have yet to be car-
ried out.
7.3.3.5 Ethyl Levulinate

Esters of LA produced from either methanol or ethanol have significant poten-
tial as blend components in diesel formulations. LA esters are similar to the
biodiesel fatty acid methyl esters (FAME) that are used in some low-sulfur die-
sel formulations but they do not have their principal drawbacks (cold flow prop-
erties and gum formation [46]). Addition of ethyl or methyl levulinate to FAME
would be expected to alleviate both these problems.
The most studied of the LA esters is a low-smoke diesel formulation devel-
oped by Biofine and Texaco that uses ethyl levulinate (made by esterifying LA
with fuel-grade ethanol) as an oxygenate additive. The 21 : 79 formulation con-
sists of 20% ethyl levulinate, 1% co-additive, and 79% diesel and can be used in
regular diesel engines. The oxygen content of ethyl levulinate (EL) is 33%, w/w,
giving a 6.9%, w/w, oxygen content in the blend, resulting in a significantly
cleaner burning diesel fuel [44].
The ethyl levulinate blend gives lower sulfur emissions than does regular die-
sel. This is because ethyl levulinate contains no sulfur. Lower sulfur emissions
can also be attributed to the high lubricity of EL blends. Fuel lubricity is used to
determine the amount of wear that occurs between two metal parts covered
with the fuel as they come into contact. Fuels of higher lubricity result in less
wear and prolong engine component life. The sulfur level of diesel is reduced
in the refinery using a hydro-treating process; this results in undesirable re-
moval of some of the lubricity components from the fuel and hence a decrease
in diesel lubricity. Addition of EL, with high lubricity, will therefore mean that
diesel blend-stocks of low lubricity, and hence lower S content, can be used
without reducing the all-over lubricity of the end product. In Europe lubricity is
measured by use of the high frequency reciprocating rig (HFRR) test with lower
values indicating higher fuel lubricity. Biofine has shown that addition of 20%
EL to a standard No 2 base fuel improves the HFRR from 410 to 275 [44]. Im-
portantly, the significant losses of engine efficiency (a decrease of up to 15% in
the distance driven per unit volume is found with other diesel oxygenates, for
example ethanol) do not occur with ethyl levulinate. This is because of the high
energy content of the 21 : 79 formulation (selected properties of EL are listed in
Table 7.1).
The levulinate esters also have potential as replacements of kerosene as a
home heating oil and as a fuel for the direct firing of gas turbines for electrical
generation [47]. The production of levulinic acid esters from LA formed in the
Biofine Process has the added advantage over conventional bioesters that there
is no coproduction of glycerol which would have to be disposed of.
7.3.3.6 Formic Acid

Formic acid (HCOOH) is a byproduct in the production of levulinic acid from
cellulose. It can be purified by distillation and sold directly as a commodity
chemical. It is conventionally produced, usually as a byproduct of acetic acid
production, by liquid phase oxidation of hydrocarbons. It is used extensively as
a decalcifier, as an acidulating agent in textile dying and finishing, and in
leather tanning [48]. It is also used in the preparation of organic esters and in
the manufacture of drugs, dyes, insecticides, and refrigerants. Formic acid can
also be converted into calcium magnesium formate for use as a road salt. In Eu-
rope, the largest single use of formic acid is as a silage additive. For example,
the AMASIL additive produced by BASF Ireland contains 85% formic acid
which is bought at a price of 1 1.35 per liter.
The catalyst-preparation sector is a very large potential future market for for-
mic acid. As well as being used in the manufacture of many catalysts, formic
acid is also used in the regeneration of catalyst metals poisoned with sulfur.
The increasing demand for low-sulfur fuels will result in increased demand for
the catalysts produced using the formates and, consequently, will require formic
acid. Additionally, esters of formic acid (e.g. methyl and ethyl formate) may also
have value as fuel components and as platform chemicals.
In 2000, world consumption of formic acid amounted to approximately
4.15 ´ 108 kg [48], roughly half of which was consumed in Europe. A Biofine
plant processing 300 dry tonnes of feedstock per day would produce approxi-
mately 9 ´ 106 kg formic acid per year (assuming a cellulose content of 40%).
Where supply of formic acid exceeds demand for its conventional uses, the mer-
chant market price may fall to around $ 0.16 per liter, which is the price needed
to open up other markets such as its use as a road salt or for formaldehyde pro-
duction [49]. If even these markets are not available, the formic acid byproduct
still has value, because it can provide energy via gasification or anaerobic diges-
tion.
7.3.3.7 Furfural
Furfural is produced from the hemicellulosic pentose fractions of biomass. Xy-
lose is the predominant pentose in most feedstocks, with hemicellulosic arabi-
nose found to a lesser extent. Furfural can be sold as a solvent or used in the
production of furfuryl alcohol, tetrahydrofuran (THF), and LA. Furfuryl alcohol
(Fig. 7.11) is a monomer for furan resins, these being used mainly as foundry
binders. It is prepared by hydrogenation of furfural. THF is produced by decar-
bonylation of furfural to furan, then catalytic hydrogenation [50]. LA is produced
by first converting furfural to furfuryl alcohol. Figure 7.11 shows the mecha-
nism involved – furfuryl alcohol, when boiled in ethyl methyl ketone in the
presence of HCl, gives rise to 90–93% levulinic acid, the reaction occurring via
hydroxy derivatives [11].
Global production of furfural in 2001 amounted to 22.5 ´ 107 kg year–1 [51].
Approximately 4 ´ 107 kg furfural was consumed in Europe in 2000, furfuryl al-
cohol being the major market. Most furfural is now produced in China, whose
total capacity is 15–20 ´ 107 kg year–1 [51]. Low labor and feedstock prices in Chi-
Fig. 7.11 Production of LA from furfuryl alcohol.

na, coupled with increasing Chinese capacity, have resulted in prices falling over
the last decade – the current market price of furfural is approximately $ 1 kg–1
compared with prices in 1990 of $ 1.74 kg–1 for furfural and $ 1.76 kg–1 for fur-
furyl alcohol [50]. EU and US import tariffs are placed on furfural from China,
these being designed to lessen this effect of this price differential, but market
prices are still highly dependent on Chinese supply. (A significant rise in price
between 1995 and 1998 was attributed to a drought in China during that peri-
od).
A Biofine plant processing 300 dry tonnes of feedstock per day would pro-
duce, from hemicelluloses, approximately 1.3 ´ 107 kg furfural per year (assum-
ing 25% pentosans by mass). This represents 32.5% of the total consumption of
furfural/furfuryl alcohol in Europe in 2000. Furfural conversion products,
whether THF or LA and their subsequent downstream products, may therefore
be more marketable final products than furfural itself in large biorefinery
schemes, especially if the fuel additive market is explored.
7.3.4
Biofine Char
The quantity of residual char from the Biofine process and its calorific value
will depend on the acid-insoluble lignin content of the biomass, the ash con-
tent, any insoluble proteins present, and the amount of degradation and rever-
sion products formed from the cellulose and hemicellulose fractions. The boil-
ing off from the char of volatiles and LA gives rise to a “cracking” of the char. It
is, therefore, difficult to predict the final composition of the char from the mass
compositions of virgin biomass. The composition would be predictable with
greater certainty if each potential feedstock was tested in the Biofine Process.
Biofine char has much promise as a fuel. Residual char from the processing
of paper sludge in the Biofine pilot plant was found to have a heating value of
approximately 25.6 MJ kg–1 (with 15% ash content), a value that was signifi-
cantly larger than that of the original feedstock (18.6 MJ kg–1). It was found that
combustion of the dry Biofine char (the mass of which was 15% of that of the
original paper sludge) yielded more energy than combustion of the entire feed-
stock (at its initial 50% moisture content).
It has been estimated that the energy provided by the residual char is greater
than that needed to completely fuel the steam and electric power needs of the
biorefinery when the scale of operation is equal to or greater than approximately
270 dry tonnes of feedstock per day (assuming a feedstock lignin content of
approximately 25%). Appropriately sized plants may therefore expect to gain
significant revenue from the marketing of surplus electricity.
Research is ongoing for alternative uses and markets for Biofine char. It is be-
lieved it may have value as a soil conditioner. Figure 7.12 compares the FTIR
spectra of lignin with those of chars obtained from paper sludge and straw feed-
stocks. It can be seen that straw char retains many of the functionalities of lig-
nin, characterized by absorbance in the 1000–1800 cm–1 region of the spectrum.
Fig. 7.12 FTIR spectra for lignin and Biofine chars from paper
sludge and straw feedstocks.
The chars from straw and from paper have significant carbonyl/carboxyl func-
tionality and the presence of significant acidic functionality was confirmed by ti-
tration data. Solid-state NMR will indicate the nature of the aromatic functional-
ity and whether or not the Biofine chars have the fused aromatic structures
characteristic of charcoals.
We have performed thermogravimetric analyses on several feedstocks and on
the chars from straw and paper. In thermogravimetry, sample weight loss is
monitored as a function of increasing temperature. Thermograms, and their
first derivatives (rate of weight loss with respect to temperature) for the renew-
able energy crop Miscanthus, for a lignin extract, and for the Biofine chars from
paper sludge, and straw feedstocks are shown in Fig. 7.13. Weight loss in the
range 50–120 8C is associated with volatilization of water whereas that resulting
from loss of low-molecular-weight compounds and other volatile organic com-
pounds (extractives in woods/grasses) occurs from 120–250 8C; degradation of
hemicelluloses occurs in the 250–300 8C range, and cellulose degrades very
sharp at approximately 300–340 8C in air (higher in nitrogen). The thermogram
for Miscanthus is strong evidence for hemicellulose, cellulose, and lignin com-
ponents. The two thermograms for the chars are similar and clearly show that
the hemicellulose and cellulose components are greatly diminished – indicated
by a significant shift of the peaks to the right compared with the Miscanthus
thermogram. This shift reflects a predominance of the ligneous type compo-
nents, this being indicated by a similarity between the char thermograms and
those of the lignin extract.
Another possible use of the char is steam gasification (thermochemical pro-
duction of hydrogen from a feedstock, for example biomass materials, or, in this
context, Biofine char) followed by upgrading of the synthesis gas produced.
Although little work has yet been done along these lines using the Biofine char,
Fig. 7.13 Thermograms and their first derivatives for lignin,

Miscanthus, and Biofine chars from paper and straw feed-
stocks.
there has been much work over the years on the gasification of equivalent mate-
rials, for example peat or coal [52, 53]. Coal gasification was originally used for
the production of syngas for use in, for example, the Fischer-Tropsch Process.
More recently, interest in connection with that process has shifted to the re-
forming of natural gas as a source of the syngas for the process (e.g. Ref. [54]
and other articles in the same volume). The steam gasification of carbon can be
represented simply by a combination of the gasification reaction:
C + H2O ? CO + H2
followed by the water-gas shift reaction:
CO + H2O = CO2 + H2
The exothermic water-gas shift reaction is favored by operation at low tempera-
tures whereas high temperatures favor the reverse reaction. Hence, a product
gas containing largely hydrogen is produced if the temperature is low and a
syngas with a CO/H2 ratio of 1 is obtained if the temperature is high. The syn-
gas can be used as a fuel (e.g. to give the energy needed for the Biofine process)
but it can also be used in a variety of reactions such as methanol synthesis:
CO + 2H2 ? CH3OH
Alternatively, it can be used in the Fischer–Tropsch process mentioned above;
this can be depicted simplistically as:
nCO + mH2 CnH2m
in which the product hydrocarbons are usually aliphatic in nature and can be
used as diesel substitutes. Much work has recently been performed on so-called
GTL technology (GTL = Gas to Liquids – technology that converts synthesis gas
(from gasification of biomass or biorefinery process chars, for example) into liq-
uid fuels), and many of these developments have been summarized recently
[55].
7.3.5
Economics of the Biofine Process
The Biofine technology is commercially viable. A commercial plant (Fig. 7.14)

processing 50 dry tonnes of feedstock per day has been constructed in Caserta,
Italy (with joint funding from the EU and private investment) and is expected
to be operational in 2005. The primary feedstocks will be paper sludge, agricul-
tural residue, and waste paper with the major products being LA and EL (for
use as a fuel). The process char will be gasified to produce a fuel gas for the
process boilers. The modular nature of the technology means that the capacity
can easily be upgraded, and there are plans for a supplemental 250 tonne per
day reactor system to be installed eventually – bringing the total capacity to 300
Fig. 7.14 Commercial plant in Caserta, Italy. Recovery vessels

(top); outside of building (bottom left); mixing tank
(top middle); second reactor (bottom middle);
arial view (right).
tonnes per day. Indeed, the Biofine process is extremely compact – a feasibility
study conducted by a marine architectural company concluded that a self-con-
tained 1000 tonne per day facility could be accommodated on an ocean-going
“Panamax” barge [56].
Significant economies of scale can accrue with increasing plant size, as
shown in Fig. 7.15 a and b. Figure 7.15 c shows, for various plant sizes, the trend
in the cost of ethyl levulinate production with feedstock cost. Table 7.2 shows a
detailed breakdown of these costs and of byproduct revenues for a plant proces-
sing 1000 dry tonnes of feedstock per day. It is assumed that the feedstock is of
composition, by mass, 50% cellulose, 20% hemicellulose, 20% lignin, and 5%
ash (comparable with many woody and herbaceous energy crops). It is also as-
sumed that the plant is operational for 350 days per year, that all of the furfural
Fig. 7.15 (a) Capital cost of Biofine plants at different scales

of operation; (b) Operating cost ($ per dry tonne of feedstock
processed) at different scales of operation; (c) Production
cost of ethyl levulinate with three plant sizes (1000, 500 and
300 dry tonnes of feedstock processed per day) and varying
feedstock cost.
Table 7.2 Data provided by Biofine on estimated operating

costs and byproduct revenues from a plant processing 1000
dry tonnes of feedstock per day for the production of ethyl
levulinate.
Scale of Operation – 1000 dry tonnes per day (350 000 dry tonnes/year)
Capital Cost – $ 150 million (Grassroots, fully self-contained and integrated)
Production Output – 133 000 tonnes per year ethyl levulinate
Raw materials:
Feedstock 350,000 dt/y @$ 40/t $ 14,000,000/y
Sulfuric acid 3,500 t/y @$ 100/t $ 350,000/y
Caustic soda 500 t/y @$ 120/t $ 60,000/y
Ethanol 35,000 t/y @$ 350/t $ 12,250,000/y
Hydrogen 120 t/y @$ 1,500/t $ 180,000/y
Others allow. $ 160,000/y
Subtotal $ 27,000,000/r
Utilities:
Steam 250,000 lb/h gen. on-site 0
Electric 14.4 MW gen. on-site 0
Electric usage 11.3 MW 0
Electric sold 3.1 MW
Water 250 gpm $ 500,000/y
Gas (boiler) 294 m BTU/h gen. on-site 0
Subtotal $ 500,000/y
Labor and Maintenance:
Operators 17 per shift @$ 20 per hr $ 2,800,000/y
Supervision 2 per shift @$ 24 per hr $ 400,000/y
Maintenance @4% of cap cost/yr $ 6,000,000/y
Subtotal $ 9,200,000/y
Overheads
Direct @30% of labor $ 1,000,000/y
General @25% of Lab. & Maintenance $ 2,300,000/y
Taxes and Insurance allow. $ 3,700,000/y
Subtotal $ 7,000,000/y
Disposal Costs:
Ash: 17,500 t/y @$ 35/t $ 600,000/y
Subtotal $ 600,000
Gross Production costs $ 44,300,000
Byproducts: Formic acid 38,500 t/y @$ 110/t $ 4,200,000
Electric sold 26,000 MWhr/y @$ 60/MWhr $ 1,500,000
Subtotal $ 5,700,000
NET PRODUCTION COST $ 38,600,000
Production cost per to Ethyl Levulinate (no capital charges) $ 291 per tonne
Equivalent Energy price 12 per GJ
7.4 Conclusion 161
is converted to LA, and that formic acid is sold for 10 c kg–1 and electrical power
for 6 c kw h–1. It can be seen that ash is the only waste product, incurring a dis-
posal charge ($ 35/tonne). Disposal need not be a problem, however. The Case-
rta plant will supply an adjacent tile factory with the ash, avoiding such costs.
The ash can also be used as a fertilizer on agricultural land. The ethyl levulinate
production cost of $ 291 tonne–1 is equivalent to a price of $ 12 GJ–1, competi-
tive with the $ 16 GJ–1 of gasoline at $ 2 gallon–1 or $ 10 GJ–1 for crude oil at
$ 50 per barrel.
7.4
Conclusion
Lignocellulosic fractionating technology offers the potential for inexpensive pro-

duction of a range of chemicals and fuels that are currently competitive only
from petrochemical reserves. The Biofine Process is among the most advanced
of this technology and provides high yields of levulinic acid (LA), furfural, and
formic acid. Furthermore, unlike most other processes aimed at harvesting
added value from carbohydrate feedstocks, the Biofine process is continuous,
compact, easily expandable and entirely chemically based. Feedstocks that con-
tain reasonable concentrations of carbohydrates can be utilized. Thus heteroge-
neous biomass reserves such as cellulosic municipal solid waste and animal ma-
nures may be processed as well as the more conventional lignocellulosic feed-
stocks such as sugar cane bagasse and high-yielding energy crops. Maximum
value is targeted from the diverse chemical constituents of biomass – the moist
nature of the biomass is exploited in the acid hydrolysis of polysaccharides to
provide platform chemicals such as LA (unlike in combustion/gasification
schemes where moisture is a barrier). The dry char residue has a significantly
higher fuel calorific value than the feedstock and it has potential for syngas pro-
duction and as a soil conditioner. The technology offers a potentially sustainable
“bio-recycling” solution in a world where rising and erratic oil prices and unpre-
dictable oil supplies are coupled with growing levels of waste production. The
extent to which this potential is realized will depend on the size of the market
for LA, furfural, and their derivatives. Mechanisms already exist for the produc-
tion of numerous saleable industrial chemicals from these, and promising re-
search indicates avenues for expansion into the potentially huge transport, agri-
cultural, and plastics sectors. It is therefore feasible that such a technology can
stimulate a transition from a hydrocarbon to a carbohydrate-based economy (i.e.
economy in which chemical, energy, fuel, and consumables requirements are
provided by carbohydrate rather than hydrocarbon feedstocks) where local self-
sustainability is possible. This concept is supported by the range of commercial-
scale Biofine plants planned for Ireland, the UK, and the US.
References
1 Szmant, H. H. (1989), Organic Building xymethylfuran-2-carbaldehyde. Croat.

Blocks of the Chemical Industry. Wiley & Chem. Acta 59: 429–438.
Sons, New York. 14 Leonard, R. H. (1956), Levulinic acid as a
2 Fitzpatrick, S. W. (1990), Lignocellulose basic chemical raw materials. Ind. Eng.
degradation to furfural and levulinic acid: Chem. 48(8): 1331–1341.
U.S. Patent 4,897,497 15 USDA (1971), Bark and its Possible Uses.
3 Fitzpatrick, S. W. (1997), Production of lev- US Department of Agriculture, Madison,
ulinic acid from carbohydrate-containing WI.
materials: U.S. Patent 5,608,105 16 Wiselogel, A. E., Agblevor, F. A., Johnson,
4 McAloon, A., Taylor, F., Yee, W., Ibsen, D. K., Deutch, S., Fennell, J. A. and San-
K. and Wooley, R. (2000), Determining the derson, M. A. (1996), Compositional
Cost of Producing Ethanol from Corn changes during storage of large round
Starch and Lignocellulosic Feedstocks, switchgrass bales. Bioresource Technology
NREL/TP-580-28893. NREL, Golden, CO. 56(1): 103–109.
5 Broder, J. D., Harris, R. A. and Ranney, 17 Susott, R. A., DeGroot, W. F. and Shafiza-
J. T. (2001), Using MSW and industrial deh, F. (1975), Heat content of natural
residues as ethanol feedstocks. Bi 8Cycle fuels. J. Fire and Flammability 6: 311–
42(10): 23–26. 325.
6 Bozell, J. J. (2001), Chemicals and Materi- 18 Nguyen, Q. (1998), Milestone Completion
als from Renewable Resources. American Report: Evaluation of a Two-Stage Dilute
Chemical Society, Washington DC. Sulfuric Acid Hydrolysis Process. Internal
7 Klass, D. L. (1981), Biomass as a nonfossil Report. National Renewable Energy Labo-
fuel source. American Chemical Society, ratory, Golden, Colorado.
Washington DC. 19 Gregg, D. and Saddler, J. N. (1996), A
8 Sjostrom, E. (1981), Wood Chemistry: techno-economic assessment of the pre-
Fundamentals and Applications. Academic treatment and fractionation steps of a
Press, Inc., London, UK. biomass-to-ethanol process. Appl. Bio-
9 Zerbe, J. I. and Baker, A. J. (1987), Inves- chem. Biotech. 57/58: 711–726.
tigation of fundamentals of two-stage, 20 Beck, M. J. and Strickland, R. C. (1984),
dilute sulphuric acid hydrolysis of wood. Production of ethanol by bioconversion
In: D. L. Klass (eds), Energy from Biomass of wood sugars derived from two-stage
and Wastes X. Institute of Gas Technol- dilute acid hydrolysis of hardwood. Bio-
ogy, Chicago, 927–947. mass and Bioenergy 6: 101–110.
10 Kooherkov, N. A., Bochkov, A. F., Dimi- 21 Cooper (1999), A Renewed Boost for
triev, B. A., Usov, A. I., Chizbov, O. S. and Ethanol. Chemical Engineering 106(2): 35.
Shibaev, Y. N. (1967), Khimiya Uglevodov 22 BioMetics Inc. (1996), Municipal Solid
(The Chemistry of Carbohydrates). Khi- Waste Conversion Project, Final Report,
miya, Moscow. Contract No: 4204-ERTER-ER-96. Bio-
11 Timokhin, B. V., Baransky, V. A. and Eli- metics Inc., Boston, MA.
seeva, G. D. (1999), Levulinic acid in or- 23 Donaldson, L. A., Wong, K. K. Y. and
ganic synthesis. Russian Chemical Re- Mackie, K. L. (1988), Ultrastructure of
views 68(1): 73–84. steam-exploded wood. Wood Science and
12 Horvat, J., Klaic, B., Metelko, B. and Technology 22: 103–114.
Sunjic, V. (1985), Mechanism of levulinic 24 Oono, T., Saito, S., Shinohara, S. and Ta-
acid formation. Tetrahedron 26: 2111– kakuwa, K. (1996), Fluxes for electric cir-
2214. cuit board soldering and electric circuit
13 Horvat, J., Klaic, B., Metelko, B. and boards: Japanese patent 08243787
Sunjic, V. (1986), Mechanism of levulinic 25 Shimizu, A., Nishio, S., Wada, Y. and
acid formation in acid-catalyzed hydroly- Metoki, I. (1996), Photographic processing
sis of 2-hydroxymethylfuran and 5-hydro- method for processing silver halide photo-
References 163
graphic light-sensitive material: European Photodynamic herbicides: Concept and

patent 704756 phenomenology. Enzyme and Microbial
26 Adams, P. E., Lange, R. M., Yodice, R., Technology 6: 390–401.
Baker, M. R. and Dietz, J. G. (1998), Inter- 36 Rebeiz, C. A., Gut, L. J., Lee, K., Juvik,
mediates useful for preparing dispersant-vis- J. A., Rebeiz, C. C. and Bouton, C. E.
cosity improvers for lubricating oils: Europe- (1995), Photodynamics of porphyric in-
an patent 882745 secticides. Crit. Rev. Plant Sci. 14: 329–
27 Isoda, Y. and Azuma, M. (1996), Prepara- 366.
tion of bis(hydroxyaryl)pentanoic acids: Ja- 37 Bedwell, J., McRoberts, A. J., Phillips, D.
panese patent 08053390 to Honshu Chemi- and Brown, S. G. (1992), Fluorescence
cal Ind. distribution and photodynamic effect of
28 Bozell, J. J., Moens, L., Elliott, D. C., ALA-induced PP IX in the DMF rat colo-
Wang, Y., Neuenschwander, G. G., Fitz- nic tumor model. Br. J. Cancer 65: 818–
patrick, S. W., Bilski, R. J. and Jarnefeld, 824.
J. L. (2000), Production of levulinic acid 38 MacDonald, S. F. (1974), Methyl 5-bro-
and use as a platform chemical for de- molevulinate. Can. J. Chem. 52: 3257–
rived products. Resources, Conservation 3258.
and Recycling 28: 227–239. 39 Ha, H.-J., Lee, S.-K., Ha, Y.-J. and Park,
29 Moore, J. A. and Tannahill, T. (2000), J.-W. (1994), Selective bromination of ke-
Homo- and co-polycarbonates and tones. A convenient synthesis of 5-ami-
blends derived from diphenolic acid. nolevulinic acid. Synth. Commun. 24:
High Performance Polymers 13: 305–316. 2557–2562.
30 Ring, K.-L., Kaelin, T. and Yoneyama, M. 40 Lucas, S. V., Loehr, D. A., Meyer, M.aE.
(2001), CEH Report: Tetrahydrofuran. and Thomas, J. J. (1993), Exhaust emis-
SRI, Menlo Park, CA. sions and field trial results of a new, oxyge-
31 Nghiem, N., Davison, B. H., Donnelly, nated, non-petroleum-based, waste-derived,
M. I., Tsai, S.-P. and Frye, J. G. (2001), gasoline blending component, 2-methyltetra-
An integrated process for the production hydrofuran. Soc. Auto. Eng. Fuels and
of chemicals from biologically derived Lubricants Sect. Mtg., Philadelphia, PA.
succinic acid. In: J. J. Bozell (eds), Chemi- 41 DoE (1999), 10 CFR Part 490: Alternative
cals and Materials from Renewable Re- Fuel Transportation Program; P-Series
sources. American Chemical Society, Fuels; Final Rule. Office of Energy Effi-
Washington DC, 160–173. ciency and Renewable Energy, Depart-
32 Gibson, H. D., Laver, W. G. and Neuber- ment of Energy (DOE).
ger, A. (1958), Initial stages in the bio- 42 Bayan, S. and Beati, E. (1941), Chemica e
synthesis of porphyrins. II. The forma- Industria 23: 432–434.
tion of 5-aminolevulinic acid from gly- 43 Thomas, J. J. (1986), Biomass Derived Lev-
cine and succinyl-CoA by particles from ulinic Acid Derivatives and Their Use as
chicken erythrocytes. Biochem. J. 70: 71– Liquid Fuel Extenders. Biomass Energy
81. Dev. [Proc. South Biomass Energy Res.
33 Beale, S. I. and Castelfranco, P. A. (1974), Conf.], Plenum.
The biosynthesis of delta-aminolevulinic 44 Texaco/NYSERDA/Biofine (2000), Ethyl
acid in higher plants. II. Formation of Levulinate D-975 Diesel Additive Test Pro-
14
C-delta-aminolevulinic acid from la- gram, Glenham, NY.
belled precursors in greening plant tis- 45 Elliott, D. C. (1999), U.S. Patent
sues. Plant Physiol. 53: 297. 5,883,266
34 Chen, J., Miller, G. W. and Takemoto, 46 Huang, C. and Wilson, D. (2000), 91st
J. Y. (1981), Biosynthesis of d-aminolevu- AOCS Annual Mtg., April 26th, 2000.
linic acid in Rhodopseudomonas spaer- 47 Erner, W. E. (1982), U.S. Patent 4,364,743
oides. Arch. Biochem. Biophys. 208: 221– 48 Bizzari, S. and Ishikawa, Y. (2001), CEH
228. Report: Formic acid. SRI, Menlo Park,
35 Rebeiz, C. A., Montazer-Zouhoor, A., CA.
Hopen, H. J. and Wu, S. M. (1984),
49 Idriss, H., Lusvardi, V. S. and Barteau, 53 Sutton, D., Kelleher, B. P. and Ross,
M. A. (1996), Two routes to formaldehyde J. R. H. (2001), Review of Literature on
from formic acid on TiO2(001) surface. Catalysts for Biomass Gasification. Fuel
Surface Science 348(1/2): 39–48. Processing Technology 73(3): 155–173.
50 Gravitis, J., Vedernikov, N., Zandersons, 54 Bakkerud, P. K., Gol, J. N., Aasberg-Peter-
J. and Kokorevics, A. (2001), Furfural sen, K. and Dybkjaer, I. (2004), Preferred
and levoglucosan production from decid- Synthesis Gas Production Routes for
uous wood and agricultural wastes. In: GTL. (eds), Studies in Surface Science and
J. J. Bozell (eds), Chemicals and Materials Catalysis. Elsevier, New York, 147: 13–18.
from Renewable Resources. American 55 Bao, X. and Xu, Y. (2004), Natural Gas
Chemical Society, Washington DC, 110– Conversion VII, Studies in Surface
122. Science and Catalysis, 147. Elsevier, New
51 Levy, J. and Sakuma, Y. (2001), CEH Re- York.
port: Furfural. SRI, Menlo Park, CA. 56 Seaworthy Systems Inc. (1997), SSI Re-
52 Rieche, A. (1964), Outline of Industrial port #403-01-01.
Organic Chemistry. Butterworths, London.
165
8
A Whole Crop Biorefinery System:
from Cereals
8.1
Introduction
Selection of the appropriate renewable raw material to supply sustainable pro-

cesses is dependent on infrastructural, economical and technological factors
(e.g. availability, skilled workforce, pretreatment technology and costs, transpor-
tation). Cereals meet most of these prerequisites and have the potential to be
used for the production of not only traditional foods but also novel functional
foods and non-food products (e.g. biodegradable plastics, chemicals, fuels).
However, cereal-based processes are currently more expensive than petroleum-
based ones. The reduction of processing costs is strongly dependent on restruc-
turing, integrating and optimizing current processes. To achieve this, the intro-
duction of contemporary low-cost unit operations, the reduction of utilities costs
and capital investment, and the creation of added-value byproducts in line with
core end-products is imperative. The starting point would be to evaluate current
cereal fractionation processes as the basis for a biorefinery and to identify focus
areas for optimization, operating/capital cost reduction and end-product/byprod-
uct production.
The concept of the whole crop biorefinery is inextricably linked to cereals as
one of the most energy intense and chemically rich groups of agricultural crops.
Cereals are also amongst the most developed crops having been progressively
’improved’ throughout the past 10 000 years. They have been continuously opti-
mized in terms of yield, with many now exceeding 10 tonnes per hectare. How-
ever, it is still the case that for every tonne of readily processable, starch-rich cer-
ISBN: 3-527-31027-4
166 8 A Whole Crop Biorefinery System
eal grain there is approximately another tonne of rather less accessible lignocel-
lulosic material such as straw and other residues. How to deal with this
material is one of the prime challenges of the whole crop biorefinery. Not only
is there less to be had from the straw component of the crop than from the
grain but it is also more difficult to get at; requiring tedious and often costly
preprocessing. In addition, there is a major impediment to transportation of
this fraction of the crop because it has a bulk density of only about a fifteenth
of that of the grain, thereby requiring vastly more voluminous vehicles to move
it around.
Ideally, of course, the whole crop biorefinery would involve harvesting the
whole of the crop and transporting it directly to the refinery where fractionation
would begin. Unfortunately, this is unlikely to be the reality and the more prag-
matic approach of separation in the field followed by utilization of the straw as
a primary energy source, through combustion, or incorporation into building
composites, is more likely to prevail for some time to come. It must also be
conceded that traditional outlets for straw such as re-incorporation into the soil,
animal feed, and animal bedding, will continue to be “first call” uses in the fore-
seeable future [1]. Thus one of the principal conclusions of a European study to
determine the profitability of a wholecrop biorefinery [2] was that “Combine har-
vesting with grain processing in a biorefinery is more profitable than wholecrop.” In
view of these observations, this chapter will consider the biorefinery concept as
one in which on-site separation of grain is carried out prior to fractionation and
conversion of the whole grains at a remote site. Should it become feasible to
process straw and other residues alongside the grain, such processing could
readily be integrated within the biorefinery.
Current cereal fractionation processes (CFP) break down the grain into macro
and micro components that are used either as end-products (e.g. gluten, oil) or
as raw materials (e.g. starch) for secondary processing in many industries (e.g.
food, pharmaceuticals, textiles, cosmetics, fermentation). The term macro com-
ponent incorporates any high molecular weight compound (e.g. starch, protein,
cellulose, hemicellulose, oil, gums), while micro components are defined as rel-
atively low molecular weight molecules (e.g. lipids, vitamins, minerals). Tradi-
tional CFP can be categorized into dry and wet milling operations. Dry milling
involves the use of successive grinding and sieving steps aiming at the maxi-
mum economic separation of bran from endosperm. Dry milling operations are
relatively inexpensive and result in incomplete macro component separation.
Wet CFP can be generally categorized into wet–aqueous and wet–nonaqueous
processes resulting in selective separation of one or more cereal components.
Wet fractionation processes could be applied to the end-products of a primary
dry milling operation.
Traditional CFP have been developed to suit the needs of the food industry
and do not exploit the potential of cereal grains for non-food applications. The
development of viable whole-crop biorefineries depends on the constructive inte-
gration of physical, chemical, thermal and biological processing resulting in var-
ious products, such as functional proteins, oils, antioxidants, polysaccharides,
fine and bulk chemicals, biofuels and biodegradable plastics. Novel cereal frac-
tionation plants could be of three kinds depending on their production capacity:
· Small-scale plants that will not focus directly on the market outlets of the tra-
ditional cereal processing plants but will target specialty industries (e.g. cos-
metics, pharmaceuticals) by extracting value-added minor constituents (e.g.
antioxidants). Such plants will utilize only one cereal grain and will leave the
majority of the raw material unprocessed, creating the need to find market
outlets for this bulk quantity of material. The use of the remaining material
for the production of fine or platform chemicals through microbial bioconver-
sion would be an attractive option.
· Intermediate-scale plants may have wider flexibility in terms of the cereal
grains that they can process. They will be able to market more end-products.
Their commercial survival though will be dependent on continuous research
and development and process improvements.
· Large-scale plants would be able to utilize any cereal grain for the simulta-
neous commercialization of traditional end-products, value-added minor com-
ponents as well as biofuels and biodegradable plastics. Extensive technical
and market research should certify high efficiency, cost-competitiveness and
customer demand for all the end-products.
In this chapter, potential whole-crop biorefineries based on wheat and oats are
presented. Future biorefineries based on cereals should aim to exploit the vast
complexity of cereal grains by extracting valuable macro and micro components
and converting the starch fraction into platform chemicals, biodegradable plas-
tics and biofuels via microbial bioconversions. This approach targets waste and
cost reductions and the creation of more market outlets.
8.2
Biorefineries Based on Wheat
8.2.1
Wheat Structure and Composition
The structure of the wheat grain consists of several layers with varying composi-
tion and functions (Fig. 8.1 and Table 8.1). The two main parts of the kernel are
the pericarp and the seed. The pericarp covers the entire wheat kernel and is di-
vided into the outer and the inner pericarp. Beneath the inner pericarp, the
seed coat and the pigment strand provide a complete covering around the seed.
The nucellar epidermis and the nucellar projection are located underneath the
seed coat and surround the endosperm and embryo. One of the most nutrition-
ally important wheat layers, the aleurone layer, is situated below the nucellar tis-
sues. This is a one-cell-thick layer that encompasses the entire endosperm and
part of the embryo. The embryo lies on the lower dorsal side of the wheat ker-
nel and its two major components are the embryonic axis and the scutellum.
Fig. 8.1 Morphology of the wheat kernel [3].
Table 8.1 Wheat nutrients and their location in the kernel.
Constituent Function Mass a) Composition
Pericarp
) 5
Seed coat, pigment Protect the grain 3 Fiber, K, P, Mg, Ca
strand and nucellus Bran
Aleurone layer Encases endosperm 7 Niacin, phytic acid, minerals (especially P)
Endosperm Stores food 82 Starch, protein, pantothenic acid, B2, minerals
Embryo Root and shoot 1 Fats, lipids, sugars

Scutellum Stores food 2 P, B vitamins (especially thiamine)
a) Mass fraction of the constituent within the kernel as % on a dry basis (db)
The scutellum is a storage organ that is considered a cotyledon. The embryo,

known as germ to a miller, when separated by traditional milling processes,
consists principally of the embryonic axis.
A detailed analysis of the chemical composition of wheat grain is given by Po-
meranz [4] and MacMasters et al. [5]. Each wheat layer has a unique composi-
tion in certain micro and/or macro components. In addition, wheat chemical
composition and distribution in grain varies between varieties. The average
chemical composition of pericarp is crude fiber (20–21%), cellulose (23.5–24%),
and arabinoxylan (25–28%). Small amounts of protein (2.5–4%) and fat (< 1%)
are also present. In comparison with the pericarp, the seed coat contains much
more protein (10–17%), less arabinoxylan (12–13.5%), much less crude fiber
(ca. 1.0%), and no cellulose. The aleurone layer contains high levels of total
phosphorus (2.7%), phytate phosphorus (2.4%), and niacin (B3) (530–640 lg g–
1
). The niacin in the aleurone layer represents about 80% of the total amount in
the entire grain. Pyridoxine (B2) occurs in a pattern similar to that of niacin,
with over 60% of all pyridoxine in the aleurone layer (30 lg g–1). The aleurone
layer is also an important contributor of pantothenic acid (B5), containing more
than 40% of the total in the wheat grain (40 lg g–1) [6]. Total free sugar ac-
counts for 10% of the aleurone materials, including sucrose 42%, raffinose
31%, neokestose 20%, and fructosyl raffinose 6% [7]. Monosaccharides, disac-
charides (maltose) and higher oligosaccharides which occur in other parts of
the grain are absent from the aleurone cells. The content of total lipids in aleur-
one cells accounts for between 8 and 11% [8], of which 70–80% are nonpolar.
Endosperm contains mainly starch (55–65%) and protein (7–11%). Peripheral
cells have the lowest starch content and the highest protein content. Values as
high as 54% protein have been found in subaleurone cells [9]. The main constit-
uents of endosperm cell walls include polysaccharides (75%) and protein (15%).
Arabinoxylan contributes 85% of the total polysaccharide content in endosperm
cell walls and b-glucan and b-glucomannan account for the remainder in equal
amounts [10]. Non-starch lipids and starch lipids contribute nearly evenly to the
1.5–2.5% total lipids in wheat endosperm. Wheat germ is rich in lipids (25–
30%), protein (21–23%), phosphorus (3%) and B vitamins including B1, B2, B3,
B5, B6. Sucrose (10%) and raffinose (7%) are the two sugars abundant in wheat
germ and no substantial amount of the other oligosaccharides has been located.
8.2.2
Secondary Processing of Wheat Flour Milling Byproducts
In the traditional wheat flour milling process, wheat is milled into various flour
fractions involving a large number of milling and sifting operations developed
originally to serve the needs of the food industry. In particular, wheat grains are
initially cleaned from impurities and tempered with water for an average of 12
hours to detach the outer bran layers from the endosperm, facilitating their sep-
aration. The tempered grains are subsequently processed through a series of
break and reduction roller mills and sifting stages. The main aim of the conven-
tional dry milling process is to produce flour fractions with the lowest possible
bran impurities. However, the nature of this process will never produce any
flour fraction 100% free from germ and bran. Current wheat flour mills operate
at 70–80% grain to flour conversion efficiency where the remaining 20–30%
constitutes various byproduct streams that contain predominantly bran as well
as lower amounts of germ and flour. The byproduct streams are mixtures of all
coarse streams from each break and reduction roll and are usually used as ani-
mal feeds or commercial bran.
As a whole, the dry milling process of wheat produces many flour streams of
varying particle sizes and compositions in endosperm, bran and germ. This
means that the separation of certain wheat layers enriched in specific value-
added chemical components is not possible. This way of processing results in
high capital investment, high operating costs for milling, conveying and sifting,
and products of low purity. Thus, traditional dry milling of wheat cannot be
considered as the basis for the development of a viable biorefinery for non-food
applications.
The most realistic option to use traditional mills for non-food applications is
through bioconversion of the significant amounts of byproduct produced. In
1999/2000, 5624 ´ 103 tonnes of wheat (83% of which was home grown) was
processed by UK flour millers resulting in 4481 ´ 103 tonnes of flour and
1148 ´ 103 tonnes of byproducts. The majority of this byproduct stream, known
as wheatfeed or middlings, contains 15–19% protein (75% of which is degrad-
able), 20–35% starch and around 1% phosphorus. However, the high crude fiber
content (7–11%), low essential amino acid content (0.6% Lys, 0.5% Thr, 0.2%
Try, 0.4% Met) and the presence of phytic acid (containing about 60–80% of the
total phosphorus) reduce its nutritional value. Consequently, its use as animal
feed is restricted only to pigs and cattle. Phytic acid, in particular, has been
identified as a certain antinutritional compound that forms complexes with iron
and zinc ions and makes these metal ions less accessible for assimilation by hu-
mans and other monogastric animals [11]. Nonruminants excrete most of it as
they do not have an efficient system for making phosphate available from phy-
tate. This forces producers to add inorganic phosphate to animal feed as a sup-
plement, leading to excessive phosphate excretion, which worsens water pollu-
tion and eutrophication [12]. The addition of enzyme preparations such as phy-
tase, xylanase and protease could increase the overall digestibility of flour
milling byproducts, allowing animals to consume phosphorus from the phytic
acid and better assimilate the iron and zinc ions. For this reason, the animal
feed industry is currently developing large-scale enzyme applications [12].
The development of an integrated biorefinery that leads to the production of
high quality wheat flour and upgrades the byproduct stream into a nutrient-en-
riched animal feed and value-added chemicals through microbial bioconversions
is an attractive option. Figure 8.2 shows such a process based around the bio-
production of succinic acid as a major potential platform chemical. The byprod-
ucts from a traditional wheat flour mill could be treated with successive wash-
ing and screening stages to separate the majority of starch and some of the pro-
Fig. 8.2 Schematic diagram of a possible biorefinery utilizing traditional wheat flour milling byproducts.
8.2 Biorefineries Based on Wheat
171
tein, as well as other soluble nutrients. The starch-rich suspension would be

used for production of the bioconversion feedstock, while bran will be used in
fungal solid state bioprocessing (SSB) for enzyme production. The crude enzy-
matic mixture produced by SSB would be used to hydrolyze wheat macromole-
cules (e.g. starch, protein) contained in the starch-rich suspension. In-situ pro-
duction of enzymes through SSB would reduce operating costs by eliminating
the purchase of unnecessarily purified commercial enzymatic solutions. SSB
would also provide a potential use for residual solids as a value-added animal
feed, enhanced by the presence of enzymes that increase digestibility and fungal
cells that contain high levels of essential amino acids (3.3% Lys, 2.82% Thr,
1.18% Try, 1.46% Met) [13, 14]. The solids could also be used for the production
of nutrient supplements similar to yeast extracts by exploiting the natural degra-
dation of fungal hyphae via the excretion of autolytic enzymes under oxygen
limiting conditions [15]. Optimization of bran utilization for enzyme production
could lead to the exploitation of the excess bran as raw material for gasification.
The synthesis gas produced in this way could be used either for energy produc-
tion to minimize the non-renewable energy requirements for the whole process
or as crude feedstock for the production of value-added chemicals via microbial
bioconversion or chemical synthesis.
The generic nature of the microbial feedstock produced from wheat flour
milling byproducts can be used in various microbial bioconversions for the pro-
duction of value-added chemicals. This specific biorefinery should target the
production of low-to-medium volume and medium-to-high value chemicals be-
cause of the relatively small amount of raw materials (milling byproducts con-
tain 20–35% starch) available for microbial bioconversion. It should be taken
into consideration that the main activity of this biorefinery is the production of
wheat flour, while only an average of 25% of the initial grain will be used for
non-food applications. In the case of high-volume, low-value chemicals, fuels
and plastics, economies of scale apply and small plants are not profitable. How-
ever, an economic analysis is necessary as the revenue derived from the primary
market outlet may lead to the creation of an overall viable biorefinery even for
commodity materials.
For this biorefinery, a potential market opportunity could be the production of
succinic acid, which has the potential to be a future intermediate molecule for
the production of a wide variety of commodities and specialties [16]. Succinic
acid is currently produced petrochemically; maleic anhydride produced via bu-
tane oxidation is hydrated to maleic acid and then hydrogenated to succinic
acid. Global production via this route exceeds 15,000 tonnes per annum. At a
selling price between $ 6–8.8 kg–1, succinic acid is a valuable commodity, but is
too expensive to be considered a feasible bulk chemical [16]. A preliminary ma-
terial balance based on reaction stoichiometries (eqs 1–3) demonstrates the po-
tential commercial and environmental impact of using wheat flour milling by-
products as the raw material for succinic acid production:
C6 H10 O5 n nH2 O ! nC6 H12 O6 1
C6 H12 O6 2CO2 4H ! 2C4 H6 O4 2H2 O 2
C6 H12 O6 ! 2C2 H5 OH 2CO2 3
The total amount of wheat flour milling byproducts (1.1 ´ 106 tonnes) available
per annum in the UK contains on average 30% starch, i.e. equivalent to
3.66 ´ 105 tonnes of glucose (eq. 1). The theoretical yield from glucose to succin-
ic acid is 1.31 kg acid kg–1 glucose (eq. 2), giving a theoretical maximum of
4.8 ´ 105 tonnes succinic acid per annum for the UK. This is far greater than
the current world production of 1.5 ´ 104 tonnes. At the same time, succinic acid
production would result in 1.8 ´ 105 tonnes per annum CO2 sequestration (eq. 2)
because mass production of the acid requires CO2 fixation in the metabolism of
the microorganism involved. This is equivalent to the CO2 released from the
production of 2.4 ´ 105 tonnes of bioethanol (eq. 3). In addition, current biopro-
cessing practices could reduce the succinic acid production cost to lower than
$ 0.6 kg–1 [17]. Thus, by utilizing a low-cost agro-industrial raw material succinic
acid could be transformed into a low-cost platform intermediate. Such alterna-
tive use of wheat flour milling byproducts would also give flexibility to farmers
and industrialists in exploring new market opportunities.
8.2.3
Advanced Wheat Separation Processes for Food and Non-food Applications
Traditional wheat processing for food (excluding breadmaking) and non-food ap-
plications concentrates mainly on the extraction of the bulk macromolecules,
starch and gluten. However, the extraction of a number of value-added compo-
nents from individual wheat layers could upgrade wheat processing into a novel
whole-crop biorefinery producing both low-volume, high-value and high-volume,
low-value products. In recent years, there has been a growing interest in the uti-
lization of advanced cereal fractionation technologies to reduce operating/capital
costs and to separate/purify value-added macro and micro components from
cereal grains. The desired functional component may be concentrated in a par-
ticular layer in the cereal grain (e.g. aleurone layer, pericarp, germ). For this rea-
son, novel CFP involve the selective separation of the outer bran layers includ-
ing the aleurone layer and the germ.
8.2.3.1 Pearling as an Advanced Cereal Fractionation Technology

In the 1990s, a new technology was commercialized for wheat dry milling,
which involves gradual removal of the outer layers (e.g. pericarp, aleurone cells)
of the wheat kernel as a means to increase wheat milling efficiency [18]. In this
process, the wheat bran layers are removed sequentially by friction and abrasion
operations, while the byproducts of pearling hold great promise as novel food
ingredients with physicochemical and nutritional properties that differ from

those of previously available cereal products [18, 19]. There are a number of
pearling devices for sequential removal of cereal outer layers [20–22]. Wheat
pearling can be used as a preprocessing stage before conventional dry milling
or non-food applications.
The Tkac and the PeriTec model Satake VBW5A pearling systems have been
presented in a previous publication [18]. The fourth generation Satake pearling
system VCW5A is presented in Fig. 8.3 [23]. Wheat is fed to the top of the pear-
ling equipment into the abrasion chamber where the wheat kernels are evenly
distributed by a rotating scroll. The abrasion chamber contains abrasive wheels,
a slotted screen and four vertically adjusted resistance bars. The wheat kernels
are rubbed between the abrasive wheels and the resistance bars resulting in the
removal of the outer kernel layers. The bran is collected through the slotted
screen by an air stream that is blown through the holes of a hollow central
shaft. The degree of debranning in the abrasion chamber is set by a weighted
flap on the outlet of the chamber. The partly pearled wheat kernels are trans-
ferred through a screw conveyor into the friction chamber. This section contains
a cast steel cylinder with two vertical agitator bars on it, which rotates around a
slotted screen. The wheat kernels are further debranned by rubbing against the
slotted screen. The friction stage is efficient only when the tough and oily testa
Fig. 8.3 The Satake VCW5A debranning apparatus.

layer has been disrupted during the abrasion stage. The bran separated during
the friction stage is removed through the screen by an air stream blown into
the center of the chamber via a perforated main shaft.
Research and commercial implementation of pearling has revealed the nu-
merous potential uses of this advanced CFP. When pearling is used prior to
conventional milling, it can improve flour quality and reduce operating costs
[24]. The application of pearling can lead to the production of bran-rich streams
that can be used as food ingredients. Cui et al. [19] reported that non-starch
polysaccharides (NSP) from a wheat bran fraction produced by the Tkac and
Timm pearling technology exhibited novel rheological properties. The NSP ex-
tracted from a pearling fraction that contained 13.4% starch, 29.2% insoluble
dietary fiber, 8.5% soluble dietary fiber and 2.6% b-glucan, exhibited shear-thin-
ning flow behavior at low concentrations in water (0.5%, 25 8C) and formed a
thermally reversible gel upon cooling at 4 8C [19].
The bran-rich fractions produced by pearling are also rich in value-added
components (e.g. antioxidants, b-glucan) in comparison to the bran-rich frac-
tions produced from conventional milling operations. This occurs because the
latter contain higher quantities of starchy endosperm, diluting the overall
amounts of added-value components [25]. In the case of wheat, Dexter and
Wood [18] used the Tkac debranning system, which produced friction fractions
rich in pericarp and enriched in dietary fiber, while the abrasion fractions were
rich in aleurone cells and enriched in protein, b-glucan and soluble fiber in
comparison to whole wheat. Marconi et al. [26] used barley pearlings enriched
in b-glucan and dietary fiber for the production of functional pastas with the
aim of meeting the Food and Drug Administration (FDA) requirements of 5 g
of dietary fiber and 0.75 g of b-glucan per serving (56 g in the US and 80 g in
Italy). It has been reported that pearling fractions from oats may contain signifi-
cant quantities of specific antioxidants [27]. It has also been reported that by-
products that are produced from the pearling stage prior to dry milling of barley
are enriched in b-glucans, tocopherols, and tocotrienols [28]. Cereal constituents
with antioxidant properties can be used in pharmaceutical and cosmetic applica-
tions.
Using pearling before wheat milling could also lead to increased gluten ex-
traction yield from pearled wheat grains [29]. The gluten extraction yield from
ground, pearled wheat (11.4% of bran and germ was removed during pearling)
was 20–25% higher than from conventionally milled flour in a process achiev-
ing 70% extraction of flour from wheat.
Apart from milling operations that produce flour streams for food, pharma-
ceutical and cosmetic purposes, the usefulness of pearling has also been recog-
nized in the case of non-food applications. Wang et al. [30] used whole grain
flour and pearled grain flour as fermentation medium for bioethanol production
and the latter increased ethanol yields by 6.5–22.5%. This occurred because the
use of pearling reduced the bran content in the fermentation feedstock, while at
the same time the starch throughput per batch increased by an average of
12% (w/v, db). By integrating the grain pearling technology in rye and triticale
with very high gravity fermentations (0.30 g solids mL–1 supernatant) the etha-
nol concentration was increased to around 16% (v/v) [31]. Partial removal of out-
er grain solids in an alcohol plant would also improve plant efficiency and de-
crease energy requirements for mash heating, mash cooling and ethanol distil-
lation. In addition, the bran byproduct fractions containing pure grain layers or
mixtures of them may be used as functional food ingredients or as raw materi-
als to extract value-added products (e.g. enzymes, phytic acid, tocopherols, oil,
proteins, wheat germ glycerides, agglutinin, arabinoxylans, b-glucan), contribut-
ing significant revenue for the development of cost-competitive biorefineries.
8.2.3.2 Air Classification

Air classifiers are rotary machines that are used in dry milling operations to
separate a particulate feed into a fine and coarse fraction, using mainly air to
entrain the fine product and a rotor to reject any airborne coarser particles [32].
The application of air classification in dry CFP can lead to the enrichment of
milling fractions with specific components. The main advantage of air classifica-
tion is that the procedure can easily be scaled up using a commercially available
large air classifier without the clogging of screens by fine particles in a sieving
method [33].
Letang et al. [34] reported that the combination of jet milling with air classifi-
cation of soft wheat could produce a starch-enriched stream with only 2% pro-
tein content and reduced lipid and pentosan contents. Wu and Doehlert [33] en-
hanced the b-glucan content (200 g kg–1) in oat bran fractions by applying a
combination of pin milling, air classification and sieving. Similar results were
obtained when air classification was applied to enrich the b-glucan content in
milled fractions of barley [35] and oat groats [36].
8.2.4
Biorefinery Based on Novel Dry Fractionation Processes of Wheat
A potential wheat-based biorefinery (Fig. 8.4) could utilize a pearling system to

produce a bran-rich fraction containing pericarp, seed coat, aleurone cells and
germ layers from the wheat kernel. This fraction could be significantly enriched
in bran by separating flour particles for further processing by the application of
air classification. The bran-enriched fraction could be then used for the produc-
tion of various coproducts, including natural polymers (e.g. arabinoxylans, b-glu-
cans), monomers (e.g. glucose, xylose, arabinose, ferulic acid) or oil components
(e.g. triglycerides, sterols). The selection of coproducts that will be eventually de-
rived from the bran-rich fraction would be dependent on process economics,
available technologies and existence of markets for their disposal.
Pearled wheat grains could be ground in a hammer mill and the resulting
bran-free flour could be used for gluten extraction by the Martin, the Batter or a
modified version of these processing schemes. The bran-free and gluten-free
flour suspension resulting from the gluten extraction stage could be used for
8.2 Biorefineries Based on Wheat
Fig. 8.4 Schematic diagram of a wheat-based biorefinery employing pearling and air classification.
177
the production of a glucose-rich solution to provide the carbon and energy

source in subsequent microbial bioconversions. The enzymes required to hydro-
lyze this stream could be produced by submerged or solid state fungal biocon-
versions on whole wheat flour and pearlings, respectively. Remaining fermenta-
tion solids could be used as animal feed, glucosamine/N-acetyl-d-glucosamine
extraction or for the production of nutrient supplements by exploiting fungal
autolysis that occurs under oxygen-limiting conditions. Unhydrolyzed solids
could be gasified for the production of energy or other value-added products.
The generic feedstock formulated by mixing the glucose-rich solution with nu-
trient supplements produced by fungal autolysis can be used in microbial bio-
conversions for the production of biofuels, platform chemicals and biodegrad-
able plastics.
8.2.4.1 Potential Value-added Byproducts from Wheat Bran-rich Fractions
Arabinoxylans Wheat bran and especially the aleurone layer contain high
amounts of arabinoxylans and lower quantities of b-glucans. Arabinoxylan is a
polysaccharide comprised of a backbone formed by xylose residues connected
together with b-(1 ? 4) bonds. The xylose residues are highly substituted with
primarily single a-(1 ? 3) and/or a-(1 ? 2)-linked l-arabinose residues as well as
short arabinooligosaccharides, d-galactose, d-4-O-methylglucuronic acid and
ferulic acid residues.
Phenolic acids, such as ferulic acid, play a significant role in the linkage of
hemicelluloses with other cell wall components, especially lignin, through ester
and ether bonds [37]. In addition, ferulic acid assists in the formation of cova-
lently crosslinked arabinoxylans with well developed networks that exert an in-
teresting water-holding capacity [38]. Different concentrations of ferulic acids
would correspond to varying gelling potential of arabinoxylans. Arabinoxylan
properties in solution are also dependent on the degree of polymerization of the
xylan backbone.
The use of enzymatic oxidizing systems such as laccase, horseradish peroxi-
dase or manganese peroxidase could dimerize the esterified ferulic acids caus-
ing gelation of the water-extractable arabinoxylans from wheat flour [39]. The
high viscosity of aqueous solutions containing water-extractable arabinoxylans
that have undergone oxidative gelation after they were treated with laccase and/
or manganese peroxidase has a positive effect in breadmaking [40]. In particular,
water-soluble arabinoxylans can absorb water, influence dough rheology and af-
fect such bread characteristics as loaf volume, crumb firmness and staling
events [41]. Arabinoxylans could also be potential ingredients for wound-care ap-
plications [42]. Sterigel is an arabinoxylan-based product that is used as wound
management aid.
Ferulic Acid Ferulic acid could be released from bran-rich fractions by the sy-
nergistic action of various enzymes, such as esterase and xylanase [43]. Extrac-
tion of arabinoxylans located at the outer bran layers, seed coat and pericarp,
may be more difficult than the ones located in the aleurone and nucellar layers
[44]. The aleurone-free wheat bran is more resistant to enzymatic hydrolysis be-
cause it contains higher amounts of lignin, cellulose and glucuronoarabinoxy-
lans than the aleurone layer. Increased crosslinking among cell wall compo-
nents creates a rigid cell wall network and decreases the effectiveness of enzy-
matic hydrolysis.
Ferulic acid could be used as a natural food preservative due to its ability to
inhibit peroxidation of fatty acids. The acid and its derivatives, steryl ferulates,
have antioxidant properties. A commercial product, c-oryzanol, with cholesterol
lowering properties containing steryl ferulates has been extracted from rice bran
[45]. Wheat bran fractions may contain up to 0.34 mg g–1 total steryl ferulates,
which is significantly higher that the 0.063 mg g–1 content in the whole wheat
grain [45]. Ferulic acid is currently used as an ingredient in many skin lotions,
sunscreens and wound dressings [43]. The antioxidant potential of ferulic acid
could be enhanced by converting it enzymatically into cafeic acid by using a mi-
crobial cell extract from the anaerobic bacterium Clostridium methoxybenzovorans
SR3 [46]. Another commercial option is the bioconversion of ferulic acid into va-
nillin by Streptomyces setonii. Muheim and Lerch [47] reported that shake flask
cultures of S. setonii resulted in the production of 6 g L–1 vanillin at a molar
conversion yield from ferulic acid to vanillin of 68%.
Hwang et al. [48] patented a process separating ferulic acid and arabinoxylan
from cereal brans by extrusion and subsequent treatment with plant cell wall
hydrolyzing enzymes.
Wheat Germ Most wheat germ currently produced by flour mills is used as an-
imal feed contributing a very low revenue. Wheat germ could be upgraded into
a useful byproduct if it is used for the extraction of various value-added materi-
als, such as proteins, oils, vitamins and enzymes [24]. Oil extraction could be
achieved simply by mechanical pressure or solvent extraction. The germ oil
could be used as a specialty food or gourmet cooking oil. A commercial applica-
tion of oil extracts has been introduced by the French company Bertin, which
extracts these oils by cold pressing at 4,000 bars in sunflower oil as solvent [49].
The purified wheat germ oil (104 kg year–1) is called heliogerme and is sold as
an ingredient in cosmetics at a price ranging between £ 5–8 kg–1. In the case
that wheat germ is processed by solvent extraction, the functional properties of
germ protein are retained enabling its commercialization as food grade protein.
The extraction of micro components (e.g. enzymes, vitamins, flavonoids) from
wheat germ may open additional market opportunities but additional research
and market/process evaluation is required [24].
The bran-rich fraction produced by wheat pearling would contain a large
amount, if not all, of the wheat germ. Thus, the utilization of the pearling tech-
nology in a wheat biorefinery may decrease the cost currently required by tradi-
tional flour mills to isolate the wheat germ fraction.
Monosaccharide Production Another potential market outlet would be the frac-

tionation of pearlings into oil components and polysaccharides, which can be
subsequently converted enzymatically into a spectrum of monomers, the most
important of which would be glucose, xylose, arabinose and ferulic acid. The
monosaccharides could be either utilized as a carbon source in microbial bio-
processes or converted catalytically into various chemicals currently derived
from petrochemicals. For instance, glucose could be hydrogenated to sorbitol,
which in turn could be catalytically converted to propylene glycol. Xylene and
arabinose could also be catalytically converted to ethylene glycol, propylene gly-
col and xylitol.
Functional Foods Supplementation of human diets with wheat bran may en-
hance the prevention of a range of cancers due to the presence of various func-
tional components, such as dietary fiber, phytic acid, lignans, oligosaccharides,
antioxidants, phytoestrogens [50]. Reddy et al. [51] reported that the lipid frac-
tion of wheat bran has strong colon tumor inhibitory properties and further
studies are required to identify biologically active constituents of wheat bran lip-
id fractions and their relative role in colon tumor inhibition. The insoluble diet-
ary fiber content of wheat bran may be responsible for their significant bile acid
binding properties that cause decrease in the plasma cholesterol level in hu-
mans [52]. In turn, Cui et al. [19] reported that bran-rich fractions enriched in
soluble fiber could be superior to AACC standard wheat bran, which contains
46.85% insoluble fiber and 2.8% soluble fiber [19]. Pearling may be employed to
produce bran-rich fractions enriched in either soluble or insoluble dietary fiber
depending on the requirements of end-product composition.
Pearling could be exploited for the sequential removal of individual wheat
layers. Fenech et al. [53] reported that pearling could lead to the production of
an aleurone-rich fraction that contains high amounts (5 lg g–1) of folate. Bran-
and germ-rich fractions produced by pearling could potentially be used to en-
rich the mineral content of pan breads [54]. In addition, the quality of wheat
bread, in terms of loaf volume, crumb structure, shelf life, starch structure and
flavor, can be significantly improved when it is supplemented with wheat bran
pre-fermented with yeast and/or lactic acid bacteria [55].
8.2.4.2 Exploitation of the Pearled Wheat Kernel
Vital Wheat Gluten Pearled wheat kernels could be used for the extraction of vi-
tal wheat gluten as a valuable co-product. Gluten can be separated from wheat
flour by mixing endosperm particles with water that initiates the formation of
protein microfibrils. The formation of dough from the mixture of flour and
water is dependent on the water to flour ratio used and mixing. The main pro-
cesses for the separation of vital wheat gluten from flour (e.g. Posner, Alfa-La-
val/Raisio, Hydrocyclone, Pillsbury Hydromilling, Far-Mar-Co., High-Pressure
Disintegration) could be categorized into those based on either the Martin or
the Batter process [56, 57]. Such processes utilize a spectrum of raw materials
(e.g. wheat, flour), solvents, equipments and wheat flour to water ratio. Weight
for weight, gluten is more valuable than starch and could therefore improve the
economics of biorefineries based on wheat. In general, 10 tonnes of wheat will
give 6–7 tonnes of starch and 1–1.5 tonnes of gluten. The major impediments
in gluten separation processes are the operational effectiveness of processing,
the high cost required for drying vital gluten and waste treatment costs.
Gluten has many applications particularly in the food industry, such as in bread-
making, specialty baked goods, pet foods, breakfast cereals, meat and cheese sub-
stitutes and pizza [57]. Other industrial uses include production of hydrolysates,
wheat protein isolate and deaminated gluten. The gradual decrease of gluten price
in the last 10 years has shown that a potential large-scale utilization of wheat for
non-food applications will saturate the current gluten market reducing its current
price even lower. Identifying novel uses for gluten could provide more commercial
outlets and higher revenue for this value-added co-product. Gluten could be used
for the production of biodegradable or edible films and packaging materials [58]. It
could also be used for the manufacture of biodegradable high-performance engi-
neering plastics and composites when thiol-terminated, star-branched molecules
are incorporated directly into the protein structure [59].
Microbial Feedstock Production and Bioconversion Processes After gluten extrac-

tion, the remaining aqueous suspension is rich in starch. Starch has many ap-
plications in numerous industries such as paper sizing/coating, thickening/gel-
ling agent in food applications, industrial glues and pastes, dusting agent, slip-
ping agent in oil drilling, water retention agent, biodegradable plastics, building
panels, cosmetic applications, encapsulating polymers, packing material, edible
packaging and feedstock for fermentation processes. This study will concentrate
on the utilization of starch as a raw material for the production of fuels, chemi-
cals and plastics by microbial bioconversion. To produce a nutrient-complete
medium for microbial fermentations, three steps are required (Fig. 8.4):
1. on-site enzyme production via submerged or solid state fermentations;
2. enzymatic hydrolysis of gluten-free and bran-free flour suspensions; and
3. production of nutrient supplements (fungal extract) via fungal autolysis.
In many existing microbial fermentations, commercial (purified) starch is enzy-

matically hydrolyzed into directly assimilable glucose by unnecessarily pure
commercial enzyme formulations. Cost reduction could be achieved by utilizing
a much less refined process involving on-site production of crude enzyme mix-
tures via fungal fermentations conducted by single or combined fungal strains
belonging to the genus Aspergilli (A. awamori, A. oryzae). The medium for the
fungal fermentations could be whole cereal flour or pearlings depending on the
bioprocess employed, which might be carried out in submerged or solid state,
respectively. The solid residue from the enzyme producing fermentation, which
contains mainly fungal cells and any undigested wheat particles, could be con-
verted into nutrient supplements via fungal cell autolysis. A similar process that
generates a generic microbial feedstock from whole wheat is described and cost
evaluated in previous publications [60–62].
The generic microbial feedstock can be used for the bioproduction of several
organic chemicals, including platform intermediates for chemical synthesis, in-
gredients for a spectrum of commercial products, biofuels, biodegradable plas-
tics and solvents. This process could lead to the production of both commodities
and specialties depending on market outlets and cost competitiveness. Potential
bioconversion products, the large-scale bioproduction of which have or will in-
cur low environmental and high societal and industrial impacts are [16]:
1. lactic acid, succinic acid and 3-hydroxypropionic acid as platform molecules
and industrial ingredients;
2. 1,3-propanediol as a monomer for the production of novel plastics;
3. butanol as platform molecule, solvent, industrial ingredient and biobased
transport fuel;
4. PHA as biodegradable plastics;
5. bioethanol as a platform intermediate and biofuel;
6. l-lysine as animal feed additive and industrial ingredient.
Remaining solids from fungal fermentation could also be used as animal feed or
for the extraction of value-added coproducts, such as glucosamine and N-acetyl-d-
glucosamine. The fungal cell walls contain chitin, which is an unbranched homo-
polymer of N-acetyl-d-glucosamine. Bohlmann et al. [63] used enzymatic and
chemical methods for the recovery of N-acetyl-d-glucosamine from fungal bio-
mass (A. niger) that was produced from a citric acid fermentation. The efficient
degradation of chitin and glucan required the synergistic action of chitinases, b-
N-acetylglucosaminidases and glucanases. Fan et al. [64] have also used various
fungal species (Aspergillus, Penicillium, Mucor) for the production of glucosamine.
Fungal autolysis might evolve into an effective and economic processing route for
releasing glucosamine or/and N-acetyl-d-glucosamine from fungal cell walls.
It is strongly believed that glucosamine provides joint health benefits and
pain relief. The US National Institute of Health is currently conducting research
in order to assess the effectiveness of glucosamine on patients with osteoarthri-
tis. The US retail market for nutritional supplements containing glucosamine is
more that $ 109/year [65]. Demand for bulk glucosamine has been growing in
excess of 20% annually and global consumption exceeds 5 ´ 106 kg [65]. Apart
from glucosamine, N-acetylglucosamine is used as health supplement as well as
in cosmetic and pharmaceutical applications.
Unhydrolyzed solids from the fungal autolysis stage and solids from the mi-
crobial bioconversion could be gasified at various combinations of high tem-
peratures, pressures and oxygen for the production of synthesis gas that is con-
stituted of various compositions of CO, CO2, H2 and CH4 depending on the
conditions applied. Synthesis gas can be subsequently used either for energy
generation or biological/chemical conversion into a spectrum of chemicals. Po-
tential bioderived products from synthesis gas are bioethanol [66], biodegradable
plastics [67], acetic acid and methanol [16].
8.3
A Biorefinery Based on Oats
8.3.1
Oat Structure and Composition
Like all cereal grains, oats have a complex structure and constitution (Table 8.2).
The oat groat, consisting of endosperm, germ and bran, is encompassed in a fi-
brous hull structure that contains approximately 50% hemicellulose, 40% crude
cellulose and 10% crude lignin. The main sections of oat groats are the tri-
chomes (hairs), pericarp, nucellar tissues, aleurone, endosperm, embryo and
scutellum. The composition of each layer varies significantly. In addition, the lo-
cation and concentration of components in oats depend upon cultivar and envi-
ronmental conditions. The chemical composition of oat groats and respective
layers has been given by Webster [68] and Wood [69].
8.3.2
Layout of a Potential Oat-based Fractionation Process
The high value of oat bran necessitates its separation prior to further processing
of the rest of the grain. Figure 8.5 presents a schematic diagram of a proposed
biorefinery based on oat groats, employing pearling and air classification. Signif-
icant information in the formulation of this processing scheme was taken from
Paton et al. [70] who used pearling to remove outer oat layers (1–15% of the oat
kernel). Pearlings were used to produce the following products:
1. a highly concentrated anti-irritant and a light oat oil extracted successively by
a volatile aqueous polar solvent and a nonpolar solvent;
2. a dark high quality oil and a highly stable lipase-active powder extracted by a
nonpolar solvent;
Table 8.2 Oat nutrients and their location in the kernel.
Constituent Function Mass a) Composition
Hull
) Encases oat groat 30 b) Fiber, some protein, some antioxidants
Pericarp Protect the grain – Fiber, protein, antioxidants, K, P, Mg, Ca
Seed coat and nucellus Bran –
Aleurone layer Encases endosperm – Protein, b-glucan, niacin, antioxidants, lipids,
phytin, aromatic amines, minerals (especially
P)
Endosperm Stores food 55–70 c) Starch, protein, b-glucan, lipids, minerals
Embryo Root and shoot 2.8–3.7 c) Similar to bran excluding niacin and aromatic
Scutellum Stores food amines
a) Mass fraction of the constituent within the kernel as % on a dry basis (db)
b) Content as related to dry weight of the complete oat kernel (hulls plus groat)
c) Content as related to the dry weight of the oat groat
184
8 A Whole Crop Biorefinery System
Fig. 8.5 Schematic diagram of a biorefinery based on oat groats employing pearling and air classification.
3. a b-glucan-rich fraction extracted by successive aqueous steeping of bran,

starch removal by enzymatic hydrolysis, bran removal by screening/centrifu-
gation and alcoholic extraction.
Pearled oat kernels could be either converted into a feedstock for subsequent
microbial bioconversions or fractionated into various coproducts. Steeping the
pearled oat groats in water would facilitate the maceration of bran and flour
fractions accomplished by aqueous ethanol extraction. The bran fraction is used
for the extraction of b-glucan, while the flour fraction is divided into starch and
protein. From the remaining pearled oat groat extract, an oat anti-irritant can be
recovered. Flour from pearled oat kernels or starch could be used in some fun-
gal bioconversions for the production of chemicals without hydrolysis. For in-
stance, Rhizopus oryzae can utilize starch for the production of lactic acid [71].
8.3.2.1 Potential Value-added Byproducts from Oat Bran-rich Fractions
Oat Gum: b-Glucan b-Glucans are linear homopolysaccharides of glucose units

linked via a mixture of (1 ? 4)- and (1 ? 3)-b-d-glycopyranosyl units. Water-solu-
ble b-glucans have potential nutritional and health benefits. b-Glucan prepara-
tions exhibit blood serum cholesterol lowering capabilities when consumed dai-
ly [72]. A potential mechanism for b-glucan cholesterol lowering capability is
their high molecular weight, which increases the luminal viscosity in the gastro-
intestinal tract [73]. Viscous water-soluble b-glucan preparations have hypoglyce-
mic effects assisting in the metabolic control of diabetes [74]. It was recently re-
ported that b-glucans could potentially be used in biomedical applications be-
cause of their abilities to:
1. activate host defense mechanisms against microbial and parasitic infections
[75]; and
2. reduce the glycemic index, making them a useful food ingredient for decreas-
ing postprandial glycemia while, at the same time, maintaining the food pa-
latability [76].
Research has also focused on the investigation of the rheological properties of

b-glucan extracts aiming at the creation of novel products for food purposes,
such as stabilizers, thickeners, textural agents and fat replacers in food prod-
ucts, and medical as well as cosmetic applications [73]. In addition, oligosaccha-
rides from b-glucan hydrolysis exhibit prebiotic properties and are considered as
food ingredients in probiotic and synbiotic preparations.
Malkki and Virtanen [77] stressed that the health effects of b-glucans on cho-
lesterol reduction, improved gastrointestinal function and glucose metabolism
would require a daily consumption of 10 g oat b-glucan. This means that the
daily digestion of adequate amounts of b-glucans requires consumption of sub-
stantial amounts of conventional oat products, which contain 35–50 g kg–1 b-glu-
can. Thus, the commercialization of concentrated oat bran products is necessary
in order to provide consumers with the daily requirements for b-glucan. The
presence of b-glucan in the pericarp, aleurone and sub-aleurone layers indicates
that the application of a pearling technology would be ideal for the separation
of b-glucan rich fractions [24]. However, distribution of b-glucan in the oat ker-
nel is dependent on the oat cultivar used. Thus, oat cultivars should be screened
so as to identify the ones that will provide b-glucan enriched fractions through
pearling.
The market price, functional properties and concentration of b-glucan com-
mercial preparations will be dependent on the final application and the fraction-
ation process employed. For instance, purified b-glucan could reach a value of
$ 55 kg–1, while its use as thickening agent would compete with guar and
xanthan gums, the market price of which is $ 0.78 kg–1 and $ 12 kg–1, respec-
tively [24].
Antioxidants Oats contain various compounds with antioxidant activities, such

as tocols, phytic acid, phenolic acids, avenanthramides, flavonoids and sterols. A
comprehensive review of these oat antioxidants has been published by [78].
Antioxidants are mainly located in the outer groat layers but each compound
with antioxidant properties can be predominantly located in a specific outer
layer, in the whole bran fraction or even in the hull. Some problems that are re-
lated to the extraction of antioxidants from cereal grains have to do with:
1. heat treatment to deactivate lipase prior to milling;
2. the localization of specific components with high antioxidant activities;
3. the production of cereal fractions containing botanical constituents enriched
in antioxidants; and
4. the selection of analytical assays that can realistically measure the antioxidant
activities of cereal fractions.
The selection of analytical assays is crucial in the detection of antioxidant activ-

ities because of the presence of both antioxidants and pro-oxidants in any cereal
fraction and the fact that it measures the inhibition of oxidation of case-specific
reactions depending on the combination of the various micro components with
antioxidant properties [79].
Several researchers have shown that pearling could be an important tool in
the production of bran-rich fractions enriched in compounds with antioxidant
activities. Handelman et al. [25] fractionated oats by either pearling or conven-
tional dry milling, with further enrichment by air classification to identify their
antioxidant activities. Pearlings exhibited the highest antioxidant content in
comparison to flour, trichome and bran fractions. Even the trichome fraction,
that is the outer (hairy) surface of the groat, exhibited relatively high antioxidant
activity. Peterson et al. [27] compared the antioxidant activity of bran-rich frac-
tions produced through pearling for between 5 and 180 s, concluding that the
antioxidant activity of 80% ethanol extract and the concentration of total phenol-
ic compounds and specific phenolic acids (e.g. ferulic acid, q-coumaric acid)
was higher in short-pearling-time fractions, and decreased at prolonged pearling
References 187
times as more endosperm tissue was included. The results reported by Peterson
et al. [27] about pearling fractions were reinforced by two other studies [79, 80].
The numerous functionalities of oat antioxidants including growth regulation,
good emulsifying properties, defense against parasites, inhibition of oxidative
degradation of unsaturated fatty acids, prevention of cardiovascular diseases,
cholesterol lowering capabilities, prevention of specific cancers, and provision of
color/aroma/stability/nutritional value in cereal food products, can impact many
industries [27, 78]. Oil-rich fractions extracted from groats or oat bran could be
used as emulsifiers or as ingredients in health foods, skin-care preparations,
specialized food oils and pharmaceuticals [81].
8.4
Summary
This chapter presents some potential cereal-based biorefineries that can be em-
ployed for the production of fuels, chemicals and plastics, as well as many val-
ue-added byproducts derived from cereal bran. Cereals are complex biological
entities and will represent a major category of the core photosynthetic raw ma-
terials that will replace petroleum in the production of commodities as well as
specialties. The creation of viable biorefineries based on cereals will require the
exploitation of all the botanical constituents of the grain as well as, if possible,
the non-grain components such as straw, stalks and hulls. Novel processing
routes for starch and protein in the production of a generic feedstock for micro-
bial bioconversions have been presented and various products that could be de-
rived from wheat and oats have been identified. The cost-competitiveness of
such cereal-based biorefineries will depend on the range of high value end prod-
ucts and their integration with the much larger volume low to mid-value prod-
ucts, and could be further improved by exploiting straw, hulls and other non-
grain components of the crop, either for the production of more value-added by-
products or simply for the generation of energy.
References
1 Morrison, R., Policy alternatives for the 3 Dobraszczyk, B. J. Wheat and flour. In
development of the cereal processing in- D. A. V. Dendy and B. J. Dobraszczyk
dustry, A report issued from Winnipeg (Eds.), Cereals and cereal products:
by Agriculture and Agri-Food Canada’s Chemistry and technology. Gaithersburg,
Policy Branch under the Grains 2000 MD: Aspen Publishers Inc. (1994) 100–
program, November, 1995. 139.
2 Audsley, E. and Sells, J. E. Determining 4 Pomeranz, Y. Chemical composition of
the profitability of a wholecrop biorefi- kernel structures. In: Wheat: Chemistry
nery. In: Campbell GM, Webb C, McKee and Technology. Vol. I. Pomeranz, Y. ed.
SL (eds) Cereals: Novel Uses and Pro- AACC: St. Paul, Minnesota, USA (1988)
cesses, Plenum press, New York, pp pp. 97–158.
191–204.
5 MacMasters, M. M., Hinton, J. J. C. and Office of the Biomass Program, Wash-

Bradbury, D. Microscopic structure and ington, DC (2003)
composition of the wheat kernel. In: 17 Zeikus, J. G., Jain, M. K., Elankovan, P.
Wheat: Chemistry and Technology, 2nd Biotechnology of succinic acid produc-
Ed. (1971) pp. 51–113. tion and markets for derived industrial
6 Lintas, C. (1988). Durum wheat vitamins products. Applied Microbiology and Bio-
and minerals. In: Durum wheat: Chem- technology 51 (1999) 545–552.
istry and Technology. Fabriani, G. and 18 Dexter, J. E. and Wood, P. J. Recent appli-
Lintas, C. ed. AACC: St. Paul, Minneso- cations of debranning of wheat before
ta, USA (1988) pp. 149–160. milling. Trends in Food Science and Tech-
7 Stevens, D. J. Free sugars of wheat aleu- nology 7 (1996) 35–41.
rone cells. Journal of Science in Food and 19 Cui, W., Wood, P. J., Weisz, J. and Beer,
Agriculture 21 (1970) pp. 31–34. M. U. Nonstarch polysaccharides from
8 Hargin, K. D., Morrison, W. R. and Ful- preprocessed wheat bran: carbohydrate
cher, R. G. Triglyceride deposits in the analysis and novel rheological properties.
starchy endosperm of wheat. Cereal Cereal Chemistry 76 (1999) 129–133.
Chemistry 57 (1980) pp. 320–325. 20 Tkac, J. J. Process for removing bran
9 Kent, N. L. Subaleurone endosperm cells layers from wheat kernels. (1992) US
of high protein content. Cereal Chemistry Patent 5,082,680.
43 (1966) pp. 585–601. 21 Wellman, W. Process for milling cereal
10 Mares, D. J., and Stone, B. A. Wheat en- grains. (1993) US Patent 5,186,968.
dosperm. I. Chemical composition and 22 Satake, S., Ishii, T. and Tokui, Y. Vertical
ultrastructure of the cell walls. Australian pearling machines and apparatus for
Journal of Biological Sciences 26 (1973) preliminary treatment prior to flour
pp. 793–812. milling using such pearling machines.
11 Kornegay, E. T. Digestion of phosphorus (1995) US Patent 5,390,589.
and other nutrients: The role of phytases 23 Gills, J. M. and McGee, B. C. Advances in
and factors influencing their activity. In: durum semolina milling. British Pasta
Enzymes in farm animal nutrition. Bed- Products Association, Technical Seminar,
ford, M., Partridge, G. (eds). Finnfeeds London (1999) http://www.satake-usa.-
International, UK (2001) com/brochures/Durum_ Milling_
12 Abelson, P. H. A potential phosphate cri- Advances.pdf
sis. Science 283 (1999) 2015 24 Anonymous. Fractionation processes:
13 Reade, A. E., Smith, R. H. and Palmer, Descriptions, merits and deficiencies.
R. M. Production of protein for nonrumi- Agriculture and Agri-Food Canada,
nant feeding by growing filamentous Threshold Technologies Company and
fungi on barley. Biochemical Journal 127 POS Pilot Plant, CANUC Database
(1972) 32p (1995).
14 Reed, G. and Nagodawithana, T. En- 25 Handelman, G. J., Cao, G., Walter, M. F.,
zymes, Biomass, Food and Feed. Wiley- Nightingale, Z. D., Paul, G. L., Prior, R. L.
VCH, Weinheim, (2001) p. 174 and Blumberg, J. B. Antioxidant capacity
15 Koutinas, A. A., Wang, R.-H. and Webb of oat (Avena sativa L.) extracts. 1. Inhibi-
C. Development of a process for the pro- tion of low-density lipoprotein oxidation
duction of nutrient supplements for fer- and oxygen radical absorbance capacity.
mentations based on fungal autolysis. Journal of Agriculture and Food Chemistry
Enzyme and Microbial Technology (in 47 (1999) 4888–4893.
press) 26 Marconi, E., Graziano, M. and Cubadda
16 Paster, M., Pellegrino, J. L. and Carole, R. Composition and utilization of barley
T. M. Industrial bioproducts: Today and pearling byproducts for making func-
tomorrow. Energetics, Incorporated for tional pastas rich in dietary fiber and b-
the US Department of Energy, Office of glucans. Cereal Chemistry 77 (2000) 133–
Energy Efficiency and Renewable Energy, 139.
References 189
27 Peterson, D. M., Emmons, C. L. and of sugar-beet pectins and maize bran

Hibbs, A. H. Phenolic antioxidants and heteroxylans. Journal of the Science of
antioxidant activity in pearling fractions Food and Agriculture 79 (1999) 396–402.
of oat groats. Journal of Cereal Science 33 39 Peyron, S., Abecassis, J., Autran, J.-C.
(2001) 97–103. and Rouau, X. Enzymatic oxidative treat-
28 Seog, H.-M., Seo, M.-S., Kim, Y.-S. and ments of wheat bran layers: Effects on
Lee, Y.-T. Physicochemical properties of ferulic acid composition and mechanical
barley bran, germ and broken kernel as properties. Journal of Agriculture and
pearling byproducts. Food Science and Food Chemistry 49 (2001) 4694–4699.
Biotechnology 11 (2002) 623–627. 40 Labat, E., Morel, M. H. and Rouau, X.
29 Posner, E. S., Seib, P. A. and Qiang, Z. Effect of laccase and manganese peroxi-
Process for dry milling of wheat to ob- dase on wheat gluten and pentosans dur-
tain gluten and starch. (1992) US Patent ing mixing. Food Hydrocolloids 15 (2001)
5,164,013. 47–52.
30 Wang, S., Sosulski, K., Sosulski, F. and 41 Rattan, O., Izydorczyk, M. S. and Bilia-
Ingledew, M. Effect of sequential abra- deris, C. G. Structure and rheological be-
sion on starch composition of five cer- havior of arabinoxylans from Canadian
eals for ethanol fermentation. Food Re- bread wheat flours. Food Science and
search International 30 (1997) 603–609. Technology (London) 27 (1994) 550–555.
31 Wang, S. Thomas, K. C., Sosulski, K., In- 42 Lloyd, L. L., Kennedy, J. F., Methacanona,
gledew, W. M. and Sosulski, F. W. Grain P., Methacanon, P., Paterson, M. and
pearling and very high gravity (VHG) Knill, C. J. Carbohydrate polymers as
fermentation technologies for fuel alco- wound management aids. Carbohydrate
hol production from rye and triticale. Polymers 37 (1998) 315–322.
Process Biochemistry 34 (1999) 421–428. 43 Sancho, A. I., Bartolome, B., Gomez-Cor-
32 Klumpar, I. V. Measuring and optimizing doves, C., Williamson, G. and Faulds,
air classifier performance. Separations C. B. Release of ferulic acid from cereal
Technology 2 (1992) 124–135. residues by barley enzymatic extracts.
33 Wu, Y. V. and Doehlert, D. C. Enrichment Journal of Cereal Science 34 (2001) 173–
of b-glucan in oat bran by fine grinding 179.
and air classification. Lebensmittel-Wis- 44 Benamrouche, S., Cronier, D., Debeire,
senschaft und Technologie 35 (2002) 30–33. P. and Chabbert, B. A chemical and his-
34 Letang, C., Samson, M.-F., Lasserre, T.- tological study on the effect of (1 ? 4)-b-
M., Chaurand, M., Abecassis, J. Produc- endo-xylanase treatment on wheat bran.
tion of starch with very low protein con- Journal of Cereal Science 36 (2002) 253–
tent from soft and hard wheat flours by 260.
jet milling and air classification. Cereal 45 Hakala, P., Lampi, A.-M., Ollilainen, V.,
Chemistry 79 (2002) 535–543. Werner, U., Murkovic, M., Wahala, K.,
35 Andersson, A. A. M., Andersson, R. and Karkola, S. and Piironen, V. Steryl phe-
Aman, P. Air classification of barley nolic acid esters in cereals and their
flours. Cereal Chemistry 77 (2000) 463– milling fractions. Journal of Agriculture
467. and Food Chemistry 50 (2002) 5300–5307.
36 Wu, Y. V. and Stringfellow, A.C. Enriched 46 Micard, V., Landazuri, T., Surget, A.,
protein and b-glucan fractions from Moukha, S., Labat, M. and Rouau, X.
high-protein oats by air classification. Demethylation of ferulic acid and feru-
Cereal Chemistry 72 (1995) 132–134. loyl-arabinoxylan by microbial cell ex-
37 Saulnier, L., Vigouroux, J. and Thibault, tracts. Lebensmittel-Wissenschaft und -Tech-
J.-F. Isolation and partial characteriza- nologie 35 (2002) 272–276.
tion of feruloylated oligosaccharides 47 Muheim, A. and Lerch, K. Towards a
from maize bran. Carbohydrate Research high-yield bioconversion of ferulic acid
272 (1995) 241–253. to vanillin. Applied Microbiology Biotech-
38 Saulnier, L. and Thibault, J.-F. Ferulic nology 51 (1999) 456–461.
acid and diferulic acids as components
48 Hwang, J., Park, B. and Yun, J. Biologi- J. J. Modified wheat glutens for use in
cally active materials such as ferulic acid fabrication of films. (1999) US Patent
and arabinoxylan obtained from cereal 5,747,648.
bran by extrusion and enzyme treat- 59 Woerdeman, D. L., Veraverbeke, W. S.,
ment. (2001) PCT International Applica- Parnas, R. S., Johnson, D., Delcour, J. A.,
tions WO 01/67891 A1. Verpoest, I., Plummer, C. J. G. Designing
49 Anonymous. Report from the state of new materials from wheat protein. Bio-
France forming part of the IENICA pro- macromolecules 5 (2004) 1262–1269.
ject. Interactive European Network for 60 Webb C., and Wang R. (1997) Develop-
Industrial Crops and their Applications ment of a generic fermentation feedstock
(IENICA). Agency for Environment and from whole wheat flour. In: Campbell
Energy Management (ADEME) (1999). GM, Webb C, McKee SL (eds) Cereals:
50 Ferguson, L. R. and Harris, P. J. Protec- Novel Uses and Processes, Plenum
tion against cancer by wheat bran: role press, New York, pp 205–218
of dietary fibre and phytochemicals. Eu- 61 Koutinas, A. A., Wang, R.–H. and Webb,
ropean Journal of Cancer Prevention 8 C. Restructuring upstream bioprocess-
(1999) 17–25. ing: Technological and economical as-
51 Reddy, B.S., Hirose, Y., Cohen, L.A., pects for the production of a generic mi-
Simi, B., Cooma, I. and Rao, C.V. Pre- crobial feedstock from wheat. Biotechnol-
ventive potential of wheat bran fractions ogy and Bioengineering 85 (2004) 524–
against experimental colon carcinogen- 538.
esis: implications for human colon can- 62 Webb, C., Koutinas, A. A. and Wang, R.-
cer prevention. Cancer Research 60 (2000) H. Developing a sustainable bioprocess-
4792–4797. ing strategy based on a generic feed-
52 Kahlon, T. S. and Woodruff, C. L. In vitro stock. In: Scheper T (Series ed), Zhong
binding of bile acids by rice bran, oat J-J (volume ed) Biomanufacturing. Ad-
bran, barley and b-glucan enriched bar- vances in Biochemical Engineering/Bio-
ley. Cereal Chemistry 80 (2003) 260–263. technology. Springer–Verlag Berlin 87
53 Fenech, M., Noakes, M., Clifton, P. and (2004) 196–268
Topping, D. Aleurone flour is a rich 63 Bohlmann, J. A., Schisler, D. O., Hwang,
source of bioavailable folate in humans. K.-O., Henning, J. P., Trinkle, J. R., An-
Journal of Nutrition 129 (1999) 1114– derson, T. B., Steinke, J. D. and Vander-
1119. hoff, A. Enzymic process for producing
54 Sidbu, J. S., Al-Saqer, J. M. and Al-Hooti, N-acetyl-d-glucosamine from spent fun-
S. N. Mineral composition of high-fiber gal biomass. (2003) PCT International
pan bread formulations containing bran Applications WO 03/13435 A2.
and germ fractions. AFS, Advances in 64 Ian, W., Bohlmann, J. A., Trinkle, J. R.,
Food Sciences 23 (2001) 108–112. Steinke, J. D., Hwang, K.-O. and Hen-
55 Salmenkallio-Marttila, M., Katina, K. and ning, J. P. Glucosamine from microbial
Autio, K. Effects of bran fermentation on biomass. (2002) US Patent 2002115639.
quality and microstructure of high-fiber 65 McCoy, M. Betting on glucosamine.
wheat bread. Cereal Chemistry 78 (2001) Chemical and Engineering News 81 (No 7)
429–435. (2003) 27–28.
56 Czuchajowska, Z. and Pomeranz, Y. Pro- 66 Datar, R. P., Shenkman, R. M., Cateni,
cess for fractionating wheat flours to ob- B. G., Huhnke, R. L., Lewis R. S. Fermen-
tain protein concentrates and prime tation of biomass-generated producer gas
starch. (1995) US Patent 5,439,526. to ethanol. Biotechnology and Bioengineer-
57 Sayaslan, A. Wet-milling of wheat flour: ing 86 (2004) 587–594
industrial processes and small-scale test 67 Weaver, P. F. and Maness P.-C. Photo-
methods. Lebensmittel-Wissenschaft und conversion of gasified organic materials
-Technologie 37 (2004) 499–515. into biologically-degradable plastics
58 Bassi, S., Maningat, C. C., Chinnaswamy, (1993) US Patent 5,250,427
R., Nie, L., Weibel, M. K. and Watson,
References 191
68 Webster, F. H. Oats: Chemistry and tech- tracted from oat, enhances disease resis-
nology. (1986) AACC, St Paul, Minneso- tance against bacterial and parasitic in-
ta, USA fections. FEMS Immunology and Medical
69 Wood, P. J. Oat bran. (1993) AACC, St Microbiology 35 (2003) 67–75.
Paul, Minnesota, USA. 76 Jenkins, A. L., Jenkins, D. J. A., Zdravko-
70 Paton, D., Reaney, M. J. T. and Tyler, N. J. vic, U., Wursch, P. and Vuksan, V. De-
Methods for processing oat groats and pression of the glycemic index by high
products thereof. (2000) US patent levels of b-glucan fiber in two functional
6,113,908. foods tested in type 2 diabetes. European
71 Tay, A. and Yang, S.-T. Production of Journal of Clinical Nutrition 56 (2002)
L(+)-lactic acid from glucose and starch 622–628.
by immobilized cells of Rhizopus oryzae 77 Malkki, Y. and Virtanen, E. Gastrointesti-
in a rotating fibrous bed reactor. Biotech- nal effects of oat bran and oat gum: A
nology and Bioengineering 80 (2002) review. Lebensmittel-Wissenschaft und -
pp. 1–12. Technologie 34 (2001) 337–347.
72 Maki, K. C., Shinnick, F. S., Marlyn, A., 78 Peterson, D. M. Oat Antioxidants. Journal
Veith, P. E., Quinn, L. C., Hallissey, P. J., of Cereal Science 33 (2001) 115–129.
Temer, A. and Davidson, M.H. Food 79 Emmons, C. L., Peterson, D. M. and
products containing free tall oil-based Paul, G. L. Antioxidant capacity of oat
phytosterols and oat b-glucan lower se- (Avena sativa L.) extracts. 2. In vitro anti-
rum total and LDL cholesterol in hy- oxidant activity and contents of phenolic
percholesterolemic adults. Journal of Nu- and tocol antioxidants. Journal of Agricul-
trition 133 (2003) 808–813. tural and Food Chemistry 47 (1999) 4894–
73 Colleoni-Sirghie, M., Kovalenko, I. V., 4898.
Briggs, J. L., Fulton, B. and White, P. J. 80 Gray, D. A., Auerbach, R. H., Hill, S.,
Rheological and molecular properties of Wang, R., Campbell, G. M., Webb C. and
water soluble (1,3) (1,4)-b-b-glucans from South, J. B. Enrichment of oat antioxi-
high-b-glucan and traditional oat lines. dant activity by dry milling and sieving.
Carbohydrate Polymers 52 (2003) 439–447. Journal of Cereal Science 32 (2000) 89–98.
74 Wursch, P. and Pi-Sunyer, E. X. The role 81 Peterson, D. M. Composition, separation
of viscous soluble fiber in the metabolic and applications. Lipid Technology 14
control of diabetes. Diabetes Care 20 (2002) 56–59
(1997) 1774–1780.
75 Yun, C.-H., Estrada, A., Van Kessel, A.,
Park, B.-C. and Laarveld, B. b-Glucan, ex-
193
9
Iogen’s Demonstration Process for Producing Ethanol
from Cellulosic Biomass
Jeffrey S. Tolan
9.1
Introduction
The production of fuel alcohol from cellulosic biomass is of growing interest

around the world. Cellulosic biomass can be used to produce transportation fuel,
with the overall process having little net production of greenhouse gases. Biomass
is available as agricultural residues or as a byproduct of many processes, or can be
potentially produced from dedicated energy crops. The technology for biomass
conversion has many significant technical and economic challenges that have de-
layed its commercialization. However, significant progress has allowed Iogen Cor-
poration of Ottawa, Canada to produce up to 2000 gallons day–1 of ethanol from
wheat straw since April 2004 to demonstrate the technology.
9.2
Process Overview
The basic process steps of Iogen’s Ottawa plant are shown in Fig. 9.1. This is a
demonstration plant that produces up to 2000 gallons day–1 of ethanol from
wheat straw. A full scale plant would produce about 170 000 gallons of ethanol
per day (60 million gallons year–1).
A cellulosic feedstock material such as wheat straw or other straws, corn
stover, or grass is subjected to pretreatment, that is, cooked in the presence of
acid to break down its fibrous structure. After pretreatment, the material has a
muddy texture. Cellulase enzymes are added to the pretreated material to hydro-
ISBN: 3-527-31027-4
194 9 Iogen’s Demonstration Process for Producing Ethanol from Cellulosic Biomass
Fig. 9.1 Iogen’s process for converting wheat straw to ethanol.
lyze the cellulose to the simple sugar glucose; this is known as cellulose hydroly-
sis. The cellulase enzymes are made at the plant site by using a wood-rotting
fungus in large fermentation vessels. This is known as cellulase enzyme produc-
tion. After cellulose hydrolysis, the sugars are separated from the unhydrolyzed
solids, which include lignin and residual cellulose. These solids are burned to
provide energy for the entire process (lignin processing). The sugars are ferment-
ed (sugar fermentation) to ethanol using recently developed recombinant Sacchar-
omyces yeast from Purdue University to ferment the glucose and the more diffi-
cult sugar to ferment, xylose. In ethanol recovery, the ethanol is recovered by con-
ventional distillation, denatured, and blended into gasoline.
The remainder of this chapter describes the process steps and related technol-
ogies in more detail.
9.3
Feedstock Selection
9.3.1
Feedstock Composition
The term “cellulosic biomass” refers to potential feedstocks that have cellulose as
a primary constituent. Other major constituents of these materials include hemi-
cellulose and lignin. Minor constituents include proteins, ash, starch, and various
other organic compounds that are not carbohydrates.
Cellulose comprises 35% to 50% of most plant material. Cellulose is a poly-

mer of glucose, of dp (degree of polymerization, or chain length) of 1000 to
10 000. Cellulose is a linear, unbranched polymer, with glucose joined together
by beta-1,4-linkages. Individual polymer chains run parallel to each other and
form hydrogen bonds with each other, up to three per monomeric glucose unit.
Several such chains form a microfibril. A microfibril region that has the full de-
gree of hydrogen bonding forms a roughly cubic, 3-dimensional lattice. Such a
region is crystalline cellulose and is very stable against attack by enzymes or
acid. Other regions are not hydrogen-bonded to nearly this extent, and in the ex-
treme are simply random configurations of glucose polymers. This is amorphous
cellulose. Most natural cellulose is primarily crystalline cellulose.
The main source of ethanol is from the glucose, originating from the cellu-
lose. However, a second source of ethanol is from the simple sugars that com-
prise the hemicellulose. Hemicellulose is a mixture of linear and branched poly-
mers of the five-carbon sugars xylose and arabinose and (less importantly) the
six-carbon sugars glucose, mannose, and galactose. Hemicellulose is readily dis-
solved and hydrolyzed to its simple sugars in dilute acid at moderate tempera-
tures, for example, 120 8C. Hemicellulose comprises 15–25% of most plant ma-
terial.
Lignin differs from cellulose and hemicellulose in that lignin is not com-
prised of carbohydrates, but rather consists of a complex three-dimensional ma-
trix of phenolic propane units. Lignin confers water resistance and stiffness to
the fiber and protection against microbial attack. Lignin does not participate in
the pretreatment or hydrolysis processes except with a decrease in the degree of
polymerization. The burning of lignin is the mode of energy generation for the
process. Lignin comprises 15–30% of most plant materials.
In normal operation, the minor constituents exert only a minor impact on
the process. The protein is degraded by pretreatment to the point where it can-
not be recovered economically. The ash must not be at too high a level as to be
abrasive to the process equipment. Such can be the case if large amounts of sili-
ca (sand) are present due to the harvest practices or the natural silica uptake by
the plant. The starch is easily hydrolyzed to glucose and increases the overall
Table 9.1 Feedstock composition (mg g–1 total solids) (from Foody et al. 1999 [1]).
Feedstock Cellulose Starch Xylan Arabinan Lignin Ash Protein
Barley straw 406 20 161 28 168 82 64

Wheat straw 455 9 165 25 204 83 64
Wheat chaff 391 14 200 36 160 121 33
Switch grass 399 3 184 38 183 48 54
Corn stover 408 3 128 35 127 60 81
Maple wood 500 4 150 5 276 6 6
Pine wood 648 1 33 14 320 0 2
ethanol yield. Table 9.1 shows the composition of several typical lignocellulosic
materials.
9.3.2
Feedstock Selection
The scope of feedstocks considered by Iogen for ethanol production are listed in
Table 9.2.
These potential feedstocks are evaluated based on the desired feedstock prop-
erties. The desired qualities of a feedstock are:
1. Low cost. Naturally, the cost of the feedstock is an important part of the over-
all cost. A desired feedstock can be obtained and transported to the plant at
low cost. This rules out primary and secondary tree growth, sawdust, and
waste paper, all of which have existing markets and high cost.
2. Availability. A feedstock must be in sufficient quantity to supply a commercial
plant. This requires perhaps 800 000 tons year–1, which is not available from
bagasse in many locations.
3. Uniformity. For a high-speed production process, foreign matter present in
municipal waste is unacceptable.
4. Cleanliness. High levels of silica can abrade equipment, as stated above. A
high degree of microbial contamination is unacceptable. High levels of toxic
or inhibitory materials are not acceptable.
5. High potential ethanol yield. The main constituents, cellulose and hemicellu-
lose, must be present in high enough levels to produce ethanol. This is a dis-
advantage of forestry waste, which is high in bark that is mostly lignin and
phenolic acids.
6. High efficiency of conversion. The efficiency of conversion to glucose (follow-
ing Iogen’s pretreatment process) is proportional to the arabino-xylan content
Table 9.2 Potential feedstocks.
Material Subclass Comments
Wood Native forest Difficult to process, especially softwood

Tree farms Too expensive due to demands of other
markets
Forest waste (bark) Cellulose/hemicellulose content is too
low
Mill waste (sawdust) Too expensive due to pulp and paper
market
Agricultural Straws (wheat, barley, oat, rice) Leading candidate feedstocks
residues Bagasse (cane) Localized feedstock of interest
Corn stover Leading candidate feedstock
Energy crops Grass Possible second generation feedstock
Waste cellulose Municipal waste Not uniform enough to process
Waste paper Too expensive due to paper demand
of the feedstock [1]. The arabino-xylan content is roughly the sum of the ara-
binan and xylan content listed in Table 9.1. Feedstocks with a low arabino-xy-
lan content, such as softwood, demand unacceptably high levels of enzyme
for conversion to cellulose.
Matching the feedstock list with the desired properties results in the feedstocks
used by Iogen, which are agricultural residues such as straws and stover and
energy crops such as grasses. The energy crops, such as grass, are not currently
harvested in a large scale, as this will require large scale demonstration of the
technology before people commit to these as crops. The grasses are therefore
second-generation feedstocks.
9.3.3
Ethanol from Starch or Sucrose
Starch-based or sucrose-based processes are already widely used to make etha-

nol. The leading starch-based material is corn, which is widely used to make
ethanol in the US. Starch is converted to glucose by grinding corn kernels (in a
dry milling process) or by steeping kernels in dilute sulfurous acid (in a wet
milling process), then using starch-degrading enzymes known as amylases. The
glucose is then fermented to ethanol. Sucrose-based feedstocks include sugar
cane (Brazil) and sugar beets (Europe). These feedstocks are pressed and
washed with water to extract the sucrose, which is then fermented to ethanol by
yeast. Other feedstocks used to make small amounts of fuel ethanol in some re-
gions include potatoes and Jerusalem artichokes.
Many cellulosic materials, including straw and grass, contain up to 10%
starch. Wheat straw fed to Iogen’s plant is 3–4% starch. This is converted to glu-
cose during pretreatment and carried through to sugar fermentation, where it is
converted to ethanol.
9.3.4
Advantages of Making Ethanol from Cellulosic Biomass
The conversion of cellulosic biomass to ethanol is more difficult than starch or su-
crose, and this has limited commercialization of the technology. However, cellulo-
sic biomass is available in much greater quantity and offers the potential for much
greater ethanol production than the other feedstocks. In addition, ethanol produc-
tion from starch and sucrose faces competition for the feedstock from the food
and cattle feed industries, which exerts pressure on the price of the ethanol. Most
cellulosic biomass is free of competition from other uses. Cellulosic biomass can
be grown in a wider variety of climates and soils than starch and sucrose and
therefore represent a potential new agricultural opportunity in many areas.
The most important advantage of making ethanol from cellulosic biomass is
that the production and use of the ethanol does not add to the emission of green-
house gases. Producing ethanol from corn, sugar cane, or sugar beets requires
large amounts of energy-intensive fertilizers. The use of these feedstocks results in

significantly less net energy generation from the lignin byproduct than that from
using cellulosic biomass. These two areas (fertilizer and process energy) require
significant input of fossil fuels in ethanol plants using corn, sugar cane, or sugar
beets. By contrast, ethanol made from cellulosic biomass is not a net user of fossil
fuels. The neutral fossil fuel usage is why ethanol from cellulose is expected to be
neutral relative to the production of greenhouse gases [2].
An additional benefit of biomass conversion arises from the fact that corn, su-
gar cane, and sugar beets all contain 5–15% cellulose and hemicellulose. Cellu-
lose conversion technology represents an opportunity to improve the ethanol
yields and decrease the wastes from these processes.
9.4
Pretreatment
9.4.1
Process
Pretreatment is the process by which surface area of the feedstock is opened up

for the subsequent enzymatic attack. In the absence of pretreatment, the re-
quirement for cellulase enzymes is too high to be practical.
The basis for Iogen’s pretreatment is steam explosion [3]. In the Iogen dem-
onstration plant, wheat straw in bales is chopped and milled, then conveyed to
the pretreatment reactor. High pressure steam and sulfuric acid are added to
the feedstock to reach 180–260 8C with 0.5–2% sulfuric acid. The material is
maintained at this condition for 0.5–5 min. The pressure is then released
rapidly.
As a result of pretreatment, the fibrous structure of the feedstock is destroyed.
The pretreated material has a muddy texture and a slightly sweet smell, with a
dark brown color. Among different feedstocks, the cooking time varies while
most of the other conditions are maintained relatively constant. The appearance
of pretreated material is similar among different feedstocks.
9.4.2
Chemical Reactions
Of the major components, the first to react is the hemicellulose. The xylan por-
tion is depolymerized and solubilized, and then hydrolyzed to xylose by the re-
action:
C5 H8 O4 n H2 O ! C5 H8 O4 n 1 C5 H10 O5 1
The presence of exogenous sulfuric acid is particularly important in the for-

mation of monomeric xylose. In the absence of exogenous acid, xylose oligo-
9.4 Pretreatment 199
mers are formed. Further, the added acid improves the uniformity of the pro-
cess, because natural acid levels vary considerably among feedstocks or batches
of a given feedstock.
If the pretreatment reaction proceeds further, the xylose is dewatered to pro-
duce furfural (Eq. 2), which is undesirable.
C5 H10 O5 ! C5 H4 O2 3H2 O 2
Arabinose undergoes analogous reactions, but more slowly than xylose. Acetic
acid is released from the hydrolysis of acetyl groups attached to the xylan.
Removing hemicellulose from the feedstock accomplishes two objectives.
First, it makes the simple sugars that can potentially be fermented to ethanol.
Second and more importantly, it opens up surface area on the feedstock, there-
by allowing the cellulase enzyme to digest the cellulose more efficiently.
A small amount of cellulose reacts to form glucose, which is degraded to hy-
droxymethylfurfural, by Eqs (3) and (4):
C6 H10 O5 n H2 O ! C6 H10 O5 n 1 C6 H12 O6 3
C6 H12 O6 ! C6 H6 O3 3H2 O 4
Only a small amount of cellulose hydrolysis (< 20%) is desired in the pretreat-
ment. More than this results in a decrease in glucose yield and accessibility of
the enzyme to the cellulose. The older acid hydrolysis process carried out a
much harsher acid hydrolysis and will be described below.
The lignin undergoes a depolymerization during pretreatment, but remains
insoluble in water or acid. Protein is destroyed and starch is hydrolyzed to glu-
cose in the pretreatment.
One can draw an analogy to cooking a turkey to describe the trade-off be-
tween time and temperature in the pretreatment: the longer the time, the lower
the temperature. Acid also acts to decrease the temperature or time required for
pretreatment.
The choice of reactor for this pretreatment is only important insofar as the
desired chemistry must be delivered to the system. Numerous guns, vessels,
and tubes have been proposed and built to carry this out.
9.4.3
Other Pretreatment Processes
Katzen et al. [4] published a detailed review of pretreatment that will only be
summarized here. Pretreatment processes may be divided into (1) those that
produce a stream directly for fermentation to ethanol and (2) those that are fol-
lowed by enzymatic hydrolysis. The former are necessarily harsher and have a
longer history.
Direct sugar production in pretreatment has been carried out using concen-
trated chemicals and dilute acid.
Concentrated chemical pretreatment represents the cellulose conversion tech-

nology with the longest history, dating back to 1890 or earlier in Germany. The
concentrated chemicals include acids, bases, and salts. The principle behind the
use of concentrated chemicals is to disrupt the crystalline cellulose structure,
thereby dissolving and depolymerizing the cellulose. Among the chemicals used
are 72% sulfuric acid, 40% hydrochloric acid, 40% sodium hydroxide, 65% zinc
chloride, 40% calcium chloride. These methods have very high yields and low
operating temperatures. Unfortunately, the economics of the process dictate that
recovery and reuse of the pretreatment chemicals is critically important. The in-
ability to obtain the necessary recoveries of order 99.9% has hindered the com-
mercialization of these processes. A second problem is the exotic materials re-
quired to handle these streams.
Dilute acid hydrolysis represents the cellulose conversion technology with the
most commercial experience. In dilute acid hydrolysis, the feedstock is treated
at perhaps 180–200 8C for 1–4 h with 1 to 4% sulfuric acid. A glucose yield of
50% of the cellulose or higher is obtained. This process was used for ethanol
production by Germany during World War II, Russia in the late 1940s, and pilot
plants in the 1950s in Switzerland and Springfield, Oregon. All of these plants
have been plagued by corrosion, low yields, high investments, and overall poor
returns. Several plants of up to 10 t day–1 built in Russia in the 1950s still oper-
ate with this process.
Pretreatment followed by enzymatic hydrolysis is newer than direct pretreat-
ment, as cellulase enzymes were only identified and worked with after World
War II. The milder pretreatments carried out in preparation for enzymatic hy-
drolysis include mechanical action, solvent-based pretreatments, alkali treat-
ments, and acid prehydrolysis.
Mechanical action was first tried at the U.S. Army laboratory in Natick, Mas-
sachusetts in the 1960s. The principle behind mechanical action is to increase
the surface area of the feedstock particles. However, beyond producing small
particles for uniform distribution of acid, there is no real advantage to further
milling of the feedstock.
In solvent-based pretreatments, organic solvents such as ethanol and metha-
nol are used to dissolve a portion of the lignin, thereby freeing up the cellulose
for enzymatic attack. However, recovery of the solvent is difficult, and partial de-
lignification is not of significant benefit in the hydrolysis.
In alkali pretreatments, such as with sodium hydroxide or ammonia, the crys-
talline cellulose is converted to a different form, cellulose II or III respectively.
These forms of cellulose can be more easily hydrolyzed than native cellulose.
However, destruction of the hemicellulose is reported in these systems, and
they are not yet used commercially.
Acid prehydrolysis is preferred by Iogen because it has fewer of these prob-
lems than the other methods. The levels of acid are low enough that recovery is
not needed and corrosion is not a problem. The process provides a selective hy-
drolysis of hemicellulose and produces a cellulosic substrate with a high surface
area suitable for enzymatic hydrolysis.
9.5 Cellulase Enzyme Production 201
9.5
Cellulase Enzyme Production
9.5.1
Production of Cellulase Enzymes
Cellulase enzymes convert cellulose to glucose, which can then be fermented to

ethanol. Cellulase enzymes are made by a wide variety of microbes, but those
best suited to cellulose hydrolysis are made by the wood-rotting fungus Tricho-
derma. This fungus was isolated during World War II in rotted U.S. Army cot-
ton tents in the South Pacific. Researchers led by Elwyn Reese and Mary Man-
dels at the U.S. Army laboratory in Natick, Massachusetts determined that the
microbe responsible for the destruction of the cotton was secreting a mixture of
enzymes that hydrolyzed the cotton. Reese and Mandels determined cultivation
conditions for production of cellulase in liquid culture. Selection of Trichoderma
strains with higher productivities of cellulase was successfully carried out by
Montenecourt at Lehigh University in the 1970s. Despite research and develop-
ment of cellulase production from other microbes, Trichoderma remains the or-
ganism of choice to produce cellulase for ethanol production.
Cellulase is made at Iogen’s commercial enzyme plant in Ottawa in a sub-
merged liquid culture, as is used by Genencor International, Novozym Biotech,
Rohm AB, and other commercial cellulase manufacturers. The fermentation
vessels are similar to those used for producing antibiotics. The fermenters are
50 000 gallons and are maintained free of contaminating microbes. The liquid
broth contains carbon source, salts, complex nutrients such as corn steep liquor,
and other nutrients in water. The most important nutrient is the carbon source.
Glucose promotes growth of the organism but not cellulase production. The car-
bon source must include an inducing sugar to promote cellulase production.
Well known inducers of cellulase include the sugars cellobiose, lactose, sophor-
ose, and other low molecular weight oligomers of glucose.
The nutrient broth is sterilized before the start of the fermentation by heating
with steam. The fermenter is inoculated with the enzyme production strain
once the liquid broth has cooled down. The operating conditions are 30 8C, pH
4 to 5, and these are maintained by the addition of cooling water in external
coils and by alkali, respectively. Trichoderma is highly aerobic and a constant
stream of air or oxygen is used to maintain aerobic conditions. A cellulase en-
zyme production run lasts about one week. At the end of the run, the broth is
filtered across a cloth to remove cells. The spent cell mass is destroyed and dis-
posed of by landfilling.
The resulting enzyme broth is a clear, light brown liquid, similar in appear-
ance to weak tea.
9.5.2
Enzyme Production on the Ethanol Plant Site
Iogen has the unique advantage of operating an ethanol plant on a cellulase

production site. In this case, the crude fermentation broth containing cellulase
is simply added to the cellulose hydrolysis tanks. If the cellulase is to be stored
for long periods before use, it must be stabilized against (1) microbial contami-
nation, which uses preservatives such as sodium benzoate, and (2) protein
denaturation, which uses compounds such as glycerol.
The production and use of cellulase on the ethanol plant site therefore has
the advantage that the cost of preservatives and stabilizers, as well as transporta-
tion of the enzyme, is avoided. This is a big cost advantage for on-site enzyme
production.
9.5.3
Commercial Status of Cellulase
There is an ongoing business in cellulase enzymes besides that used for cellu-
lose hydrolysis. Cellulase sales are roughly $ 100 million annually to the textiles,
detergent, animal feed, beverage, and pulp and paper industries [5].
These commercial cellulase enzymes are made by several microbes, including
Humicola, Aspergillus, and Penicillium fungi, and Bacillus bacteria in addition to
Trichoderma. Ethanol production requires aggressive action of cellulase to de-
stroy cellulose, for which Trichoderma cellulase is superior. The other industries
often require milder action and/or specific conditions better suited to other cel-
lulases. For example, the textile industry uses cellulase to soften denim blue
jeans. Humicola cellulases can be less aggressive in this application than Tricho-
derma cellulases, though Trichoderma cellulases can be modified to improve
their performance. Detergents require alkaline conditions that are not easily ac-
cessible to Trichoderma enzymes.
9.6
Cellulose Hydrolysis
9.6.1
Process Description
In cellulose hydrolysis, cellulase enzymes convert the cellulose to glucose. The

pretreated feedstock is conveyed to the hydrolysis tanks in a slurry that is 5–
15% total solids (as high as can be handled). The slurry is adjusted to pH 5 with
alkali and maintained at 50 8C. A single hydrolysis tank has a volume of 200,000
gallons. Crude cellulase broth is added as a liquid at a dosage of 100 liters per
tonne of cellulose. The contents of the tank are agitated to move the material
and keep it dispersed, but not nearly as agitated as a fermentation vessel.
9.6 Cellulose Hydrolysis 203
The hydrolysis proceeds for 5–7 days. As it proceeds, the viscosity of the slur-
ry drops, and the remaining insoluble particles, which are lignin in increasing
proportion, diminish in size. At the end of the hydrolysis, 90% to 98% of the
cellulose is converted to glucose. The remainder is insoluble and contained
within the unhydrolyzed particles, which are mostly lignin.
9.6.2
Kinetics of Cellulose Hydrolysis
Trichoderma cellulase is a mixture of three types of enzymes: cellobiohydrola-

se(CBH), endoglucanase (EG), and beta-glucosidase (BG). CBH enzymes act se-
quentially along the cellulose. Trichoderma cellulase includes two CBH enzymes,
CBHI and CBHII, which together account for 80% of the total cellulase protein.
EG enzymes act to cut random locations on the fiber. Trichoderma makes at
least 4 different EG enzymes: EGI, EGII, EGIII, and EGV. The EG enzymes ac-
count for about 20% of the cellulase protein. The third type of enzyme, beta-
glucosidase, hydrolyzes the glucose dimer cellobiose to glucose.
The enzymatic hydrolysis of cellulose proceeds as two consecutive reactions:
C5 H10 O5 n H2 O ! C5 H10 O5 n 2 C12 H22 O11 5
C12 H22 O11 H2 O ! 2C6 H12 O6 6
Eq. (5) depicts the hydrolysis of cellulose to its soluble dimer, cellobiose. This re-
action is catalyzed by the CBH and EG enzymes. The CBH and EG enzymes
work synergistically to hydrolyze cellulose. Reaction (6) is the hydrolysis of cello-
biose to glucose. This reaction is catalyzed by the soluble enzyme beta-glucosi-
dase and proceeds according to standard Michaelis-Menten kinetics. BG ac-
counts for less than 1% of the total cellulase protein. Hydrolysis of the cello-
biose is important because glucose can be readily fermented to ethanol while
cellobiose is not. In addition, cellobiose is a potent inhibitor of CBH and EG, so
the accumulation of even 5 g L–1 cellobiose slows down the hydrolysis signifi-
cantly.
The properties of the Trichoderma cellulase enzymes are summarized in Ta-
ble 9.3.
There are several inherent difficulties in cellulose hydrolysis that have been
the focus of much research. The first is the inherent shortage of BG, both be-
cause of its low concentration and because it is inhibited by its product glucose.
With a shortage of BG, cellobiose accumulates, thereby inhibiting the action of
CBH and EG in hydrolyzing cellulose.
Three approaches have been proposed to overcoming the shortage of BG. The
first is to produce BG in a separate fermentation by Aspergillus spp. The disad-
vantage of this is the added process cost of a second fermentation.
The second and most widely discussed approach is to carry out a simulta-
neous saccharification and fermentation (SSF) process. In SSF, the enzymatic
Table 9.3 Trichoderma cellulase enzymes.
Enzyme Mol. wt. Isoelectric point Family Concn. (%) Ref.
CBHI 63 000 4.3 7 50–60 6

CBHII 58 000 6.0 6 15–18 7
EGI 53 000 4.6 7 12–15 8
EGII 50 000 5.3 5 9–11 9
EGIII 25 000 7.4 12 0–3 10
EGV 23 000 3.7 45 0–3 11
hydrolysis and glucose fermentation are run simultaneously, with the notion
that the yeast consumes the glucose to prevent inhibition of BG, which can
then hydrolyze cellobiose and lift the inhibition of CBH/EG. In Iogen’s re-
search, SSF systems have achieved poor results, because the enzyme’s optimum
temperature is 50 8C and the yeast runs best at 28 8C, so a compromise tempera-
ture of 37 8C is used. In addition to the loss of rate, microbial contamination is
observed at 37 8C. We have put this approach aside.
The third option is to develop Trichoderma strains with high beta-glucosidase
production included in the CBH/EG. This has been carried out at Iogen [12]
and has largely overcome the shortage of BG.
Other important difficulties in the hydrolysis are the decrease in rate as hy-
drolysis proceeds and the diminishing returns with enzyme dosage, i.e. dou-
bling the amount of enzyme does not double the extent of conversion achieved.
These issues, which are probably linked, are not well understood and differ sub-
stantially from Michaelis-Menten kinetics. These effects can, however, be charac-
terized empirically.
The enzyme components initially must adsorb to the surface of the insoluble
substrate cellulose. An equilibrium corresponding roughly to a Langmuir ad-
sorption isotherm is reached within a few minutes. The adsorbed enzyme then
acts on the cellulose at a rapid initial rate. The rate declines significantly after
the first few minutes of hydrolysis, and after 24 h is less than 2% of the initial
rate. The hydrolysis continues over several days at ever decreasing rates. De-
pending on the enzyme dosage used and the duration of the hydrolysis, the fi-
nal cellulose conversion is 90% to 98%.
The reason the rate slows down is not fully known. Speculation in the litera-
ture has centered on: end-product inhibition; an increasingly difficulty in hydro-
lyzing the substrate (substrate recalcitrance); and denaturation of the cellulase
protein over time. However, straightforward experiments demonstrate that none
of these factors account for more than a small fraction of the drop in rate.
Further research continues in this area.
9.7 Lignin Processing 205
9.6.3
Improvements in Enzymatic Hydrolysis
Despite many years of research, cellulose hydrolysis remains the least efficient
part of the process. To illustrate the problem, hydrolysis of pretreated cellulose
requires 100-fold more enzyme than hydrolysis of starch. The enzyme manufac-
turing cost is still sufficiently high that the trade-off between enzyme dosage
and hydrolysis time favors longer times and lower dosages.
One approach to potentially decrease the cost of enzymatic hydrolysis is to recycle
enzyme and/or substrate. These ideas have not been fully explored. The cellulase
enzyme adsorbs to the substrate, so recycle of unconsumed cellulose would be one
way to reuse the enzyme. Reuse of the enzyme in solution is also a possibility. The
current configuration of Iogen’s plant does not carry out these recycles, but up-
grades are under way to allow the demonstration plant to evaluate these options.
Another approach to reducing the cost of hydrolysis is to use fed-batch of sub-
strate or of enzyme. Again, these are ideas with a sound basis that have not
been explored. Fed batch systems are especially interesting because they might
allow high cellulose concentrations to be maintained at all times.
The development of superior cellulase enzymes has been explored extensively
at several research labs, with as yet no cellulases found that are superior to Tri-
choderma cellulase. However, the new tools of molecular biology, such as protein
engineering, can be brought to bear on this problem and might lead to success.
Large development efforts are under way at Iogen and at Genencor Interna-
tional, Novozym Biotech, and Diversa to address this area.
Another area that has been widely explored is novel hydrolysis reactors, such
as trickling bed, high shear, etc. Some improvements in efficiency have been re-
ported, but not easily generalized across feedstocks and enzymes. A better un-
derstanding of the nature of the enzyme’s action will be helpful in evaluating
these reactors.
Taking a wider point of view, better hydrolysis would be a direct benefit of
better pretreatment. Pretreatment has been widely studied, but there are always
new ideas on the horizon.
9.7
Lignin Processing
9.7.1
Process Description
The hydrolysis slurry contains glucose, xylose, arabinose, and other compounds
dissolved in the aqueous phase, and insoluble lignin and unconsumed cellulose.
The insoluble particles are separated by a plate and frame filter, with the cake
washed with water to obtain a high sugar recovery. The sugar stream is pumped
to the fermentation tanks. The lignin cake is disposed of. In a full-scale ethanol
plant, the lignin would be burned on site to generate power and steam to run
the plant, with excess electricity sold to the power grid.
9.7.2
Alternative Uses for Lignin
Lignin is a lattice of phenolic propane units. Lignin has been the object of
much study, and a good review of applications of lignin is provided by Chum et
al. [13]. The potential applications of lignin fall into the broad categories of in-
soluble and chemically modified. Insoluble lignin is limited to high-volume,
low value applications such as an ingredient in roads or cement. In these appli-
cations, lignin is a filler and competes with corn cobs, gravel, and ground bark.
Chemically modified lignin has a much wider variety of potential markets.
The major types of chemical modifications are to solubilize the lignin, by reac-
tion with i.e. sulfurous acid, or to crosslink the lignin, by reaction with i.e. phe-
nol. Crosslinked lignin is suitable for resins, glues, and other such materials,
where it competes with phenol–formaldehyde resins. Solubilized lignin, i.e. so-
dium lignosulfonate, is used in surfactants, detergents, and biocides.
9.8
Sugar Fermentation and Ethanol Recovery
The hydrolysis sugars, which consist of glucose, xylose, arabinose, and various
organic impurities in aqueous solution, are pumped to the sugar fermenters for
ethanol production. The fermenters are large tanks with gentle agitation, suffi-
cient to keep the contents moving. The fermenters are inoculated with Saccharo-
myces yeast, which readily ferment the glucose to ethanol, and have been geneti-
cally modified for metabolism and fermentation of xylose. These yeast strains
have been developed at Purdue University [14]. The advantage of these strains
over others is that the plant operations (contamination control, cell recycle, and
markets for spent cells) involving Saccharomyces yeast are well developed, and
the ethanol tolerance of the strains are good. The areas for further improvement
include developing the inability to ferment arabinose to ethanol and increasing
the yields and rates of xylose fermentation.
In addition to Saccharomyces yeast, other microbes are under development to
ferment xylose to ethanol, which is not carried out naturally in high efficiency.
Pichia yeast have a natural ability for xylose uptake, but require genetic modifi-
cation to ferment the xylose to ethanol. This strategy is carried out at the Uni-
versity of Wisconsin [15]. In addition, Zymomonas bacteria have been genetically
modified for xylose uptake and metabolism. This strategy is carried out by the
National Renewable Energy Laboratory in Boulder, Colorado [16]. Two other
strategies for pentose fermentation include genetically modified enteric bacteria
such as E. coli and Klebsiella, carried out at the University of Florida [17–19],
and thermostable Bacillus strains developed at Imperial College.
References 207
After fermentation, the broth containing ethanol and unfermented sugar is

pumped to a distillation column. The ethanol is distilled off the top and dehy-
drated. The ethanol yield is about 75 gallons per tonne wheat straw. The ethanol
is denatured with 1% gasoline. The denatured ethanol is then blended into ga-
soline in 10% or 85% ethanol mixtures. The still bottoms are disposed of. Tests
are under way to determine whether the still bottoms can be recovered for by-
products.
References
1 Foody, B., Tolan, J. S., Bernstein, J., Pre- 10 Ward, M., Clarkson, K. A., Larenas, E. A.,
treatment process for conversion of cellulose Lorch, J. D., Weiss, G. L., DNA Sequence
to fuel ethanol, US Patent 5,916,780 is- Encoding Endoglucanase III Cellulase, US
sued June 29, 1999. patent 5,475,101 issued Dec. 12, 1995.
2 Singh, L., Scenarios of U.S. Carbon Re- 11 Saloheimo et al., A novel, small endogluca-
ductions: Potential Impacts of Energy Tech- nase gene, egl5, from Trichoderma reesei iso-
nologies by 2010 and Beyond, Interlabora- lated by expression in yeast, Molecular Mi-
tory Working Group on Energy Efficient crobiology, 1993, 61, 1090–1097.
and Low-Carbon Technologies (ORNL, 12 White, T. C., Hindle, C., Genetic constructs
LBNL, PNNL, NREL, ANL), 1997. and genetically modified microbes for en-
3 Foody, P., Method for Increasing the acces- hanced production of beta-glucosidase. US
sibility of cellulose in lignocellulosic materi- patent 6,015,703 issued January 18,
als, particularly hardwoods, agricultural res- 2000.
idues, and the like, US patent 4,461,648 13 Chum, H. L., Parker, S. K., Feinberg,
issued July 24, 1984. D. A., Wright, J. D., Rice, P. A., Sinclair,
4 Katzen, R., Madsen, P. W., Monceaux, S. A., Glasser, W. G., The Economic Con-
D. A., Bevernitz, K., Use of Cellulosic Feed- tribution of Lignins to Ethanol Production
stocks for Alcohol Production, Chapter 5 of from Biomass, National Renewable En-
The Alcohol Textbook, edited by T. P. ergy Lab, Golden, Colo., 1985.
Lyons, D. R. Kelsall, and J. E. Murtagh, 14 Ho, N. W. Y., Chen, Z.-D., Stable Recombi-
1990. nant Yeasts for Fermenting Xylose to Etha-
5 Godfrey, T., Industrial Enzymology, nol, PCT patent application WO 97/
Chapter 1, 1996. 42307, 1997.
6 Shoemaker et al., Molecular cloning of 15 Cho, J., Jeffries, T. J., Pichia stipitis Genes
exo-cellobiohydrolase I derived from Tricho- for Alcohol Dehydrogenase with Fermenta-
derma reesei strain L27, Bio/technology, tive and Respirative Functions, Appl. Envi-
1983, 1, 691–696. ron. Microbiol., 1998, 64(4), 1350–1358.
7 Chen et al., Nucleotide sequence and de- 16 Mohagheghi, A., Evans, K., Finkelstein,
duced primary structure of cellobiohydrolase M., Zhang, M., Cofermentation of Glucose,
II from Trichoderma reesei, Bio/technol- Xylose, and Arabinose by Mixed Cultures of
ogy, 1987, 5, 274–278. Two Genetically Engineered Zymomonas
8 Penttila et al., Homology between cellulase mobilis Strains, Applied Biochem. Bio-
genes of Trichoderma reesei: complete nu- technol., 1998, 70–72, 285.
cleotide sequence of the endoglucanase I 17 Ingram, L. O., Conway, T., Clark, D. P.,
gene, Gene, 1986, 45, 253–263. Sewall, G. W., Preston, J. F., Genetic Engi-
9 Saloheimo et al., EGIII, a new endogluca- neering of Ethanol Production in Escheri-
nase from Trichoderma reesei: the character- chia coli, Appl. Environ. Microbiol., 1987,
ization of both gene and enzyme, Gene, 53(10), 2420–2425.
1988, 63, 11–21. 18 Ohta, K., Alterthum, F., Ingram, L.O.,
Effects of Environmental Conditions on Xy-
lose Fermentation by Recombinant Escheri- Production: chromosomal integration of Zy-

chia coli, Appl. Environ. Micro., 1990, momonas mobilis genes encoding pyruvate
56(2), 463–465. decarboxylase and alcohol dehydrogenase
19 Ohta, K., Beall, D. S., Mejia, J. P., Shan- II, Appl. Environ. Micro., 1991, 57(4),
mugam, K. T., Ingram, L. O., Genetic Im- 893–900.
provement of Escherichia coli for Ethanol
209
10
Sugar-based Biorefinery –
of Poly(3-hydroxybutyrate), Sugar, and Ethanol
10.1
Introduction
Poly(3-hydroxybutyric acid) polymer (PHB) and related copolymers such as

poly(3-hydroxybutyric-co-3-hydroxyvaleric) are natural polyesters synthesized by a
wide range of organisms, particularly some bacterial strains. They have very in-
teresting properties, for example they are totally and rapidly biodegraded to car-
bon dioxide and water by many different microorganisms and are biocompati-
ble. These polyesters can be compounded to thermoplastic resins whose physi-
cochemical and mechanical properties are quite similar to petrochemical-based
polymers such as polyethylene and polypropylene. PHB can be produced in an
environmentally safe way integrated to a sugar mill. This context, with its large
quantities of readily available and comparatively low-cost sugar, and accessible
thermal, mechanical, and electrical energy obtained from renewable agricultural
sources, could be the optimum place to introduce a large-scale facility for its
production.
10.2
Sugar Cane Agro Industry in Brazil – Historical Outline
10.2.1
Sugar and Ethanol Production
During the last quarter of the 20th century, Brazil’s sugar cane agroindustry un-
derwent major changes. This initially traditional sector, previously devoted spe-
cifically to the production of sugar and occasionally to by-products, recovering
or producing very few new products from available sugar or molasses, shifted
its scope to diversify its production lines. In 1975, after the international oil cri-
sis and the rise of oil prices, the Brazilian government launched the National
ISBN: 3-527-31027-4
210 10 Sugar-based Biorefinery
Fuel Ethanol Program, with the objective of replacing oil imports with sugar
cane-based ethanol as an alternative fuel. Many mills began to produce aqueous
ethanol, for direct use in converted car engines, or anhydrous ethanol to add to
gasoline as a fuel enhancer. The scale of production increased substantially,
agricultural and industrial productivity grew, and production costs dropped.
This new situation, which led to a favorable change in the country’s energy
balance, caused the production of an excess of bagasse, which has been used as
primary fuel by the mills or has been sold to third parties. Today, Brazil is the
largest worldwide producer of sugar and sugar cane-based ethanol and, on the
basis of cultivated area and income, sugar cane is one of the most important
agricultural activities. This statement is confirmed by figures from the 2003/
2004 season – sugar cane production was 338 316 619. metric tons. Ethanol pro-
duction was 14 107 873 m3, 8 577 410 m3 of which was anhydrous fuel grade and
the remaining 5 530 468 m3 aqueous fuel grade. Sugar production rose to
23 404 703 tons, of which 12 914 468 tons were exported, accounting for revenues
of US $ 2 140 002 217 [1].
10.2.2
The Sugar Cane Agroindustry and the Green Cycle
The modern sugar agroindustry, which tends to operate as a “green cycle”, can
achieve a unique condition as a source of renewable raw materials if a continu-
ous effort is made to keep its production practices environmentally friendly.
The basis of this sector is sugar cane (Saccharum officinarum), a subtropical gra-
mineous plant cultivated on a large scale in Brazil’s south central region with
productivity averaging 85 tonnes per hectare per year and for which production
costs are low. Sugar cane captures solar energy and converts atmospheric carbon
dioxide into biomass. Macedo [2] produced an inventory of carbon dioxide re-
leased during sugar cane processing into final products and, after fuel ethanol
combustion, concluded that the net balance was positive and that atmospheric
carbon dioxide is effectively removed by cane crops. Macedo also demonstrated
that the balance between energy available from final products such as fuel etha-
nol, bagasse and electrical power generated versus energy consumption during
cane culture and industrial processing is positive [3, 4].
The sugar mills operating in Brazil today are very large units functioning as
autonomous industrial complexes. Their main products are sugar and fuel etha-
nol, and bagasse – the fibrous fraction of sugar cane – is the primary fuel. Ba-
gasse burned in high-pressure boilers provides mechanical power for driving
mill tandems, electrical power to meet the requirements of sugar and ethanol
production, and the heat demand involved in processing cane into final prod-
ucts. Mills whose energy production is well planned and optimized exceed their
energy requirements, generating excess electrical power and bagasse. Modern
mills have large facilities for processing and cooling water, and systems for recy-
cling liquid and effluent to irrigate and/or fertilize crops. Vinasse from ethanol
distillation, condensed water from sugar processing, filter sludge resulting from
10.2 Sugar Cane Agro Industry in Brazil – Historical Outline 211
Fig. 10.1 Sugar cane processing to sucrose, ethanol, by-products, and new products.
juice treatment, and ashes and carbon residue captured in the treatment of boil-
er exhaust gases are disposed of in this way.
The idea of integrating the production of new products with that of sugar is
not new. Sugar cane is a renewable agricultural resource and its processing
yields sugar, cane juice, syrup, and molasses, a very rich substrate that is con-
vertible by fermentation into higher value chemicals such as organic acids, ami-
no acids, and biopolymers, etc. Sugar, a highly reactive molecule, can be chemi-
cally converted into valuable products, as indicated by Kahn [5]. Ethanol, the
other main product of Brazilian sugar mills, is the starting molecule of ethanol
chemistry, whereby important basic chemicals such as aldehyde, acetic acid, and
acetates are produced. Paturau [6] describes some successful examples of inte-
gration at sugar mills whose processing involves products such as citric acid,
fodder yeast and yeast products, acetic acid, vinegar and cellulose, paper, and ba-
gasse-based fiberboard.
The block diagram in Fig. 10.1 illustrates the context of green cycle produc-
tion from renewable agricultural raw materials.
10.3
Biodegradable Plastics from Sugar Cane
10.3.1
Poly(3-Hydroxybutyric Acid)
10.3.1.1 Biodegradable Plastics and the Environment

A wide variety of synthetic plastics have been applied in many products, replac-
ing metal, wood, natural rubber, and other materials, and developing into a base
industry in industrialized countries since the early 20th century. Today, the pro-
duction of plastics amounts to 150 million tonnes year–1 and the trend is still
upward [7, 8]. The main polymeric materials are oil-derived, and the world’s
growing population has led to increasing oil consumption, which may lead to
its depletion as a natural resource [7]. Another problem is the environmental
pollution resulting from the disposal of polymeric materials, which may take
hundreds of years to decompose. This, with the world’s growing environmental
awareness, renders the characteristic of nondeterioration desired for a material’s
use an inconvenience during its disposal.
Although attempts have been made to solve this environmental problem by
recycling techniques, despite its wide acceptability, recycling alone has proved
insufficient to solve environmental problems, because it is impossible to recover
all discarded plastic by this process. The recycling of loaded materials and mul-
ticomponents is usually a more complex process. One relevant aspect is that
such processes usually consume substantial amounts of energy [7, 8]. Another
means of treating solid residues (discarded plastics) is by incineration; this also
generates environmental problems, for example air and water pollution, because
it releases aggressive chemical agents and causes global warming by release of
carbon dioxide. These effects are not restricted to the places where such tech-
niques are employed but are spread around the globe [7, 8].
In this context, the search for a material that is durable while in use and deg-
radable after disposal has led to the emergence of biodegradable plastics – mate-
rials that decompose into low molar mass compounds as a result of the action
of microorganisms (bacteria, fungi). Biodegradable materials were initially used
in medical applications such as sutures, prostheses, controlled drug-release sys-
tems, vascular grafts, etc., owing to their biocompatibility, their ability to dis-
solve and be absorbed by the body, and because their mechanical properties
were appropriate for such applications. More recently, biodegradable plastics
have been applied elsewhere, including packaging and agriculture (plant con-
tainers, controlled release of chemical substances, etc.) [10, 11]. According to
U. J. Hänggi [12], because of their higher costs, biodegradable plastics should
not be used as a substitute for traditional materials but in applications where
traditional plastics are inappropriate. It is worth noting that biodegradable plas-
tics cannot yet compete with traditional plastics because of their higher cost,
and that some types are limited to products which are stored for less than one
year [12].
10.3.1.2 General Aspects of Biodegradability

It is important to clearly define the concepts of degradation and biodegradation.
According to the ASTM D-833 there are [13]:
· degradable plastic which, under specific environmental conditions, undergoes
significant changes in chemical structure, resulting in loss of some of its
properties, and
· biodegradable plastic, degradation of which also results from the action of
naturally occurring microorganisms such as bacteria, fungi, and algae.
Biodegradable plastic must initially be broken down into fragments of low-mo-

lecular-mass by chemical reactions, after which they can be absorbed by micro-
organisms, because they are inert to microorganism attack in their original
form [8]. These reactions can be induced by oxidative enzymes, which cause
superficial erosion, or by abiotic mechanisms (without the presence of living or-
ganisms), through hydrolytic or oxidative reactions. In the former process a bac-
terial or fungal colony on the surface of the material releases an extracellular
degrading enzyme which breaks down the polymer into smaller units (mono-
mers or oligomers) which are then absorbed by the microorganisms’ cell walls
and metabolized as a source of nutrients (carbon). It has been proposed that
this mechanism first hydrolyzes the chains of the amorphous phase of PHB
and then proceeds to attack the chains in the crystalline state. The enzymatic
degradation rate decreases as the crystallinity increases [14, 15]. In the latter sit-
uation, hydrolytic and oxidative mechanisms occur in the absence of living mi-
croorganisms and are restricted to the amorphous phase and the borders of the
crystals, because the crystalline regions are almost impermeable to water and
oxygen. The action of the microorganisms begins after the polymeric chains
have been broken down.
Although the phenomenon of biodegradation seems simple, it is actually
quite complex, for it is affected by several factors which are often interrelated.
The rate of biodegradation of these materials may vary over time and depends
on the material and on environmental factors, for example the type of repeating
unit (nature of the functional group and its complexity), morphology (crystallini-
ty, size of the spherulites), hydrophilicity, surface area, presence of additives,
and environment (humidity, temperature, pH, etc.) [9, 15]. A complete under-
standing of the degradation process will enable us to optimize the entire life cy-
cle of the materials obtained [16]. The products resulting from the biodegrada-
tion process are carbon dioxide and water in aerobic environments and carbon
dioxide and methane in anaerobic environments [17].
10.3.2
Poly(3-Hydroxybutyric Acid) Polymer
10.3.2.1 General Characteristics of Poly(3-hydroxybutyric acid)

and its Copolymer Poly(3-hydroxybutyric Acid-co-3-hydroxyvaleric Acid)
PHB is an environmentally degradable material belonging to the polyhydroxyal-
kanoates (PHA) family, alkanoic acid polyesters which were first described in
1926 by Lemoigne [18]. Figure 10.2 shows the general chemical structure of the
PHA and the biodegradable PHB plastic.
This is a material with a unique characteristic among thermoplastics because
it presents a complete cycle starting from sugar cane and bacterial fermentative
synthesis. PHB is produced, molded, and, after a period of use, discarded and
transformed into compost, thus completing the natural cycle. In the absence of
microorganisms biodegradability the hydrolysis of PHB in aqueous environ-
ments is slow, because of its hydrophobicity. Thus, in principle, the lifetime of a
stored PHB product is unlimited; after its disposal, however, PHB becomes
clearly biodegradable in domestic effluent-treatment systems [19].
PHB is a biocompatible, biodegradable, thermoplastic, hydrophobic, and
stereospecific material. It has a high molecular mass, high crystallinity (55 to
75%), good chemical resistance, and its barrier properties enable practical
packaging applications [20, 21]. Table 10.1 lists the physical and thermal charac-
teristics of this polymer.
This material has unit cells with an orthorhombic structure, with the lattice pa-
rameters a = 5.76 Å, b = 13.20 Å, c = 5.96 Å [22]. Many of the physical and mechanical
characteristics of PHB are similar to those of polypropylene (PP), among them [14]:
Fig. 10.2 Chemical structure of the repetition unit (mer)

present in macromolecules of (a) PHA and (b) biodegradable
PHB plastic.
Table 10.1 Physicochemical properties of PHB [14, 18, 21, 22].
Properties Units Reference values
Mean weighted molar mass Dalton (Da) 200 000 to 600 000
Crystalline melting temperature (Tm) 8C 165 to 175
Vitreous transition temperature (Tg) 8C 0 to 5
Theoretical density of 100% crystalline PHB kg m–3 1260
Density of amorphous PHB kg m–3 1180
· Young’s modulus 2.5–3.5 GPa (comparable with PP or PET)

· tensile strength 20–40 MPa
· Elongation at rupture 3–5%.
The processibility, high crystallinity, and high Tm value of PHB can be altered by
bacterial fermentation or use of polymer blends. The various copolyesters (PHBV)
include random copolyester (R)-3-hydroxybutyrate with (R)-3-hydroxyvalerate,
P(3HB-co-3HV), and random copolyester (R)-3-hydroxybutyrate with (R)-4-hydro-
xybutyrate, P(3HB-co-4HB). The copolyesters are characterized by their lower crys-
talline melting point, hardness, tensile strength, and crystallinity (hence, greater
ductility and elasticity) than pure PHB. Their physical and thermal properties
can be adjusted by varying the HV content of P(3HB-co-3HV) and the 4-HB con-
tent of P(3HB-co-4HB). Simply varying the copolymer content [15, 21] enables pro-
duction of a material with elastomeric characteristics from one which is rigid and
highly crystalline. For P(3HB-co-3HV) the crystallinity usually decreases with in-
creasing HV content. The Tm of PHB of approximately 175 8C drops to 71 8C for
P(3HB-co-3HV) with a 3HV molar mass of 40%. The Tg of P(3HV) varies from
–10 to –12 8C and the Tm varies from 107 to 112 8C [14].
Although copolymers such as PHBV can be used to prevent thermal degrada-
tion (lower melting point), the comonomer must be inserted homogeneously
into the polymeric chains. For example, for a comonomer content of 16 mol%
HV inserted homogeneously into the polymeric chains the corresponding Tm is
approximately 140 8C and a single melting peak is obtained for the copolymer
in differential scanning calorimetry (DSC) thermal curves. If the comonomer is
not inserted uniformly into the PHB chains, however, multiple melting peaks
will appear, and the highest-melting peak among these multiple peaks will be
located at a temperature exceeding that at which the material is thermally stable
(below 160 8C). The HV content is therefore important for the reduction of the
system’s Tm; equally important is reduction of the heterogeneity of the HV con-
tent in the copolymer [23].
The physical properties of copolymers can be altered by varying the comonomer
content, but the use of polymeric blends and incorporation of plasticizers are pre-
ferable alternatives, because they enable greater versatility in the modification of
properties. The synthesis of copolymers is also a more complex process.
10.3.2.2 Processing of Poly(Hydroxybutyrates)

Poly(hydroxybutyrate) can be processed as a conventional thermoplastic in most
industrial transformation processes, including extrusion, injection, and thermo-
pressing. By extrusion, PHB can be transformed into rigid shapes (for example
pipes) and films for packaging. PHB can also be modified by extrusion by incor-
poration of additives (stabilizers, plasticizers, and pigments), immiscible addi-
tives (e.g. wood and starch powder), or by mixing with other plastics. Because
poly(hydroxybutyrate) undergoes thermal degradation at temperatures above
190 8C, the extruder’s temperature profile must be as low as possible (approx.
150 8C), and the screw speed must be appropriate. Because of the need for strict
control, the processing window of PHB is narrow in comparison with that of
conventional plastics. It should be pointed out that virgin PHB is supplied in
the powder form, requiring an appropriate screw profile to enable it to be pro-
cessed efficaciously.
Poly(hydroxybutyrate) is highly versatile when used in the injection process
and is easily molded into pieces of varying shapes and sizes, which may range
from a few grams to several kilograms. The main uses of this material include
injectable packaging for cosmetics and food, agrotoxic packaging and tubettes,
and medical and veterinary implants. As in extrusion, injection of poly(hydroxy-
butyrate) should be conducted with a nonaggressive temperature profile and a
low injection pressure to prevent the appearance of burrs.
PHB can also be thermopressed into sheets or other flat shapes. Because
characteristics that affect the speed of biodegradation (for example crystallinity,
physical shape, molar mass distribution, and compaction of the material) can
be altered during processing, PHB samples that have undergone different types
of processing may not biodegrade at the same rate. When using most types of
conventional equipment, without inclusion of special modifications such as
screws or matrixes with specific designs, several operating procedures must be
observed if products with satisfactory end quality are to be obtained. Extrusion
or injection processes can use typical polyolefin processing screws (L/D 20 : 1)
designed to minimize the exposure time of the melted biopolymer. After use
the equipment can be purged using low-density polyethylene resin as vehicle.
To prevent marked thermal degradation poly(3-hydroxybutyric acid) should not
be exposed to temperatures exceeding 160 to 170 8C for more than 5 min. The
polymer’s “window of processibility” is relatively narrow compared with that of
olefinic polymers, so precise control of the processing temperature is required
to prevent temperature peaks in the heating resistances, which would lead to
rapid degradation of the material. The “window of processibility” can be moni-
tored by observation of the surface characteristics of the molded component, for
example rugosity and shine, which are highly indicative for this polymer.
When using multistage injection equipment, it is advisable to fill the mold
cavity rapidly, using high injection velocities combined with high pressures, and
ensuring that the injection pressure applied does not lead to the appearance of
defects such as burrs and rugosity, because the fluidity index of poly(3-hydroxy-
butyric acid) drops rapidly when the mass is subjected to high shearing rates.
Poly(3-hydroxybutyric acid) does not usually require long cooling times in the
mold in comparison with polyolefins. To minimize PHB-molding “cycle times”
it is advisable to use injection molds heated to approximately 50 8C rather than
injection molds with cold water systems at approximately 10 8C; this enables
faster crystallization, with direct consequences on the thermomechanical proper-
ties of the molded component. This mold temperature is appropriate, because it
is approximately 50 8C higher than the polymer’s vitreous transition tempera-
ture. The use of this procedure improves the characteristics of ejectability of the
molded component, resulting in a gain in “cycle times”.
10.4
Poly(3-Hydroxybutyric Acid) Production Process
Poly(3-hydroxybutyric acid) is produced in an aerobic fermentation process in

which the sugar carbon source is converted into biopolymer by means of the mi-
croorganism Ralstonia eutropha. Biopolymer stored in cells as a carbon reserve is
recovered by an extraction and purification process described by Derenzo et al.
[24]. A description of the fermentation process, microorganism strains, culture
media, and the fermentation procedure has been given by Braunegg [25].
10.4.1
Sugar Fermentation to Poly(3-Hydroxybutyric acid) by Ralstonia eutropha
The fermentation process follows the procedure described by Bueno et al. [26].
A strain of Ralstonia eutropha is grown under aerobic conditions to furnish a
high-cell-density culture. After this step the culture conditions are altered and
the metabolic pathway is shifted to biosynthesis and intracellular storage of
poly(3-hydroxybutyric acid). The block diagram in Fig. 10.3 illustrates the fer-
mentation process; its main characteristics are listed in Table 10.2.
Industrial strains of the Ralstonia genus, for example those described by Brau-
negg [25], are employed in fermentation. Some varieties are highly productive
and can store up to 80% of their dry weight as biopolymer. When cultured in a
medium containing sugar, ammonium, phosphorus, and salts with a low car-
bon-to-nitrogen ratio they produce mainly biomass; when cultured with a high
carbon to nitrogen ratio, however, their growth phase stops and they begin
synthesizing biopolymer.
Fig. 10.3 Fermentation for PHB production.

Table 10.2 Fermentation process data.
Biomass concentration in final culture (dry basis) 150 kg m–3
Poly(3-hydroxybutyric acid) content of biomass 75%

Fermentation yield 98%
Fermentation productivity 1.89 kg m–3 h–1
Remaining reducing sugars in culture 10 kg m–3
Fermentation temperature 32–34 8C
Fermentation pH 6.8
Molecular weight of poly(3-hydroxybutyric acid) obtained 1200 kDa
Fermentation is by a fed-batch procedure at temperatures of 32–34 8C and

pH 6.8. Vigorous aeration and stirring must be provided to accommodate the
high oxygen uptake of the biomass. The cell growth stage is highly exothermic
and requires the assistance of an efficient cooling system. The growth phase
lasts from 24 to 28 h and is followed by the polymer biosynthesis stage, which
lasts 30–35 h. At the end of fermentation, when the poly(3-hydroxybutyric acid)
content has reached 75% of the dry biomass weight, the microorganisms are in-
activated by heating to 105 8C for 5 min to prevent polymer loss, because starved
Ralstonia use it as a carbon source in the absence of reducing sugars. The final
culture is then ready for the polymer recovery stage.
10.4.2
Downstream Processing for Recovery and Purification
of Intracellular Poly(3-Hydroxybutyric Acid)
10.4.2.1 Processes for Extraction and Purification of Poly(hydroxyalkanoates)

Recovery of poly(hydroxyalkanoates) in biomass from fermentation involves several
complex steps, for example microorganism cell breakdown, removal of impurities,
and purification of the final product. These steps are critical from the standpoint of
production cost and polymer quality [28]. Use of organic solvents, some of them
chlorinated, therefore is unavoidable, at least during the final steps of purification.
These solvents are usually hazardous to human health and the environment. The
large-scale production of a biodegradable and biocompatible product using envir-
onmentally aggressive procedures is contrary to common sense. Poly(hydroxyalk-
anoate) production should be conducted according to a green cycle, using renewable
materials and energy, to avoid impact on the environment [27]. The product recovery
process can follow two main routes – biomass digestion and solvent extraction [29].
10.4.2.2 Chemical Digestion

Chemical digestion of microorganism cells using strong oxidants such as sodium
hypochlorite was one of the initial procedures for recovering intracellular poly(hy-
droxyalkanoates) [31]. The granules obtained were washed with solvents such as
diethyl ether or methanol to increase their purity. A drawback of this procedure is

the possibility of partial breakage of the polymer, reducing its molecular weight
and the purity of the final product. Improvements introduced in this procedure,
mainly an organic solvent wash of the polymer granules [32–34], yielded a 95%
pure product with a molecular weight of 600 kDa. The main disadvantage of
chemical digestion is the resulting harmful and heavily polluting effluents.
10.4.2.3 Enzymatic Digestion

This procedure, which was introduced by Imperial Chemical Industries [35–37]
for commercial production of poly(3-hydroxybutyrate-co-valerate), consists of en-
zymatic digestion of biomass to recover polymer granules. Biomass cells are
subjected to thermal treatment at 100–150 8C and pH 6.8 to dissolve the nucleic
acid and denature proteins. After this pretreatment, the material is digested
with a mixture containing enzymes such as phospholipidase, proteinase, and al-
kalase. The granules thus obtained are 90% pure, containing 6–7% of proteinac-
eous material and 3–4% of peptidoglycan as impurities. To obtain a polymer
with the required purity, the procedure must include a solvent-treatment step.
The solvent step increases production costs and requires the use of environmen-
tally harmful solvents.
10.4.2.4 Solvent Extraction

Many extraction and purification procedures using solvents have been reported
[30]. The dry biomass is treated with a chlorinated solvent such as chloroform
[38–43], dichloroethane [45, 46], 1,1,2-trichloroethane [46], dichloromethane [47,
48], and/or propylene carbonate [48]. The polymer is dissolved by the solvent,
suspended insoluble material is then removed by filtration, and the poly(hy-
droxyalkanoates) are recovered from the solution by precipitation with metha-
nol, diethyl ether, or hexane. This procedure yields a highly pure final product,
but requires the use of undesirable toxic compounds.
Extraction Process in Brazilian Patent PI 9302312-0 Extraction using a renew-

able, biodegradable solvent is described in the Brazilian patent PI 9302312-0.
Figure 10.4 illustrates the solvent extraction and purification of poly(3-hydroxy-
butyric acid) with isoamyl alcohol, a byproduct of ethanol fermentation [50].
Thermally inactivated fermentation liquor is diluted with water and then
coagulated by adding phosphoric acid and lime. A polyelectrolyte is added to
flocculate particles, which are recovered by settling and centrifugation. A sludge
containing 25 to 35% solids is recovered and sent to the extraction stage. Extrac-
tion is performed in a set of multistage continuous stirred-tank reactors coupled
with hydrocyclones. Vaporized solvent and liquid are fed continuously counter-
current to the biomass flow. Extraction is performed at the boiling point of the
binary mixture. This condition makes it possible to remove excess water, break
the cell wall, and remove the polymer with the aid of the solvent.
Fig. 10.4 Downstream processing of PHB.
Raw extract of Poly(3-hydroxybutyric acid) leaving the extraction stage con-

tains suspended material such as cell debris and calcium salts, which are re-
moved by deep filtration in a bed of diatomaceous material. The clarified extract
is cooled to precipitate the polymer and the debris cake is sent to the solvent-re-
covery section. The solvent-free cake, consisting of biomass debris and filter aid,
is composted and applied to cane crops.
The poly(3-hydroxybutyric acid) suspended in isoamyl alcohol is preconcen-
trated by membrane microfiltration, raising the concentration from 1.5 to 4%.
The solvent permeate is recovered by distillation and the main stream of the
polymer suspension is concentrated in a multistage evaporation system to
which steam and water are added to distil the solvent as a binary mixture. A fi-
nal stripping step completes the removal of the solvent. The poly(3-hydroxybuty-
ric acid) is washed, dewatered, and carefully dried to avoid thermal breakage of
the polymer. The moisture content after drying and cooling is less than 0.3%. A
white powder of 100 to 200 mesh size and 500 to 800 kDa molecular weight is
obtained. The fraction of degraded polymer of molecular weight < 300 kDa is
less than 3%.
This procedure yields a highly pure polymer by solvent extraction, avoiding
the negative environmental impacts of other processes.
10.4.3
Integration of Poly(3-Hydroxybutyric Acid) Production in a Sugar Mill
Production of poly(3-hydroxybutyric acid) is integrated with that of sugar and

ethanol and with the generation and consumption of energy at the sugar mill.
The production of poly(hydroxybutyrates) from sugar cane is conceived by us as
a process to be integrated into sugar mill operations, using not only sugar sub-
strate but also all the facilities the mill can advantageously offer, for example
heating and cooling, electric power, water, and effluent treatment and disposal.
Figure 10.5 shows a flow diagram for simultaneous processing of sugar cane to
sugar, ethanol, and PHB in a typical Brazilian sugar mill in which a milling ca-
pacity of 12 000 tons of sugar cane per day is used for production of sugar (55%
of total milling) and the rest for fuel ethanol, and where PHB production is to
be installed. The milling season last 180 days whereas PHB facilities work all
year round, a total of 330 working days.
Fig. 10.5 Mass and energy balance for sugar, ethanol, and PHB.
Sugar cane processing begins with extraction of cane juice by mill tandems,
leaving behind bagasse, the fibrous material that is sent to the boiler house and
to storage. The cane juice is treated physically and chemically to purify it. Most
of this juice is used for production of sugar and the rest goes to the fermenta-
tion plant to produce ethanol. In the sugar-processing sector, this juice is con-
centrated in multiple effect evaporators, yielding thick syrup consisting of nearly
65% soluble solids. The syrup is then boiled in vacuum pans until sugar crys-
tals are produced by evaporation–crystallization, which is usually performed in
two stages. White sugar of various standards is recovered in this step; the mo-
lasses by-product, left over noncrystallizable thick impure sugar syrup, is nor-
mally fermented and converted into ethanol.
Part of the sugar production will be used to produce PHB, and sent to the
PHB plant for direct use or stored for use during the off-season. At the cane
juice fermentation plant, the molasses and sugar syrup are blended to formulate
the fermenting must. The must then undergoes ethanol fermentation, yielding
the final liquor, which is distilled into aqueous and anhydrous fuel ethanol. By-
products such as fusel oil and yeast are recovered in the ethanol production sec-
tor. Meanwhile, at the PHB production facility, medium quality standard sugar
is processed into PHB by the aforementioned procedure.
Sugar cane milling and ethanol and PHB processing are energy-intensive pro-
cesses that require mechanical and electrical power and thermal energy in the
form of low-pressure steam. Recovered bagasse is burned in a high-pressure
boiler, producing superheated steam at 60 bar and 450 8C. This primary steam
is expanded in high efficiency multistage turbines equipped with electricity gen-
erators, providing the electricity consumed by the manufacturing complex. Me-
dium pressure steam at 20 bar is extracted from multistage turbines and used
as the primary mover for mechanical power generation in the milling step in-
volving cane cutters, defibrators, and extraction tandems. Low-pressure steam
extracted from the turbines is used as the source of thermal energy for sugar,
ethanol, and PHB processing.
The solid effluents from the PHB process will be composted and spread over
sugar cane fields as filtering mud from the juice treatment. Liquid effluents, for
example the final fermentation liquor and the washing water left behind after
removal of microorganism cells containing PHB, will be sprayed on the cane
crops like vinasse from ethanol distillation.
10.4.4
Investment and Production Cost of Poly(3-Hydroxybutyric Acid) in a Sugar Mill
A preliminary analysis has been made of the investments required for, and the
production costs involved in, sugar-derived PHB. The purpose of the work was
to determine the economical feasibility of biodegradable production integrated
with an existing sugar mill. The analysis was based on two scenarios – an
autonomous unit producing 10 000 tons of PHB per year, located outside the
mill site, and an integrated unit having the same production capacity. Construc-
Table 10.3 Investment and production cost of the PHB process.
Investment in US $ %
Buildings and civil works 3 000 000.00 7.9

Fermentation unit 15 000 000.00 39.5
Downstream processing unit 15 000 000.00 39.5
Utilities 5 000.00 13.2
Total 38 000 000.00
Production cost breakdown
Depreciation over buildings and equipment 11
Sugar substrate 29
Other raw materials and chemicals 15.5
Bagasse for energy needs 4.9
Salaries 12.3
Maintenance 5.1
Others 8.6
tion of a manufacturing unit for a production capacity of 10 000 tons per year
requires a large investment. Table 10.3 gives a breakdown of investments and
costs, with installed equipment totaling US $ 38 000 000. The production cost of
PHB is highly dependent on the price of sugar, which is the major factor, ac-
counting for almost 29% of the final cost.
The production cost for a basic 99% pure poly(3-hydroxybutyrate) chemical is
estimated at US $ 2.25 to 2.75 kg–1, depending on the price of sugar, other chemi-
cals, and bagasse. It is worth noting that a similar calculation for an autonomous
PHB unit shows the production cost rises to US $ 2.50 to 3.00 kg–1, indicating the
advantages of integrating PHB production with an existing mill. Comparing our
cost data with Lee’s [51] estimate of $US 2.65 kg–1 and Bertrand’s [52] of
US $ 5.85 kg–1, we conclude that our proposed scenario is feasible.
10.5
Expectations of the development of industrial poly(3-hydroxybutyric acid) pro-

duction by the sugar cane agroindustry are high. There is a large margin for
improvement of the current production process, which will result in lower capi-
tal and production costs, less generation of solid and liquid effluents, and lower
consumption of energy. Poly(hydroxyalkanoate) production technology will un-
dergo significant improvements when novel microorganisms are obtained by se-
lection or by genetic engineering. One target is microorganisms that can direct-
ly assimilate more complex carbon sources, for example sucrose, or even meta-
bolize pentose sugar. Other targets are simultaneous growth and polymer pro-
duction steps and larger percentages of intracellular stored PHB. Efficient
synthesis of heteropolymers other than poly(3-hydroxybutyrate) and fermenta-
tion at higher temperatures and lower medium pH will also affect the fermenta-
tion technology favorably.
Steinbuchel [53] reviewed bacterial strains and possible genetically modified
organisms to improve PHB production. Vicente [54, 55] gave examples of the
construction of genetically modified bacteria for poly(3-hydroxybutyric acid) bio-
synthesis. Poly(3-hydroxybutyric acid) production costs could be significantly re-
duced by optimizing production processes. Moreover, fermentation could be im-
proved to increase the PHB content of the cells to 80%. There is also a margin
for increasing the concentration of biomass in the final liquor to 200 kg m–3, as-
suming the oxygen transfer rate in fomenters is improved. The technology avail-
able today limits fermenter capacity to less than 500 m–3; overcoming this obsta-
cle will lead to increasing gains resulting from scale. The fermentation cycle
can be optimized by reducing the content of reducing sugars in the final liquor
from the current 1 to 0.2%.
There is a broad range of possibilities for optimizing and reducing the cost of
technology available for the extraction and purification of poly(3-hydroxybutyrate).
The consumption of thermal energy and the power requirements in downstream
processing could be reduced. Polymer extraction and purification should be re-
viewed to improve the processing stages and to obtain a purer product of higher
molecular weight and a lower poly dispersion index. A survey of new extraction
solvents is required to identify nontoxic and environmentally friendly solvents that
can dissolve more polymer and less undesirable impurities.
A milestone in poly(hydroxyalkanoate) production will be reached when it be-
comes possible to replace sucrose with carbohydrates contained in the lignin–
cellulose biomass component of sugar cane. The bagasse or trash left behind
after cane harvesting are lower-cost substrates than sugar and, according to Ma-
cedo [3], will be available in large quantities. Depending on the extent of optimi-
zation of energy production and available consumption cycles, the amount of
bagasse will range from 39 to 67 kg ton–1 sugar cane. Biomass availability in
trash is estimated to be 56 to 98 kg ton–1 cane, depending on the harvesting
method employed. In the near future, when the hydrolytic process is commer-
cially ready, this lignin cellulose-based material will be transformed into hexose
and pentose sugars, replacing sucrose in PHB fermentation. Da Silva [56] re-
ports on the biosynthesis of poly(3-hydroxybutyric acid) by strains of Bhukolderia
Spp, using hydrolysis liquor from an organosolve process that yields 65 to 75%
total reducing sugars from bagasse. The Bhukolderia strain can ferment the pen-
tose and hexose sugar contained in bagasse.
Other poly(hydroxyalkanoates) unlike poly(3-hydroxybutyric acid) and with su-
perior functional properties will lead to improvement of biodegradable plastics.
Long chain hydroxyacids and heteropolymer alkanoates can be produced by dos-
ing specific carbon substrates in the polymer-biosynthesis step. Thus, the bio-
logical process could be engineered to produce new polymers with desirable
properties.
References 225
References
1 ÚNICA, www.unica.com.br, 2004. 20 M. K. Cox, International Scientific Work-

2 I. Macedo, São Paulo State Government shop on Biodegradable Polymers and
Report. March, 2004. Plastics, 2. Proceedings, Royal Society of
3 I. Macedo, Copersucar Technical Bulle- Chemistry, Osaka, Japan, 1994, 120–135.
tin. 1985, 31:22–27. 21 H. Sudesh, Progress in Polymer Science
4 I. Macedo, Biomass and Bioenergy. 1996, 2000, 25, 1503–1555.
14:77–81. 22 A. El-Hadi. Polymer Testing, 2002, 21,
5 R. Kahn, H. F. Jones, Chemistry and pro- 665–674.
cessing of sugar beet and sugar cane. 23 M. Yamaguchi, Yokkaichi Research Labo-
M. A. Clarke, M. A. Godshall editors, ratory Report. 2004.
1988, Elsevier Science Publishers B. V., 24 S. Derenzo, Brazilian Patent PI 9302312-
Netherlands. 0, 1993.
6 J. M. Paturau, By-products of the cane 25 Braunegg, J. Biotechnol. 1998, 65, 127–
sugar industry. 1989, Elsevier Science 161.
Publishers B. V., Netherlands. 26 C. L. Bueno, Brazilian Patent PI
7 M. Okada, Progress in Polymer Science. 9103116-8, 1993.
2002, 27, 88–103. 27 R. V. Nonato, Appl. Microbiol. Biotech-
8 W. M. Pachekoski, Ms Sc. Thesis. UFS- nol. 2002, 57, 1–5G.
CAR, SP, Brazil, 2001 28 A. J. Asenjo, Separation Processes in Bio-
9 S. Karlsson, A. C. Alberton, Polymer En- technology, Marcel Dekker, Inc., New
gineering and Science. 1998, 38, 1251– York, USA, 1990.
1254. 29 G. J. L. Griffin, Chemistry and Technol-
10 R. Chandra, R. Rutsgi, Progress in Poly- ogy of Biodegradable Polymers, Blackie
mer Science. 1998, 23, 1273–1335. Academic and Professional, Glasgow,
11 K. Van de Velde, P. Kiekens, Polymer Scotland, 1994.
Testing, 2002, 21, 433–442. 30 E. Berger, Dr. Sc. Thesis, Ecole Polytech-
12 U. J. Hänggi, International Symposium nique, Universite de Montreal, Canada,
on Natural Polymer and Composites, 2. 1990.
Proceedings, Embrapa, SP, Brazil. 1998, 31 D. H. Williamson, J. F. Wilkinson, J.
309–310. Gen. Microbiol., 1958, 19, 198.
13 ASTM. D883: Terminology relating to 32 M. P. Nuti, Can. J. Microbiol., 1972, 18,
plastics. Filadélfia, 2002, vol. 08.01. 1257.
14 C. Ha, W. J. Cho, Progress in Polymer 33 E. Berger, Biotechnol. Tech., 1989, 3 (4),
Science. 2002, 27, 759–809. 227.
15 M. K. Cox, International Scientific Work- 34 B. A. Ramsay, Biotechnol. Technol. 1989,
shop on Biodegradable Polymers and 4 (4), 221.
Plastics, 2. Proceedings, Royal Society of 35 J. M. Merrick, M. Dourdoroff, J. Bacte-
Chemistry, Montpellier. 1992, 95–100. riol. 88(1), 60–71, 1964.
16 P. Gatenholm, A. Mathiason, Journal of 36 P. A. Holmes, European Patent Applica-
Applied Polymer Science 1994, 51, 1231– tion EP 46 335, 1985.
1237. 37 P. A. Holmes, G. B. Lim, European Pat-
17 G. Swift, International Scientific Work- ent Application EP 145 233, 1985.
shop on Biodegradable Plastics and Poly- 38 Y. Inoue, N. Yoshie, Prog. Polym. Sci,
mers. Proceedings, Tokio, Japan. 1994, 1992 17, 517–610.
228–236. 39 M. Lemoigne, C. R. Acad. Sci. 1925,
18 Y. Doi, International Scientific Workshop 180,1539.
on Biodegradable Polymers and Plastics, 40 M. Lemoigne, Ann. Inst. Pasteur. Sci.
2. Proceedings, Royal Society of Chemis- 1925, 39, 144.
try, Montpellier. 1992, 139–148. 41 M. Lemoigne, Bull. Soc. Chim. Biol,
19 G. J. M. Koning, Prospects of Bacterial 1925, 8, 770.
Poly(R)-3-Hydroxyalkanoates, 1993, 5–26.
42 M. Lemoigne, Ann. Inst. Pasteur Sci. 49 J. N. Baptist, US Patent Application

1925, 41 ,148. 3 044 942, 1962.
43 J. Walker, European Patent Application 50 S. Y. Lee, J. Choi, Polym Deg Stab, 1998,
EP 0 046 017, 1982. 59, 387–393.
44 Solvay and Co, European Patent Applica- 51 J. L. Bertrand, Dr. Sc. Thesis, Université
tion EP 14 490, 1979. de Montreal, Montreal, Canada, 1992.
45 P. J. Barham, A. Selwood, US Patent Ap- 52 A. Steinbuchel, B. Fuchttenbusch, Tib-
plication 4 391 766, 1982. tech. 1998, 16, 419–427.
46 P. J. Barham, A. Selwood, European Pat- 53 E. J. Vicente, Brazilian Patent PI
ent Application EP 0 058 480, 1982. 9806581-5, 1998.
47 J. N. Baptist, US Patent Application 54 E. J. Vicente, Brazilian Patent PI
3 036 959, 1962. 9805116-4, 1998.
48 J. N. Baptist, US Patent Application 55 L. F. Da Silva, J. Ind. Microb. Biotech-
3 044 942, 1962. nol., 2004, 31, 245–254.
227
11
Biomass Refineries Based on Hybrid
Thermochemical-Biological Processing – An Overview
Robert C. Brown
11.1
Introduction
The Biomass Research and Development Technical Advisory Committee (2002)

of the US Departments of Energy and Agriculture defines a biorefinery as: “A
processing and conversion facility that (1) efficiently separates its biomass raw
material into individual components and (2) converts these components into
marketplace products, including biofuels, biopower, and conventional and new
bioproducts.” Implicit in this definition is the assumption that grain will be
fractionated into starch, oils, proteins, and fiber and lignocellulosic crops will be
fractionated into cellulose, hemicellulose, lignin, and terpenes before these com-
ponents are converted into market products. Certainly, this is the approach of
modern wet corn milling plants and wood pulp and paper mills.
Another possibility for high fiber plant materials is, however, thermochemical
processing into a uniform intermediate product that can be biologically con-
verted into a biobased product. This alternative route to biobased products is
known as hybrid thermochemical-biological processing or simply hybrid pro-
cessing of biomass. There are two distinct approaches to hybrid processing:
· gasification followed by fermentation of the resulting gaseous mixture of car-
bon monoxide (CO), hydrogen (H2) and carbon dioxide (CO2), and
· rapid pyrolysis followed by hydrolysis and/or fermentation of the anhydrosu-
gars found in the resulting bio-oil.
ISBN: 3-527-31027-4
228 11 Biomass Refineries Based on Hybrid Thermochemical-Biological Processing – An Overview
11.2
Historical Outline
The history of hybrid thermochemical-biological processing is brief. The concept

only emerged in the 1980s and no such processes have been commercially in-
troduced. Although hybrid refining has potential for overcoming some of the
shortfalls of conventional fractionation of biomass, it has not drawn wide attrac-
tion, possibly because it crosses the disparate fields of high-temperature thermo-
chemistry and biology. The following paragraphs give brief histories of the gasi-
fication and fast pyrolysis approaches to hybrid refining.
11.2.1
Origins of Biorefineries Based on Syngas Fermentation
Traditionally, the feedstocks for the manufacture of biotechnology products have

been carbohydrates. Several anaerobic bacteria use C1 compounds such CO,
CO2, and methanol (CH3OH) and hydrogen as sources of carbon and energy
for growth and metabolite production, however. Syngas, a mixture rich in CO,
CO2, and H2, is produced by heating carbon-rich solids under high-temperature
(600–1000 8C), oxygen-starved conditions. Its name derives from the fact that
this gas mixture is used to synthesize a variety of industrially important organic
compounds, for example acetic acid and methanol, by the application of metal
catalysts and elevated temperatures and pressures. Zeikus and his colleagues
(1985) at the Michigan Biotechnology Institute were among the first to propose
substituting biocatalysts for the metal-based catalysts currently used to convert
syngas into industrial chemicals. A few years later Gaddy and coworkers at the
University of Arkansas published a series of papers detailing how a variety of
products, including methane, acetic acid, and ethanol, might be fermented from
syngas (Ko et al. 1989; Vega et al. 1989 a, b, c, d). Because of the US Department
of Energy’s interest in developing alternative transportation fuels from biomass,
much of the early work in syngas fermentation focused on alcohol production.
By 1991, the Michigan Biotechnology Institute had identified the Gram-positive,
nonmotile, rod-shaped anaerobe Butyribacterium methylotrophicum as a possible
candidate for production of alcohol from syngas (Worden et al. 1991) whereas
the University of Arkansas focused on the Gram-positive, motile, rod-shaped
anaerobe Clostridium ljungdahlii (Vega et al. 1989 d). These two groups published
several papers in the 1990s although by 1993 Gaddy had started a company to
commercialize syngas fermentation technology (Tobler 1994) and his group
ceased publishing the results of their investigations.
Only a few other groups have explored syngas fermentation. Elmore and co-
workers at Louisiana Tech University (Madhukar et al. 1996) isolated three un-
identified rod-shaped, Gram-positive cultures that used mixtures of CO, CO2,
and H2 (that is, simulated syngas) as their primary carbon source to produce
acetate, ethanol, methanol, and smaller quantities of other alcohols and organic
acids. Maness and Weaver at the National Renewable Energy Laboratory (1994)
opened up new opportunities for syngas fermentation by exploring the conver-

sion of CO and H2 into poly(3-hydroxybutyrate) by photosynthetic bacteria.
More recently, a team at the University of Oklahoma (Datar et al. 2004) has
demonstrated the production of ethanol from clean syngas derived from a bio-
mass gasifier and Iowa State University (Brown et al. 2003) is exploring the pro-
duction of both hydrogen and polyesters from the purple non-sulfur bacteria
Rhodospirillus rubrum under dark reaction conditions.
11.2.2
Origins of Biorefineries Based on Fermentation of Bio-oils
Bio-oil is a liquid mixture of oxygenated organic compounds produced by heating

finely divided biomass in the absence of oxygen. The process, known as fast
pyrolysis, involves lower temperatures and shorter reaction times than gasification
with the result that it produces mostly liquid product (as much as 70% w/w)
whereas gasification yields essentially all gas. The technology was developed in
the 1980s with the goal of using bio-oil as a substitute for (petroleum-derived)
fuel oil (Bridgwater and Peacocke 2000).
Scott and his coworkers at the University of Waterloo, Ontario (1989) discov-
ered that the amount of anhydrosugars, particularly levoglucosan, in bio-oil
could be dramatically increased by demineralizing the biomass before pyrolyz-
ing it. Because levoglucosan is readily hydrolyzed to glucose, they suggested
that fast pyrolysis be used as an alternative to acid or enzymatic hydrolysis for
recovery of sugars from lignocellulosic biomass.
Recognizing that elimination of the hydrolysis step would improve the pro-
spects for using bio-oil as substrate, Zhuang and coworkers (2001 a) at the Chi-
nese Academy of Sciences adapted a mutant of A. niger CBX-209 to directly fer-
ment levoglucosan to citric acid. They demonstrated that levoglucosan was the
sole source of carbon and energy for this fermentation.
So and Brown at Iowa State University (1999) performed an economic assess-
ment that found the cost of ethanol from this fast pyrolysis route to be, within
the uncertainty of the analysis, comparable to the cost of ethanol from acid hy-
drolysis or enzymatic hydrolysis of woody biomass. The prospects of producing
both power and chemical products by fast pyrolysis of fibrous biomass, which
would constitute a biorefinery, was recently evaluated by Sandvig and his collab-
orators (2004). One manifestation of this biorefinery included recovery, hydroly-
sis, and fermentation of levoglucosan. Although fast pyrolysis is a commercial
technology, it has yet to be employed as part of a hybrid thermochemical-biolog-
ical biorefinery.
11.3
Gasification-Based Systems
11.3.1
Fundamentals of Gasification
Gasification is the high-temperature (750–850 8C) conversion of solid, carbonac-

eous fuels into flammable gas mixtures, sometimes known as synthesis gas or
syngas, consisting of CO, H2, CO2, methane (CH4), nitrogen (N2), and smaller
quantities of higher hydrocarbons (Reed 1981). Not only can syngas be used for
generation of heat and power, it can serve as feedstock for production of liquid
fuels and chemicals. Because of this flexibility of application, gasification has
been proposed as the basis for refineries that would provide a variety of energy
and chemical products, including electricity and transportation fuels.
Gasification consists of several distinct processes: heating and drying of the
fuel; pyrolysis of solid fuel to gases, condensable vapors, and char; solid-gas re-
actions that consume char; and gas-phase reactions that adjust the final chemi-
cal composition of the syngas. Pyrolysis, which begins between 300 8C and
400 8C, may convert up to 80% w/w of solid biomass into gases and vapors. The
pyrolytic gases include CO, CO2, H2, H2O, and CH4 and the condensable vapors
include a variety of hydrocarbons and oxygenated organic compounds. The sol-
id-gas reactions produce CO, H2, and CH4 by the following reactions (Reed
1981):
Carbon-oxygen reaction:
1
C O2 $ CO DHR 110:5 MJ kmol 1
2
Boudouard reaction:
C CO2 $ 2CO DHR 172:4 MJ kmol 1
Carbon-water reaction:
C H2 O $ H2 CO DHR 131:3 MJ kmol 1
Hydrogenation reaction:
C 2H2 $ CH4 DHR 74:8 MJ kmol 1
Two important gas-phase reactions also influence the overall gasification pro-
cess:
Water-gas shift reaction:
CO H2 O $ H2 CO2 DHR 41:1 MJ kmol 1

Methanation:
CO 3H2 $ CH4 H2 O DHR 206:1 MJ kmol 1
The final gas composition is highly dependent on the amount of oxygen and
steam admitted to the reactor and the time and temperature of reaction. For
sufficiently long reaction times chemical equilibrium is achieved and the prod-
ucts are essentially limited to the light gases CO, CO2, H2, and CH4 (and nitro-
gen if air was used as a source of oxygen).
Gasifiers are usually classified according to the method of contacting fuel and
gas. The three main types suitable for biomass gasification are illustrated in
Fig. 11.1 (Brown 2003). Updraft gasifiers are the simplest, consisting of little
more than a grate with chipped fuel admitted from above and insufficient air
for complete combustion entering from below. This countercurrent flow of fuel
and air results in producer gas with large quantities of undesirable tars. In
downdraft gasifiers, fuel and gas move in the same direction with contemporary
designs usually adding an arrangement of tuyeres that admit air or oxygen di-
rectly into a region known as the throat where combustion forms a bed of hot
char. This design assures that condensable gases released during pyrolysis are
forced to flow through the hot char bed, where tars are cracked. A disadvantage
is the need for tightly controlled fuel properties (particles sizes between 1 and
30 cm, low ash content, and moisture less than 30%) and an upper bound on
gasifier size of approximately 2 MW thermal. In fluidized bed gasifiers a gas
Fig. 11.1 Common types of biomass gasifier:

(a) updraft, (b) downdraft, (c) fluidized bed (Brown 2003).
stream passes vertically upward through a bed of inert particulate material to

form a turbulent mixture of gas and solid. They are able to process a wide vari-
ety of fuels and are easily scaled to large sizes. Disadvantages include moderate
tar loadings, high particulate loadings, and relatively high power consumption
to run the air blower.
Overall, gasification is endothermic and requires either simultaneous burning
of part of the fuel or an external source of heat to drive the process. Addition of
air is called air-blown gasification and has the disadvantage of admitting nitro-
gen to the syngas, which dilutes the concentration of reactive compounds in the
syngas and reduces its chemical enthalpy. Substitution of oxygen for air, known
as oxygen-blown gasification, eliminates nitrogen as a diluent but is an extreme-
ly expensive solution to the problem.
Several researchers have developed methods for bringing sufficient heat to
the gasifier without admitting air or oxygen, a process known as indirectly
heated gasification (Bridgwater 1995). Chemical enthalpy from such gasifiers is
typically 200% higher than from air-blown gasifiers.
Often gasifier temperatures and reaction times are not sufficient to achieve
chemical equilibrium and the raw syngas contains various amounts of light hy-
drocarbons such as C2H2 and C2H4 and up to 10% w/w heavy hydrocarbons
that condense to a black, viscous liquid known as “tar.” Furthermore, two kinds
of particulate matter often contaminate syngas. The first, known as ash, is
mineral matter in the raw biomass that remains on completion of gasification.
The second is char formed during pyrolysis but not consumed by solid-gas reac-
tions. Taken together, ash and char are sometimes referred to as ash or gasifica-
tion residue.
Typical gas concentrations and chemical enthalpies for syngas are compared
in Table 11.1 for air-blown and indirectly heated gasifiers. Clearly, the higher
concentrations of CO and H2 from an indirectly heated gasifier reduce the size
of bioreactors needed to convert these gases into organic compounds.
Table 11.1 Syngas composition from various kinds of gasifiers (Brown 2003).
Gasifier type Gaseous constituents (% v/v, dry) HHV Gas quality

(MJ m–3)
H2 CO CO2 CH4 N2 Tars Dust
Air-blown updraft 11 24 9 3 53 5.5 High Low

(*10 g m–3)
Air-blown downdraft 17 21 13 1 48 5.7 Low Medium
(*1 g m–3)
Air-blown fluidized bed 9 14 20 7 50 5.4 Medium High
(*10 g m–3)
Oxygen-blown downdraft 32 48 15 2 3 10.4 Low Low
(*1 g m–3)
Indirectly-heated fluid bed 31 48 0 21 0 17.4 Medium High
(*10 g m–3)
11.3.2
Fermentation of Syngas
Traditional fermentations rely on carbohydrates as the source of carbon and en-

ergy in the growth of microbial biomass and the production of commercially
valuable metabolites. Several microorganisms, however, can use less expensive
substrates for growth and production. These include autotrophs, which use C1
compounds as their sole source of carbon and hydrogen as their energy source,
and unicarbonotrophs, which use C1 compounds as their sole source of both
carbon and energy. Among suitable C1 compounds are CO, CO2, and methanol
(CH3OH), all of which can be produced by thermochemical processing of bio-
mass.
Although both aerobic and anaerobic microorganisms can use C1 substrates,
anaerobes offer the most promising route to chemicals and fuels from syngas,
because they employ very energy-efficient metabolic pathways – most of the
chemical energy of the substrate appears in the products of fermentation.
Organisms that form the metabolic intermediary acetyl-CoA from carbonyl or
carboxyl precursors are known as acetogens. Although many acetogens con-
sume alcohols or fatty acids to produce acetate, CO2, and H2, some are able to
utilize CO2 and hydrogen (autotrophic acetogens) or CO (unicarbonotrophic
acetogens) as substrates for growth and production of organic acids and, occa-
sionally, alcohols (Grethlein and Jain 1993).
As illustrated in Fig. 11.2, metabolism of CO begins with the reaction of CO
and H2O via CO dehydrogenase to yield CO2 and H2; this is the biologically-
mediated water-gas shift reaction. Subsequent steps include production of for-
mate from CO2 via formate dehydrogenase, several tetrahydrofolate-mediated
dehydrogenase transformations resulting in a methyl-corrinoid complex, reac-
tion of CO with CO dehydrogenase to form an enzyme-bound carbonyl moiety,
and synthesis of acetyl-CoA from methyl and carbonyl groups bound to the CO
dehydrogenase complex (Zeikus et al. 1985).
Metabolism of H2 and CO2 occurs by the same mechanism. As shown in
Fig. 11.2, H2 reduces CO2 to a bound form of CO in a ferredoxin-dependent re-
action. The resulting carbonyl group reacts with the methyl-corrinoid complex
described previously via CO dehydrogenase to form acetyl-CoA (Zeikus et al.
1985). Acetyl-CoA is the chemical intermediate in the subsequent formation of
biomass (growth) and metabolites (production), as subsequently described.
The autotrophs and unicarbonotrophs that convert single-carbon compounds
into higher-molecular-weight products are dependent on enzymes and co-en-
zymes that contain nickel, cobalt, iron, tungsten, molybdenum, selenium, zinc,
or combinations of these metals (Zeikus et al. 1985). Similarly, the petrochemical
industry is dependent on metal-based catalysts to convert syngas into products.
This includes nickel catalysts for steam reforming of hydrocarbons, iron-chro-
mium and copper-zinc catalysts to produce hydrogen via the water-gas shift reac-
tion, copper catalysts to produce methanol, and iron or chromium catalysts to pro-
duce hydrocarbons via the Fischer-Tropsch reaction (Spath and Dayton 2003).
Fig. 11.2 Metabolic pathways for acetogenic following enzymic activities: 1, hydrogenase;
bacteria that synthesize acetate or butyrate 2, CO dehydrogenase; 3, formate dehydro-
during growth on C1 substrates or H2 and genase; 4, formyl-THF synthetase; 5, other
CO2. The symbols [CH3OH] and [HCOOH] THF enzymes; and 6, one or more enzymes
represent two oxidation states of C1 units required for the synthesis of acetyl-CoA from
bound to tetrahydrofolate (THF) carriers [CO] and a methyl-corrinoid (Zeikus et al.
whereas the chemical nature of [CO] remains 1985).
undetermined. Numbers indicate the
Biological routes to syngas fermentation have several potential advantages over

conventional catalytic routes (Grethlein and Jain 1993). Most catalysts used in the
petrochemical industry are readily poisoned by sulfur-bearing gases whereas syn-
gas-consuming anaerobes are sulfur-tolerant; thus, expensive sulfur-gas clean-up
can be eliminated by using biological catalysts. In conventional catalytic processing
the CO/H2 ratio of the syngas is critical to commercial operations. Because CO/H2
ratios depend on the quality of the gasified feedstocks, water-gas shift reactors are
required to make this adjustment. Biological catalysts are not sensitive to this ratio;
indeed, the water-gas shift reaction is implicit in the metabolism of autotrophic
and unicarbonotrophic anaerobes. Gas-phase catalysts typically employ tempera-
tures of several hundreds of degrees Centigrade and at least ten atmospheres of
pressure whereas syngas fermentation proceeds at near ambient conditions. Final-
ly, biological catalysts tend to be more product specific than inorganic catalysts.
11.3.2.1 Production of Organic Acids

As illustrated in Fig. 11.2, the primary metabolites from the conversion of C1
compounds by autotrophic and unicarbonotrophic anaerobes are organic acids.
The chemical intermediate acetyl-CoA can produce either acetate via acetyl
phosphate or butyryl-CoA and subsequently butyrate. The relative yields of these

two organic acids depend on the type of organism and the substrate. For exam-
ple, in studies with Butyribacterium methylotrophicum Worden et al. (1989)
showed that the fraction of electrons going from CO into butyrate production
could be increased from 6 to 70% at the expense of acetate production by reduc-
ing the pH from 6.9 to 6.0.
Representative species of acidogenic (acid-forming) anaerobes include Clostri-
dium thermoaceticum, Clostridium ljungdahlii, Peptostreptococcus productus, Aceto-
bacterium woodii, Eubacterium limosum and Butyribacterium methylotrophicum
(Grethlein and Jain 1993), with some of these also forming alcohols, as subse-
quently described.
The metabolism of B. methylotrophicum is given as an example of the molar
stoichiometric yields that can be expected (Bredwell et al. 1999). For CO sub-
strate and acetate production, the molar stoichiometry, balanced for carbon and
available electrons, is:
4CO ! 2:17CO2 0:74CH3 COOH 0:45Cell 1
where Cell indicates C-moles (carbon equivalents) in the cell mass produced. At
least half of the CO must be oxidized to CO2 to provide enough electrons to re-
duce the remaining CO to acetate and cell mass. For CO2 and H2 substrate and
acetate production, the molar stoichiometry is:
2H2 1:03CO ! 0:43CH3 COOH 0:13Cell 2
11.3.2.2 Production of Alcohols

Although production of organic acids seems to dominate the metabolites from
wild strains of autotrophic and unicarbonotrophic anaerobes, alcohols have also
been produced from some organisms. For example, the wild strain of Clostri-
dium ljungdahlii, a Gram-positive, motile, rod-shaped anaerobe, originally furn-
ished ethanol/acetate ratios of only 0.05 with a maximum ethanol concentration
of 0.1 g L–1 (Vega et al. 1989 d). Adjustment of the fermentation conditions, no-
tably reducing the pH, was reported by Gaddy and coworkers to essentially
eliminate acetate production and produce ethanol concentrations as high as
48 g L–1 (Phillips et al. 1993).
Similarly, in continuous-culture experiments with B. methylotrophicum, Wor-
den and coworkers (Grethlein et al. 1990) found increasing quantities of ethanol
and butanol in the fermentation products as pH decreased. More generally, they
found a trend toward more reduced products (acids with longer chain lengths
and alcohols) as fermentation pH decreased. For example, at pH 6.8 the molar
stoichiometry of the continuous fermentation of B. methylotrophicum was:
4CO ! 2:09CO2 0:63CH3 COOH 0:043C3 H7 COOH 0:027C2 H5 OH

0:43Cell 3
whereas reducing the pH to 6.0 changed the molar stoichiometry to:
4CO ! 2:27CO2 0:30CH3 COOH 0:161C3 H7 COOH 0:032C2 H5 OH

0:029C3 H7 OH 0:31Cell 4
Worden et al. (1991) noted that acetone, butanol, and ethanol were once pro-
duced commercially from glucose by use of a biphasic process consisting of an
acidogenic phase, which produced organic acids and H2, followed by a solvento-
genic phase, in which the organic acids were reduced to alcohols. In this pro-
cess acid production generates ATP but consumes no electrons while alcohol
production consumes electrons but produces no ATP. They propose a similar
scheme for syngas fermentation, with the first phase producing acetate and bu-
tyrate from CO (acidogenic phase) followed by alcohol production in a second
phase, with reducing equivalents to convert the acids to alcohols coming from
hydrogen in the syngas.
11.3.2.3 Production of Polyesters

Acetyl-CoA is the chemical intermediate not only for production of organic acids
and alcohols by anaerobes but for growth of cell mass, including polyesters that
serve as energy stores for the organism. Under conditions of stress, such as an
imbalance in the supply of nutrients, many microorganisms synthesize polyhy-
droxyalkanoates (PHA), which are stored in the cells as discrete granules, as il-
lustrated in Fig. 11.3, that can accumulate to levels as high as 30 to 80% of their
cellular dry weight (Kim and Lenz 2001).
Most known polyhydroxyalkanoates are polymers of 3-hydroxyalkanoic acids,
the monomeric unit of which is illustrated in Fig. 11.4. Although a wide range
of alkanes of different carbon chain length between 4 and 14 carbon atoms can
Fig. 11.3 Photomicrograph showing the accumulation of PHA

in Rubrivivax gelatinosus (Maness and Weaver 1994).
Fig. 11.4 Monomeric unit of poly(3-hydroxybutyrate).
be incorporated into PHA, the most commonly occurring in nature is poly(3-hy-

droxybutyrate) (PHB). PHB can be synthesized by a variety of prokaryotes, in-
cluding Gram-positives and Gram-negatives, aerobic and anaerobic chemo-orga-
no-heterotrophs, chemo-litho-autotrophs, and aerobic and anaerobic phototrophs
(Babel et al. 2001).
Although glucose is commonly used as the substrate for PHA production, a
variety of carbon and energy sources, including CO and CO2 and H2, have been
exploited by bacteria in production of PHA. The key enzyme in CO utilization,
CO dehydrogenase, functions to oxidize CO to CO2, synthesize acetyl-CoA, and
cleave acetyl-CoA in a variety of energy-yielding pathways, depending on the mi-
croorganism (Ferry 1995). Ralstonia eutropha is an autotrophic bacterium that
produces PHB from CO2, H2, and O2 (Schlegel et al. 1961). Synechococcus sp.
MA19 is a cyanobacteria capable of producing up to 20% PHB when cultivated
in a nitrogen-free inorganic medium aerated with CO2 (Miyake et al. 1996). The
photosynthetic bacterium Rubrivivax gelatinosus has produced similar yields of
PHA copolymers from syngas (Maness and Weaver 1994). Rhodopseudomonas ge-
latinosa can utilize CO as a sole carbon and energy source in the dark to pro-
duce PHA (Uffen 1983) and Rhodospirillum rubrum can accomplish this in
either the presence or absence of light (Kerby et al. 1995).
Irrespective of the carbon source, synthesis of PHA is initiated from acetyl-
CoA. The process is illustrated in Fig. 11.5 for synthesis of PHB (labeled
poly(3HB)). The route from acetyl-CoA to PHB is thought to involve three steps
(Babel et al. 2001). The first step, catalyzed by 3-ketothiolase, links two acetyl-
CoA moieties to acetoacetyl-CoA. The second step produces d-(–)-3-hydroxybu-
tyryl-CoA either through a single reaction catalyzed by reductase or a sequence
of three reactions involving reductase and two hydratases. The final step,
mediated by a polymerase, adds hydroxybutyryl monomer to the growing poly-
mer chain to form PHB. Other steps shown in Fig. 11.5 are associated with the
decomposition of PHB.
PHB was thought to be the only polyester produced by microorganisms. In
1974, however, other 3-hydroxyalkanoates, including 3-hydroxyvalerate (PHV)
and 3-hydroxyhexanoate, were isolated from microorganisms in sewage sludge
(Wallen and Rohwedder 1974). Since then a variety of polyesters containing 3-,
4-, and 5-hydroxyalkanoate units have been found to be synthesized by bacteria
(Steinbuchel 2001). Most of these are obtained only if precursor substrates
structurally related to the resulting PHA are provided to the bacteria as carbon
sources, however. Although this might seem to preclude the synthesis of all but
a few PHA from syngas, the volatile organic acids that can be generated from
syngas by some anaerobes might prove suitable substrates for production of
longer-chained hydroxyalkanoates.
Fig. 11.5 Metabolic pathways to PHB reductase; (5), (6) enolases; (7) depoly-
synthesis and degradation (1) ketothiolase; merize; (8) D-(–)-3-hydroxybutyrate dehyro-
(2) NADPH-dependent acetoacetyl-CoA genase; (9) acetoacetyl-CoA synthetase;
reductase; (3) poly(3HB) synthase; (10) succinyl-CoA transferase; (11) citrate
(4) NADH-dependent acetoacetyl-CoA synthase (Babel et al. 2001).
Yields of PHB and cell biomass from a given substrate can be determined by
experiment or by calculation based on the known metabolic pathways involved.
Recovery of this water-insoluble polymer can proceed by one of several methods.
Solvent extraction gives high recovery but requires high capital investment and
large quantities of solvent. Non-solvent alternatives disintegrate cells by heat
shock followed by enzymatic and detergent digestive processes to solubilize the
non-PHA components (Anderson and Dawes 1990).
PHB is structurally similar to polypropylene and has similar crystallinity and
glass transition temperature. Their chemical properties are completely different,
however, and PHB is stiffer and more brittle than polypropylene. These physical
properties can be changed by forming copolymers from monomeric units of
PHB and PHV. Thus, a range of properties can be engineered from copolymers,
ranging from hard and brittle to soft and tough (Anderson and Dawes 1990).
Polyhydroxyalkanoates are attractive as biobased and biodegradable polymers.
Specialty applications of PHA include hydrophobic coatings, specialty elasto-
mers, medical implants, functionalized polymers for chromatography, microgra-
nules for use as binders in paints or in blends that incorporate latexes, and as
sources of chiral monomers (Kessler et al. 2001).
11.3.3
Biorefinery Based on Syngas Fermentation
One possible manifestation of a biorefinery based on syngas fermentation is il-

lustrated in Fig. 11.6. Fibrous feedstock, for example switchgrass, woodchips, or
cornstover, is fed into an oxygen-blown or indirectly heated gasifier followed by
gas clean-up to remove particulates and char from the gas stream. Removal of
trace contaminants, for example sulfur, chlorine, ammonia, and alkali, is prob-
ably unnecessary because they are not thought to poison the anaerobes used in
the fermentation process. The cleaned gas is cooled and passed through a bio-
reactor where CO is dissolved in the fermentation media and taken up by a
suitable unicarbonotroph, for example Rhodospirillum rubrum. In the analysis
that follows, it is assumed that 20% w/w CO is used for growth (formation of
cellular biomass) and 80% w/w of CO goes to metabolite production (H2 gen-
eration via the biologically-mediated water-gas shift reaction). It is further as-
sumed that 40% w/w of the cellular biomass is in the form of storage polymer
(PHA). The actual values for these yields are not well known but these assumed
values are within the expected ranges (Dispirito 2004). Although the diagram
shows gas recycle, it is not clear whether this will be necessary, because the pro-
cess may not be thermodynamically limited as it is for Fischer-Tropsch and
other types of syngas-to-chemicals catalytic reactors. PHA is recovered and the
hydrogen-rich gas-stream leaving the reactor is used for either on-site or distrib-
uted power production via fuel cells.
Brown et al. (2003) have performed a preliminary economic assessment of a
biorefinery producing 20 tpd of PHA and 50 tpd of hydrogen. The capital costs
for this biorefinery, detailed in Table 11.2, are estimated to be $103 million, with
60% of the cost associated with the fermentation equipment. The operating
costs for this plant are detailed in Table 11.3. In this analysis, PHA is taken to
be the primary product with hydrogen a co-product, providing a credit of
Fig. 11.6 Conceptual schematic diagram of biorefinery to

produce hydrogen and PHA coproducts from fibrous biomass
(Brown et al. 2003).
Table 11.2 Estimated capital costs for a biorefinery to produce

hydrogen and PHA coproducts from fibrous biomass
Gasifier $ 18.6 million Estimated from: Larson and Svenningsson (1990)

Fermenter $ 59.1 million Estimated as 25% of total cost of an ethanol
plant
Separation equipment $ 25.3 million Estimated as 30% of total costs of a fermentation
plant
Basic Capital $ 103 million
Table 11.3 Estimated operating costs for biorefinery to pro-

duce hydrogen and PHA coproducts from fibrous biomass
Annual H2 output 16.4 ´ 106 kg Assumes 20% CO to cell mass;

40% cell mass to PHA
Annual PHA output 6.6 ´ 106 kg
Annual input (switchgrass) 192 ´ 106 kg 90% capacity factor
Total Capital $ 119 million
Raw materials (switchgrass) $ 9.6 million Purchased at $ 0.05 kg–1

Credit for H2 ($ 42.8 million) Assumed to sell for $ 2.60 kg–1
(DOE target price)
Labor, utilities, maintenance $ 16.0 million
Indirect costs $ 11.5 million
Annual capital charges $ 13.9 million 10% interest, 20 years
Annual operating costs $ 8.2 million
PHA Production costs $ 1.24 kg–1
$ 2.60 kg–1, which is based on a US Department of Energy target price for this
fuel. The cost of producing PHA through syngas fermentation is estimated to
be $ 1.24 kg–1, which is in the range of many petroleum-derived polymers and
considerably cheaper than the production cost of PHA from glucose, which
may be as much as $ 5–7 kg–1. Until more complete information is available on
yields of H2 and PHA from syngas, however, this cost of production should be
considered an approximate estimate.
11.3.4
Enabling Technology
In a comprehensive review on the prospects of obtaining ethanol from cellulosic

biomass, Lynd (1996) noted that syngas fermentation represents an “end run”
with regard to acid or enzymatic hydrolysis of biomass, because it avoids the
costly and complicated steps of extracting monosaccharide from lignocellulose.
Syngas fermentation, by virtue of reducing all feedstocks to a common set of
low-molecular-weight building blocks, is able to accept a wide variety of biomass

feedstocks, irrespective of their chemical composition. It also has the potential
for being more energy-efficient, because it effectively utilizes all the constituents
of the feedstock, whether cellulose, hemicellulose, lignin, starch, oil, or protein.
Nevertheless, as described by Grethlein and Jain, syngas fermentation has
several barriers to overcome before it can be commercialized (1993). Among
these are relatively low rates of growth and production by anaerobes, difficulties
in maintaining anaerobic fermentations, product inhibition by acids and alco-
hols, and difficulties in transferring relatively insoluble CO and H2 from the
gas phase to the liquid phase, where the anaerobes can utilize the gas. Of these,
mass-transfer limitations are probably the main bottleneck to commercializing
this technology. Studies by Worden and coworkers (1997), however, give encour-
agement that the use of non-toxic surfactants and novel dispersion devices can
enhance mass transfer through the generation of microbubbles to carry syngas
into bioreactors.
11.4
Fast Pyrolysis-based Systems
11.4.1
Fundamentals of Fast Pyrolysis
Fast pyrolysis is the rapid thermal decomposition of organic compounds in the ab-
sence of oxygen to produce liquids, gases, and char (Bridgwater and Peacocke
2000). The distribution of products depends on the biomass composition and
the rate and duration of heating. The yield of pyrolytic liquid, also known as
bio-oil, depends on pyrolysis conditions, including relatively short residence times
(0.5–2 s), moderate temperatures (400–600 8C), and rapid quenching at the end of
the process. Rapid quenching is essential if high-molecular-weight liquids are to
be condensed rather than further decomposed to low molecular weight gases. Typ-
ical product yields for two kinds of wood are given in Table 11.4.
Bio-oil from fast pyrolysis is a low viscosity, dark-brown fluid containing up to
15 to 20% water, which contrasts with the black, tarry liquid resulting from slow
pyrolysis or gasification (Piskorz et al. 1988). As indicated by Table 11.4, the bio-
oil is a mixture of many compounds although most can be classified as acids,
aldehydes, sugars, and furans, derived from the carbohydrate fraction, and phe-
nolic compounds, aromatic acids, and aldehydes, derived from the lignin frac-
tion. The liquid is highly oxygenated, approximating the elemental composition
of the feedstock, which makes it highly unstable.
Despite the high water content of bio-oil, no appreciable phase separation is
apparent (Scott et al. 1999). If an equal volume of water is added to the liquid,
however, the high-molecular-weight, largely aromatic compounds, are precipi-
tated. Because most of the aromatic compounds can be traced to the lignin con-
tent of the biomass, this precipitate is widely known as “pyrolytic lignin”.
Table 11.4 Analysis of products from fast pyrolysis (Piskorz et al. 1988).
White Spruce Poplar Type of compound
Moisture content, % w/w 7.0 3.3

Particle size, lm (max) 1000 590
Temperature 500 497
Apparent residence time 0.65 0.48
Product yields, % w/w, m.f.
Water 11.6 12.2
Char 12.2 7.7
Gas 7.8 10.8
Pyrolytic liquid (bio-oil) 66.5 65.7
Gas composition, % w/w, m.f.
H2 0.02 –
CO 3.82 5.34
CO2 3.37 4.78
CH4 0.38 0.41
C2 hydrocarbons 0.20 0.19
C3+ hydrocarbons 0.04 0.09
Pyrolytic liquid composition, % w/
w, m.f.
Organic liquid 66.5 65.7
Oligosaccharides – 0.70 Saccharides
Glucose 0.99 0.41
Other monosaccharides 2.27 1.32
Levoglucosan 3.96 3.04 Anhydrosugars
1,6-anhydroglucofuranose – 2.43
Cellobiosan 2.49 1.30
Glyoxal 2.47 2.18 Aldehydes
Methylglyoxal – 0.65
Formaldehyde – 1.16
Acetaldehyde – 0.02
Hydroxyacetaldehyde 7.67 10.03
Furfural 0.30 – Furans
Methylfurfural 0.05 –
Acetol 1.24 1.40 Ketones
Methanol 1.11 0.12 Alcohols
Ethylene glycol 0.89 1.05
Acetic acid 3.86 5.43 Carboxylic acids
Formic acid 7.15 3.09
Water-soluble – Total above 34.5 34.3
Pyrolytic lignin 20.6 16.2
Amount not accounted for (losses, 11.4 15.2
water soluble phenols, furans, etc.)
Bio-oil has several undesirable characteristics (Oasmaa and Czernik 1999).

The low pH of bio-oil, which arises from organic acids derived primarily from
the hemi-cellulosic content of the feedstock, makes the liquid highly corrosive.
The oil contains large quantities of non-volatile carbohydrates and oligomeric
phenolic compounds, which prevents complete distillation of the bio-oil. The
highly oxygenated product is chemically unstable, with polymerization of dou-
ble-bonded compounds and etherification and esterification reactions proceed-
ing over time. The liquid contains fine-particulate char, which is thought to pro-
mote polymerization.
The higher heating values of pyrolysis liquids range between 17 MJ kg–1 and
20 MJ kg–1 with liquid densities of about 1280 kg m–3. Assuming conversion of
72% of the biomass feedstock to liquid on a weight basis, yield of pyrolysis oil
is approximately 560 L ton–1.
The mechanism by which cellulose, hemicellulose, and lignin in biomass are
converted into liquids is not fully understood. Rapid pyrolysis of pure cellulose
yields levoglucosan, an anhydrosugar with the same empirical formula as the
monomeric building block of cellulose: C6H10O5 (Evans and Milne 1987). Addi-
tion of a small amount of alkali inhibits the formation of levoglucosan and pro-
motes the formation of hydroxyacetaldehyde (glycolaldehyde). Pyrolysis of pure
cellulose at slower heating rates and lower temperatures favors the formation of
char rather than liquids. These observations suggest the multiple reaction path-
ways for pyrolysis of cellulose illustrated in Fig. 11.7 (Bridgwater and Peacocke
2000). Low temperatures and slow heating rates favor dehydration reactions that
ultimately convert the cellulose to char and water. At higher temperatures, depo-
lymerization dominates, yielding levoglucosan as the primary product. The pres-
ence of alkali, however, catalyzes the dehydration route but yields hydroxyacetal-
dehyde instead of char and water if reaction products are removed fast enough.
Similarly, hemicelluloses form furanoses and furans as primary reaction prod-
ucts whereas lignin forms monocyclic aromatics and non-condensed bicyclic
aromatic materials with high phenolic content.
The mechanism by which reaction products are transported from the reaction
zone and recovered as liquids is also uncertain (Daugaard and Brown 2004).
Many of the reaction products, including levoglucosan, have very low vapor
pressures, making vapor transport problematic, but this possibility has not been
definitively disproved. Alternatives include transport of low-molecular-weight
compounds that condense to higher molecular weight compounds outside the
reactor and the elutriation of fine liquid droplets (aerosols) from the reactor.
Fig. 11.7 Reaction pathways in fast pyrolysis.

Fig. 11.8 Schematic diagram of fast-pyrolysis plant (Brown 2003).
Production of pyrolysis oils and its co-products is illustrated in Fig. 11.8

(Brown 2003). Lignocellulosic feedstocks, such as wood or agricultural residues,
are milled to fine particles of less than 1 mm diameter to promote rapid reac-
tion. The particles are entrained in an inert gas stream, which transports the
material to the pyrolysis reactor, shown here as a fluidized bed, which provides
the prerequisite high heat transfer rates. Within the reactor, the particles are
rapidly heated and converted into condensable vapor, non-condensable gas, and
solid charcoal. These products are transported out of the reactor into a cyclone
operating above the condensation point of pyrolysis vapor where the charcoal is
removed. Vapor and gas is transported to a direct-contact quench vessel where a
spray of pyrolysis liquid cools and condenses the vapor. The non-condensable
gas, which include the flammable gases CO, H2, and methane (CH4), are
burned in air to provide heat for the pyrolysis reactor. A number of schemes
have been developed for indirectly heating the reactor, including transport of
solids into fluidized beds or cyclonic configurations to bring the particles into
contact with hot surfaces.
There are several problems with bio-oil. Phase-separation and polymerization
of the liquids and corrosion of containers make storage of these liquids difficult.
The high oxygen and water content of bio-oil makes it incompatible with con-
ventional hydrocarbon fuels. Furthermore, bio-oil is of much lower quality than
even Bunker C heavy fuel oil, so upgrading is highly desirable.
11.4.2
Fermentation of Bio-oils
One possibility for upgrading bio-oil is to change the processing conditions to

yield a product that is compatible with biochemical processing. Scott and co-
workers (1989) at the University of Waterloo in Ontario, Canada recognized that
alkali and alkaline earth metals in the biomass serve as catalysts that degrade
lignocellulose to char. If these cations are removed by soaking the feedstock in
dilute acid before pyrolysis, the lignocellulose is depolymerized to anhydrosu-
Fig. 11.9 Chemical structure for 1,6-anhydro-beta-D-glucose.
gars at very high yields. Anhydrosugar is a sugar from which one or more mol-
ecules of water have been removed, resulting in the formation of an internal
acetal structure. The prevalent anhydrosugar from the fast pyrolysis of biomass
is 1,6-Anhydro-beta-d-glucose. The chemical structure of this compound, com-
monly known as levoglucosan, is illustrated in Fig. 11.9. A dimer of levogluco-
san, cellobiose, is also produced during fast pyrolysis, but in much lower con-
centrations. Anhydrosugar has prospects as a platform for chemical synthesis or
as a substrate for fermentation.
In studies on woody biomass with and without cation removal Piskorz et al.
(1997) found that levoglucosan increased from 3.04% in pyrolysis liquid from
untreated poplar wood to 30.42% in bio-oil from pretreated poplar wood. In-
creases were more modest for cellobiose.
Brown and his collaborators (2001) evaluated the effect of alkali removal on
the pyrolytic products of cornstover, an important herbaceous biomass. Three
pretreatments were evaluated: acid hydrolysis, washing in dilute nitric acid, and
washing in dilute nitric acid with addition of (NH4)2SO4 as a pyrolytic catalyst.
Although alkali compounds in plant materials are generally water-soluble, at-
tempts to remove alkali by water washing did not prove effective in this study.
On the other hand, all three acid treatments were able to substantially increase
the yield of anhydrosugars, as shown in Table 11.5. Acid hydrolysis of this anhy-
drosugar yielded 5% solutions of glucose and other simple sugars.
The resulting glucose solutions can be fermented, as demonstrated by Prosen
et al. (1993). However, the substrate derived from the bio-oil contains fermenta-
tion inhibitors that must be removed or neutralized by chemical or biological
methods. Chemical methods that have been evaluated on bio-oil-derived sub-
strate include solvent extraction, hydrophilic extraction, and adsorption extrac-
tion (Brown et al. 2000). The most effective of the chemical methods employed
activated carbon. As a less expensive alternative, Khiyami (2003) explored biolog-
ical treatments. He found that biofilms of Pseudomonas putida and Streptomyces
setonii were able to remove toxins from substrates derived from bio-oil.
As an alternative to hydrolyzing levoglucosan to glucose, several microorgan-
isms have been identified that directly ferment levoglucosan (Kitamura et al.
1991; Nakahara et al. 1994; Zhuang et al. 2001 a, b). This would eliminate the
hydrolysis step and probably improve the economics of producing fermentation
products from bio-oil.
Table 11.5 Products of pyrolysis for several different pretreat-

ments of cornstover (Brown et al. 2001).
No Acid Demineral- Demineralization

pretreatment hydrolysis ization with catalyst
Pyrolysis products (% w/w maf)

Char 15.8 13.2 13.2 15.9
Water 2.57 10.6 10.4 7.96
Organics 59.1 67.2 68.5 67.7
Gases 22.6 9.02 7.88 8.44
Organics (% w/w)
Cellobiosan Trace 4.55 3.34 4.97
Levoglucosan 2.75 17.69 20.12 23.10
Hydroxyacetaldehyde 11.57 5.97 3.73 3.93
Formic acid 2.61 Trace Trace 0.73
Acetic acid 3.40 1.51 1.26 0.40
Acetol 4.53 Trace Trace Trace
Formaldehyde 2.75 1.63 trace 0.70
Pyrolytic lignin 33.40 16.89 17.74 20.08
11.4.3
Biorefineries Based on Fast Pyrolysis
One manifestation of a biorefinery based on fermentation of bio-oil is illustrated

in Fig. 11.10. Fibrous biomass is pretreated with dilute acid to simultaneously
remove alkali and hydrolyzes the hemicellulose fraction to pentose. The remain-
ing fraction, containing cellulose and lignin, is pyrolyzed at 500 8C to yield char,
gas, and bio-oil. The bio-oil is separated into pyrolytic lignin and levoglucosan-
rich aqueous phase. The char, gas, and lignin are burned to generate steam for
distillation and other process heat requirements of the plant and the levogluco-
san is hydrolyzed to hexose. The pentose and hexose are fermented to ethanol.
So and Brown (1999) compared the cost of producing ethanol from cellulosic
biomass using fast pyrolysis combined with a fermentation step to acid hydroly-
sis and enzymatic hydrolysis technologies. The azeotropic ethanol production
capacity used in this case study was 95 million L year–1 and the assumed cost
for biomass was $ 46 ton–1 (1997 US dollars). As summarized in Table 11.6, to-
tal capital investment for a plant based on fermentation of bio-oil was estimated
to be $ 69 million, while the annual operating cost was about $ 39.2 million, re-
sulting in an ethanol selling price of $ 0.42 L–1. This is about 23% higher than
ethanol from plants based on acid hydrolysis and enzymatic hydrolysis of bio-
mass, but well within the uncertainty of the analysis (30%).
A more advanced concept for a biorefinery based on fast pyrolysis is illus-
trated in Fig. 11.11 (Sandvig et al. 2004). This biorefinery integrates pyrolysis
with combined cycle (IPCC) power. Biomass is first washed to remove alkali be-
fore it is pyrolyzed.
Fig. 11.10 Schematic diagram of cellulosic biomass-to-ethanol based on fast pyrolysis.
Table 11.6 Comparing production cost of ethanol from cellulo-

sic biomass for three conversion technologies (1997 US $).
Fast pyrolysis SSF a) Acid hydrolysis
Annual ethanol output 95 million L 95 million L 95 million L

Annual biomass input 240 ´ 106 kg 244 ´ 106 kg 238 ´ 106 kg
Total capital $ 69 million $ 64 million $ 67 million
Raw materials $ 11.1 million $ 11.3 million $ 11.0 million
Labor, utilities b), maintenance $ 6.18 million $ 0.9 million $ 2.13 million
Indirect costs $ 8.07 million $ 7.13 million $ 7.21 million
Annual capital charges $ 13.8 million $ 12.8 million $ 13.3 million
Annual operating costs $ 39.2 million $ 32.1 million $ 33.7 million
Production cost of ethanol $ 0.42 L–1 $ 0.34 L–1 $ 0.35 L–1
a) Simultaneous saccharification and fermentation (enzymatic hydrolysis)

b) Includes credit for steam generation for SSF and acid hydrolysis processes
Pyrolytic char is recovered for additional processing to activated carbon or

marketed as a soil amendment. Pyrolytic gas is used to heat the pyrolysis reac-
tor. Bio-oil is recovered in a specially designed quencher intended to fractionate
the bio-oil into different chemicals, including levoglucosan. The levoglucosan-
rich fraction is either purified or used as substrate for fermentation to addi-
tional chemical products (not illustrated). The other fractions of bio-oil are used
to fire the gas turbine cycle to produce electricity. The waste heat from the gas
turbine is directed to a waste heat recovery generator, which provides power to a
Rankine steam turbine bottoming cycle.
Advantages of the biomass-fueled IPCC system include: cycle efficiency exceed-
ing that of biomass-fired Rankine cycles; avoids need for high-pressure thermal
Fig. 11.11 Biorefinery based on fast pyrolysis incorporating

combined cycle power and chemical recovery (Sandvig et al.
2004).
reactors; reduces the strong coupling between fuel processing and power genera-
tion typical of integrated power systems; and provides opportunities for recovering
value-added products. The proposed IPCC system is estimated to have a total pro-
ject cost of between $ 2300 and $ 2500 kW–1 based on a net combined cycle output
of 7655 kW. This cost does not include the additional equipment required for pro-
duction of value-added chemicals. This capital cost compares favorably with that of
conventional biomass power systems in this range, which cost around $ 2000 for
basic systems to over $ 3000 kW–1 for systems designed for higher efficiency and
reliability. The cost of electricity for the IPCC plant is projected to be similar to the
costs for conventional biomass power systems, approximately $ 0.02 kWh–1.
11.4.4
Enabling Technologies
When Scott and coworkers (1989) first proposed a pyrolytic route to cellulosic
ethanol, it was offered as a way of leapfrogging the barriers to fractionating bio-
mass. In a simple, rapid process, fast pyrolysis was able to separate lignocellu-
lose into a pyrolytic lignin and a carbohydrate-rich aqueous phase. The process
introduces its own set of technical barriers that have yet to be fully solved, how-
ever.
Fast pyrolysis of pure cellulose produces, in principle, levoglucosan as its sole
reaction product. In practice, the presence of lignin and a variety of inorganic
compounds in fibrous biomass results in more than a hundred chemical prod-
ucts, many of which are not only unsuitable as a carbon and energy source for
fermentation but are actually toxic to the microorganisms to be cultivated. Im-
proved selectivity of pyrolytic reactions will be important to achieving high
yields of fermentable carbohydrate. Understanding reaction pathways will be
the key to success in this endeavor.
Like many of the pre-treatment processes used to facilitate fractionation by
acid or enzymatic hydrolysis, fast pyrolysis generates biological inhibitors that
must be removed before the bio-oil is used as a fermentation substrate. Meth-
ods that are more cost-effective than adsorption with activated carbon must be
developed.
Like any process for the production of ethanol from biomass, efficient use of
the hemicellulosic and lignin fractions of the lignocellulose will be essential to
economic viability. In principle, the pentoses released during demineralization
of the biomass can be fermented and the pyrolytic lignin can be used in the
production of process steam. Effective use of these coproducts will require more
attention to integrating the individual processes making up a system for pyrolyt-
ic production of cellulosic ethanol.
11.5
Thermochemical processing of biomass to produce substrates suitable for fer-

mentation is a relatively new and unexplored approach to biobased products.
Two distinct routes for hybrid thermochemical-biological processing have been
offered in this paper: (1) gasification then fermentation of the syngas, and (2)
fast pyrolysis then hydrolysis and/or fermentation of the anhydrosugars in the
resulting bio-oil. The syngas route, by transforming all the plant constituents
into CO and H2, is attractive for its efficient use of biomass. The fast pyrolysis
route, by yielding a storable carbohydrate-rich liquid, enables processing of the
solid biomass to be decoupled from fermentation and offers prospects for dis-
tributed processing of widely dispersed biomass resources. Both have an advan-
tage over hydrolytic methods in that they are able to process a wider variety of
feedstocks, although this is especially true for the gasification route.
Compared with acid and enzymatic hydrolysis, relatively few resources have
been devoted to developing hybrid thermochemical-biological routes to biobased
products. The reason for this circumstance is easy to understand – the original
feedstocks of the fermentation industry were naturally occurring sugars and
starches that were easily hydrolyzed to sugar. The fact that starch and cellulose
are both polymers of glucose encouraged similar approaches to depolymerizing

these two carbohydrates. In fact, cellulose is not only more recalcitrant than
starch but it is imbedded in a matrix of lignin, which makes the process of re-
leasing sugar from lignocellulose much more difficult than for starch. Consider-
ing these difficulties, hybrid thermochemical-biological approaches to biobased
products deserves increased attention.
References
Anderson, A. J. and E. A. Dawes (1990). “Oc- Brown, R. C., D. Radlein and J. Piskorz
currence, Metabolism, Metabolic Role, and (2001). Pretreatment Processes to Increase
Industrial Uses of Bacterial Polyhydroxy- Pyrolytic Yield of Levoglucosan from Her-
alkanoates.” Microbiological Reviews 54: baceous Feedstocks. Chemicals and Materi-
450–472. als from Renewable Resources: ACS Sympo-
Babel, W., J. Ackermann and U. Breuer sium Series No. 784. Washington, D.C.,
(2001). “Regulation, and Limits of the Syn- American Chemical Society: 123–132.
thesis of Poly(3HB) Physiology.” Advances Datar, R. P., R. M. Shenkman, B. G. Cateni,
in Biochemical Engineering/Biotechnology R. L. Huhnke and R. S. Lewis (2004). “Fer-
71. mentation of biomass-generated producer
Biomass Research and Development Techni- gas to ethanol.” Biotechnology and Bioengi-
cal Advisory Committee of the US Depart- neering 86(5): 587–594.
ments of Energy and Agriculture (2002). Daugaard, D. E. and R. C. Brown (2004). The
Roadmap for Biomass Technologies in the transport phase of pyrolytic oil exiting a fluid-
United States, Biomass Research and De- ized bed reactor. Science in Thermal and
velopment Technical Advisory Committee. Chemical Biomass Conversion Confer-
Bredwell, M. D., P. Srivastava and R. M. ence, Victoria, British Columbia, Canada.
Worden (1999). “Reactor design issues for Dispirito, A. (2004). Personal communica-
synthesis-gas fermentations.” Biotechnology tion.
Progress 15(5): 834–844. Evans, R. J. and T. A. Milne (1987). “Molecu-
Bridgwater, A. V. (1995). “The technical and lar characterization of the pyrolysis of bio-
economic feasibility of biomass gasifica- mass.” Energy and Fuels 1: 123–137.
tion for power generation.” Fuel 74: 631– Ferry, J. G. (1995). “CO dehydrogenase.” An-
653. nual Review of Microbiology 49: 305–333.
Bridgwater, A. V. and G. V. C. Peacocke Grethlein, A. J. and M. K. Jain (1993). “Bio-
(2000). “Fast pyrolysis processes for bioprocessing of coal-derived synthesis gases
mass.” Renewable and Sustainable Energy by anaerobic bacteria.” Trends in Biotech-
Reviews 4: 1–73. nology 10: 418–423.
Brown, R. C. (2003). Biorenewable Resources: Grethlein, A. J., R. M. Worden, M. K. Jain
Engineering New Products from Agriculture. and R. Datta (1990). Applied Biochemistry
Ames, IA, Blackwell Publishing. and Biotechnology 24/25: 875.
Brown, R. C., T. Heindel, A. Dispirito and B. Kerby, R. L., P. W. Ludden and G. P. Roberts
Nikolau (2003). Production of biopolymers (1995). “Carbon monoxide-dependent
and hydrogen via syngas fermentation. Na- growth of Rhodospirillum rubrum.” J.
tional ACS Meeting, Anaheim, California. Bacteriology 177: 2241–2244.
Brown, R. C., A. L. Pometto, T. L. Peeples, M. Kessler, B., R. Weusthuis, B. Witholt and G.
Khiyami, B. Voss, J. W. Kim and S. Fischer Eggink (2001). “Production of Microbial
(2000). “Strategies for pyrolytic conversion Polyesters: Fermentation and Downstream
of herbaceous biomass to fermentation Processes.” Advances in Biochemical Engi-
products.” Proceedings of the Ninth Biennial neering/Biotechnology and Bioengineering
Bioenergy Conference, Buffalo, New York. 71: 159–182.
References 251
Khiyami, M. A. (2003). Biological methods Applied Biochemistry and Biotechnology

for detoxification of corn stover and corn 39/40: 559–571.
starch pyrolysis liquors, Ph.D. Thesis, Piskorz, J., P. Majerski, D. Radlein, D. S.
Iowa State University. Scott, Y. P. Landriault, R. P. Notarfonzo
Kim, Y. B. and R. W. Lenz (2001). “Polyesters and D. K. Vijh (1997). Economics of the Pro-
from Microorganisms.” Advances in Bio- duction of Fermentable Sugars from Biomass
chemical Engineering/Biotechnology 71: 51– by Fast Pyrolysis. Third Biomass Confer-
79. ence of the Americas, Montreal, Ontario,
Kitamura, Y., Y. Abe and T. Yasui (1991). Canada.
“Metabolism of levoglucosan (1,6-Anhy- Piskorz, J., D. S. Scott and D. Radlein
dro-b-d-glucopyranose) in microorgan- (1988). Pyrolysis Oils from Biomass. ACS
isms.” Agric. Biol. Chem. 55: 515–521. Symposium Series 376. E. J. Soltes and T. A.
Ko, C. W., J. L. Vega, E. C. Clausen and J. L. Milne. Washington, DC, American Chemi-
Gaddy (1989). “Effect of high-pressure on cal Society: 167–178.
a co-culture for the production of methane Prosen, E. M., D. Radlein, J. Piskorz, D. S.
from coal synthesis gas.” Chemical Engi- Scott and R. L. Legge (1993). “Microbial
neering Communications 77: 155–169. utilization of levoglucosan in wood pyroly-
Larson, E. D. and P. Svenningsson (1990). sate as a carbon and energy source.” Bio-
Development of biomass gasification systems technol. and Bioengineering 42: 538–541.
for gas turbine power generation. Energy Reed, T. (1981). Biomass Gasification: Princi-
from Biomass and Wastes XIV, Lake Bue- ples and Technology. Park Ridge, N.J., Noyes
na Vista, FL, USA. Published by Inst Gas Data Corp.
Technology, Chicago, FL, USA, pp. 1–19. Sandvig, E., G. Walling, D. E. Daugaard,
Lynd, L. R. (1996). “Overview and evaluation R. J. Pletka, D. Radlein, W. Johnson and
of fuel ethanol from cellulosic biomass: R. C. Brown (2004). “The prospects for in-
technology, economics, the environment, tegrating fast pyrolysis into biomass power
and policy.” Annu. Rev. Energy Environ. 21: systems.” International Journal of Power
403–465. and Energy Systems 24(3): 228–238.
Madhukar, G. R., B. B. Elmore and H. K. Schlegel, H. G., G. Gottschalk and R. von
Huckabay (1996). “Microbial conversion of Bartha (1961). Nature 191: 463.
synthesis gas components to useful fuels Scott, D. S., S. Czernik, J. Piskorz and D.
and chemicals.” Applied Biochemistry and Radlein (1989). Sugars from biomass cellu-
Biotechnology 57/58: 243–251. lose by a thermal conversion process. Energy
Maness, P. C. and P. F. Weaver (1994). “Pro- from Biomass and Wastes XIII, New Or-
duction of Poly-3-Hydroxyalkanoates from leans, LA, USA, Published by Inst of Gas
CO and H2 by a Novel Photosynthetic Bac- Technology, Chicago, IL, USA, pp. 1349–
terium.” Applied Biochemistry and Biotech- 1362.
nology 45/46: 395–406. Scott, D. S., P. Majerski, J. Piskorz and
Miyake, M., M. Erata and Y. Asada (1996). D. Radlein (1999). “A second look at fast
J. Fermentation Bioengineering 82: 512. pyrolysis of biomass – the RTI process.”
Nakahara, K., Y. Kitamura, Y. Yamagishi J. Analytical and Applied Pyrolysis 51: 23–37.
and H. Shoun (1994). “Levoglucosan dehy- So, K. and R. C. Brown (1999). “Economic
drogenase involved in the assimilation of analysis of selected lignocellulose-to-etha-
levoglucosan in Arthrobacter sp. I-552.” nol conversion technologies.” Applied Bio-
Biosci. Biotech. Biochem. 58: 2193–2196. chemistry and Biotechnology 77–79: 633–
Oasmaa, A. and S. Czernik (1999). “Fuel oil 640.
quality of biomass pyrolysis oils – State of Spath, P. L. and D. C. Dayton (2003). Prelim-
the art for the end users.” Energy and Fuels inary Screening – Technical and Economic
13: 914–921. Assessment of Synthesis Gas to Fuels and
Phillips, J. R., K. T. Klasson, E. C. Clausen Chemicals with Emphasis on the Potential
and J. L. Gaddy (1993). “Biological produc- for Biomass-Derived Syngas. National Re-
tion of ethanol from coal synthesis gas.” newable Energy Laboratory, Technical Re-
port NREL/TP-510-34929, Golden, CO, sis gas.” Applied Biochemistry and Biotech-
USA. nology 20/21: 781–797.
Steinbuchel, A. (2001). “Perspectives for bio- Wallen, L. L. and W. K. Rohwedder (1974).
technological production and utilization of “Poly-beta-hydroxyalkanoate from activated
biopolymers: metabolic engineering of sludge.” Environ Sci Technol 8: 576–579.
polyhydroxyalkanoate biosynthesis path- Worden, R. M., M. D. Bredwell and A. J.
ways as a successful example.” Macro- Grethlein (1997). Engineering Issues in
molecular Bioscience 1: 1–14. Synthesis-Gas Fermentations. Fuels and
Tobler, C. (1994). Genesis gives Birth to Chemicals from Biomass. Washington,
another company. Arkansas Business, D. C., American Chemical Society. ACS
February 28. Symposium Series No. 666: 320–336.
Uffen, R. L. (1983). “Metabolism of carbon Worden, R. M., A. J. Grethlein, M. K. Jain
monoxide by Rhodopseudomonas gelatinosa: and R. Datta (1991). “Production of buta-
cell growth and properties of the oxidation nol and ethanol from synthesis gas via fer-
system.” J. Bacteriology 155: 956–965. mentation.” Fuel 70(5): 615–619.
Vega, J. L., G. M. Antorrena and E. C. Clausen Worden, R. M., A. J. Grethlein, J. G. Zeikus
(1989 a). “Study of gaseous substrate fer- and R. Datta (1989). Applied Biochemistry
mentations: Carbon monoxide conversion and Biotechnology 20/21: 687.
to acetate. 2. Continuous culture.” Biotech- Zeikus, J. G., R. Kerby and J. A. Krzycki
nology and Bioengineering 34: 785–793. (1985). “Single-carbon chemistry of aceto-
Vega, J. L., E. C. Clausen and J. L. Gaddy genic and methanogenic bacteria.” Science
(1989 b). “Study of gaseous substrate fer- 227: 1167–1173.
mentations: Carbon monoxide conversion Zhuang, X. L., H. X. Zhang and J. J. Tang
to acetate. 1. Batch culture.” Biotechnology (2001 a). “Levoglucosan kinase involved in
and Bioengineering 34(6): 774–784. citric acid fermentation by Aspergillus ni-
Vega, J. L., V. L. Holmberg, E. C. Clausen ger CBX-209 using levoglucosan as sole
and J. L. Gaddy (1989 c). “Fermentation pa- carbon and energy source.” Biomass and
rameters of peptostreptococcus-productus Bioenergy 21: 53–60.
on gaseous substrates (CO, H2/CO2).” Ar- Zhuang, X. L., H. X. Zhang, J. Z. Yang and
chives of Microbiology 151(1): 65–70. H. Y. Qi (2001 b). “Preparation of levoglu-
Vega, J. L., S. Prieto, B. B. Elmore, E. C. cosan by pyrolysis of cellulose and its cit-
Clausen and J. L. Gaddy (1989 d). “The bio- ric acid fermentation.” Bioresource Technol-
logical production of ethanol from synthe- ogy 79(1): 63–66.
253
Green Biorefineries
12
The Green Biorefinery Concept – Fundamentals and Potential
12.1
Introduction
Green biorefineries are integrated technologies and technology systems for pro-
duction of materials and energy processing of green plants and parts of green
plants. Above all, green biorefinery technologies are based on traditional tech-
nologies of green forage preservation, leaf-protein extraction, chlorophyll pro-
duction, and modern biotechnological and chemical conversion methods.
The main raw material of green biorefineries are green plants, for example
grass, alfalfa, and immature (green) grain or green plant parts, for example
leaves. Green plant parts are a virtually inexhaustible raw material reservoir,
which is fast-growing, available world-wide, and may have ecological advantages.
Considering alfalfa alone, 32 million hectares are currently cultivated and con-
verted into green pellets or forage flour worldwide.
By means of primary photosynthesis green C3 plants can yield up to 20 tons
dry matter with up to four tons of proteins in temperate climates each year. C4
plants in tropical zones can, however, produce up to 80 tons dry matter with six
tons of proteins per hectare per year. Green plants are, moreover, rich in carbo-
hydrates, proteins, lipids, lignin, and a group of secondary plant substances and
phytochemicals. Green plants and green plant parts may therefore be seen as a
chemical plant with a huge potential. In comparison with other vegetable bio-
mass green plants are characterized by a high content of aqueous cell juice with
carbohydrates of low molecular weight, a large amount of enzymes (proteins)
for photosynthesis and a relatively low content of lignin in the cell walls.
For these reasons all technological concepts of green biorefineries include the
separation of the cell juice from the plant framework. Both fractions, the cell
ISBN: 3-527-31027-4
254 12 The Green Biorefinery Concept – Fundamentals and Potential
juice and the cellulose-containing leaf cells are subjected to different biotechno-
logical and physicochemical conversion methods.
12.2
Historical Outline
12.2.1
The Inceptions
The green parts of plants have been used by the human being for ages. Primar-
ily ruminants use green plants as fodder. The relevance of green crop cultiva-
tion was already apparent from the occupation of the sons of Adam and Eve,
Cain and Abel – Abel became a shepherd, Cain a farmer [1]. The direct use of
green plants in the diet of humans is ancient. We are able to distinguish be-
tween use as nutrition or for salvation or as a natural stimulant. In Europe the
consumption of spinach (Spinacia oleracea), stinging-nettle (Urtica), sorrels (Ru-
mex acetosa), curly kale (Brassica oleraceae var. Sabellica), and leek (Allium ampe-
lobrasum) is very common.
The chemical and biochemical scientific exploration of green plant leaves can
be traced back to the 18th century. The French chemists G. F. and H. M. Roulle
reported obtaining protein extracts from alfalfa leaves in 1773. Hillaire Marin
Roulle in particular, a demonstrator at the royal garden in Paris since 1770,
proved that the juices obtained by pressing the vegetable alfalfa contain a sub-
stance which coagulated when heated into a “cheesy” substance similar to ani-
mal material. The new coagulate, found by Roulle in the heated juice obtained
by pressing different plants, was therefore called “vegato-animale” substance (to-
day it is called leaf protein) [2].
Mild warming yielded a green colored fraction, further heating delivered an
almost colorless precipitate. Chemical analysis of the colorless substance re-
vealed it contained a larger amount of that new substance than the green coagu-
late. Also, by extraction of the green pigments with alcohol the green precipitate
could be removed [2]. It is impressive that key procedures of modern protein
separation of green biomass in green biorefineries were researched as early as
1773 [3–7]. The functional characteristic of thermal coagulation of proteins was,
moreover, the first criterion for later definition of proteins as a class of sub-
stance [3].
12.2.2
First Production of Leaf Protein Concentrate
In the 20th century research on leaf protein concentrate (LPC) focused on use
of the proteins for human nutrition [4–6], because of the widespread availability,
high nutritional value, and high protein productivity in green plants, with its
valuable spectrum of essential amino acids and the intensive growth of green
plants under climatically favorable conditions. Technical manipulations are nec-

essary to separate the leaf protein in a form digestible by humans, because the
human being is not equipped with the opportunity to digest leaf cells and, par-
ticularly, their cellulose membrane as ruminants do.
Of special economic interest is a fraction expressed in the chloroplasts of
green plants. The so called fraction-I-protein is the photosynthesis enzyme ribu-
lose-1,5-biphosphatecarboxylase/oxygenase or rubisco, which can be regarded as
the most widespread protein in the world. In spinach leaves, rubisco accounts
for 75% of soluble proteins; in wheat and barley it is 53–75% and in corn and
sweet sorghum (sugar millet) (Sorghum dochna) 15% [8, 9]. It must, however, be
stressed that by means of electrophoresis approximately 250 to 300 different
proteins and polypeptides can be detected in green-plant extracts [10, 11]. In al-
falfa (Medicago sativa L.) rubisco amounts for 30–70% of the soluble proteins,
depending on genotype and vegetation cycle [9, 12]. On the basis of the protein
harvest from one hectare of arable land, alfalfa provides a 3 to 10-fold higher
yield than oilseeds, grain legumes, or grain [9].
In times of crises, especially, the topic of leaf protein for nutrition has re-
peatedly been put on the nutrition agenda. For example, in 1917 the use of al-
falfa flour for bread manufacture was reported in the “Literary digest”. In 1920
and 1921 Osborne and Chibnell published their results of examinations of pro-
teins in green leaves. Ereky proposed the utilization of leaf proteins for public
nutrition in 1925 and Slade renewed this suggestion in 1937. Slade and Birkin-
shaw were the first to patent the utilization of grass and other green plants in
1939 [13].
These developments were favored by results from examinations which proved
that green forage supplies ruminants with more proteins and essential amino
acids than they can actually utilize. Ruminants like cows, oxen, and sheep need,
on a dry matter basis, only 16% of crude protein in their feed. Alfalfa and
grasses, however, contain 22–28% crude protein on dry matter basis [14]. The
opportunity to simultaneously provide benefits for both animals and humans
from green plants would result from removal of the “surplus” protein before
feeding the green fodder to ruminants. This idea evolved for the first time dur-
ing World War II. Because of the manure-nitrogen problem, due to the indus-
trialization of livestock farming, the idea underwent a revival.
During World War II, researchers and developers stressed the importance of
leaf protein concentrate for providing the population with sufficient protein be-
cause of food-supply shortages in Europe. After the occupation of France by the
Germans in 1940, Great Britain in particular, was cut off from the food supply
on the continent. Thus, the United Kingdom enforced large-scale developments
and put priority on nutrition by use of green-plant proteins.
All large-scale developments revealed technical problems and were not very
profitable, and development was stopped because of the American-British Land
Lease Agreement in 1941. Nevertheless, young Pirie, Scientist at the Rotham-
sted Experimental Station in Hertfordshire, UK, was able to accomplish impor-
tant pioneering work for later industrial production of leaf protein [15–17].
In the sixties development of production plants utilizing green leaves began

again. Five main reasons can be identified for the relaunch of leaf protein
plants. First, forecasts suggested a lack of protein-rich products worldwide. Sec-
ond, the industrial livestock farming which was developing demanded standard-
ized and metered feeding. Third, the interest of the industrialized world in the
nutritional problems of the developing countries was increasing. Fourth, the
member states of the Warsaw Pact wanted to separate themselves from the
world market and generated their own supply of food and feed protein. Finally,
the rising cost of energy (the later oil price shock) focused interest on extraction
of leaf protein as alternative to other means of green forage conservation.
The first modern industrial process for leaf protein extraction was called the
Rothamsted process, developed by Pirie. The procedure based on heat coagula-
tion of green plant juice at 70 8C, resulted in leaf protein concentrates with 60%
Fig. 12.1 Flow chart of the fractionation of green plant parts

for extraction of leaf protein concentrate.
protein content and a lipid content of 20 to 25% (lipid–protein concentrate) [4,

18]. Later, processes were developed which were based on heat coagulation at
80 8C [19, 20]. Finally, procedures based on two-step heating of the green press
juice enabled fractionated extraction of proteins, leading to products of different
composition [21, 22].
The basic fractionation process is the same for all concepts, as shown in
Fig. 12.1.
Delays between harvesting in the field and processing in the factory should
be reduced to a minimum. After grinding and crushing in mills the green mat-
ter is pressed. Wet fractionation (pressing) leads to two fractions – the press
cake and the press juice. The press cake is rich in crude fiber and the press
juice contains proteins, water-soluble sugars (WSC), ash and other interesting
substances, for example lutein.
The developments mentioned above resulted throughout the seventies in mar-
ket-leading technologies such as the Proxan procedure and the Alfaprox proce-
dure, which are used for generation of protein–xanthophyll concentrates, includ-
ing utilization of the by-products, although predominantly in agriculture [14,
23].
12.2.3
First Production of Leaf Dyes
Chlorophylls, often termed the “pigments of life”, are green colored macrocyclic
pigments which are the primary photosynthetic pigments in nature. The term
chlorophyll, coined by Berzelius in 1838, is derived from Greek roots and indi-
cates the green of leaves [24]. In fact, as green pigments they are responsible
for the primary biochemical energy generation in nature and give the only indi-
cations of life on earth visible from outer space. Reduction of the chlorophyll
(leaf green) gives the xanthophylls (leaf yellow) and carotenes that are not dis-
solved and remain in the leaf, resulting in showy yellow and orange tinges. The
red color pigments are derived from anthocyan, created by metabolic alteration
of the leaves [25].
Although scientists had previously studied the green plant pigment it was
only in 1913 that the first significant research on its structure, separation, and
properties was reported. This work, which won the 1915 Nobel Prize for the
German chemist Willstätter, serves as the basis for subsequent production of
chlorophyll [26, 27].
In the United States, commercial production of chlorophyll and carotene by ex-
traction from alfalfa leaf meal has conducted since 1930 [28, 29]. Chlorophyll, in
various forms, was reported to be present in 1000 products that consumed 10 000
pounds of the green material per month in 1952, with a market value of 50 Million
USD [30]. For example, Strong, Cobb and Company obtained 0.5 ton of chloro-
phyll per day from alfalfa in 1952. The water-soluble chlorophyll, or chlorophyllin,
found use as a deodorizing agent in toothpastes, soaps, mouthwashes, shampoos,
chewing gums, candies, deodorants, and pharmaceuticals [31].
Since 1990 chlorophyll has also been used for conversion of light energy into
electric energy. Electrochemical solar cells, e.g. the Graetzel cell a TiO2–chloro-
phyll-SnO2 solar cell, use organic dyes (not a semiconductor material), for ex-
ample the leaf dye chlorophyll, for absorption of light [32].
12.3
Green Biorefinery Raw Materials
12.3.1
Raw Materials
The major raw material of green biorefineries is “green biomass”, including the
large group of green plant materials (green grass from meadows, willow, exten-
sive willow management, other natural resources), wild fruit and crops, alfalfa
and clover, and immature cereals and plant shoots. The green plant material
contains complex natural and valuable materials in the form of carbohydrates,
proteins, fibers, fragrances, dyes, fats, hormones, amino acids, enzymes, and
other important substances [5, 7, 33, 34].
Ecologically friendly agriculture is based on primary production by photo-
synthesis in green plants during the whole growth season. During a vegetation
period successive harvest and re-growth of one and the same crop can give a
maximum yield of dry matter and protein per area. Green grasses and imma-
ture cereals are excellent for this purpose. Especially, grasses can be grown on
most types of soil in most types of climate, on both normal agricultural land
and marginal land. Thus C3 species in temperate climates can yield up to 20
tonnes of dry matter and 4 tons of protein per ha (hectare = 10 000 m2) per year
whereas C4 species in tropical climates can produce 80 tonnes of dry matter
and 6 tonnes of protein [33, 34].
The yield of dry matter and protein from grass and the quality of the leaf pro-
tein concentrate (LPC) obtained is affected by the type of photosynthesis [35].
Leaf anatomy and cell structure are different for the two types of plant adapted
to different climates. The C4 species have a more efficient carbon dioxide fixa-
tion mechanism and are grown on soils poor in nitrogen. Thus C4 species have
a very high dry-matter production per soil area, but have a low protein content
of the dry matter. The C3 species lose fixed carbon dioxide by a process called
photorespiration. Thus C3 plants produce less dry matter per unit area. In C3
species more leaf cells are rich in protein (FI protein/rubisco protein). There-
fore, a relatively high proportion of the dry matter of C3 species consists of pro-
tein. Subsequently, LPC from C3 species is rich in protein. Both temperate
grass species, including green cereals [34, 35] and tropical grasses [36], have
been investigated for LPC production (Table 12.1).
The second important raw material source is the green harvesting residue
material from agricultural cultivated crops. In particular the vegetables of im-
portance are those with green foliage. This includes, e.g., not insignificant
Table 12.1 Grasses and green cereals investigated for green

crop fractionation [34, 35].
Avena sativa Holcus lanatus Pennisetum purpureum

Bromus arvensis Hordeum vulgare Phalaris arundinacea
Cynodon dactylon Lolium multiflorum Secale cereale
Dactylis glomerata Lolium perenne Tricum aestivum
Festuca arundinaceae Paspalum dilitatum Zea mays
Festuca pratensis
amounts of sugar beet leaves (sugar beet for the sugar industry), hemp scrapes
and leaves (hemp for fiber production), residues from flax processing, and resi-
dues from the fresh vegetable production.
Further potential refinery raw materials are the little standardized juice-rich
waste biomass. This should contain moisture and mainly natural and valuable
materials or have a substantial conversion grade. According to coupling effects
of material and energy use, the constitution can strongly vary. Such waste bio-
mass is not yet standardized, but is a renewable natural waste resource that
must be managed. Such biomass can be residues from plant production (mixed
and ripe harvest residuals), potato juice, hydroxycarboxylic acid-rich waste as si-
lage seepage, juices from the canned food industry, or residues from the sugar
industry or animal production.
The fourth large group is the little standardized dried biomass and waste bio-
mass. These often contain a large amount of plant cellulose and will therefore
be supplied as raw material to press-cake-using production lines. This can be re-
sidual straw, hay, and all kinds of dried foliage (e.g. maize hay). Residues from
in-plant waste paper and wood, e.g. for energy production or cardboard produc-
tion, are also included in this category. This group also includes modern con-
cepts of dry crop fractionation, for example immature cereals [37].
It should be mentioned that transitions between raw material types will and
should be fluid [38].
12.3.2
Availability of Grassland Feedstocks for Large-scale Green Biorefineries
In Europe, grassland amounts to about 45 Mio ha, which is approximately 35%

of the arable land (basis: 15 member states without new member states). A
large part of this grassland is regarded as absolute grassland habitat which can-
not legally be converted into plain arable land. Based on an average yield of
10 tons dry matter per ha per year, however, the European grassland produces
about 450 Mio tons dry matter each year [39]. The main purpose of grassland
cultivation is still the production of forage for animal farming. The use of grass-
land for feed production is dropping, because limitation of production quotas
and the increasing efficiency of animal breeding (especially dairy farming) is
leading a decrease in livestock numbers. Other uses for grassland must there-
Table 12.2 Production of green pellets or powder in Europe.
Country Amount dry matter (t a–1) Country Amount dry matter (t a–1)
Germany 320 000 The Netherlands 214 300

Austria 1 292 Ireland 5 337
Belgium 3 600 Italy 704 000
Denmark 170 868 Portugal 3 734
Spain 2 100 678 Great Britain 56 539
Finland 1518 Sweden 11 571
France 1 398 445 Czech Republic 29 158
Greece 48 848 Europe 5 069 888
fore be found, for example the supply of raw material for the bio-industry. This
trend is apparent throughout Europe [40].
Because of its ability to fix nitrogen from the air and enrich the soil with this
element, alfalfa is the most important forage crop in the world, cultivated on ap-
proximately 32 million hectares. The plant contains the protein rubisco as ap-
proximately 20% of the total dry matter [41].
Green forage drying plants, especially, offer a very good possibility for use in
biorefinery systems. These plants can be seen as agro–industrial knots in grass-
land farming. More than 300 green forage drying plants are used to produce
over 5 Mio tons of dried pellets and powder (Table 12.2) [42].
In the USA the Alfalfa New Products Initiative (ANPI) has the objective of ex-
tending the cultivation and utilization of alfalfa. The ANPI consists of five
states: North Dakota, South Dakota, Minnesota, Wisconsin, and Michigan.
Prominent technology in this context is dehydration and fractionation (dry or
wet) [43].
12.3.3
Key Components of Green and Forage Grasses
The research literature on forage grasses is mainly concerned with their nutri-
tive aspects as fodder grass, hay, or silage. From a biochemical perspective the
composition of forage grasses is well described. Thus, a comprehensive inven-
tory of forage grass chemical/material constituents is available in the literature.
The components can be conveniently categorized according to their location
within the grass, either as a cell wall constituent or as a component within the
cell.
12.3.3.1 Structural Cell Wall Constituents

Cell wall constituents comprise structural polysaccharides (hemicellulose, cellu-
lose), lignin, and pectin substances. The qualitative and quantitative composi-
tion of constituents within the cell walls varies during the growing season.
Hemicellulose, Cellulose, and Lignin The hemicellulose, cellulose, lignin, and

crude fiber content of fresh herbage, hay, and silage from meadow grasses has
been compared [44]. The crude fiber content of grass harvested as fresh herbage
was 24.0–35.5% of the dry matter (DM). The crude fiber content increased with
delay in harvesting and was higher in hay and silage than in the fresh herbage.
The combined total hemicellulose, cellulose, and lignin content was twice that
of crude fiber in grasses. During ensiling of grasses the hemicellulose content
was reduced by an average of 3–11%, the cellulose remained unchanged, and
the lignin content increased by 23%.
With advancing maturity, the concentrations of cellulose, hemicellulose, and
lignin, in grasses increase. In general, the digestibility of cellulose decreases
during the growing season; this commonly attributes to an increasing lack of
accessibility of the polymer to be attacked by microorganisms [45]. This varia-
tion should be considered when assessing grasses as feedstock for industrial
processes, particularly when a decision to harvest a forage grass specifically for
its fiber content must be made.
Chemical composition and digestibility were studied in vivo and in vitro [45].
The main finding was that although the grass species studied had similar gross
chemical composition, the digestibility varied substantially at comparable stages
of maturity. Thus a rapid decrease in digestibility was observed between the two
first cuts whereas only small changes were observed between the two times of
harvesting the re-growth. Although digestibility in this study was studied with
reference to ruminants, variation in this property may become significant when
considering the use of grass as an industrial fermentation feedstock, for exam-
ple xylitol or lactic acid production.
The more digestible rye grasses have two to three times more (1 ? 4)-linked
d-xylose units without branch points at the O-2 and O-3 positions, the propor-
tions of those branch points being substantially reduced. The changes were
greater in the early cut samples [46].
Similarly, it has been noted that re-growth has a lower nutritional value than
the first cut at a comparable stage of growth. Dactylis glomerata and Lolium pe-
renne were cut 1 to 3 times and analyzed chemically. The material from the first
cutting had the highest total digestible nutrient content, 55.96%. Protein utiliza-
tion value was lowest in the third cut grass [47]. The amounts of d-galactose
and other carbohydrates were much lower in the re-growth [48].
Åman and Lindgren studied [49] the change in the chemical composition and
degradability of six grasses including Festuca pratensis, Festuca arundinacea, and
Dactylis glomerata which were harvested at two stages of maturity in both the
first and second cuts. The results are shown in Tables 12.3 and 12.4.
Composition studies have also been driven by recognition of the effect of
covalent binding between the cell wall polymers on utilization of the cell wall as
a nutrient source.
Morrison investigated [45] variations in the hemicellulose and lignin composi-
tion of grasses over the growing season. It is known that these two cell compo-
nents are covalently bonded and it is believed that the lignin has a substantial
Table 12.3 Composition of grasses harvested at early first cut

and late first cut (% of DM of unextracted material, sugar
residues given as anhydrosugars).
Dactylis glomerata Festuca pratensis Festuca arundinacea
Early first cut

80% Ethanol extract 31.5 29.2 28.2
Crude protein 16.6 14.2 14.3
Polysaccharides 37.5 38.8 40.5
Rhamnose 0.1 0.1 0.1
Arabinose 2.6 2.6 2.8
Xylose 11.2 10.6 13.1
Mannose 0.2 0.1 0.2
Galactose 1.4 1.0 0.9
Glucose 19.1 21.3 20.0
Uronic acids 2.9 3.1 3.4
Glu/Xyl + Ara 1.4 1.6 1.3
Klason lignin 7.7 10.3 9.1
Ash 10.0 10.2 10.0
NDF 51.4 55.4 55.4
ADF 28.5 31.4 30.8
Permanganate lignin 4.2 5.2 4.8
Residual organic matter
in vitro 12.1 15.8 16.5
in vivo 25.8 22.8 27.1
Leaf percent 48.0 41.0 42.0
Late first cut
80% Ethanol extract 28.1 24.9 26.6
Crude protein 10.6 9.8 9.8
Xylose 12.1 15.5 14.4
Mannose 0.2 0.2 0.2
Glucose 25.3 25.4 22.9
Glu/Xyl + Ara 1.7 1.4 1.4
Ash 8.6 8.5 8.5
NDF 57.9 62.3 62.4
ADF 32.6 35.6 34.3
in vitro 21.1 24.6 28.9
in vivo 31.8 32.2 35.7
Table 12.4 Composition of grasses harvested at early second

cut and late second cut (% of DM of unextracted material,
sugar residues given as anhydrosugars).
Dactylis glomerata Festuca pratensis Festuca arundinacea
Early second cut

80% Ethanol Extract 22.2 22.7 25.6
Crude Protein 9.9 10.0 10.0
Xylose 11.9 10.2 12.5
Mannose 0.2 0.2 0.2
Glucose 26.3 26.2 24.0
Glu/Xyl + Ara 1.7 2.0 1.5
Ash 9.7 11.5 10.9
NDF 65.1 60.5 61.2
ADF 40.0 37.0 34.0
in vitro 20.5 19.4 18.4
in vivo 30.9 28.4 29.2
Late second cut
80% Ethanol Extract 24.3 22.0 24.8
Crude Protein 9.0 8.7 9.7
Xylose 10.5 11.8 11.2
Mannose 0.2 0.4 0.2
Glucose 26.7 26.4 23.4
Glu/Xyl + Ara 2.0 1.8 1.7
Ash 9.8 10.6 11.5
NDF 63.5 62.3 60.6
ADF 41.2 39.2 34.7
in vitro 19.1 19.9 16.8
in vivo 32.9 29.6 30.1
effect on the digestibility of the hemicellulose moiety. In this study, ten varieties
of temperate grass were studied by harvesting at five stages of maturity, taking
only a first cut. The lignin and hemicellulose content were measured, with the
hemicellulose being further fractionated into linear and branched hemicellulose
by iodine treatment. The hemicelluloses were analyzed for the neutral sugars l-
arabinose, d-xylose, d-galactose and d-glucose. The results are shown in Ta-
ble 12.5.
Lignin–carbohydrate complexes from Lolium perenne contained high propor-
tions of d-glucose residues (ca 50%). Leaf tissue complexes had the highest d-
glucose content, whereas stem and leaf sheath were very similar. The other neu-
tral sugar residues present in these complexes were mainly l-arabinose and d-
xylose. The polysaccharide components of the lignin-hemicellulose complexes
contained mainly d-xylose (63–77%) and l-arabinose (19–28%) [50].
Forage grass lignin was more extensively solubilized by acid detergent than
forage legume lignin. Forage plant lignins were characterized by guaiacyl–syrin-
gyl lignin with p-hydroxyphenylpropane units. The number of ferulic acid cross-
linkages in the cell wall matrices of forage grasses increased with plant matura-
tion [51].
Two classes of phenolic–carbohydrate complexes were purified from the water-
soluble products obtained from digestion of Lolium perenne cell walls with a cellu-
lase preparation [52]. They contained d-glucose, d-xylose, l-arabinose, d- galactose,
and d-mannose in the ratios 3.6 : 10 : 6.3 : 1.4 : 2.3 and 5.3 : 10 : 3.0 : 1.1 : 2.1, respec-
tively. The complexes were based on (1 ? 4)-b-d-xylan chains to which were
attached residues of l-arabinofuranose and d-galactopyranose. Mixed linkage
(1 ? 3),(1 ? 4)-b-d-glucan chains also seemed to be integral components of these
complexes.
The principle outcomes of these studies were:
1. The quantities of hemicellulose increased with increasing maturity with the
increase being larger in stem tissue compared with leaf tissue. For example,
the hemicellulose content of Lolium perenne leaf tissue increased from 7.6 to
Table 12.5 Hemicellulose concentrations (g kg–1 DM) in the

leaf and stem tissue of forage grasses.
Leaf Stem
Cut no. 1 2 3 4 5 1 2 3 4 5
Lolium perenne S24 83 114 162 101 133 244

Lolium perenne Reveille 79 104 140 97 136 211
Lolium perenne S23 76 120 167 183 211 99 137 204 228 291
Lolium perenne Barpastra 78 111 153 180 199 89 136 183 194 272
Festuca pratensis 113 159 202 147 177 270
Festuca arundinacea S170 124 172 194 162 201 193
21.1% of dry matter over the study period. The d-xylose content of the linear
hemicellulose increased concomitantly from 69 to 85%.
2. The hemicellulose content of stem tissue increased from 9.9 to 29.1% with
the linear hemicellulose increasing from 74 to 91%.
3. The linearity of hemicelluloses tended to increase with crop age.
4. Higher lignin content was associated with hemicellulose of a higher linear:
branched ratio.
5. Hemicellulose also had higher d-xylose : l-arabinose ratios.
In summary:
1. Time of harvesting has a significant effect on the sugar composition.
2. Hemicellulose sugars increase during the growth season.
3. Stem tissue contains greater amounts of hemicellulose (xylans) than leaf tis-
sue.
4. Digestibility of grasses decreases with age, which may affect yields of fermen-
tation-derived products such as xylitol and lactic acid.
Although, overall, at higher maturity, the absolute quantities of potentially fer-

mentable sugars (e.g. d-xylose, present as hemicellulose) are greater, their ac-
cessibility to fermentation media may be reduced.
Pectin Substances Extraction of mesophyll cell walls from the leaves of Lolium per-
enne afforded 25 mg of a uronic acid polymer per gram of material [53]. The poly-
mer was identified as a 1,4-linked homogalacturonan, essentially free from neutral
sugar residues, with a low degree of acetylation (3.6%) and methyl esterification
(3.3%). Thus, the pectin was similar to the pectins of dicotyledons but the amounts
found were substantially lower than in most dicotyledonous plants. On that basis
there seems little scope for industrial end uses of forage grass pectins.
12.3.3.2 Cell Contents

The cell contents of forage grasses contain sugars, fructans, amino acids, pro-
teins, silica, alkanes, starches, minerals, nucleic acids, lipids, and alkaloids. Pro-
tein and sugars are the most abundant components. The concentration of non-
structural carbohydrates in leaves and stems is highest in winter for Lolium pe-
renne – 13% of dry matter. Seasonal variations in element concentration are
small [54]. Festuca pratensis and Dactylis glomerata are characterized by high cell
wall contents.
Sugars d-Glucose, d-fructose, d-sucrose, and fructans are the main nonstruc-
tural carbohydrates in Lolium perenne tissues [55]. The d-glucose, d-fructose, d-
sucrose, and d-xylose, d-mannitol, d-sorbitol, glycerol, and d-maltose content of
Dactylis glomerata, Lolium perenne, and Festuca pratensis cut three times on differ-
ent dates without interim harvesting have been recorded [56]. The results are re-
ported in Table 12.6. Significant findings were:
Table 12.6 Changes in water-soluble carbohydrate content and

mono- and disaccharide content (% on dry matter basis) of
Lolium perenne and Festuca pratensis.
Monosaccharides Sugar alcohol Disaccharides Mono + WSC

Dis-
acch.
Species Glc Fru Xyl Mann Sorb Glyc Sucr Malt
Lolium perenne
June 6 3.11 5.43 0.00 0.05 0.05 0.24 0.15 0.10 9.12 27.0
June 21 3.82 4.62 0.05 0.00 0.00 0.24 0.14 0.14 9.02 16.1
July 6 2.89 3.92 0.08 0.04 0.04 0.20 0.12 0.16 7.45 18.1
Aug 6 4.36 6.25 0.26 0.13 0.00 0.46 0.26 0.26 11.98 9.2
Aug 21 2.43 5.10 0.23 0.08 0.00 0.32 0.00 0.16 8.32 9.8
Sep 3 1.46 1.87 0.11 0.00 0.00 0.19 0.11 0.08 3.83 7.8
Sep 30 5.00 7.47 0.14 0.07 0.00 0.40 0.27 0.20 13.53 16.0
Festuca pratensis
June 6 2.38 4.91 0.00 0.00 0.00 0.24 1.42 0.10 9.04 13.8
June 21 2.93 3.48 0.00 0.00 0.00 0.19 0.12 0.12 6.85 9.0
July 6 2.13 3.33 0.07 0.00 0.00 0.18 0.00 0.21 5.95 12.5
Aug 6 2.07 3.30 0.15 0.25 0.00 0.20 0.30 0.40 6.66 4.9
Aug 21 1.74 2.8 1 0.07 0.07 0.00 0.34 0.00 0.20 5.22 7.6
Sep 3 1.53 0.47 1.80 0.19 0.12 0.19 0.19 0.27 4.78 6.6
Sep 30 4.56 6.56 0.13 0.13 0.06 0.44 0.19 0.19 12.25 10.3
Glc: d-glucose; Fru: d-fructose; Xyl: d-xylose; Mann: d-mannitol;

Sorb: d-sorbitol; Glyc: glycerol; Sucr: d-sucrose; Malt: d-maltose;
Mono + disacch: monosaccharides + disaccharides; WSC: water-sol-
uble carbohydrate
· Lolium perenne contained the most water-soluble carbohydrate (27%) in the

early season, compared with Festuca pratensis (13.8%).
· This figure decreased steadily through the growing season to 7.8% in early
September although it increased to 16% at the end of the month.
· Lolium perenne also contained the most xylose (0.26%) in mid-season.
· In Lolium perenne, glucose levels peaked in early August and again in late
September. Although xylose levels similarly peaked in early August, there was
no corresponding peak in late September. Similar trends were observed for
Festuca pratensis.
In analogous work, Fales and colleagues [57] reported results for stems of Festu-
ca arundinacea. The stems were extracted with 95% ethanol and water to afford
d-glucose, d-fructose, d-sucrose, and fructans. The fructan extract was hydro-
lyzed with sulfuric acid and shown to contain d-glucose and d-fructose. A hemi-
cellulose fraction was hydrolyzed and found to contain d-xylose, l-arabinose and
small amounts of d-glucose.
Fructans In grasses, fructan reserves are mobilized from vegetative plant parts
during seasonal growth, after defoliation during grazing. In expanding leaves,
fructans are accumulated in cells of the elongation zone [58]. Fructan structures
have been characterized in Lolium perenne as belonging to essentially three se-
ries: the inulin series, the inulin neoseries, and the levan neoseries [59]. Festuca
arundinacea contains an inulin and neokestose based series of oligosaccharides
[60].
Fructans are an important class of carbohydrate of substantial biotechnologi-
cal importance [61]. First, they are a potential source of d-fructose, for which
there is a growing market in the food industry as a sweetener. The use of fruc-
tans has been reviewed by Fuchs [62]. Fructans are mainly used in the food sec-
tor. More pertinently, fructans could be chemical feedstocks from which a vari-
ety of chemicals can be produced. Hydrolysis to d-fructose and subsequent de-
hydration leads to hydroxymethyl furfural which, like lactic acid, is regarded as
key chemical intermediate for chemistry based on renewable raw materials.
Similarly, hydrolysis of inulin to d-fructose followed by catalytic hydrogenation
yields d-mannitol/d-sorbitol mixtures from which d-mannitol can be easily crys-
tallized. d-Mannitol, like xylitol, is a valuable, non-cariogenic low-calorie sweet-
ener. Other chemicals that could be derived from fructans include ethanol,
other organic solvents, and chemicals such as furans [63].
Inulin is the best known sub-class of the fructans. Inulin is colorless and
odorless, and has a pleasant slightly sweet taste; it is moderately soluble in
water and acts as a gel-forming agent at concentrations > 30%; it is also a foam
stabilizer and texturing agent. Its calorific value is 4 kJ g–1, but it acts as dietary
fiber. It suppresses putrefying bacteria and selectively supports bifidobacteria
and lactobacilli in the colon. In food applications its main functions are to re-
place fat and sugar, to enrich with dietary fiber, to activate bifidobacteria, and to
reduce cariogenicity. It is classified as a foodstuff [64].
Amino Acids The amino acid composition of Festuca pratensis, Dactylis glomera-
ta and Lolium perenne has been studied. There were no significant differences
between the species. As the grasses aged, amounts of aspartic acid, glutamic
acid, alanine, tyrosine, and phenylalanine decreased and amounts of threonine,
serine, and proline increased. Lysine, histidine, arginine, glycine, valine, methio-
nine, isoleucine, and leucine did not change with plant age nor did the total
amino acid content [65].
In amino acid analysis of six crops and the corresponding juice, the amino
acid composition of the juice deviated only slightly from that of the crop but
the amounts of glutamic acid and aspartic acids were somewhat higher and cor-
respondingly the amounts of other amino acids, particularly arginine, glycine,
alanine, tyrosine, and phenylalanine were somewhat lower [66].
Degradation of protein and amino acids in juice extracted from ryegrass can
be reduced by adding hydrochloric acid. For complete preservation, the pH
must be less than 3. Heating to 80 8C also has a preservative effect [67].
Proteins Leaf protein concentrate is obtained by green crop fractionation. The

acceptability of leaf protein concentrate in the human diet has been discussed
by McDougall [68]. Lesnitski has also advocated [69] the manufacture of high
protein feeds from green mass for partial replacement of, for example, soybean
protein and dried skim milk.
Silica In comparison with elements commonly associated with the nutrition of

higher plants, silicon has received relatively little attention. Biogenic amorphous
silica (BAS) is a natural constituent of living matter. In some plants some of
the BAS occurs externally as pointed or irregularly shaped fibers, and these
have been implicated as human toxicants [70]. Silica deposits commonly called
phytoliths occur in cell walls, cell lumens or in extracellular locations. Silicifica-
tion occurs in roots and shoots, including leaves, culms, and in grasses, most
heavily in the inflorescence. Biogenic silica structures provide support and pro-
tection [71].
Grasses are heavy accumulators, but substantial variation occurs between and
within species. Deposition is heaviest in inflorescence bracts [72]. Silica has
been detected in the leaf mesophyll of Lolium multiflorum at an estimated con-
centration of 1–2%. Samples were also subjected to a range of techniques for re-
moval of organic matter, to confirm the presence of silica throughout the cell
walls [73]. In Lolium perenne, only the epidermal cell walls of the leaf edges and
the trichomes contained silica [74]. The Lolium perenne variety Fortis, which has
some resistance to stem borer, had many silica bodies between the veins of the
leaf sheath [75].
The silica content of each of four cuts of 3 Dactylis glomerata, 4 Festuca praten-
sis, 5 Lolium perenne, 5 Lolium multiflorum are reported by Puffe and colleagues
[76]. Silica content is lower in legumes than in grasses.
Alkanes The total n-alkane (C27–C35) content of Dactylis glomerata, Lolium

multiflorum, and Lolium perenne was found to be 143, 681, and 531 mg kg–1 dry
matter, respectively. The concentrations of C29 and C31 were always highest [77].
These levels do not seem to be high enough for commercial exploitation.
Starch The starch content of forage grasses is low – a maximum of 3% starch

is accumulated in field-grown grasses. Cocksfoot contains more starch than Lo-
lium perenne or Lolium multiflorum [78]. This low level rules out industrial use
of forage grass starches.
Minerals The dry matter of extracted juice of Lolium perenne has a high
mineral content [66] that may be exploitable as plant fertilizer.
Alkaloids Perloline, perlolidine, and loline are alkaloids of the Lolium spp. The
toxicity of lolium species is because of to a symbiotic fungal infection of the
plants [79].
Antifreeze Protein A plant antifreeze protein from Lolium perenne has been re-
ported [80]. Present in organisms enduring freezing environments, antifreeze pro-
teins have the ability to inhibit damaging ice-crystal growth. The macromolecular
antifreeze protein present in Lolium perenne has superior ice recrystallization inhi-
bition activity compared with fish and insect antifreeze proteins [81].
12.4
Green Biorefinery Concept
12.4.1
Fundamentals and Status Quo
Green biorefineries are complex systems based on ecological technology for

comprehensive (holistic), substantial, and energy utilization of renewable re-
sources and natural materials in the form of green and waste biomass from
focused sustainable regional land utilization. Such green biomass is, e.g., grass
from cultivation of permanent grassland, fallow land cultivation, nature re-
serves, or green crops, for example alfalfa, clover, and immature cereals from
extensive land cultivation. Thus, green plants are a natural chemical factory and
food plant. Careful wet fractionation technology is used as a first step (primary
refinery) to isolate the substances in their native form. Thus, the green crop (or
humid organic waste goods) is separated into a fiber-rich press cake (PC) and a
nutrient-rich green juice (GJ). Besides, cellulose and starch, the press cake con-
tains valuable dyes and pigments, crude drugs, and other organic substances.
The green juice contains proteins, free amino acids, organic acids, dyes, en-
zymes, hormones, minerals, high quality crude drugs, and other organic sub-
stances. By use of this biotechnology, ecotechnology, and “soft” and “green”
chemistry, these valuable materials can be isolated in their natural form or, by
mild careful conversion, can be utilized economically [7].
Activity in the green biorefinery field is increasing and developing into an in-
dependent aspect of the large field of biomass technology. Raw material and
technological aspects of this system are particularly characterized by considera-
tion of sustainability criteria and incorporation of technology from regional and
rural living and business (sustainable economy, sustainable agriculture, and sus-
tainable regional development).
The term “green biorefinery” is, on the one hand, used for model processes
but, on the other hand, also used for an entire program. To “refine” is originally
French (raffiner) and means “to improve something, to purify”. A refinery is, by
definition, a technical facility for purification, separation, and refining of materi-
als and products. “Green” in the field of plants means, simultaneously, high
concentrations of chlorophyll, nutrients, and water, “bio” is Greek (bios) and
means “live”, something biological and natural. Programmatically, the green
biorefinery stands for technology (refinery) formed by imitation of nature (bio-
logically) with the target being soft and sustainable [7].
Fig. 12.2 Green biorefinery combined with a green crop-drying

plant. Concept of the Havelland biorefinery, Selbelang, State
of Brandenburg, Germany [82, 83].
In principle, the primary conversion technology is set up on the basis of the

water content of the different raw materials [84]. It is reasonable to use mechan-
ical pressing for green nature-wet biomass in the first step. Traditionally green
crop fractionation was regarded as providing edible protein for humans. Frac-
tionation separates the crop into pressed matter, which can be used to feed
ruminants, and leaf protein concentrate (LPC) that can be eaten directly by hu-
mans [15–17, 68]. Different choppers and shredders, e.g. hammer-mills and dif-
ferent presses, e.g. screw-presses, roller-presses, and piston-presses, are used as
technical equipment for fractionation of green biomass [85–88]. The literature
also mentions the separation of proteins [89, 90]. The company “France Lu-
zerne” has realized a process for separation of proteins on an industrial scale.
The resources, mainly alfalfa, are treated by four main process steps:
· pressing,
· heat coagulation,
· centrifugation, and
· drying.
The technical specifications of the product and, especially, its high xanthophyll
content make it very useful in poultry farming for egg yolk and broilers (Ta-
ble 12.7) [91].
Table 12.7 Industrial production of PX (protein–xanthophylls)

and their specification [91].
Product Production Quantity Specification

year
PX 1 1977 1300 T 50% crude protein and 1.000 ppm of total xanthophylls
PX 1 1980 6200 T 50% crude protein and 1.000 ppm of total xanthophylls
PX Super 1997 13000 T 52% crude protein and 1.250 ppm of total xanthophylls
12.4.2
Wet Fractionation and Primary Refinery
The special first feature of the green biorefinery is the wet fractionation of
green biomass (Fig. 12.3 A).
This is also called first fractionation step or primary refinery step. (It includes,
for example, the harvest, fractionation, conservation, and storage of the primary
fraction.) Here, fresh harvest and waste goods are treated. Thus, the plant com-
pounds are mostly unadulterated; the green goods should, in any case, be
treated immediately, however. This processing step, usually performed by means
of an industrial press, produces a fiber-rich, water-insoluble, solid material,
press cake (PC), and a nutrient-rich green juice (GJ) or brown juice (BJ). The
wet fractionation is based on soft separation of water-soluble and water-insoluble
components of the green biomass.
The silage wet-fractionation is a form of the primary refinery technology
(Fig. 12.3 C). The green goods are conserved by organic acids or fermentation
processes before treatment by the procedure shown. Treatment of silage from
green resources has many advantages (decentralized raw material preparation,
simple and low price conservation and storage, reasonable whole year operation
of the biorefinery, etc.) [92]. The end products of silage are different from that
of the substances in the green juice, because silage fermentation degrades the
cell walls and modifies or converts substances because of the biotechnological
processes involved.
The so called “decomposition” methods are the third category of primary refin-
ery technology (Fig. 12.3 B). “Decomposition” methods are mainly applied to the
humid or dry whole plant. The processes work with enzymatic, fermentative, hy-
drolytic, chemical, thermal, or combined thermal and fractionation methods. The
strength (depth of operation) of the decomposition varies, and ranges from low
(enzymatic, fermentative) to high intensity (chemical, hydrolytic). For every step
classification is needed to check if it belongs to green biorefinery technology. A
high single yield of products can be achieved if the complete plant decomposition
occurs at the primary refinery step (e.g. saccharification, which increases the total
amount of sugars in the raw charge). But these procedures reduce the level of na-
tive product diversity. Nevertheless decomposition methods have been regarded as
feasible technologically and economically. This is also true for the secondary prod-
Fig. 12.3 The green biorefinery – primary refinery. Methods for fractionation of green crops [7].
uct lines. Problems with the green biorefinery system may be solved by further
development of new biotechnological decomposition methods.
All primary fractionations after the secondary refinery steps contain processes
for substantial and energy utilization of the fractionation products. The kind
and number of the secondary fractionation steps are determined by the compo-
sition and energy potential of the input green biomass and waste biomass, the
type of technology, and the marketability of potential products of the refinery.
The company Avebe operates a pilot plant at Veendam (The Netherlands) and
has formed a consortium with other partners to explore the potential of grass to
supply a range of fiber, protein, and nutraceutical products. The current pilot
plant processes 3 tons of fresh grass per hour. Depending on the outcome, a
full-scale (200 tons per hour, 50 000 tons per year) factory may be commis-
sioned. The intended full-scale plant requires 1000 ha per annum of mixed
grass sources harvested from a radius of up to 50 km [93].
The concept of the Havelland biorefinery, Selbelang, State of Brandenburg,

Germany is the fractionation of 25 000 tons of fresh green biomass per year, in
its first stage of expansion, and the production of proteins and fermentation
medium from green juice. Production of feed, tech-paper, and chlorophyll from
press cake [94] (Fig. 12.2) will also be established.
The Austrian-Concept is based on a decentralized system to take into account
small scale agriculture. The system is, however, built around grass silage fer-
mentation and the production of lactic acid, amino acids (hydrolyzed proteins),
and fiber [92].
The problem of storage of fresh green biomass must be solved, i.e. green bio-
mass must be preserved such that it is available for longer periods of time to
enable continuous year-round operation of the plant.
The development of sustainable green biorefinery systems requires a combi-
nation of central and decentralized/localized units, i.e. large-scale conversion
and processing plants that take advantage of economies of scale must be com-
bined with smaller and localized units as close to biomass feedstock as possible,
resulting in improving rural economies and reducing the environmental impact
of transportation [95].
12.5
Processes and Products
The kind and number of products from a green biorefinery is nearly unlimited,
if the fractal character of the biosynthesis and biochemistry of green plant mate-
rials is considered [96]. Characterization of a plant by a new analytical method
usually discovers, in addition to the main components, innumerable new side
products. Not all substances have been discovered and technologically obtained
from natural products even from plants with great trading importance, e.g. al-
falfa [97]. For the green biorefinery the main products, side-products and im-
purities are of interest [98]. Economic aspects reduce the diversity of products of
interest, however. Soft technology such as biotechnology is used to reduce the
complex molecules of natural materials. Nevertheless, the scientific field of eco-
technology develops new methods, preferring a reduction of technological
strength (depth of operation). This can be done by using, e.g., biodiversity be-
fore molecular modification or applying less intensive methods, etc. [99].
12.5.1
The Juice Fraction
12.5.1.1 Green Juice

In the (especially) freshly pressed (Fig. 12.3 A) green juice (GJ) we can find pro-
teins, lipids, glycoproteins, lectins, sugars, free amino acids, dyes (carotenes),
hormones, enzymes, minerals, and other materials. The GJ can be fractionated
by heat, treatment with organic and inorganic acids, acid anaerobic fermenta-
tion, centrifugation, and gel filtration into a leaf nutrient concentrate (LNC) and
a brown juice (BJ). The LNC consists of a mixture of chloroplast and other orga-
nell membranes plus denatured soluble plant-cell proteins. The composition of
a LNC is: true protein (60–70%); lipid (especially palmitic acid, linoleic acid,
and linolenic acid) (20–30%), starch (5–10%), ash (1–10%), carotenoide/polyene
dyes: b-carotene (1–2 g kg–1), and xanthophyll [6, 100]. The LNC is mainly used
for nonruminant feed to enhance the color (red b-carotene, or lutein) of chicken
skin or egg yolk. It also produces tender meat in chickens, ducks, and pigs.
Feeding pigs with LNC results in pork with an increased content of healthy
oleic and linoleic fatty acids in the fat [101].
Sugars (in Particular Glucose, Fructose, and Fructans) GJ and BJ contains valu-
able special sugars and have highly valuable and sometimes expensive applica-
tions. Other sugars in GJ are erythrose, rhamnose, xylose, galactose, mannose,
mannitol, maltose, and derivatives, for example myoinositol and glycerin. Before
these compounds are fermented they are studied with regard to their potential
characterization, and isolation [98, 102].
Dyes and Vitamins Green leaf nutrient concentrate (GLNC) enriched in b-caro-
tene may have anti-cancer effects. b-Carotene (provitamin A) and xanthophyll
are used in cosmetic drugs and as food, textiles, and toy-coloring agents (see
also chlorophyll) [28, 29, 31]. Green juice contains further vitamins, for example
vitamin B1, vitamin B2, and vitamin E [103].
Fatty Acids GLNC is also rich in oleic and linoleic fatty acids, especially palmit-
ic acid, linoleic acid, and linolenic acid. The lipids provide good health value.
The lipids can be separated by steam distillation. They are also of interest to the
cosmetic industry [100].
Crude Drugs/Ingredients Because the BJ contains specific secondary plant sub-

stances, for example saponins and nicotine, these can be separated from the
juice for pharmacological or pesticide purposes (isolation is described elsewhere
[97]).
Proteins The proteins in the green juice can be fractionated by advanced tech-
nology, in a second step, into a green leaf nutrient concentrate GLNC. The
main protein is the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase,
also known as fraction-I (F-I) protein (rubisco EC 4.1.1.39). In alfalfa leaves pro-
teins account for 30 to 70% of total nitrogen, depending on the physiological
stage or genotype [106]. Rubisco has a molecular weight (MW) between 500 000
and 600 000 Daltons and is composed of eight large and eight small subunits
with MW of approximately 55 000 and 12 500, respectively. The sedimentation
coefficient is close to 18.5 S. Rubisco has a compact, tightly folded three-dimen-
sional structure typical of globular proteins. Because of its amino acid composi-
tion Rubisco is mildly acidic and is negatively charged at neutral pH (isoelectric
Table 12.8 Comparison of the amino acid composition of the

different protein fractions of lucerne [113, 114], according to
[124].
Protein Amino acids (parts per thousand, by weight)
Hydrophilic (H) Charged Apolar (A) Small H/A
Rubisco 414 289 285 180 1.45

White protein 421 303 272 183 1.55
Green protein 432 286 268 180 1.61
Soluble protein 491 333 239 143 2.10
Oligomeric soluble protein 451 310 275 166 1.70
Membranous protein 427 288 299 169 1.40
Hydrophilic: Asp + Glu + Ser + Thr + Arg + Lys + His

Charged: Asp + Glu + Arg + Lys
Apolar: Val + Ile + Leu + Phe + Met
Small: Gly + Ala
point pH 4.4–4.7) [107]. It also has a relatively high average hydrophobic value
of 1275 cal/residue, calculated according to Bigelow [108]. Native Rubisco from
alfalfa contains 90 sulfhydryl groups, of which eight are “free” (one per proto-
mer), 36 are exposed after denaturation by SDS, and 46 are involved in the for-
mation of disulfide bonds within the Rubisco subunits [109]. The denaturation
temperature of alfalfa rubisco varies between 70 (pH 7.5) and 61 8C (pH 10.3)
[110]. More details about Rubisco are available in reviews [106, 111].
Fraction-II protein consists of a mixture of proteins originating from the
chloroplasts and cytoplasm with molecular weights from 10 000 to 300 000 Dal-
tons and sedimentation constants from 4 S to 10 S [112]. On the basis of amino
acid composition, Rubisco and the green and white fraction of leaf proteins are
regarded as hydrophobic (Table 12.8).
The F I protein can be used in medical diets to enhance recovery from brain
damage, where a high calorie/high protein diet is needed. People with kidney
problems can easily digest F I protein with no negative effects on body metabo-
lism. Both F I and F II proteins are advocated for solid foods and drinks as a
supplement. The nutritive value and functional properties of LNC and white
leaf protein isolated for incorporation in human diets have been reviewed [68,
69, 115–118].
12.5.2
GJ Drinks/Alternative Life
Young green cereal leaves are used for production of health food grass juices and
cosmetics. Dried, finely ground, and resolved young leaves are used as “green tea”
and added to health-food drinks [119, 120]. Those familiar with folk medicine also
know much about the effects of wild mixed grasses, herbs, and herb teas [97].
12.5.2.1 Silage Juice

To facilitate continuous year-round operation of a green biorefinery it may be
very reasonable to introduce common agricultural technology. For that reason
the concept involves not only processing of directly cut grass, but also of silage,
which can be prepared in the growing season and stored in a silo. Silage is the
product formed when grass or other material of sufficiently high moisture con-
tent (grass optimum of 28 to 35% dry matter; maize 25–30% dry matter) liable
to spoilage by aerobic microorganisms is stored anaerobically. It is formed by
the process referred to as ensilage which occurs in a vessel or structure called a
silo. Normally during ensilage the fodder undergoes acid fermentation in which
bacteria produce lactic, acetic, and butyric acids from sugars present in the raw
material. The net result is a reduction in pH (to approximately pH 4 to 4.5)
which prevents the growth of spoilage microorganisms, most of which are in-
tolerant of acid conditions [121].
Table 12.9 Physicochemical characteristics of grass silage juice [123].
Property Value
Conductivity (20.5 8C) 35.8 mS

pH (20.5 8C) 4.04
Dry matter 13.6%
Color Dark brown
Cations (g L–1)
K+ 15.63
Na+ 0.15
NH4+ 1.22
Ca2+ 1.78
Mg2+ 0.50
Anions (g L–1)
Lactate 37.54
Acetate 2.08
Cl– 6.41
NO–3 2.13
PO3–4 4.38
SO2–
4 2.55
Sugars (g L–1)
Glucose 8.88
Fructose 14.99
Saccharose 5.36
Arabinose 1.72
Xylose 1.44
Galactose 2.86
Mannitol 3.09
Amino acid 26.13
The silage juice (Fig. 12.3 C) contains a relatively high concentration of lactic
acid, amino acids, sugars, and inorganic salts. Protein and peptide degradation
occurs during ensiling. In silage juices only 5 to 10% of the crude proteins (or-
ganic nitrogen compounds) are peptides > 1.2 kD (*15 amino acids). At least
18 amino acids are found in the juice with a total amino acid content of
26.13 g L–1. Among the most important are alanine, leucine, lysine, GABA (c-
aminobutyric acid), aspartic acid, and isoleucine (all in the l form) [122, 123]
(Table 12.9).
12.5.3
Ingredients and Specialities
12.5.3.1 Proteins/Polysaccharides
The polysaccharide and protein components of Festuca spp cell walls have been
transformed into emulsifiers by extraction and treatment with xylan-hydrolyzing
enzyme preparations. The emulsifiers are useful, for example, for food, cos-
metics, pharmaceuticals, and industrial chemicals applications [125].
12.5.3.2 Cholesterol Mediation

It has been demonstrated that polysaccharide–lignin complexes from fodder
grasses are active sorbents of cholic acid, a metabolite of cholesterol [126]. Equa-
tions have been derived for calculating the sorption of cholic acid by the grass
material. The equations can be used to construct dietary fiber with the desired
properties, for example for cholesterol metabolism in humans and animals.
12.5.3.3 Antifeedants
Festuca arundinacea and Lolium perenne can become infected with fungal endo-
phytes (Neotyphodium spp). The symbiosis between plant and fungus leads to
the synthesis of alkaloids that have been shown to be either toxic or act as feed-
ing detergents against insect pests. Alkaloid production/accumulation in Festuca
arundinacea and Lolium perenne is enhanced by reduced mowing frequency
[132]. Such alkaloids may have a role as insecticides for agrochemical use [104,
105] or in the clinic as a result of their pharmacology [79].
12.5.3.4 Silica
A process has been described for manufacture of high-purity amorphous silica
from biogenic materials [127]. Rice hulls are given as the example. The hulls
are finely divided, screened, subjected to a surfactant wash, rinsed, and soaked
in water to accelerate and enhance penetration of an oxidizing solution. The oxi-
dizing solution removes organic compounds, and volatile impurities are re-
moved by heated oxidation to leave silica. The remaining silica may be rinsed
with water, acid solution, or other solution to remove even trace impurities. At
the end of the process, a fine white amorphous silica of extremely high purity
is produced.
12.5.3.5 Silicon Carbide

Rye grass has been proposed as a raw material for the production of polytypi-
cally pure b-silicon carbide in an economically effective and ecologically compat-
ible procedure. When the particle size of starting raw material is defined then
the particle size of the developing silicon carbide is also controlled [128]. Silicon
carbide has many industrial applications and is a valuable chemical used for
cutting, grinding, and polishing applications. Silicon carbide is also used in the
electronics industry in hostile environments where its ability to function in
high-temperature, high-radiation conditions overcomes the limitations of con-
ventional silicon-based systems. It is used as a component of blue and violet
light-emitting diodes.
12.5.3.6 Filter Aids

Highly purified biogenic silica has an intricate and diatomaceous SiO2 structure
and a high-SiO2 specific volume. These products are extremely bright and can
be used in filtration processes [129]. In one example the adsorbent has been
used for the removal of proteins in chillproofing of beer [130].
12.5.3.7 Zeolites
Artificial zeolites are manufactured by heating a mixture of grass husks and
plants with aqueous alkali solutions to elute silicic components. These are
mixed with aluminum-enriching agents and treated under heat and pressure.
The zeolites have high cation-exchange properties and are be useful as fertiliz-
ers [131].
12.5.4
The Press-Cake (Fiber) Fraction
The products and product groups described below are technologically possible
after fractionation in accordance with Fig. 12.3 A and C. Use of the PC as feed
(silage, bale press food, green pellets) is well known [133]. Furthermore, extrac-
tion of plant dyes (chlorophyll, carotenes, xanthophyll) [25, 28] and applications
in the food and candle industry [31], in environmental analysis [134] or, after re-
fining, in cosmetics, medicine, biochemistry [135], electronics (nematic liquid
crystals) [136], and photovoltaics (organic dyes [32]) have also been described in
the literature.
Because of the structural similarity of chlorophyll and blood hemoglobin one
can expect interesting developments in the field of plant dyes and colorants.
The resulting fraction will, substantially, be thermally treated analogous to unex-
Fig. 12.4 Classification of the major components of grass

press cake, hay, and late hay crop (by analogy with Ref. [143]).
tracted PC. The suitability (and applicability) as feed depends mainly on the cor-
responding extraction compounds and has to be tested.
The PC fraction can be separated by analogy with wood raw materials into its
main components (Fig. 12.4). On the one hand, this green plant fractionation
does not seem to make much economic sense today (because of wood competi-
tion). There are, on the other hand, interesting applications for special vegetable
celluloses, hemicelluloses, and lignin. The “green plant” polyoses (hemicellu-
loses) are nutrient-physiologically valuable [137]. Furthermore, they can be used
(similarly to plant rubber) as protecting colloids, emulsifiers in cosmetics, thick-
eners in the food industry [138 a], adhesives, additives in the pulp and paper in-
dustry, stabilizers for environmentally friendly inks and dyes [138 b], or as thick-
eners for crude oil drilling [139].
Lignin is one component of press cake. Isolated lignin can be used as a dis-
persant in the food industry, as stabilizer for foams and bitumen, or as an envi-
ronmental friendly adhesive [140–142].
12.5.4.1 Fibers
Non-wood fibers have been used to manufacture all kinds of paper, including
printing, writing, and packaging. Such feedstock is expected to play an impor-
tant role in improving the sustainability of the pulp and paper industry [144],
by enabling more rational utilization of forest resources. Non-wood fiber pulps
can be used effectively in combination with recycled papers, improving many of
their attributes and enabling overall cost reduction because of a decrease in the
use of starch [145].
Semi-chemical Pulping The process starts with atmospheric alkali cooking in a

continuous digester [145]. The semi-chemical pulp obtained is washed, refined,
screened, and sent to the paper mill for corrugated paper manufacture. From
the black liquor obtained in the pulping process, lignin is initially recovered by
precipitation then given a post-treatment to improve its filterability. Most of the
silica remains with the filtrate and the resulting lignin cake is high in purity
and contains less than 1% silica and less than 3.5% sugars. Lignin sales in-
crease overall mill revenues and lead to a possible reduction of the minimum
plant scale required for economic operation. The filtrate after lignin recovery
can be processed in a biological treatment plant. Alternatively, oxygen-based wet
oxidation of the filtrate can be used to generate energy and green liquor. From
the latter, a precipitate that typically accounts for 70–90% of the silica in the
wet-oxidation feed can be filtered, effectively purging silica from the cycle. The
filtered green liquor can be made caustic to generate white liquor for re-use in
pulping. A study of the pulping characteristics and mineral composition of 16
field crops grown in Finland showed that the most suitable species for alkali
cooking were the grass and cereal crops, which gave the highest pulp yields and
the lowest amounts of rejects. On the basis of the test results, Festuca arundina-
cea, Festuca pratensis, reed canary grass, and spring barley were selected for
further study [146]. Further work selected Festuca arundinacea and reed canary
grass as worthwhile candidates [147].
Steam Explosion Steam explosion of ryegrass straw has been reported in a

patent application to yield separate portions of usable straw pulp and a usable
aqueous by-product comprising lignins and hemicellulose. The pulp was
blended with Kraft pulp and old corrugated containers to make linerboard [148].
Mechanical Pulping A recent Chinese patent describes the production of non-

polluting grass pulp and a method for reclaiming its by-product [149]. Grass is
processed into refined grass chip and refined grass residue, the refined grass
chips are treated by soaking with water, softening, washing, and pulping to pro-
duce high-quality grass pulp, and the refined grass residue is mixed with an ad-
ditive containing functional preparation (organic selenium, organic calcium)
and carrier (refined grass powder) to produce a high-quality fiber feed.
Downstream Processing of the Grass Fiber Fraction (PC) The biorefinery primary
process generates a fibrous press cake (Fig. 12.3 A and C). This fraction can
either be further processed while wet – by applying technology used in the pulp
und paper industry – or it can be dried. After mechanical fractionation grass
and silage press cake particles are typically less than 3 cm in length. The struc-
ture of grass stems and leaves is mostly eliminated and the bulk has a fibrous
appearance.
Basic Properties of Grass Fibers Sfiligoj et al. [150] evaluated the fundamental
physical properties of press cake fibers (from Ryegrass (Lolium hybridum), wheat
(Triticum aestivum L.), red clover (Trifolium pratense), and lucerne (Medicago sati-
va L.) after mechanical separation. Investigation involved isolation of elementary
fibers or fiber bundles from the press cake fraction using chemical or biological
retting. For the resulting samples density, size, and strain-stress-behavior were
analyzed in wet conditions (Table 12.10).
There are no significant differences between mechanical properties of fiber
bundles of different origin (green or ensiled grass). For the press cake of trefoil,
ryegrass and alfalfa tenacity values of stem fiber bundles were measured in the
range 11.4 to 21.4 cN/tex, leaf fiber bundles reached 6.8 to 13.1 cN/tex [150].
The geometrical properties (length and diameter) of press cake fibers were simi-
lar to those of soft wood fibers and the mechanical properties (e.g. tenacity and
elongation) of elementary fibers were comparable with those of bast fibers (jute,
hemp). Grass fibers, especially the dry fibers, have poor bending strength and
are characterized by brittleness. They are, therefore, used for non-woven textiles,
preferably for technical applications. Chemical analysis of different press cake
fractions gave the average results (% dry matter content): *5.8–7.6% cellulose,
14.7–28.7% hemicellulose, and 27.5–31.6% lignin. Crude fiber was 29.1–32.9%
and crude ash 6.3–9.1%. These values are for the first cut of grass and may alter
for later harvests.
Table 12.10 Basic properties of grass fibers from press cake – green and ensiled.
Property Unit Measured value
Fiber content, stem [%] 20.2–39.5

Fiber content, leaves [%] 6.9–10.2
Fiber length, stem a) [mm] 0.8–3.2
Fiber length, leaves a) [mm] 0.6–1.3
Fiber diameter [lm] 15–18
Linear density b) [dte x] 12–105
Tenacity [cN/tex ] 6–21 c)
Elongation [%] 1–6
a) Elementary fibers
b) Fiber bundles (technical fiber)
c) Coir 15 cN/tex; jute 23–31 cN/tex; hemp 29–47 cN/tex
Products From Grass Fiber (PC) Generally speaking, the grass and silage fibers
fraction of the green biorefinery may be used as raw material for:
· insulation material (mats, boards, loose fill material)
· building panels (fiber and chip boards)
· products used in horticulture and landscaping (mulch fleece, erosion-control,
peat substitution)
· bio-composites
· packaging material
· pore forming additives (e.g. brick and tile industry)
· gypsum boards
· pulp and paper [151]
· thermoplastics [156].
Paper and Cardboard Paper has been manufactured from PC of lucerne [152,
153] and from reed canary grass, wild mix grass, and Cock’s foot [7, 154]. It has
also been shown that the quality of grass cardboards is the same or even better
(for the paper re-working industry) than analog waste paper and that they are
also less expensive [7, 155].
Thermoplastics Adhesive films have been produced from grass fiber by prepa-
ration of alkali cellulose and then film-forming. The adhesive film can be used
as agricultural mulching film or packing material [156]. Grass fiber has been
proposed as a component of a biodegradable protein/starch-based thermoplastic
composition. The grass fibers function as reinforcement filler. The composition
is processed by conventional methods, for example extrusion and injection
molding, into packaging material or articles that are low density and have high
compressive strength and tensile strength and good resilience [157].
12.5.4.2 Chemicals
It is reasonable to combine the primary refining of green biomass with fermenta-
tion processes for production of chemicals. This assumption is based on the high
water content of the raw material and the occurrence of many different substances
in green biomass, important for biotechnological processes. Green juice, brown
juice, and silage juice (Fig. 12.3 A and C) contain all the necessary macroelements
(minerals, peptides, amino acids, sugar) for making fermentation products [103,
123, 158–160, 168, 170]. After enrichment with further sources of carbohydrates
(sugars from lignocellulosic feedstock, for example press cake) it should be possi-
ble to produce a variety of biotechnologically basic chemicals [7]. Chemicals which
provide two or three functional groups and can be integrated into the product trees
of the chemical industry are of most interest [162, 163]. An assortment of indus-
trial biotechnologically produced chemicals are:
· (C2) chemicals such as ethanol [160, 161, 166, 167] and acetic acid,
· (C3) chemicals such as lactic acid [48, 159, 164, 165], acetone [167], and 1,3-
propandiol [171],
12.6 Green Biorefinery – Economic and Ecological Aspects 283
· (C4) chemicals such as n-butanol [167],

· (C5) chemicals such as itaconic acid [171], and
· (C6) chemicals such as lysine [158, 168, 170].
Products from lactic acid include, e.g., polylactic acid and ethyl lactate [83, 164,
172]. The biotechnological production of polyhydroxybutyrate from switchgrass
is currently in a stage of industrial development, and chemical or combined
chemical/biotechnological decomposition of lignocellulosic press cake or switch-
grass can be used to produce basic chemicals [169].
Basic chemicals which can be used as precursors in genealogical trees are fur-
fural [174] and xylitol [175, 176] from the hemicellulose line and hydroxymethyl-
furfural [177] and levulinic acid [178] from the cellulose line. Esparto grass is a
particularly important source of xylitol [179], and grasses with a high concentra-
tion of fructan, for example Lolium perenne are a source of hydroxymethylfur-
fural. The same is true of chicory roots [180].
12.5.4.3 Residue Utilization

Green juice, brown juice, and silage juice can be used as bio-fertilizer (soil
bioactivators) to return to the soil the macro and micro mineral nutrients which
were removed by harvesting the green crop [101]. The low-molecular-mass sub-
stances in this juice are quickly transformed into methane in fermentation
units [183, 184]. This has been developed a production process for biogas, heat,
and electricity in a combined green biorefinery–animal-breeding complex [7, 94].
Silage residues have been used as sources of natural chelates to improve the
ecological and economical balance of leaching techniques for remediation of
metal-polluted soils. Silage effluent containing a variety of aliphatic carboxylic
acids, sugar acids, and amino acids has been used to remove approximately
75% of the cadmium and more than 50% of the copper and zinc from contami-
nated soils [181]. A trial led to the conclusion that biomass residues have poten-
tial to serve as extractants in remediation techniques.
Porous carbon fibers have been obtained from cut grass by baking in an oven
in a roped form for formation of coiled carbonized fibers. The porous carbon fi-
bers are useful for sound absorbers, adsorbents, purification materials, and
radio wave absorbers [182]. The press cake has also been used as a medium for
growing mushrooms, as a mulch/green crop enhancer, and as a fertilizer [34].
12.6
Green Biorefinery – Economic and Ecological Aspects
Plant biomass is the only foreseeable sustainable source of organic fuels, chemi-
cals, and other materials. A variety of forms of biomass, notably many ligno-cel-
lulosic feedstocks, are potentially available on a large scale and are cost-competi-
tive with low-cost petroleum whether considered on a mass or energy basis, in
terms of price defined on a purchase or net basis for both current and projected
mature technologies, or on a transfer basis for mature technology [185]. Green
plant biomass and lignocellulosic feedstocks are the dominant source of feed-
stocks for biotechnological processes for production of chemicals and materials
[7, 158, 162, 163, 173]. The development of integrated technology for conversion
of biomass is essential for the economic and ecological production of products.
The biomass industry or bio-industry produces basis chemicals such as ethanol
(15 Mio tons per year), amino acids (1.5 Mio tons a year of which l-lysine
amounts to 500 000 tons per year [186]), and lactic acid (200 000 tons per year).
The target of a biorefinery is to establish a combination of a biomass–feedstock
mix with a process and product mix [162, 163]. A life cycle assessment (LCA) is
available for production of polylactic acid (capacity 140 000 tons per year) [187].
In total assessment of the utilization of biomass one must consider that plant
cultivation must fulfill economic and ecological criteria. Agriculture both creates
pressure on the environment and plays an important role in maintaining many
cultural landscapes and semi-natural habitats [188]. Green crops, especially, are
available in large quantities. Additionally, grassland can be cultivated sustainably
[92]. European grassland experiments have shown that species-rich grassland
cultivation has both ecological and economic advantages. With plant diversity
grassland is more productive and protects the soil against nitrate leaching.
Seventy-one species have been examined, of which 29 had significant influ-
ence on productivity. In particular, Trifolium pratense has an important function
with regard to productivity. On sites where this species occurs more than 50%
of the total biomass has been used. Legumes, like clover and herbs, also play an
important role, as do fast-growing grasses [189]. An initial assessment of the
concept of a green biorefinery was performed by S. Schidler et al. for the Aus-
trian system approach [190]. An Austria-wide concept for use of biomass and
cultivable land for renewable resources has yet to be developed; the same is true
for Europe [191]. The size of such plants depends on the rural structures of the
different regions. Concepts with more decentralized units would have a size of
about 35 000 tons raw material per year [192] and central plants could have sizes
of approximately 300 000 to 600 000 tons per year [174].
The synthetic method used for modeling biorefinery systems [192] is based
on combinatorial acceleration of separable concave programming developed by
Nagy et al. [193].
Table 12.11 Cost calculation for production of grass in com-

parison to straw, including intermediate storage [195].
Raw material Yield t Work

DM/ha
Person-h ha–1 Person-h t–1 DM Euro ha–1 Euro t–1 DM
Late cut Grass 5.0 4.46 0.89 297 60

Straw 2.1 1.58 0.75 77 36
References 285
Cost calculation for raw materials in the green biorefinery are based on the
supply of agricultural products. For late-cut grass and straw the costs of cultiva-
tion, harvest, and intermediate storage have been calculated for Germany (Ta-
ble 12.11 [194]).
Currently, the costs are US $ 30 per ton for corn stover or straw [196]. The
prices for green pellets are also available and range from 80 1 per ton in Ger-
many to 160 1 per ton in Sweden [42]. Production of pellets from silage has
been calculated to be approximately 110 to 155 Euro per ton in Austria [190].
12.7
Technology and research challenges associated with converting plant biomass

into commodity products must be considered in relation to green biomass in
combination with lignocellulosic biomass [196] (converting biomass into reactive
intermediates) and product diversification (converting reactive intermediates
into useful products). After isolation of the valuable products (chlorophyll, caro-
tenoids) biomass precursors such as carbohydrates and proteins must be consid-
ered. Their isolation as functional products and their biotechnological or chemi-
cal conversion into derivatives such as O-chemicals and N-chemicals must be
included in the development of the relevant technologies. Biotechnological con-
version methods must be integrated into the concepts of utilization of nature –
wet biomass. It is necessary to establish green biorefinery demonstration plants
which are best suited for the different regional rural structures of grassland
agriculture and the cultural landscape.
Acknowledgment
The authors thank Michael Mandl and Niv Graf, Joanneum Research, Graz, Aus-
tria, for investigations silage fibers, and Werner Koschuh, University of Natural
Resources and Applied Life Sciences, Vienna, for investigations of silage juice.
References
1 The Bible – Old Testament, First Book 5 Pirie, N. W. (ed.). Leaf Protein: its agron-
Moses, Genesis 4.2 omy, preparation, quality and use. IBP
2 Roulle, H. M.; J. Med. Chirurg. Pharm., Handbook 20 [Blackwell, Oxford, 1971]
40 (1773) 59–67 6 Pirie, N. W.; Leaf protein: a beneficiary of
3 Schwenke, K. D.; Leaf Proteins in the tribulation. Nature, 253 (1975) 239–241
history of Protein research. Ernährungs- 7 Kamm, B.; Kamm, M.; The green biore-
forschung, 37 (1993) 1–11 (germ.) finery – Principles, Technologies and
4 Pirie, N. W.; Leaf protein as human food. Products, 2nd International Symposium
Science, 152 (1966) 1701–1705. Green Biorefinery, October, 13–14, 1999,
SUSTAIN, Verein zur Koordination von
Forschung über Nachhaltigkeit (Hrsg.), damentals and Potential of Chlorophylls.

Feldbach, Austria, 1999, S. 46–69 In this book, 2005
8 Pheloung, P.; Brady, C. J. J. Sci. Food Ag- 26 Willstätter, R.; Stoll, A.; Untersuchungen
ric., 30 (1979) 246–250 über Chlorophyll. Methoden und Ergeb-
9 Schwenke, K. D.; Leaf proteins, Ernäh- nisse. [Verlag Julius Springer, Berlin,
rungsforschung, 28 (1983) 125–129 1913]
(germ.) 27 Willstätter, R.; Stoll, A.; Investigations on
10 Douillard, R.; Porcheron, A.; Lola, M.; Chlorophyll [Science Press, Lancaster
Guy, P.; Genier, G.; Agronomie, 10 (1991) Pa., 1928] transl. from [28]
273–284 28 Schertz, F. M.; Isolation of Chlorophyll,
11 Hari, V.; Anal. Biochem., 113 (1981) 332– Carotene and Xanthophyll by improved
335 methods, Ind. Eng. Chem., 30 (1938)
12 Hirano, H.; Phytochemistry, 21 (1982) 1073–1075
1513–1518 29 Shearon, W. H.; Gee, O. F.; Ind. Eng.
13 Slade, R. E.; Birkinshaw, J. H. (ICI ); Im- Chem., 41 (1949) 218–226
provement in or related to the utilization 30 Stenerson, H.; Chem. Eng. News, 30
of grass and other green crops. Brit. Pat. (1952) 2040
BP 511,525 (1939) 31 Judah, M. A.; Burdick, E. M.; Carroll,
14 Ullmanns –Encyklopedie der tech- R. G.; Chlorophyll by solvent extraction.
nischen Chemie; Proteine., 4. Aufl. Ind. Eng. Chem., 46 (1954) 2262–2271
Bd. 19 [Verlag Chemie, Weinheim, Flori- 32 Graetzel, M.; Liska, P.; “Photoelectro-
da Basel] chemical Cells and Process for Making
15 Pirie, N. W.; Chemy Ind., 61 (1942) 45 Same”, US Patent 5,084,365 (1992)
16 Pirie, N. W.; Nature, 149 (1942) 251 33 Carlsson, R.; An ecological better
17 Tilley, J. M. A.; Raymond, W. F.; Herb. adapted agriculture. Wet-fractionation of
Abstr., 27 (1957) 235 biomass as green crops, macro-alga, and
18 Davys, M. N. G.; Pirie, N. W.; Batch pro- tuber crops, Proc. 2nd Int. Conf. Leaf
duction of protein from leaves. J. Agric. Protein Res., Nagoya, Japan, 1985,
Eng. Res., 8 (1963) 70–73. pp. 93–100
19 Kohler, G. O.; Knuckles, B. E.; Edible pro- 34 Carlsson, R.; Status quo of the utilization
tein from leaves. Food Technol., 31 (1977) of green biomass, In: The Green biore-
191–195 finery, Kamm B, Kamm M, Soyez K
20 Telek, L.; Graham, H. D.; Leaf protein (eds); The Green Biorefinery, Concept of
concentrates. [AVI Publishing Comp., technology, 1st International Symposium
Westport, Connecticut, 1983] Green Biorefinery, Oct. 1997, Neuruppin,
21 Defremery, D.; Miller, R. E.; Edwards, Society of ecological technology and sys-
R. H.; Knuckles, B. E.; Bickoff, E. M.; tem analysis, Berlin, 1998, 39–45
Kohler, G. O.; Centrifugal separation of 35 Carlsson, R.; A tentative list of plant for
white and green protein fractions from commercial production of leaf protein
alfalfa juice following controlled heating. concentrates, Proc. 3rd Int. Leaf Protein
J. Agric. Food Chem., 21 (1973) 886–889. Res. Conf., Pisa–Perugia–Viterbo, Italy,
22 Edwards, R. H.; Miller, R. E.; Defremery, 1989, 350–353
D.; Knuckles, B. E.; Bickoff, E. M.; Koh- 36 Telek, L.; Graham, H. D. (Eds.) Leaf pro-
ler, G. O.; Pilot plant production of an tein concentrates, AVI Publ., Co., Inc.,
edible white fraction leaf protein concen- Westport, Conn., USA, 1983, 81–116
trate from alfalfa. J. Agric Food Chem., 23 37 Coombs, J.; Hall, K.; The potential of
(1975) 620–626. cereals as industrial raw materials: Legal
23 Knuckles, B. E.; Bickoff, E. M.; Kohler, technical, commercial considerations. In:
G. O.; J. Agric. Food Chem., 20 (1972) Cereals – Novel Uses and processes
1055 [G. M. Campbell, C. Webb and S. L.
24 Krasnovsky, A. A.; Jr. Photosynth. Res., 76 McKee (eds.), Plenum, New York, 1997]
(2003) 389–403. 1–12.
25 Senge, O. M.; Richter, J.; Adding Color to 38 Kamm, M. et al., Product family trees:
Green Chemistry? An Overview on Fun- Lignocellulose – Hemicellulose and Cel-
References 287
lulose based chemical products, In this stages of maturity. Swedish Journal of

book, 2005 Agricultural Research, 13, (1983) 221–227.
39 Statistisches Jahrbuch über Ernährung, 50 Morrison, I. M. Lignin-carbohydrate com-
Landwirtschaft und Forsten, Ed.: Bun- plexes from Lolium perenne. Phytochem-
desministerium für Ernährung, Land- istry, 13, (1974) 1231–1235.
wirtschaft und Forsten), Landwirtschafts- 51 Kondo, T.; Chemical and physical studies
verlag GmbH, Münster, Germany, 2004 on characteristics of forage lignins. Bulle-
40 Pickert, J.; Grassland Economy in Ger- tin of the Tohuku National Agricultural Ex-
many – Potentials for Green Biorefinery, periment Station, 85, (1993)103–214.
In: biorefinica 2004, Proceedings and Pa- 52 Tanner, G. R. and Morrison, I. M.; Pheno-
pers, October, 27–28, Osnabrück, Eds.: lic-carbohydrate complexes in the cell
Kamm, B.; Hempel, M.; Kamm, M; bio- walls of Lolium perenne. Phytochemistry,
pos e.V., Teltow 2004, p. 26, ISBN 3-00- 22, (1983) 2133–2139.
015166-4 53 Chesson, A., Gordon, A. H. and Scobbie,
41 http://europa.eu.int/comm/research/in- L.; Pectic polysaccharides of mesophyll
focentre/export/0331e_374.html cell walls of perennial ryegrass leaves.
42 Production of dry-crop in Europe: CIDE- Phytochemistry, 38, (1995) 579–583.
Comision Intersyndicale des Déhydra- 54 Thom, E. R., Sheath, G. W. and Bryant,
teurs Européen, 2004/2005, Brussels A. M. Seasonal variations in total non-
43 http://www.hayconference.com; http:// structural carbohydrate and major ele-
www.auri.org/research/alfproce.htm ment levels in perennial ryegrass and
44 Jagiello, R. and Wojcik, S. A.; Compari- paspalum in a mixed pasture. New Zea-
son of the contents of hemicellose, cellu- land Journal of Agricultural Research, 32,
lose, lignin and crude fibre in fresh her- (1989) 157–235.
bage, hay and silage from meadow 55 Prud’homme, M. P., Gonzalez, B., Bill-
grasses and lucerne. Annales Universitatis ard, J. P. and Boucaud, J.; Carbohydrate
Mariae Curie-Sklodowska, E, 31, (1976), content, fructan and sucrose enzyme ac-
525–535. tivities in roots, stubble and leaves of
45 Morrison, I. M.; Changes in lignin and ryegrass (Lolium perenne L.) as affected
hemicellulose concentrations of ten vari- by source/sink modification after cut-
eties of temperate grasses with increas- ting. Journal of Plant Physiology, 210,
ing maturity. Grass and Forage Science, (1992) 282–291.
35, (1980) 287–293. 56 Masuko, T., Kodama, I., Uematsu, H.,
46 Gordon, A. H., Lomax, J. A. and Chesson, Kuboi, S., Maeda, Y. and Yamanaka, Y.
A. Glycosidic linkages of legume, grass Changes in mono and disaccharide con-
and cereal straw cell walls before and tents of temperate grass cut at three
after extensive degradation by rumen mi- stages of growth in Hokkaido. Nippon
croorganisms. Journal of the Science of Sochi Gakkaishi, 40, (1994) 230–233.
Food and Agriculture, 34, (1983) 1341– 57 Fales, S. L., Holt, D. A., Lechten-
1350. berg, V. L., Johnson, K., Ladisch, M. R.
47 Kolarski, D., Koljajić, V., Koljajić, V., Po- and Anderson, A. Fractionation of forage
pović, J. and Popović, Z.; Potential bio- grass carbohydrates using liquid (water)
logical value of some roughages. Krmiva, chromatography. Agronomy Journal, 74,
32, (1990) 101–106. (1982) 1074–1077.
48 Seyfarth, W., Knabe, O. and Arnold, H. 58 Simpson, R. J. and Bonnett, G. D. (1993).
Changes in the carbohydrate fractions of Fructan exohydrolase from grasses. New
grasses during growth and effects on fer- Phytologist, 123, (1982) 453–469.
mentability. Proceedings of the 13th Inter- 59 Pavis, N., Chatterton, N. J., Harrison,
national Grassland Congress. Sectional Pa- P. A., Baumgartner, S., Praznik, W., Bou-
pers, Sections 8-9-10 (1977) pp 238–243. caud, J. and Prud’homme, M. P.; Struc-
49 Åman, P. and Lindgren E. Chemical ture of fructans in roots and leaf tissues
composition and in vitro degradability of of Lolium perenne. New Phytologist, 150,
individual chemical constituents of six (2001) 83–95.
Swedish grasses harvested at different
60 Spollen, W. G.; Fructan composition and 72 Parry, D., Hodson, M. J. and Sangster,
physiological roles in wheat, tall fescue, A. G.; Some recent advances in studies
and timothy. Dissertation Abstracts Inter- of silicon in higher plants. Philosophical
national B, Sciences and Engineering, 51, Transactions of the Royal Society of Lon-
(1990) 523. don, B, 304, (1984) 537–549.
61 Azis, B. H., Chin, B., Deacon, M. P., 73 Dinsdale, D., Gordon, A. H. and George,
Harding, S. E. and Pavlov, G. M.; Size S.; Silica in mesophyll cell walls of Ita-
and shape of inulin in dimethyl sulphox- lian ryegrass (Lolium multiflorum Lam.
ide solution. Carbohydrate Polymers, 38, Cv. RvP). Annals of Botany, 44, (1979)
(1999) 231–234. 73–77.
62 Fuchs, A., In M. Suzuki and N. J. Chat- 74 Bode, E., Kozik, S., Kunz, U. and Leh-
terton, Science and technology of fructans. mann, H.; Comparative electron micro-
N. J. Boca Raton, Florida: CRC Press, scopic studies for the localization of sili-
p. 319 (1993). ca in leaves of two different grass spe-
63 Pontis, H. G.; Fructans. Methods in Plant cies. Deutsche Tierärztliche Wochenschrift,
Biochemistry, 2, (1990) 353–369. 101, (1994) 367–372.
64 Teeuwen, H., Thoné, M. and Vandorpe, 75 Moore, D.; The distribution of silica
J.; Inulin – From traditional food source bodies in leaf sheaths of two perennial
to an all-round raw material. ZFL, Inter- ryegrass cultivars differing in their sus-
nationale Zeitschrift für Lebensmittel-Tech- ceptibility to attack by dipterous stem-
nik, Marketing, Verpackung und Analytik, borers. Grass and Forage Science, 39,
43, (1992) 732, 734, 737–738. (1984) 205–208.
65 Mela, T. and Rand, H.; Amino acid com- 76 Puffe, D., Morgner, F. and Zerr, W.; In-
position of timothy, meadow fescue, vestigations on the contents of different
cocksfoot and perennial ryegrass at two constituents in important forage plants.
levels of nitrogen fertilisation and at suc- 2. Mineral and silicic acid contents.
cessive cuttings. Annales Agriculturae Wirtschaftseigene Futter, 30, (1984) 52–70.
Fenniae, 18, (1979) 246–251. 77 Malossini, F., Piasentier, E. and Bovolen-
66 Pedersen, E. J. N., Witt, N. and Morten- ta, S.; n-Alkane content of some forages.
sen, J.; Fractionation of green crops with Journal of the Science of Food and Agricul-
expression of juice and preservation of ture, 53, (1990) 405–409.
pressed crop and juice. 3. Relationship 78 Jones, D. I. H. and Hayward, M. V.;
between chemical composition of the Starches in forages. UK, Welsh Plant
crop and that of the juice. Tidsskrift for Breeding Station: Report for 1977, (1990)
Planteavl, 88, (1984) 25–36. 109–110.
67 Pedersen, E. J. N., Witt, N., Mortensen, J. 79 Rizk, A. F. M. and Hussiney, H. A.;
and Soerensen, C. Fractionation of green Chemistry and toxicity of Lolium spe-
crops and preservation of pressed crop cies. In A. F. M. Rizk, Poisonous plant con-
and juice. 2. Preservation of juice. Tids- tamination of edible plants (pp. 95–106),
skrift for Planteavl, 85, (1981) 13–30. (1991) Boca Raton: CRC Press Inc.
68 McDougall, V. D. Support energy and 80 Sidebottom, C., Buckley, S., Pudney, P.,
green crop fractionation in the United Twigg, S., Jarman, C., Holt, C., Telford,
Kingdom. Agricultural Systems, 5, (1980) J., McArthur, A., Worrall, D., Hubbard,
251–266. R. and Lillford, P. Heat-stable antifreeze
69 Lesnitski, V. R. High-protein feeds from protein from grass. Nature, 406 (2000)
green mass. Kormoproizvodstvo, 1/2, 256.
(1997) 61–62. 81 Kuiper, M. J., Davies, P. L. and Walk-
70 Rabovsky, J. Biogenic amorphous silica. er, V. K. A theoretical model of a plant
Scandinavian Journal of Work, Environ- antifreeze protein from Lolium perenne.
ment and Health, 21 (1995) 108–110. Biophysical Journal, 81, (2001) 3560–
71 Sangster, A. G., Hodson, M. B. and Tubb, 3565.
H. J.; Silicon deposits in higher plants. 82 Kamm, B.; Kamm, M.; Principles of
Studies in Plant Science, 8, (2001)85–113. biorefineries, Appl. Microbiol. Biotechn.
64 (2004) 137–145
References 289
83 Kamm, B.; Kamm, M.; Biorefinery-Sys- 93 (a) Ketelaars, J. J. M. H., Bio-raffinage

tems. Chem. Biochem. Eng. Q., 18 van natuurgras, Netherland, 2001,
(2004) 1–6 www.precisievoeding.nl/projecten/
84 Okkerse, C.; v. Bekkum, H.; From fossil p07_10316.htm; (b) Hulst, A. Bio-refin-
to green, Green Chemistry, 1 (2) 1999, ery of grass and other raw materials
111 from vegetable sources and product ap-
85 Heier, W.; Das Fraktionieren von Gras, plications; GreenTech 2002, Amster-
In: Grundlagen der Landtechnik, 33 dam, 24–26 April, 2002: Abstract book,
(1983) 2 p. 37; http://www.ienica.net/greentech/
86 Sundberg, M.; Mekanisk avvattning av hulst.ppt
vallföder – En litteraturöversikt (Mechan- 94 Kamm, B.; et al Green biorefinery
ical dewatering of forage crops – a litera- Brandenburg, Article to development
tur review); Jordbrukstekniska institutes, of products and of technologies and as-
Rapport 134, Uppsala, 1991 sessment. Brandenburg. Umweltber. 8,
87 Kasper, G. J.; Fractioning of grass and lu- (2000) 260–269.
cerne. Landbouwmechanisatie, 49, (1998) 95 Biomass R&D, Technical Advisory
47–48. Committee, Roadmap for Biomass
88 Kaczmarek, J.; Lange, B.; Ohm, H.-D.; Technology in the United States, Dec.
Investigation for fractionation of biore- 2002, Washington D.C., www.biopro-
finery-raw material, In: The Green biore- ducts-bioenergy.gov/pdfs/FinalBiomass-
finery, Kamm B, Kamm M, Soyez K Roadmap.pdf, 30
(eds) The Green Biorefinery, Concept of 96 (a) Peitgen, H.-O., Richter, P. H.; The
technology, 1st International Symposium beauty of fractals [Berlin, 1986], (b)
Green Biorefinery, Oct. 1997, Neuruppin, Mandelbrot, B.; Die fraktale Geometrie
Society of ecological technology and sys- der Natur [Basel, 1987]
tem analysis, Berlin, 1998, 126–130 97 Hagers Handbuch der pharmazeu-
89 Bruhn, H. D.; Straub, R. J.; Koegel, R. G.; tischen Praxis (Hagers handbook of
A systems approach to the production of pharmaceutical practice) band 1–5,
plant protein concentrate; Proceedings of chemicals and drugs, band 1–5 [W.
the International Grain and Forage Har- Kern et al. (eds), Springer Verlag, Ber-
vesting Conference, 1978, (Ed.) Ameri- lin, Heidelberg, New York, 1972–78] (a)
can Society of Agricultural Engineers bd 1, p. 732–736; (b) bd 2, lipides
90 Kamo, M. and Nakagawasai, H.; Studies 98 Starke, I., Holzberger, A., Kamm, B.,
on the development of effective grass Kleinpeter, E.; Qualitative and quantita-
utilization techniques. 1. The separation tive analysis of carbohydrates in green
and concentration of leaf protein from juices (wild mix grass and alfalfa) from
grass juice by centrifuging. Bulletin of the a green biorefinery by gas chromato-
National Grassland Research Institute, Ja- graphy/ mass spectrometry, Fresenius
pan, 31, (1985) 81–92. J. Anal. Chem., 367, (2000) 65–72
91 France Luzerne Alfalis – Dossier PX. 99 Moser, A.; (ed); Eco-tech The technical
France Luzerne, 2000. Development [Verlag TU Graz, Austria,
92 (a) Kromus, S.; Wachter, B.; Koschuh, 1997]
W.; Mandl, M.; Krotschek, C.; Narodo- 100 Schwenke, K. D.; Eiweisquellen der Zu-
slawsky, M.; The green biorefinery Aus- kunft [Aulis-Verlag Deubner, Köln,
tria – Development of an Integrated Sys- 1985, ISBN 3-7614-0858-7] pp. 82.
tem for Green Biomass Utilization, 101 Carlsson R.; Food and Non-food uses
Chem Biochem Eng Q 18 (1) (2004) 13– of immature cereals. In: Cereals – Nov-
19; (b) Kromus, S.; Die Grüne Bioraffi- el Uses and processes [G. M. Campbell,
nerie Österreich – Entwicklung eines in- C. Webb and S. L. McKee (eds.), Ple-
tegrierten Systems zur Nutzung von num, New York], (1997) 159–167.
Grünlandbiomasse. Dissertation, 2002, 102 Kirk, R. E.; Othmer, D. F. (ed.).; Ency-
TU Graz (German). clopedia of chemical technology
[John Wiley and Sons, New York,
1994].
103 Keller, K.; Technology for two-steps 112 Jones, W. T., Mangan, J. L. Large-scale
conservation of green biomass, In: The isolation of fraction 1 leaf protein (18S)
Green biorefinery, Kamm B, Kamm M, from lucerne (Medicago sativa L). J.
Soyez K (eds); The Green Biorefinery, Agr. Sci. 86 (1976) 495–501.
Concept of technology, 1st Interna- 113 Hatch, F. T., Bruce, A. L.; Amino acid
tional Symposium Green Biorefinery, composition of soluble and membra-
Oct. 1997, Neuruppin, Society of eco- nous lipoproteins. Nature 218 (1968)
logical technology and system analysis, 1166–1168.
Berlin, 1998, 120–125 114 Douillard, R.; Biochemical and physico-
104 Simmonds, M. S. J.; Molecular- and chemical properties of leaf proteins. In
chemo-systematics: do they have a role Proteines Veg, Publisher Tech. Doc. La-
in agrochemical discovery? Crop Protec- voisier, Paris, France, (1985) 211–244.
tion, 19, (2000) 591–596. 115 Betschart, A. A.; The incorporation of
105 Wang, Y. R. and Li, Q.; Investigation leaf protein concentrates and isolates
and evaluation of native plant rein human diets, In “Green Crop Frac-
sources in Loess Plateau of Gansu Pro- tionation” [Ed. R. J. Wilkins (ed.) The
vince. In J. Z. Ren, Proceedings of the In- British Grassland Society, c/o Grass-
ternational Conference on Farming Sys- land Research Institute, Hurley, Mai-
tems on the Loess Plateau of China denhead, SL6 5LR, UK, 1977] pp. 83–
(1992) (pp. 246–250). Lanzhou: Gansu 96.
Science and Technology Press 116 Carlsson, R.; White leaf protein prod-
106 Douillard, R., de Mathan, O.; Leaf pro- ucts for human consumption. A global
tein for food use: potential of Rubisco. review on plants and processing meth-
In New and developing sources of food ods. In “Tobacco Protein Utilization
proteins (Ed. Hudson BJF), Publisher Perspectives”, Proc. Round Table Conf.
Chapman and Hall, London, UK, At 1st Int. Congr. Food and Health
(1994) 307–342. [(P. Fantozzi (ed.), CNR/Italian Na-
107 Bahr, J. T., Bourque, D. P., Smith, H. J.; tional Res. Council, Special Project
Solubility properties of fraction I pro- IPBR, Subproject L. EEC Agrimed Pro-
teins of maize, cotton, spinach, and to- ject, 1985] pp. 125–145
bacco. J. Agric. Food. Chem. 25 (1977) 117 Kung, S. D.; Saunders, J. A.; Tso, T. C.;
783–789. Vaughan, D. A.; Womack, M.; Staples,
108 Bigelow, C. C. On the average hydro- R. C.; Beechers, G. R.; Tobacco as a po-
phobicity of proteins and the relation tential food sources and smoke materi-
between it and protein structure. J. al: Nutritional evaluation of tobacco
Theor. Biol. 16 (1967) 187–211. leaf protein. J. Food Sci., 45 (1980)
109 Hood, L. L.; Cheng S. G.; Koch, U.; 320–332, 327.
Brunner, J. R.; Alfalfa proteins: Isola- 118 Linnemann, A. R. and Dijkstra, D. S.
tion and partial characterization of the Toward sustainable production of pro-
major component – fraction I protein. tein-rich foods: appraisal of eight crops
J Food Sci 46 (1981) 1843–1850. for Western Europe. Part 1. Analysis of
110 Burova, T. V.; Soshinskii, A. A.; Danilen- the primary links of the production
ko, A. N.; Antonov, YuA; Grinberg, Vya; chain. Critical Reviews in Food Science
Tolstoguzov, V.B.; Conformation stabili- and Nutrition, 42 (2002) 377–401.
ty of ribulose diphosphate carboxylase 119 Wolfrum, C.; Wheat grass – The power
of alfalfa green leaves according to the in the green Juice [Gräfe und Unzer
data of differential scanning microca- Verlag GmbH, München, 1998, ISBN:
lorimetry, Biofizika 34 (1989) 545–549. 3-7742-4072-8] (German)
111 Barbeau, W. E.; Kinsella, J. E.; Ribulose 120 Gaynor, M. L. and Hickey, G. P.; Green
bisphosphate carboxylase/oxygenase nutritional powder composition con-
(Rubisco) from green leaves – potential taining natural food and herbal prod-
as a food protein, Food. Rev. Int. 41 ucts. US Patent Application 5904924
(1988) 93–127. (1999).
References 291
121 Messman, M. A., Weiss, W. P., and 130 Palm, S. K., Smith, T. R., Shiuh, J. C.
Koch, M. E.; Changes in total and indi- and Roulston, J. S.; Filterable compo-
vidual protein during drying, ensiling site adsorbents with adsorptive and fil-
and ruminal fermentation of forages. J. terable components as filter aids suit-
Dairy Sci. 77 (1994) 492–500. able for beer chillproofing. PCT Inter-
122 Koschuh, W., Povoden, G., Vu Hong, national Application, 9830324 (1998).
T., Kromus, S., Kulbe, K. D., Novalin, 131 Itsumi, A., Sakagami, E.; Manufacture
S., Krotscheck, C.; Production of leaf of artificial zeolites from grass. Japa-
protein concentrate from ryegrass (Lo- nese Patent Application, 2001323109
lium perenne ´ multiflorum) and alfalfa (2001).
(Medicago sativa subsp. Sativa). Compar- 132 Salminen, S. O. and Grewal, P. S.; Does
ison between heat coagulation/centrifu- decreased mowing frequency enhance
gation and ultrafiltration. Desalination alkaloid production in endophytic tall
163 (2004) 253–259. fescue and perennial ryegrass? Journal
123 Vu Hong, T., Koschuh, W., Kulbe, of Chemical Ecology, 28 (2002) 939–950.
K. D., Kromus, S., Krotscheck, C., No- 133 Fechner, M.; Grassland economy in
valin, S. Desalination of high salt con- Brandenburg. In: Proceedings of the
tent mixture by two-stage electrodialy- 1st. Int. Symposium on Green Biore-
sis as the first step of separating valu- finery, Neuruppin, Germany 1997
able substances from grass silage. De- [K. Soyez, B. Kamm, M. Kamm (eds.),
salination 162 (2004) 343–353 Berlin, Germany, 1998, ISBN 3-929672-
124 Kamm, B.; Kamm, M.; Scherze, I.; 06-5] (German) 47–52
Muschiolik, G.; Bindrich, U.; Biobased 134 Wantanabe, T.; Fujishima, A.; Honda,
polymers by chemical valorization of K.; Dye-sensitive electrodes. In: Energy
biomass components, In: Vasile, C.; Resour. Photochem. Cat. [M. Grätzel
Zaikov (Eds.) Polymer reactions and (ed.), Academic Press, 1983] 359
properties, Publishing House Nova 135 Cohen, B. S., et al.; J. Agric. Food
Science – New York (in press) Chem. 32 (1984) 516
125 Oyama, K., Kobayashi, T., Imazato, Y.; 136 Leblanc, R. M., et al.; Photobiochem.
Kumasawa, Y.; Polysaccharides from Photobiophys. 7 (1984) 41
fescue plant cell walls, emulsifiers con- 137 Girio, F. M. F. Development of xylo-oli-
taining them, and method for emulsifi- gosaccharides and xylitol for use in
cation. Japanese Patent Application, pharmaceutical and food industries
10237107 (1998). (XYLOPHONE) (1997) FAIR-CT97-
126 Cherno, N. K., Adamovskaya, K. D. and 3811.
Lobotskaya, L. L.; Polysaccharide–lignin 138 Aspinall (ed.); The polysaccharides.
complexes of raw materials not tradi- Vol. 2 [Academic Press, New York,
tional for the food industry, and their 1983] (a) p. 98–193, (b) p. 411–490
properties. Khimiya Drevesiny, 3 (1991) 139 Davidson (ed.); Handbook of Water sol-
95–98. uble Gums and Resins. [McGraw-Hill,
127 Shipley, L. W. Manufacture of high pur- New York, 1980]
ity amorphous silica from biogenic ma- 140 Perl (ed.); The Chemistry of Lignin
terial. US Patent Application, 6406678 [Dekker, New York, 1967]
(2002). 141 Crawford (ed.); Lignin Biodegradation
128 Noeske, R. and Horn, I. Procedure for and Transformation. [John Wiley and
the production of silicon carbide from Sons, New York, 1981]
renewable raw materials. German Pat- 142 Hofrichter, M.; Steinbüchel, A.; (ed.)
ent Application, 10020626 (2001). Biopolymers: Lignin, humic substances
129 Shiuh, J. C., Palm, S. K., Nyamekye, and coal; 2001, Wiley-VCH, ISBN 3-
G. A., Smith, T. R., Taniguchi, J. D. and 527-30220-4
Wang, Q. Highly purified biogenic sili- 143 Nay, W. H. and Fuller, W. S.; Method
ca product and its preparation. Europe- for processing straw into pulp and ani-
an Patent Application, 758560 (1997). mal feed byproduct and paper product
therefrom. PCT International Applica- Landwirtschaft, 11 (1994) 29–31 (in Ger-

tion, (1999), WO9918285. man)
144 Sonnenfeld, D. A.; Logging vs recy- 155 Hille, C.; Prenacell-process, UVER
cling: Problems in the industrial ecol- GmbH, Germany, 1999
ogy of pulp manufacture in south-east 156 Jiang, Z., Zang, W. and Feng, S.; Pro-
Asia. Greener Management International, duction of grass fiber adhesive film.
22 (1998) 108–122. Chinese Patent Application, 1235735
145 Lora, J. H. and Escudero, E. Soda pulp- (1997).
ing of agricultural fibres for boardmak- 157 Wang, S. H.; Biodegradable protein/
ing applications. Paper Technology, 41 starch-based thermoplastic composi-
(2000) 37–42. tion. PCT International Application,
146 Pahkala, K. A., Mela, T. J. N. and Laama- 9956556 (1999).
nen, L.; Pulping characteristics and 158 Thomsen, M. H.; Bech, D.; Kiel, P.;
mineral composition of 23field crops Manufacturing of Stabilised Brown
cultivated in Finland. In J. F. Kennedy, Juice for l-lysine Production – from
G. O. Phillips and P. A. Williams, The University Lab Scale over Pilot Scale to
Chemistry and Processing of Wood and Industrial Scale, Chem. Biochem. Eng.
Plant Fibrous Materials [Cellucon ’94] Q. (CABEQ) 18(1) (2004) 37–46
(1996) (pp. 119–125). Cambridge: 159 Kamm, B.; Neue Ansätze in der Orga-
Woodhead. nischen Synthesechemie – Verknüp-
147 Saijonkari-Pahkala, K.; Non-wood fung von biologischer und chemischer
plants as raw material for pulp and pa- Stoffwandlung am Beispiel der Bioraf-
per. Agricultural and Food Science in finerie-Grundprodukte Milchsäure und
Finland, 10, (2001) 1–101. Carnitin, Habilitationsschrift, Univer-
148 Janes, R. L.; In: The pulping of wood. sity of Potsdam, Germany, 2004 (Ger-
2nd edn, vol 1 [R. G. MacDonald (ed), man)
McGraw-Hill, New York, 1969] p. 34 160 Grass, S.; Hansen, S.; Sieber, M.; Mül-
149 Fan, W., Xu, J., Guo, Y. and Xing. Q. ler, P. H.; Production of Ethanol, Pro-
Production of nonpolluting grass pulp tein concentrate, and Technical Fibres
and method for reclaiming its by-prod- from Clover/Grass; In: Proceedings of
uct. Chinese Patent Application, (2001) the fourth biomass conference of
1298984. Americas, Biomass, A Growths Oppor-
150 Sfiligoj Smole, M.; Kleinschek, K. S.; tunity in Green Energy and Value
Kreze, T.; Strnad, S.; Mandl M.; Wach- added Products, Vol. 1, Overend, R. P.;
ter, B.; Physical properties of grass fi- Chornet, E. (ed.); Elsevier Science Ltd.,
bers; Chem. Biochem. Eng. Q. (CA- 1999, 911–914, ISBN 008 0430198
BEQ) 18(1) (2004): 47–53 161 Dale, B. E.; Biomass Refining, protein
151 Bartl, A.; Mihalyi, B.; Marini, I.; Appli- and ethanol from alfalfa. Ind. Eng
cations of renewable fibrous materials; Product Research and Development, 22
Chem. Biochem. Eng. Q. (CABEQ) (1983) 446
18(1) (2004): 21–28 162 Werpy, T.; Petersen, G.; (eds.); Top Val-
152 Holm-Christensen; The dehydration ue Chemicals under the refinery con-
plant as producer for the cellulose in- cept: A Phase II Study [US Depart-
dustry. Proc. Dry-Crops 89, 4th Int. ment of Energy, Office of scientific and
Green Crop Drying Congr., Cambridge technical information], 2004, No.:
[Agra Europe Ltd., London, UK, 1989] DOE/GO-102004-1992, www.osti.gov/
pp. 91–94 bridge
153 Carlsson, R.; Pressed crop for possible 163 Kamm, B.; Kamm, M.; Gruber, P. R.;
production of paper crop. Proc. 4th Int. Kromus, S.; Biorefinery-Systems – An
Conf. Leaf Protein Res., New Zealand– Overview; The Role of biotechnology,
Australia 1993, pp. 69–74 In this book, 2005
154 Fechner, M.; Hertwig, F.; Paper made 164 Gruber, P. R.; Henton, D. E.; Starr, J.;
of grass very much in the future. Neue Polylactic acid from Renewable Re-
sources, In this book, 2005
References 293
165 Neureiter, M.; Danner, H.; Madzingaid- tives on new crops and new uses.
zo, L.; Miyafuji, H.; Thomasser, C.; ASHS Press, Alexandria, VA, USA,
Bvochora, J.; Bamusi, S.; Braun, R.; 1999, 114–123
Lignocellulose Feedstocks for the pro- 175 Jiang, Z., Chen, R. and Wu, P.; Hydro-
duction of lactic acid, Chem. Biochem. genation of xylose to xylitol in trickle-
Eng. Q. (CABEQ) 18(1) (2004) 55–63 bed reactor. Riyong Huaxue Gongye, 6,
166 McGrath, D.; Italian ryegrass as a (1998) 6–8.
source of fermentable sugars and pro- 176 Nigam, P. and Singh, D.; Processes for
tein feedstuff. Luxembourg: Commis- fermentative production of xylitol – a
sion of the European Communities, sugar substitute. Process Biochemistry,
(1990). 30, (1995) 124–124.
167 Quereshi, N. and Blaschek, H. P.; ABE 177 Bhatt, S. and Shukla, R. P. HMF – a
production from corn: a recent eco- new route to industrial chemicals from
nomic evaluation. Journal of Industrial sugars. Taiwan Sugar, 48, (2001) 4–15.
Microbiology and Biotechnology, 27, 178 Elliott, D. C.; Fitzpatrick, S. W.; Bozell,
(2001) 292–297. J. J.; Jarnefeld, J. L.; Bilski, R. J.; Moens,
168 Starke, I.; Kamm, B.; Kleinpeter, E.; L.; Frye, Jr. J. G.; Wang, Y.; Neuen-
Separation, identification and quantifi- schwander, G. G.; Production of levuli-
cation of amino acids in l-lysine fer- nic acid and use as a platform chemi-
mentation potato juices by gas chroma- cal for derived products; In: Proceed-
tography/ mass spectrometry, Fresenius ings of the fourth biomass conference
J. Anal. Chem., 371 (2001) 380–384 of Americas, Biomass, A Growths Op-
169 Kamm, B.; Kamm, M.; Schmidt, M.; portunity in Green Energy and Value
Starke, I.; Kleinpeter, E.; Chemical and added Products, Vol. 1, Overend, R. P.;
biochemical generation of carbohy- Chornet, E. (ed.); Elsevier Science Ltd.,
drates from lignocellulose-feedstock 1999, 911–914, ISBN 008 0430198
(Lupinus nootkatensis), Quantification of 179 Rothschild, Z., Silva, H. C., Braga,
glucose, Chemosphere (in press) G. L., Carlomagno, D. N., Prado,
170 Kiel, P.; Thomsen, M. H.; Andersen, I. M. R., de Carvalho, R., Polizello,
M.; Plant Juice in the Biorefinery – A. C. M. and Spadaro, A. C. C.; Xylitol a
Use of plant juice as fermentation me- non-cariogenic sugar obtained from
dium, In this book grasses of the genus Aristida and re-
171 Vorlop, K. D.; Willke, Th.; Prüße, U.; lated species. Arquivos de Biologica e
Biocatalytic and catalytic routes for the Tecnologia, 34, (1991) 61–71.
production of bulk and fine chemicals 180 de Baynast, R. and Renard, C.; Pro-
from renewable resources, In this book cesses for extraction and transforma-
172 Datta, R.; Tsai, S.-P.; Lactic acid Pro- tion of products with high fructose
duction and potential uses: A Technol- content from chicory roots (Cichorium
ogy and Economics Assessment. In: intybus). Comptes Rendus de l’Académie
Fuels and chemicals from biomass, d’Agriculture de France, 80, (1994) 31–
B. C. Sara, J. Woodward (ed.), ACS 46.
Symposium series, ISSN 0097- 181 Fischer, K., Bipp, H.-P., Riemschneider,
6156 666, ACS, Washington D. C., P., Leidmann, P., Bienek, D. and Ket-
1997, pp. 224–236 trup, A.; Utilization of biomass resi-
173 Tullo, A.; Renewable Materials, Two dues for the remediation of metal-pol-
Pacts May Help Spur Biomass Plastics; luted soils. Environmental Science and
Chemical and Engineering News, Technology, 32, (1998) 2154–2231.
March 28, 2005, www.CEN- 182 Kato, H.; Porous carbon fibers from
ONLINE.org plant waste materials manufactured by
174 Van Dyne, D. L.; Blasé, M. G.; Clem- baking the waste materials in an oven
ents, L. D.; A Strategy for returning in the roped form for formation of
agriculture and rural America to long- coiled carbonized fibers and reuse of
term full employment using biomass plant waste materials therefor. Japanese
refineries. In: J. Janeck (ed.) Perspec- Patent Application, 2000303266 (2000).
183 Linke, B.; Schelle, H.; Anaerobe Ver- Kromus, S., Will, M., 2003, Technikfol-
fahren zur Reststoffbehandlung, In: gen-Abschätzung der Grünen Bioraf-
The Green biorefinery, Kamm B, finerie, Teil II: Materialsammlung, In-
Kamm M, Soyez K (eds) The Green stitut für Technikfolgen-Abschätzung,
Biorefinery, Concept of technology, 1st Österreichische Akademie der Wis-
International Symposium Green Biore- senschaften, 2003 (German)
finery, Oct. 1997, Neuruppin, Society 191 Narodoslawsky, M.; Kromus, S.; Devel-
of ecological technology and system opment of decentral green biorefinery
analysis, Berlin, (1998) 196–207 in Austria; In: biorefinica 2004, Pro-
184 Verstrate, W.; Debeer, D.; Pena, M.; Let- ceedings and Papers, October, 27–28,
tinga, G.; Lens, P.; Anaerobic Biopro- Osnabrück, Eds.: Kamm, B.; Hempel,
cessing of Organic Wastes, World Jour- M.; Kamm, M; biopos e.V., Teltow
nal of Microbiology and Biotechnology, 2004, p. 24; ISBN 3-00-015166-4
12 (1996) 221–223 192 Halasz L., G. Povoden, M. Narodo-
185 Fowler, P. A.; McLaughlin, A. R.; Hall, slawsky: Process Synthesis for Renewable
L. M.; The potential industrial uses of Resources, presented at the PRES 03,
forage grasses including miscanthus; Hamilton, Canada, 2003
BioComposites Centre, University of 193 Nagy, A. B., F. Friedler, and L. T. Fan,
Wales, Bangor, Gwynedd 2003; Combinatorial Acceleration of Separ-
www.nnfcc.co.uk/library/reports/ able Concave Programming for Process
download.cfm?id=60 Synthesis, presented at the AIChE An-
186 Elements, Degussa Science Newsletter nual Meeting, Miami Beach, FL, USA,
7 (2005) 35 November 15–20, 1998.
187 Vink, E. T. H.; Rabago, K. R.; Glassner, 194 Fechner, M.; Hertwig, F.; Nachwach-
D. A.; Gruber, P. R.; Applications of life sende Rohstoffe auch vom Grünland,
cycle assessment to NatureWorks poly- Neue Landwirtschaft 11, (1994) 29–31
lactide (PLA) production, Polymer deg- 195 Dale, B.; Encyclopedia of physical
radation and stability 80 (2003) 403– sciences and technology, vol 2, 3rd
419 edn. McGraw-Hill, New York, 2002,
188 IEEP, Contribution to the background pp. 141–157
study agriculture, The Institute for Eu- 196 (a) Lynd, L. R., Wyman, C. E. and Gern-
ropean Environmental Policy, 2004. gross, T. U.; Biocommodity engineer-
189 Hector, A.; et al.; Plant Diversity and ing. Biotechnology Progress, 15, (1999)
Productivity Experiments in European 777–793. (b) Lynd, L. R.; Biomass Pro-
Grasslands, Science, 286 (1999) 1123– cessing in Response to sustainability
1127 and Security Challenges – A Vision for
190 (a) Schidler, S., Technikfolgenabschät- What is possible, In: biorefinica 2004,
zung der Grünen Bioraffinerie, Teil I: Proceedings and Papers, October, 27–
Endbericht, Institut für Technikfolgen- 28, Osnabrück, Eds.: Kamm, B.; Hem-
Abschätzung, Österreichische Akade- pel, M.; Kamm, M.; biopos e.V., Teltow
mie der Wissenschaften, 2003; Schid- 2004, p. 20; ISBN 3-00-015166-4
ler, S., Adensam, H., Hofmann, R.,
295
13
Plant Juice in the Biorefinery –
Use of Plant Juice as Fermentation Medium
Mette Hedegaard Thomsen, Margrethe Andersen, and Pauli Kiel
13.1
Introduction
Biotechnological utilization of waste and residues from agriculture and the agri-
cultural industry has been the common goal for AgroFerm A/S and University
of Southern Denmark. The concept of the green biorefinery has been described
elsewhere [8]. Additionally, much experimental work has been performed on re-
alizing these ideas for industrial purposes. This paper describes the possibility
of using brown juice from the green crop-drying industry and potato juice from
the potato starch industry as raw materials in Danish lactic acid production, as
a basis for the production of bio-based polylactate to be used as packaging mate-
rials for fruit and vegetables. One single green crop-drying factory producing
50 000 tons of fodder pellets a year has enough brown juice to supply a 6 000
ton lactic acid factory with fermentation medium, and a 50 000 ton potato starch
factory produces enough potato juice to supply a 35 000 ton lactic acid factory.
13.2
Historical Outline
Since 1997 work has been performed on a research project on the production
and testing of bio-based packaging materials for food. The work has been per-
formed as co-operation between The Technical University of Denmark, The
Danish Technological Institute, The Royal Danish Veterinary and Agricultural
University, Risø National Laboratory, The University of Aarhus, and The Univer-
sity of Southern Denmark. Bio-based materials are defined as materials originat-
ing from agricultural sources, i.e. produced from renewable and biological raw
materials. The advantages of such materials are that they are CO2-neutral and
biodegradable.
ISBN: 3-527-31027-4
296 13 Plant Juice in the Biorefinery – Use of Plant Juice as Fermentation Medium
13.3
Biobased Poly(lactic Acid)
Poly(lactic acid) (PLA) is a bio-based, biodegradable polymer with much poten-

tial as a material for food packaging because of its mechanical properties. Be-
cause it is a moisture and gas barrier and can be used to produce flexible water-
resistant films, PLA is suitable for packaging of respiring fruit and vegetables
and for liquid food applications, e.g. juice. PLA can be used [1, 2] as a pure
product or it can be used in combination with other polymers. It may contain
natural extracts/components e.g. lignin and waxes acting as preservatives or
antioxidants preventing oxidation-sensitive products from deteriorating [3].
For PLA to compete with conventional packaging materials it must be pro-
duced from cheap raw materials and by feasible processes. Our objective in this
collaborative research project was to find the cheapest and most suitable raw
materials for Danish production of lactic acid for polylactate production.
13.3.1
Fermentation Processes
PLA is produced by polymerization of lactic acid. Lactic acid can be produced by

chemical synthesis or by a fermentation process. Fermentation is a process where-
by carbohydrates such as sucrose, glucose, fructose, or lactose are converted by mi-
croorganisms into a desired product. Most residues from the agricultural industry
contain such carbohydrates either free or bound in long chains of polysaccharides.
Molasses – a waste product from sugar beet production – contains saccharose;
whey – a waste product from cheese making – contains lactose; potato waste –
from the potato starch or French fries industry – contains starch, which can be me-
tabolized by certain strains of microorganisms or hydrolyzed to glucose. Straw
contains cellulose and hemicelluloses. Cellulose can be hydrolyzed to glucose
and hemicelluloses can be hydrolyzed to xylose, which also can be metabolized
by some strains. Green and brown juice from the green crop-drying industry con-
tains several types of carbohydrate and other nutrients. Some of the waste prod-
ucts are available free, others can be purchased at very favorable prices and could
be used as cheap raw materials in feasible production of PLA.
13.3.2
The Green Biorefinery
In the green biorefinery, jointly described by the University of Southern Den-

mark and AgroFerm A/S, crops are converted by means of mechanical and bio-
technological methods into useful materials such as food and feed products and
additives, and into materials and organic chemical compounds and bio-energy.
The crops used in the green biorefinery are crops that give a high yield, fit in
with normal crop rotation, absorb large amounts of organic fertilizers, are ro-
bust and require a minimum of pesticides.
13.3 Biobased Poly(lactic Acid) 297
Fig. 13.1 The principles of the green biorefinery.
The crops are separated in a liquid fraction – the juice containing the soluble
compounds and the press cake containing particles and insoluble high-molecu-
lar-weight compounds. Vitamins, colors, enzymes, and other phytochemicals
can be isolated directly from the juice or press cake. The press cake can be used
as animal feed or, after drying, as solid fuel.
After extraction of the high-value compounds from the juice, it can be used
as a substrate for fermentation [4]. The fermentation products can be any organ-
ic compounds, for example enzymes, antibiotics, biodegradable plastics, organic
acids, alcohols and amino acids.
13.3.3
Lactic Acid Fermentation
Lactic acid fermentation is normally performed by lactic acid bacteria. Lactic

acid bacteria comprise several genera with similar physiology, metabolism, and
nutritional needs. A primary similarity is that they all produce lactic acid as a
major or sole end product and that they do not use oxygen in their metabolism
(anaerobes). Nutritionally, lactic acid bacteria are extremely fastidious. A medi-
um that will support their growth must contain many growth factors such as
amino acids, peptides, nucleic acid derivatives, vitamins, salts, and fatty acids or
fatty acid esters. When all these nutrients are present in the fermentation medi-
um, lactic acid bacteria will convert carbohydrates to lactic acid.
13.3.4
Brown Juice as a Fermentation Medium
The green crop-drying industry in Denmark uses Italian rye grass, clover, and
alfalfa as raw materials for production of green pellets. Approximately 300 000
tons of green pellets are produced in Denmark each year. The green crop-drying
industry solves its energy-economical problems by pressing the green crop be-
fore drying. The by-product produced, green juice, typically has a dry-matter
content of 3 to 8%. At some factories the green biomass is heated to 80 8C by
steam before pressing, this causes the plant cells to burst and the protein to
coagulate. The by-product produced is called brown juice. Typically brown juice
has a dry-matter content of 4 to 8%. Approximately 200 000 m3 of brown juice
is produced each year in Denmark. Although the green and brown juice are
spread on the fields as fertilizer, pollution of ground water, particularly with ni-
trate, in the late autumn has led to stringent regulations on the use of plant
juice as a fertilizer. In Denmark plant juice can be spread on green fields only
in the autumn and not in the period between October 1st and February 1st [5].
The problem with the plant juice can be solved by storing the juice in large la-
goons from October 1st. Because storage can easily result in foul-smelling waste
products, we have studied other possibilities.
It is common knowledge that grass and almost all kinds of crop decay if
stored inappropriately, but it can be conserved by ensiling either by a sponta-
neous process in which the microorganisms present in the crops convert the
carbohydrates to acid, or by adding a culture of lactic acid bacteria. Therefore
the obvious solution is to use the brown juice as a fermentation medium for
lactic acid fermentation [6].
13.4 Materials and Methods 299
13.4
Materials and Methods
13.4.1
Analytical Methods
13.4.1.1 Sugar Analysis

Sugar analysis was performed by HPLC using a Bio-Rad IG Carbo C pre-col-
umn and a Bio-Rad Aminex HPX-87C column. Before HPLC analysis samples
were subjected to ion exchange through a cation-exchange column (Amberlite
CG-120) and an anion-exchange column (Dowex 1(4)).
13.4.1.2 Analysis of Organic Acids

Analysis of organic acids by HPLC was performed using a Bio-Rad Aminex
HPX-87H column. Before analysis of organic acids by HPLC, samples were sub-
jected to ion exchange through a cation-exchange column (Amberlite IR-120)
and an anion-exchange column (DEAE Sephadex A-25).
13.4.1.3 Analysis of Minerals

Cations, anions and trace minerals were kindly analyzed by the central laborato-
ry of the Danish Co-operative Farm Supply, DLG, using EU-approved methods.
13.4.1.4 Analysis of Vitamins

Vitamins are analyzed by use of Bacto Assey Methods (Difco Manual, 11th edn,
1998).
13.4.1.5 Analysis of Amino Acids

Analysis of free amino acids was performed by EU-approved method 98/64
1EC.
13.4.1.6 Analysis of Protein

Nitrogen (N) analysis was performed by the Kjeldahl method and crude protein
was calculated as N ´ 6.25.
13.4.2
Fed Batch Fermentation of Brown Juice with Lb. salivarius BC 1001
A 2-L continuously stirred tank reactor containing 1.5 L fresh (non-heat treated)
brown juice (2% DM) was used for the fed-batch experiment. The bioreactor
was equipped with a Mettler Toledo pH meter, heat sensor (pT100, MJK auto-
mation) and a heating element (50 W, 220 V), to keep pH and temperature con-
stant. pH was controlled automatically by addition of 4 m NaOH by a peristaltic
pump, the computer program used for control and regulation of pH and tem-
perature was Genesis 4.2.
Growth of the strain was followed by measurement of the dry cell mass. This
was achieved by centrifuging samples and washing twice with saline water
(0.9%) before drying the samples in an oven at 190 8C overnight. During fer-
mentation 2% glucose (40% solution) was added successively.
13.4.3
Pilot Scale Continuous Fermentation with Lb. salivarius BC 1001
The pilot scale experiment with continuous lactic acid fermentation of brown juice
was performed at the green crop-drying factory – Dangrønt Products, Ringkøbing,
Denmark. The brown juice produced from the pressing of the crops was cooled to
30 to 40 8C and fed into an 8 m3 tank and the tank was inoculated with 4 m3 bac-
terial culture (Lb. salivarius BC 1001). Fermentation was initiated as batch fermen-
tation, and after the pH in the tank had dropped to approximately 4.5, substrate
flow was started. The initial flow rate was 3.5 m3 h–1. The acidified BJ was led
to a sedimentation tank from where the supernatant was pumped to a storage/buf-
fer tank before evaporation to approximately 40% DM.
13.4.4
Study of Potato Juice Quality During Aerobic and Anaerobic Storage
Experiments were performed to investigate the quality of potato juice stored un-
der aerobic and anaerobic conditions. The investigations were carried out in
plastic containers with a volume of 25 L. The containers were filled with 10 L
fresh potato juice from the Karup, Denmark, potato starch factory. For anaerobic
storage the surface was covered with 1 to 2 cm corn oil. Containers were kept at
a temperature between 15 and 20 8C.
13.5
Brown Juice
13.5.1
Chemical Composition
The chemical composition of the green and brown juice was analyzed to investi-
gate the suitability of the brown juice as fermentation medium. The average
composition of evaporated fresh brown juice determined from 42 different
batches collected from June 9th 1998 to November 11th 2000 is shown in Ta-
ble 13.1.
Table 13.1 Average composition of evaporated fresh brown

juice determined from 42 different batches of brown juice col-
lected from June 9th 1998 to November 11th 2000, and the
calculated composition of average fresh brown juice with a
dry-matter content of 4%.
Component Average evaporated fresh brown juice Average fresh

brown juice
Content Maximum Minimum St. dev. Content Content
(g kg–1 DM) (%) (g L–1) (g L–1)
(Calculated)
DM (%) 32.4 52.6 14.7 28.0 4.0

Density (kg L–1) 1.2 1.3 1.1 4.0
pH 5.2 5.9 3.9 9.0
Sugar
Glucose 75.7 149.7 27.7 34.4 28.3 3.5
Fructose 104.5 168.1 60.2 25.6 39.0 4.8
Sucrose 50.1 123.1 4.4 57.6 18.7 2.3
Free sugars 229.7 355.7 96.4 26.7 85.8 10.6
Fructan 92.2 224.7 6.8 58.5 34.4 4.3
1-Ketose 9.1 29.0 0.0 67.5 3.4 0.4
Total 325.7 573.4 169.1 31.6 121.7 15.0
Acids
Citric acid 17.5 27.4 6.5 28.0 6.5 0.8
Malic acid 24.0 50.2 0.0 41.5 9.0 1.1
Malonic acid 13.1 24.8 0.0 40.3 4.9 0.6
Succinic acid 13.2 22.9 1.7 37.5 4.9 0.6
Lactic acid 33.1 103.6 0.0 65.8 12.4 1.5
Acetic acid 17.8 42.9 0.0 62.9 6.7 0.8
Total 118.8 271.8 65.1 48.9 44.4 5.5
Cations
Calcium 10.6 17.2 5.4 29.6 4.0 0.5
Magnesium 5.2 8.3 3.0 25.5 1.9 0.2
Sodium 9.5 16.8 4.6 40.4 3.5 0.4
Potassium 73.5 112.5 27.3 35.4 27.4 3.4
Ammonium 5.9 17.9 1.4 54.6 2.2 0.3
Total 98.7 149.9 28.3 31.1 36.9 4.6
Anions
Phosphate 29.4 39.4 17.7 18.1 11.0 1.4
Chloride 52.8 83.1 28.5 24.8 19.7 2.4
Nitrate 5.2 14.7 0.0 88.2 2.0 0.2
Total 83.1 115.2 49.7 19.8 31.1 3.8
Trace-minerals
Cobber 0.013 0.020 0.000 33.2 0.005 0.001
Zinc 0.106 0.216 0.048 46.6 0.040 0.005
Manganese 0.077 0.156 0.017 42.3 0.029 0.004
Iron 0.392 1.023 0.126 47.4 0.146 0.018
Total 0.585 1.277 0.289 37.4 0.219 0.027
Table 13.1 (continued)
Component Average evaporated fresh brown juice Average fresh

brown juice
Content Maximum Minimum St. dev. Content Content
(g kg–1 DM) (%) (g L–1) (g L–1)
(Calculated)
Vitamins
Pantothenic acid 0.060 0.101 0.035 27.139 0.023 0.003
Niacin 0.133 0.190 0.068 17.676 0.050 0.006
Thiamin 0.015 0.028 0.011 23.158 0.006 0.001
Total 0.209 0.276 0.154 14.865 0.078 0.010
Amino acids
Lysine 5.0 7.2 3.2 17.5 1.9 0.2
Serine 6.3 12.1 4.2 25.8 2.4 0.3
Glutamine 17.2 27.2 2.9 24.8 6.4 0.8
Glycine 6.2 16.4 4.0 33.8 2.3 0.3
Alanine 13.1 21.8 8.3 20.5 4.9 0.6
Asparagine 20.9 51.7 10.3 42.1 7.8 1.0
Methionine 1.2 1.8 0.6 22.5 0.4 0.1
Cystine 1.3 1.9 0.8 24.0 0.5 0.1
Threonine 6.0 7.9 3.8 18.0 2.2 0.3
Valine 7.3 10.4 4.6 19.7 2.7 0.3
iso-Leucine 4.6 6.6 2.6 20.7 1.7 0.2
Leucine 7.0 9.6 4.2 20.7 2.6 0.3
Tyrosine 2.3 5.9 0.0 70.6 0.9 0.1
Phenylalanine 4.4 6.8 2.3 24.7 1.6 0.2
Histidine 2.9 5.0 0.5 28.1 1.1 0.1
Arginine 4.0 8.8 0.5 32.5 1.5 0.2
Total 124.0 19.1 167.5 20.7 46.3 5.7
Crude protein 203.0 279.1 133.2 18.3 75.9 9.4
Mono-, di-, and trisaccharides are called free carbohydrates because they can
be metabolized by most lactic acid bacteria. Fructans (including 1-ketose) are
polymeric carbohydrates consisting of variable numbers of fructose molecules
and terminal sucrose. Fructans can be decomposed to free carbohydrates both
by enzymes in the crops, fructan fructohydrolases, and by some strains of lactic
acid bacteria, especially in the genera Lactobacillus plantarum and Lactobacillus
paracasei subspecies paracasei [7]. The enzymes in the crops are activated after
harvesting, carving, and pressing. Alfalfa does not contain fructans.
13.5.2
Seasonal Variations
Table 13.1 also shows the highest and the lowest value for a component found
in the 42 different batches of brown juice and the standard deviation. For most
compounds there are very high deviations between batches. The composition of
Fig. 13.2 Seasonal variation in free sugars and fructan in

different batches of brown juice harvested from June 1st to
November 6th 2000.
green crops is known to be influenced by many factors. These include climatic

influences such as light and night temperature, the fall of rain, management
practices, nitrogen application level, genotype, and relative proportions of leaf
and stem. The composition of the brown juice is further more influenced by
the type of crops harvested. Figure 13.2 shows the seasonal variation in free su-
gars and fructan in different batches of brown juice harvested from June 1st to
November 6th 2000.
The amount of free sugars in BJ has been found to vary between 135 and
340 g kg–1 DM and fructans between zero and 200 g kg–1 DM. The highest total
amount of carbohydrate was found at the beginning of the season in June and
lowest total amount of carbohydrate was found short periods in the beginning
of August and September when the fructan content, especially, drops dramati-
cally. Because alfalfa contains no fructan and is harvested only in this period it
is likely that the drop is caused by a considerable increase in juice derived from
alfalfa in the mixed brown juice. A high content of organic acid in the BJ dur-
ing this period indicates that some of the sugars are converted to organic acid
before pressing (Fig. 13.3).
Figures 13.4 and 13.5 shows the variation in minerals and amino acid/protein
in the brown juice.
The amounts of cations and anions in the brown juice seem to increase to-
ward the end of the season whereas amounts of trace minerals are more stable.
The amount of amino acids and crude protein in the juice fluctuate through-
out the harvesting season, but no great seasonal variations are found.
Fig. 13.3 Seasonal variation in organic acids in different

batches of brown juice harvested from June 1st to November
6th 2000.
Fig. 13.4 Seasonal variation in minerals in different batches of

brown juice harvested from June 1st to November 6th 2000.
Fig. 13.5 Seasonal variation in amino acids and crude protein

in different batches of brown juice harvested from June 1st to
November 6th 2000.
13.5.3
Lactic Acid Fermentation of Brown Juice
Different lactic acid bacteria have been tested for their ability to utilize the most
common carbohydrates. Several strains have been found suitable, but despite
the fact that Lactobacillus salivarius is unable to metabolize fructan, it has been
chosen for lactic acid fermentation of brown juice because of its high growth
rate. This high growth rate makes L. salivarius BC 1001 robust and capable of
competing with other microorganisms for an unsterile substrate [6]. By choos-
ing a robust, fast-growing microorganism that can compete with unwanted mi-
croorganisms in the brown juice, sterilization of the substrate can be avoided.
The brown juice contains both carbohydrates and amino acids, which in combi-
nation can form Maillard compounds during heat sterilization. This will cause a
decrease in the nutrient-content and reduce the quality of the juice, because of
the toxicity of some Maillard compounds.
Fermentation experiments with fresh non-heat-sterilized brown juice have
shown that fructan is used in fermentations with L. salivarius even though this
strain is unable to produce fructan-degrading enzymes. This is probably because
of the activity of plant enzymes in the brown juice. Under monoseptic fermen-
tation conditions fructan is not metabolized because enzymes in the juice are
destroyed by heat sterilization [8].
Fed batch fermentation of fresh brown juice (2% DM) has shown that brown
juice with only 2% DM contains enough nutrients in the substrate to achieve
high lactic acid production. In this experiment the specific growth rate of Lb.
Fig. 13.6 Growth and titration curve for fed batch fermenta-
tion with L. salivarius BC 1001 in fresh non-heat-sterilized
brown juice with 2% DM, to which glucose syrup, 40%, was
added successively.
salivarius BC 1001 was 0.9 h–1 and a cell mass concentration of approximately
3 g L–1 was achieved (Fig. 13.6).
The composition of nutrients in the brown juice causes the growth of cell mass to
stop at a certain concentration, whereupon the energy in the substrate is used ex-
clusively for maintenance and production of lactic acid. This characteristic makes
the brown juice suitable as a substrate for continuous lactic acid fermentation.
It has been shown that the growth rate of L. salivarius BC 1001 is higher in
brown juice than in MRS-bouillon, which is a substrate commonly used for
lactic acid fermentation [8].
13.5.4
The Green Crop-drying Industry as a Lactic Acid Producer
The green crop-drying industry in Denmark is located in Jutland. There are five
factories with a total production of approximately 200 000 m3 green and brown
juice 5% DM. It is shown in Fig. 13.6 that 2% DM is enough to achieve good
conversion of carbohydrates to lactic acid. A single green crop-drying factory
produces enough juice for lactic acid production of approximately 8000 tons per
year if a carbohydrate source is added to achieve a lactic acid concentration of
8% in the fermentation broth.
Experiments with continuous lactic acid fermentation of brown juice have
been performed at the green crop-drying factory: Dangrønt Products, Ringkø-
bing, Denmark. Figure 13.7 shows the process.
This pilot scale continuous lactic acid fermentation of brown juice was suc-
cessful. The pH of brown juice was reduced to 4.7 and much of the sugar in
the brown juice was converted to lactic acid. Table 13.2 shows the dry-matter
content, density, and pH of the brown juice after the process. Table 13.3 shows
results from analysis of the lactic acid fermented brown juice.
Fig. 13.7 The brown juice produced from the mately 4.5, substrate flow was started. The
pressing of the crops was cooled to initial flow rate was 3.5 m3 h–1. The acidified
35 8C ± 5 8C and fed into a 8 m3 tank. The BJ was fed into a sedimentation tank, from
tank was inoculated with 4 m3 bacterial where the supernatant was pumped to a
culture (Lb. salivarius BC 1001). Fermentation storage/buffer tank before evaporation to
was initiated as batch fermentation, and approximately 40% DM.
after pH in the tank had dropped to approxi-
Table 13.2 Dry matter, density, and pH of lactic acid ferment-

ed and evaporated brown juice in pilot scale continuous fer-
mentation at Dangrønt Products, Ringkøbing 16/10 2001.
Dry matter Density (kg L–1) pH
45.3 1.2 4.7
The analysis of the lactic acid fermented brown juice shows that efficient lac-
tic acid fermentation has occurred. The amount of lactic acid produced is
280 g kg–1 DM, leaving only small amounts of sugar in the medium. The strain
used for this fermentation (Lb. salivarius BC 1001) is a homofermentative strain,
and only small amount of acetic acid and succinic acid is present in the fermen-
308
Table 13.3 Composition of lactic acid fermented and evaporated brown juice (45.3% DM) produced in pilot scale continuous
fermentation at Dangrønt Products, Ringkøbing 16/10 2001.
Crude Cations Anions Trace Free sugars Fructan Total sugars Succinic- Lactic- Acetic- Total Amino acids
protein minerals acid acid acid org. acids
(g kg–1 DM) (g kg–1 DM) (g kg–1 DM) (g kg–1 DM) (g kg–1 DM) (g kg–1 DM) (g kg–1 DM) (g kg–1 DM) (g kg–1 DM) (g kg–1 DM) (g kg–1 DM) (g kg–1 DM)
245.2 143.4 103.5 0.818 12.5 3.4 16.0 33.5 280.8 23.5 337.8 117.3
Crude Cations Anions Trace Free sugars Fructan Total sugars Succinic Lactic Acetic Organic Amino acids
protein minerals acids
(g L–1) (g L–1) (g L–1) (g L–1) (g L–1) (g L–1) (g L–1) (g L–1) (g L–1) (g L–1) (g L–1) (g L–1)
132.0 77.2 55.7 0.440 6.8 1.8 8.6 18.0 151.2 12.7 181.9 63.2
13 Plant Juice in the Biorefinery – Use of Plant Juice as Fermentation Medium
13.6 Potato Juice 309
tation product. Substantial amounts of nutrients (minerals and amino acids) re-
main in the brown juice after lactic acid fermentation, indicating that adding
more carbohydrate could increase the yield of lactic acid. This would be neces-
sary in the production of PLA to make the process feasible. This will be dis-
cussed in Section 13.7.
13.6
Potato Juice
13.6.1
Potato Juice as Fermentation Medium
At potato starch factories the potatoes are sorted, washed, and grated. After grat-
ing, the potato pulp is centrifuged and the starch, pulp, and potato juice are
separated. Two waste products are produced: potato pulp and potato juice. The
pulp is sold for animal feed and the juice is spread on fields as fertilizer. The
potato starch industry thus faces the same problem as the green crop-drying in-
dustry – a waste product that must be stored in large lagoons from October 1st
or be used for other purposes.
Experiments have been performed to investigate the quality of potato juice
with regard to pH, lactic acid, and acetic acid formation, when stored under
aerobic and anaerobic conditions. Figures 13.8 and 13.9 show the results of
these experiments.
The figures show that aerobic and anaerobic storage of the juice causes a con-
spicuous drop in the pH of the juice within the first 7 days. This is because of
lactic acid and acetic acid fermentation of the carbohydrates and some organic
Fig. 13.8 pH, lactic acid and acetic acid during aerobic stor-
age of potato juice at a temperature between 15 and 20 8C.
Fig. 13.9 pH, lactic acid and acetic acid during anaerobic
storage of potato juice at a temperature between 15 and
20 8C.
acids in the potato juice by naturally occurring microorganisms in the juice.

Further storage of the juice induces the pH level in the aerobic container to in-
crease to over 6 whereas the pH in the anaerobic container remains stable at ap-
proximately 4.5. Accompanying this, a drop in lactic acid concentration and an
increase in acetic acid concentration can be observed.
At Karup potato starch factory the potato juice is fermented by adding lactic
acid bacteria and molasses to achieve a pH of approximately 4.5. Continuous
fermentation is performed in a 6000 m3 tank before it is led to big lagoons
(140 000 m3) where it is stored. During storage of the potato juice in the big la-
goons it can be difficult to maintain a low pH for a long time, primarily be-
cause of access to air. A rise in pH provides good growth conditions for bacteria
other than lactic acid bacteria, resulting in obnoxious smells. The results in
Figs. 13.8 and 13.9 show that prevention of access to air by (anaerobically) cover-
ing of the lagoons could be a way of maintaining the quality of the potato juice
and thus prevent obnoxious smells.
13.6.2
The Potato Starch Industry as Lactic Acid Producer
There are four potato starch factories in Denmark situated in Northern Jutland,
Karup, Brande, and Toftlund, manufacturing a total of approximately 1 million
tons of potatoes each year. This gives a total of approximately 200 000 tons of
potato starch, 150 000 tons of potato pulp (12 to 13% DM) and between 1 and
1.5 million tons of potato juice (2 to 4% DM) depending on the production
methods. At the factory in Karup 300 000 tons of potato juice (2% DM) are pro-
duced each year. On that basis Karup potato starch factory could produce ap-
proximately 35 000 tons of lactic acid a year if a sufficient amount of carbohy-
drate is added to the juice.
13.7 Carbohydrate Source 311
13.7
Carbohydrate Source
To profitably utilize brown juice from the green crop-drying industry or potato
juice from the potato starch industry, a carbohydrate source must be added. The
carbohydrate source could be another waste product from the agricultural indus-
try such as molasses from beet sugar production, whey from the dairy industry,
or soy meal extract from the production of soy protein. It could also be refined
sugar (white sugar), hydrolyzed straw, or hydrolyzed cereal grains, e.g. wheat.
Wheat has advantages over other carbohydrate sources. Cereals, being much
lower in moisture than molasses, are more energy intensive and have the ad-
vantage that they can be stored and transported easily. The sugar is in the form
of starch and in addition cereal grains contain nutrients which can be separated
easily from the grain and sold as lucrative by-products – bran, gluten, and A-
Wet separation
(pressing)
Fig. 13.10 A green biorefinery based on green crops will be

able to produce products based on organic acids and amino
acids made by fermentation [4].
starch. Gluten – a protein used in the baking industry – is the most profitable
of these by-products.
Webb and Wang describe a process for utilization of wheat as a carbohydrate
source for lactic acid fermentation in which gluten is separated from the grain
before hydrolysis of the starch. The filamentous fungus Aspergillus awamori is
capable of producing enzymes to break down the starch to glucose [9].
As shown in Fig. 13.10, there are many possibilities of producing fermenta-
tion products on the basis of on green crops and potatoes and thereby changing
the green crop-drying factories and potato starch factories to green biorefineries.
13.8
Purification of Lactic Acid
The lactic acid must be recovered and purified from the fermentation broth.
The classical method is the calcium lactate process in which the lactic acid is
precipitated as calcium lactate and treated with sulfuric acid. The resulting cal-
cium sulfate is separated from the lactic acid solution by filtration and the liq-
uid is evaporated. The process gives residual amounts of gypsum, and waste
gypsum disposal can be a problem.
The latest developments in lactic acid purification have been towards mem-
brane processes such as ultrafiltration and electrodialysis. Other methods such
as ion-exchange and solvent extraction involve heavy initial costs and operating
costs; they are, therefore, not very suitable for low-cost production of lactic acid.
Electrodialysis is a membrane process in which the membranes allow passage
only of either anions or cations (ion-exchange membranes), the driving force
being potential difference. Because the membranes are easily fouled if the fer-
mentation broth contains large impurities and particles, these are removed by
ultrafiltration before electrodialysis. Problems with membrane processes arise if
the fermentation broth contains large amounts of organic particles and in-
organic ions such as calcium and magnesium, which damage the membrane.
Birgit and Michael Kamm use ultrafiltration, nanofiltration, and electrodialy-
sis for purification of lactic acid from fermentation broth. They have developed
a process whereby lactic acid is neutralized with piperazine, an amine that com-
bined with two molecules of lactic acid makes piperazinium dilactate. The pi-
perazinium dilactate can be converted to dilactid (a building block in the pro-
duction of polylactate) without the production of undesired by-products. Birgit
and Michael Kamm did not experience problems with fouling of the ultrafiltra-
tion membrane but some optimization of the nanofiltration and electrodialysis
processes is still needed [10].
At the Technical University of Denmark a process has been developed in
which the lactic acid is continuously removed and purified from the fermenta-
tion broth using various membrane processes (Donnan dialysis, electrodialysis
with bi-polar membranes, and electrodialysis). In this process problems with
fouling of the membranes are minimized or avoided [11].
References 313
13.9
Conclusion and Outlook
Waste products from the green crop-drying industry and the potato starch in-
dustry, brown juice and potato juice, contain all the nutrients necessary for lac-
tic acid bacteria to convert carbohydrates to lactic acid. Brown juice 2% DM in a
substrate contains enough nutrients to achieve good lactic acid production in
continuous fermentation by addition of a carbohydrate source.
It is possible to use other cheap waste products from the agricultural industry,
for example molasses or hydrolyzed straw, or purer products such as refined su-
gar or hydrolyzed starch, as a carbohydrate source. Hydrolyzed B-starch from
wheat has several advantages compared with other carbohydrate sources. The
most important is that the grain contains nutrients, for example bran, gluten
and A-starch, which can be separated easily from the grain and sold as lucrative
by-products.
At the Technical University of Denmark a very promising process has been
developed for purification of lactic acid produced from agricultural waste prod-
ucts. This process can substantially reduce the cost of lactic acid production.
The experiments reported in this article show it is possible to produce lactic
acid from brown juice or potato juice on an industrial scale using non-sterile
brown juice or potato juice for fermentation. The fermented juice can be stored
under anaerobic conditions and used as a substrate for all-year round produc-
tion of lactic acid, lysine, and many other fermentation products.
Acknowledgments
This work was supported by the Danish Ministry of Food, Agriculture and Fish-
eries under the program “Increased Utilization of Renewable Resources for In-
dustrial Non-food Purposes” (1997–2001). We thank Vagn Hundelbøll, Agro-
Ferm A/S, and Jens Mikkelsen, the potato starch factory in Karup for the inter-
esting co-operation.
References
1 Shogren, R., 1997, Water Vapour Perme- 3 Petersen, K. et al., 1999, Potential of Bio-
ability of Biodegradable Polymers, J. En- based Materials for Food Packaging,
viron. Polym. Degrad. 5(2). Trends Food Sci. Tech. 10, 52–68.
2 Weber, C. J., Biobased Packaging Materials 4 Andersen, M., Kiel, P., 1999, Method for
for the Food Industry Status and Perspec- Treating Organic Waste Materials, Euro-
tives – A European Concerted Action, KVL pean Patent Application, 19 March 1999,
Department of Dairy and Food Science, WO 00156912.
Frederiksberg, Denmark. Trio Design, 5 The Danish Ministry of Environment
Copenhagen and Energy’s order no. 823 of 16 Sep-
tember 1996. Order of the use of waste 9 Webb, C., Wang, R., 1997, Development
products for agricultural purpose. of a Generic Fermentation Feedstock
6 Thomsen, M. H., Bech, D., Kiel, P., from Whole Wheat Flour. In: Campel,
2004, Manufacturing of Stabilized Brown G. M., Webb, C., McKee, S. L. (Eds) Cer-
Juice for l-lysine production – from Uni- eals: Novel Uses and Processes, Plenum
versity Lab Scale over Pilot Scale to In- Press, New York, pp. 205–218.
dustrial Production. Chem. Biochem. Eng. 10 Kamm, B., Kamm, M., Richter, K., Rei-
Q. 18, 37–46. mann, W., Siebert, A., 2000, Formation
7 Müller, M., Steller, J., 1995, Comparative of Aminium Lactates in Lactic Acid Fer-
Studies of the Degradation of Grass mentation. Acta Biotechnol. 20(3/4), 289–
Fructans and Inulin by Strains of Lacto- 304.
bacillus paracasei subsp. paracasei and 11 Garde, A., Rype, J. U., Jonsson, G., 2000,
Lactobacillus plantarum. J. Appl. Bacteriol. A Method and Apparatus for Isolation of
78, 229–236. Ionic Species from a Liquid. Not yet pub-
8 Andersen, M., Kiel, P., 2000, Integrated lished patent. Application no. PA 2000
Utilisation of Green Biomass in the 01862.
Green Biorefinery, Ind. Crops Prod. 11,
129–137.
Part III
Biomass Production and Primary Biorefineries
ISBN: 3-527-31027-4
317
14
Biomass Commercialization and Agriculture Residue Collection
James Hettenhaus
14.1
Introduction
In the next ten year biorefineries may be processing 100 million metric dry tons
(dt) biomass annually for production of fuels and chemicals if a stretch goal set
by the US Department of Energy is met [1–3]. Initially, feedstocks with no col-
lection cost like paper mill sludge, bagasse, and rice hulls may be used to vali-
date the conversion technology. They have no associated transport cost. Their
quantities may also serve niche markets for high-value chemicals [4]. For larger
markets like transportation fuels, their conversion is too small and costly to
compete. The biorefining industry’s growth is predicated mostly on corn stover,
supplemented with straw and grasses with a delivered price of $ 30 to $ 35 dt–1
[5, 6]. The quantity and ready availability makes stover an early feedstock choice
for initial biorefineries (Table 14.1), US Ag residue feedstock availability.
When the feedstock market is established with stover, other biomass feed-
stock, prairie grasses and energy crops, from wide geographic areas will emerge
to supply biorefineries.
While great strides have been made in improving the conversion process,
there remains much uncertainty regarding the feedstock supply. Economies of
scale require a biorefinery size of 500 to 2000 dt feedstock per day. Supplying
these large quantities raises major issues including:
Table 14.1 US Ag residue feedstock availability, metric dry tons (millions).
Corn stover 200

Cereal straw 70
Energy crops 74
Bagasse 6
Corn fiber 4
Rice hulls 1
Total dry tons 355
ISBN: 3-527-31027-4
318 14 Biomass Commercialization and Agriculture Residue Collection
· agronomic systems for sustainable removal – maintaining soil quality;

· economic and environmental benefits for the farmer and other stakeholders;
· methods for improved feedstock collection, storage and transport; and
· infrastructure required for reliable feedstock supply.
More information is needed for the farmer to assess the opportunity for supply-
ing the emerging biorefinery market for biomass feedstock. What is the value
of the biomass to the soil and to the farmer? What cropping practice is needed
for sustainable removal, balancing environmental and economic requirements?
Previous studies of crop residue removal on soil quality are limited to small re-
search plots that often do not align with current best practice. Although existing
models provide guidelines for residue management to limit soil erosion, robust
models that address soil quality and residue removal are just emerging.
Bulky crop residues are collected to meet small needs, mostly for local farm
use–bales for animal bedding. There is negligible collection infrastructure to serve
large markets like biorefineries. Sizable investment – $ 50 to $ 100 million – is
probably needed in the supply systems for biomass harvesting, collection, storage
and transport to supply a 1 million dt year–1 to a biorefinery [1]. Who will make
these investments – the farmer, the potential processor, or a biomass supplier that
aggregates individual growers? Current supply systems for grain like corn and
wheat are highly evolved and efficient infrastructures and may provide valuable
extensions. Existing grain elevators may serve as collection points for biomass.
Local conditions determine the potential feedstock quantities. Areas capable
of providing large supplies have been identified – but like commercializing an
idea, much work remains between knowing the location of promising biorefin-
ery sites and reliably supplying them with economic, sustainable feedstock.
14.2
Historical Outline
Commercial ventures that require large quantities of crop residues have a mixed
history of success. Collected material has found more use as co-products than
those that remain in the field after harvest. For example, corn fiber is mixed
with steepwater and sold as corn gluten feed with a nutrient value 1.1 times
cracked corn for ruminant animals [8]. Bagasse has a long history and is dis-
cussed in Case Study 2.2. Small quantities like rice hulls offer little economic
incentive to develop markets, therefore most remains to be disposed of, often
by burning as a fuel or simply land applied [9].
For biomass left in the field – stover, straw and grasses – collection and trans-
portation cost increases the economic hurdle. In areas of the world lacking
trees, non wood fiber is pulped, producing quality papers [10]. They make a rel-
atively poor animal feed because of low protein content [8] and their use as a
fuel is limited by their composition, especially the low thermal content com-
pared with coal and natural gas.
During the last decade the list of unsuccessful ventures using bagasse, grass,
straw, and stover for particle board has grown by more than a dozen [11, 12].
This mixed record hinders enthusiasm among many growers and others to con-
sider feedstock collection as a viable route for biomass commercialization.
14.2.1
Case Study: Harlan, Iowa Corn Stover Collection Project
The largest recent corn stover collection project was undertaken in Harlan, Iowa
in 1996 by Great Lakes Chemical. Modern collection methods – self loading and
unloading wagons and high speed, over the road tractors – were used to reduce
feedstock cost for the production of furfural [13, 14]. The stover revenue to the
farmer was $ 3 to $ 12 dt–1, depending on the hauling distance (Table 14.2).
Total feedstock requirements were approximately 100 000 dt per year. A collec-
tion center for approximately 50% of the total was constructed in Harlan, Iowa,
USA, in 1996. The facility sampled and weighed all deliveries, then stacked
bales for storage. During the year bales were removed from storage, milled, pel-
letized, and loaded into trailers and trucked 90 km to the furfural plant.
The first year was a learning experience for all. Meetings with local producers
were held, and many showed interest in collecting stover for added income, and
as a way to remove most of it so the soil would warm in the spring for seed ger-
mination without having to plow. The amount collected complied with soil ero-
sion guidelines. But at the conclusion little stover was delivered. Great Lakes
had left the collection with the farmers who mostly used their resources for har-
vesting the corn grain, not baling.
The second year Great Lakes employed contract balers and haulers, matching
them up with the farmers and the result was an overwhelming collection suc-
cess. More than 400 farmers committed 20 000 ha of their corn fields. Thirty
plus custom harvesters were contracted to perform the baling.
Self loading and unloading wagons pulled by high speed tractors were used
for bale transport (Fig. 14.1). The bales were collected in the field – 17 round
bales, approximately 9.5 dt – in less than 20 min. The wagon traveled at high-
way speeds, up to 90 km h–1, en route to the collection center. At the collection
center the load was weighed, sampled for moisture, and unloaded. In less than
10 min the driver was on the way to the next field.
Table 14.2 Corn stover pricing summary.
Revenue payments, dollars per dry ton (mkg)
Radius, km 0–25 26–49 50–80 81–164

Producers revenue $ 12.00 $ 9.05 $ 6.12 $ 3.19
Baler’s revenue $ 16.06 $ 16.06 $ 16.06 $ 16.06
Hauler’s revenue $ 6.71 $ 9.65 $ 12.58 $ 15.51
Total, delivered price $ 34.76 $ 34.76 $ 34.76 $ 34.76
Fig. 14.1 Load-and-go wagon with high-speed tractor.
a)
b)
Fig. 14. 2 Corn stover bale storage.
Start-up problems were mostly baler-related: working to achieve a dense bale,

with a minimum 550 kg dry weight. Bales are often sold as a unit, not on a dry
weight process. Low bale density made transporting a losing proposition. These
difficulties were worked out and several weeks of productive baling occurred before
the first blizzard on October 27. Afterward, the field conditions were too wet for
baling. Approximately 12 000 ha were actually collected, because of the wet, unusu-
ally warm winter. The season ended with a waiting list of more than 40,000 ha,
more than enough cropland to meet the total plant requirements IF weather per-
mitted.
Meanwhile, the price of imported furfural dropped below the plant manufac-
turing cost, and the operation was bought out by another firm to make hydro-
mulch. Economic, process, and product-quality problems slowed sales. Finally a
bale fire destroyed most of their inventory, forcing a liquidation of their assets
in 2002.
The major findings from the collection operation include the following [15]:
· Farmers are willing to sell excess stover when the economics fit.
· Collection practices have to meet farmers’ requirements for success.
· Logistics are a major factor for successful collection.
· Contract operators are essential to meet the extra workload.
· Shortening the harvest window is essential to reduce collection risk due to
weather.
· Bale storage is costly and adds no value.
· One-pass harvest, with the grain, can reduce cost and harvest risk.
· Bale fires are a serious hazard.
14.2.2
Case Study: Bagasse Storage – Dry or Wet?
Bagasse, the biomass remaining from sugar cane after the sucrose-containing
juice has been extracted, has been used by the pulp and paper industry for
more than a century [10]. It incurs no collection cost and competes favorably
with wood pulp in a global market. The bagasse exits the mill with about 50%
moisture. For stable storage it must be below 20% or above 60%.
In the early 1920s the sugar cane industry investigated ways to store bagasse
for processing to particleboard and pulp. Particleboard processors preferred dry
material. Pulp mills readily accepted wet material.
14.2.2.1 Dry Storage

Celotex produced insulation board, a dry process, and pursued ways to improve dry
feedstock storage. Their effort resulted in using the heat from microbial fermenta-
tion to dry bales from 50% to less than 20% moisture [16]. The bales were sized and
stacked to dissipate heat and acid fumes. Sheltered from the weather, bales kept for
several years without serious deterioration (Figs. 14.3 and 14.4).
Fig. 14.3 Bale stacking, circa 1930.
Fig. 14.4 Bale storage, 1930–1960.
Although this dry storage method was used for more than 40 years [17], a
change to wet storage occurred in the 1960s because of increasing recognition
of its advantages:
· The bales were relatively small, weighing 115 kg “as is”.
· Mechanical handling was slow and costly.
· The bales had to be precisely stacked to vent fumes and dissipate heat.
· Procedures were labor-intensive.
· Several months were required to dry bales from 50 to 20% moisture.
· Fire loss and increasing fire insurance costs.
14.2.2.2 Wet Storage

Whereas Celotex pursued dry storage, other companies in the pulp and paper
industry continued investigating wet storage, because pulping is a wet process.
The results were more successful than dry storage. Wet storage of non-wood
fibers was first commercialized by E. A. Ritter in 1950. Wet storage of bagasse
has been in widespread use on a commercial scale since 1960 for both wet and
dry downstream processes [18].
Figure 14.5 shows a typical collection area with a pile under construction in
the foreground. Major studies on a commercial scale showed pulping the stored
bagasse was superior to pulping dry bagasse and fresh, wet bagasse. The wet
feedstock contained less solubles, required less chemical treatment, produced
higher yields and had better processing characteristics compared with dry feed-
stock and fresh, wet bagasse [19]. In addition, wet feedstock stored for 6 months
was superior to “green” bagasse, the fresh material from the current harvest in
processing characteristics, with 80% less solubles and higher holocellulose com-
position. As a result, green bagasse is held over, for processing the following
year [20]. Section 14.5.2 discusses storage further.
Fig. 14.5 Wet storage pile construction.

14.3
Biomass Value
Lacking large uses of crop residues, studies have not addressed the impact of re-
moval of stover [21] and straw [22] on soil quality, although several implications
can be drawn [23]. An environmental and economic balance is required for suf-
ficient retention of residues to avoid erosion losses and maintain soil quality,
while economically removing excess residue as biomass feedstocks. The impact
of different levels of surface removal depends on local conditions and practices.
Maintaining this balance is key for success.
14.3.1
Soil Quality
Agricultural residues provide a key role in maintaining soil quality. Surface cov-
er is needed to prevent wind and water erosion, retain soil moisture, recycle nu-
trients from the plant back to the soil, and support assorted life. When residue
is removed, reduced inputs from the residue to the soil can result in a negative
flux from the soil and a loss of soil organic matter, SOM, and other nutrients
leading to a breakdown of soil structure.
The amount of excess is a complex question. It depends on local factors like
soil type, cropping practice, weather, and topography. For example, in some dry
areas surface cover is required to retain moisture, mostly in western areas of
the grain belt [24]. Further east, surface cover is given as a major reason for til-
ling, because the cover prevents the cold, wet soils from warming in the spring,
delaying planting and reducing yield [25]. Recycling nutrients from the plant,
especially P and K, back to the soil reduces the need for replacement. Converse-
ly, in areas where manure is used, the P contained in the soil may already be
too high, resulting in excessive run-off that contributes to algae formation in
ponds and streams [26]. Surface cover provides shelter and food for many or-
ganisms – microbial and larger. Their contribution to the humic pool is impor-
tant, but they also shelter destructive weed seeds, pests, and toxins that can
harm the next crop [27].
Models are under development to better measure soil quality. For example,
agricultural ecosystem models like Century, DayCent, and Cstore show some
beneficial effects of removing residue while still meeting constraints of soil and
wind erosion. Namely, nitrate leaching decreases tenfold in some situations and
nitrous oxide emissions (a potent greenhouse gas) are also significantly reduced.
These models can be used with actual field measurements for guidance in se-
lecting among alternatives that best balance economic and environmental bene-
fits [28]. Using field measurements with the soil conditioning index will show if
the crop practice is correctly managing soil carbon and is recommended [29].
14.3.2
Farmer Value
Most farmers are forced to manage crop residues in place. Present markets are
negligible for straw and corn stover. Less than 5% is used for animal bedding
and feed, with the major portion used on the site and then recycled as soiled
bedding or manure. Some growers have historically rented harvested corn fields
for grazing, charging $ 12 to $ 25 ha–1. This practice is declining as combines
have become more efficient, lodging is less with Bt corn, and cattle ranching
has grown more “factory-like” with heavy dependence on large feedlots.
If residue were removed, the phosphorus (P) and potassium (K) content in
straw and stover will eventually need to be replaced. The composition is typical-
ly 0.1% P and 1% K, valued at $ 3.50 dt–1 [6]. The N fertilizer value is more
complex, and depends on crop rotation and local conditions. Reduced field
operations are estimated to reduce inputs $ 24 ha–1 for preparation of the seed
bed [30].
Carbon credits are likely to add additional economic incentive for US farmers.
Reducing tillage or no-till sequesters about 0.3 to 0.5 metric tons C equiv ha–1.
The increased soil carbon improves yields, and this benefit continues with each
crop year. Eventually, over decades, soil carbon equilibrium is achieved. In the
European Union, carbon is currently trading for about $ 35 per ton C equiv. A
small, voluntary greenhouse gas trading market has been established for agri-
cultural carbon sequestration as part of the Chicago Board of Trade, the Chicago
Climate Exchange [31]. Recent efforts to move US policy in this direction call
for a $ 26 per ton of C-equivalent credit that would fund renewable fuels re-
search and development [32].
Reducing N fertilizer use is also possible, depending on crop rotation. Mi-
crobes desire a 10 : 1 ratio of C/N for breaking down residue. Because the C/N
ratio of straw and stover is 40 to 70 : 1 , 10 kg N fertilizer addition per ton of re-
sidue is typically recommended to avoid denitrification of the next crop. For
250 ha of 9 dt ha–1 corn (170 bu acre–1), 30 to 40 tons of N fertilizer may be
avoided. In addition to the out-of-pocket costs, environmental benefits include
reducing N run-off to streams and groundwater, and reducing greenhouse gas –
0.17 to 3.5 tons of N2O/100 tonnes applied – 5 to 100 tonnes C equiv ha–1 [33].
The economic benefit from selling stover to the farmer is summarized for
three production yields – 6.9 dt ha–1, 9.9 dt ha–1, and 10.6 dt ha–1 (130, 170, and
200 bu acre–1) in Table 14.3. More details are presented in Section 14.5. The ex-
ample uses the following values:
· delivered sale price is $ 33 dt–1
· moisture is 15% to adjust to dry basis
· harvest index of 0.5, a 1 : 1 ratio of grain to stover
· surface cover of 2.2 dt ha–1 left in the field,
· P and K fertilizer value of $ 3.50 dt–1 removed with the stover
· reduced field operations, $ 24 ha–1
· no credit for carbon sequestration or other inputs like N fertilizer.
Table 14.3 Stover field value before transportation and collection cost, $ ha–1.
Production case 1 2 3
Stover production, dt ha–1, 1 : 1 ratio 6.9 9.1 10.7

Surface cover, 2.2 dt ha–1, left in field (2.2) (2.2) (2.2)
Stover sales, dt ha–1 4.7 6.9 8.5
Stover revenue, $ 33 dt–1 $ 155 $ 225 $ 278
P & K nutrient credit ($ 3.50 dt–1) (16) (24) (29)
Reduced field operations, $ 24 ha–1 24 24 24
Net value, bulk in field, $ ha–1 $ 163 $ 226 $ 273
The net value in the field is $ 163 to $ 273 ha–1 before collection and transporta-
tion cost.
The net margin after Collection and transportation costs are determined is
given for two examples – typical baled stover collection (Table 14.4), and one-
pass collection (Table 14.5). Other costs like shrinkage or change in properties
of the feedstock in storage, storage investment, and operation, and relative qual-
ity of final feedstock processed are not included in the examples. There is a
large variation in these factors depending on the local situation.
Baled stover, Table 14.4, is trucked within a 50 km radius – an average 70 km
round trip using high speed tractors and “load and go wagons” as in Harlan,
IA. Baling and transport cost adds $ 25 dt–1, leaving the farmer a net margin of
$ 41 to $ 54 ha–1. A minimum $ 50 ha–1 return is most often cited to raise
grower interest [34, 35].
Table 14.4 Stover sale net to farmer, $ ha–1 W/custom bale and 50 km radius collection site.
Case 3.1 3.2 3.3
Net margin, bulk in field, $ ha–1 $ 163 $ 226 $ 273

Less custom bale, $ 16.00 dt–1 (75) (109) (135)
Hauling, 50 km radius, $ 9.9 dt–1 (47) (68) (83)
Net margin to farmer after delivery, $ ha–1 $ 41 $ 49 $ 54
Table 14.5 Sale net to farmer, $ ha–1, W/one-pass harvest and 3–25 km radius collection sites.
Case 3.1 3.2 3.3
Net margin, bulk in field, $ ha–1 $ 163 $ 226 $ 273

Less one-pass harvest, $ 40 ha–1 (40) (40) (40)
Field to collection site, $ 6.00 dt–1 (28) (41) (51)
Hauling, 50 km radius, $ 9.9 dt–1 (35) (51) (63)
Net margin to farmer after delivery, $ ha–1 $ 59 $ 93 $ 119
One pass collection of grain and stover, with stover trucked from field to one
of three collection centers within a 25 km radius – 35 km average round trip,
stored above 60% moisture, then transported via rail to the biorefinery – offers
more opportunity to reduce cost and reduce harvest risk [36]. Even with higher
transport cost, $ 13.50 vs $ 9.90 dt–1, the farmer’s net margin after delivery
ranges from $ 59 to $ 119 ha–1 (Table 14.5). The difference is greatest for fields
with high yields, with nearly twice the margin compared with baling.
One-pass harvest prototypes are under development, and further discussed in
Section 14.5.
14.3.3
Processor Value
Carbohydrate feedstock is being seriously considered as an alternative to petro-

leum and natural gas feedstocks by the chemical and plastic materials industry.
The escalating cost of fossil feedstocks and their pricing instability have contrib-
uted significantly to the industries low margins. Added environmental concerns,
especially emissions that contribute to global warming have generally shrunk
their market value.
Low cost fermentation sugars coupled with rapid advances in biotech tools of-
fers these industries a potentially economic and sustainable feedstock for pro-
duction of transportation fuels, chemicals, and materials. For example, E85 fuel,
85% ethanol in gasoline, reduces greenhouse gases by 64% compared with ga-
soline. The effective benefit is 170 kg C equiv mitigated per dt corn stover pro-
cessed compared with regular gasoline [37].
Sourcing low-cost feedstock is a common critical success factor. Successful
crop residue-processing plants wish to achieve an economy of scale well below
the local feedstock supply limits to accommodate expansion. Most biorefineries
plan to have a business model that offers the grower an option to participate in
the value chain to help ensure a win–win business relationship. An example is
corn growers who are also shareholders in dry mill plants now growing hybrids
with traits that produce more liters of ethanol kg–1 corn. They have no grain
yield drag and processing this corn adds value to their stake in the dry mill [35].
Sustainable supply is of prime importance to the processors. Environmental con-
cerns are a major driver for the industry to move from fossil feedstocks. Assurances
will be needed that the long-term effect on the soil quality is neutral or better to avoid
the claim that removal of residues is depleting the soil of needed nutrients [35].
Feedstock pricing has a major effect. The National Renewable and Energy
Laboratory uses $ 33 dt–1 delivered cost to the biorefinery in the base case of
their process. The result is based on extensive experimentation, process model-
ing and industry peer reviews. A $ 5 dt–1 change in feedstock price affects etha-
nol 1 c/ to 1.5 c/ L–1, depending on process yield [7].
Wet or dry feedstock can be processed, but if dry, some prefer bulk delivery of
milled product to avoid the multi-million dollar investment in equipment and
dust-explosion-proof installation [38].
Clean feedstock with consistent composition and properties are desired, but
more information remains to be provided by the processors. Because dirt, inorgan-
ics and ash add to the processing cost, and higher cellulose and hemicellulose con-
tent can increase yields, feedstock pricing based on composition and ease of pro-
cessing is expected to emerge, not paying just per dry ton. Rapid analytical meth-
ods for compositional analysis are under development and are expected to be va-
lidated and available for incorporating in a feedstock payment program [39].
14.4
Sustainable Removal
Removing residues from the soil depletes the amount available for replenish-
ment for nutrients and increases the possibility of erosion. Compensating for
this will probably require revising some present agronomic practices. For exam-
ple, leaving anchored stubble, 20 cm or more above the crown may suffice in
some areas to control erosion, especially with narrow planting rows, e.g. 40 cm
compared with 80 cm, the most prevalent practice for corn rows. This and other
contemplated needs are discussed in this section.
With a biorefinery, the excess is removed, processed into fuels – and “di-
gested” by the autos – the CO2 exits from the auto’s exhaust, instead of being
exuded from microbes in the field. The benefits are huge, BUT only when this
is conducted in a sustainable manner by controlling erosion and maintaining
soil quality, especially soil organic material (SOM).
14.4.1
Soil Organic Material
Tilling causes loss of SOM, an important measure of soil quality. If too much
stover is removed or a cover crop is not planted that adds to the biomass, SOM
can be depleted. SOM is more strongly affected by below-ground residues (i.e.
roots), with above-ground residue contributing less to SOM formation. Studies
at the National Soil Tilth Laboratory shows 80% or more of the surface material
is lost as CO2 within months, and three times the amount of SOM comes from
roots compared with surface material [40].
The tillage effect on soil carbon loss after corn harvest is shown for various
field operations in Fig. 14.6 [41].
The CO2 flux emitted from the soil is shown for various cases over time. The
amount of loss depends on the amount of disturbance – more exposure, more
oxidation of the organic material and more lost soil carbon. The bottom line in
the figure shows normal soil respiration as microbes and other organisms in
the soil and on the soil surface emit CO2 as they digest biomass carbohydrates
and lignin. The top line shows the highest loss when plowing the soil – an ini-
tial burst of CO2 occurs as the plow rips open the soil and the anaerobic soil en-
vironment is exposed to oxygen in the air.
Fig. 14.6 Fall tillage effect on soil carbon.
No-till or reduced-till practices are needed to maintain soil organic material

when stover is removed – if not, the carbon loss from tillage will deplete the
soils’ carbon pool, resulting in negative effects on soil quality, including aggre-
gate instability, less water-holding capacity, and an associated drop in productiv-
ity.
14.4.2
Soil Erosion Control
Loss of topsoil remains a concern and continues to be a severe problem in

many areas. Not only does much soil get lost in waterways, erosion also de-
pletes soil fertility. The SOM is greatest in the topsoil, and up to 20% of the
SOM is estimated to eventually be lost to the atmosphere as a result of cropping
practices [42].
For adequate feedstock supply and efficient collection less tillage will be re-
quired to control erosion in addition to maintaining SOM. With present tillage
practices all of the residue must remain in 60% the corn fields and 70% of the
wheat fields to comply with USDA erosion control guidelines. The USDA
guidelines are based on extensive studies that indicate all the residue should re-
main in fields with conventional till, i.e. less than 30% of the surface is covered.
The guidelines are based on “tolerable” soil loss, a judgmental value [21].
Less tillage, referred to as mulch till, has more than 30% of the surface cov-
ered, allows some residue removal while no-till or ridge till permits most of the
surface material to be removed. Ridge-till or strip till, involves tilling a strip
through the field, clearing away the residue in a space adequate for planting
and warming the soil in the seed area for early germination. The ridge is fa-
vored with furrow irrigation. Without the ridge, it is referred to as strip till.
For example, using the USDA water-erosion model, revised universal loss
equation (RUSLE), and wind erosion equation (WEQ), the variation in required
erosion cover for selected counties in the top corn-producing states – Illinois,
Iowa, and Nebraska – are shown in Table 14.6. Wheat is included, because irri-
gated wheat yields enough to warrant straw collection in Peoria County, IL, Jas-
per County, IA, and Rock County, NE [43]. Unless wheat is irrigated, much of
the wheat yield on dry land is below 2.2 dt ha–1, negating any straw removal.
Even with no till, economic quantities are difficult unless yields approach corn,
8 to 10 dt ha–1.
The historic tillage practices for corn and wheat for the US are summarized
in Tables 14.7 and 14.8. The values are based on a nationwide survey conducted
by the Conservation Technology Information Center (CTIC) [44]. Table 14.7
shows about 20% of corn is no-tilled or ridge-tilled, enabling efficient collection.
More than 80% of farmers employ some form of tillage to manage surface
residues, with 60% of corn fields conventionally tilled. This has become a larger
task as yields have increased. In 1960, corn belt states averaged 3.6 tons ha–1. In
2003 the yield had increased to 10.0 tons ha–1.
Adapting to no-till corn will be easier in many areas when some of the stover
is economically collected, especially in the northern parts of the corn belt where
cold moist soils can delay germination in the spring. Each day delayed is 25 kg
corn grain lost according to local lore. Most corn growers till for removing, i.e.,
burying, corn stover to induce spring soil warming.
Table 14.6 County-level stover and straw cover required for water and wind erosion.
State County Corn, dt ha–1 Wheat, dt ha–1
No-till Mulch till No-till Mulch till
IL McLean 1.1 2.2 NA NA

IL Peoria 2.1 4.6 NA NA
IA Grundy 1.6 2.7 NA NA
IA Jasper 2.8 6.7 NA NA
NE Rock 2.4 4.0 1.1 2.3
NE Dawson 1.6 2.4 1.1 1.7
Table 14.7 Corn tillage practice, % total.
Year 1994 1996 1998 2000 2002
No till 18 17 16 18 19
Mulch 22 23 23 19 16
Ridge till 3 3 3 2 2
Conventional 60 60 61 63 64
Table 14.8 Wheat tillage practice, % total.
Year 1994 1996 1998 2000 2002
No till 5 7 9 10 11
Mulch 25 24 23 20 16
Conventional 69 69 68 70 73
For wheat, less than 10% of the acres are no-till (Table 14.8). Weed control of
wheat is the primary reason for tillage. Changing to no-till wheat is slowly gain-
ing favor, increasing from 5% in 1994 to 11% in 2002. But conventional till has
increased 4% over the same period, despite higher fuel costs for field opera-
tions. Evidently, the new investment in no-till equipment, $ 70 000 to $ 150 000,
coupled with low commodity prices is a significant obstacle to changing.
14.4.3
Cover Crops
Cover crops can offset the erosion effect from collecting residues by restoring
the surface cover, reducing wind and water erosion. Cover crops can also im-
prove soil quality: their additional biomass builds SOM, especially from their
roots. Cover crops also choke out weeds and may retain N in its root system
over the winter, thereby reducing N2O emissions and nitrate leaching [45].
Cover crops require a higher level of management. Important factors for con-
sideration include:
· Selecting appropriate cover crops to fit in the rotation to avoid allelopathic ef-
fects, inhibition of growth in one species of plants by chemicals produced by
other species
· Planning to ensure enough growth occurs to provide the above benefits.
Broadcast seeding may be used for the current crop to give it a timely start,
and care must be taken not to hinder its growth during that crop’s harvest
· Cover crop harvesting or killing growth needs to consider soil moisture condi-
tions. It can deplete needed soil moisture in a dry spring, or if left to grow
longer in a wet spring, deplete excess soil moisture
· Growth must be stopped for some cover crops like rye before it goes to seed
or it will interfere with future cash crops.
Establishing local, credible test plots to investigate the impact of residue re-
moval for various cropping practices is important to provide information to both
the grower and the processor.
14.5
Innovative Methods for Collection, Storage and Transport
Most previous studies focus on collecting and baling dry material after the grain
harvest. Grain is the cash crop and is at risk until safely collected and stored.
Collecting, storing, and transporting bulky straw and stover is secondary. Addi-
tional field operations add cost and increase risk.
Straw is normally dry enough to bale, but corn stover must dry from 30–50%
moisture to less than 20%. Feedstock drying and densification methods can re-
duce collection delays and increase density. These approaches may be appropri-
ate when a dry, compacted material is desired for co-firing or for thermo-chemi-
cal processes. These operations increase cost to $ 50 dt–1 or more, however [46].
Densification inhibits wet processing and pellets need to be “reconstituted” by
soaking in water to shorten digestion time for hydrolysis.
One-pass harvest of corn grain and stover, wet storage, and transport to the
processor seem to be advantageous where wet feedstock is acceptable, as already
shown in Tables 14.4 and 14.5. Custom bailing and transporting bales from the
field to a processing point within a 50 km has a relative return of $ 41–$ 54 ha–1
depending on the production. One-pass harvest and transport to 3–25 km collec-
tion centers for storage and then supplying the processing plant from these
sites is estimated to increase margins to $ 59–$119 ha–1, a 44 to 118% im-
provement. The relative difference between custom baling and one-pass harvest
with wet storage and transport for the farmer is shown in Table 14.9.
These cost comparisons are relative. Other costs like shrinkage or change in
properties of the feedstock in storage, storage investment and operation, and
relative quality of final feedstock processed are not included. There is a large
variation in these factors depending on the local situation. These are addressed
in the following sub-sections.
14.5.1
Collection
Collection choices are divided into two general categories, baling the residue fol-
lowing harvest or one-pass collection of both grain and residue.
Table 14.9 Farmers net margin, stover sale comparison.
Case 1 2 3
Stover yield, dt ha–1 6.9 9.0 10.6

A. Custom bale net margin, $ ha–1 $ 41 $ 49 $ 54
B. One-pass, bulk, net margin, $ ha–1 $ 59 $ 93 $ 119
% Improvement 44% 92% 118%
14.5.1.1 Baling
Many studies have been made of baling biomass on a large scale. For straw, bal-
ing is an option. Cereal grains, like winter wheat, are grown in dryer areas and
mature earlier than corn, enabling a longer harvest window. When cereal grain
is ready to harvest, straw moisture is usually suitable for collection. In contrast,
stover is typically too high in moisture, 30 to 50%. It must remain in the field
to dry and be collected later. To speed drying, some flail the stalk. Raking is
then required before baling, adding more cost, increasing the foreign matter,
especially dirt, in the bales and compacting the soil. A wet harvest season can
prevent its collection entirely, because of wet residue [13].
For industrial feedstock, baling only adds cost, $ 15 dt–1 or more at the field
[30]. Bales also add cost at the processor for additional equipment and disposal
of twine, wrap and foreign contamination [38].
14.5.1.2 One-pass Collection

Achieving the target of $ 33 dt–1 with adequate farmer margins will probably
exclude baling. This can be achieved with many variations. There are more
choices for separation, i.e. ear and stalk, grain-stalk-cob, remove leaves and
husk, or just leaves. Prototypes for one-pass harvest of straw and stover are un-
der development, adapting existing equipment and examining new designs.
One-pass systems have been compared with multi-pass alternatives for
wheat [47] and stover [36, 48–50]. Results indicate one-pass harvest can deliver
$ 33 dt–1 target price with $ 70 ha–1 or more margin to the farmer when the
following conditions are met:
· 9.0 dt ha–1 (175 bu acre–1) or more yield when 2.2 dt ha–1 or less cover main-
tains soil quality
· 50 km or less collection radius
· bulk delivery
· 400 hours (700 ha) or more utilization of stover collection equipment.
Table 14.10 summarizes the harvest cost as a function of harvester utilization

and yield, adjusted for 4th Q 2004 fuel pricing [51].
The key stover collection equipment issue resolves around one question:
“What does customer need?” For example:
· Corn with ear and stalk? Or grain and stover?
· Number of harvester take-offs: 1, 2 or 3?
– Component separation? Which parts?
– Size reduction? How much?
– Other pre-processing?
· On-farm storage or at a collection site?
Local requirements will vary. In areas where soybeans and corn rotate, the needs
are different from wheat and corn or other crop rotations. For wheat, stripper
headers work well now, leaving a standing stalk that is clean and readily harvested.
Table 14.10 Collection cost for forage harvester.
Annual collection Harvest cost per ha Harvest price per dt
ha Hours Fixed Direct Total cost Case 4.1, Case 4.2, Case 4.3,
1.8 ha h–1 cost cost +15% 7 dt ha–1 9 t ha–1 11 dt ha–1
250 140 $ 70 $ 28 $ 113 $ 9.60 $ 7.03 $ 5.86

500 281 $ 35 $ 28 $ 72 $ 6.20 $ 4.52 $ 3.76
1000 561 $ 18 $ 28 $ 52 $ 4.50 $ 3.26 $ 2.71
1500 842 $ 12 $ 28 $ 45 $ 3.90 $ 2.84 $ 2.36
Some areas may be satisfied with one take off, separating the grain and other
components of the field. Others may desire to use existing equipment, but at
the same speed, separating the grain from the stover in the field. One design of-
fers three field separation possibilities as cobs offer commercial value as a car-
rier for herbicides and other chemicals, cat litter, and metal-polishing applica-
tions.
Development cost for a new design is $ 2 million or more for prototypes, with
millions more required to bring to market. The definition of the customer’s
needs, regional differences, market size uncertainty and the cyclical economic
performance of this industry makes significant up-front development beyond
paper studies difficult to justify until the market for machine sales exist. With-
out subsidizing development, a “chicken and egg” dilemma exists.
14.5.2
Storage
Maintaining quality of feedstock in storage is a major concern. Stable storage is

possible only when the material is dry, less than 20% moisture, or wet, greater
than 60% moisture. In the US most industrial experience is with dry storage,
mostly bales, and the associated need to keep dry and protect from fire. Pest in-
festations have not been noted with corn stover, but some report straw is more
susceptible.
Wet storage of large quantities is also familiar for silage, fermented forage
crops. Ensiling green sorghum and corn plants has been practiced for years as
a means of preserving feedstock for ruminant animals. These crops are chopped
and stored in bags, bunkers, or silos at 65% moisture. The storage life can ex-
tend to years, with losses less than 3%. In addition, in many tropical areas “Rit-
ter” wet storage is practiced to supply pulp mills – 250 000 to 500 000 dt piles
are built via circulating liquor, saturating the feedstock to 80% moisture. Resid-
ual sugar results in fermentation that drops the pH below 4, halting microbial
activity. Less area is required; fire is eliminated when stored above 60% mois-
ture. A comparison of dry bale and wet bulk storage is shown in Table 14.11.
Table 14.11 Dry and wet storage comparison [36].
Property Dry (bales) Wet storage
Dry density, lb ft–3 7 to 10 12 to 14

Storage area 10 ´ 1´
Storage loss > 10% < 5%
Foreign matter and soil nutrients High Low
Non-volatile solubles removal Process residue Storage liquor
Weather risk Rain Extreme cold
Fire hazard High None
Investment Low to high Medium to high
Storage quantity Small, mostly farm use Large, bagasse for pulp
14.5.2.1 Density
The dry density of bales is about half that of wet stored material, ranging be-
tween 112 kg m3 for round bales to 160 kg m–3 for square bales. Wet storage
density depends on the stack height, with 40 meters achieving 200 kg m–3 aver-
age pile density [52].
14.5.2.2 Storage Area

Square bales require about ten times the wet storage space because of bale
stacking limits, access corridors, and a measure of fire protection. The total area
required for 1 million dry tons is approximately 500 acres for square bales. With
wet storage, a 333 000 dt pile requires 15 acres. For 1 million dt storage, just 3
piles or about 50 acres is needed. The equivalent land rent depends on the local
situation, adding zero to $ 2 or more per dt.
14.5.2.3 Storage Loss

Bales are adversely affected by wet weather and without shelter can decompose and
break apart. For 6'' dia ´ 5' bales, 30% of the mass is in the outer 4 inches and 25%
weight loss can easily occur in one season. Stored inside barns, both round and
square bales had 14% weight loss over 10 months in eastern Canada [53].
For wet storage, the major losses are the 5 to 8% solubles removed during
storage. Typical cellulose and hemicellulose losses reported by the pulp and pa-
per industry are 1 to 3% [54]. Surface loss depends on the total surface exposed
relative to the stored tons. The higher the pile, the smaller the exposed surface
and the surface loss. While there may be aesthetic or zoning limits, wet storage
piles height is limited by design considerations for pump head and recovery,
varying between 30 and 40 m.
The lignin, pentosans, and holocellulose increased in proportion to decreases
of the solubles in water, alcohol–benzene, and 1% caustic soda. The absolute
quantity of lignin, pentosans, and holocellulose remained constant during the
Fig. 14.7 Solubles removal in wet storage.
storage period. The loss in solubles during storage is shown in Fig. 14.7, declin-
ing from 10 to 3% within weeks, and fresh bagasse having 7% less solubles for
the five trials.
The pentosans and holocellulose, which consists of the alpha-cellulose plus the
hemicelluloses, continue to increase over a longer period (Figs. 14.8 and 14.9).
Fig. 14.8 Pentosan change in wet storage.
Fig. 14.9 Holocellulose change in wet storage.

14.5.2.4 Foreign Matter and Solubles

Bales contain soluble soil nutrients along with foreign material that can be dele-
terious during storage and processing. Wet storage has proven to remove dirt,
foreign matter and solubles over time. Removing the nutrients during storage,
returning them to the fields is much preferable to processing the bales and dis-
posing of the process ash. Fewer solubles in the wet feedstock, with the abso-
lute values of holocellulose and pentosans unchanged, increases the plant capac-
ity up to the distillation step: 7% removal opens up 7% more pretreatment,
hydrolysis, and fermentation capacity.
14.5.2.5 Storage Investment

Storage investment cost must be investigated and evaluated as part of the sup-
ply chain – from collection through the disposition cost of the residue and stor-
age liquors. Investment can vary widely for both types of storage. An uncovered
stack of round bales on the ground requires negligible investment. Sheltered
bale storage investment can be high, depending on the degree of automation.
Wet storage investment may consist of a conveyor for building the pile and a
water spray system to raise the pile moisture to above 60%. A more elaborate
system is shown in Fig. 14.5 can add $ 1 million or more.
Bale-storage systems were recently estimated for rice straw [55]. Short term,
tarps were favored at a cost of $ 6 to $ 11 dt–1. Longer term, pole barns are fa-
vored, costing 50% less with the upfront investment. For a 1 million dt plant,
the pole barn investment would range between $ 6 and $ 11 million.
At the plant, the bale unloading, interim storage and handling system de-
signed for NREL’s model has a $ 12.9 million capital cost, $ 2.94 dt–1. The oper-
ating cost is $ 2.62 dt–1 based on twelve operators, twenty-four hours a day,
seven days a week, to handle the bales, adding a total of $ 5.56 dt–1 to the feed-
stock cost [38]. Truck or rail car unloading of wet or dry bulk material can be
automatic, with minimum additional plant labor.
14.5.3
Transport
Transport is divided into two components:

· harvest period for the annual crop, typically 20 to 40 days
· biorefinery supply requirements: 2 000 to 3 000 dt day–1.
During harvest, existing resources are stretched just collecting and transporting
the grain in the field while keeping the combines operating continuously. In-
creasing the resources to accommodate up to 2.5 to 3 times that quantity from
the filed is a serious increase.
14.5.3.1 Harvest Transport

Transportation distance from the field to a storage site during harvest needs to
be short for truck requirements to remain manageable. Leaving the feedstock at
the edge of the field or at multiple collection sites 20 to 30 km in diameter can
reduce the trucking requirements, potentially reduce cost by better utilization of
equipment, and reduce traffic congestion.
Delivering 1 million dt to one site during harvest cause much congestion. For
a 20-h delivery day, 100 trucks per hour rumble past, in and out of the location.
Sugar cane harvest approaches this, with deliveries to some mill sites reaching
22 000 dt day–1, 1 000 or more truck deliveries.
In areas with high yields, three collection sites with a 25 km radius (200 000
ha) can supply 1.5 million dt assuming 30% of the land is harvested. The aver-
age round trip is 35 km for each site. Increasing to four sites further reduces
congestion during harvest and shortens trips by 35%, to 23 km.
The simplest and most efficient logistics occur when the ear and stover are trans-
ported together, Weight and volume limits are met using standard trailer dimen-
sions with the resulting bulk density. Baled or bulk stover loads generally reach a
maximum volume before reaching the weight limit. Milling the stover or straw
increases the bulk density, more closely approaching the maximum weight.
14.5.3.2 Biorefinery Supply

Because an annual feedstock supply, 1 million dt, is not expected to be stored
on the biorefinery sites, reliable supply is required by the biorefinery through-
out the year. Processors have indicated a minimum one to two week supply is
desired on site, with deliveries made daily, 5 to 6 days per week. Truck transpor-
tation is most common. Other transportation modes previously considered in-
clude rail, pipelines, and dirigibles.
The viable transport options are truck and rail, and wet or dry feedstock.
Truck transportation is most common. Rail is most efficient when prompt ser-
vice is available. Pipelines are possible but, because of the high absorption of
water by the fiber (80%), slurries of more than 96% water are desired – huge
volumes that may require a 2nd pipeline for water return [56].
Truck Transport Truck transport has long been favored for both wet and dry ma-
terials for short distances. Dry material is bulky and flammable. Flaming trailers
in transit are a serious hazard, especially in populated areas. Depending on bio-
mass density and local regulations, the dimensional limits for road transport
may be exceeded before the weight limit is reached. Because trucking cost is based
on distance, $ 1.00 to $ 1.50 km–1, less than a full weight load results in higher
cost. For example, round bale transport using load-and-go wagons was just 9 dt
per trailer load, a transport cost twice that of a full load, normally 18.2 tons.
Wet material from storage can be passed through a dewatering press and re-
duced to 50% moisture before transporting. It is perishable with this moisture
content and requires timely delivery. Full truck loads are readily achieved, but
14.6 Establishing Feedstock Supply 339
50% water results in the same pay load as the load-and-go wagon. The water
volume requires management.
Rail Transport Rail shipments for short distances have become feasible as a result
of improved practices, especially when shipments remain on the same line of a
regional railroad. With multiple collection points along the rail line, the collection
area for the biorefinery can be more than doubled. Moving a rail car 100 to 300
miles is $ 150 to $ 250 with little regard to weight [57]. The cost of rail transit
was estimated to be $ 3.00 to $ 5.00 dt–1 for transporting feedstock several hundred
miles. Modeling shows conversion costs drop 30% for a 4 million dt plant.
Rail shipment of dry material has the associated fire liability issue. The high-
er weight limits make wet feedstock shipments possible at lower cost. Maxi-
mum load per car is usually 114 tonnes, 91 tonnes net, to accommodate grain
shipments.
Truck traffic increases with increased plant size while rail car shipments are
more manageable. Assuming 50 car unit trains traveling at 50 km h–1 (off the
main line), approximately 1.5 min is required for the train to pass a road cross-
ing. In contrast, when the truck delivery is limited to just 10 hours each day
and 6 days per week, the truck traffic just from feedstock delivery is 50 to 60
trucks in and out every hour for a 1 million dt plant. Storing 1/3 of the feedstock
on site reduces the value accordingly, but the disruption is still substantial.
Trucks are assumed to carry maximum loads (Table 14.12).
14.6
Establishing Feedstock Supply
At the present time in the US, the dairy herd feed lots in Jerome County Idaho
purchase about 1 million dt of straw from the surrounding irrigated barley and
wheat fields with yields often exceeding 10 dt grain ha–1. Grower participation
and infrastructure evolved over years. It is 20 times the largest corn stover col-
lection effort in Harlan, IA. Establishing a reliable feedstock supply system re-
quires substantial investment, planning and outreach to the growers and other
stakeholders.
Table 14.12 Plant feedstock requirements. Rail and truck traf-

fic volume, units day–1.
Plant, dt (000) Units day–1 (60 h week–1 truck delivery)
Mode Moisture 1000 2000 4000 6000
Rail cars Up to 70% 70 143 275 418

Trucks 50% 308 608 1230 1850
Trucks 15% 240 396 960 1920
14.6.1
Infrastructure
In addition to the biorefinery investment, farmers and biomass suppliers will

be faced with investment decisions in the range of $ 70 million or more for
equipment, excluding feedstock storage facilities. The storage facilities could
add on more millions.
14.6.1.1 Infrastructure Investment

For the participating grower, new cropping practices may require investment in
seed drills for planting, modifying existing equipment for less tillage and possi-
bly narrow rows, managing cover crops, all exclusive of harvesting. Based on
CTIC tillage survey, at least 50% of farmers require new tillage equipment. The
new equipment can range up to $ 150 000 per farmer. If half of conventional till
farmers make a collection commitment, their investment exceeds $ 50 million.
Assuming a net margin of $ 10 dt–1, the grower needs to sell 15 000 dt for a
simple pay back, the equivalent of 1500 high-yielding ha. Even half this amount
is a significant hurdle for the grower assuming they offer 200 to 400 ha each,
25 to 50% of their crop, the payback time on the new equipment is 4 to 8 years.
Other benefits, such as improved yields, may shorten this time [41].
In addition another $ 20 million investment is required for approximately 100
collection units needed for the annual campaign based on Section 14.5.2 – esti-
mating a unit and the peripheral cost is equivalent to silage harvesters,
$ 200 000 each, operating 40 days, averaging 1.8 ha h–1 and 14 hours per day.
Collection sites may require additional investment in land, material handling
equipment and year-round facilities to transfer the material to the biorefinery.
Trucking during the harvest nearly triples to remove the stover along with the
grain for one-pass harvest.
Because of feedstock transportation demands, additional investment may be
required to improve roads and bridges, rail track, and crossing to meet the in-
creased traffic – an additional 2.5 million km of truck traffic from field to the
three collection sites, with more for delivery to the biorefinery, depending on
the use of rail.
14.6.1.2 Organization Infrastructure

For a corn processor, procuring grain is as simple as a phone call to purchase
feedstock. Standards for grain quality are in place. Delivery schedules are rou-
tine to establish. Payment is usually made to local grain elevators that in turn
pay the growers. If needed, grain is readily obtained from more distant suppli-
ers. None of this is in place for biomass.
To supply 1 million dt, commitments from 1000 to 2000 farmers totaling
200 000 ha is required to insure adequate feedstock. Enlisting them is a time-
consuming task. One likely business model is to mimic the “grain elevator”
14.7 Perspectives and Outlook 341
model. Local growers already deal with them for their grain business [14]. Ac-
counts are in place, and their managers are skilled in logistics issues. Feedstock
from other areas could also be sourced.
14.7
Perspectives and Outlook
Industry segments are beginning to move toward carbohydrate feedstocks as al-

ternatives to fossil fuels. With improved biotech tools their processing cost are
becoming competitive with present methods of chemical synthesis. Price insta-
bility and higher prices of petroleum and natural gas, global warming policies,
and higher liability insurance costs are accelerating interest in a move to more
sustainable feedstocks.
Global warming is driving policies like the Kyoto agreement, adding econom-
ic incentives in the form of carbon credits. Liability insurance companies are
considering the potential for claims from policy holder emissions as they set
rates for corporate coverage – all positive activities for the rural economy.
Feedstock supply must be given significantly more attention if it is to serve as
a sustainable platform for this industrial shift to have significance. Sourcing the
feedstock quantity has a high risk without improved methods for delivery to the
processor from the field. There are many areas for economic and environmental
improvement before it enters the processing plant. The target price, $ 33 dt–1,
seems achievable with one-pass harvest and bulk delivery of clean feedstock.
Farmers are the key determinate for supplying the feedstock. Improved agro-
nomic systems with more crop removal can also maintain soil quality. A busi-
ness model with an option for the grower to participate in the value chain is
important, because the bulky biomass is inherently local. Partnering with the
processor insures a win–win for both.
Short term, two to three years, dry feedstock is most likely to be chosen to
supply biorefineries. Straw collection will be favored, because of to reduced sup-
ply risk compared to stover.
Mid term, four to seven years, the economic and environmental advantages of
one-pass harvest and wet storage will be validated. Chemical, biochemical, and
microbial treatments will emerge, improving the feedstock “processability” from
the collection centers before delivery to biorefineries.
Long term, 2014 and beyond, other feedstocks – especially energy crops
grown on marginal croplands – will emerge as the processing technology is
more proven. Plant science will enhance the feedstock value and co-products
from the biorefinery will become more significant in their economic impact on
the product mix.
References
1 J. DiPardo, Outlook for Biomass Ethanol Number: 01-16075, 2001 ASAE Annual
Production and Demand, DOE EIA, International Meeting, http://www.mea-
2002,http://www.eia.doe.gov/oiaf/analy- dowoodindustries.com/asae_paper.htm
sispaper/biomass.html 12 D. Lengel, Ag-Fiber Dot Gone: A Litany
2 The Vision for Bioenergy and Biobased of Failure. Panel World July 2001: 8–9,
Products in the United States, October 34–38.
2002, http://www.bioproducts-bioenergy.- 13 D. Glassner, David, James Hettenhaus,
gov/pdfs/BioVision_03_Web.pdf Tom Schechinger, Corn Stover Collection
3 USDA National Agriculture Statistics Project, Bioenergy ’98, Expanding Bioe-
Service nergy Partnerships, Madison, WI, 1998
4 S.W. Fitzpatrick, Commercialization of 14 J. Hettenhaus, T. Schechinger, Corn
the Biofine technology for levulinic acid Stover Harvest: Grower, Custom Opera-
production from paper sludge, DOE/CE/ tor and Processor Issues and Answers,
41178, April 2002 www.osti.gov/gpo/serv- Oak Ridge National Laboratory Contract
lets/purl/771246-zVDe5D/native/ 4500008274, 1999.
5 M. E. Walsh, R. L. Perlack, A. Turhollow, 15 J. Hettenhaus, T. Schechinger, Improved
D. de la Torre Ugarte, D. A. Becker, R. L. Corn Stover Harvest & Collection Meth-
Graham, S. E. Slinsky, and D. E. Ray, Bio- ods, 3rd Annual AgFiber Technology
mass Feedstock Availability in the Unit- Showcase, Memphis, TN, October 2000
ed States: 1999 State Level Analysis, Jan- 16 E. Lathrop, C. Elbert and B. Treadway,
uary, 2000 Ind. Eng. Chem. 26:594–598, 1934.
6 Biobased Industrial Products: Priorities 17 H. Hay and Lathrop, E. C. Storage of
for Research and Commercialization, Crop Fibers and Preservation of their
National Research Council, National Properties. TAPPI Technical Association
Academy Press, Washington, D.C. 1999 Papers, Series XXIV. P 412–418, 1941.
7 A. Aden, A., M. Ruth, K. Ibsen, J. Je- 18 J. Salaber and Maza. Ritter Biological
chura, K. Neeves, J. Sheehan, B. Wallace, Treatment Process for Bagasse Bulk Stor-
L. Montague, A. Slayton, J. Lukas, Ligno- age. TAPPI Non-wood Plant Fiber Pulp-
cellulosic Biomass to Ethanol Process ing Progress Report, No 2, October
Design and Economics Utilizing Co-Cur- 1971.
rent Dilute Acid Prehydrolysis and Enzy- 19 J. Moebius, The Storage and Preserva-
matic Hydrolysis for Corn Stover, Na- tion of Bagasse in Bulk Form, Without
tional Renewable Energy Laboratory, Baling. Pulp and Paper Development in
NREL/TP-510-324328, June 2002. Africa and Near East, United Nations,
8 T. Klopfenstein (1996) Crop residue as N.Y. Volume II, 1966.
an animal feed. p. 315–340. In: P. Unger 20 J. Bruin, Gonin, C., McMaster, L., and
(ed.), Managing agricultural residues. Morgan, R., Wet Bulk Storage of Ba-
Lewis Pub., Boca Raton, FL gasse. Proc. Intern, Soc. of Sugar Cane
9 Cooperative Research Centre for Sustain- Tech. (ISSCT) XV Congress: 1793–1820,
able Rice Production Annual Report, 1974.
2004 21 L. Mann, Tolbert, V. and Cushman, J.
10 J. Atchison, Review of Progress with Ba- (2002) Potential environmental effects of
gasse for Use In Industry (A review of corn (Zea mays L.) stover removal with
progress in purchasing, handling, stor- emphasis on soil organic matter and ero-
age and preservation of bagasse). J. E. sion. Agriculture, Ecosystems and Envi-
Proc. Intern. Soc. Sugar Cane Technolo- ronment 89:149–166.
gists 14:1202–1217 (1971) 22 M. Stumborg, L. Townley-Smith, and E.
11 K. E. Gorzell, Finding an Economic and Coxworth (1995) Crop residue export-
Environmental Balance to the Technol- issues and concerns. Innovations in
ogy of Producing Building Materials Straw Utilization Symp, Oct 23–25.
from Agricultural Crop Residue, Paper 1995, Winnipeg, Manitoba. Agriculture
References 343
and Agri-Food Canada Research, Swift newable Energy Laboratory, NREL/ACO-

Current, Sask. 1-31051-01, January 2002.
23 W. Wilhelm, and Cushman, J. (2003) Im- 35 Roadmap for Agriculture Biomass Feed-
plications of Using Corn Stalks as A Bio- stock Supply in the United States, No-
fuel Source: A Joint ARS and Doe Pro- vember, 2003, Document Number: DOE/
ject [abstract]. Eos. Trans. Agu. 84(46), NE-ID-11129 http://devafdc.nrel.gov/
Fall Meet. SUPPL., Abstract B51b-05. pdfs/8245.pdf
24 J. Doran, W. Wilhelm and J. Power, Crop 36 J. Atchison, J. Hettenhaus, Innovative
residue removal and soil productivity Methods for Corn Stover Collecting,
with no-till corn, sorghum and soybean, Handling, Storing and Transporting, Na-
Soil Sci. Soc. of Am. J., 48, 3, 1984. tional Renewable Energy Laboratory Sub-
25 D. Linden, C. Clapp and R. Dowdy, Long contract No. ACO-1-31042-01, March
term corn grain and stover yields as a 2003, www.afdc.doe.gov/pdfs/7241.pdf
function of tillage and residue removal 37 Levelton Engineering Ltd. and (S&T)2
in east central Minnesota. Soil Till. Res. Consultants Inc., Assessment of Net
56, 167–174, 2000. Emissions of Greenhouse Gases From
26 V. Beri, Sidhu, B. S., Bahl, G. S. and Ethanol-Blended Gasoline in Canada:
Bhat, A. K. (1995) Nitrogen and phos- Lignocellulosic Feedstocks, 2000.
phorus transformations as affected by 38 Harris Group Inc., Process Design and
crop residue management practices and Cost Estimate of Critical Equipment in
their influence on crop yield. Soil Use the Biomass to Ethanol Process, Report
and Management 11: 51–54. No. 99-10600/13, Baled Feedstock Han-
27 P. Unger, Ed., Managing Agricultural dling System, Revision 1w, Subcontract
Residues, 1994. CRC Press No. aco-9-29067-0, October 11, 2000
28 K. Paustian, J. Brenner, K. Killian, J. Ci- 39 B. Hames, S. Thomas, A. Slitter, C. Roth
pra, S. Williams, E. T. Elliott, M. D. Eve, and D. Templeton, Rapid Biomass Anal-
T. Kautza, and G. Bluhm (2002) State ysis: New Tools for Compositional Analy-
level analyses of C sequestration in agri- sis of Corn Stover Feedstocks and Pro-
cultural soils. Pp. 193–204 in: J. M. Kim- cess from Ethanol Production, 24th Bio-
ble, R. Lal, and R. F. Follett (eds.), Agri- tech Symp for Fuels and Chemicals,
culture Practices and Policies for Carbon 2002.
Sequestration in Soil. Lewis Pub, CRC 40 W. Gale, C. Gambardella, and T. Bailey
Press, Boca Raton, Fl. (2000) Surface residue- and root-derived
29 Soil quality web site: http://csltest.ait.ias- carbon in stable and unstable aggregates.
tate.edu/SoilQualityWebsite/home.htm Soil Sci Soc Am J 64(1):196–201.
30 Iowa Farm Custom Rate Survey, 2004, 41 D. Reicosky, Kemper, W. D., Langdale,
http://www.extension.iastate.edu/Publica- G. W., Douglas, C. L., Jr. and Rasmussen,
tions/FM1698.pdf P. E. (1995) Soil organic matter changes
31 Agricultural carbon sequestration, Chica- resulting from tillage and biomass pro-
go Climate Exchange, 2004, duction. J. Soil and Water Conservation
http://www.chicagoclimatex.com May–June: 253–261.
32 National Commission on Energy Policy, 42 R. Lal, J. Kimble, R. Follett and C. Cole,
“Ending the Energy Stalemate: A Biparti- The potential of US Cropland to Seques-
san Strategy to Meet America’s Energy ter Carbon and Mitigate the Greenhouse
Challenges,” 2004, Effect, 1998, Sleeping Bear Press.
http://www.energycommission.org 43 R. Nelson (2002) Resource assessment
33 CAST 1992. Preparing US Agriculture and removal analysis for corn stover and
for global climate change. Council for wheat straw in the Eastern and Midwes-
Agricultural Science and Technology, tern United States – rainfall and wind-
Task Force Report 199. induced soil erosion methodology. Bio-
34 J. Hettenhaus, R. Wooley, J. Ashworth, mass and Bioenergy 22: 349–363.
Sugar Platform Colloquies National Re-
44 Conservation Technology Information tom Rate Guide–Machinery Cost Esti-

Center, Purdue University, 2004, mates, 2003, http://www.ace.uiuc.edu/
www.ctic.purdue.edu fbfm/farmmgmt.htm
45 Managing Cover Crops Profitably, 2nd 52 J. Moebius, The Storage and Preserva-
ed., 1998, Sustainable Agriculture Net- tion of Bagasse in Bulk Form, Without
work. II. Series, USDA. www.sare.org/ Baling. Pulp and Paper Development in
publications/covercrops/covercrops.pdf Africa and the Near East, United Na-
46 S. Sokhansanj and A. Turhollow, Bio- tions, Volume II, 1966.
mass Densification – Operations and 53 J. Billy, Corn stover in eastern Canada as
Costs, BioEnergy 2002 Proceedings, Uni- raw material for production of ethanol,
versity of ID, Moscow, ID Natural Resources Canada and Georges
47 PAMI, Prairie Agriculture Machinery In- Lê of “Resources naturelles Québec”
stitute, Modeling and Comparing Whole 2001.
Crop Harvest Systems, Research Up- 54 J. Salaber, and Maza. Ritter Biological
Date 739, 1998, Treatment Process for Bagasse Bulk Stor-
http://www.pami.ca/PDFs/Pami739.pdf age. TAPPI Non-wood Plant Fiber Pulp-
48 R. Quick, and T. J. Tuetken (2001) Har- ing Progress Report, No 2, October
vest, handling, and densification for 1971.
commercial processing of biomass feed- 55 W. Huisman, B. M. Jenkins and M. D.
stock. DOE/EE/10595-4. Iowa State Univ Summers, Cost Evaluation of Bale Stor-
49 A. Turhollow, M. Downing, J. Butler, age Systems for Rice Straw, BioEnergy
The cost of silage harvest and transport 2002, www.bionergy2002.org
systems for herbaceous crops, Proc., 56 A. Kumar, J. Cameron, P. Flynn, Pipe-
BIOENERGY ’96, September 15–20, line Transport of Biomass, 25th sympo-
1996, http://bioenergy.ornl.gov/papers/ sium on Biotechnology for fuels and
bioen96/turhllw.html Chemicals, 2003.
50 K. Shinners, P. Savoie, Single pass 57 Roof, R., Farmrail Systems, Inc., Perso-
Whole-Plant Corn Harvesting for Bio- nal Communications, March 2004.
mass, 25th Symp. Fuels and Chem, 2003
51 G. Schnitkey, D. Latz and J. Siemens, Il-
linois Farm Business Management Cus-
345
15
The Corn Wet Milling and Corn Dry Milling Industry –
A Base for Biorefinery Technology Developments
Donald L. Johnson
15.1
Introduction
15.1.1
Corn – Wet and Dry Milling – Existing Biorefineries
Corn dry milling has existed for hundreds of years – maize was ground with
stones into flour for food consumption by early American populations. Modern
corn refining, however, began in the mid 1800s when Thomas Kingsford started
up his corn refining plant in Oswego, New York [1].
Corn refining is distinguished from corn milling in that the refining process
separates the corn grain into its components, starch, fiber, protein, and oil, and
further processes the starch into a substantial number of products. Corn “wet
milling” is the aqueous slurry process by which the corn grain is separated into
its component parts. Corn “dry milling”, in contrast, physically alters moist corn
granules into composite products such as flakes, grits, meal, flour, and hominy
feed, although some operations do separate germ and recover oil. The dry
milling process produces food and industrial products based on flakes, meal,
and flour, and also fermentation ethanol.
Specialty products such as white corn flour for food uses and yellow corn
flour-based adhesives, produced by what are termed the flour millers, are a
small part, less than ten percent, of the industrial market [2] and will not be dis-
cussed further here. Those interested in the topic are referred to texts available
on the subject [3].
Corn refining has been the fastest growing market for US agriculture over
the past 25 years. This is attributed to the burgeoning high-fructose corn sweet-
ener market early in the period, followed by rapid growth in fuel alcohol, and,
more recently, by fermentation products. Corn refiners now use over 14% of
the annual corn crop [4], exceeding 39 million metric tons (MT) of corn refined
each year.
ISBN: 3-527-31027-4
346 15 The Corn Wet Milling and Corn Dry Milling Industry
15.2
The Corn Refinery
15.2.1
Wet Mill Refinery
A corn refinery can be described succinctly in five process steps, but is substan-
tially more complicated in operation. Corn grain that has been received by truck
and/or rail is inspected for moisture content and debris, passed through clean-
ers to remove foreign material and steeped in large tanks. Steeped corn is
coarsely ground to loosen and free the low-density germ which is removed by
centrifugation from the starch and fiber slurry. A second grind releases starch
and gluten from the fibrous hulls, the fiber is further washed to remove resid-
ual starch and sent to a feedhouse. The low-density gluten is removed by centri-
fugation and a battery of cyclone cleaners washes residual protein from the
starch. The 99.5+ percent pure “refined” starch is ready for further processing.
Germ, which was removed early in the process, is subjected to further proces-
sing to remove the oil, which can be refined or sold as crude corn oil. The spent
germ is combined with the fiber from the second grind, dried, and sold as corn
gluten feed. The gluten is dried and sold as a 60% protein-feed supplement.
“Wet milling” is so named because the corn is steeped in slightly acidic warm
water and is processed as an aqueous slurry until dried or solubilized in down-
stream processing.
15.2.2
Dry Mill Refinery
A dry mill, or “mash,” ethanol plant, is a simpler process. Corn is received and
cleaned as in the wet mill, but the clean corn is tempered with steam, ground,
and wetted to a free flowing “mash,” from which the name is derived. The
mash is superheated in a continuous cooker, to which acid and/or enzymes are
added to solubilize the starch in the corn meal. Additional water is added to ad-
just the solids and temperature, and saccharifying enzymes are added. This
mash is added to a fermenter, adjusted to appropriate solids and temperature,
and yeasts are added to convert the sugars to alcohol. When the fermentation is
complete, alcohol is removed by distillation and the residual “still bottoms” are
recovered for animal feed. In such a plant, the only two products are ethanol
and distillers dried grains and solubles (DDGS). Some plants now separate the
germ before “mashing” – the added cost is justified by the value of the oil recov-
ered.
A wet mill corn refinery and a dry mill ethanol plant are compared in a pro-
cess flow schematic diagram of Fig. 15.1. As might be expected, the capital in-
vestment for a mash ethanol plant is significantly lower than that of a corn re-
finery. The operating profits from the multitude of products normally oversha-
dow the investment cost differences, however; this will be discussed later.
15.2 The Corn Refinery 347
Fig. 15.1 Process flow diagrams comparing wet to dry milling.
15.2.3
Waste Water Treatment
Another common feature of wet and dry mills is, as indicated in Fig. 15.1, the
waste water treatment required. Both operations, but especially wet milling, are
water-intensive processes. A corn refinery may require two hundred to two hun-
dred fifty gallons per bushel of corn (a bushel is defined as 56 pounds (25.45
kilograms) of corn at 15.5 percent moisture) processed, most of it as process
water that is vaporized, condensed, heated, and cooled needing very little treat-
ment before being returned to its source. But five to ten percent contains organ-
ic material which must be treated before discharge into the environment. Meet-
ing local, state and federal regulations for liquid wastes may add more than
10% to the total plant investment [5]. The corn milling industry and its equip-
ment suppliers work diligently to minimize water usage.
15.3
The Modern Corn Refinery
15.3.1
Background and Definition
The modern corn refinery is a model for developing future biorefineries. Corn
refining has been compared with petroleum refining wherein flexibility of
downstream processing is used to maximize profitability [6]. Thus the focus of
this chapter is on wet milling, with some reference for comparative purposes to
mash ethanol plants.
Corn refining produces several products in large volume. As already men-
tioned, wet milling refines the corn into four components, starch (carbohy-
drate), gluten (protein), hull (fiber), and germ (corn oil). Moreover, the carbohy-
drate fraction, which is nearly 70 percent of the corn composition, is further
processed and refined into products such a native starch, modified starch, dex-
trose, high fructose corn sweetener (HFCS), ethanol, glucose syrup, and special
hydrolyzates.
15.3.2
Technologies and Products
In the United States, the largest producer and miller of corn in the world, plant
capacities are rated in bushels per day. Refineries in the US range in grind ca-
pacity from 55 000 bushels/day (14 000 MT/day) to more than 550 000 bushels/
day (14 000 MT/day) [6]. A metric ton of corn grain yields, on average, 684 kg
starch, 237 kg corn gluten feed (CGF), 45 kg gluten meal (60% protein) and
34 kg corn oil in a typical wet milling operation. Thus the largest corn refinery
produces more than 5 million metric tons of pure carbohydrate per year to be
sold as such and further processed into a myriad of refined products.
The wet milling process, as alluded to earlier, is a countercurrent aqueous
slurry process. The aqueous stream, called mill water, begins as fresh water en-
tering the final washing step of the pure granular starch stream, flows backward
through several recycle loops in intermediate processes, and exits from the
freshest corn steeping tank as heavy steep water. The solid phase, beginning as
corn kernels, flows from a steep tank forward through the separation processes
as components are removed and exits the final washing step as a pure granular
starch slurry.
Steeping is accomplished by conveying corn grain into large steep tanks
where it is exposed to warm water (circa 122 8F, 50 8C) for 30 to 40 h. A small
amount of sulfur dioxide is added to maintain approximately 0.2% concentra-
tion to control bacterial growth. The corn kernels soften, loosening the hulls
and disrupting gluten–starch bonds as the slightly acidic water diffuses into the
swelling kernels. Steeping is currently a semi-continuous countercurrent pro-
cess. Process water to which sulfur dioxide is added enters the first of a train of
15.3 The Modern Corn Refinery 349
steep tanks, each holding as much as 3000 to 5000 bushels (76 to 127 MT) of
corn kernels. The steepwater flows continuously through the train while tanks
of steeped corn are sequentially removed from the front end and fresh corn
tanks added at the back. Flow rates are adjusted to provide the appropriate
steeping time. Water exiting the final tank has been exposed to the corn the
longest whereas the corn in that tank has been exposed to the steepwater the
shortest time. The fully steeped tanks are drained and the kernels are sluiced to
the first, or coarse, grind.
Steeping process improvements have been proposed with the objective of
shortening steeping time, eliminating sulfur dioxide, and other cost reductions
[7] but none has yet been incorporated to any extent.
Steeping yields an additional product, corn-steep liquor (CSL). This nutrient
rich product can be concentrated for sale as condensed fermented corn extractives,
more commonly called concentrated steep liquor. Steep liquor that is not used
internally (called thin steep liquor if it has not been concentrated) as a fermen-
tation nutrient or sold to other users as CSL, is combined with corn germ meal
(germ from which the oil has been extracted) and hulls, the composite consti-
tuting CGF.
Hydrocyclones remove the low-density corn germ from the coarsely ground
slurry in the germ separation process. The germ of each kernel contains about
85% of the kernels’ oil. Germ is thoroughly washed over bent screens to remove
residual starch, then dried and subjected to mechanical and chemical proces-
sing to remove the oil. The oil is further processed into either crude or refined
corn oil, and the spent germ combined with hulls and steep liquor as described
above.
The degermed slurry is subjected to more extensive grinding in attrition mills
to free the starch and gluten from the fibrous hull of the kernels. Slurry from
the grinding mills flows over concave “bent” screens. The slotted screens enable
starch and gluten particles to flow through, but not the fiber, effectively separat-
ing the fiber from the starch–gluten slurry. The fiber is rewashed to optimize
starch–gluten recovery and combined with spent germ and CSL as already de-
scribed.
The starch–gluten slurry is centrifuged to remove the gluten (light phase)
from the starch water slurry. Gluten, containing 60% protein is dried, and mar-
keted primarily as a premium animal feed ingredient. The starch is subjected to
exhaustive countercurrent washing to remove protein to less than 0.5% in the
starch, commonly approximately 0.3% range. The last stage, in which the starch
is washed to 99.5% purity (some oil and trace minerals also remain), is the only
point in the milling process where fresh water is added. This essentially pure
carbohydrate stream is ready for further processing into starch products, syrups,
or fermentation products.
15.3.3
Refinery Economy
15.3.3.1 Refinery Economy of Scale and Location Considerations

A large corn refinery produces a multitude of products from the pure carbohy-
drate stream and strives to minimize the cost per unit of production of the car-
bohydrate and subsequent products also. Unit production cost consists of the
cost of the corn and costs related to plant investment, labor, and conversion.
The first two costs decrease as capacity increases whereas conversion cost is
usually insensitive to capacity, on a unit production cost basis. Investment-re-
lated costs include maintenance, insurance, property tax, and general plant
costs, which are a direct function of plant size. Doubling the plant capacity
usually increases the investment required by only approximately 50%. Thus, the
investment-related cost per unit of production decreases with plant size. Labor
cost per unit of production also decreases as production capacity increases. Dou-
bling the capacity may require very little added labor. Unit conversion cost in-
cludes charges for utilities such as steam, electricity, and water and are sensitive
to volume. That is, doubling the capacity usually doubles the steam, electrical,
and water requirements.
As plant capacity increases, the investment per unit production decreases and
asymptotes at some minimum. The investment in dollars/annual gallon plotted
against annual capacity for a mash alcohol plant, for example, has been shown
to level off at about the 70 000 bushel/day grind capacity [8]. The generally ac-
cepted size of an “economy of scale” corn refinery is 60 000 to 80 000 bushels/
day grind (1525 to 2033 MT/day).
The size and location of a scale plant is also influenced by availability of raw
material corn, water, electricity, transportation (rail, water and highway), and la-
bor. To put this in context, a 100 000 bushel/day plant uses more than a square
mile of corn crop production per day (based upon national average yield of 142
bushels/acre) [9]. Each days grind requires 28 railcar loads of corn. If the grind
were split evenly between two products, corn syrup and ethanol, 25 rail cars of
products, including the CGF and the oil would be transported from the plant.
Transportation considerations are not trivial.
Most large corn refineries grew from one or two primary products, aside from
the attendant co-products, by adding finishing capacity to support a new prod-
uct with an attendant grind expansion to support the new capacity. For example,
a burgeoning HFCS market supported new syrup refining capacity with the at-
tendant grind expansions at existing syrup manufacturers. For HFCS, dramatic
growth also spurred green-field plant construction dedicated solely to HFCS
production on the scale mentioned above. Even those plants, however, have now
been expanded to furnish additional products.
15.4 Carbohydrate Refining 351
15.4
Carbohydrate Refining
The flexibility in operating a corn refinery is in the downstream processing of

the primary product, the carbohydrate. The granular starch slurry can be dried
to produce native, or “pearl” corn starch. Alternatively, some or all of the granu-
lar starch can be processed using chemical and/or physical methods, into modi-
fied starch products. Although important, the over nine billion pounds (4.1 mil-
lion MT) of corn starch forecasted to be used in the year 2004 [10] is still a dis-
tant third to the quantity used for producing corn sweeteners or fuel alcohol.
The greatest portion of the wet milled cornstarch is converted to sweeteners or
ethanol. In this process the granular corn starch slurry is solubilized and sacchari-
fied using a combination of heat, acid, and enzymes. Large thermal cookers intro-
duce high-pressure steam into the slurry exposing the granules to heat and shear
to disrupt the granular structure. Acid and enzymes begin depolymerizing the
high-molecular-weight glucose polymer chains. Large vessels, called saccharifica-
tion tanks, provide long residence time for the continuous flowing starch suspen-
sion. Starch-hydrolyzing enzymes are used to saccharify the glucose polymer, con-
verting it to short-chain hydrolyzates. The extent of hydrolysis determines whether
it is a corn syrup (20 to 70 dextrose equivalent, DE) or dextrose syrup (94 to 98 DE).
(Dextrose equivalent, DE, is a measure of the total reducing sugars in the syrup
calculated as dextrose and expressed as a percentage of the total dry substance
of the solution. The higher the DE, the greater the extent of hydrolysis.) Corn
sweeteners are sold as a wide range of glucose or dextrose syrups depending upon
DE and purity. These hydrolyzates are mechanically clarified and refined with car-
bon and ion exchange to produce the desired syrup.
Converting to 95 or higher DE glucose syrup provides the refiner with three op-
tions. It can be refined as dextrose or 95 DE corn syrup, fermented to ethanol, or
isomerized to HFCS. Having the high DE glucose stream also provides flexibility
in the choice of ethanol fermentation. Batch, semi-continuous, or continuous fer-
mentation are options available with a 95 DE syrup stream. Large refiners produc-
ing both HFCS and ethanol provide a common stream which enables production
swings, sometimes as much as 50%, from HFCS to ethanol and vice-versa as de-
mand fluctuates. In such circumstances, some of either capacity is idle at times.
That “idle capacity” investment is a small part of the overall plant investment,
however, and considered a small price to pay for the flexibility.
HFCS requires a 95 DE glucose or higher feed to the isomerization reactors,
the higher the better (while avoiding isomaltose reversion, which is discussed
later). Fixed isomerizing enzymes convert some of the glucose to fructose, a
very sweet carbohydrate monomer. (Sucrose is a dimer of a molecule of glucose
and a molecule of fructose). A 42% fructose stream issues from the isomerizing
reactors, the balance being glucose and any higher sugars which were present
in the feed stream. The product is refined with carbon and ion exchange and
concentrated for shipment. Large chromatographic columns are also used in an
enriching process in which the components fructose, glucose, and higher su-
gars are separated. An 85 to 90% fructose product results, which is blended

with 42% fructose syrup to produce a range of fructose compositions. The pre-
ponderance of product is 55% fructose syrup, an extensively used syrup in the
huge soft drink industry.
Corn refiners produce large volumes of glucose syrups. This covers a broad
range of products ranging from 20 DE to 70 DE, as already mentioned. Such
products are widely used in the baking, canning, and confectionary industries.
If a hydrolysate is to be converted to ethanol, some refiners will provide a
somewhat lower DE stream to the fermenter to avoid isomaltose, a non-fer-
mentable reversion sugar of glucose which occurs as a result of an equilibrium
reaction at high DE. The higher sugars (oligomers) are hydrolyzed, by enzymes
added to the fermenter, to hydrolyzates that are used by the glucose-fermenting
organism. The fermenting organism consumes the glucose and other ferment-
able sugars, reversion conditions are avoided in the saccharification process,
and overall yields are improved.
Having appropriate converter capacity also enables the option to produce other
fermentation products. Some vitamins and amino acids may require a highly pure
dextrose whereas a simultaneous saccharification–fermentation process can read-
ily utilize a low-DE syrup. The range of choices is broad and, as new industrial
organisms are introduced, even more options will become available.
15.5
The modern corn refinery is a model for the application of biotechnology to the
production of fuels, chemicals, and materials from abundantly available natural
resources in a sustainable, environmentally acceptable manner. New commodity
chemicals and materials, capable of replacing non-renewable petroleum-derived
products are being developed and manufactured now from corn-derived glucose
in such refineries. Other biomass sources of glucose that are equally or more
abundantly available and potentially less expensive than corn can readily be in-
corporated into such a process environment as the technology for such utiliza-
tion is developed.
References
1 B. W. Peckham 2000, The First Hundred 3 See for example, P. White and L. A.
Years of Corn Refining in the United Johnson (eds.) 2004, Corn: Chemistry and
States, in Corn Annual 2000, Corn Refin- Technology, 2nd edn, American Associa-
ers Association, Washington, DC. tion of Cereal Chemists, Eagan Press.
2 United States Department of Agriculture 4 Corn Refiners Association 2003, Corn
2003, Economic Research Service, Feed Annual 2003, Washington, DC.
Outlook, January 2003 5 P. W. Madson, and J. E. Murtagh 1991,
Fuel Ethanol in USA: Review of Reasons
References 353
for 75% Failure Rate of Plants Built, In- ics, National Corn Growers Association
ternational Symposium on Alcohol Fuels, Corn Utilization Conference V, June
Firenze, 1991, available from Katzen In- 1994, unpublished.
ternational, Cincinnati, Ohio. 9 US Department of Agriculture, NASS,
6 L. R. Lynd 2002, Principal Investigator, Agricultural statistics board.
Strategic Biorefinery Analysis, NREL 10 US Department of Agriculture, Econom-
Subcontract ADZ-2-31086-01. ic Research Service.
7 J. Randall et al. 1978, USP 4,106,487.
8 R. Katzen et al. 1994, Ethanol from Corn
– State of the Art Technology and Econom-
Part IV
Biomass Conversion: Processes and Technologies
ISBN: 3-527-31027-4
357
16
Enzymes for Biorefineries
16.1
Introduction
The total amount of carbon and nitrogen biologically processed by photosyn-

thesis into polymeric organic material is referred to as biomass. Plants are the
main source of biomass and it is estimated that the total amount of biomass
worldwide is approximately 1012 tons. Biomass is a renewable source of carbon
building blocks, and there is great potential for converting this resource into a
diverse array of biobased products.
Just over a hundred years ago, 90% of our energy needs were supplied by bio-
mass, largely by combustion of wood. Most non-fuel industrial products, includ-
ing dyes, inks, paints, medicines, chemicals, fibers, and plastics, were made
from trees, vegetables, and crops. By the 1970s, 70% of US energy and 95% of
industrial products were derived from petroleum rather than from renewable
sources. The finite petroleum resources, a host of environmental issues, coupled
with a growing interest in reducing the US dependence on foreign energy and
industrial feedstock sources, have fueled research and development into alterna-
tive energy sources and the economic utilization of biomass as a source of bio-
based products [1].
The conversion of agricultural commodities such as corn into fuel-grade etha-
nol is one successful alternative energy initiative. Gasoline containing 10% etha-
nol was introduced as a fuel for automobiles. Today, ethanol in gasoline con-
tinues as an additive replacement for the oxygenate methyl tertiary butyl ether
(MBTE). Fuel ethanol production from sugar/starch biomaterials, for example
corn and sugar cane, has become an economically viable industry. In North
America, approximately 11 billion liters of ethanol are currently produced an-
nually, with a projected growth rate of *20% for the foreseeable future.
Comparing with sugar and starch based agricultural products, biomass has a
much larger potential to become the renewable energy source of the future. Bio-
mass includes agro/forest byproducts such as corn stover (corn leaves and
stalks) and wood pulp and paper residues. It is estimated that in the US alone,
ISBN: 3-527-31027-4
358 16 Enzymes for Biorefineries
biomass is capable of yielding *100 billion liters of ethanol annually. In the

US corn production generates *100 000 dry metric tons of corn stover [2], an
excellent raw biomaterial containing a huge amount of under-utilized, energy-
rich lignocellulosics.
The concept of the biorefinery is analogous to the concept of the petroleum
refinery in the sense that an abundant feedstock is converted into many differ-
ent products. Unlike a petroleum refinery, a biorefinery utilizes renewable feed-
stocks, such as plant-based starch or residual biomass (cellulose, hemicellulose,
and lignin) which can be harvested and re-planted year after year. In one scenar-
io, the feedstock is harvested, delivered to the refinery, and pretreated to make
the cellulose accessible for enzymatic conversion to fermentable sugars. The re-
sulting sugars are then fermented into primary products such as ethanol, lactic
acid, or several other materials for industrial use. Unused residual materials are
burned as fuel to generate heat and electricity. Biorefineries exist today, to a
small but growing extent, where, for example, corn starch feedstocks are con-
verted to high-fructose syrups, animal feed, oil, and various organic acids (for
example citric, gluconic, itaconic and lactic acids).
Like the petroleum refinery, it is expected that the process and products from
a biorefinery will evolve to meet society’s demand. The first refineries in the
1860s produced kerosene for oil lamps to replace a dwindling supply of whale
oil, and the byproducts, naphtha and tar, had few low value uses. With the de-
velopment of the combustion engine, gasoline and light oil became dominant
products and spurred the development of industries utilizing chemical by-
products of fuel production as chemical feedstocks for plastics, synthetic fibers,
elastomers, drugs, and synthetic rubber. Although the primary focus of the bio-
refinery concept today is on fuels and energy production, large-scale implemen-
tation will stimulate development of a feedstock industry similar to that seen
from the petrochemical refinery.
With recent developments in biotechnology, many petrochemical-derived
products can be replaced with industrial materials made from renewable re-
sources. Biotechnology has had a positive impact on the cost-efficiency of enzy-
matic conversion of biomass to fermentable sugars and has increased the range
of products that can be produced by genetic engineering of fermentative organ-
isms. Biotechnological improvements may soon enable us to produce plants
with altered properties that make them more amenable to refining.
Biobased products have the potential to dramatically alter our world. The
availability of technology enabling the refining of biomass will reduce the re-
lease of petroleum-originated greenhouse gases into the atmosphere, will largely
decentralize fuel and chemical production, with concomitant improvement of
rural economies, and will positively impact US national security by reduction of
its dependence on foreign oil imports.
In this chapter we consider the biorefining of agricultural residue materials,
primarily focusing on recent advances in enzymatic catalysis in the conversion
of biomass to fermentable sugars. We conclude with a short discussion of the
prospects for various biorefinery products.
16.2
Biomass as a Substrate
16.2.1
Composition of Biomass
A vast carbon source for biobased products is locked up in plant matter, the
most abundant source of biomass on earth. The principal components of bio-
mass are cellulose (30–50%), hemicellulose (20–30%), and lignin (20–30%);
with starch, protein, and oils as minor components. The exact composition of
each biomass varies depending both on the plant and on the residue collected
(Table 16.1). The composition, in turn, determines the ease with which the bio-
mass can be converted to useful products and/or intermediates and affects the
functionality of the final product.
The complex polymeric structure of crystalline bundles of cellulose embedded
in a covalently linked matrix of hemicellulose and lignin poses a formidable
challenge for solubilization and conversion to monomeric sugars. As can be
seen in Table 16.1, the relative lignin, cellulose, and hemicellulose content of a
variety of potential feedstocks is quite similar, yet even a 5% increase in lignin
or hemicellulose content can significantly alter accessibility to enzymatic attack.
Thus, the variation in the composition of a given biomass requires some tailor-
ing of the conversion method.
16.2.1.1 Cellulose
Cellulose is abundant in plant cell walls and comprises a linear beta-(1 ? 4) an-
hydroglucopyranose polymer (six-carbon sugars). The molecular weights of dif-
ferent celluloses can range from 200–2 000 kDa where the number of glucose
Table 16.1 Composition of representative biomass samples.
Samples Variety % Mass
Total Lignin Cellulose Hemicellulose
Monterey pine Pinus radiata 25.9 41.7 20.5

Hybrid poplar DN-34 24 40 22
Sugarcane bagasse Gramineae saccharum var. 24 43 25
65-7052
Corn stover Zea mays 18 35 22
Switchgrass Alamo 18 31 24
Wheat straw Thunderbird 17 33 23
Barley straw Hordeum vulgare sp. 14 40 19
Rice straw Oryza sativa sp. 10 39 15
Source: http://www.eere.energy.gov/biomass/feedstock_databases.html
residues can exceed 15 000 per polymer molecule. Cellulose has such extensive
hydrogen bonding, because of the formation of overlapping, staggered flat
sheets, that it is a highly recalcitrant and water-insoluble crystalline material.
Within a cellulose chain, each d-glucosyl residue is rotated by approximately
1808 relative to its nearest neighbor residue, making cellobiose the repeating
unit present in cellulose. Only agents that can attack the glycosidic linkages
between the glucose residues or which can disrupt the hydrogen bonding can
solubilize cellulose.
16.2.1.2 Hemicellulose
Hemicelluloses are plant cell wall heteropolymeric sugars and sugar acids with
a backbone of 1,4-linked b-d-pyranosyls in which O4 is in the equatorial orienta-
tion. They are usually shorter than celluloses, typically containing fewer than
200 1,4 linkages, highly branched, and easily hydrolyzed by strong acid or base.
Hemicellulose serves as an interface between cellulose and lignin in plant cell
walls, and may form covalent and non-covalent linkages with other cell-wall
constituents, for example pectin, glucans and proteins. One major hemicellu-
lose is xyloglucan, a beta-(1 ? 4) linked polymer of xylose with mono-, di-, or tri-
glycosyl side-chains, via O6, composed of a variety of substituents, for example
acetyl, arabinosyl, or glucuronosyl units. Other hemicelluloses include xylan,
glucuronoxylan, arabinoxylan, mannan, glucomannan, and galactoglucoman-
nan.
16.2.1.3 Lignin
Lignin is a highly complex, amorphous and heterogeneous complex of substi-
tuted phenolic compounds, often comprising syringyl, guaiacyl, and p-hydroxy-
phenol components. It binds to hemicellulose and cellulose. Lignin is highly re-
sistant to enzymatic, chemical, and microbial hydrolysis because of its extensive
cross linking. It can, however, be pyrolyzed to form oil for fuel and resins.
16.2.1.4 Starch
Starch is composed of glucose, as a mixture of amylose and amylopectin in
varying ratios. Amylose is a linear alpha-(1 ? 4) d-glucopyranose polymer
whereas amylopectin has a similar structure but with additional side branches
of more than 20 glucose residues with alpha-(1 ? 6) linkages. Currently, corn
starch is the primary raw material of several major grain-based products, for ex-
ample ethanol, polylactide, plastics, some packing materials, and adhesives.
16.2.1.5 Protein
Proteins are polymers of amino acids. In plants, proteins serve as structural,
functional, and regulatory agents. Catalytic proteins, the enzymes, are essential
for a variety of plant physiological activity.
16.2.1.6 Lipids and Other Extracts

Lipids are esters of moderate to long-chain fatty acids, either saturated or un-
saturated. Acidic or basic hydrolysis yields the component fatty acid and alcohol.
Triglycerides, esters of fatty acids with glycerol, constitute fats and oils. Esters of
fatty acids with monohydric alcohols, often mixed with hydrocarbons, constitute
waxes. Phospholipids are important components of cell membranes. Another
major plant extract is terpene, made from isoprene (isopentane) units.
16.2.2
Biomass Pretreatment
In a natural setting, lignocellulosic biomass is broken down over a period of

years by the accumulated action of physical disruption from the forces of nature
(wind, rain, snow, heat, sunlight) and by the action of microbes that chemically
and enzymatically degrade it into compounds they can use for their growth.
The components of the plant cell wall that give it strength and rigidity, namely
the intertwined network of cellulose, hemicellulose, and lignin, also make it re-
sistant to breakdown, whether on a forest floor or in a biorefinery reactor. In
contrast with natural decomposition of lignocellulose in nature, breakdown of
the feedstock in a biorefinery must occur in a matter of hours or days rather
than years. To accomplish this requires coordinated steps of physical disruption,
chemical modification, and enzymatic action. The recalcitrant cellulose is rela-
tively resistant to breakdown by microbial hydrolytic enzymes in its natural
form, with only approximately 20% of the cellulose present in untreated bio-
mass being hydrolyzed to glucose after treatment with high doses of enzymes.
In pretreatment, plant materials are physically disrupted, under the action of
stress/tear, temperature, pressure, and/or pH. To convert biomass into ferment-
able sugars, the purpose of the pretreatment is to disorder or remove cellulose–
hemicellulose–lignin interactions and thereby improve access of hydrolytic en-
zymes to sugar polymers in subsequent steps in the biorefinery.
Physical disruption usually begins with a reduction in the size of the plant
material by milling, crushing, and/or chopping. For example, in the processing
of sugar cane, the cane is first cut into segments and fed by conveyor into con-
secutive roller presses that both extract the cane juice (rich in sucrose) and
physically crush the cane, producing a fibrous bagasse that has the consistency
of sawdust. In corn-stover processing, the stover is initially chopped with knives
or ball-milled to increase the exposed surface area and improve wetability.
After physical disruption, pretreatment may continue with a chemical extrac-
tion designed to maximize subsequent enzymatic hydrolysis of the cellulose.
This usually means modifying or removing lignin, which not only acts as a
block to enzyme action by coating cellulose microfibrils in untreated biomass,
but also interferes with enzymatic hydrolysis by directly absorbing some cellu-
lose-active enzymes. The result of pretreatment is a cellulose with both im-
proved solvent accessibility and reduced lignin interference with enzyme action.
Numerous methods of biomass pretreatment have been described in the litera-
ture and are summarized below.
16.2.2.1 Dilute Acid Pretreatment

This process is perhaps the most thoroughly studied of the pretreatment meth-
ods and consists of mixing the biomass with a solution of dilute strong acid
(e.g. 5% sulfuric) in a pressurized reactor at high temperature (e.g. 160–200 8C)
for 1–10 min then rapidly releasing the pressure. This method effectively hydro-
lyses most (up to 95%) of the hemicellulose to its constitutive C5 sugars (xylose,
arabinose, etc.) [3]. Little lignin is removed by the process, but it is thought to
melt and be “redistributed”, enabling improved cellulose hydrolysis. Pretreat-
ment conditions must be adjusted, depending on the source of the biomass, to
maximize hemicellulose hydrolysis, while at the same time minimizing the for-
mation of compounds such as furfural and hydroxymethyl furan that are toxic
to fermenting yeast and probably also to other organisms of industrial interest
(a review on this topic is given elsewhere [4]). In literature reports the solids re-
maining after pretreatment are often washed before enzyme digestion, but this
may not be economically practical in a biorefinery.
16.2.2.2 Ammonia Fiber Explosion

This pretreatment utilizes ammonia mixed with biomass in 1:1 ratio under high
pressure (1.4–3 atm) at temperatures of 60–110 8C for 5–15 min, then explosive
pressure release. Because of it volatility, ammonia can be recycled with near quan-
titative recovery. Little (10–20%) lignin is removed, but enzymatic cellulose hydro-
lysis is reported to proceed to as much as 98% of theoretical yield at relatively high
cellulase loadings (15 FPU g–1 glucan) [5]. Hemicellulose depolymerization is
highly variable and depends on the moisture content of the biomass, but is typi-
cally quite low. Enzymatic cellulolysis therefore requires the presence of at least
some hemicellulase activity to increase cellulose accessibility during hydrolysis.
16.2.2.3 Hot-wash Pretreatment

This method involves passing hot water through a heated stationary biomass
bed and, like dilute acid, has been reported to result in solubilization of more
than 90% of the hemicellulose fraction [6]. The hemicellulose is largely con-
verted to pentose oligomers which must be enzymatically converted to monosac-
charides before fermentation. The performance of the pretreatment depends on
temperature and flow rate, and requires washing for ca. 8–16 min. At high flow
rates and temperatures, the lignin content is reduced by as much as 46% and
the process produces no significant amounts of compounds inhibitory to fer-
mentation [7, 8]. Although the hydrothermal process does not require the acid-
resistant reactor materials of acid pretreatment, this advantage may be offset by
increased water use and recovery costs.
16.2.2.4 Wet Oxidation

Here molecular oxygen and water are applied to the biomass at high tempera-
ture and pressure. In a series of experiments reported by Varga [9], 60 g L–1 bio-
mass incubated at 195 8C for 15 min under 12 atm O2 and containing 2 g L–1
Na2CO3 solubilized 10% of the cellulose, 60% of the hemicellulose, and 30% of
the lignin present in corn stover. Enzymatic hydrolysis of the remaining solids
after hydrolysis at 50 8C for 24 h using 25 FPU enzymes per gram of dry bio-
mass achieved an 85% conversion of cellulose to glucose.
Other methods of pretreatment involve the use of sodium hydroxide, lime, or-
ganic solvent extraction, or lime steam explosion, but are, in general, less stud-
ied than the methods described above.
The critical issues in selecting a pretreatment for lignocellulosic biomass are
sugar yield and composition, the cost of the process both in terms of energy
and chemical costs, and the capital cost of building the system. The pretreat-
ment system selected has an impact on all the downstream processes in the
biorefinery and must be evaluated carefully. As described above, increased en-
zyme digestibility may be accompanied by an increase in byproducts that inhibit
downstream fermentation, whereas less severe conditions may produce a sub-
strate requiring excessive enzyme loadings for cellulose hydrolysis. This inter-
play between pretreatment, enzymatic digestion, and fermentation is the crux of
current research projects to develop an integrated biorefinery.
16.3
Enzymes Involved in Biomass Biodegradation
Biomass degradation is required for the survival of many organisms including

bacteria, fungi, plants, protozoa, insects, and herbivores (through symbiotic mi-
crobes). Investigation into the action of these enzymes on biomass or its compo-
nents has been active for over sixty years [10–16]. With the advent of large-scale
genome sequencing, the complexity of biomass degradation has become more
apparent. Bacteria such as Clostridium thermocellum, Cytophaga hutchinsonii, Mi-
crobulbifer degradans, Rubrobacter xylanophilus, and Thermobifida fusca, and the
fungi Trichoderma reesei and Phanerochaete chrysosporium have all been se-
quenced to at least draft form, revealing a diverse array of enzymes involved in
carbohydrate degradation. Organisms devote much of their resources to degrad-
ing plant material, with well over fifty genes targeting polysaccharide degrada-
tion even in relatively simple bacteria.
16.3.1
Glucanases or Cellulases
On the basis of sequence homology, cellulases can be grouped in the glycoside

hydrolase (GH) family classification system of Coutinho and Henrissat (Carbo-
hydrate-Active Enzymes server at URL: http://afmb.cnrs-mrs.fr/*cazy/CAZY/
index.html). Cellulases fall into families GH5–9, 12, 44, 45, 48, 61, and 74 [17].
Cellulases are often modular, comprising a catalytic core, a linker, and one or
more cellulose-binding modules (CBM, which can be grouped into *13 fami-
lies, part of *39 families of carbohydrate-binding domains). Such modular or-
ganization is found with other enzymes active on other insoluble polysaccha-
rides, for example amylases and chitinases. The CBM can be at the N or C-ter-
minus of the catalytic domain and is attached via a linker domain rich in pro-
line, serine, and threonine. The 3D structure of many catalytic domains has
been solved [18]. In general, the active site in exoglucanases is enclosed by two
surface loops forming a tunnel, believed to be vital for conferring directionality
in enzyme action on linear cellulose microfibrils. In contrast, the active site in
endoglucanases has an open groove, enabling it to act in the middle of a cellu-
lose chain. The 3D structure of many CBM has also been solved [19]. In gener-
al, a CBM has a flat surface with exposed aromatic side-chains that mediate
binding to the hydrophobic cellulose surface.
Cellulase is a general term encompassing a diverse set of enzymes that parti-
cipate in the hydrolysis of cellulose into glucose. Cellulases include exo-1,4-b-d-
glucanases or cellobiohydrolases (CBH, EC 3.2.1.91), endo-1,4-b-d-glucanases
(EG, EC 3.2.1.4), and b-glucosidases (BG, EC 3.2.1.21). EG act internally within
a cellulose chain at amorphous cellulose regions to cleave glycosidic bonds,
thereby fragmenting the cellulose polymer. CBH are believed to “processively”
degrade a cellulose chain from either the reducing or non-reducing ends, and
can cleave glycosidic bonds within crystalline cellulose regions to release the
disaccharide cellobiose. Because EG create new reducing and non-reducing ends
within cellulose chains for CBH attack, these two enzyme classes act synergisti-
cally in cellulose degradation. At high concentrations cellobiose or other cellooli-
gosaccharides can inhibit CBH activity. Thus BG, which hydrolyses these solu-
ble sugars to glucose, is often required in a “complete”, effective cellulolytic sys-
tem.
In addition to cellulase, phosphorylase can also cleave b-glycosidic bonds,
especially those in cellodextrin, by phosphorylating a glucosyl unit [20].
16.3.2
Hemicellulases
Hemicellulases are involved in degrading hemicellulose. Some cellulases, for ex-

ample GH7 and GH74 EG, have significant xylanase or xyloglucanase side-activ-
ity. The structural similarity enables many hemicellulases and cellulases to be
grouped into the same GH family [17].
On the basis of the products they form xylanases can be classified as endoxy-
lanases (EC 3.2.1.8) and b-xylosidases (EC 3.2.1.37). On the basis of sequence,
endoxylanases belong to the GH10 and GH11 families whereas b-xylosidases be-
long to GH3. Galacto/glucomannan-active mannanases can also be classified as
endomannanases (EC 3.2.1.78) and b-mannosidases (EC 3.2.1.25). Other hydro-
lases are active on other polysaccharides commonly found in biomass, for exam-
ple pectinase/polygalacturonase, arabinofuranohydrolase, arabinase, galactanase,
glucoronidase, and acetylesterase [21].
16.3.3
Nonhydrolytic Biomass-active Enzymes
In addition to hydrolases, various proteins and enzymes are active on lignocellu-

losics. Expansin and swollenin are not cellulolytic, but seem able to disrupt the
hydrogen bond between cellulose chains, leading to structurally weakened cellu-
lose [10]. Polysaccharide lyases cleave glycosidic bond by b-elimination, resulting
in a double bond at the newly formed non-reducing end. Laccase, lignin peroxi-
dase, and Mn peroxidase can oxidize lignin, thus loosening lignin–polysaccharide
interactions or relieving lignin inhibition of polysaccharide hydrolases [22, 23].
16.3.4
Synergism of Biomass-degrading Enzymes
The selection pressure on organisms to feed efficiently on biomass has led to

the evolution of biological cellulolytic systems integrating highly specialized yet
synergistic enzymes. Two distinct, effective systems have developed – the multi-
component, secreted, and non-complexed fungal/bacterial (aerobic) cellulases
and the multi-component, scaffold-assembled, complexed bacterial (anaerobic)
cellulosomes.
One representative fungal cellulolytic system is that of Trichoderma reesei (syn.
Hypocrea jecorina) [24]. One of the most effective cellulose degraders known, T.
reesei secretes an array of cellulases, including CBH I or Cel7A (*60%), CBH
II or Cel6A (*15%), EG I or Cel7B, EG II or Cel5A (*20%), and other minor
components (for example as EG III or Cel12A, EG IV or Cel61A, EG V or
Cel45A, EG VI or XG74A, BG I or Cel3A, Xyn I or Xyn11A, and swollenin).
CBH I and CBH II differ in their affinity for reducing and non-reducing ends.
The subtle yet significant difference among the various EG can accommodate
the need to degrade a heterogeneous substrate such as cellulose. Commercial T.
reesei cellulase preparations, for example Novozymes’s Celluclast 1.5L and Gen-
encor International’s Spezyme, are widely used in a variety of applications.
One representative bacteria cellulosome is that of Clostridium thermocellum. This
cellulase system is organized around a *200 000 molecular mass scaffolding, a
protein equipped with a dockerin, a CBM, and many cohesin domains. The dock-
erin attaches the assembly to the cell surface, and the cohesins anchor a variety of
enzyme molecules (CBH, EG, etc.) by interacting with their dockerin domains [10,
12]. Cellulosomes range from 0.5 to 50 megadaltons, and many cellulosomes can
further aggregate into polycellulosomes. The catalytic domains of cellulosomic en-
zymes are very similar to those of their non-complexed counterparts. Co-localiza-
tion of enzymes in a cellulosome may improve synergism by bringing necessary
components together within the same vicinity, but the organization gives the hy-
drolases limited mobility in comparison with the non-complexed cellulases. Never-
theless, the cellulolytic activity of the supramolecular cellulosome is comparable
with that of the multi-component, non-complexed fungal cellulase system [10].
An effective industrial cellulase preparation should include enzymes that can
“multi-task”, and the enzyme function should be collaborative and synergistic.
Several fungal cellulase preparations are available commercially but no bacterial
cellulase preparation has yet been produced industrially. Available cellulase
products, developed for detergent, textile, and other industries, are too expensive
for a viable biorefinery. The economic considerations of protein production led
us to choose Novozymes’s Celluclast 1.5L, produced by large scale batch fermen-
tation of T. reesei, as a starting point for developing the next generation of bio-
mass-targeting cellulase product. We focused on improving the activity of the T.
reesei cellulase system while maintaining its already high protein productivity
during fermentation. All studies were performed using acid-pretreated corn
stover (PCS) as the substrate.
16.4
Cellulase Development for Biomass Conversion
16.4.1
Optimization of the CBH-EG-BG System
16.4.1.1 BG Supplement
A “complete” cellulase system requires BG to hydrolyze cellobiose, a potent in-
hibitor of CBH and a precursor of fermentable glucose. Balancing the ratio of
CBH, EG, and BG is vital for improved cellulose hydrolysis. T. reesei secretes at
least two enzymes with BG activity at a very low level during normal cellulose-
induced growth. We observed that Celluclast hydrolyzed cellulose with improved
performance when assayed in a diafiltration–saccharification device which en-
ables continuous removal of small sugars by filtration, compared with a closed
vessel. Accumulation of CBH-inhibiting cellobiose in a closed vessel resulted in
a slowing down of the overall reaction [25]. We exogenously supplemented Cel-
luclast with an Aspergillus oryzae BG (belonging to the GH3 family). Addition of
small amounts of BG, present as a few percent of total protein, enabled us to
achieve equivalent conversion of cellulose in PCS with half the enzyme dosage
of the unsupplemented Cellulase mix (Fig. 16.1). By expressing the A. oryzae
BG in the T. reesei strain used to produce Celluclast 1.5L we were able to elimi-
nate the need to ferment BG separately.
Fig. 16.1 Improvement of PCS-hydrolyzing cellulases by addition of BG.
16.4.1.2 Novel Cellulases with Better Thermal Properties

One focus of our research has been to obtain a collection of CBH, EG, and BG
from a taxonomically diverse group of mesophilic, thermotolerant, and thermo-
philic fungi. Extending the number of cloned and characterized cellulases be-
yond currently reported enzymes could lead to discovery of enzymes with novel
Fig. 16.2 Phylogenetic tree comparing the catalytic core

sequences of the novel GH7 CBH I genes to the catalytic core
sequences of published genes.
properties. In addition to T. reesei we have isolated other cellulolytic fungi that

effectively degrade complex lignocellulosic substrates such as PCS, and sought
novel CBH I enzymes (from the GH7 family), particularly those from thermo-
philic filamentous fungi. Thermally stable enzymes could enable cellulose hy-
drolysis at elevated temperature, which in theory should result in thermal en-
hancement of the enzymatic rate. We attempted to find a CBH I superior to T.
reesei CBH I, which has an unfolding temperature Tm of 62.5 8C [26] and is
known to be inactivated in hydrolysis reactions above 50 8C [27]. Many genes
with a large amount of sequence identity with T. reesei CBH I (Cel7A) have been
deposited in public databases. We used homology search tools, for example the
Smith–Waterman, FASTA, BLAST (gap penalties and scoring matrices), Clustal
W (multiple alignment), MEME, and MAST (motif searching) algorithms, to
discover numerous GH family 7 genes with wide phylogenetic diversity
(Fig. 16.2). They provided a resource for understanding the functional diversity
of cellobiohydrolases. Of these genes, 15 were expressed in fungal hosts, puri-
fied and characterized. In hydrolyzing various cellulose substrates, a few en-
zymes from thermophilic fungi had thermal stability/activity superior to that of
T. reesei CBH I.
Fig. 16.3 Improved CBH I (Cel7A) variants the wild type (wt) is marked as a triangle
with enhanced activity at high temperatures. (s). The open circle (*) marks the position
Activity at moderate temperature (at which of a variant obtained by protein design.
the wild-type enzyme is stable) is plotted Closed diamonds (^) denote variants
against the ratio of activity at a thermally obtained from primary screens whereas open
challenging temperatures (at which the wild- squares (`) show variants obtained by
type is unstable) divided by activity at the rounds of shuffling from pools of primary
moderate temperature. The performance of variants.
Because T. reesei CBH I has high specific activity on PCS at moderate tem-
perature, we tried to improve its thermal stability, using structure-based design
and directed molecular evolution. For structure-based rational design, we com-
pared the coding sequences of thermostable CBH with their less stable counter-
parts and modeled their structures, informed by the published structures of gly-
cosyl hydrolase domains [28–31]. This comparison revealed specific residues
that could be mutated to enhance thermostability, and several of these muta-
tions were created by site-directed mutagenesis. In addition, we generated ran-
dom mutations in the CBH I gene and identified variants with improved activ-
ity at elevated temperature. As a result of both approaches we identified several
substitutions in the CBH I gene that led to enhanced performance in hydrolyz-
ing a soluble cellulase substrate at high temperature. DNA shuffling, the pro-
cess of using recombination between genes with partial sequence identity, was
used to find favorable combinations of the identified substitutions and to elimi-
nate detrimental mutations [32] (Fig. 16.3).
We expressed several of our thermally improved CBH I variants in Trichoder-
ma host strains that lacked the native CBH I gene. Expression of the improved
variants was achieved at levels that approximated those found in the wild-type
parent strain, and their expression did not noticeably alter the levels of other
proteins in the T. reesei secretome. We assayed complete broths containing vari-
ant CBH I enzymes for hydrolysis of PCS at temperatures higher than the opti-
mal temperature for T. reesei native cellulases. Figure 16.4 shows that the pres-
ence of the variant CBH I improved the high-temperature saccharification of
Fig. 16.4 High-temperature hydrolysis by cel- lines, 55 8C hydrolysis; dotted lines, 60 8C hy-
lulase mix including variant CBH I. Sacchari- drolysis. Cellulases were used at equivalent
fication of pretreated corn stover by two Tri- protein loadings. Cellulose conversion was
choderma broths, one expressing the wild determined by measurement of reducing su-
type CBH I (“WT CBH I”) and one express- gars.
ing a CBH I variant (“Variant CBH I”). Solid
Fig. 16.5 Thermostabilization of A. oryzae BG by directed

evolution. Residual activity is the ratio of BG activity after heat
treatment at 68 8C for 10 min compared with the activity of an
untreated sample. Activity was measured using 4-nitrophenyl
b-D-glucopyranoside at pH 5.
PCS over the wt strain. The selected variants failed to surpass wt T. reesei cellu-
lase mix at its optimum temperature (50 8C), however (data not shown).
In addition to CBH I, we also improved the thermal stability of A. oryzae BG
by directed molecular evolution. We screened for stabilized variants by measur-
ing enzyme residual activity after brief thermal denaturation at temperatures
that partially denature the wt enzyme. Several improved variants had better ther-
mal stability, as measured by assessing residual activity after a ten-minute ther-
mal challenge at 68 8C (Fig. 16.5).
16.4.1.3 Structure–Function Relationship of EG

For efficient cellulose conversion, it is crucial that CBH and EG act synergisti-
cally; thus, EG activity and substrate specificity is important for maximizing cel-
lulose hydrolysis. Microbial EG are grouped into many families according to the
extent of their sequence identity [17]. T. reesei secretes at least six EG, with EG I
and EG II making up as much as 15% of the total secreted protein. Although
the substrate specificity of several individual representative EG has been thor-
oughly studied, a systematic, comparative investigation of EG from different
GH families has yet to be made [33]. We assayed *20 EG from GH5, 7, and 45
with a variety of representative substrates to further define their substrate pre-
ferences (Fig. 16.6). Cel5 EG had significant mannanase activity, in addition to
their cellulase activity. Cel7 EG had significant xyloglucanase activity, in addition
to their cellulase activity. In contrast, Cel45 EG were “strict” cellulases, acting al-
most exclusively on b-1,4 linked glucose polymers. Among the EG, only Cel7
were active on p-nitrophenyl-b-d-cellobioside, a commonly used chromogenic
surrogate substrate for cellulases. These results suggest that Cel45 have an ac-
tive site groove that is more defined, or specifically “tuned” to accommodate a
b-1,4-d-cellodextrin unit, compared with the active sites found in Cel5 and Cel7.
Fig. 16.6 Substrate specificity of endoglucanases. Substrates:

PNPC, p-nitrophenyl-b-D-cellobioside; PASC, phosphoric acid-
swollen cellulose. T. reesei Cel7B is marked with an arrowhead
(Tr Cel7B).
Elucidation of this structure–function relationship could assist us in tailor-mak-

ing cellulase systems specific toward biomass with different cellulose–hemicel-
lulose compositions [34].
16.4.2
Other Proteins Potentially Beneficial for Biomass Conversion
16.4.2.1 Secretome of Cellulolytic Fungi

It is known that the number of cellulase-encoding genes in cellulolytic microbes
can far exceed the number (3 to 4) of components in the simplest “complete” cel-
lulase system. The need for a microbe to have an extensive cellulase array might
be related to the diversity/heterogeneity of its carbon source, or the requirement of
other proteins, beyond the “canonical” composition (reducing-end-preferred CBH
I, nonreducing-end-preferred CBH II, EG and BG), for its cellulolytic function.
Different cellulolytic fungi can secrete sets of proteins with significantly dif-
ferent 2D electrophoretic patterns, as exemplified in Fig. 16.7. The explanation
for this difference might be twofold. Different fungi use at least one of the four
Fig. 16.7 Two-dimensional gels of proteins secreted

by T. reesei and three other cellulolytic fungi grown on PCS.
The “canonical” cellulases are marked.
“canonical” cellulases, but the cellulase(s), although highly similar in terms of

amino acid sequence identity, can differ by posttranslational processing (i.e. IEF
isoforms). In addition to the major cellulase(s), different fungi can secrete var-
ious minor proteins under cellulose-induced growth. The presence of these mi-
nor components indicates that other proteins (in addition to the four canonical
ones) may play complementary/beneficial roles in cellulosic degradation.
Among the proteins secreted by T. reesei, Cel12A, Cel61A, Cel45A, XG74A,
Cel3A, Xyn11A, swollenin, and other proteins have been identified in addition
to Cel7A, Cel6A, Cel7B, and Cel5A. Microarray cDNA/genomic DNA analysis,
Expressed-Signal-Tags (EST), and other molecular biology tools have unveiled
many unknown genes whose expression were induced by PCS (Fig. 16.8). Given
this evolutionary diversity, we can expect other cellulolytic fungi/bacteria to uti-
lize different sets of proteins. Some organisms may utilize novel component(s)
in the enzyme mix; others might be unique with regard to the stoichiometry of
the cellulases present.
16.4.2.2 Hydrolases
The two CBH, six EG, and one BG known to be secreted by T. reesei belong to
eight GH families, representing approximately half of the GH families known
to include cellulases. It is possible that cellulases from other GH families could
be beneficial to T. reesei cellulolytic system, by providing either complementary
specificity, stronger synergism, reduced inhibition, enhanced reactivity, or in-
Fig. 16.8 Schematic of fungal genes induced by growth on

PCS. The action of gene products on lignocellulose is
depicted.
creased stability. For example, cellulases derived from hyperthermophilic mi-

crobes are of particular interest because of their superior thermal profile.
One example of the benefits of pairing the T. reesei cellulolytic system with
another quite different fungal cellulolytic system is shown in Fig. 16.9. A 1 : 1
mixture of the two cellulase preparations performed as well as the individual
system dosed at twice as much as the T. reesei cellulolytic system alone, indicat-
ing a significant synergism between the two systems. Identification and transfer
of those enzymes responsible for this synergism into the T. reesei host should
create a single organism with significantly improved cellulose-hydrolysis activity.
Fig. 16.9 Effect of adding fungus Z proteins to T. reesei broth

in hydrolyzing PCS at 50 8C.
Thus, studying non-canonical cellulases is of interest for improving enzymatic

PCS hydrolysis.
16.4.2.3 Nonhydrolytic proteins

As shown in Fig. 16.8, several non-hydrolytic proteins seem to be up-regulated
under cellulose induction. Some of these can be attributed to cellular processes
directly relevant to cellulose utilization, but others apparently cannot. The con-
tributions of these additional components to microbial biomass conversion
should be an indispensable part of “cellulolyteomics” – understanding the global
network of all cellulosics-active biomolecules. Investigating the mechanism and
extent of action of the components on different feedstocks will be an important
part of improving biomass conversion. Swollenin and phenolic oxidases are cur-
rently attracting attention because of their ability to disrupt cellulose and lignin,
respectively; the two reactions are thought to be beneficial to biomass hydrolysis
[10].
16.5
Expression of Cellulases
Many individual enzymes acting synergistically form an effective cellulase mix

for conversion of lignocellulosics to sugars. Production of cellulase components
individually is not economically feasible; instead, all the proteins necessary
should be expressed and secreted by a single fungal host. The protein composi-
tion must be well balanced to take advantage of the optimum mix for cellulase
synergy. At the same time, the overall total protein yield must be high. Consid-
ering these factors, it becomes clear that an important aspect of biorefinery re-
search is the technology of protein expression in fungi.
Enhancement of a cellulase production strain can be performed on two differ-
ent levels; first, classical strain mutagenesis can be used, as has been reported
previously for T. reesei [35]. Second, genetic engineering can be used to modify
levels of endogenous gene expression and to introduce genes for heterologous
expression of novel cellulase components.
The use of genetic engineering to introduce and manipulate specific gene ex-
pression in T. reesei has been indispensable to our cellulase research program.
We used several selective genetic markers to follow gene integration into the
host and developed a variety of promoter elements to enable variable levels of
gene expression. Because we required the ability to introduce several novel
genes into the host strain simultaneously, we investigated transformation effi-
ciency, and developed procedures for simultaneous co-transformation of differ-
ent transgenes. These technological improvements enabled us to investigate,
rapidly and efficiently, the effect of introducing different enzymes into the T. re-
esei cellulase mix.
Fig. 16.10 Signal peptide effect on BG secre- secreted fraction, using 4-nitrophenyl b-D-
tion in T. reesei. T. reesei strains were geneti- glucopyranoside at pH 5. B. SDS–PAGE of
cally modified to heterologously express A. secreted proteins from the two T. reesei
oryzae BG, either behind the native A. oryzae strains. The positions of molecular weight
BG signal peptide, or behind a signal markers are labeled; the position of A. oryzae
peptide from the H. insolens Cel45A. BG is marked with an arrow. The gels were
A. Relative BG activity measured in the stained with Coomassie Brilliant Blue.
In addition to controlling gene expression transcriptionally, by using pro-

moters of different strengths, we focused on enhancing individual protein yield
by optimizing protein secretion. One example is replacement of the A. oryzae
BG signal sequence with a signal peptide from Humicola insolens Cel45A EG,
which improved the BG secretion in T. reesei (Fig. 16.10).
The objective of the research discussed here is to produce a single fermenta-
tion product with improved capability for converting the cellulose in pretreated
corn stover to glucose. The economic impact of these improvements on a corn-
stover-based biorefinery that produces fuel ethanol will be discussed below.
16.6
Range of Biobased Products
The goal of the biorefinery is to utilize renewable feedstocks in the production

of power, a variety of fuels, and chemicals. Several biobased products are already
on the market, including fuel, industrial and potable ethanol, sweeteners (high-
fructose syrups and sorbitol), organic acids (citric and lactic acids), MSG, lysine,
enzymes, polymers (xanthan gums), food and feed products, and specialty
chemicals, with annual multi-billion dollar sales. Biochemicals produced from
today’s biorefineries find their utility in diverse products such as adhesives, wall-
boards, resins, paper coatings and additives, textile sizing agents, foam packag-
ing materials, solvents, cosmetics, toiletries, paints, plastics, food, animal feed,
and pharmaceuticals.
In deriving products from raw biomaterials, biorefineries employ two funda-
mental technological platforms – the syngas platform based on thermochemical
gasification and the sugar-platform based on biochemical conversion to simple

sugars. Biotechnology is currently being applied to the sugar platform, with ex-
tensive focus on enzymatic saccharification and whole-cell microbial fermenta-
tion of the resulting sugars.
Oxygenated biomass-derived products, for example ethanol and other fer-
mented organic compounds, will be key precursors to many industrial chemi-
cals traditionally dependent on petroleum feedstocks. The tractability of a sugar-
platform for biosynthesis of products is apparent in comparison with a petro-
leum-based industry. Biorefinery-based products have an advantage in that oxy-
genated intermediates, for example adipic acid or ethylene glycol, can be pro-
duced more readily, because of the presence of oxygen in the sugar and starch
backbone. Because the raw materials can vary depending on the local source of
fermentable sugars, the fermentation of such products is more flexible.
16.6.1
Fuels
In the US, fuels make up approximately 70% of the carbon consumed annually
(more than 1.8 billion tons). Biobased ethanol and biodiesel are currently pro-
duced at higher cost than gasoline, and these renewable fuels account for less
than 2% of total liquid fuel consumption. Developing biobased liquid fuels will
require production cost reduction, including use of low-cost carbon sources, for
example agricultural/forestry byproducts and urban wastes.
As a direct result of research and development programs worldwide, fuel
ethanol production from sugarcane/beet (direct fermentation of material ob-
tained by crushing) and corn/wheat starch (by starch saccharification) has be-
come a viable industry. Yeast-fermentation of simple sugars, particularly sucrose
and glucose, has been used economically to produce ethanol, in amounts of
more than 20 million tons per year [36]. In Brazil, fermentable sugars are ob-
tained from mechanically processed sugar canes. In the US the sugars come
mainly from enzyme-degraded corn and wheat starch. To extend this industry
by tapping into inexpensive, readily available biomass materials such as corn
stover, wheat straw, and other agro/forestry byproducts as sources for ferment-
able sugars, intensive research is being conducted to develop enzymes which
convert lignocellulosics and other polysaccharides to simple sugars. In addition,
research efforts are being focused on obtaining and improving microbes for fer-
menting diverse sugars [37, 38].
Production of fermentable sugars from cellulose is currently more expensive
than their production from amylose (starch). This can partly be explained by
the relative recalcitrance of cellulose – amylase hydrolysis is intrinsically faster
and the kinetics of cellulase action require relatively higher loadings of enzyme.
As a result of significant funding by the US Department of Energy and col-
laboration between Novozymes and the US National Renewable Energy Labora-
tory (NREL), however, substantial progress has been made toward reducing en-
zyme cost for conversion of cellulose to fermentable sugars. After the research
EtOH)
Fig. 16.11 Progress in enzyme cost reduction for corn stover-

based ethanol production. Symbols denote milestones
achieved in reducing enzyme cost for production of ethanol
from pretreated corn stover, as validated by the National Re-
newable Energy Laboratory (NREL.) Costs are specific to the
corn stover feedstock (PCS) and NREL cost models.
and development advances mentioned above, more than 20-fold cost reduction
has been achieved (Fig. 16.11).
Importantly, the reduction of enzyme cost achieved in the last few years has
had a major affect on the estimated cost of producing fuel ethanol from corn
stover in a biorefinery. In 1999, the total expected cost for producing bioethanol
was dominated by enzyme cost; today enzyme cost is comparable with the esti-
mated costs of biomass feedstock collection or depreciation of capital. Using a
2004 “state of the technology” process cost estimate supplied by NREL, and an
enzyme cost of $0.50 per gallon ethanol produced, ethanol derived from bio-
mass has a total cost of about $2.50 per gallon [39]. Comparing costs for pro-
ducing ethanol from corn starch saccharification (by amylase) and fermentation
to projected costs for lignocellulosic-rich biomass-based ethanol production indi-
cates that the biomass based industry is becoming economically viable.
In addition to fuel ethanol, biomass-derived sugars can be fermented into
combustible “biogas.” Mainly methane, the fermentation is accomplished by
anaerobic bacteria, a technology already developed on small to medium-scale.
Fuel ethanol seems to be the future for a biomass-based energy industry, how-
ever. Future research and development effort will probably be focused on cellu-
lases with improved reactivity and stability, and on microbes with expanded su-
gar specificity (e.g. novel yeasts or pathway-engineered microbes capable of fer-
menting a variety of pentoses, hexoses, or oligomeric sugars).
16.6.2
Fine/Specialty Chemicals
Biomass is abundant in C6 sugars (hexoses), of which glucose is the most com-

mon, and C5 sugars (pentoses), of which xylose is predominant. Several organic
acids are currently produced from these starting sugars via fermentation by mi-
croorganisms. As noted in Fig. 16.12, lactic acid and succinic acid fermentation
platforms can lead to numerous value-added products. The C5 sugars can also
be fermented into a variety of xylose derivatives including itaconic acid, furfural,
furfuryl alcohol, and 2-hydroxymethyltetrahydrofuran (Table 16.2). Among var-
ious biomass-derived organic acids being studied, lactic acid and, to a lesser ex-
tent, 3-hydroxybutyric acid have attracted the most attention, because of their
use in the manufacture of plastics. In addition to biodegradability, reduced CO2
emission is another benefit of making these biopolymer/plastics [40].
16.6.3
Fuel Cells
A clean, efficient, and easily rechargeable energy source, fuel cells have been ac-
tively studied in the past few decades. In fuel cells, chemical energy is converted
into electricity by electrochemistry rather than combustion. Biofuel cells, in
which enzymes or entire microbial cells serve as electron-transfer catalysts, use
Fig. 16.12 Schematic diagram of biorefinery products based

on lactic and succinic acid fermentation platforms.
Table 16.2 Industrial bioproduct opportunities.
Technology Chemical Applications

platform
Sugar Lactic acid (currently biobased) Acidulant (food, drink), electroplating bath
fermentation additive, mordant, textile/leather auxiliary
Polylactide (currently biobased) Film and thermoformed packaging, fiber,
fiberfill
Ethyl lactate (currently Solvent, chemical intermediate
biobased)
1,3-Propanediol Apparel, upholstery, specialty resins, other
applications
Succinic acid Surfactants/detergents, ion chelators, food,
pharmaceuticals, antibiotics, amino acids,
vitamins
Succinic acid derivatives Surfactants, adhesives, printing inks,
magnetic tapes, coating resins, plasticizer/
emulsifiers, deicing compounds, herbicide
ingredients, chemical and pharmaceutical
intermediates
Bionolle 4,4 polyester Thermoplastic polymer applications
3-Hydroxypropionic acid Acrylates, acrylic fibers, polymers, resins
n-Butanol Solvent, plasticizers, polymers, resins
Itaconic acid Aluminum anodizing reagent, methyl acryl
relatively inexpensive, safe, and available “feeds”, for example alcohol or sugar,
instead of hydrogen gas or its volatile derivatives used in conventional fuel cells
[41]. Because of the safety and cost issues of conventional fuel cells, biofuel cells
are quickly emerging; in the near future, mini and micro-scale biofuel cells
could replace conventional batteries that power a variety of consumer and medi-
cal-implant devices. In the more distant future, scaled-up biofuel cells could
serve as a major industrial energy source.
Current research on biofuel cells focuses on how to improve performance in
terms of speed, output, reliability and durability. Enzyme-mediated electron-
transfer between feeds and electrodes is a focus of research and development.
For example, alcohol dehydrogenase and sugar oxidase are being studied as fa-
cilitators for electron-donation from alcohols or sugars (e.g. glucose) to an an-
ode, and laccase is being studied to facilitate the electron-accepting of O2 (air)
from a cathode. In the future, the focus of biofuel cell research may shift to
cheaper, more readily available feedstocks. Because both ethanol and glucose
can be generated from biorefineries, we may envisage a next-generation biofuel
cell that is powered by biomass. In such a fuel cell, biomass would be enzymati-
cally converted into glucose (e.g. by cellulase), which would then be enzymati-
cally oxidized on an electrode (e.g. by glucose oxidase). The extracted electrons
would run through a wire, perform electric work, and then be used to enzymati-
cally reduce O2 (e.g. by laccase). Biofuel cells may be set up as part of a biore-
finery plant that generates electric power from biomass, or be used for wilder-
ness exploration or military operations where fuel transport is logistically costly.
16.7
Biorefineries: Outlook and Perspectives
16.7.1
Potential of Biomass-based Material/Energy Sources
The goal of biorefinery is to utilize renewable feedstocks in the production of

power, a variety of fuels, and chemicals. One goal for the next generation of
biorefineries will be to more fully integrate facilities that can process a variety
of biomass feedstocks into a full range of biochemical products. This integration
would enable us to take advantage of the different product streams that might
emanate from renewable feedstocks by bioconversion of products (Fig. 16.13).
More than 100 million metric tons of fine, specialty, intermediate, and com-
modity chemicals are produced annually in the US. Today, only 10% of these
Fig. 16.13 The next biorefineries: Integrating a variety of

feedstocks for a multitude of end products.
16.7 Biorefineries: Outlook and Perspectives 381
are biobased. Thus, there is tremendous growth potential for biorefineries, if

the economics are competitive, if the environmental impact is favorable, and/or
if novel products are created.
16.7.2
Economic Drivers Toward Sustainability
The chemical industry currently consumes approximately 8% of total petroleum

and natural gas output to produce approximately 2500 products worth approxi-
mately $215 billion [36]. An attempt to replace part of the petrochemical pro-
cesses with biomass-based biotechnological processes is driven by the need to
curb greenhouse gas emission, upgrade agro/forestry industry value production,
and reduce reliance on fossil resources.
For biobased production to be economically feasible, the cost of producing the
biobased product must compete favorably with the comparable petroleum-de-
rived product. An additional or alternative economic driver for the biorefinery
could be synthesis of novel products that provide unique utility, and that are un-
available or uneconomical via petroleum-based chemistry. With regard to im-
proving economy, there are several technologies where cost efficiency should be
improved. These include:
· harvesting, collection and pretreatment of the biomass, which serve to unlock
the fermentable sugars and increase the carbon conversion to the desired
products;
· enzymatic conversion of the polysaccharides in the pretreated biomass stover
into glucose and other fermentable sugar monomers; and
· microbial fermentation of the sugars to the desired products.
Efficient enzyme catalysis is one of the primary economic barriers in the chal-
lenge to design an overall cost-effective process for converting biomass into fer-
mentable sugars. The research efforts described here, and research efforts in
similar work performed at Genencor International, have specifically focused on
improving the efficiency of enzymatic hydrolysis, and progress thus far looks
quite promising. Further work on metabolic engineering of microbial produc-
tion strains should continue, as should efforts to better integrate biomass pre-
treatment, enzyme hydrolysis, and fermentation to avoid complications result-
ing from isolated efforts.
Several major problems must be solved to enable commercialization of var-
ious biorefineries. To satisfy the optimum operating conditions of enzymes and
microbes, raw biomass materials collected from diverse regions under diverse
climates must be examined to assess the impact of biomass feedstock variability
on pretreatment, enzymatic conversion, and fermentation. Optimizing these
steps and integrating them into a robust, low cost, efficient, sustainable, and
value-generating material–energy cycle is highly challenging, yet offers great so-
cial, economic, and environmental promise.
References
1 Finlay, M. Old Efforts at New Uses: A lulosomes. Curr Opin Struct Biol 8, 548–
Brief History of Chemurgy and the 57 (1998).
American Search for Biobased Materials. 13 Mosier, N. S., Hall, P., Ladisch, C. M. and
J. Ind. Ecology 7, 33–46 (2004). Ladisch, M. R. Reaction kinetics, molecu-
2 Rooney, T. Lignocellulosic Feedstock Re- lar action, and mechanisms of cellulolyt-
source Assessment. (National Renewable ic proteins. Adv Biochem Eng Biotechnol
Energy Laboratory, Golden, CO, 1998). 65, 23–40 (1999).
3 Lee, Y. Y., Lyer, P. and Torget, R. W. Di- 14 Maheshwari, R., Bharadwaj, G. and
lute-Acid hydrolysis of Lignocellulosic Bhat, M. K. Thermophilic fungi: their
Biomass. Advances in Biochemical Engi- physiology and enzymes. Microbiol Mol
neering and Biotechnology 65, 93–115 Biol Rev 64, 461–88 (2000).
(1999). 15 Schulein, M. Protein engineering of cel-
4 Zaldivar, J., Nielsen, J. and Olsson, L. lulases. Biochim Biophys Acta 1543, 239–
Fuel ethanol production from lignocellu- 252 (2000).
lose: a challenge for metabolic engineer- 16 Lynd, L. R., Weimer, P. J., van Zyl, W. H.
ing and process integration. Appl Micro- and Pretorius, I. S. Microbial cellulose
biol Biotechnol 56, 17–34 (2001). utilization: fundamentals and biotechnol-
5 Teymouri, F., Laureano-Perez, L., Aliza- ogy. Microbiol Mol Biol Rev 66, 506–77
deh, H. and Dale, B.E. Ammonia fiber (2002).
explosion treatment of corn stover. Appl 17 Bourne, Y. and Henrissat, B. Glycoside
Biochem Biotechnol 113–116, 951–63 hydrolases and glycosyltransferases: fam-
(2004). ilies and functional modules. Curr Opin
6 Allen, S. G., Kam, L. C., Zemann, A. J. Struct Biol 11, 593–600 (2001).
and Antal, M. J. J. Fractionation of Sugar 18 Davies, G. J. Structural studies on cellu-
Cane with Hot, Compressed, Liquid lases. Biochem Soc Trans 26, 167–73
Water. Ind Eng Chem Res 35, 2709–2715 (1998).
(1996). 19 Shimon, L. J. et al. Structure of a family
7 Liu, C. and Wyman, C. E. Impact of fluid IIIa scaffoldin CBD from the cellulo-
velocity on hot water only pretreatment some of Clostridium cellulolyticum at 2.2
of corn stover in a flowthrough reactor. A resolution. Acta Crystallogr D Biol Crys-
Appl Biochem Biotechnol 113–116, 977–87 tallogr 56 Pt 12, 1560–8 (2000).
(2004). 20 Zhang, Y. H. and Lynd, L. R. Kinetics and
8 Nagle, N. J. et al. Efficacy of a hot wash- relative importance of phosphorolytic
ing process for pretreated yellow poplar and hydrolytic cleavage of cellodextrins
to enhance bioethanol production. Bio- and cellobiose in cell extracts of Clostri-
technol Prog 18, 734–8 (2002). dium thermocellum. Appl Environ Micro-
9 Varga, E., Schmidt, A. S., Reczey, K. and biol 70, 1563–9 (2004).
Thomsen, A. B. Pretreatment of corn 21 de Vries, R. P. and Visser, J. Aspergillus
stover using wet oxidation to enhance enzymes involved in degradation of
enzymatic digestibility. Appl Biochem Bio- plant cell wall polysaccharides. Microbiol
technol 104, 37–50 (2003). Mol Biol Rev 65, 497–522 (2001).
10 Wilson, D. and Irwin, D. Genetics and 22 Kirk, T. K. and Farrell, R. L. Enzymatic
properties of cellulases. Adv Biochem Eng “combustion“: the microbial degradation
Biotechnol 65, 1–21 (1999). of lignin. Annu Rev Microbiol 41, 465–
11 Tomme, P., Warren, R. A. and Gilkes, 505 (1987).
N. R. Cellulose hydrolysis by bacteria and 23 Gronqvist, S. et al. Lignocellulose proces-
fungi. Adv Microb Physiol 37, 1–81 sing with oxidative enzymes. in Applied
(1995). Enzymology to Lignocellulosics (eds. Mans-
12 Bayer, E. A., Chanzy, H., Lamed, R. and field, S.D. and Saddler, J.N.) 46–65 (Am.
Shoham, Y. Cellulose, cellulases and cel- Chem. Soc, Washington, DC, 2002).
References 383
24 Kubicek, C., Eveleigh, D. E., Esterbauer, 32 Stemmer, W. P. Rapid evolution of a pro-

E., Steiner, W. and Kubicek-Pranz, E. Tri- tein in vitro by DNA shuffling. Nature
choderma reesei Cellulases, (Royal Society 370, 389–91 (1994).
of Chemistry, Cambridge, UK, 1990). 33 Lawoko, M., Nutt, A., Henriksson, H.,
25 Sternberg, D., Vijayakumar, P. and Gellerstedt, G. and Henriksson, G.
Reese, E. T. beta-Glucosidase: microbial Hemicellulase activity of aerobic fungal
production and effect on enzymatic cellulases. Holzforschung 54, 497–500
hydrolysis of cellulose. Can J Microbiol (2000).
23, 139–47 (1977). 34 Vlasenko, E., Xu, F. and Cherry, J. R.
26 Boer, H. and Koivula, A. The relationship Thermostability, substrate specificity and
between thermal stability and pH opti- hydrolysis of cellulose by endoglucanases
mum studied with wild-type and mutant from families 5,7, and 45 of glycoside
Trichoderma reesei cellobiohydrolase hydrolases. in 25th Symposium on Bio-
Cel7A. Eur J Biochem 270, 841–848 (2003). technology for Fuels and Chemicals (Breck-
27 Baker, J. O. et al. Thermal denaturation enridge, CO, 2003).
of Trichoderma reesei cellulases studied by 35 Eveleigh, D. E. and Montenecourt, B. S.
differential scanning calorimetry and Increasing yields of extracellular en-
tryptophan fluorescence. Appl Biochem zymes. Adv Appl Microbiol 25, 57–74
Biotechnol 34/35, 217–231 (1992). (1979).
28 Kraulis, P. J. et al. Determination of the 36 Danner, H. and Braun, R. Biotechnology
three-dimensional solution structure of for the production of commodity chemi-
the C-terminal domain of cellobiohydro- cals from biomass. Chem Soc Rev 28,
lase I from Trichoderma reesei. A study 395–405 (1999).
using nuclear magnetic resonance and 37 van Wyk, J. P. H. Biotechnology and the
hybrid distance geometry-dynamical sim- utilization of biowaste as a resource for
ulated annealing. Biochemistry 28, 7241– bioproduct development. Trends in Bio-
7257 (1989). technology 19, 172–177 (2001).
29 Divne, C., Stahlberg, J., Teeri, T. T. and 38 Himmel, M. et al. Advanced bioethanol
Jones, T.A. High-resolution crystal struc- production technologies: A perspective.
tures reveal how a cellulose chain is in Fuels and Chemicals from Biomass, Vol.
bound in the 50 A long tunnel of cello- 666 (eds. Saha, B. C. and Woodward, J.)
biohydrolase I from Trichoderma reesei. 2–45 (American Chemical Society, Wash-
J Mol Biol 275, 309–25 (1998). ington, DC, 1997).
30 Mattinen, M. L. et al. Three-dimensional 39 Ibsen, K. Personal Communication. (Na-
structures of three engineered cellulose- tional Renewable Energy Laboratory,
binding domains of cellobiohydrolase I Golden, CO, 2004).
from Trichoderma reesei. Protein Sci 6, 40 Ohara, H. Biorefinery. Appl Biochem Bio-
294–303 (1997). technol 62, 474–477 (2003).
31 Mattinen, M. L., Linder, M., Drakenberg, 41 Wong, T. S. and Schwaneberg, U. Protein
T. and Annila, A. Solution structure of engineering in bioelectrocatalysis. Cur-
the cellulose-binding domain of endoglu- rent Opinion in Biotechnology 14, 590–596
canase I from Trichoderma reesei and its (2003).
interaction with cello-oligosaccharides.
Eur J Biochem 256, 279–86 (1998).
385
17
Biocatalytic and Catalytic Routes for the Production of Bulk
and Fine Chemicals from Renewable Resources
17.1
Introduction
17.1.1
Renewable Resources
The most important sources of renewable resources for industry are oil plants,
starch plants, sugar plants, energy plants, and wood, but also waste and resi-
dues from agriculture and industry. The corresponding substrates for conver-
sion processes are manifold and belong to such heterogeneous substance
classes as oils, fats, glycerol, lignocellulose, cellulose, starch, inulin, sugar, com-
plex biomass, etc. Fats and oils are already being used as feedstock in industry
at a level of 15 million t a–1. The corresponding products are mainly applied in
plastics, paints, lacquers, biotensides, and energy (as biodiesel). The potential of
carbohydrates (starch, sugar, cellulose) is far from being fully exploited. In the
year 2002/2003, approximately 143 million tons of sugar were produced world-
wide (Germany 4 million tons). Of these, only 70 000 t (1.7%) were industrially
employed as renewable resources in the pharmaceutical and chemical sector in
Germany [1].
Because of the substantial quantities of (ligno)cellulose available, utilization
of the material of biomass (wood, straw, waste, and residues) has huge poten-
tial. Countries with large amounts of wood, for example Canada, the USA,
Scandinavia, or Austria, invest much effort to utilize this potential. There is
much need for research. If it were possible to establish highly efficient enzy-
matic pulping processes, numerous bulk products (ethanol, butanol, lactic acid,
etc.) could be produced at competitive prices.
ISBN: 3-527-31027-4
386 17 Biocatalytic and Catalytic Routes for the Production of Bulk and Fine Chemicals
17.1.2
Products
In 2001 the organic chemicals produced worldwide were estimated to be worth

almost 1,900 billion Euros. Asia leads, with 586 billion Euros, followed by the
European Union (519 billion Euros) and North America (508 billion Euros). The
German chemical industry, with approximately 25% of the total European
amount, recorded sales in 2002 of 133 billion Euros.
In respect of price and amount, the products can be roughly classified into two
sectors: (1) bulk chemicals or commodities (> 1 million t a–1, < US$2–4 kg–1), and
(2) fine and specialty chemicals (< 1 million t a–1, > US$2–4 kg–1). Worldwide the
sales of fine and specialty chemicals is approximately 250 billion US$ (Europe
120 billion US$), which, for Europe, is approximately 20% of all the chemical
products sold.
17.1.2.1 Bulk Chemicals and Intermediates

In bulk chemical synthesis the use of renewable resources as feedstock is rather
limited. Relevant future applications include biobased synthesis gas, and prod-
ucts thereof, and a few selected products, for example ethanol. The potential
use of renewable resources for the production of intermediates, for example,
monomers or plasticizers for polymers, is mainly a question of the price of
crude oil. At a crude oil price (2004) of approximately 50 US$ per barrel
(0.25 1 L–1), biotechnological processes can hardly compete. With rising crude
oil prices, chemical and biotechnological processes based on renewables become
increasingly competitive with petrochemical-derived products, especially for
product prices > 2 US$ kg–1.
17.1.2.2 Fine Chemicals and Specialties

For fine chemicals the price of the product is not so critical – product function-
ality and purity, and quality assurance, are much more important. Extremely
high prices are charged for pharmaceuticals, followed by cosmetics and food in-
gredients and some intermediates for industry. If such high prices are accepted
by the market, sustainability forces the chemical industry to use economical
and environmentally friendly processes. Thus largest chances of broader appli-
cation of renewable resources, either by biotechnological or chemical processes,
are in fine chemicals. Often, biotechnology is the only way to obtain the re-
quired product.
17.2
Historical Outline
Until 1930 many important bulk products, for example fuels (ethanol, butanol),
organic acids (acetic acid, citric acid, lactic acid), and other basic chemicals, were
mainly produced from renewable resources. Process engineering was limited to
fermentation by use of fungi or bacteria. In the past some basic chemicals (for ex-
ample butanol and acetone) were produced exclusively by fermentation. With the
development of petroleum chemistry, however, they were replaced by chemical–
technical products. In the production of other chemicals (ethanol, citric acid, lactic
acid, and acetic acid), biotechnological techniques have always been predominant,
because the chemical–technical alternatives are not economical.
There are several prognoses of the amount and range of worldwide petroleum
reserves. Most of the experts predict that maximum production will be achieved
in the next few decades [2] (Fig. 17.1). Countries with high energy demands and
limited resources, for example the USA, have already realized this and are in-
vesting much effort in appropriate research [3]. In the USA, for example, substi-
tution of fossil fuel with renewable resources in the production of liquid fuels
and organic chemicals is envisaged to be 50% and 90%, respectively, by 2090
[4]. More realistic prognoses expect an increase to 25% in the next 30 years [5].
Shell Oil, for example, intends to provide 30% of the world‘s chemical and en-
ergy needs by use of biomass by 2050, corresponding to nearly US$ 150 billion
[6]. DuPont, one of the largest manufacturers of plastics, intends to produce
25% of its products from renewable resources by the year 2010 [7]. Figure 17.2
summarizes several prognoses for the USA.
Fig. 17.1 Prognoses of crude oil production and estimated ultimate recovery (EUR) [2].
Fig. 17.2 Biotechnologically produced liquid fuels and organic

chemicals in the USA as share of total production [3]
(estimated data for 2090).
17.3
Processes
Large-scale chemical processes are usually very sophisticated, because environ-

mentally friendly catalytic conversions are used almost exclusively. Although
they are usually performed at high temperatures and pressures, and although
substrates and products are often detrimental to health or are environmentally
significant, they can be regarded as clean processes. Nevertheless, chemical pro-
cesses with low annual production volumes and/or multi-step synthetic path-
Table 17.1 Typical problems of biotechnological processes and possible solutions.
Problem Solution
Appropriate biocatalyst not available Cell screening

Strain optimization, mutation/selection
Metabolic pathway design (MPD)
Productivity too low High-cell density fermentation
Cell recycle
Immobilization
Substrates too expensive Cell screening, substrate screening
Cheap substrates often not suitable Genetic engineering, MPD
Unwanted by-products Strain optimization, mutation/selection
Metabolic pathways not optimum
Low product concentration (production
inhibition)
Product recovery In situ processes
Process control On-line analysis
17.3 Processes 389
ways, especially in the fine and specialty segment, are less efficient and less en-
vironmental friendly. In this segment more classic organic chemistry is used
than catalytic processes, resulting in significant waste generation, emissions,
and energy consumption.
Biotechnological processes usually occur under mild conditions. Biocatalysts,
substrates, intermediates, and by-products, and the product itself, are biodegrad-
able. Water is usually used as a solvent. There are also frequent disadvantages,
however, including low product concentration, low productivity and, hence, high
recovery costs. Table 17.1 lists some problems and possible solutions. Some
products are only accessible chemically, whereas biotechnology is more appro-
priate for others (chiral substances, some vitamins and amino acids, highly se-
lective transformations with polyfunctional substrates, such as sugars). Numer-
ous syntheses are conducted exclusively using enzymes (lipases, amylases, pro-
teases, and, also, increasing in the future, cellulases). To establish a large-scale
process based on a biochemical reaction it is preferable to have means available
to hold back the catalyst (i.e. enzyme) in the bioreaction vessel. By immobilizing
catalysts, for example growing, resting, or dead cells or enzymes, it is possible
to retard them.
17.3.1
Immobilization
Different types of immobilization procedure have been developed for this pur-
pose, as is shown in Fig. 17.3 [8]. Besides the advantage of easy retention, im-
mobilized catalysts are also often more stable with regard to, for example, pH
and temperature. When entrapped the catalysts are, moreover, protected against
other bacteria and thus processes can run under non-sterile conditions, because
potential contamination is washed out while the favored catalyst is specifically
protected.
Encapsulation of catalysts also has disadvantages, however: during the immo-
bilization process the catalyst may be inactivated by physicochemical or physio-
logical effects. Even if this does not happen, the overall activity of the immobi-
lized system could be lower than that of the free catalyst, because of diffusion
Fig. 17.3 Different ways of im-

mobilizing biocatalysts [8].
limitations. To minimize this effect the particles should be small and applicable
for the later application.
Examples of processes developed, investigated, and optimized at the Institute
of Technology and Biosystems Engineering are discussed in the sections below.
17.3.2
Biocatalytic Routes from Renewable Resources to Solvents or Fuels
17.3.2.1 Ethanol Production with Bacteria or Yeasts?
Introduction Ethanol can be used as a liquid energy carrier, fuel additive, and
feedstock in the chemical industry. Because of the costs – except for use in
foods or stimulants – biotechnological production in Europe has not yet been
profitable. In May 2003 an EU directive on the use of biofuels set a minimum
level of 5.75% bio-fuels (including bioethanol) for all transport fuels sold by
2010 [9]. Thus ethanol will be more highly in demand in the future. The bio-
technological production of ethanol – at the beginning of the 20th century
mainly for fuel – is currently experiencing a renaissance.
In the year 2001, worldwide annual ethanol production amounted to more
than 30 billion liters. The leading role played by Brazil, where bioethanol from
sugar cane has been used for more than 25 years, has currently been taken over
by the USA, which will probably continue to occupy first place. In the USA,
mainly corn or wheat starch is used. In the EU, with a total of approximately
2 billion liters, France (approx. 0.8 billion liters) is the largest producer; Ger-
many produces only approximately 0.3 billion liters [10].
The Process Ethanol is produced by fermentation with yeasts or bacteria

(Fig. 17.4). The classic route by use of yeasts has been known for thousands of
years and is one of the oldest biotechnological processes used by mankind. Sub-
strates are sugar-based feedstocks (sucrose, glucose, molasses) and also starch
hydrolyzates. A study recently promoted by the German Research Foundation
(DFG) determined costs based on grain starch for the German market. Accord-
ing to this study, substrate costs account for up to 50% of total production costs
[11].
To find cheaper sources of renewable raw materials, various routes are being
followed:
· A search for organisms which, in addition to glucose, can utilize other sugars,
for example pentoses (xylose, arabinose). Such sugars are the products of hy-
drolysis of hemicellulose, the basic constituent of wood, straw, and a variety
of other plant residues [12–14].
· Screening for enzymes which split the raw materials into usable sugars as ef-
ficiently as possible, either directly or in coupled processes [15].
· Alteration of known or new organisms by genetic engineering (genetically
modified microorganism, GMO) to enlarge their substrate spectrum [16, 17].
In the USA a new technique is currently being implemented on a pilot scale
17.3 Processes 391
Fig. 17.4 Process scheme for the production of ethanol from renewable resources.
[18]. The process uses genetically modified Zymomonas mobilis, which was
given the ability to use pentoses (xylose) in addition to hexoses (glucose, fruc-
tose). This enables inexpensive substances which could not previously be
used, for example, rice straw, to be transformed into ethanol with high yields
[19].
Reduction of the process costs can be achieved by several methods:
· Screening or genetic engineering of microorganisms with the goal of increas-
ing productivity, ethanol tolerance (and hence achievable final product concen-
tration), and yield. For example, use of the bacterium Zymomonas mobilis in-
stead of conventional yeasts (Saccharomyces spp.) also results in increased
product yields, besides the fivefold higher productivity. In addition, immobili-
zation boosts volumetric productivity and, again, product yield. Furthermore,
product tolerance, and with it the final ethanol concentration, is enhanced.
Essential data for the process are listed in Table 17.2.
Table 17.2 Ethanol production with yeast or bacteria.
Biocatalyst Productivity in kg ethanol m–3 h–1
Suspended cells Immobilized cells
Yeast 0.5–2 10–30

Saccharomyces cerevisiae
Bacterium 4–5 50–80
Zymomonas mobilis
Fig. 17.5 Cell-Immobilization procedure for the production of

lens-shaped particles (LentiKats).
· Development of new fermentation methods which, in particular, include the

use of immobilized (entrapped) cells, so that the process can be run under
non sterile conditions or the biocatalyst can be easily recycled. The latter is
particularly important for high-performance strains or genetically engineered
microorganisms.
Fig. 17.6 Pilot plant for the continuous ethanol production with immobilized cells.
17.3 Processes 393
Fig. 17.7 Process design of an ethanol production plant.

Effect of type of biocatalyst on plant size. Source:
BMA–Starcosa 2001, Brunswick, Germany, capacity 60 000 L
ethanol day–1.
For example, new immobilization technology based on lens-shaped particles

(Fig. 17.5) [20] was adapted for production of ethanol from molasses in coopera-
tion with the company BMA–Starcosa (Braunschweig, Germany). Continuous
and stable ethanol production from untreated molasses was achieved over several
months, even under nonsterile conditions. A pilot plant (Fig. 17.6) has been run-
ning at BMA–Starcosa, under the described conditions, since 2003. Figure 17.7
shows clearly the extent to which plant size, and consequently investment costs,
can be reduced solely by introduction of immobilized cell systems for the biotech-
nological ethanol production. In combination with the above-mentioned improve-
ments ethanol could be produced economically in the future.
17.3.3
Biocatalytic Route from Glycerol to 1,3-Propanediol
17.3.3.1 Introduction
One of the applications of 1,3-propanediol (PD) is its use as a diol component
in the plastic polytrimethyleneterephthalate (PTT), a new polymer with proper-
ties comparable with those of Nylon. It is preferably used for carpets (Corterra
by Shell) or special textile fibers (Sorona by DuPont). Further applications are
appearing in polyester resins, mainly in the paint industry.
17.3.3.2 The Process

PD can be produced biotechnologically from glycerol with the aid of bacteria
(Fig. 17.8). Glycerol is mainly a by-product of fat splitting and biodiesel produc-
tion. Further growth of the biodiesel market would result in a fall in the price
of glycerol. One of the largest biodiesel producers in Germany (Nevest,
Schwarzheide) filed for insolvency in December 2003, partly because of the de-
cline in the price of glycerol from 1000 1/t to nearly half [21]. Some companies
even pay to dispose of their glycerol. Glycerol–water from RME production, in
particular, would be an interesting raw material if it could be used in fermenta-
tion without further pretreatment. Another method would be the utilization of
glucose instead of glycerol, which would provide independence from the fluctu-
ating glycerol market. Because no microorganisms are known which directly
convert glucose into 1,3-propanediol, however, this technique requires mixed
cultures or microorganisms designed by genetic engineering. Both possibilities
have been examined. The gene-technological variant is favored by DuPont in co-
operation with Genencor and is on the verge of technical implementation [22].
Under some conditions, however, the classic technique based on glycerol can
be quite interesting technically and economically. A concerted, extensive search
for new microorganisms (screening) and improved process design (fed-batch
with pH-controlled substrate dosage) enabled product concentrations, which
were relatively low at a maximum of 70 to 80 g L–1 as a result of product inhibi-
tion (Fig. 17.9), to be increased to more than 100 g L–1 (Fig. 17.10). Another ad-
vantage of the new technique and the new isolated strains is the use of low-
priced crude glycerol or glycerol–water (Fig. 17.11). This is a factor which
should not be underestimated and has a direct effect on product costs
Fig. 17.8 Process scheme for 1,3-propanediol production from

plant oil via glycerol or glycerol–water as a byproduct of
biodiesel production.
17.3 Processes 395
Fig. 17.9 1,3-Propanediol production from pharma glycerol by

use of a commercially available strain (pH-controlled fed
batch, mineral medium with YE, 328C, pH 7.2).
Fig. 17.10 1,3-Propanediol production from pharma glycerol

with a new strain from screening (35 8C, pH 7.0, other
conditions as for Fig. 17.9).
(Fig. 17.12). Further on, use of immobilized cells (LentiKats; Section 17.3.2.1,
above) rather than freely suspended cells, enables productivity to be increase
from approximately 2 to 30 gPD L–1 h–1.
Comparison of current (chemical) techniques with the new biotechnical tech-
niques based on different substrates and glycerol qualities (= raw glycerol costs)
Fig. 17.11 1,3-Propanediol production from raw glycerol–water

with a new strain from screening (conditions as for
Fig. 17.10).
Fig. 17.12 Cost comparison of industrial 1,3-propanediol

production, effect of substrate costs, (*) glucose or glycerol,
respectively (Data from PERP 1998, substituted).
17.3 Processes 397
shows that biotechnology might be competitive with chemical techniques if

crude glycerol is used (PERP 1998).
17.3.4
Biocatalytic Route from Inulin to Difructose Anhydride
Inulin is a linear b-2,1-linked polyfructane terminated with a glucose residue.
Large amounts of inulin are contained in the roots and tubers of crops like dah-
lia, chicory, and Jerusalem artichoke. Inulins have a very limited market thus
far, mainly because of the high cost of expensive separation and purification
steps. For 1.5 to 2 1 kg–1 it is approximately four times as costly as competing
glucose, starch, or sucrose. This also explains why short oligofructoses are
synthesized enzymatically from sucrose for probiotic products rather than by
partial hydrolysis of inulins. Future use of inulin-derived products is thus either
in high-value markets, for example the functional food segment, or by convert-
ing inulin into intermediates which can be separated and purified at lower cost.
One promising compound derived from inulin for this purpose is difructose an-
hydride (DFA III, Fig. 17.13).
DFA III can be the basis for plastics and tensides. It can be crystallized as
easily as sucrose after an ion-exchange step and hence can be produced at a
price well below that of inulin. So far DFA III has not been introduced to the
market, because no efficient enzyme and biotechnical process was available for
the necessary bioconversion of inulin.
To produce DFA III on a technical and industrial scale, large amounts of en-
zyme are needed. The following sections introduce a strategy showing how this
problem can be solved [23, 24].
Chicory
Chemical feedstock
Fig. 17.13 Process for production of DFA III from inulin.

17.3.4.2 Enzyme Screening

From 65 tested samples from extremophile locations, approximately 400 bacteri-
al strains were obtained and investigated further. Four strains were found to
produce DFA III and one strain (Buo141) identified as an Arthrobacter sp. ex-
pressed an enzyme, stable for weeks at elevated temperatures of 60 8C whereas
the activity of the other strains declined within a few hours (Fig. 17.14). The
new strain grows aerobically at ambient temperatures and secreted inulase II
extracellulary but only with low activity, too low for economical industrial pro-
cess. To overcome these limits, genetic engineering was used.
17.3.4.3 Genetic Engineering

To increase production of the enzyme the gene encoding for the inulase II (ift
gene) should be transferred to and expressed in an E. coli host. To gain access
to the bacterial gene suitable primers for a polymerase chain reaction (PCR)
were needed. For primer design, only two highly divergent sequences of inulase
proteins were known.
A special primer design based on phylogenetic analysis substantially acceler-
ated isolation of the ift gene. The complete ift gene was obtained by screening
the genomic library with this probe. As a result a plasmid was constructed
which expressed an enzyme of 477 amino acids when transferred to E. coli. A
cell-free extract of such a culture had an activity of 3000 U L–1, whereas most of
this activity was detected intracellularly. In Arthrobacter the inulase II enzyme is
expressed as an extracellular enzyme. Transport through the cell membrane is
accomplished by means of a specific signal-transfer peptide which is part of the
ift gene. Because of phylogenetic differences between the species Arthrobacter
and Escherichia the transfer-peptide does not work in E. coli. In E. coli the en-
zyme remains intracellular, as was shown by analysis of the activity of disrupted
cells and the supernatant of cultivations. Exact removal of the complete transfer-
peptide resulted in a one-hundredfold increase in activity. A further increase in
Fig. 17.14 Screening of inulinase II producers: comparison of long-term stability.

17.3 Processes 399
Fig. 17.15 Increase of inulinase II activity by genetic engineering.
activity of approx. 35% was possible because of a point-mutation induced by er-

ror-prone PCR. In position 221 of the enzyme a glycine was exchanged with ar-
ginine. (Fig. 17.15). To obtain sufficient large quantities of the enzyme the ge-
netically modified organism was fermented.
17.3.4.4 Fermentation of the Recombinant E. coli

The recombinant E. coli pMSiftOptR was fermented using an inexpensive tech-
nical medium. During the fermentation inulase activity was monitored. A final
biomass concentration of 11 g L–1 (dry weight) and an overall activity of
1 760 000 U L–1 was measured (Fig. 17.16). Because high-density fermentations
of E. coli are known to reach biomass concentrations of approximately 100 g L–1
it seems possible that after optimizing the fermentation step activity of at least
15 ´ 106 U L–1 is possible.
Fig. 17.16 Fermentation of the redombinant E. coli pMSiftOptR for production of inulinase II.
17.3.4.5 Enzyme Immobilization and Scale-up

For immobilization of inulinase II, the molecular weight of the enzyme must
be increased, otherwise the enzyme will be lost by diffusion out of the particle.
Inulase II was therefore flocculated from a cell-free extract by co-crosslinking
with glutardialdehyde and chitosan (Fig. 17.17). Fortunately, the enzyme is not
damaged by glutardialdehyde. The crosslinked enzyme was then immobilized
in alginate beads of different diameter in the range 500 to 800 lm and analyzed
to compare their activity with regard to bead diameter (Fig. 17.18.) Although
beads 500 lm in diameter had 54% activity compared with the value when the
same beads were dissolved, beads of 600 lm had only 42% of the activity. For
850-lm beads only one third of the original activity was observed. This shows
once again the benefits of using sufficiently small particles when working with
encapsulated systems.
Fig. 17.17 Enzyme-immobilization: cross-linking of inulase II

for molecular-weight enhancement to prevent enzyme loss by
diffusion.
Fig. 17.18 Effect of bead diameter on the activity of encapsu-

lated inulase II. Dissolved beads means that the formerly im-
mobilized enzyme is released to prevent diffusion limitation.
17.3 Processes 401
a)
b)
Fig. 17.19 Principle of JetCutting, and high-speed-motion pic-
ture of the cutting process showing the effect of correct ad-
justment.
To accomplish the task of producing the desired small droplets from the very
viscous alginate–enzyme solution, a novel JetCutter technology was used. In
comparison with other techniques, for example blow-off devices, vibrating noz-
zles or electrostatic forces, the JetCutter uses mechanical cutting of a continu-
ous jet of liquid to produce small droplets; this is shown in Fig. 17.19 [25].
17.3.4.6 Summary
Complete process development has been shown, starting from screening for an
enzyme with the desired properties – an inulase II converting inulin to DFA III
at elevated temperatures (60 8C). After screening and successful optimization of
the enzyme by genetic engineering and construction of a genetically modified
organism which expresses the enzyme in very high numbers, this strain was
fermented to furnish large amounts of enzyme for immobilization. After immo-
bilization by entrapment of the enzyme in hydrogel particles, an industrially
applicable enzyme formulation with an activity of 196 U g–1 (wet matter) is ob-
tained.
17.3.5
Chemical Route from Sugars to Sugar Acids
The increasing use of low-molecular-mass carbohydrates (sugars) for production
of chemical building blocks is of great interest both economically and ecologically.
Besides the biotechnological routes presented above, chemical catalysis has great
potential for functionalization of sugars to furnish fine chemicals or building
blocks for the chemical industry. Sugar oxidation in the presence of supported no-
ble metal catalysts has always been an attractive subject. The products are biode-
gradable compounds and have many potential applications. Gluconic acid, for ex-
ample, is of great industrial interest. Annual production amounts to nearly
80 000 t; this is used as a noncorrosive and biologically degradable complexing
agent in industry, and in numerous applications in food, pharmaceuticals, and
cosmetics. Lactobionic acid and maltobionic acids – the oxidation products of
the corresponding sugar monomers lactose and maltose, respectively – can be
used in the detergent and pharmaceutical and food industries.
Gluconic acid is mainly produced biotechnologically, because chemical cata-
lysts are inadequate. With gold catalysts, this could be changed in the future
(Fig. 17.20).
Table 17.3 gives an overview of some important milestones in chemical cata-
lytic glucose oxidation. The work of Prati and Rossi describes an charcoal-sup-
ported gold catalyst for glucose oxidation, which exceeds the previous Pt- and
Fig. 17.20 Chemical catalytic conversion of carbohydrates from renewable resources.

17.3 Processes 403
Table 17.3 Milestones in the chemical catalytic oxidation of carbohydrates.
Topic Description Working group
Beginnings “Mannitol acid” with platinum-Mohr Group-Besanz (1861)

Oxidation of carbohydrates on platinum Wieland (1912)
interpreted as oxidative dehydration
Systematic Reactivity sequence of the moieties in carbo- Heyns and Paulsen
hydrates on platinum (1950s/60s)
Bimetal catalysts Doping of platinum and palladium catalysts Kuster/Bekkum
with bismuth or lead (problem: long-term (1980s)
stability, Bi/Pb leaching)
Gold catalysts Au/C for oxidation of glucose to gluconic Prati and Rossi (2001)
acid highly active and selective (problem:
long-term stability)
Enhancement of long-term stability by new Mirescu and Prüße
preparations on TiO2 (2003)
Pd-based catalysts in activity and selectivity, which is nearly 100% to gluconic

acid [26]. Nevertheless long-term stability is still far too low for industrial appli-
cation. A breakthrough could be achieved by the use of titania- or alumina-sup-
ported gold catalysts, which combine high activity and selectivity with long-term
stability, which seems sufficient for industrial applications.
17.3.5.2 Gold Catalysts

For glucose oxidation a comparative study of catalysts used industrially for the
same reaction shows the high selectivity of the new gold catalysts (Fig. 17.21).
This gold catalyst is highly active over a wide range of pH, temperature, and
glucose concentrations. Except for formation of small amounts of fructose by
isomerization at temperatures above 70 8C and pH > 10, selectivity to glucose al-
ways exceeds 99.5%. These results confirm the outstanding properties of the
gold catalysts in this reaction.
Whereas the charcoal-supported catalysts of Prati and Rossi lose 50% activity
after four replicate batches, the TiO2-supported catalysts remain stable. After 17
replicate batches no activity loss was observed. The mean activity was
425 mmolglucose gmetal–1 min–1, corresponding to an overall productivity of
5 kggluconic acid ggold–1 h–1.
In addition to glucose, several other carbohydrates were tested with both TiO2
and Al2O3-supported gold catalysts. Fast and complete reaction occurs with pen-
toses (arabinose, ribose, xylose, and lyxose), hexoses (mannose, rhamnose, galac-
tose, and n-acetylglucosamine), and some di- and oligosaccharides (lactose, cello-
biose, melibiose, maltose, maltotriose, maltotetrose) each with a selectivity of vir-
tually 100% (Fig. 17.22, Table 17.4). No reaction occurred with ketoses or blocked
aldoses (fructose, isomaltulose, methylglucose, sorbose, sucrose, and trehalose).
Fig. 17.21 Catalytic oxidation of glucose: screening of catalysts and selectivity proven by HPLC.
Fig. 17.22 Catalytic oxidation of other carbohydrates:

galactose, xylose, maltobiose and maltotriose with Au/TiO2
catalyst.
References 405
Table 17.4 Catalytic oxidation of carbohydrates (conditions:

cat: 0.6% Au/Al2O3, 40 8C, pH 9, 100 mmol substrate, conver-
sion > 99%).
Type Substrate Activity Activity Selectivity (%)

(mmol min–1 (kgproduct* h–1
gMe–1) gMe–1) a)
Pentose (C5) Arabinose 334 4.1 > 99.9

Lyxose 145 1.8 > 99.9
Ribose 234 2.8 > 99.9
Xylose 251 3.1 > 99.9
Hexoses (C6) Galactose 338 4.8 > 99.9
Glucose 165 2.3 > 99.9
N-Acetylglucose 295 4.8 > 99.9
Mannose 172 2.4 > 99.9
Rhamnose 180 2.3 > 99.9
Disaccharides (C12) Cellobiose 282 6.7 > 99.9
Lactose 84 1.9 >99.9
Maltose 177 4.0 > 99.9
Melibiose 54 1.3 > 99.9
a) Potassium salt of acid
17.3.5.3 Summary
The newly developed Au/TiO2 catalysts have outstanding properties, including
pronounced substrate selectivity and extremely high product selectivity. Combi-
nation of these with very high activity and excellent long-term stability results
in catalysts that meet all the requirements demanded from a “Synzyme” (syn-
thetic enzyme). In the future, gold catalysts may be an alternative to the bio-
technological process for industrial production of gluconic acid.
References
1 WVZ, Wirtschaftliche Vereinigung Zu- 5 BRDTAC, Vision for Bioenergy and Bio-
cker, Informationen zum Zuckermarkt based Products in the United States.
Stand 12/2003. 2003, http://www.zucker- 2002, Biomass R&D Technical Advisory
wirtschaft.de Committee.
2 K. Hiller and P. Kehrer, Erdöl Erdgas 6 OECD, Biotechnology for clean indus-
Kohle, 2000, 116, 9, 427. trial products and processes, p. 30. 1998,
3 BRDTAC, Roadmap for Biomass Tech- OECD Publications, Paris Cedex.
nologies in the United States. 2002, Bio- 7 Dupont, Press release: Genencor Inter-
mass R&D Technical Advisory Commit- national and Dupont expand R&D colla-
tee. boration to make key biobased polymer.
4 NRC, Biobased industrial products: pri- 2001, http://www1.dupont.com/NA-
orities for research and commercializa- SApp/dupontglobal/corp/index.jsp?-
tion. In: National Research Council (ed.), page=/content/US/en_US/news/product/
2000, Washington, DC. 2001/pn03_12_01.ht ml
8 J. Klein and K. D. Vorlop, Immobiliza- 18 US-Department of Energy: The DOE

tion techniques: Cells. In: M. Moo-Young ethanol pilot plant – a tool for commer-
(ed), Comprehensive Biotechnology, cialization. 2000. http://www.ott.doe.gov/
1985, 203–224. Pergamon Press, Oxford. biofuels/pdfs/28397.pdf
9 EU, Directive 2003/30/EC of the Europe- 19 D. de Jesus and N. P. Nghiem, Student
an Parliament and of the Council of 8 Abstracts: Chemistry at ORNL – Abstract
May 2003 on the promotion of the use Ethanol Production from Rice-Straw Hy-
of biofuels or other renewable fuels for drolyzate Using Zymomonas mobilis in a
transport. Official Journal of the European Continuous Fluidized-Bed Reactor
Union, 2003, 46, L123, 42–47. (FBR). 2002, http://www.scied.science.
10 C. Berg, World ethanol production 2001. doe.gov/scied/abstracts2000/
2001. ornlchem.htm
11 A. Rosenberger, H. P. Kaul, T. Senn, W. 20 P. Wittlich, E. Capan, M. Schlieker, K.-D.
Aufhammer, Costs of bioethanol produc- Vorlop, U. Jahnz, Entrapment in Lenti-
tion from winter cereals: the effect of Kats. In: V. A. Nedovic and R. Willaert
growing conditions and crop production (ed.), Fundamentals of Cell Immobilisa-
intensity levels. Industrial Crops and tion Biotechnology, 2004, 53–63. Focus
Products, 2002, 15, 2, 91–102. on Biotechnology. Hofman, M. and Anné,
12 J. Zaldivar, J. Nielsen, L. Olsson, Fuel Jozef. Kluwer Academic Publishers, Dor-
ethanol production from lignocellulose: drecht.
a challenge for metabolic engineering 21 VDI, Ökosprit mit Makel. VDI-Nachrich-
and process integration. Applied Micro- ten, 2004, 9, 11.
biology and Biotechnology, 2001, 56, 1–2, 22 S. K. Ritter, Green Reward – Presidential
17–34. honors recognize innovative syntheses,
13 Q. A. Nguyen, J. H. Dickow, B. W. Duff, process improvements, and new prod-
J. D. Farmer, D. A. Glassner, K. N. Ibsen, ucts that promote pollution prevention.
M. F. Ruth, D. J. Schell, I. B. Thompson, Chemical and Engineering News, Science
M. P. Tucker, NREL/DOE ethanol pilot- and Technology, 2003, 81, 26, 30–35.
plant: Current status and capabilities. 23 U. Jahnz, M. Schubert, H. Baars-Hibbe,
Bioresource Technology, 1996, 58, 2, 189– K. D. Vorlop, Process for producing the
196. potential food ingredient DFA III from
14 H. G. Lawford and J. D. Rousseau, Cellu- inulin: screening, genetic engineering,
losic fuel ethanol – Alternative fermenta- fermentation and immobilisaton of inu-
tion process designs with wild-type and lase II. International Journal of Pharma-
recombinant zymomonas mobilis. ceutics, 2003, 256, 199–206.
Applied Biochemistry and Biotechnology, 24 U. Jahnz, M. Schubert, K. D. Vorlop,
2003, 105, 457–469. Effective development of a biotechnical
15 Iogen, EcoEthanol. 2003, process: Screening, genetic engineering,
http://www.iogen.ca/ and immobilization for the enzymatic
16 L. O. Ingram, P. F. Gomez, X. Lai, M. conversion of inulin to DFA III on in-
Moniruzzaman, B. E. Wood, L. P. Yoma- dustrial scale. Landbauforschung Völken-
no, S. W. York, Metabolic engineering of rode, 2001, 51, 3, 131–136.
bacteria for ethanol production. Biotech- 25 U. Prüße and K. D. Vorlop, The Jetcutter
nology and Bioengineering, 2002, 58, 2–3, technology. In: V. A. Nedovic and R.
204-214. Willaert (eds.), Fundamentals of Cell
17 J. Zaldivar, A. Borges, B. Johansson, Immobilisation Biotechnology, 2004,
H. P. Smits, S. G. Villas-Boas, J. Nielsen, 295–309. Focus on Biotechnology. Hof-
L. Olsson, Fermentation performance man, M. and Anné, J., Kluwer Academic
and intracellular metabolite patterns in Publishers, Dordrecht.
laboratory and industrial xylose-ferment- 26 S. Biella, L. Prati, M. Rossi, Selective oxi-
ing Saccharomyces cerevisiae. Applied dation of D-glucose on gold catalyst.
Microbiology and Biotechnology, 2002, 59, Journal of Catalysis, 2002, 206, 2,
4–5, 436–442. 242–247.
Biorefineries –
Industrial Processes and
Products
Edited by
Birgit Kamm,
Patrick R. Gruber,
and Michael Kamm
ISBN: 3-527-31027-4
Related Titles
Elvers, B. (Ed.)
Handbook of Fuels
Energy Sources for Transportation
2006
ISBN 3-527-30740-0
Olah, G. A., Goeppert, A., Prakash, G. K. S.
Beyond Oil and Gas: The Methanol Economy

2006
ISBN 3-527-31275-7
Shahidi, F. (Ed.)
Bailey’s Industrial Oil and Fat Products

6 Volume Set
2005
ISBN 0-471-38460-7
Ocic, O.
Oil Refineries in the 21st Century

Energy Efficient, Cost Effective, Environmentally Benign
2005
ISBN 3-527-31194-7
Biorefineries –
Industrial Processes and Products
Status Quo and Future Directions
Volume 2
Edited by
Birgit Kamm, Patrick R. Gruber, and Michael Kamm
The Editors n All books published by Wiley-VCH are carefully
produced. Nevertheless, authors, editors, and
Dr. Birgit Kamm publisher do not warrant the information contained
Research Institute in these books, including this book, to be free of
Bioactive Polymer Systems errors. Readers are advised to keep in mind that
biopos e.V. statements, data, illustrations, procedural details or
Kantstr. 55 other items may inadvertently be inaccurate.
14513 Teltow
Germany
Dr. Patrick R. Gruber

President and CEO
Outlast Technologies Inc. Library of Congress Card No.: applied for
5480 Valmont Road British Library Cataloguing-in-Publication Data:
Boulder, CO 80301 A catalogue record for this book is available
USA from the British Library.
Bibliographic information published by
Michael Kamm
Die Deutsche Bibliothek
Biorefinery.de GmbH
Die Deutsche Bibliothek lists this publication in
Stiftstr. 2 the Deutsche Nationalbibliografie; detailed
14471 Potsdam bibliographic data is available in the Internet at
Germany <http://dnb.ddb.de>
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA,

Weinheim, Germany
All rights reserved (including those of translation

into other languages). No part of this book may
be reproduced in any form – by photoprinting,
microfilm, or any other means – nor transmitted
or translated into a machine language without
written permission from the publishers.
Registered names, trademarks, etc. used in this
book, even when not specifically marked as such,
are not to be considered unprotected by law.
Typsetting K+V Fotosatz GmbH, Beerfelden

Printing Betz-Druck GmbH, Darmstadt
Binding Litges & Dopf Buchbinderei GmbH,
Heppenheim
Printed in the Federal Republic of Germany

Printed on acid-free paper
ISBN-13: 978-3-527-31027-2
ISBN-10: 3-527-31027-4
V
Contents
Volume 1
Part I Background and Outline – Principles and Fundamentals
1 Biorefinery Systems – An Overview 3

2 Biomass Refining Global Impact –

The Biobased Economy of the 21st Century 41
3 Development of Biorefineries –
Technical and Economic Considerations 67
4 Biorefineries for the Chemical Industry –

A Dutch Point of View 85
Ed de Jong, René van Ree Rea, Robert van Tuil, and Wolter Elbersen
Part II Biorefinery Systems
5 The Lignocellulosic Biorefinery –

and Industrial Organic Chemicals 115
6 Lignocellulosic Feedstock Biorefinery:

History and Plant Development for Biomass Hydrolysis 129
ISBN: 3-527-31027-4
VI Contents
7 The Biofine Process – Production of Levulinic Acid, Furfural,

and Formic Acid from Lignocellulosic Feedstocks 139
Daniel J. Hayes, Steve Fitzpatrick, Michael H. B. Hayes,
and Julian R. H. Ross
8 A Whole Crop Biorefinery System:

from Cereals 165
9 Iogen’s Demonstration Process for Producing Ethanol

from Cellulosic Biomass 193
Jeffrey S. Tolan
10 Sugar-based Biorefinery –
of Poly(3-hydroxybutyrate), Sugar, and Ethanol 209
11 Biomass Refineries Based on Hybrid

Thermochemical-Biological Processing – An Overview 227
Robert C. Brown
Green Biorefineries
12 The Green Biorefiner Concept – Fundamentals and Potential 253

13 Plant Juice in the Biorefinery –

Use of Plant Juice as Fermentation Medium 295
Margrethe Andersen, Pauli Kiel, and Mette Hedegaard Thomsen
Part III Biomass Production and Primary Biorefineries
14 Biomass Commercialization and Agriculture Residue Collection 317

James Hettenhaus
Contents VII
15 The Corn Wet Milling and Corn Dry Milling Industry –

A Base for Biorefinery Technology Developments 345
Donald L. Johnson
Part IV Biomass Conversion: Processes and Technologies
16 Enzymes for Biorefineries 357

17 Biocatalytic and Catalytic Routes for the Production of Bulk and Fine
Chemicals from Renewable Resources 385
Subjcet Index 407
Volume 2
Editor’s Preface XXIII
Foreword XXV
Henning Hopf
Foreword XXVII
Paul T. Anastas
List of Contributors XXIX
Part I Biobased Product Family Trees
1 The Key Sugars of Biomass: Availability, Present Non-Food Uses and

Potential Future Development Lines 3
1.1 Introduction 3
1.2 Availability of Mono- and Disaccharides 4
1.3 Current Non-Food Industrial Uses of Sugars 7
1.3.1 Ethanol 7
1.3.2 Furfural 8
1.3.3 D-Sorbitol (: D-Glucitol) 9
1.3.4 Lactic Acid ? PolylacticAcid (PLA) 10
1.3.5 Sugar-based Surfactants 11
1.3.6 ‘Sorbitan’ Esters 11
1.3.7 N-Methyl-N-acyl-glucamides (NMGA) 12
VIII Contents
1.3.8 Alkylpolyglucosides (APG) 12

1.3.9 Sucrose Fatty Acid Monoesters 13
1.3.10 Pharmaceuticals and Vitamins 14
1.4 Toward Further Sugar-based Chemicals:
Potential Development Lines 14
1.4.1 Furan Compounds 16
1.4.1.1 5-Hydroxymethylfurfural (HMF) 16
1.4.1.2 5-(Glucosyloxymethyl)furfural (GMF) 17
1.4.1.3 Furans with a Tetrahydroxybutyl Side-chain 19
1.4.2 Pyrones and Dihydropyranones 20
1.4.3 Sugar-derived Unsaturated N-Heterocycles 24
1.4.1.4 Pyrroles 24
1.4.1.5 Pyrazoles 26
1.4.1.6 Imidazoles 27
1.4.1.7 3-Pyridinols 28
1.4.1.8 Quinoxalines 28
1.4.4 Toward Sugar-based Aromatic Chemicals 29
1.4.5 Microbial Conversion of Six-carbon Sugars into Simple Carboxylic
Acids and Alcohols 32
1.4.5.1 Carboxylic Acids 34
1.4.5.2 Potential Sugar-based Alcohol Commodities Obtained by Microbial
Conversions 36
1.4.6 Chemical Conversion of Sugars into Carboxylic Acids 37
1.4.7 Biopolymers from Polymerizable Sugar Derivatives 40
1.4.7.1 Synthetic Biopolyesters 41
1.4.7.2 Microbial Polyesters 44
1.4.7.3 Polyamides 45
1.4.7.4 Sugar-based Olefinic Polymers (“Polyvinylsaccharides”) 47
1.5 Conclusion 49
References 51
2 Industrial Starch Platform – Status quo of Production,

Modification and Application 61
2.1 Introduction 61
2.1.1 History of Starch 61
2.1.2 History of Industrial Starch Production 62
2.1.3 History of Starch Modification 62
2.2 Raw Material for Starch Production 63
2.3 Industrial Production of Starch 65
2.3.1 Maize and Waxy Maize 66
2.3.2 Wheat 66
2.3.3 Potato 69
2.3.4 Tapioca 70
Contents IX
2.3.5 Other Starches 71

2.4 Properties of Commercial Starches 71
2.5 Modification of Starch Water 76
2.5.1 Modification Technology 76
2.5.1.1 Slurry Process (Heterogeneous Conditions) 76
2.5.1.2 Dry Reactions 77
2.5.1.3 Paste Reactions (Homogeneous Conditions) 77
2.5.1.4 Extrusion Cooking 77
2.5.2 Types of Starch Modification 78
2.5.2.1 Physical Modification 78
2.5.2.2 Degraded Starches 79
2.5.2.3 Chemical Modification 80
2.6 Application of Starch and Starch Derivatives 82
2.6.1 The Paper and Corrugating Industries 83
2.6.1.1 Use of Starch in the Paper Industry 83
2.6.1.2 Use of Starch in the Corrugating Industry 85
2.6.2 The Textile Industry 85
2.6.2.1 Sizing Agents 85
2.6.2.2 Textile-printing Thickeners 86
2.6.2.3 Finishing Agents 86
2.6.3 Adhesives 87
2.6.4 Building Chemistry 87
2.6.5 Pharmaceuticals and Cosmetics 88
2.6.6 Laundry Starches 89
2.6.7 Bioconversion of Starch 89
2.6.8 Other Applications of Starch 91
2.7 Future Trends and Developments 92
2.7.1 Tailor-made Starches by Use of Biotechnological Tools 92
2.7.2 New Modification Technologies for New Properties 93
2.7.3 New Fields of Application 94
Bibliography 95
3 Lignocellulose-based Chemical Products

and Product Family Trees 97
Birgit Kamm, Michael Kamm, Matthias Schmidt, Thomas Hirth,
and Margit Schulze
3.1 Introduction 97
3.2 Historical Outline of Chemical and Technical Aspects of Utilization
Lignocellulose in the 19th and 20th Century 98
3.2.1 From the Beginnings of Lignocellulose Chemistry Until 1800 98
3.2.2 Lignocellulose Chemistry in the Eighteenth Century 99
3.2.2.1 Cellulose Saccharification 99
3.2.2.2 Oxalic Acid 99
3.2.2.3 Xyloidin and Nitrocellulose 99
3.2.2.4 Cellulose 100
X Contents

3.2.2.6 Lignin 101
3.2.2.7 Hemicellulose (Polyoses) and Furfural 101
3.2.2.8 Lignocellulose 102
3.2.3 Industrial Lignocellulose Utilization in the 19th and Beginning
of the 20th Century 102
3.3 Lignocellulosic Raw Material 103
3.3.1 Definition 103
3.3.2 Sources and Composition 105
3.3.2.1 Sources 105
3.3.2.2 Chemical Composition of Lignocelluloses 106
3.3.2.3 Carbohydrates in Lignocelluloses 108
3.4 Lignocelluloses in Biorefineries 110
3.4.1 Background 110
3.4.1.1 Example 1 110
3.4.1.2 Example 2 110
3.4.2 LCF Biorefinery 111
3.4.3 LCF Conversion Methods 113
3.4.3.1 Pretreatment Methods 113
3.4.3.2 Chemical Pulping Methods 114
3.4.3.3 Enzymatic Methods 115
3.5 Lignin-based Product Lines 116
3.5.1 Isolation and Application Areas 116
3.5.2 A Lignin-based Product Family Tree 117
3.6 Hemicellulose-based Product Lines 119
3.6.1 Isolation and Application Areas 119
3.6.2 A Hemicellulose-based Product Family Tree 119
3.6.2.1 Mannan/Mannose Product Lines 119
3.6.2.2 Xylan/Xylose Product Line 120
3.6.3 Furfural and Furfural-based Products 122
3.6.3.1 Furfural 122
3.6.3.2 A Furfural-based Family Tree 127
3.7 Cellulose-based Product Lines 127
3.7.1 Isolation, Fractionation and Application Areas 127
3.7.2 Cellulose-based Key Chemicals 128
3.7.2.1 Glucose 128
3.7.2.2 Sorbitol 129
3.7.2.3 Glucosides 130
3.7.2.4 Fructose 131
3.7.2.5 Ethanol 132
3.7.2.6 Hydroxymethylfurfural 133
3.7.3 An HMF and Levulinic Acid-based Family Tree 135
References 139
Contents XI
4 Lignin Chemistry and its Role in Biomass Conversion 151

Gösta Brunow
4.2 Historical Overview 152
4.3 The Structure of Lignin 152
4.3.1 Definition 152
4.3.2 The Bonding of the Phenylpropane Units 153
4.3.3 Bonding Patterns and Functional Groups 156
4.3.3.1 General 156
4.3.3.2 Survey of Different Types of Lignin Unit 156
4.4 Role of Lignin in Biomass Conversion 159
4.4.2 Low-molecular-weight Chemicals from Lignin 160
4.4.3 Polymeric Products 160
4.4.4 Biodegradation 160
References 160
5 Industrial Lignin Production and Applications 165

E. Kendall Pye
5.2 Historical Outline of Lignin Production and Applications 168
5.2.1 Lignosulfonates from the Sulfite Pulping Industry 168
5.2.2 Lignin from the Kraft Pulping Industry 169
5.2.3 Lignin from the Soda Pulping Industry 170
5.3 Existing Industrial Lignin Products 172
5.3.1 Lignosulfonates 172
5.3.1.1 Chemical Characteristics of Lignosulfonates 172
5.3.1.2 Lignosulfonate Producers 173
5.3.1.3 Markets for Lignosulfonates 174
5.3.2 Kraft Pulping and Kraft Lignin Recovery 175
5.3.2.1 Producers of Kraft Lignin 175
5.3.2.2 Markets for Kraft Lignin 175
5.3.3 Lignins Produced from the Soda Process 176
5.3.4 Lignin from Other Biomass Processing Operations 176
5.3.5 Comparisons of the Physical and Chemical Properties
of Commercially Available Lignins 176
5.4 Lignin from Biorefineries 177
5.4.1 Advantages of Lignin and Hemicellulose Removal on Saccharification
and Fermentation of Cellulose 177
5.4.2 Lignin from an Organosolv Biorefinery 179
5.5 Applications and Markets for Lignin 181
5.5.1 Phenol–Formaldehyde Resin Applications 181
5.5.2 The Potential Use of Biorefinery Lignin in Phenolic Resins 181
XII Contents
5.5.3 Panelboard Adhesives 183

5.5.4 Thermoset Resins for Molded Products 184
5.5.5 Friction Materials 184
5.5.6 Foundry Resins 184
5.5.7 Insulation Materials 185
5.5.8 Decorative Laminates 185
5.5.9 Panel and Door Binders 185
5.5.10 Rubber Processing 186
5.5.11 The Opportunity for Lignin in Phenol–Formaldehyde Resin
Markets 187
5.6 Lignin as an Antioxidant 187
5.6.1 Antioxidants in Animal Feed Supplements 188
5.6.2 Antioxidants in the Rubber Industry 188
5.6.3 Antioxidants in the Lubricants Industry 188
5.7 Applications for Water-soluble, Derivatized Lignins 189
5.7.1 Concrete Admixtures 189
5.7.2 Dye Dispersants 190
5.7.3 Asphalt Emulsifiers 192
5.7.4 Agricultural Applications 192
5.7.5 Dispersants for Herbicides, Pesticides and Fungicides 193
5.8 New and Emerging Markets for Lignin 194
5.8.1 Printed Circuit Board Resins 194
5.8.2 Animal Health Applications 195
5.8.3 Animal Feed Supplement 196
5.8.4 Carbon Fibers for Mass-produced Vehicles 196
5.9 Conclusions and Perspectives 198
References 199
6 Towards Integration of Biorefinery and Microbial Amino Acid

Production 201
6.2 Present State of the Industry 202
6.2.1 Microbial Amino Acid Production 202
6.2.2 Biorefinery and the Building-block Concept 202
6.2.3 Metabolic Engineering and the Building-block Concept 204
6.3 Environmental and Commercial Consideration of Microbial
Amino Acid Production Integrated in a Biorefinery 205
6.4 Technical Constraints for Integration of Microbial Amino Acid
Fermentation into a Biorefinery 209
6.4.1 Mono-septic Operation 209
6.4.2 Carbon Sources 209
6.4.3 Nitrogen Source 211
Contents XIII
6.4.4 Phosphorus Source 211

6.4.5 Mixing and Oxygen Supply 212
6.4.6 Toxicity 212
6.4.7 Cultivation Temperature 213
Acknowledgment 214
References 215
7 Protein-based Polymers:
Mechanistic Foundations for Bioproduction and Engineering 217
Dan W. Urry
7.1.1 Definitions 217
7.1.1.1 Proteins and Protein-based Polymers 217
7.1.1.2 Two Basic Principles for Protein-based Polymer Engineering 217
7.1.2 Proteins in Aqueous Media 218
7.1.3 Thermodynamics of Proteins in Water 218
7.1.3.1 Exothermic Hydration of Apolar Groups 218
7.1.3.2 The Change in Gibbs Free Energy of Hydrophobic Association 218
7.1.3.3 The Apolar–Polar Repulsive Free Energy of Hydration, DG8ap 218
7.1.4 The Inverse Temperature Transition for Hydrophobic
Association 219
7.1.5 The Role of Elasticity in the Engineering of Protein-based
Polymers 219
7.1.5.1 Near Ideal Elasticity Provides for Efficient Energy Conversion 219
7.1.5.2 Mechanism of Near Ideal Elasticity 220
7.1.6 Many of the Advantages of Protein-based Polymeric Materials 220
7.2.1 Historical Beginnings of (Elastic) Protein-based Polymer
Development 221
7.2.2 Mechanistic Foundations: Fundamental Engineering Principles 222
7.2.2.1 The Hydrophobic Consilient Mechanism 222
7.2.2.2 The Elastic Consilient Mechanism 223
7.2.3 Highlights of Bioproduction 223
7.3 Bioproduction 224
7.3.1 Gene Construction using Recombinant DNA Technology 225
7.3.1.1 Preparation of Monomer Genes and the PCR Technique 225
7.3.1.2 Transformation, Monomer Gene Production and Sequence
Verification 226
7.3.1.3 Monomer Gene Concatenation Produces Multimer Genes
of Monomer 226
7.3.2 E. coli Transformation for Protein-based Polymer Expression 227
7.3.3 Fermentation using Transformed E. coli 227
7.4 Purification of Protein-based Polymers 227
XIV Contents
7.4.1 Use of the Inverse Temperature Transition as a Method

of Purification 228
7.4.1.1 Purification by Phase Separation as Demonstrated
by SDS–PAGE 228
7.4.1.2 Purification by Phase Separation Shown by Carbon-14-labeled
E. coli 228
7.4.2 Physical Characterization and Verification of Product Integrity 229
7.4.2.1 Gross Visualization of the Phase Separated Product 229
7.4.2.2 Sequence Integrity and Purity Evaluated by Nuclear Magnetic
Resonance 229
7.4.2.3 Mass Spectra Reaffirm Size of Expressed Polymer 229
7.4.3 Biocompatibility 230
7.4.3.1 The Challenge of Using E. coli-produced Protein
as a Biomaterial 230
7.4.3.2 Removal of Endotoxins and Determination of Levels 230
7.4.3.3 Western Immunoblot Technique to Demonstrate Level
of Purity 230
7.4.3.4 Western Immunodotblot Technique to Demonstrate
Medical Grade Purity 231
7.4.3.5 Subcutaneous Injection in the Guinea-pig 231
7.4.3.6 ASTM Tests 232
7.5 Mechanistic Foundations for Engineering Protein-based
Polymers 232
7.5.1 Phenomenological Axioms 232
7.5.2 The Change in Gibbs Free Energy for Hydrophobic Association,
DGHA 232
7.5.2.1 The Change in Gibbs Free Energy Attending a Phase Transition,
dDGt(v) 234
7.5.2.2 The DGHA-based Hydrophobicity Scale for Amino Acid
Residues 234
7.5.2.3 DG8HA-based Hydrophobicity Scale of Prosthetic Groups, etc. 235
7.5.2.4 Comprehensive Hydrophobic Effect: dGHA Responds
to all Variables 237
7.5.2.5 The Apolar–Polar Repulsive Free Energy of Hydration, DGap 237
7.5.3 The Coupling of Hydrophobic and Elastic Mechanisms 237
7.6 Examples of Applications 238
7.6.1 Soft Tissue Restoration 238
7.6.1.1 Prevention of Post-surgical Adhesions 238
7.6.1.2 Soft Tissue Augmentation 238
7.6.1.3 Soft Tissue Reconstruction: The Concept of Temporary Functional
Scaffoldings 239
7.6.2 Controlled Release Devices for Amphiphilic Drugs
and Therapeutics 240
7.6.2.1 The Use of DGap in the Design of Controlled-release Devices 240
Contents XV
7.6.2.2 Prevention of Pressure Ulcers by Means of Elastic Patches

for Drug Delivery 240
7.6.3 Fibers of Improved Elastic Moduli and Break Stresses and Strains 241
7.6.4 Programmably Biodegradable Thermoplastics 241
7.6.5 Acoustic Absorption 242
7.7.1 List of Gene Constructions and Expressed Protein-based Polymers 242
7.7.2 Efforts Toward Low-cost Production in other Microbes
and in Plants 242
7.8 Patents 245
7.8.1 Patents of D.W. Urry on Protein-based Polymers 245
7.8.2 Result of Ex Parte Patent Reexamination Request
to the USPTO 245
Acknowledgment 249
References 249
8 New Syntheses with Oils and Fats as Renewable

Raw Materials for the Chemical Industry 253
Ursula Biermann, Wolfgang Friedt, Siegmund Lang, Wilfried Lühs,
Guido Machmüller, Jürgen O. Metzger, Mark Rüsch gen. Klaas,
Hans J. Schäfer, Manfred P. Schneider
8.2 Reactions of Unsaturated Fatty Compounds 254
8.2.1 Oxidations 254
8.2.1.1 New Methods for the Epoxidation of Unsaturated Fatty Acids 254
8.2.1.2 Oxidation to vic-Dihydroxy Fatty Acids 257
8.2.1.3 Oxidative Cleavage 258
8.2.2 Transition Metal-Catalyzed Syntheses of Aromatic Compounds 259
8.2.3 Olefin Metathesis 259
8.2.4 Pericyclic Reactions 260
8.2.5 Radical Additions 261
8.2.5.1 Solvent-Free, Copper-Initiated Additions of 2-Halocarboxylates 262
8.2.5.2 Addition of Perfluoroalkyl Iodides 263
8.2.5.3 Thermal Addition of Alkanes 264
8.2.6 Lewis Acid-Induced Cationic Addition 264
8.2.7 Nucleophilic Addition to Reversed-Polarity Unsaturated Fatty
Acids 265
8.3 Reactions of Saturated Fatty Compounds 266
8.3.1 Radical C–C Coupling 266
8.3.1.1 Oxidative Coupling of C2 Anions of Fatty Acids 266
8.3.1.2 Anodic Homo- and Heterocoupling of Fatty Acids
(Kolbe Electrolysis) 267
8.3.2 Functionalization of C–H Bonds 269
XVI Contents
8.3.2.1 Oxidation of Nonactivated C–H Bonds 269

8.3.2.2 Oxidation of Allylic C–H Bonds 269
8.4 Enzymatic Reactions 270
8.4.1 Lipase Catalyzed Transformations 270
8.4.1.1 Lipase-Catalyzed Syntheses of Monoglycerides and Diglycerides 270
8.4.1.2 Lipase-Catalyzed Syntheses of Carbohydrate Esters 272
8.4.2 Microbial Transformations 272
8.4.2.1 Microbial Hydration of Unsaturated Fatty Acids 272
8.4.2.2 Microbial x- and b-Oxidation of Fatty Acids 273
8.4.3 Microbial Conversion of Oils/Fats and Glucose into Glycolipids 274
8.5 Improvement in Natural Oils and Fats by Plant Breeding 275
8.5.1 Gene Technology as an Extension of the Methodological Repertoire
of Plant Breeding 275
8.5.2 New Oil Qualities by Oil Designed with Available Agricultural
Varieties 276
8.5.3 Overview of Renewable Raw Materials Optimized by Breeding 277
8.5.3.1 Soybean 277
8.5.3.2 Rapeseed 277
8.5.3.3 Sunflower 280
8.5.3.4 Peanut 281
8.5.3.5 Linseed 281
8.5.4 Concluding Remarks on the Use of Gene Technology 281
8.6 Future Prospects 282
Acknowledgments 282
References 282
9 Industrial Development and Application of Biobased

Oleochemicals 291
Karlheinz Hill
9.2 The Raw Materials 292
9.3 Ecological Compatibility 293
9.4 Examples of Products 294
9.4.1 Oleochemicals for Polymer Applications 295
9.4.1.1 Dimerdiols Based on Dimer Acid 297
9.4.1.2 Polyols Based on Epoxides 298
9.4.2 Biodegradable Fatty Acid Esters for Lubricants 299
9.4.3 Surfactants and Emulsifiers Derived from Vegetable Oil 301
9.4.3.1 Fatty Alcohol Sulfate (FAS) 303
9.4.3.2 Acylated Proteins and Amino Acids (Protein–Fatty Acid
Condensates) 304
9.4.3.3 Carbohydrate-based Surfactants – Alkyl Polyglycosides 305
9.4.3.4 Alkyl Polyglycoside Carboxylate 307
9.4.3.5 Polyol Esters 307
9.4.3.6 Multifunctional Care Additives for Skin and Hair 309
Contents XVII
9.4.4 Emollients 310

9.4.4.2 Dialkyl Carbonate 311
9.4.4.3 Guerbet Alcohols 311
9.5 Perspectives 312
9.6 Trademarks 312
References 312
10 Phytochemicals, Dyes, and Pigments in the Biorefinery Context 315

George A. Kraus
10.3 Phytochemicals from Corn and Soybeans 317
10.3.1 Phytosterols 317
10.3.2 Lecithin 318
10.3.3 Tocopherols 319
10.3.4 Carotenoids 320
10.3.5 Phytoestrogens 321
10.3.6 Saponins 321
10.3.7 Protease Inhibitors 322
References 323
11 Adding Color to Green Chemistry?

An Overview of the Fundamentals and Potential of Chlorophylls 325
11.3 Chlorophyll Fundamentals 326
11.3.1 Occurrence and Basic Structures 326
11.3.2 Principles of Chlorophyll Chemistry 327
11.3.3 Isolation of Chlorophylls 328
11.4 Chlorophyll Breakdown and Chemical Transformations 330
11.4.1 Biological Chlorophyll Catabolism 330
11.4.2 Geological Chlorophyll Degradation – Petroporphyrins 331
11.4.3 Chemical Degradation of Chlorophylls 333
11.5 Industrial Uses of Chlorophyll Derivatives 335
11.6 A Look at “Green” Chlorophyll Chemistry 337
Acknowledgment 341
References and Notes 341
XVIII Contents
Part II Biobased Industrial Products, Materials and Consumer Products
12 Industrial Chemicals from Biomass – Industrial Concepts 347

12.3 Basic Principles 349
12.3.1 Primary Conversion Technologies of Biomass 350
12.3.1.1 Gasification 350
12.3.1.2 Hydrothermolysis 351
12.3.1.3 Fermentation to Ethanol 351
12.4 Current Status 351
12.4.1 Europe 351
12.4.2 United States 352
12.4.3 Products 353
12.5 Industrial Concepts 354
12.5.2 Biorefinery Concepts 355
12.5.3 Classes of Bioproduct 356
12.5.4 Opportunities for Industrial Bioproducts 357
12.5.5 Product Categories Based on C6-Carbon Sugars to Bioproducts 358
12.5.6 Product Categories Based on C5-Carbon Sugars to Bioproducts 358
12.5.7 Thermochemical Conversion of Sugars to Bioproducts 360
12.5.8 Thermochemical Conversion of Oils and Lipid Based
Bioproducts 361
12.5.9 Bioproducts via Gasification 361
12.5.10 Bioproducts via Pyrolysis 362
12.5.11 Biocomposites 362
References 364
13 Succinic Acid – A Model Building Block for Chemical Production

from Renewable Resources 367
13.2 Economics of Feedstock Supply 368
13.3 Succinic Acid Fermentation 369
13.4 Succinic Acid Catalytic Transformations 372
13.5 Current Petrochemical Technology 373
13.5.1 1,4-BDO, THF, GBL, and NMP 373
13.6 Current Biobased Technology 375
13.6.1 1,4-BDO, GBL, and NMP 375
13.6.2 Derivatives of Diammonium Succinate 376
13.7 Conclusions 378
References 378
Contents XIX
14 Polylactic Acid from Renewable Resources

Patrick Gruber, David E. Henton, and Jack Starr 381
14.2 Lactic Acid 382
14.2.1 Lactic Acid Production Routes 382
14.2.1.1 Chemical Synthesis 382
14.2.1.2 Fermentation 383
14.2.2 Production by Fermentation 384
14.2.2.1 Microorganisms 384
14.2.2.2 Sugar Feedstock 385
14.2.2.3 Nutrients 385
14.2.2.4 Neutralizing Agent 385
14.2.3 Acidification 386
14.2.3.1 Strong Acid Addition 386
14.2.3.2 Salt Splitting Technology 387
14.2.4 Purification 388
14.2.4.1 Cell Removal 388
14.2.4.2 Separation of Residual Sugars, Nutrients and Fermentation
By-products 388
14.3 PLA Production 390
14.3.1 Polymerization of Lactide 392
14.4 Control of Crystalline Melting Point 394
14.5 Rheology Control by Molecular Weight and Branching 396
14.5.1 Melt Rheology of Linear PLA 397
14.5.2 Melt Rheology of Branched PLA 397
14.5.3 Branching Technology 398
14.5.3.1 Multi-functional Polymerization Initiators 398
14.5.3.2 Hydroxy Cyclic Ester and/or Carbonate Polymerization
Initiators 398
14.5.3.3 Multi-cyclic Ester, Multi-cyclic Carbonate and/or Multi-cyclic Epoxy
Comonomers 398
14.5.3.4 Free Radical Cross-linking 399
14.6 Melt Stability 399
14.7 Applications and Performance 400
14.8 PLA Stereocomplex 401
14.9 Fossil Resource Use and Green House Gases 402
14.10 Summary 402
Abbreviations 403
References 404
15 Biobased Consumer Products for Cosmetics 409

Thomas C. Kripp
15.1 Introduction and Historical Outline 409
15.1.1 Cosmetics Past and Present 409
15.1.2 Bionics: Learning from Nature 410
XX Contents
15.2 Betaine, The Conditioner Made from Sugar Beet 410

15.2.1 Occurrence 410
15.2.2 Chemical Properties 411
15.2.3 Production 411
15.2.4 Use and Fields of Application 412
15.2.5 Innovation Through Combination: Betaine Esters 414
15.2.6 Summary and Prospects 415
15.3 Chitosan, Hair-setting Agent from the Ocean 415
15.3.1 Chitin, a Precursor of Chitosan 415
15.3.2 Occurrence of Chitin 415
15.3.3 Production 416
15.3.3.1 Purification of Chitin 416
15.3.3.2 Production of Chitosan 417
15.3.4 Chitosan in cosmetic products 419
15.3.5 Summary and Prospect 421
15.4 From Energy Reserve to Shampoo Bottle: Biopol 422
15.4.1 Biodegradable Packages 422
15.4.2 What is “Biopol”? 423
15.4.3 Biodegradability of Biopol 424
15.4.4 The Long Way to the Shampoo Bottle 426
15.4.4.1 Product Development 426
15.4.4.2 Market Launch 427
15.4.5 Quo vadis, Biopol? 428
15.5 Natural Apple-peel Wax: Protection for Hair and Skin 429
15.5.1 Raw Material Source 429
15.5.2 Apple-peel Wax 430
15.5.3 Observations 430
15.5.4 Production of Apple-peel Wax 432
15.5.5 Chemical Composition 433
15.5.6 Mode of Action and Uses 433
15.5.6.1 Skin Cosmetics 434
15.5.6.2 Hair Care 434
15.5.7 Market Launch 436
15.6 Ilex Resin: From Shiny Leaves to Shiny Hair 437
15.6.1 Holly 437
15.6.2 Extraction of a Resin Fraction 438
15.6.3 Effects in Cosmetics 439
15.6.3.1 Skin Care 439
15.6.3.2 Hair Care 439
15.6.3.3 Styling 440
References 441
Contents XXI
Part III Biobased Industry:

16 Industrial Biotech –
Setting Conditions to Capitalize on the Economic Potential 445
16.2 Time to Exploit the Potential 446
16.2.1 How Far Can it Go? 446
16.2.2 Better Technology, Faster Results 447
16.2.3 Environmentally and Balance-sheet Friendly 448
16.2.4 Rekindling Chemicals Innovation 450
16.2.5 Increasing Corporate Action in all Segments 451
16.3 The Importance of Residual Biomass 452
16.3.1 Why Waste Biomass Works 452
16.3.2 Economic Benefits and Regulation 452
16.3.3 Still a Long Way to Go 454
16.3.4 Collaboration Will Push Biomass Conversion Forward 454
16.4 Overcoming the Challenges Ahead 455
16.4.1 Internal Obstacles 455
16.4.2 External Challenges 456
16.5 Overcoming Challenges 457
16.5.1 Case 1: Building a Biotech Strategy 457
16.5.2 Case 2: Identifying the Right Opportunities 458
16.5.3 Case 3: Managing Uncertainties 459
16.5.4 Case 4: Preparing the Launch and Market Development 460
16.5.5 Case 5: Building a Favorable External Environment 461
16.6 More Needs to be Done 461
Subject Index 463

XXIII
Editor’s Preface
In the year 2003 when the idea for this set of books “Biorefineries, Biobased In-
dustrial Processes, and Products” arose, the topic of biorefineries as means of
processing industrial material and efficient utilization of renewable products
had been primarily a side issue beyond the borders of the United States of
America. This situation has changed dramatically over the last two years. Today
in almost every developed and emerging nation much work is being conducted
on biorefinery systems, driven by the rising cost of oil and the desire of to move
away from petrochemical-based systems.
In these books we do not claim to describe and discuss everything that be-
longs or even might belong to the topic of biorefineries – that would be impos-
sible. There are many types of biorefinery, and the state of the technology is
changing very rapidly as new and focused effort is directed toward making bio-
refineries a commercial reality. It is a very exciting time for those interested in
biorefineries – technologies for bio-conversion have advanced to a state in which
they are becoming practical on a large scale, economics are leaning more fa-
vourably to the direction of renewable feedstocks, and chemical process knowl-
edge is being applied to biobased systems.
As the editors of the first comprehensive biorefinery book we saw it as our
duty to provide, first of all, a general framework for the subject – addressing
the main issues associated with biorefineries, the principles and basics of biore-
finery systems, the basic technology, industrial products which fall within the
scope of biorefineries, and, finally, technology and products that will fall within
the scope of biorefineries in the future.
To provide a reliable description of the state of biorefinery research and devel-
opment and of industrial implementations, strategies, and future developments
we asked eighty-five experts from universities, research and development insti-
tutes, and industry and commerce to present their views, their results, their im-
plementations, and their ideas on the topic. The results of their contributions
are thirty-three articles organized into seven sections. Our very special thanks
go to all the authors.
We are especially indebted to Dr. Hubert Pelc from Wiley-VCH publishing,
who worked with us on the concept and then, later, on the development and
implementation of the book. Thanks go also to Dr. Bettina Bems from Wiley-
ISBN: 3-527-31027-4
XXIV Editor’s Preface
VCH publishing, who managed with admirable professionalism and very much
patience, and to the three editors and eighty-five authors from three different
continents. We are also indebted to Hans-Jochen Schmitt, also of Wiley-VCH
publishing, who had the not always easy task of arranging the manuscripts in a
form ready for publication.
Maybe in 2030, when a biobased economy utilizing biorefinery technology
has become a fundamental part of national and globally connected economies,
someone will wonder what had been thought and written about the subject of
biorefineries at the beginning of the 21st century. Hopefully this book will be
highly representative. Until then we hope it will contribute to the promotion of
international biorefinery developments.
Teltow-Seehof (Germany) Birgit Kamm

Boulder, CO (USA) Patrick R. Gruber
Potsdam (Germany) Michael Kamm
November 2005
XXV
Foreword
One-hundred-and-fifty years after the beginning of coal-based chemistry and 50
years after the beginning of petroleum-based chemistry industrial chemistry is
now entering a new era. In the twenty-first century utilization of renewable raw
materials will gain importance in the chemical conversion of substances in in-
dustry. Partial or even complete re-adjustment of whole economies to renewable
raw materials will require completely new approaches in research, development,
and production. Chemical and biological sciences will play a leading role in the
building of future industries. New synergies between biological, physical, chem-
ical, and technical sciences must be elaborated and established and special re-
quirements will be placed on raw material and on product-line efficiency and
sustainability. The necessary change from chemistry based on a fossil raw mate-
rial to biology-based modern science and technology is an intellectual challenge
for both researchers and engineers. Chemists should support this change and
collaborate closely with their colleagues in adjoining disciplines, for example
biotechnology, agriculture, forestry, and the material sciences.
The German Chemical Society will help direct this necessary development by
supporting within its structure new kinds of organization for chemists to work
on this subject in universities, research institutes, and industry.
This two-volume book is based on the approach developed by biorefinery-sys-
tems – transfer of the logic and efficiency of today’s petrochemical product lines
and product family trees into manipulation of biomass. Raw biomass materials
are mechanically separated into substances for chemical conversion into other
products by different methods, which may be biotechnological, thermochemical,
and thermal. Review of biomass processes and products developed in the past
but widely forgotten in the petroleum age will be as important as the presenta-
tion of new methods, processes, and products that still require an enormous
amount of research and development today.
Henning Hopf
President of the German Chemical Society
Frankfurt (Germany)
November 2005
ISBN: 3-527-31027-4
XXVII
Foreword
On October 5, 2005, the Nobel Prize Committee made an interesting and im-
portant statement with regard to the prize in chemistry. It said, “This represents
a great step forward for ‘green chemistry’, reducing potentially hazardous waste
through smarter production. [This research] is an example of how important ba-
sic science has been applied for the benefit of man, society and the environ-
ment.” By making this statement, the Nobel committee recognized what a new
generation of scientists has known for quite some time, that by working at the
most fundamental level – the molecular level – we are able to design our prod-
ucts, processes, and systems in ways that are sustainable.
There is general recognition that the current system by which we produce the
goods and services needed by society is not sustainable. This unsustainability
takes many forms. It would be legitimate to note that in our current system of
production we rely largely on finite feedstocks extracted from the Earth that are
being depleted at a rate that cannot be sustained indefinitely. It is equally legiti-
mate to recognize that our current production efficiency results in more than
90% of the material used in the production process ending up as waste, i.e. less
than 10% of the material ends up in the desired product. Yet another condition
of unsustainability is in our current energy use; this not only relies largely on fi-
nite energy sources but also results in degradation of the environment that can-
not be continued as the growing population and demands of the developing
world emerge over the course of the twenty-first century. Finally, the products
and processes we have designed since the industrial revolution have accom-
plished their goals without full consideration of their impact and consequence
on humans and the biosphere, with many examples of toxic and hazardous sub-
stances being distributed throughout the globe and into our bodies.
If we are to change this unsustainable path, it will need the direct and com-
mitted engagement of our best scientists and engineers to design the future dif-
ferently from the past. We will need to proceed with a broader perspective such
that when we design for efficiency, effectiveness, and performance, we now
must recognize that these terms include sustainability – a minimized impact
on humans and the environment.
An essential part of meeting the challenge of designing for sustainability will
be based on the nature of the materials we use as starting materials and feed-
stocks. Any sustainable future must ensure that the materials on which we base
ISBN: 3-527-31027-4
XXVIII Foreword
our economic infrastructure are renewable rather than depleting. The rate of re-
newability is also important because certainly one could argue that petroleum is
renewable if you have a few million years to wait. Serious analysis would, how-
ever, necessitate that the rate of renewability is connected to the rate of use.
There are options for how to approach this technological challenge, for example
using waste products from one process as a feedstock for another, that are well
thought through in industrial ecology models. There is, however, recognition
that an essential part of a sustainable future will be based on appropriate and
innovative uses of our biologically-based feedstocks.
This book addresses the essential questions and challenges of moving toward
a sustainable society in which bio-based feedstocks, processes, and products are
fundamental pillars of the economy. The authors discuss not only the important
scientific and technical issues surrounding this transition but also the necessary
topics of economics, infrastructure, and policy. It is only by means of this type
of holistic approach that movement toward genuine sustainability will be able
to occur where the societal, economic, and environmental needs are met for the
current generation while preserving the ability of future generations to meet
their needs.
While it will be clear to the reader that the topics presented in this book are
important, it is at least as important that the reader understand that these topics
– and the transition to a sustainable path that they address – are urgent. At this
point in history it is necessary that all who are capable of advancing the transi-
tion to a more sustainable society, engage in doing so with the level of energy,
innovation, and creativity that is required to meet the challenge.
Paul T. Anastas
Director of the Green Chemistry Institute
Washington, D.C.
November, 2005
XXIX
List of Contributors (Volume 1 and 2)
José A. M. Agnelli Robert C. Brown

Universidade Federal de São Carlos Center for Sustainable Environmental
Departamento de Engenharia Technologies
de Materiais Iowa State University
Rodovia Washington Luis (SP-310) 286 Metals Development Building
São Carlos, São Paulo Ames, IO 50011
Brazil USA
Margrethe Andersen Gösta Brunow

AgroFerm A/S Department of Chemistry
Limfjordsvej 4 University of Helsinki
6715 Esbjerg N A. I. Virtasen aukio 1
Denmark 00014 Helsinki
Finland
Rolf Bachmann
McKinsey and Company Inc Stefan Buchholz
Zurich Office Degussa AG
Alpenstrasse 3 Creavis
8065 Zürich Projecthouse ProFerm
Switzerland Rodenbacher Chaussee 4
63403 Hanau-Wolfgang
Ursula Biermann Germany
Fachbereich Chemie
Carl von Ossietzky Universität Rainer Busch
Oldenburg Dow Deutschland GmbH & Co. OHG
Postfach 2603 Industriestrasse 1
26111 Oldenburg 77836 Rheinmünster
Germany Germany
ISBN: 3-527-31027-4
XXX List of Contributors
Grant M. Campbell Tim Dodge

Satake Centre for Grain Process Genencor International
Engineering 925 Page Mill Road
School of Chemical Engineering and Palo Alto, CA 94304
Analytical Science USA
The University of Manchester
Sackville Street Donald L. Van Dyne
Manchester M60 1QD Agricultural Economics
UK University of Missouri – Columbia
214c Mumford Hall
Joel R. Cherry Columbia, MO 65211
Novozymes Biotech Inc USA
1445 Drew Ave
Davis, CA 95616 Wolter Elbersen
USA Agrotechnology and Food Innovations
B. V.
Gopal Chotani P.O. Box 17
Genencor International 6700 AA Wageningen
925 Page Mill Road The Netherlands
Palo Alto, CA 94304
USA Steve Fitzpatrick
Biofine
L. Davis Clements 245 Winter Street
Renewable Products Development Waltham, MA 02154
Laboratories USA
3114 NE 45th Ave.
Portland, OR 97213 Paul Fowler
USA The BioComposites Centre
University of Wales
Bruce E. Dale Bangor
Department of Chemical Engineering Gwynedd LL57 2UW
and Materials Science UK
East Lansing, MI 48824 Wolfgang Friedt
USA Institut für Pflanzenbau
und Pflanzenzüchtung 1
Bill Dean Justus-Liebig-Universität Giessen
Genencor International Heinrich-Buff-Ring 26–32
925 Page Mill Road 35392 Giessen
Palo Alto, CA 94304 Germany
USA
List of Contributors XXXI
John Frye James R. Hettenhaus

Pacific Northwest National Laboratory CEA Inc
P.O. Box 999/K2-12 3211 Trefoil Drive
Richland, WA 99352 Charlotte, NC 28226
USA USA
Patrick R. Gruber Karlheinz Hill

President and CEO Cognis Deutschland GmbH & Co. KG
Outlast Technologies Incorporated Paul-Thomas-Straße 56
5480 Valmont Road 40599 Düsseldorf
Suite 200 Germany
Boulder, CO 80301
USA Thomas Hirth
Fraunhofer-Institut
Dietmar R. Grüll Chemische Technologie
Südzucker Aktiengesellschaft Joseph-von-Fraunhoferstraße 7
Mannheim/Ochsenfurt 76327 Pfinztal
Wormser Strasse 11 Germany
67283 Obrigheim/Pfalz
Germany John Holladay
Daniel J. Hayes P.O. Box 999/K2-12
Department of Chemical Richland, WA 99352
& Environmental Sciences USA
Limerick Franz Jetzinger
Ireland Zuckerforschung Tulln
Gesellschaft mbH
Michael H. B. Hayes Josef-Reither-Strasse 21–23
Department of Chemical 3430 Tulln
& Environmental Sciences Austria
Limerick Donald L. Johnson
Ireland Biobased Industrial Products
Consulting
David E. Henton 29 Cape Fear Drive
Nature Works LLC Hertford, NC 27944
(former Cargill Dow LLC) USA
15305 Minnetonka Blvd
Minnetonka, MN 55345 Ed de Jong
USA Agrotechnology and
Food Innovations B.V.
P.O. Box 17
6700 AA Wageningen
The Netherlands
XXXII List of Contributors
Birgit Kamm Apostolis A. Koutinas

Research Institute Bioactive Polymer Satake Centre for Grain Process
Systems (biopos e.V.) Engineering
Research Centre Teltow-Seehof School of Chemical Engineering and
Kantstraße 55 Analytical Science
14513 Teltow The University of Manchester
Germany Sackville Street
Manchester M60 1QD
Michael Kamm UK
Biorefinery.de GmbH
Stiftstraße 2 Martin Kozich
14471 Potsdam Zuckerforschung Tulln
Germany Gesellschaft mbH
and Josef-Reither-Strasse 21–23
Laboratories Teltow 3430 Tulln
Kantstraße 55 Austria
14513 Teltow
Germany George A. Kraus
Department of Chemistry
Raphael Katzen Iowa State University
9220 Bonita Beach Road 1605 Gilman Hall
Suite 2000 Ames, IA 50011-3111
Bonita Springs, FL 34135 USA
USA
Thomas C. Kripp
Ralf Kelle Wella AG
Degussa AG Abt. FON
R & D Feed Additives Berliner Allee 65
Kantstrasse 2 64274 Darmstadt
33790 Halle/Westfalen Germany
Germany
Stefan Kromus
Pauli Kiel BioRefSYS-BioRefinery Systems
Biotest Aps Innovationszentrum Ländlicher Raum
Gl. Skolevej 47 Auersbach 130
6731 Tjæreborg 8330 Feldbach
Denmark Austria
Seungdo Kim
Department of Chemical Engineering
and Materials Science
East Lansing, MI 48824
USA
List of Contributors XXXIII
Siegmund Lang Achim Marx

Institut für Biochemie Degussa AG
und Biotechnologie Creavis
Technische Universität Projecthouse ProFerm
zu Braunschweig Rodenbacher Chaussee 4
Spielmannstraße 7 63403 Hanau-Wolfgang
38106 Braunschweig Germany
Germany
Jürgen O. Metzger
Frieder W. Lichtenthaler Fachbereich Chemie
Institute of Organic Chemistry Carl von Ossietzky Universität
Darmstadt University of Technology Oldenburg
Petersenstraße 22 Postfach 2603
64287 Darmstadt 26111 Oldenburg
Germany Germany
Wilfried Lühs Michael Narodoslawsky

Institut für Pflanzenbau Graz University of Technology
und Pflanzenzüchtung 1 Institute of Resource Efficient and
Justus-Liebig-Universität Giessen Sustainable Systems (RNS)
Heinrich-Buff-Ring 26–32 Inffeldgasse 21 B
35392 Giessen 8010 Graz
Germany Austria
Guido Machmüller Jefter Nascimento

FB 9 – Organische Chemie PHB Industrial SA
Bergische Universität Fazenda da Pedra
GH Wuppertal s/n – C. Postal 02
Gaußstraße 20 CEP 14150 Servana
42097 Wuppertal São Paulo
Germany Brazil
Paulo E. Mantelatto Glenn E. Nedwin

Centro de Tecnologia Canavieira Novozymes Biotech Inc
(formerly Centro de Tecnologia 1445 Drew Ave
Copersucar) Davis, CA 95616
Fazenda Santo Antonio USA
CP 162
13400-970 Piracicaba
Brazil
XXXIV List of Contributors
Ulf Prüße Carlos Eduardo Vaz Rossell

Federal Agricultural Research Centre Centro de Tecnologia Canavieira
(FAL) (formerly Centro de Tecnologia
Institute of Technology and Copersucar)
Biosystems Engineering Fazenda Santo Antonio
Bundesallee 50 CP 162
38116 Braunschweig 13400-970 Piracicaba
Germany Brazil
E. Kendall Pye Mark Rüsch gen. Klaas

Lignol Innovations Corp. Department Technology
3650 Westbrook Mall University of Applied Sciences
Vancouver, BC Neubrandenburg
V6S 2L2 Brodaer Straße 2
Canada 17033 Neubrandenburg
Germany
René van Ree Rea
Energy research Centre Hans J. Schäfer
of the Netherlands (ECN) – Organisch-Chemisches Institut
Biomass Department Universität Münster
P.O. Box 1 Corrensstraße 40
1755 ZG Petten 48149 Münster
The Netherlands Germany
Julia Richter Daniel J. Schell

Institut für Chemie National Bioenergy Center
Universität Potsdam National Renewable Energy
Karl-Liebknecht-Str. 24–25 Laboratory
14476 Golm 1617 Cole Blvd.
Germany Golden, CO 80401-3393
USA
Jens Riese
McKinsey and Company Inc Matthias Schmidt
Munich Office Biorefinery.de GmbH
Prinzregentenstraße 22 Stiftstraße 2
80538 München 14471 Potsdam
Germany Germany
Julian R. H. Ross Manfred P. Schneider

University of Limerick FB 9 – Organische Chemie
Department of Chemical Bergische Universität
& Environmental Sciences GH Wuppertal
Limerick Gaußstraße 20
Ireland 42097 Wuppertal
Germany
List of Contributors XXXV
Margit Schulze Robert van Tuil

FB Angewandte Naturwissenschaften Agrotechnology and Food Innovations
FH Bonn-Rhein-Sieg B.V.
Grantham-Allee 20 P.O. Box 17
53754 Sankt Augustin 6700 AA Wageningen
Germany The Netherlands
Mathias O. Senge Dan W. Urry

SFI Tetrapyrrole Laboratory BioTechnology Institute
School of Chemistry University of Minnesota
Trinity College Dublin Twin Cities Campus
Dublin 2 1479 Gortner Avenue
Ireland Suite 240
St. Paul, MN 55108-6106
Jack Starr USA
Cargill Dow LLC and
15305 Minnetonka Blvd Bioelastics Inc.
Minnetonka, MN 55345 2423 Vestavia Drive
USA Vestavia Hills, AL 35216-1333
USA
Sarah A. Teter
Novozymes Biotech Inc Fernando Valle
1445 Drew Ave Genencor International
Davis, CA 95616 925 Page Mill Road
USA Palo Alto, CA 94304
USA
Johan Thoen
Dow Europe GmbH Klaus-Dieter Vorlop
Bachtobelstrasse 3 Federal Agricultural Research Centre
8810 Horgen (FAL)
Switzerland Institute of Technology and
Biosystems Engineering
Mette Hedegaard Thomsen Bundesallee 50
Risø National Laboratory 38116 Braunschweig
Biosystems Department Germany
Frederiksbovgvej 399
4000 Roskilde Rouhang Wang
Denmark Satake Centre for Grain Process
Engineering
Jeffrey S. Tolan School of Chemical Engineering and
Iogen Corporation Analytical Science
8 Colonnade Road The University of Manchester
Ottawa Sackville Street
Ontario K2E 7M6 Manchester M60 1QD
Canada UK
XXXVI List of Contributors
Marnik M. Wastyn Thomas Willke

Zuckerforschung Tulln Federal Agricultural Research Centre
Gesellschaft mbH (FAL)
Josef-Reither-Strasse 21–23 Institute of Technology and
3430 Tulln Biosystems Engineering
Austria Bundesallee 50
38116 Braunschweig
Colin Webb Germany
Satake Centre for Grain Process
Engineering Robert Wittenberger
School of Chemical Engineering and Zuckerforschung Tulln
Analytical Science Gesellschaft mbH
The University of Manchester Josef-Reither-Strasse 21–23
Sackville Street 3430 Tulln
Manchester M60 1QD Austria
UK
Feng Xu
Volker F. Wendisch Novozymes Biotech Inc
Institute of Biotechnology 1 1445 Drew Ave
Research Center Juelich Davis, CA 95616
52425 Juelich USA
Germany
Todd Werpy
P.O. Box 999/K2-12
Richland, WA 99352
USA
Part I
Biobased Product Family Trees
ISBN: 3-527-31027-4
3
1
The Key Sugars of Biomass: Availability, Present Non-Food
Uses and Potential Future Development Lines
1.1
Introduction
Because our fossil raw materials derived from prehistoric organic matter are ir-
revocably decreasing – the end of cheap oil is realistically predicted to occur in
the next 2–3 decades, i.e. 2040 at the latest [1–3] – and because pressure on our
environment is building up, the progressive change-over of chemical industry to
renewable feedstocks emerges as an inevitable necessity [3–5].
The terrestrial biomass which Nature graciously provides us on an annual ba-
sis is considerably more complex than fossil raw materials, constituting a multi-
faceted accumulation of low- and high-molecular-weight products, exemplified
by sugars, hydroxy and amino acids, lipids, and biopolymers such as cellulose,
hemicelluloses, chitin, starch, lignin, and proteins. By far the most important
class of organic material in terms of volume produced is carbohydrates, which
represent approximately 75% of the annually renewable biomass of about 180
billion tons (Fig. 1.1). Of these, only a minor fraction (ca. 5%) is used by man,
the rest decays and recycles along natural pathways.
Thus, carbohydrates, a single class of natural products are – aside from their tradi-
tional uses for food, lumber, paper, and heat – the major biofeedstocks from which to
develop industrially and economically viable organic chemicals and materials to re-
place those derived from petrochemical sources.
ISBN: 3-527-31027-4
4 1 The Key Sugars of Biomass
Fig. 1.1 Distribution of types of natural products in biomass.
1.2
Availability of Mono- and Disaccharides
The bulk of the annually renewable carbohydrate biomass is polysaccharides,

yet their nonfood utilization is confined to the textile, paper, and coating indus-
tries, either as such or in the form of simple esters and ethers. Organic com-
modity chemicals, however, are of low-molecular-weight, hence they are more
expediently acquired from low-molecular-weight carbohydrates than from poly-
saccharides. Accordingly, the constituent repeating units of these polysaccha-
rides – glucose (cellulose, starch), fructose (inulin), xylose (hemicelluloses), etc.,
or disaccharide versions thereof – are the actual carbohydrate raw materials for
organic chemicals with tailor-made industrial applications: they are inexpensive,
ton-scale accessible and provide an ensuing chemistry, better worked out and
more variable than that of their polymers.
Table 1.1 lists the availability and bulk-quantity prices of the eight least expen-
sive sugars, some sugar-alcohols and sugar-derived acids – all well below
1 10 kg–1 – compared with some basic chemicals and organic solvents from pet-
rochemical sources. The result is stunning, because the five cheapest sugars,
their alcohols, and some important sugar-derived acids are within the same
price range as basic organic bulk chemicals such as naphtha, ethylene, acetalde-
hyde, or aniline. Actually, the first three of these sugars, sucrose, glucose, and
lactose, are in the price range of some of the standard organic solvents.
The uniqueness of this situation becomes even more imposing when looking
at the availability of these sugars. Sucrose, “the royal carbohydrate”, has for cen-
turies been the worlds most abundantly produced organic compound, current
annual production being an impressive 140 million tons [6]. Similarly bulk
scale-accessible are its component sugars d-glucose, produced by hydrolysis of
starch [7], and d-fructose, generated either from glucose by base-induced isomeri-
zation or from inulin or sucrose by hydrolysis [8]. Isomaltulose, an a(1 ? 6) iso-
mer of sucrose, has recently become accessible on an industrial scale through
enzymatic transglucosylation [9], lactose and maltose are available in large quanti-
ties from whey [10] and starch [11], d-xylose, the cheapest pentose, from wood-
or straw-derived xylans. l-Sorbose is the cheapest, large-scale accessible l-sugar,
because of its production from d-sorbitol (= d-glucitol) in the Vitamin C fabrica-
tion process [12]. The sugar alcohols d-sorbitol, erythritol [13], d-xylitol, and d-
mannitol [14], each of comparatively high yearly production via hydrogenation of
1.2 Availability of Mono- and Disaccharides 5
Table 1.1 Annual production volume and prices of simple su-

gars, sugar-derived alcohols and acids compared with some
petrochemically derived basic chemicals and solvents.
World production a) Price b)

(metric t year–1) (1 kg–1)
Sugars Sucrose 140 000 000 0.20

d-Glucose 30 000 000 0.30
Lactose 295 000 0.60
d-Fructose 60 000 1.00
Isomaltulose 70 000 2.00
Maltose 3 000 3.00
d-Xylose 25 000 4.50
l-Sorbose 60 000 7.50
Sugar alcohols d-Sorbitol 650 000 1.80
Erythritol 30 000 2.25
Xylitol 30 000 5.00
d-Mannitol 30 000 8.00
Sugar-derived acids Citric acid 1 500 000 1.00
d-Gluconic acid 100 000 1.40
l-Lactic acid 150 000 1.75
l-Tartaric acid 35 000 6.00
l-Ascorbic acid 80 000 8.00
Amino acids l-Glutamic acid 1 500 000 1.20
l-Lysine 740 000 2.00
Petrochemicals Ethylene 90 000 000 0.40
Propylene 45 000 000 0.35
Benzene 23 000 000 0.40
Terephthalic acid 12 000 000 0.70
Aniline 1 300 000 0.95
Acetaldehyde 900 000 1.10
Adipic acid 1 500 000 1.70
Solvents Methanol 25 000 000 0.15
Toluene 6 500 000 0.25
Acetone 3 200 000 0.55
a) Reliable data are available for world production of sucrose

only, the figure given referring to the crop cycle 2004/2005
[6]. All other data are average values based on estimates
from producers and/or suppliers as the production volume
of many products is not publicly available.
b) Prices given are those attainable in early 2005 for bulk deliv-
ery of crystalline material (where applicable) based on pric-
ing information from sugar industry (sugars) and the Chemi-
cal Market Reporter 2005 (acids, basic chemicals, and sol-
vents). The listings are intended as a benchmark rather than
a basis for negotiations between producers and customers.
Quotations for less pure products are, in part, sizeably lower,
e.g. the commercial sweetener “high fructose syrup”, which
contains up to 95% fructose, may readily be used for large-
scale preparative purposes.
their parent aldoses, are mainly used as food ingredients, because of their
sweetening properties, yet also have potential as inexpensive raw materials for
broad-scale preparative purposes. The same holds for d-gluconic acid [15] and
the other sugar-derived acids listed.
Despite their large-scale accessibility at comparatively low cost, it must seem
surprising that the chemical industry currently utilizes these mono- and disac-
charides to a minor extent only as feedstock for organic chemicals – a state of
affairs amply documented by the fact that of the 100 major organic chemicals
manufactured in the US in 1995 [16], only seven were derived from biofeed-
stocks, and five of these – ethanol, sorbitol, citric acid, lysine and glutamic acid
– used carbohydrates as the raw material source. Intense efforts within the last
decade [17–26] to boost the acquisition of organic chemicals from the sugars in
Table 1.1 have not basically changed this picture.
There are a variety of reasons for this. Current use of fossil raw materials is
more economic and, as important, the process technology for conversion of pet-
rochemical raw materials into organic chemicals is exceedingly well developed
and basically different from that required for transforming carbohydrates into
products with industrial application profiles. This situation originates from the
inherently different chemical structures of the two types of raw material, of
which the essence is manifested in their structure-based names (Fig. 1.2). Our
fossil resources are hydrocarbons, distinctly hydrophobic, oxygen-free and devoid
of functionality, thus, organic functional groups such as hydroxyl, amino, alde-
hyde, acid, ester or halo functionalities have to be introduced – usually into ole-
finic hydrocarbons such as ethylene, propylene, and butane – to obtain the in-
dustrially important intermediate chemicals. In contrast, annually renewables
are carbohydrates, overfunctionalized with hydroxyl groups and pronouncedly hy-
drophilic. Needless to say, that the methods required for converting carbohy-
Fig. 1.2 Hydrocarbons vs. carbohydrates: more than a play on

words, because their names, taken literally, reveal the basic
differences in their utilization as organic raw materials.
drates into viable industrial chemicals – reduction of oxygen content with intro-
duction of C = C and C = O unsaturation – are diametrically opposed to those
prevalent in petrochemical industry.
As higher oil prices, environmental issues, and regulations begin to adversely
affect the manufacture of chemicals from fossil raw materials, the transition to
a biobased production system is unavoidable, strongly emphasizing the need for
systematically elaborating appropriate chemical and microbial process methods
to convert carbohydrates – they are the major biofeedstocks to fill the gap be-
tween dwindling oil supply and demand – into industrially useful products, be
it bulk, intermediate, and fine chemicals, pharmaceuticals, agrochemicals, high-
value-added specialty chemicals, or simply enantiomerically pure building
blocks for organic synthesis.
1.3
Current Non-Food Industrial Uses of Sugars
Current utilization of carbohydrates as a feedstock for the chemical industry –

be it for bulk, commodity, intermediate, fine, or high-value-added specialty
chemicals – is modest when considering their ready availability at low cost and
the huge as yet unexploited potential. The examples presently realized on an in-
dustrial scale are outlined briefly.
1.3.1
Ethanol
With production of about 24 million tons in 2004 (300 mill hL [27]), fermenta-
tion ethanol (“bioethanol”) is the largest-volume biobased chemical today. The
principal organism for fermentation is Saccharomyces cerevisiae, an ascomycetous
yeast that can grow on a wide variety carbohydrate feedstocks – sugar crops,
and sugar-containing by-products such as sugar cane, sugar beet, sorghum, mo-
lasses, and – after hydrolysis to glucose – starchy crops such as corn, potatoes,
and grain, or cellulosic materials, e.g. wood pulping sludges from pulp and pa-
per mills [28 a]. Recent developments [28 b] replace the conventional yeast by
bacteria (Zymomonas nobilis) and/or genetically engineered organisms, which
seems to improve productivity significantly.
The manufacturing costs are said to be approximately the same as those for
its production from ethylene in a plant of comparable size [28 c]. The large
growth in production of industrial-grade fermentation ethanol within recent
years is less because of its use as a solvent and starting material for follow-up
chemicals such as acetaldehyde, ethyl esters (e.g. EtOAc) and ethers (Et2O) –
these mostly result from ethylene-based processing lines – but because of its
high potential as a fuel additive. It is either directly mixed with standard gaso-
line at a level of 5%, or indirectly in the form of ETBE (ethyl t-butyl ether) in
proportions of up to 15%; a hefty government subsidy is, however, required (re-
moval of gasoline tax) if it is to remain competitive. The growth opportunities

for fuel-grade bioethanol are enormous and are predicted to increase substan-
tially within the next five years.
1.3.2
Furfural
With an annual production of approximately 250 000 tons, furfural (2-furfuralde-

hyde) seems to be the only unsaturated large-volume organic chemical prepared
from carbohydrate sources. The technical process involves exposure of agricul-
tural or forestry waste – hemicellulose content up to 25% d-xylose polysaccha-
rides (xylosans) – to aqueous acid and fairly high temperatures; the xylosans are
first hydrolyzed then undergo acid-induced cyclo-dehydration [29].
The chemistry of furfural is well developed, providing a host of versatile in-
dustrial chemicals by simple straightforward operations (Scheme 1.1): furfuryl
alcohol (2) and its tetrahydro derivative 1 (hydrogenation), furfurylamine 3 (re-
ductive amination), furoic acid 4 (oxidation), and furanacrylic acid 5 (Perkin re-
Scheme 1.1 Furan commodity chemicals derived from pento-

sans in agricultural wastes (corn cobs, oat hulls, bagasse,
wood chips).
action), or furylidene ketones 6 (aldol condensations). Furfural is also the key

chemical for the commercial production of furan (by catalytic decarbonylation)
and tetrahydrofuran (8) (hydrogenation), thereby providing a biomass-based al-
ternative to its petrochemical production by dehydration of 1,4-butanediol [29].
Further importance of these furan chemicals stems from their ring-cleavage
chemistry [30], which has led to a variety of other established chemicals, for ex-
ample fumaric, maleic, and levulinic acids, the last a by-product of production
of furfural [31].
The susceptibility of the furan ring in these compounds to electrophilic sub-
stitution at C-5 has been widely exploited. Mineral acid-promoted condensation
with aldehydes or ketones converts 3 into difurfural diamine 9 [32], whereas es-
ters of 2-furoic acid afford the respective difurfuryl dicarboxylate (10 ? 11). Both
– the latter on saponification – are relevant monomer components for the gen-
eration of polyesters and polyamides [33].
Most of the furfural currently produced is used as a selective solvent in the re-
fining of lubricating oil and, with furfuryl alcohol in condensations with formal-
dehyde, phenol, acetone, or urea, to yield resins with complex, ill-defined struc-
tures, yet excellent thermosetting properties, most notably high corrosion resis-
tance, low fire hazard, and extreme physical strength [29]; they are extensively
used in the foundry industry as cores for high-quality castings.
1.3.3
D-Sorbitol (: D-Glucitol)
Although the main consumer of its sizable annual production (Table 1.1) is the
food industry, primarily as a non-calorific sweetening agent and as a key inter-
mediate in the production of ascorbic acid (vitamin C) [12], it has important
non-food applications, because of its moisture conditioning, softening, and plas-
tifying properties. These result in its use in adhesives, paper, printing, textiles,
cellulose-based foil, and pharmaceutical formulations. Other non-food applica-
tions of d-sorbitol result from etherification and polycondensation reactions
which provide biodegradable polyetherpolyols used for soft polyurethane foams
and melamine–formaldehyde or phenol resins [34]. Sizable amounts of d-sorbi-
tol are also used for production of the sorbitan ester surfactants (cf. below).
1.3.4
Lactic Acid ? Polylactic Acid (PLA)
Large amounts of d-glucose – in the crude form as obtainable from corn, pota-
toes, or molasses by acid hydrolysis – are used in industrial fermentation pro-
cesses for production of lactic acid (Scheme 1.2), citric acid, and different amino
acids, for example l-lysine or l-glutamic acid. Although the major use of these
products is in food and related industries, recent non-food exploitation of lactic
acid have made it a large-scale, organic commodity chemical. Most of it is sub-
sequently polymerized via its cyclic dimer (lactide) to polylactic acid [36, 37], a
high molecular weight polyester.
Because of its high strength, PLA can be fabricated into fibers, films, and
rods that are fully biodegradable (? lactic acid, CO2) and compostable, having
degraded within 45–60 days. Accordingly, PLA and copolymers of lactic and gly-
colic acid are of particular significance for food packaging and in agricultural or
gardening applications, but are highly suitable materials for surgical implants
and sutures, because they are bioresorbable.
Cargill, since 1989, has invested some $ 750 million to develop and commer-
cialize polylactic acid (tradename “NatureWorks”), its Nebraska plant with an
annual capacity of 140 000 metric tons opened in 2002 [36, 37]. Thus, polylac-
tides, because they combine favorable economics with green sustainability, are
poised to compete in large-volume markets that are now the domain of thermo-
plastic polymers derived from petrochemical sources.
Another green development based on lactic acid is its ethyl ester (“Vertec”)
that has been marketed for applications in specialty coatings, inks, and directly
Scheme 1.2 Production and uses of lactic acid.

for cleaning because of its high performance and versatility [38]. As a most be-
nign solvent – green, readily biodegradable, and excellent toxicology – it has the
potential to displace a variety of petrochemically based solvents such as acetone,
DMF, toluene or N-methylpyrrolidone in industrial processes.
1.3.5
Sugar-based Surfactants
Utilization of cheap, bulk-scale accessible sugars as the hydrophilic component

and fatty acids or fatty alcohol as the lipophilic component provides non-ionic
surfactants which are nontoxic, non-skin-irritating and fully biodegradable. Typi-
cal examples of such industrially relevant surfactants are fatty acid esters of sor-
bitol (sorbitan esters) and of sucrose, fatty acid amides of 1-methylamino-1-
deoxy-d-glucitol (NMGA) and, most pronounced in terms of volume produced,
fatty alcohol glucosides, the so-called alkyl polyglucosides (APGs) [39].
1.3.6
‘Sorbitan’ Esters
Bulk-scale accessible d-sorbitol (cf. Table 1.1) readily undergoes dehydration on

exposure to mineral acid at fairly high temperatures to give anhydrosorbitol or
sorbitan, de facto a mixture of sorbitol and its 1,4-anhydro and 1,4 : 3,6-dianhy-
dro derivatives, the exact composition depending on the conditions employed
[34] (Scheme 1.3). Esterification of this mixture with C16/C18 fatty acid chlor-
ides/base or transesterification with their methyl esters leads to either sorbitan
monoesters (SMS for sorbitan monostearate), or di- and tri-esters.
Because of their favorable hydrophilic/hydrophobic balance (HLB) values, sor-
bitan esters find use as non-ionic surfactants and as solubilizers and emulsifiers
in cosmetics, pharmaceuticals, textile processing, and a variety of other formula-
Scheme 1.3 Dehydration of D-sorbitol to “sorbitan”, giving

the ‘sorbitan monoester’ surfactant on esterification with
C16/C18 fatty acids.
tions [34]. Having been commercially available since the 1940s, they de facto
constitute one of the first fully green synthetic surfactants, presently produced
at an estimated 20 000 t a–1.
1.3.7
N-Methyl-N-acyl-glucamides (NMGA)
Reductive amination of d-glucose with methylamine smoothly generates the

aminoalditol, 1-methylamino-1-deoxy-d-glucitol, which, on amidation with fatty
acids, gives the corresponding fatty acid amides, carrying a methyl group and a
pentahydroxylated six-carbon chain on the amido nitrogen:
The NMGA have highly advantageous ecological and toxicological properties

which brought about their use as surfactants, cleansing agents, and in cos-
metics applications [39, 40].
1.3.8
Alkylpolyglucosides (APG)
Commercially produced by several companies – most notably by Cognis with a

capacity in the 50 000 t a–1 range, Kao, Seppic, and ICI – APG’s are by far the
most important non-ionic surfactants. They are fatty alcohol glucosides with an
alcohol chain length normally between C8 and C14. Their industrial synthesis
entails either direct acid-catalyzed Fischer glycosidation of glucose (in the form
of a syrupy starch hydrolysate) or starch itself. The alternative process consists
of two stages, the first being Fischer glycosidation with n-butanol to butyl glyco-
sides which are subsequently subjected to acid-promoted transacetalization [35]
(see also Chapter 9 in this volume, Hill).
The resulting product mixtures contain the a-d-glucosides predominantly, as
designated in the formula (Scheme 1.4) and are marketed as such. APG are not
skin-irritating, have good foaming properties, and are completely biodegradable,
hence are widely used in manual dishwashing detergents and in the formula-
tion of shampoos, hair conditioners, and other personal care products [35].
Scheme 1.4 Synthesis of alkyl polyglucosides (APG).
1.3.9
Sucrose Fatty Acid Monoesters
These are currently produced at an approximate 5000 t · a–1 only, and are mostly
used in cosmetic and personal care formulations because of their attractive der-
matological properties. Produced by transesterification of fatty acid methyl es-
ters or fats, the resulting sucrose monoester (if 1 : 1 molar ratios have been used
in the process) is not a defined product acylated exclusively at the primary glu-
cose-6-OH, as indicated in the formula, but at the other primary and some sec-
ondary OH groups also [41]:
1.3.10
Pharmaceuticals and Vitamins
Aside the enormous amount of sugars, mostly glucose and sucrose, that flows into
the fermentative production of amino and hydroxy acids (cf. Table 1.1), of which a
substantial part is for food use, a significant volume of these sugars is used in fer-
mentation processes furnishing high-value-added products – antibiotics and vita-
mins, much too complex in their structures as to be generated by chemical synthe-
sis. Figure 1.3 lists several representative examples – penicillins and cephalospor-
ins with an estimated world production in the 70 000 t a–1 range, the aminogly-
coside antibiotics of the kanamycin and spectinomycin type, or the recently
optimized bioprocesses for bulk-scale production of vitamin C and B6.
Some sugar-derived drugs obtained by chemical means have also reached
some importance, e.g. ranitidine (Zantac), an inhibitor of gastric acid secretion
– one of the top 30 drugs based on sales [42] – isosorbide dinitrate, a coronary
vasodilatator [43], or topiramate, a fructose-derived anticonvulsant drug with
high antiepileptic efficacy [44].
1.4
Toward Further Sugar-based Chemicals: Potential Development Lines
Considering the large-scale, low-cost availability of the basic biomass-sugars

listed in Table 1.1, most notably sucrose, glucose and fructose, their present
non-food utilization by chemical industry is modest indeed, i.e. the huge poten-
tial as the raw material for further viable industrial chemicals and materials is
largely untapped. In view of the need for the chemical industry to somehow
bring about the changeover from fossil raw materials to biofeedstocks, most no-
tably carbohydrates because they are more readily accessible from agricultural
crops and waste materials than any other natural products, their further exploi-
tation to produce industrially viable products is one of the major “green” chal-
lenges. The attempt to trace those sugar-based development lines along which
the further exploitation of the key sugars of biomass is likely to proceed, implies
assessment of many imponderables, particularly with regard to current dy-
namics in exploiting genetically engineered enzymes and the products resulting
from them. Hence, some important directions along which relevant develop-
ments are likely to proceed are surely to be missed. Nevertheless, an “inventory”
based on the present status may be expedient for focusing efforts on those areas
where useful methods leading to promising products already exist and wait to
be further developed.
1.4 Toward Further Sugar-based Chemicals: Potential Development Lines 15
6)
Fig. 1.3 Sugar-derived high-value-added products –

antibiotics, vitamins, and pharmaceuticals.
1.4.1
Furan Compounds
In addition to furfural, an established sugar-based five-carbon commodity with

versatile industrial applications (cf. above), several other furan compounds, read-
ily prepared from sugars, hold high promise as industrial intermediate chemi-
cals, albeit – for purely economic reasons – they are not (yet) produced on an
industrial scale.
1.4.1.1
5-Hydroxymethylfurfural (HMF)
Like many petroleum-derived basic chemicals, e.g. adipic acid or hexamethyl-
enediamine, HMF is a six-carbon compound with broad application potential,
inasmuch it is readily accessible from fructose or inulin hydrolysates by acid-in-
duced elimination of three moles of water [45]. Even a pilot-plant-size process
has been elaborated [46].
HMF has been used for the manufacture of special phenolic resins of type
12, because acid catalysis induces its aldehyde and hydroxymethyl group to react
with phenol [47].
12
Of high industrial potential as intermediate chemicals are the various HMF-de-

rived products for which well worked-out, large-scale-adaptable production pro-
cedures are available (Scheme 1.5). Of these, the 5-hydroxymethylfuroic acid 13,
the 2,5-dicarboxylic acid (19), the 1,6-diamine (15), and the respective 1,6-diol
(17) are most versatile intermediate chemicals of high industrial potential, be-
cause they are six-carbon monomers that could replace adipic acid, or alkyldiols,
or hexamethylenediamine in the production of polyamides and polyesters.
Indeed, an impressive series of furan polyesters and polyamides has been pre-
pared [30] in which the furandicarboxylic acid 19 replaces terephthalic and iso-
phthalic acids in current industrial products (cf. below). None has yet proved
economically competitive with existing products, however. Thus, HMF is not yet
produced on an industrial scale. Tentative assessment of its economics compared
with those of petrochemical raw materials clearly reveals the underlying reasons –
ton prices of naphtha and ethylene are in the 150–400 1 range; those of aniline
(500 1 t–1) and fructose (*1000 1 t–1) , in particular, are substantially higher, en-
tailing an HMF-marketing price of at least 2500 1 t–1 – too expensive at present
for a bulk-scale industrial product. Accordingly, as long as the economic situation
favors fossil raw materials, applications of HMF lie in high value-added products,
for example pharmaceuticals or special niche materials.
Scheme 1.5 Versatile intermediate chemicals from HMF [48–52].
1.4.1.2 5-(Glucosyloxymethyl)furfural (GMF)

The industrial production of isomaltulose from sucrose (Table 1.1) – for food
reasons, because it is hydrogenated to an equimolar mixture of glucosyl-
a(1 ? 6)-glucitol and -mannitol [9], the low calorie sweetener isomalt [53] – has
made it a lucrative target for generating disaccharide intermediates of industrial
potential. For example, air oxidation in strongly alkaline solution smoothly pro-
vides the glucosyl-a(1 ? 5)-d-arabinonic acid in the form of its lactone 20 in ex-
cellent yield [54]. Alternatively, acid-induced dehydration of the fructose portion
gives a-d-glucosyloxymethylfurfural (a-GMF, 21) (Scheme 1.6). As this process
is also feasible in a continuous flow reactor [55 a], the compound may be readily
produced on large scale.
As a glucosylated HMF, 20 provides a rich chemistry toward products with
broad application profiles. Oxidation with chlorite gives the glucosylfuroic acid
21; when Pt/O2 is used in aqueous medium the dicarboxylic acid 22 is gener-
Scheme 1.6 Conversion of sucrose into products with

industrial potential [54–56].
ated [56]. Both of these reactions proceed in excellent yield. Aldol-type condensa-
tions deliver derivatives with polymerizable double bonds, most notably the
methylenation product 24, which polymerizes spontaneously, and the acrylic
acid 25 [55], expected to yield novel, hydrophilic polymers with interesting per-
formance profiles. Reductive amination provides GMF-amine; N-acylation of
this with fatty acid chlorides gives compounds of type 26, non-ionic surface-ac-
tive agents in which hydrophobic fat-alkyl residue and hydrophilic glucose part
are separated by a quasi-aromatic spacer. They also have useful liquid-crystalline

properties [57], as do esters of glucosylfuroic acid 21 with long-chain alcohols
(? 27), which combine high surface activity with biocompatibility, rendering
them promising candidates for biomedical applications.
1.4.1.3 Furans with a Tetrahydroxybutyl Side-chain

Another simple, one-step entry from hexoses to more highly substituted furans
is their ZnCl2-mediated reaction with 1,3-dicarbonyl compounds such as 2,4-
pentanedione or ethyl acetoacetate. Because only the first two sugar carbons
contribute to the formation of the furan, a distinctly hydrophilic tetrahydroxybu-
tyl side-chain is produced. Thus, d-glucose smoothly provides furans 28 and 29
with the d-arabino configuration in the polyol fragment [58]; these can be short-
ened oxidatively to the dicarboxylic acid (29 ? 30) or a variety of other furan
building blocks (Scheme 1.7). Of biological relevance seem to be ensuing prod-
ucts of type 32, i.e. amino acids with the carboxyl group in the hydrophobic por-
tion of the molecule and the amino group in the hydrophilic portion, because
they have been used to prepare hetarylene-carbopeptoid libraries by combinator-
ial techniques [59].
In contrast, under mildly basic conditions (aqueous bicarbonate at 85 8C),
d-glucose reacts with pentane-2,4-dione in an entirely different manner, produc-
ing, via C-addition and subsequent retroaldo-type elimination of OAc–, the 2-C-
glucosylpropanone 31 [60]. Because this conversion can be performed with the
unprotected sugar and in aqueous solution with simple reagents, it may legiti-
mately be referred to as a prototype of green and/or sustainable sugar transfor-
mations. The procedure is equally feasible with other aldohexoses and with
d-fructose [61], thus, is one of the cleanest and most efficient preparative entries
Scheme 1.7 One-pot conversions of D-glucose into hydrophilic

furans [58] or, alternatively, into C-glucosides by reaction with
acetylacetone [60].
into the area of C-glycosides, which, as stable “mimics” of the usual O-glyco-
sides, command major interest as glycosidase inhibitors [62].
1.4.2
Pyrones and Dihydropyranones
The bulk-scale-accessible mono- and disaccharides of Table 1.1 invariably adopt

pyran cyclohemiacetal forms, from which well-elaborated, efficient reaction
channels lead to an unusually large variety of unsaturated pyran building
blocks, for example pyrones, dihydropyrans, and dihydropyranones, of which
the last two have the additional advantage of being enantiomerically pure. They
are treated only cursorily in this context, because their potential as ideally func-
tionalized six-carbon building blocks, particularly for the preparation of pharma-
ceutical targets, has not been utilized comprehensively. They may, however, be
regarded as high-value-added specialty chemicals.
Pyrones of type 33 (kojic acid) and 34 are readily obtained from d-glucose,
the former either enzymatically by growing Aspergillus oxyzae on steamed rice
[63] or chemically via pyran 3,2-enolones [64, 65], or the a-pyrone 34 by oxida-
tion to d-gluconic acid [15] and acetylation [66]. Both, at present, are of little sig-
nificance as six-carbon building blocks, despite a surprisingly effective route
from 34 to cyclopentanoid products of type 35 [67] which is surmised to have
industrial potential.
Other, potentially useful derivatizations of d-glucose – entry reactions with
which the tautomeric forms are fixed – were elaborated before the turn of the
19th century – thiation to the acyclic dithio acetals, isopropylidenation to fura-
noid systems, or generation of pyran structures, such as glucosides, glucals, and
Asp. oryzae 1. Oxid. DMAP

! !
2. Ac2O/D 90%
hydroxyglucalesters (Scheme 1.8) [68]. Of particular interest in the context of

versatile sugar-based six-carbon building blocks are the pyranoid glycal and hy-
droxyglycal esters, because they not only have the oxygen content of d-glucose
reduced – a precondition for elaboration of industrially viable products – but
carry olefinic unsaturation in the pyran ring.
Despite the ready accessibility of these glucal and hydroxyglucal esters, and
their well-developed ensuing chemistry, their exploitation as industrial inter-
mediates is exceedingly modest. Nevertheless, to emphasize their potential to-
ward industrial intermediates, whether as enantiomerically pure building blocks
Scheme 1.8 Readily accessible, tautomerically fixed D-glucose

derivatives which can be used to produce versatile building
blocks [68].
for the synthesis of non-carbohydrate natural products [65, 69] or for agrochem-
icals and/or high-value-added pharmaceuticals, a particularly versatile array of
six-carbon dihydropyranones is listed in Fig. 1.4; all are accessible from d-glucose
(via the glucal and hydroxyglucal esters) in no more than three to five straight-
forward steps.
Fig. 1.4 Pyranoid six-carbon building blocks accessible from D-

glucose via glucal (upper half) or hydroxyglucal esters (lower
entries) intermediates. All products require no more than three
to five straightforward steps from D-glucose [70–78].
A bicyclic dihydropyranone, levoglucosenone, is accessible even more directly

by vacuum pyrolysis of waste paper [79]. Although the yield achievable is rela-
tively low – levoglucosan is also formed, their proportions depending on the ex-
act conditions (Scheme 1.9) – relatively large amounts can be amassed quickly;
levoglucosenone has been used for synthesis of a diverse variety of natural prod-
ucts in the enantiomerically pure form [80].
Scheme 1.9 High-vacuum pyrolysis of cellulose [79].
Similarly convenient are the acquisition of the three dihydropyranones 36–38

(Fig. 1.5), requiring two and three steps from maltose and sucrose, respectively
[81], their “left-over” glycosyl and fructosyl residues serving as acid-labile block-
ing groups, in contrast with the alkali-sensitive ester functions.
Fig. 1.5 Simple, disaccharide-derived dihydropyranones [81].
All these pyran building blocks are enantiomerically pure and have a unique,
highly diverse array of functional groups to which the armory of preparative or-
ganic methodology can directly be applied. The enolone esters in Fig. 1.4, for ex-
ample, have three differently functionalized carbonyl groups, one being free,
the other two masked in enol ester and acetal form. In addition, the enolone
structural unit is flanked by chiral centers, so any addition reaction to either car-
bonyl or enolic double bond proceeds with high stereoselectivity. As for the dis-
accharide-derived building blocks 36–38, they feature functionality in one of the
units and – in the form of the intact glycosyl moiety – a cheap acid-sensitive
protecting group in the other.
In addition to their extensive use in the total synthesis of enantiomerically
pure non-carbohydrate natural products [65, 69] these pyran building blocks
have found little use as high-value-added chemicals. If suitable targets and ap-
propriate preparative outlets are found, however, particularly toward pharmaceu-
tically promising compound libraries via combinatorial techniques, these pyran
building blocks are likely to become a plethora of attractive, industrially relevant
specialty chemicals.
1.4.3
Sugar-derived Unsaturated N-Heterocycles
Although transformation of sugars into trace amounts of N-heterocycles occurs ex-

tensively on exposure of foodstuffs to heat (Maillard reaction [82]), and although a
variety of nitrogen heterocycles have been generated from saccharide derivatives
[83], procedures meeting preparative standards are exceedingly scarce. Recent im-
provements of existing procedures and the development of new methods has led
to the more ready acquisition of various N-heterocycles from carbohydrates, e.g.
imidazoles, pyrroles, pyrazoles, pyridines, and quinoxalines which, because of
their sugar derivation, have hydrophilic side-chains – a favorable asset particularly
in pharmaceutical applications. A brief overview on those N-heterocycles readily
accessible from the basic sugars in practical, large-scale adaptable procedures
should enhance their utilization as solid intermediate chemicals.
1.4.3.1 Pyrroles
The generation of pyrroles by heating a glycerol solution of lactose-derived am-
monium salt of galactaric acid over a free flame [84] seems to be the highest-
yielding acquisition (40%) from a carbohydrate source – a process that, in this
or a modified form, does not seem to be utilized industrially.
2,5-Disubstituted pyrroles are accessible from carbohydrate sources via HMF in a

preparatively straightforward reaction sequence involving photooxidative furan ring
opening and cyclization of the saturated 2,5-diketones with ammonia or amines [85]:
These reaction sequences can directly be transferred to GMF, leading to pyr-

roles carrying an additional glucosyl residue [85]. Pyrroles with an equally hy-
drophilic tetrahydroxybutyl substituent, e.g. 39, are available from d-glucosa-
mine by exposure to acetylacetone or ethyl acetoacetate under mildly basic con-
ditions [86] or in a one-pot reaction from d-fructose by heating with acetylace-
tone and ammonium carbonate in DMSO [87].
The hydroxylated side-chain can, of course, be oxidatively shortened to give a

variety of simple pyrrole building blocks, for example carboxylic acid 40, or cy-
clized to a furanoid ring (39 ? 41) [86], compounds that may be regarded as
C-nucleosides.
1.4.3.2 Pyrazoles
Expeditious four-step approaches to 1-phenylpyrazol-3-carboxaldehydes with 5-
hydroxymethyl, 5-dihydroxyethyl, or 5-glucosyloxymethyl substituents has been
elaborated starting from d-xylose [88], d-glucose, and isomaltulose [89], respec-
tively.
As illustrated for d-xylose, its osazone, nearly quantitatively formed on reaction

with phenylhydrazine, readily gives the pyrazole when added to acetic anhydride
under reflux. Subsequent removal of the N-acetylphenylhydrazone residue with
formaldehyde/acetic acid and de-O-acetylation provides a pyrazole-aldehyde
(57% overall yield from d-xylose), a versatile heterocyclic building block, useful
for preparation of pharmaceuticals or monomers for the generation of poly-
amides and polyesters, e.g. the diamino and diol derivatives [88]:
1.4.3.3 Imidazoles
A variety of imidazoles carrying hydrophilic substituents in the 4-position
are readily accessible in one-pot procedures from standard monosaccharides.
Of those, the formation of 4-hydroxymethylimidazole by Cu(II)-promoted reac-
tion with formaldehyde and conc. ammonia [90] is rather unique, because ob-
viously retroaldolization to glyceraldehyde and dihydroxyacetone is involved
(Scheme 1.10). The retroaldol fission can be partially suppressed, however, on
heating d-fructose with formamidinium acetate in liquid ammonia in a pres-
sure vessel [91] or with formamidinium acetate in the presence of boric acid
and hydrazine, obviously proceeding via a boric acid complex of the bishydra-
zone of d-glucosone [56].
Scheme 1.10 D-Fructose-derived hydrophilic imidazoles.

A: CH2O, aq. NH3, CuCO3/Cu(OH)2, 2 h, 100 8C [90]; B: for-
mamidine · HOAc/liq. NH3, 15 h, 75 8C [91]; C: N2H4/forma-
midine · HOAc, H3BO3, 3 h reflux [56].
These conditions can be readily applied to pentoses or disaccharides with ac-

ceptable yields, as exemplified with d-xylose [91] and isomaltulose [56] in one-
pot procedures:
1.4.3.4 3-Pyridinols
The conversion of pentosans or pentoses into 3-pyridinol can be effected in a
practical three-step sequence, involving acid-induced dehydration to furfural,
reductive amination to furfurylamine, and subsequent oxidation with hydrogen
peroxide [51, 92], the last step conceivably proceeding through the stage of a
2,5-dihydroxy-2,5-dihydrofurfurylamine, which forms the pyridine nucleus by
dehydration to a 5-aminopentenal intermediate and intramolecular aldimine for-
mation. The pyridinol is a prominent intermediate chemical for the preparation
of herbicides and insecticides [93], and cholinergic drugs of the pyridostigmine
type.
For conversion of furfurylamines with readily oxidizable hydroxyl groups, e.g.

those derived from fructose via HMF/bromine in water/methanol, the entire
multistep process to the hydroxymethylpyridinol is effected in a one-pot proce-
dure [94]:
1.4.3.5 Quinoxalines
Useful one-pot procedures are also available for the conversion of various mono-
saccharides into tetrahydroxybutyl-substituted quinoxalines, the preparatively
most favorable conditions seem to be reaction of fructose with hydrazine, o-phe-
nylenediamine, and boric acid in dilute acetic acid by bubbling oxygen through
the solution [95], the decisive intermediate being the bis-hydrazone of d-gluco-
sone:
On briefly heating the quinoxaline under reflux in aqueous acid with excess
hydrazine or phenylhydrazine, a surprising oxidative cyclization occurs with for-
mation of the trihydroxypropyl-substituted flavazols [96].
1.4.4
Toward Sugar-based Aromatic Chemicals
On the basis of a 1995 compilation [16], twenty of the 100 major organic chemi-
cals in the US were aromatic compounds, invariably manufactured from fossil
raw materials, mostly from the BTX (benzene–toluene–xylene) fraction derived
from naphthas in refineries. There are very few alternatives. The direct thermo-
chemical conversion of biomass to an equivalent BTX product is not realistically
feasible, because only small amounts of monocyclic aromatic hydrocarbons –
phenols of the catechol and pyrogallol series – are formed on pyrolysis or ther-
mal cracking of woody feedstocks. The same is true for exposure of simple
sugars, for example d-xylose, d-glucose, or d-fructose, to either basic or slightly
acidic aqueous conditions at 100–160 8C [97]. Vanillin, however, is a by-product
of the manufacture of cellulose pulp by the action of alkali on basic calcium
lignosulfonate and may be isolated in yields of up to 25%.
An entirely different, highly promising approach from sugars to industrially

relevant aromatic compounds is based on microbial conversions along the shiki-
mic acid pathway by using genetically modified biocatalysts. By incorporation of
the genomic portion encoding the synthesis of 3-dehydroquinic and 3-dehy-
droshikimic acid into Escherichia coli constructs, the carbon flow is channeled
into accumulation of large amounts of either quinic or shikimic acid [98]
(Scheme 1.11), rendering their availability independent of often difficult isola-
tion from plant sources. These improvements are likely to lead to pronounced
expansion of the synthetic utilization of these enantiomerically pure carbocycles,
not only in the pharmaceutical industry, where quinic acid is already used as
the starting material for the synthesis of the anti-influenza drug Tamiflu (oselta-
mir phosphate [99]), but for production of bulk-scale commodity chemicals such
as hydroquinone [100] or phenol [101] by application of simple chemical trans-
formations.
The powerful potential of metabolic engineering is similarly manifested in
the E. coli biocatalyst-promoted conversion of d-glucose into protocatechuic acid,
which can be readily decarboxylated to catechol [102]. This two-step process (cf.
Scheme 1.11), feasible in a 24% overall yield, may replace the present process
used to manufacture of this 25 000 t a–1 petrochemical commodity [103]. Of sim-
ilar significance seems the genetically modified microbe-catalyzed conversion of
d-glucose into gallic acid and pyrogallol [104], the accessibility of which cur-
rently relies on isolation from plant sources, despite a spectrum of uses, partic-
ularly as educts for pharmaceuticals [105].
The recent unraveling of the biosynthesis of phloroglucinol could also pave
the way to its production from glucose. By expressing the enzyme from Pseudo-
monas fluorescens that assembles three molecules of malonyl coenzyme A into
an activated diketoheptanediote which subsequently undergoes cyclization and
conceivably spontaneous aromatization, in Escherichia coli, phloroglucinol can
be generated from glucose in yields of up to 10 g L–1 [106].
Scheme 1.11 Metabolic engineering of the (c) E. coli KL3/pWL2.46B [101], (d) E. coli
shikimic acid pathway intermediates toward KL7/pSK6.161 [102]. Abbreviations: PEP = -
aromatic chemicals: chemical phosphoenolpyruvic acid, EHP = erythrose-4-
transformations; ? bioconversions with phosphate, DAHP = 3-deoxy-D-arabino-heptu-
biocatalysts. (a) E. coli QP1.1/pKD12.138 losonic acid 7-phosphate, DHQ = 3-dehydro-
[98], (b) E. coli SP1.1PTS–/pSC6.090B [99], quinic acid, DHS = 3-dehydroshikimic acid.
Scheme 1.12 Potential generation of C7 plant acids from D-glucose [107].
Whereas protocatechuic and gallic acid are generated via the shikimic acid
pathway, other C7 plant acids of c-pyrone structure are biosynthesized from
d-glucose via 3-deoxy-manno-octulosonate 8-phosphate (KDO 8-P), as a result of
aldol-type addition of PEP to d-arabino-5-phosphate (Scheme 1.12) [107].
Although widespread throughout the plant kingdom, daucic, chelidonic, and
meconic acids are of limited availability because of their capricious isolation
from the individual plants. Given their utility for industrial purposes when read-
ily accessible, metabolic engineering of the genes involved in the respective en-
zymes is likely to be the proper approach to their large-scale acquisition.
1.4.5
Microbial Conversion of Six-carbon Sugars
into Simple Carboxylic Acids and Alcohols
Some experts predict that biotechnology will produce up to 20% of the indus-
trial chemicals by 2010 – from the current level of 5% [108, 109]. Undoubtedly,
such an increase will receive its major thrust from the various genetically engi-
neered bioprocesses currently under industrial investigation, most notably those
that involve bioconversion of sugars into industrially relevant bio-alcohols other
than ethanol and into simple C3–C5 carboxylic acids other than those already
exploited (i.e. lactic, citric, and tartaric acids, and a variety of amino acids – cf.
Table 1.1). Table 1.2 lists a variety of acids and alcohols that can be obtained by
Table 1.2 C3–C5 carboxylic acids and alcohols producible by

microbial fermentation a).
Product b) Substrate Microorganism
Acids Propionic acid Various sugars Clostridium sp.

Propionibacterium shermanii
Pyruvic acid Glucose Pseudomonas aeruginora
3-Hydroxypropionic acid Glucose E. coli constructs
Butyric acid Clostridium butyricum
3-Hydroxybutyric acid Glucose Alcaligenes eutrophus
Succinic acid Various sugars Actinobacillus succinogenes
Mannheimia succiniciproducens
Fumaric acid Various sugars Rhizopus nigricans
Rhizopus arrhizus
l-Malic acid Various sugars Parcolobactrum sp.
Brevibacterium sp.
Itaconic acid Various sugars Aspergillus terreus
Aspergillus itaconicus
2-Oxoglutaric acid Glucose Pseudomonas fluorescens
Alcohols n-Propanol Glucose Clostridium fallax
i-Propanol Various sugars Clostridium sp.
1,2-Propandiol Glucose E. coli constructs
1,3-Propanediol Glucose, glycerol Clostridium pasteurianum
E. coli mutants
Glycerol Glucose yeasts
n-Butanol Various sugars Clostridium butylicum
2,3-Butanediol Glucose, xylose Klebsiella pneumoniae
Bacillus polymyxa
1,2,4-Butanetriol Xylose (xylonic acid) E. coli construct
a) Compiled from Ref. [110]

b) Acids already exploited industrially, i.e. lactic, tartaric, and
citric acid (cf. Table 1.1) are not listed here.
microbial production and substantial activity in this field is to be expected.

Which of these products, however, are likely to enter industrial production will
be determined by a variety of factors – demand, availability of a genetically engi-
neered biocatalyst, and, not least, by economics, with rising oil prices increas-
ingly providing more favorable conditions.
The products with significant industrial potential marked in bold in Table 1.2
are briefly discussed below in respect of the current status of their microbial
production and future prospects.
1.4.5.1 Carboxylic Acids

The US Department of Energy (DOE) recently published a list of twelve sugar-
derived chemicals worthy of industrial exploitation [111], of which five are sim-
ple carboxylic acids accessible by biotransformation of sugars, mostly glucose:
· 3-hydroxypropionic acid (3-HPA)
· four-carbon 1,4-diacids (malic, fumaric, and succinic acids)
· itaconic acid (IA)
· aspartic acid (Asp)
· glutamic acid (Glu).
Each of these are considered to have high “building block potential”, either as
monomers for the production of novel polyesters and polyamides [112] or as a
starting material for a variety of commodity chemicals, currently produced pet-
rochemically. Accordingly, these five carboxylic acids are to become economically
viable products if low-cost fermentation routes can be developed and implemen-
ted on an industrial level. Even for glutamic acid – for which several fermenta-
tion processes have been industrially realized to meet the need for its sodium
salt as a flavor enhancer – the productivity of the organism and the final fer-
mentation titer must be improved if it is to become an attractive candidate for
chemical and microbial follow-up transformations [111 a]. The same is true for
aspartic acid, for which Krebs cycle pathway engineering of biocatalytic organ-
isms to overproduce oxaloacetate (cf. Scheme 1.13) are required to provide a
Scheme 1.13 Glycolytic pathway leading to the L-malic, fumaric, and succinic acids [114].
product competitive with the (racemic) one obtained currently from petrochem-
ical-derived fumaric acid by amination [111 b].
3-Hydroxypropionic Acid (3-HPA) Structurally isomeric to lactic acid, 3-HPA

represents a three-carbon building block with the potential to become a key in-
termediate for a variety of high-volume chemicals – malonic and acrylic acids,
methacrylate, acrylonitrile, 1,3-propanediol, etc. [111 c]. Cargill is developing a
low-cost fermentation route by metabolic engineering of the microbial biocata-
lyst that produces 3-HPA under anaerobic conditions [113 a], yet it will take an-
other one or two years for the process to reach commercial viability [113 b].
Unlike a product such as lactic acid, one of the attractions of 3-HPA is that it is
not manufactured commercially, either by chemical or biological means.
Fumaric Acid Fumaric acid, a metabolite of many fungi, lichens, mosses and
some plants, which is mainly used as the diacid component in alkyd resins
[115], is produced commercially to some extent by fermentation of glucose with
Rhizopus arrhizus [116], yet productivity improvements seem essential for the
product to be an option for replacing its petrochemical production by catalytic
isomerization of maleic acid.
Malic Acid Most of the malic acid produced, approximately 10 000 t a–1, is race-
mic, because it is derived from petrochemically produced fumaric acid. The L-
form can also be produced from fumaric acid by hydration with immobilized
cells of Brevibacterium or Corynebacterium species.
Succinic Acid Succinic acid is used to produce food and pharmaceutical prod-
ucts, surfactants and detergents, biodegradable solvents and plastics, and ingre-
dients to stimulate animal and plant growth. Although it is a common metabo-
lite formed by plants, animals, and microorganisms, its current commercial pro-
duction of 15 000 tons per year is from petroleum, i.e. by hydrogenation of
malic acid. The major technical hurdles for succinic acid as green, renewable
bulk scale commodity chemical – 1,4-butanediol, THF, c-butyrolactone, or pyrro-
lidones are industrially relevant products – entail the development of very low-
cost fermentation routes from sugar feedstocks. Currently available anaerobic
fermentations of glucose include use of an organism genetically cloned from As-
pergillus succinoproducens, an engineered E. coli strain developed by DOE labora-
tories [111 d], and several others [117]. The processes are currently under active
development. Production costs must be at or below $ 0.25 lb–1 to match petro-

chemical production [111d].
Itaconic Acid (IA) Structurally an a-substituted methacrylic acid, IA is a C5

building block with significant market opportunities. It is currently produced by
fungal fermentation at approximately 10 000 t a–1 [118] and mainly used as a
specialty co-monomer in acrylic or methacrylic resins, e.g. incorporation of
small amounts of IA into polyacrylonitrile significantly improves its dyeability.
To become a commodity chemical, productivity improvements with the cur-

rently used fungi Aspergillus terrous and Aspergillus itaconicus are required, and
progress seems to be encouraging [119]. To be competitive to analogous com-
modities, the crucial production price of about 0.25 $ lb–1 possesses to be
reached [111 e] – a significant technical challenge still to be solved.
1.4.5.2 Potential Sugar-based Alcohol Commodities

by Microbial Conversions
1,3-Propanediol Both the diol and the dicarboxylic acid components of poly(tri-
methylene terephthalate), a high performance polyester fiber with extensive ap-
plications in clothing textiles and carpeting, are currently manufactured from
petrochemical raw materials.
For the diol portion of the polyester, 1,3-propanediol, however, biobased alterna-
tives have been developed which rely on microbial conversion of glycerol [122],
a by-product of biodiesel production, or of corn-derived glucose [123]. For the
latter conversion, DuPont has developed a biocatalyst, engineered by incorporat-
ing genes from bakers’ yeast and Klebsiella pneumoniae into E. coli, which effi-
ciently converts corn-derived glucose in 1,3-propanediol [120, 123]. The biopro-
cess, currently being implemented on an industrial scale in a Tennessee manu-
facturing plant by a DuPont/Tate & Lyle joint venture will be providing bulk
quantities from 2006 [124].
1,2-Propanediol In its racemic form, 1,2-propanediol is a petroleum-based

high-volume chemical with an annual production of over 500 000 t, mostly used
for manufacture of unsaturated polyester resins, yet also with excellent anti-
freeze properties. Enantiomerically pure (R)-1,2-propanediol accumulates along
two different pathways via DAHP (cf. Scheme 1.11) and methylglyoxal, which is
then reduced with either hydroxyacetone or lactaldehyde as intermediates. Both
routes have been examined for microbial production from glucose by means of
genetically engineered biocatalysts, obtained either by expressing glycerol dehy-
drogenase genes or by overexpressing the methylglyoxal synthase gene in E. coli
[125]. This work provides a basis for further strain and process improvement.
Another approach entails inoculating silos of chopped whole-crop maize with
Lactobacillus buchneri. After storage for four months yields of 50 g · kg–1 were re-
ported [126]. Thus prospects for elaborating an economically sound bioprocess
look promising.
1,2,4-Butanetriol Used as an intermediate chemical for alkyd resins and rocket

fuels, 1,2,4-butanetriol is presently prepared commercially from malic acid by
high-pressure hydrogenation or hydride reduction of its methyl ester. In a novel’
environmentally benign route to this chemical, wood-derived d-xylose is micro-
bially oxidized to d-xylonic acid, followed by multistep conversion to the product
by use of a biocatalyst specially engineered by inserting Pseudomonas putida
plasmids into E. coli [127]. Although further metabolic engineering is required
to increase product concentration and yields, microbial generation of 1,2,4-but-
anetriol is a clear alternative to its acquisition by chemical procedures.
1.4.6
Chemical Conversion of Sugars into Carboxylic Acids
The sugar-derived carboxylic acids mentioned in Table 1.1, i.e. gluconic, citric,
lactic, tartaric, and ascorbic acid, are accessible in bulk by fermentation pro-
cesses and may be considered (and used as) commodity chemicals despite being
mostly used for food purposes. In addition to these, however, there are several
industrially attractive carboxylic acids obtainable from sugars by chemical
means which have high potential as versatile building blocks.
In the 2004 DOE report [111], four of these sugar-derived carboxylic acids
have already been singled out as suitable candidates for further development:
· furan-2,5-dicarboxylic acid
· glucaric acid
· levulinic acid, and
· 3-hydroxybutyrolactone
yet there are many others that equally merit the development and implementa-
tion of low-cost preparative procedures to become competitive products. They
are addressed briefly here.
The high industrial potential of furan-2,5-dicarboxylic acid (19) (cf. Scheme 1.5),
accessible from fructose or fructosans (inulin) via HMF [45], has already been
emphasized, because it could replace petroleum-derived diacids such as adipic
or terephthalic acid in the production of polyesters and polyamides. The aldaric
acids of the key hexoses and pentoses as highly hydrophilic diacids also have
much potential, similar to that of the sugar platform – d-glucaric acid, a direct
nitric acid oxidation product of glucose or starch [128], usually isolated as its
1,4-lactone, galactaric and xylaric acid, accessible from lactose [129] and from d-
xylose or hemicellulosic xylans. The technical barriers to their large-scale pro-
duction are mainly development of efficient and selective oxidation technology
for these sugars to eliminate the need for nitric acid as oxidant. Investment in
Pt-catalyzed oxidation with oxygen seems to be a promising approach.
Because sucrose is cheaper than its component sugars d-glucose and d-fructose,
and available in unprecedented quantities (cf. Table 1.1) and exceptional purity,
carboxylic acids resulting from selective oxidation of its primary hydroxyl groups
are likely of even higher industrial relevance. Because of the persistence of an
intersaccharidic water-bridge of the 2g-HO H2O HO-1–f type in aqueous

solution [130], oxidation of sucrose with air in the presence of 0.5% Pt/C at
35 8C gives a 9 : 9 : 1 ratio of the 6g-, 6f- and 1f-saccharonic acids [131]. On further
oxidation, particularly when using large amounts of the Pt catalyst and higher
temperatures (80–100 8C), the formation of sucrose-6g,6f-dicarboxylic acid 42 has
been observed [132], yet a preparatively useful procedure for its acquisition was
developed only recently [133] by combining Pt/air-oxidation with continuous
electrodialytic removal of 42, thereby protecting it from further oxidation. Other-
wise, on letting the reaction proceed, the sucrose-tricarboxylate is obtained [134].
An useful alternative oxidant to the tricarboxylate 43 is the NaOCl/TEMPO sys-
tem [135], particularly when applying high-frequency ultrasound; this results in
yields in the 70% range [136] (Scheme 1.14).
Levulinic acid (“LA”) and formic acid are end products of the acidic and ther-
mal decomposition of lignocellulosic material, their multi-step formation from
the hexoses contained therein proceeding through HMF as the key intermedi-
ate; the hemicellulose part, mostly xylans, furnishes furfural [31, 45 b]. A com-
mercially viable fractionation technology for the specific, high-yield acquisition
of LA has been developed (Chapter 7 in the first volume of this book, “The Bio-
fine Process”), rendering it an attractive option as an important biorefinery plat-
form chemical [111 g].
Levulinic acid is a starting material for a large number of higher-value products,
because it can be converted by established procedures into products such as acrylic
and succinic acids, pyrrolidines, diphenolic acid (44), which has the potential of
Scheme 1.14 Catalytic oxidation of sucrose [133, 134].

replacing bisphenol A in the manufacture of polycarbonate resins, or 5-aminole-

vulinic acid (45), applied in agriculture as a herbicide and as a growth-promoting
factor for plants. Another asset is its convertibility into 2-methyltetrahydrofuran
(46), which is used as a liquid fuel extender. The esters of LA, e.g. ethyl levulinate,
have similar industrial potential as oxygenated additives to diesel fuels.
3-Hydroxybutyrolactone (3-HBL) is now a specialty chemical for fairly high value

pharmaceuticals. The 2004 DOE report [111h] places it in the list of the top
twelve sugar-based candidates worthy of industrial exploitation. Its generation
from starch by oxidative (H2O2) degradation is regarded as “messy”, because it
involves multiple steps and results in a variety of side-products. Thus, this pro-
cess must be improved, or alternatives found; one such alternative is reduction
and cyclization of microbially produced l-malic acid (cf. Section 1.4.5.1) – to
fully utilize the potential of this four-carbon building block for the production
of a variety of industrially important tetrahydrofuranoid derivatives.
1.4.7
Biopolymers from Polymerizable Sugar Derivatives
Today, biocompatibility and biodegradability are key functional requirements in

the design of new polymeric materials, whether polyesters, polyamides, or poly-
urethanes [137]. If composed of sugar-derived monomer components, such poly-
mers are non-toxic and biodegradable and, hence, have minimal impact on
waste management. They can be safely incinerated and, by composting, can be
returned to the ecosystem harmlessly in a carbon dioxide-neutral process.
1.4.7.1 Synthetic Biopolyesters

Polyester production worldwide is estimated to be approximately 20 million t a–1,
of which only a small fraction is based on renewable monomers such as polyols,
dicarboxylic acids, or hydroxyalkanoic acids, despite the fact that a huge variety
of these building blocks are amenable to either chemical or biotechnological
production. As amply illustrated by the large variety of sugar-derived di- and
polyols, hydroxyacids, and dicarboxylic acids in Tables 1.3 and 1.4 – only those
which are reasonably accessible are listed – the number of possible polyesters is
immense, and not all conceivable combinations have been implemented and
evaluated for their application profiles. The only one of industrial relevance to-
day is Cargill’s polylactic acid (PLA), used as a benign, biodegradable material
for packaging, for disposable single use items, and for medical devices (vide su-
pra). On the verge though of becoming an industrial bioproduct is DuPont’s
poly(trimethylene terephthalate) – its high-performance fiber Sorona – because
one of its components, the presently petroleum-derived 1,3-propanediol, is being
replaced by one obtained from glucose by microbial fermentation.
Of the vast number of polyesters prepared from the monomers listed in the
tables, those containing furan residues have attracted particular interest [30, 33,
138]. 5-Hydroxymethyl-2-furoic acid, for example, gave a mixture of linear (47)
and cyclic products (48) on polycondensation [139, 140] whereas the 2-furoyl-
acrylic acid analog afforded polyester 49; 2,5-furandicarboxylic acid has been
polyesterified with a series of aliphatic diols (? 50 [140]), with dianhydrosorbitol
(? 52 [141]), or with bisphenols (? 53 [142]) (cf. Fig. 1.6). Even the all-furan
polyester 54 has been successfully prepared from its respective monomeric com-
ponents [30] – like polyesters 47–50, 52, and 54, a “fully green”, naturally re-
sourced product. The same applies to polyester 51, composed of 1,3-propanediol
and the furan-2,5-diacid – in fact an analog of DuPont’s Sorona in which the
terephthalic acid portion is replaced by a bio-counterpart. Given the same fiber
properties it would rightfully deserve the “clothing from a cornfield” attribute
[143].
Despite the versatile application profiles of these polyesters – and a vast num-
ber of others have been synthesized – they have been prepared only on the labo-
ratory scale and used in experimental applications and so currently are “aca-
demic curiosities” rather than industrially promising products. They will remain
that, however, only as long as the economics are in favor of the production of
the monomeric components from fossil raw materials – a scenario that will
change within the next 25–30 years [1].
Table 1.3 Sugar-based alcohols and diamines suitable as

monomers for polyesters, polyamides, or polyurethanes.
Compound Derivation
1,2-Propanediol D-Glucose
1,3-Propanediol D-Glucose
Glycerol Fats (D-Glucose)
2,5-Bis(hydroxymethyl)furan D-Fructose
1,6-Dianhydro-D-sorbitol D-Glucose
3,5-Bis(hydroxymethyl)pyrazole D-Xylose
Xylitol D-Xylose
D-Sorbitol D-Glucose
1,6-Diamino-1,6-dideoxy-D-glucitol D-Glucose
2,5-Diamino-1,4 : 3,6-dianhydrosorbitol D-Glucose
2,5-Bis(aminomethyl)furan D-Fructose
Table 1.4 Sugar-derived carboxylic acids for potential use as

monomers for polyesters and polyamides.
Compound Derivation
Lactic acid D-Glucose
3-Hydroxybutyric acid D-Glucose
5-Hydroxymethylfuroic acid D-Fructose
3-Hydroxyvaleric acid D-Glucose
Xylaric acid D-Xylose
D-Glucaric acid D-Glucose
D-Galactaric acid Lactose
Furan-2,5-dicarboxylic acid D-Fructose
Pyrazol-3,5-dicarboxylic acid D-Xylose
Sucrose-6,6'-dicarboxylic acid Sucrose
GMF-6,6'-dicarboxylic acid Sucrose
5-Aminomethylfuroic acid D-Fructose
6-Amino-D-gluconic acid lactam D-Glucose

(R = H, CH3)
Fig. 1.6 Polyesters containing furan moieties.
1.4.7.2 Microbial Polyesters

Microbial polyesters, or PHA for poly(hydroxyalkanoates) [144, 145], constitute
a large and versatile family of polyesters produced by various bacteria in
which they are deposited within the bacterial cell wall as a storage polymer. In-
dustrial processes have been developed for poly(3-hydroxybutyrate) [poly(3HB)]
and a polymer consisting of 3-hydroxybutyric and 3-hydroxyvalerianic acid
[ ? poly(3HB)-co-3(HV), tradename Biopol]. Both polyesters have outstanding
properties in respect of thermoplastic behavior, biocompatibility, and biodegrad-
ability, and hence have wide applications in cosmetics, hygiene, and agricultural
materials, in drug delivery systems, and in medical surgery.
Other representative PHA’s synthesized by microorganisms contain 3-hydroxy-

hexanoic, 3-hydroxyoctanoic, and malic acid as repeating units. It is to be expected
that improvement of fermentation strategies, e.g. by recombinant E. coli harboring
the microbial PHA biosynthesis genes, will enhance the economic viability of
PHA production, currently on an approximate 3000 t a–1 level, as they have the
potential to replace numerous chemosynthetic polymers in many applications.
1.4.7.3 Polyamides
More than 90% of the polyamides produced worldwide, amounting to approxi-
mately 5.8 million tons in 1998 [146], are based on six-carbon monomers, i.e.
caprolactam (Nylon-6), and adipic acid/hexamethylenediamine (Nylon-66), the
manufacture of which is based exclusively in petroleum-based pathways [146].
When considering the large variety of aminocarboxylic acids, dicarboxylic acids,

and diamines reasonably well accessible from common six-carbon sugars
[147, 148] – expedient examples are listed in Tables 1.3 and 1.4 – substitution of
petroleum-based monomers of these polyamides by those derived from sugars
seems particularly obvious and promising. Of the myriad of possible combina-
tions of these sugar-derived monomers either with themselves or with the com-
mon, petrochemically-derived diamines and dicarboxylic acids, an immense
number have been realized. Here only a few of these polyamides are covered as
examples.
Solution or interfacial polycondensation of galactaric acid dichloride in its
acetylated form with a variety of aliphatic and aromatic diamines resulted in a
series of polyamides [149], the one resulting from 1,6-diaminohexane resem-
bling a Nylon-6,6 in which half of the methylene hydrogen atoms of the usual
adipic acid are substituted by acetoxy groups (R = Ac). These can be deacylated
with aqueous ammonia to give the tetrahydroxylated Nylon-66:
Use of the lactone monomethyl ester of d-glucaric acid proved advantageous in

the generation of stereoregular polyglucaramides, effected with an impressive
array of aliphatic and aromatic diamines [149]:
Of similar practical utility is the sucrose-6,6'-dicarboxylic acid (42), readily ob-

tained from sucrose by Pt-catalyzed oxidation with oxygen in aqueous medium
(cf. Scheme 1.14), which on amidation of its dimethyl ester with fat-amines pro-
vided surface-active diamines of type 55 with remarkable tensidometric proper-
ties [150], whereas reaction with hexamethylenediamine furnished the interest-
ing, highly hydrophilic polyamide 56 [151].
Sugar-based “quasi-aromatic” monomers for polyamides, i.e. the furan-2,5-dicar-

boxylic acid, seem particularly relevant because they have the potential to re-
place petrochemically derived terephthalic or isophthalic acid in current indus-
trial products. The furan-1,6-diamine has similar potential as a substitute for p-
phenylenediamine. Indeed, a series of such furan polyamides has been pre-
pared [152] using the dicarboxylic acid and aliphatic and aromatic diamines. Of
these, the polyamide resulting from condensation with p-phenylenediamine,
which de facto is an analog of the commercially introduced Kevlar, has particu-
larly promising decomposition and glass temperature properties [153], distinctly
better than those of the all-furan polyamides:
Despite of the impressive array of highly useful products – their application profiles
compare favorably with those of well known commercial polyamides – none of these
sugar-derived polyamides is currently produced on an industrial scale; the reasons
are purely economic, because the products derived from fossil raw material sources
are still cheaper, on average by a factor of five. Eventually, however, with the end of
cheap oil predicted for 2040 [1], and the pressure on our environment increasing,
this untoward situation for products from carbohydrate feedstocks will change.
1.4.7.4 Sugar-based Olefinic Polymers (“Polyvinylsaccharides”)

The synthesis of sugars carrying O-linked residues with polymerizable double
bonds (“vinylsaccharides”) and their radical or cationic co-polymerization has
been extensively pursued over the last 50 years, the first example apparently
going back to Reppe in 1939 [154]. The following list (Fig. 1.7) gives relevant ex-
amples of the huge number of monosaccharide-derived “olefins” that have been
prepared and subjected to polymerization as such, and to co-polymerizations
with a variety of the petroleum-based standard monomers (methyl methacrylate,
methyl acrylate, acrylonitrile, styrene, etc.). The products obtained invariably
consist of a C–C backbone with pendant sugar residues:
Because the sugar groups along the carbon chain may be freed from their O-
protection by smooth simple reactions, highly hydrophilic, in most cases water-
soluble polymers are obtained. These are not accessible from the respective
monomers with free OH groups, because these usually give polymers of low
molecular weight only, due to the increasing insolubility of oligomers.
Fig. 1.7 Selected examples of pyranoid, furanoid, and acyclic

sugar derivatives carrying ether-, ester-, amide- or C–C-linked
residues with polymerizable C = C double bonds. Their poly-
mers and various copolymers with styrene, acrylonitrile, and/
or methacrylate have been characterized.
Sucrose, the most readily accessible bulk-scale sugar, has received particular
attention in this context. A large variety of mono- and disubstituted ethers and
esters have been prepared – mostly not clearly defined with respect to attach-
ment of the residues to one or two of the eight OH groups – and subjected to
radical or ionic homo- and co-polymerizations – acryloyl, and methacryloyl es-
ters, styryl ethers, ethers with primary amino groups, etc. [162–165]. Some of
the respective polymers are depicted schematically in Fig. 1.8.
1.5 Conclusion 49
Fig. 1.8 Idealized structures of a linear polymer resulting from

radical polymerization of a mono-O-methacroylsucrose (left)
and a 1 : 1 copolymerization product with styrene. Di-O-substi-
tuted “vinylsucroses” are deemed to lead to crosslinked poly-
mers (right).
Despite the broad and versatile application profiles of these polymers, mainly
because of their enhanced hydrophilicity (compared with their hydrophobic pet-
roleum-derived counterparts), none seems to be produced commercially; one
reason is that only the sugar portion of these polymers is biodegradable, leaving
an extended carbon chain. Because biodegradability is currently a major issue
[137], these polyvinylsaccharides are unlikely to become petrochemical substitu-
tion options in the near future.
1.5
Conclusion
The non-food utilization of inexpensive, bulk scale-accessible, low-molecular-

weight carbohydrates – sucrose, glucose, xylose and fructose being the most read-
ily accessible – is at a rather modest level in terms of large-scale manufactured
commodities currently on the market. The unusually diverse stock of readily ac-
cessible products described in this account, which cover a wide range of industrial
application profiles, lies mostly unexploited – for economic reasons mainly, be-
cause equivalent products based on petrochemical raw materials are distinctly
cheaper. Notwithstanding, a basic change in this scenario is clearly foreseeable.
As depletion of our fossil raw materials progresses, chemical products derived
from them will inevitably increase in price, such that biobased products will be-
come competitive. Realistic predictions [1–3] expect this by 2040 at the latest.
In the meantime, it is imperative that carbohydrates are systematically further
exploited toward efficient, environmentally benign, and economical process
methods for their large-scale conversion into industrially viable products,
whether bulk, intermediate, or fine chemicals, pharmaceuticals, or polymeric or-
ganic materials. General conceptual formulations toward this goal are available
[4, 5, 166], yet their straightforward implementation is currently exceedingly
slow. To enhance this, it is essential that national and supranational funding in-
stitutions – in Europe the corresponding EU bodies (in the European Commis-
sion’s 7th framework program?) and/or the European Renewable Resources and
Materials Association [167] – play a substantially more dynamic role than hither-
to, e.g. by the elaboration of a conclusive funding strategy for the material utili-
zation of biorenewables rather than for biofuels and bioenergy only – a funding
strategy capable of providing and maintaining a judicious balance between applied
and basic research.
Applied research ordinarily has predefined goals usually applying known scien-
tific facts to the solution of practical problems, and has constraints that require
results in a short time. It certainly should be funded and implemented on a
broad basis, yet its short-term pressures, e.g. the expectation of obtaining mar-
ketable products within a three-year time frame, are often counter-innovative
and tend to dodge research projects too complex to be solved in a few years.
Basic (fundamental) research, in contrast, is driven by the interest and curiosity
of an investigator usually following his intuition, be it an unresolved phenome-
non, conflicting literature data, or the unconstrained exploration of a new inter-
Fig. 1.9 Ronald Searle’s notion [172] of miners searching for

gold with conventional means along much-(re)searched main
roads.
References 51
face between existing methods; it frequently creates its aims and specific targets
along the way. Basic research of this sort inspires project proposals “for which
it is infeasible or impractical to express a performance goal in an objective,
quantifiable, and measurable form” [168]. Because Europe’s national granting
agencies, e.g. the FNR [169] or AGRICE [170], do not have provisions enabling
funding for such proposals, they have no chance of being supported [171].
Nevertheless, it is the author’s firm conviction that new, industrially viable, su-
gar-based chemicals and materials are likely to emerge as much from basic re-
search as from mission-oriented applied investigations. Thus, there is an urgent
need to create an instrument in our granting agency system that gives innova-
tive basic research projects an equal chance of support.
In this context, a pertinent cartoon (Fig. 1.9) seems to catch the essence of
the present scenario – “biogold”-bearing seams are not only to be found along
the common, much (re)searched main roads with conventional, generally ap-
plied means, but also – maybe even more likely – in remote, virgin, basic terri-
tory with unconventional, innovative methodology, and a healthy measure of in-
tuition and serendipity.
For full utilization of the “biogold” lying in carbohydrates – widely available
from the plant kingdom and easily recovered at low cost – any promising, inno-
vative research project, irrespective of involving mission-oriented, applied inves-
tigations or non-predefined basic explorations, should receive generous support
either by funding institutions or by the chemical industry and/or both. Econom-
ically sound biobased alternatives to petrochemicals – various potential exam-
ples are contained in this review – will then become available as a matter of
course.
References
1 (a) C. J. Campbell, J. H. Laherrére, “The 4 National Research Council, USA. Bio-

end of cheap oil”, Sci. Am., March 1998, based Industrial Products: Priorities for Re-
60–65. (b) J. Attarian, “The coming end search and Commercialization, Natl. Acad.
of cheap oil: Hubbert’s peak and be- Press: Washington, 2000. http://www.
yond”, The Social Contracts 2002, 12, nap.edu/books/0309053927/html/
276–286. (c) C. J. Campbell, “Industry 5 (a) Biomass Research & Development
urged to watch for regular oil production Technical Advisory Committee, Roadmap
peaks, depletion signals”, Oil & Gas J. for Biomass Technologies in the US, Dec.
2003, 101, 38–45. 2002; http://www.bioproducts-bioenergy.
2 D. H. Klass, “Fossil fuel reserves and de- gov/pdfs/FinalBiomassRoadmap.pdf.
pletion”, Biomass for Renewable Energy, (b) Vision for bioenergy & biobased products
Fuels, and Chemicals, Acad. Press, San in the US, Oct. 2002; http://
Diego, 1998; pp. 10–19. www.bioproducts-bioenergy.gov/pdfs/
3 R. H. Hirsch, R. Bezdek, R. Wendling, BioVision_03_-Web.pdf
“Peaking of world oil production: Im- 6 UN Food & Agriculture Organization.
pacts, mitigation, and risk management”, World Sugar Production, 2004/05. http://
US National Energy Technology Laboratory www.fao.org/es/esc/en/20953/21032/
Report, Febr. 2005, 91 pp.; http://www.oil- highlight_25853en.html
crisis.com/US/NETL/OilPeaking.pdf
7 F. W. Schenck, “Glucose and glucose- 22 F. W. Lichtenthaler, S. Mondel, “Perspec-

containing syrups”, Ullmann’s Encyclo- tives in the use of low-molecular-weight
pedia of Industrial Chem. 1989, A12, 457– carbohydrates as organic raw materials”,
476. Pure Appl. Chem. 1997, 69, 1853–1866.
8 W. Wach, Fructose. Ullmann’s Encyclo- 23 F. W. Lichtenthaler, “Towards improving
pedia Industrial Chem. 2004, http:// the utility of ketoses as organic raw ma-
www.mrw.interscience.wiley.com/ueic/ terials”, Carbohydr. Res. 1998, 313, 69–89.
articles/a12_047/frame.html 24 Chemicals and Materials from Renewable
9 H. Schiweck, A. Bär, R. Vogel, E. Resources, J. J. Bozell, ed., ACS Sympo-
Schwarz, M. Kunz, “Isomaltulose, treha- sium Series No. 784, 2001, 226 pp.
lulose and isomalt”, Ullmanńs Encyclope- 25 F. W. Lichtenthaler, “Unsaturated O- and
dia Industrial Chem. 1994, A25, 426–429. N-heterocycles from carbohydrate feed-
10 W. A. Roelfsema, B. F. M. Kuster, M. C. stocks”, Acc. Chem. Res. 2002, 35, 728–
Heslinga, H. Pluim, M. Verhage, “Lac- 737.
tose and derivatives”, Ullmann’s Encyclo- 26 F. W. Lichtenthaler, S. Peters, “Carbohy-
pedia Industrial Chem. 1990, A15, 107– drates as green raw materials for the
114. chemical industry”, Comptes Rendue Chi-
11 G. Tegge, “Maltose”, Ullmann’s Encyclo- mie 2004, 7, 65–90.
pedia Industrial Chem. 1983, 24, 770–772. 27 Wirtschaftliche Vereinigung Zucker,
12 B. Oster, W. Fechtel, “Vitamin C”, Ull- “Biokraftstoffe aus Zuckerrüben und Ge-
manńs Encyclopedia Industrial Chem. treide”, www.zuckerwirtschaft.de/pdf/
1996, A27, 547–559. Broschuere_BioE.pdf
13 (a) http://www.eridex.com/html. 28 (a) For a pertinent overview, see: M. E.
(b) http://www.micchem.com/products/ Himmel, W. A. Adney, J. O. Baker, R.
Erythritol.htm. (c) P. De Cock, C.-L. Be- Elander, J. D. McMillan, R. A. Nieves, J. J.
chert, “Erythritol in non-caloric func- Sheehan, S. R. Thomas, T. B. Vinzant, M.
tional beverages”, Pure Appl. Chem. 2002, Zhang, “Advanced bioethanol production
74, 1281–1289. technologies”. In: Fuels and Chemicals
14 A. Bär, R. Vogel, E. Schwarz, “Xylitol, from Biomass, B. C. Saha, J. Woodward,
sorbitol and mannitol”, Ullmann’s Ency- eds., ACS Symp. Ser. No 666, 1997,
clopedia Industrial Chem. 1994, A25, 416– pp. 2–45. (b) H. G. Lawford, J. D. Rous-
426. seau, “Cellulosic fuel ethanol – Alterna-
15 H. Hustede, H.-J. Haberstroh, E. Schin- tive fermentation process design with
zig, “Gluconic Acid”, Ullmann’s Encyclo- wild-type and recombinant Zymomonas
pedia Industrial Chem. 1989, A12, 449 ff. mobilis”, Appl. Biochem. Biotechnol. 2003,
16 D. L. Klass, “Organic commodity chemi- 105, 457–469. (c) O. Goebel, “Compari-
cals from biomass”, Ref. 2, pp. 495–546. son of process economics for synthetic
17 Carbohydrates as Organic Raw Materials I, and fermentation ethanol”, Ullmann’s
F. W. Lichtenthaler, ed., VCH, Weinheim, Encyclopedia Industrial Chem. 2001,
1991, 367 pp. http://www.mrw.interscience.wiley.com/
18 Carbohydrates as Organic Raw Materials ueic/articles/a09_587/frame.html
II, G. Descotes, ed., VCH, Weinheim, 29 (a) W. J. Mc Killip, G. Collin, H. Höke,
1993, 278 pp. K. J. Zeitsch, “Furan and derivatives”,
19 Carbohydrates as Organic Raw Materials Ullmanńs Encyclopedia Industrial Chem.
III, H. Van Bekkum, H. Röper, A. Vora- 2000, http://www.mrw.interscience.
gen, eds., VCH, Weinheim, 1996, 315 wiley.com/ueic/articles/a12_119/
pp. frame.html. (b) K. J. Zeitsch, The Chemis-
20 Perspectiven nachwachsender Rohstoffe in try and Technology of Furfural and its
der Chemie, H. Eierdanz, ed., VCH, many Byproducts, Elsevier, Amsterdam,
Weinheim, 1996, 358 pp. 2000, 374 pp.
21 Carbohydrates as Organic Raw Materials 30 A. Gandini, M. N. Belgacem, “Furans in
IV, W. Praznik, A. Huber, eds., Wiener polymer chemistry”, Prog. Polym. Sci.
Universitätsverlag 1997, 288 pp. 1997, 22, 1203–1379.
References 53
31 F. D. Klingler, “Levulinic acid”, Ullmann’s 43 L. A. Silviri, N. J. DeAngelis, “Isosorbide

Encyclopedia Industrial Chem. 2000, dinitrate”, Anal. Profiles Drug Subs. 1975,
http://www.mrw.interscience.wiley.com/ 4, 225–244.
ueic/articles/a18_313/frame.html. 44 B. E. Maryanoff, S. O. Nortey, J. F. Gar-
32 M. S. Holfinger, A. H. Conner, D. R. docki, R. P. Shank, S. P. Dodgson, Antic-
Holm, C. G. Gill, Jr., “Difurfuryl dia- onvulsant sulfamates. 2,3 : 4,5-Di-O-iso-
mines by the acidic condensation of fur- propylidene-b-d-fructopyranose sulfa-
furylamine with aldehydes”, J. Org. mate. J. Med. Chem. 1987, 30, 880–887.
Chem. 1995, 60, 1595–1598. 45 For pertinent reviews, see: (a) J. Lew-
33 C. Moreau, M. N. Belgacem, A. Gandini, kowski, “Synthesis, chemistry and appli-
“Substituted furans from carbohydrates cations of 5-hydroxymethylfurfural and
and ensuing polymers”, Topics in Cataly- its derivatives”, ARKIVOC 2001, 17–54.
sis 2004, 27, 11–30. (b) B. F. M. Kuster, “Manufacture of 5-hy-
34 R. Vogel, “Sorbitol”, Ullmann’s Encyclope- droxymethylfurfural”, Starch/Stärke 1990,
dia Industrial Chem. 2000, http:// 42, 314–321. Newer developments:
www.mrw.interscience.wiley.com/ueic/ (c) M. Bicker, J. Hirth, H. Vogel, “Dehy-
articles/a25_413/frame.html. dration of fructose to 5-hydroxymethyl-
35 W. von Rybinski, K. Hill, “Alkyl polygly- furfural in sub- and supercritical acet-
cosides – properties and applications of one”, Green Chemistry 2003, 5, 280–284.
a new class of surfactants”, Angew. Chem. (d) C. Lansalot-Matras, C. Moreau, “De-
1998, 110, 1394–1412; Angew. Chem. Int. hydration of fructose to 5-hydroxymethyl-
Ed. 1998, 37, 1328–1345. furfural in ionic liquids”, Catalysis Com-
36 (a) M. McCoy, “Seeking Biomaterials”, mun. 2003, 4, 517–520.
Chem. Eng. News 2003, 81 (8), 18; 2003, 46 H. Schiweck, M. Munir, K. Rapp, M. Vo-
81 (45), 17–18. (b) S. K. Ritter, “Green gel, “New developments in the use of su-
chemistry progress report”, Chem. Eng. crose as an industrial bulk chemical”. In:
News 2002, 80 (47), 20. F. W. Lichtenthaler, ed., Carbohydrates as
37 http://www.cargilldow.com/nature-works. Organic Raw Materials, VCH, Weinheim,
asp 1991, pp. 57–94; Zuckerind. (Berlin)
38 http://www.vertecbiosolvents.com/ 1990, 115, 555–565.
39 K. H. Hill, O. Rhode, “Sugar-based sur- 47 H. Koch, J. Pein, “Condensations be-
factants for consumer products and tech- tween 5-hydroxymethylfurfural, phenol,
nical applications”, Fett/Lipid 1999, 101, and formaldehyde”, Polym. Bull. (Berlin)
27–33. 1985, 13, 525–532; Starch/Stärke 1983,
40 P. Jürges, A. Turowski, “Vergleichende 35, 304–313.
Untersuchung von Zuckerestern, N- 48 T. El Haj, J. A. Masroua, J.-C. Martin, G.
Methylglucamiden und Glycosiden am Descotes, “5-Hydroxymethylfurfural and
Beispiel von Reinigungsprodukten”, in derivatives”, Bull. Soc. Chim. Fr. 1987,
Perspektiven Nachwachsender Rohstoffe in 855–860.
der Chemie, VCH, Weinheim, 1996, 49 C. Larousse, L. Rigal, A. Gaset, “Thermal
pp 61–70. degradation of 5-hydroxymethylfurfural”,
41 (a) N. B. Desai, “Esters of sucrose and J. Chem. Technol. Biotechnol. 1992, 53,
glucose as cosmetic materials”, Cosmetics 111–116.
& Toiletries 1990, 105, 99–107. (b) Mitsu- 50 V. Schiavo, G. Descotes, J. Mentech,
bishi-Kagaku Foods Corp., “Sugar esters”. “Hydrogenation of 5-hydroxymethylfur-
http://www.mfc.co.jp/english/whatsse. fural”, Bull. Soc. Chim. Fr. 1991, 128,
htm 704–711.
42 J. L. McGuire, “Pharmaceuticals: General 51 N. Elming, N. Clauson-Kaas, “6-Methyl-
Survey and Development”, Ullmann’s 3-pyridinol from 2-hydroxymethyl-5-ami-
Encyclo_pedia Industrial Chem. 2000, nomethyl-furan”, Acta Chem. Scand.
http://www.mrw.interscience.wiley.com/ 1956, 10, 1603–1605.
ueic/articles/a195_273/frame.html. 52 (a) E. Leupold, M. Wiesner, M. Schling-
mann, K. Rapp, “Catalytic oxidation of 5-
hydroxymethylfurfural”, Ger. Offen. DE 65 F. W. Lichtenthaler, “Building blocks

3.826.073 (1988). (b) M. L. Ribeiro, U. from sugars and their use in natural
Schuchardt, “Cooperative effect of cobalt product synthesis”, In: Modern Synthetic
acetylacetonate and silica in the catalytic Methods, Vol. 6, R. Scheffold, ed., VCH,
cyclization and oxidation of fructose to Weinheim, 1992, pp. 273–376.
2,5-furandicarboxylic acid”, Catalysis 66 C. Nelson, J. Gratzl, “Conversion of
Commun. 2003, 4, 83–86. d-glucono-1,5-lactone into an a-pyrone”,
53 Südzucker 2005: http://www.isomalt.de Carbohydr. Res. 1978, 60, 267–273.
54 F. W. Lichtenthaler, R. Klimesch, V. Mül- 67 K. Tajima, “Cyclopentenones from 3-ace-
ler, M. Kunz, “Disaccharide-building toxy-6-acetoxymethyl-2-pyrone”, Chem.
blocks from isomaltulose: Glucosyl-a Lett. 1987, 1319–1322.
(1?5)-d-arabinonic acid and ensuing 68 For useful preparative procedures, see:
products”, Liebigs Ann. 1993, 975–980. Methods Carbohydr. Chem. 1963, 2, 318,
55 F. W. Lichtenthaler, D. Martin, T. Weber, 326, 405, and 427.
H. Schiweck, “5-(a-d-Glucosyloxymethyl)- 69 F. W. Lichtenthaler, “Sugar-derived build-
furfural: Preparation from isomaltulose ing blocks for the synthesis of non-car-
and exploration of its ensuing chemis- bohydrate natural products”, In: Carbohy-
try”, (a) Ger. Offen. 3.936.522 (1989); drate Synthons in Natural Product Chemis-
(b) Liebigs Ann. 1993, 967–974. try (S. J. Witczak, K. Tatsuta, eds.), ACS
56 F. W. Lichtenthaler, A. Brust, T U Darm- Symp. Series 841, 2003, 47–83. (b) S.
stadt, unpublished results. Hanessian, Total Synthesis of Natural
57 T. Hanemann, W. Haase, F. W. Lichten- Products: The Chiron Approach, Perga-
thaler, “Disaccharide-derived liquid crys- mon, Oxford, 1983.
tals”, Liquid Cryst. 1997, 22, 47–50. 70 R. J. Ferrier, N. Prasad, “Synthesis of 2,3-
58 F. García-Gonzáles, “Reactions of mono- dideoxy-a-d-erythro-hex-2-enopyranosides
saccharides with b-ketoesters”, Adv. Car- from tri-O-acetyl-d-glucal”, J. Chem. Soc.
bohydr. Chem. 1956, 11, 97–143. C, 1969, 570–575.
59 A. J. Moreno-Vargas, J. Fuentes, J. Fer- 71 F. W. Lichtenthaler, S. Rönninger, P. Jar-
nández-Bolanos, I. Robina, “Synthesis of glis, “Expedient approach to pyranoid
hetarylene-carbopeptoids”, Tetrahedron ene and enol lactones”, Liebigs Ann.
Lett. 2001, 42, 1283–1285. 1989, 1153–1161.
60 (a) F. Rodrigues, Y. Canac, A. Lubineau, 72 (a) S. Hanessian, A. M. Faucher, S. Leger,
“A convenient, one-step synthesis of b-C- “Total synthesis of meroquinene”, Tetra-
glycosidic ketones in aqueous media”, hedron 1990, 46, 231–234. (b) N. Ohyabu,
Chem. Commun. 2000, 2049–2050. T. Nishikawa, M. Isobe, “First asym-
(b) I. Riemann, M. A. Papadopoulos, metric total synthesis of tetrodotoxin”,
M. Knorst, W.-D. Fessner, “C-Glycosides J. Am. Chem. Soc. 2003, 125, 8798–8805.
by aqueous condensation of b-dicarbonyl 73 S. Czerneckí, K. Víjayakuraman, G. Ville,
compounds with unprotected sugars”, “Convenient synthesis of hex-1-enopyran-
Aust. J. Chem. 2002, 55, 147–154. 3-uloses: Selective oxidation of allylic
61 S. Peters, F. W. Lichtenthaler, H. J. Lind- alcohols using pyridinium dichromate”,
ner, “A C-Fructosyl-propanone locked in J. Org. Chem. 1986, 51, 5472–5475.
a 2,7-Dioxabicyclo[3.2.1]octane frame- 74 B. Fraser-Reid, A. McLean, E. W. Usher-
work”, Tetrahedron: Asymmetry 2003, 14, wood, M. Yunker, “Synthesis and proper-
2475–2479. ties of some alkyl 2,3-dideoxy-2-enopyra-
62 Y. Chapleur, ed., Carbohydrate Mimics, nosid-4-uloses”, Can. J. Chem. 1970, 48,
Wiley-VCH, Weinheim/New York 1998, 2877–2884.
p. 604 ff. and references cited therein. 75 R. J. Ferrier, “Modified synthesis of 2-hy-
63 A. Beélik, “Kojic acid”, Adv. Carbohydr. droxyglycalesters”, Methods Carbohydr.
Chem. 1956, 11, 145–183. Chem. 1972, 6, 307–311.
64 F. W. Lichtenthaler, “Sugar enolones and 76 F. W. Lichtenthaler, U. Kraska, “Prepara-
c-pyrone-formation”, Pure Appl. Chem. tion of sugar enolones”, Carbohydr. Res.
1978, 50, 1343–1362. 1977, 58, 363–377.
References 55
77 F. W. Lichtenthaler, S. Nishiyama, T. Wei- 87 A. Rozanski, K. Bielawski, J. Boltryk, D.

mer, “2,3-Dihydropyranones with contig- Bartulewicz, “Simple synthesis of 3-ace-
uous chiral centers”, Liebigs Ann. 1989, tyl-5-(tetrahydroxybutyl)-2-methylpyrrole”,
1163–1170. Akad. Med. Juliana Marchlewskiego Bia-
78 F. W. Lichtenthaler , S. Ogawa, P. Heidel, lymstoku 1991, 35–36, 57–63; Chem.
“Synthesis of unsaturated hexopyrano- Abstr. 1992, 118, 22471m.
sid-4-uloses”, Chem. Ber. 1977, 110, 88 V. Diehl, E. Cuny, F. W. Lichtenthaler,
3324–3332. “Conversion of d-xylose into hydrophili-
79 (a) J. Gravitis, N. Vedernikon, J. Zander- cally functionalized pyrazoles”, Hetero-
sons, A. Kokorevics, “Furfural and levo- cycles 1998, 48, 1193–1201.
glucosan production from deciduous 89 M. Oikawa, C. Müller, M. Kunz, F. W.
wood and agricultural wastes”, in J. J. Lichtenthaler, “Hydrophilic pyrazoles
Bozell, ed., Chemicals and Materials from from sugars”, Carbohydr. Res. 1998, 309,
Renewable Resources, ACS Symp. Ser. No. 269–279.
784, 2001, pp. 110–122. (b) R. C. Brown, 90 R. Weidenhagen, R. Hermann, “4-Hy-
D. Radlein, J. Piskorz, “Pretreatment droxymethylimidazole”, Ber. Dtsch. Chem.
processes to increase pyrolytic yield of le- Ges. 1937, 70, 570–583; Org. Synth., Coll.
voglucosan from herbaceous feedstocks”, Vol. III, 1955, 460–462.
in: Chemicals and Materials from Renew- 91 J. Streith, A. Boiron, A. Frankowski, D.
able Resources, J. J. Bozell, ed., ACS Le Nouen, H. Rudyk, T. Tschamber,
Symp. Ser. No. 784, 2001, pp. 123–132. “One-pot synthesis of imidazolosugars”,
(c) F. Shafizadeh, R. Furneaux, T. Ste- Synthesis 1995, 944–946.
venson, “Reactions of levoglucosenone”, 92 N. Elming, S. V. Carlsten, B. Lennart,
Carbohydr. Res. 1979, 71, 169–191. I. Ohlsson, “Improvement in preparing
80 Z. J. Witczak, “Synthesis of natural prod- 3-pyridinols”, Brit. Pat. 862,581 (1961);
ucts from levoglucosenone”, Pure Appl. Chem. Abstr. 1962, 56, 11574g.
Chem. 1994, 66, 2189–2192. 93 V. Koch, L. Willms, A. Fuss, K. Bauer,
81 F. W. Lichtenthaler, S. Immel, D. Martin, H. Bieringer, H. Buerstell, “Heterocyclic
V. Müller, “Disaccharide-building blocks phenoxypyridines as herbicidal agents
of potential industrial utility”, Zuckerind. and insecticides”, Eur. Pat. 227045,
(Berlin) 1992, 44, 445–456. 227046 (1987); Chem. Abstr. 1987, 107,
82 F. Ledl, E. Schleicher, “The Maillard re- 175892, 134217.
action in foodstuffs and in the human 94 C. Müller, V. Diehl, F. W. Lichtenthaler,
body”, Angew. Chem. 1990, 102, 597–626; “3-Pyridinols from fructose and isomal-
Angew. Chem. Int. Ed. 1990, 29, 565–594. tulose”, Tetrahedron 1998, 54, 10703–
83 (a) H. ElKhadem, “N-Heterocycles from 10712.
saccharide derivatives”, Adv. Carbohydr. 95 H. Ohle, M. Hielscher, “Darstellung von
Chem. 1970, 25, 351–405. (b) E. S. H. Tetraoxybutyl-chinoxalin”, Ber. Dtsch.
ElAshry, N. Rashad, “Carbohydrate hy- Chem. Ges. 1941, 74, 13–19.
drazones and osazones as raw materials 96 (a) H. Ohle, G. A. Melkonian, “Flavazol,
for nucleosides and N-heterocycles”, ein neuer Heterocyclus aus Zuckern”,
Curr. Org. Chem. 2000, 4, 609–657. Ber. Dtsch. Chem. Ges. 1941, 74, 279–285.
84 S. M. McElvain, K. M. Bolliger, “Pyrrole”, (b) H. Ohle, A. Iltgen, “Flavazole”, Ber.
Org. Synth., Coll. Vol. 1, 1941, 473–474. Dtsch. Chem. Ges. 1943, 76, 1–14.
85 F. W. Lichtenthaler, A. Brust, E. Cuny: 97 (a) I. Forsskahl, T. Popoff, O. Theander,
“Hydrophilic pyrroles, pyridazines and “Reactions of d-xylose and d-glucose in
diazepinones from d-fructose and iso- alkaline aqueous solutions”, Carbohydr.
maltulose”, Green Chemistry 2001, 3, Res. 1976, 48, 13–21. (b) T. Popoff,
201–209. O. Theander, “Formation of aromatic
86 F. García-Gonzáles, A. Gómez Sánchez, compounds from d-glucose and d-fruc-
“Reactions of amino sugars with b-dicar- tose in slightly acidic aqueous solu-
bonyl compounds”, Adv. Carbohydr. tion”, Acta Chem. Scand. 1976, B30,
Chem. 1965, 20, 303–355. 397–402.
98 K. M. Drahts, D. R. Knop, J. W. Frost, implications”, Angew. Chem. Int. Ed.

“Shikimic acid and quinic acid: Replac- 2003, 42, 5838–5843; Tetrahedron:
ing isolation from plant sources with Asymmetry 2003, 14, 3973–3986.
recombinant microbial catalysis”, J. 108 E. K. Wilson, “Engineering cell-based
Am. Chem. Soc. 1999, 121, 1603–1604. factories”, Chem. Eng. News 2005, 83
99 J. C. Roloff, K. M. Kent, M. J. Postich, (12), 41–44.
M. W. Beeker, H. H. Chapman, D. E. 109 J. Riese, R. Bachmann, “Industrial bio-
Kelly, W. Lew, M. S. Louie, L. R. technology: Turning the potential into
Mc Gee, E. J. Prisbe, L. M. Schultze, profits”, Chemical Market Reporter 2004
R. H. Yu, L. Zhang, “Practical total syn- (Dec. 1.); http://www.mckinsey.com/-
thesis of the anti-influenza drug GS- clientservice/chemicals/potentialprofit.
4104”, J. Org. Chem. 1998, 63, 4545– asp
4550. 110 Fundamentals of Biotechnology, P. Präve,
100 N. Ran, D. R. Knop, K. M. Drahts, J. W. U. Faust, W. Sittig, D. A. Sukatsch,
Frost, “Benzene-free synthesis of hy- eds., VCH, Weinheim, 1987.
droquinone”, J. Am. Chem. Soc. 2001, 111 Doe, Energy Efficiency and Renewable
123, 10927–10934. Energy, “Top value added chemicals
101 J. M. Gibson, P. S. Thomas, J. D. Tho- from biomass: Screening for potential
mas, J. L. Barker, S. S. Chandran, M. K. candidates from sugars and synthesis
Harrup, K. M. Drahts, J. W. Frost, “Ben- gas”, http://www.nrel.gov/docs/
zene-free synthesis of phenol”, Angew. fy04osti/35523.pdf, 67 pp. –
Chem. Int. Ed. 2001, 40, 1945–1948. (a) “Glutamic acid”, pp. 39–42;
102 (a) K. M. Drahts, J. W. Frost, “Environ- (b) “Aspartic acid”, pp. 31–35; (c) “3-
mentally compatible synthesis of cate- Hydroxypropionic acid (3-HPA)”,
chol from d-glucose”, J. Am. Chem. pp. 29–31; (d) “Four-carbon 1,4-dia-
Soc. 1975, 117, 2395–2400. (b) W. Li, cids”, pp. 22–25; (e) “Itaconic acid”,
D. Xie, J. W. Frost, “Benzene-free syn- pp. 42–44; (f) “Glucaric acid”, pp. 36–
thesis of catechol: Interfacing microbial 38; (g) “Levulinic acid”, pp. 45–48;
and chemical synthesis” J. Am. Chem. (h) “3-Hydroxybutyrolactone”, pp. 49–
Soc. 2005, 127, 2874–2882. 51.
103 T. Hamamoto, S. Uemura, “Catechol”, 112 S. Y. Lee, S. H. Park, S. H. Hong, Y.
Ullmann’s Encyclopedia of Industrial Lee, S. H. Lee, “Fermentative produc-
Chem., Wiley–VCH 2000; http:// tion of building blocks for chemical
www.mrw.interscience.wiley.com/ueic/ synthesis of polyesters”, Biopolymers,
articles/a19_313/frame.html Wiley-VCH, Weinheim 2001, Vol. 3b,
104 S. Kambourakis, K. M. Drahts, J. W. chapter 10, p. 265 ff.
Frost, “Synthesis of gallic acid and py- 113 (a) D. C. Cameron, 2003, “3-Hydroxy-
rogallol from glucose: Replacing natu- propionic acid: A new platform chemi-
ral product isolation with microbial cat- cal from the biorefinery”, http://www.-
alysis”, J. Am. Chem. Soc. 2000, 122, ibeweb.org/meetings/2005/proceed-
9042–9043. ings/abstracts/industryªbstracts.html.
105 T. Iwata, H. Miki, Y. Fujita, Trihydroxy- (b) A. Tullo, Chem. Eng. News 2005, 83
benzenes. Ullmann’s Encyclopedia of In- (26), 11.
dustrial Chem., 2000; http:// 114 (a) For pertinent reviews, see: (a) S. Y.
www.mrw.interscience.wiley.com/ueic/ Lee, S. H. Hong, S. H. Lee, S. J. Park,
articles/a19_313/frame.html “Fermentative production of chemicals
106 J. Achkar, M. Xian, H. Zhao, J. W. that can be used for polymer synthe-
Frost, “Biosynthesis of phloroglucinol”, sis”, Macromol. Biosci. 2004, 4, 157–
J. Am. Chem. Soc. 2005, 127, 5332– 164. (b) G. T. Tsao, N. J. Cao, J. Du,
5333. C. S. Gong, “Production of multifunc-
107 F. W. Lichtenthaler, K. Nakamura, J. tional organic acids from renewable re-
Klotz, “(–)-Daucic acid: Revision of con- sources”, Adv. Biochim. Eng. Biotechnol.
figuration, synthesis, and biosynthetic 1999, 65, 243–280.
References 57
115 K. Lohbeck, H. Haferkorn, W. Fuhr- duction and new trends”, Adv. Biochem.
mann, “Maleic and fumaric acids”, Ull- Engineering/Biotechn. 2002, 74, 239–
manńs Encyclopedia Industrial Chem., 259.
Wiley–VCH, 2000; http://www.mrw.in- 124 DuPont 2004, press release: http://
terscience.wiley.com/ueic/articles/ www.dupont.com/sorona/news/
a16_053/frame.html 052604.html
116 F. S. Carta, C. R. Soccol, L. P. Ramos, 125 (a) N. E. Altaras, D. C. Cameron, “Meta-
J. D. Fontana, “Production of fumaric bolic engineering of a 1,2-propanediol
acid by fermentation of enzymic hydro- pathway in E. coli”, Appl. Environ. Micro-
lyzates derived from cassava bagasse”, biol. 1999, 65, 1180–1185. (b) “En-
Bioresour. Technol. 1999, 68, 23–28. hanced production of (R)-1,2-propane-
117 (a) G. N. Vemuri, M. A. Eiteman, E. Alt- diol by metabolically engineered
man, “Succinate production in dual- E. coli”, Biotechnol. Progr. 2000, 16, 940–
phase E. coli fermentations depends on 946. (c) N. E. Altaras, M. R. Etzel, D. C.
the time of transition from aerobic to Cameron, “Conversion of sugars to 1,2-
anaerobic conditions”, J. Ind. Microbiol. propanediol by Thermoanaerobacterium
Biotechnol. 2002, 28, 325–332. (b) “Ef- thermosaccharolyticum”, Biotechnol.
fects of growth mode and pyruvate car- Progr. 2001, 17, 52–56.
boxylase on succinic acid production 126 N. Nishino, M. Yochida, H. Shiota, E.
by metabolically engineered E. coli Sakaguchi, “Accommodation of 1,2-pro-
strains”, Appl. Environ. Microbiol. 2002, panediol and enhancement of aerobic
68, 1715–1727. (c) R. R. Gokarn, M. A. stability in whole crop maize silage in-
Eiteman, J. Sridhar, “Production of suc- oculated with Lactobacillus buchneri”,
cinate by anaerobic microorganisms”, J. Appl. Microbiol. 2003, 94, 800–807.
in Fuels and Chemicals from Biomass, 127 W. Niu, M. N. Molefe, J. W. Frost, “Mi-
B. D. Saha, J. Woodard, eds., ACS crobial synthesis of the energetic mate-
Symp. Series 666, 1997, pp. 237–279. rial precursor 1,2,4-butanetriol”, J. Am.
118 J. H. Kane, A. Finlay, P. F. Amann Chem. Soc. 2003, 125, 12998–12999.
(Chas. Pfizer, Merck), “Itaconic acid”. 128 C. L. Mehltretter, “d-Glucaric acid”,
US Pat. 2 385–283 (1945); Chem. Abstr. Methods Carbohydr. Chem. 1963, 2, 46–
1946, 40, 995. 48.
119 (a) C. S. Reddy, R. P. Singh, “Enhanced 129 B. A. Lewis, F. Smith, A. M. Stephen,
production of itaconic acid from corn “Galactaric acid and its derivatives”,
starch and market refuse fruits by ge- Methods Carbohydr. Chem. 1963, 2, 38–
netically manipulated Aspergillus terreus 46.
SKR 10”, Bioresour. Technol. 2002, 85, 130 S. Immel, F. W. Lichtenthaler, “The
69–71. (b) T. Willke, K. Welter, K. D. conformation of sucrose in water: A
Vorlop, “Biotechnical production of ita- molecular dynamics approach”, Liebigs
conic acid from sugar”, Appl. Microbiol. Ann. Chem. 1995, 1938–1947.
Biotechnol. 2001, 56, 289–295. 131 M. Kunz, H. Puke, C. Recker, L.
120 DuPont 2004: http://www.dupont/soro- Scheiwe, J. Kowalczyk (Südzucker AG),
na/biobasedinitiative.html “Process and apparatus for the prepara-
121 SHELL 2004: http://www.shellchem- tion of mono-oxidized products from
icals.com/corterra/1,1098,280,00.html carbohydrates”, Ger. Offen. DE 4 307 388
122 M. M. Zhu, P. D. Lawman, D. C. Ca- (1994); Chem. Abstr. 1995, 122, 56411.
meron, “Improving 1,3-propanediol 132 L. A. Edye, G. V. Meehan, G. N. Ri-
production from glycerol in a metaboli- chards, “Platinum-catalyzed oxidation
cally engineered E. coli by reducing ac- of sucrose”, J. Carbohydr. Chem. 1991,
cumulation of glycerol-3-phosphate”, 10, 11–23; 1994, 13, 273–283.
Biotechnol. Prog. 2002, 18, 694–699. 133 M. Kunz, A. Schwarz, J. Kowalczyk
123 For an informative review, see: A. N. (Südzucker AG), “Process for continu-
Zeng, H. Biebl, “Bulk chemicals from ous manufacture of di- and higher-oxi-
biomass: Ease of 1,3-propanediol pro- dized carboxylic acids from carbohy-
drates”, Ger. Offen. DE 19 542 287 Adv. Biochem. Engineering/Biotechnol.

(1996); Chem. Abstr. 1997, 127, 52504. 2001, 71, 125–127. (c) B. Kessler, R.
134 W. Fritsche-Lang, E. I. Leupold, M. Westhuis, B. Witholt, G. Eggink, “Pro-
Schlingmann, “Preparation of sucrose duction of microbial polyesters: Fer-
tricarboxylic acid”, Ger. Offen. DE mentation and downstream processes”,
3 535 720 (1987); Chem. Abstr. 1987, Adv. Biochem. Engineering/Biotechnol.
107, 59408. 2001, 71, 159–182.
135 TEMPO is an established acronym for 146 H.-P. Weiss, W. Sauerer, “Polyamides”,
2,2,6,6-tetramethyl-1-piperidinyloxy. Kunststoffe 1999, 89, 68–74.
136 S. Lemoine, C. Thomazeau, D. Joan- 147 (a) J. Thiem, F. Bachmann, “Carbohy-
nard, S. Trombotto, G. Descotes, A. drate-derived polyamides”, Trends
Bouchu, Y. Queneau, “Sucrose tricar- Polym. Sci. 2 1994, 425–432. (b) E. M. E.
boxylate by sonocatalyzed TEMPO- Mansur, S. H. Kandil, H. H. A. M. Has-
mediated oxidation”, Carbohydr. Res. san, M. A. E. Shaban, “Synthesis of car-
2000, 326, 176–184. bohydrate-containing polyamides and
137 E. Chiellini, R. Solaro, “Biodegradable study of their properties”, Eur. Polym. J.
polymeric materials”, Advanced Materi- 1990, 26, 267–276.
als 1996, 8, 305–313. 148 O. Varela, H. A. Orgueira, “Synthesis of
138 J. A. Moore, J. E. Kelly, “Polyesters de- chiral polyamides from carbohydrate-
rived from furan and tetrahydrofuran derived monomers”, Adv. Carbohydr.
nuclei”, Macromolecules 1978, 11, 568– Chem. Biochem. 1999, 55, 137–174.
573. 149 (a) L. Chen, D. E. Kiely, “Synthesis of
139 J. A. Moore, J. E. Kelly, “Poly(hydroxy- stereoregular head/tail hydroxylated
methylfuroate)”, J. Polym. Sci., Polym. Nylons derived from d-glucose”, J. Org.
Chem. Ed. 1984, 22, 863–864. Chem. 1996, 61, 5847–5851; US Pat.
140 H. Hirai, “Synthesis of macrocyclic oli- 5,329,044 (1994); Chem. Abstr. 1994,
goesters from 5-hydroxymethyl-2-furan- 122, 56785. (b) D. E. Kiely, “Carbohy-
carboxylic acid”, J. Macromol. Sci., drate diacids: Potential as commercial
Chem. Part A 1984, 21, 1165–1179. chemicals and hydrophobic polyamide
141 R. Storbeck, M. Ballauf, “Synthesis and precursors”, in: J. J. Bozell, ed., Chemi-
thermal analysis of copolyesters de- cals and Materials from Renewable Re-
rived from 1,4:3,6-dianhydrosorbitol”, sources, ACS Symp. Ser. 784, 2001,
J. Appl. Polymer Sci. 1996, 59, 1199– pp. 64–80.
1202. 150 S. Mondel, Dissertation, TU Darm-
142 J. A. Moore, J. E. Kelly, “Polymerization stadt, 1997.
of furandicarbonyl chloride with bis- 151 A. Vlach, Dissertation, TU Darmstadt,
phenol A”, Polymer 1979, 20, 627–628. 2001.
143 DuPont 2003, press release: “Clothing 152 A. Gandini, M. N. Belgacem, “Furanic
from cornfields”, http://www.dupont.- polyamides chemistry”, Prog. Polym.
com/sorona/news/021403.html Sci. 1997, 22, 1238–1246.
144 (a) Y. Doi, A. Steinbüchel, eds., Biopoly- 153 A. Mitiakoudis, A. Gandini,
mers, Vols. 3a, 3b: Polyesters – Properties “Poly(p-phenylene)-2,5-furandicarbona-
and Chemical Synthesis, Wiley-VCH, mide and conversion into filaments
2001, 468 pp. (b) A. Steinbüchel, Y. and films”, Macromolecules 1991, 24,
Doi, eds., Biopolymers Vol. 4: Polyesters – 830–835.
Applications and Commercial Products, 154 (a) W. Reppe, O. Hecht, Ger. Pat.
Wiley–VCH, 2002, 398 pp. 715 268 (1936); US Pat 2 157 347
145 (a) Y. B. Kim, R. W. Lenz, “Polyesters (1939); Chem. Abstr. 1939, 33, 44456.
from microorganisms”, Adv. Biochem. (b) B. Helferich, H.-J. Hofmann,
Engineering/Biotechnol. 2001, 71, 51–79. “p-Oxystyrol-b-d-glucosid”, Chem. Ber.
(b) W. Babel, J.-U. Ackermann, U. 1952, 85, 175–180.
Breuer, “Physiology, regulation, and 155 B. I. Mikhantév, V. L. Lapenko, “Vinyla-
limits of the synthesis of poly (3HB)”, tion of d-glucose and its acetone deri-
References 59
vatives”, Zhur. Obshch. Khim. 1957, 27, characterization of polymers containing

2840–2841. linear sugar moieties as side groups”,
156 W. A. P. Black, E. T. Dewar, D. Ruther- Eur. Polymer J. 2001, 38, 273–280.
ford, “Polymerization of unsaturated 162 V. Kollonitsch, Sucrose Chemicals, The
derivatives of 1,2:5,6-di-O-isopropyli- Internat. Sugar Research Foundation,
dene-d-glucose, J. Chem. Soc. 1963, Washington, D. C., 1970.
4433–4439. 163 H. Gruber, G. Greber, “Reactive su-
157 Y. Iwakura, Y. Imai, K. Yagi, “Prepara- crose derivatives”, in Carbohydrates as
tion of polymers containing sugar resi- Organic Raw Materials, F. W. Lichten-
dues”, J. Polym. Sci. 1968, A1, 1625– thaler, ed., VCH, Weinheim, 1991,
1632. pp 95–116.
158 (a) E.-J. Yaacoub, S. Wick, K. Buchholz, 164 D. Jhurry, A. Deffieux, M. Fontanille,
“Synthesis and polymerization of I. Betremieux, J. Mentech, G. Descotes,
methyl 5-deoxy-2,3-O-isopropylidene-b- “Linear polymers with sucrose side-
d-erythro-pent-4-enofuranoside”, Macro- chains”, Makromol. Chem. 1992, 193,
mol. Chem. Phys. 1995, 196, 3155–3170. 2997–3007. (b) E. Fanton, C. Fayet,
(b) E.-J. Yaacoub, B. Skeries, K. Buch- J. Gelas, A. Deffieux, M. Fontanille,
holz, “Synthesis and polymerization of D. Jhurry, “Synthesis of 4-O- and 6-O-
exo-glucal derivatives”, Macromol. monoacryloyl derivatives of sucrose.
Chem. Phys. 1997, 198, 899–917. Polymerization and copolymerization
(c) S. Wick, E.-J. Yaacoub, “Polymeriza- with styrene”, Carbohydr. Res. 1993,
tion of a 5,6-unsaturated fructofura- 240, 143–152.
nose derivative”, Macromol. Chem. Phys. 165 D. Jhurry, A. Deffieux, “Sucrose-based
2000, 201, 93–101. (d) A. Glümer, polymers: polyurethanes with sucrose
E.-J. Yaacoub, “Synthesis and polymer- in the main chain”, Eur. Polym. J. 1997,
ization of 1,2-unsaturated fructopyra- 33, 1577–1582.
nose derivatives”, Macromol. Chem. 166 M. Eissen, J. O. Metzger, E. Schmidt,
Phys. 2000, 201, 1521–1531. U. Schneidewind, “Concepts on the
159 R. L. Whistler, H. P. Panzer, J. L. Goat- contribution of chemistry to a sustain-
ley, “Preparation and polymerization of able development”, Angew. Chem. 2002,
6-O-vinyl-1,2:3,4-di-O-isopropylidene-d- 114, 402–424; Angew. Chem. Int. Ed.
galactose”, J. Org. Chem. 1962, 27, 2002, 41, 414–436.
2961–2962. 167 Errma, http://www.errma.com
160 J. Klein, D. Herzog, “Synthesis of some 168 US Government Results Act, 1993;
poly(vinylsaccharides) of the amide http://www.whitehouse.gov/omb/
type and their solution properties”, mgmt-gpra/gplaw2m.html
Macromol. Chem. 1987, 188, 1217–1237. 169 Fachagentur Nachwachsende Roh-
161 (a) G. Wulff, J. Schmid, T. Venhoff, stoffe; http:///www.fnr.de
“The preparation of new types of poly- 170 Agriculture pour la Chimie et l’Ener-
merizable vinyl sugars with C–C bonds gie; http://www.ademe.fr/partenaires/
between sugar and double bond”, agrice/htdocs/present01.htm
Macromol. Chem. Phys. 1996, 197, 259– 171 The sugar-based projects presently
274, 1285–1299. (b) G. Wulff, S. Bell- funded by the FNR qualify, without ex-
mann, J. Schmid, S. Podzimek, “Prepa- ception, for mission-oriented applied
ration and polymer-analogous reactions research.
of a polyvinyl sugar”, Macromol. Chem. 172 R. Searle, From frozen north to filthy
Phys. 1997, 198, 763–775. (c) R. Narain, lucre, Viking Press, New York, 1964.
D. Jhurry, G. Wulff, “Synthesis and
61
2
Industrial Starch Platform – Status quo of Production,
Modification and Application
2.1
Introduction
Starch is the most abundant storage carbohydrate and is generated in many

plants by photosynthesis. The plant assimilates carbon dioxide from the atmo-
sphere and transforms it into glucose, the basic molecule for the synthesis of
starch. These molecules are stored in tubers, roots, seeds, and fruits. Starch is
composed of small granules which are insoluble in cold water. The sizes of
these granules is characteristic of the origin of the starch and varies from 1 to
more than 100 lm.
2.1.1
History of Starch
Although the early history of starch use is mainly unrecorded, some interesting
examples of its industrial uses are known. Starch paste was used by the ancient
Egyptians to cement strips of papyrus stem together for use as writing paper as
early as 4000 B.C. In a treatise Cato described a process used by the Romans
for separating starch from grain in 170 B.C., and 300 years later Celsus, a Greek
physician, described starch as a wholesome dietary product. Pliny the Elder (23–
74 A.D.) stated that the Romans used wheat starch to make papyrus documents,
to stiffen and whiten cloth and to powder their hair. In his Greek Herbal,
Dioscorides (1st century A.D.) recommended the use of wheat starch for the
treatment of ulcers, sores and eye inflammations. Around 312 A.D. starch was
shown to provide resistance to ink penetration in Chinese paper, so starches
from rice, wheat and barley came to be commonly used at that time. In 975
A.D. Abu Mansur, an Arab pharmacologist, described a method to treat wounds
with an artificial honey made by mixing starch with saliva. Starch was used in
northern Europe to stiffen linen, possibly as early as in the 13th to 14th centu-
ries. Colored starches were used as cosmetics, and uncolored starches were pri-
ISBN: 3-527-31027-4
62 2 Industrial Starch Platform – Status quo of Production, Modification and Application
marily used as hair powder. The Puritans used blue starch until it was banned
by Queen Elizabeth in 1596.
2.1.2
History of Industrial Starch Production
The most common sources of starch are maize, potato, wheat, tapioca, and rice.
Starches are isolated by wet grinding of the grain or tuber, followed by wet sift-
ing, centrifugation, and drying. Wheat starch is an exception in that it may also
be extracted from flour. These steps remove most of the other plant compo-
nents, for example proteins, oil, and fiber. Because of their availability, wheat
and barley were the first sources of starch to be commercially wet-milled. The
first American wheat starch plant was founded in Utica, New York, in 1807.
Wet milling of potato starch began in Hillsborough County, New Hampshire, in
1824, and in Europe at about the same time in Foxhol, The Netherlands, where
Willem Albert Scholten founded his first potato-starch mill. Wet milling of corn
began in 1844, when the Colgate wheat starch mill in Jersey City, New Jersey,
was converted to processing corn using Thomas Kingford’s alkaline purification.
2.1.3
History of Starch Modification
Searching for substitutes for gum Arabic and tragacanth, Bouillon-Lagrange

first reported the production of dextrin by roasting starch in 1804. The indus-
trial use of dextrins, however, only became widespread after the accidental dis-
covery of the torrefaction method for producing dextrins (now called British
gums). In 1821 a fire broke out in an Irish textile factory where starch was used
for sizing the yarns. After the fire was extinguished it was found that a part of
the stored starch had turned brown due to the action of the heat and was solu-
ble in cold water. So it could be used to produce a thick, adhesive paste.
As access to cane sugar from the West Indies was limited during the Napo-
leonic wars, alternative technologies were investigated to produce sweeteners. In
1811, Kirchoff discovered that sugar could be produced from potato starch by
hydrolysis with sulfuric acid. Concurrent with the opening of large potato starch
mills, the use of sugar from starch in wines began in Germany in 1830. In
1874 Naegeli found that partial acid hydrolysis of starch granules produced a re-
sidue containing short linear molecules. His products and those of Lintner
twelve years later formed the basis for today’s fluid starches. Bellmas in Ger-
many and Duryea in the US both filed patents for acid-thinned starches at the
beginning of the 20th century.
2.2 Raw Material for Starch Production 63
2.2
Raw Material for Starch Production
Virtually all plants contain carbohydrates. We are primarily interested in those

plants that store these carbohydrates in the form of starch granules which can
be extracted. Worldwide, starch is mainly derived from cereals, but it is also ob-
tained in significant quantities from tubers and roots, especially in Europe and
Asia. The main sources of starch are maize, wheat, potatoes, rice, and cassava,
from which tapioca starch is derived (Fig. 2.1). Many other crops are also used
for starch production, such as sorghum, sweet potato, arrowroot, taro, barley,
oat, rye, peas, beans, lentils, and yam, but the quantities are small. Even exhaus-
tive research on agronomic and phenotypic properties of tropical starch crops
(e.g. arrowroot, sago, taro, sweet potato, and yam) has not made them competi-
tive on an international scale. As a result, maize, wheat, potatoes, and tapioca
dominate world markets for starches in both food and non-food industries.
Worldwide, maize is the dominating crop for starch production, in particular in
the US, where, apart from maize starch, only 1% of wheat starch and minor
amounts of potato starch are produced. In 2000, the US starch production
amounted to 24.6 million tons. The primary starch crops in the EU, including
the new member states, are maize, wheat, and starch potatoes, so the European
starch industry is more widely diversified in its raw material sources than other
Fig. 2.1 Starch production by raw material, worldwide, in 2000.
Fig. 2.2 Starch production by raw material in the EU in 2000.

Fig. 2.3 Starch production worldwide in 2000.
Fig. 2.4 Development of EU starch production.
starch industries elsewhere. The different raw materials each supply a large
share of total starch output, as shown in Fig. 2.2.
In 2000, the world starch market was estimated to be approximately 48.5 mil-
lion tons, of which the EU holds a share of approximately 17% or 8.5 million
tons (Fig. 2.3). During the past 20 years the quantities of starch produced and
the demand in food and non-food areas have increased continually. Whereas the
increase worldwide was about threefold (Fig. 2.5) in the past 20 years, the in-
crease in the EU was some 2.5-fold (Fig. 2.4); this was mainly based on an in-
crease in maize and wheat-starch production capacity.
Fig. 2.5 Development world starch production.
2.3
Industrial Production of Starch
Starch production technologies depend on the type of raw material, because

there are some basic differences between cereals, roots, and tubers. Due to its
suitability for large-scale storage, cereal-starch may be produced throughout the
year, whereas tuber and root-starch is produced only immediately after harvest.
The composition of the most important raw materials used as sources of starch
is summarized in Table 2.1.
Table 2.1 Composition of starch raw materials (median values).
Maize Potato Tapioca Wheat Rice
Moisture (%) 16 75 70 13 14
Starch (%) 62 19 24 60 77
Protein (%) 8.2 2 1.5 13 7
Minerals/Ash (%) 1.2 1.2 2 1.7 0.5
Sugars (%) 2.2 1.1 0.5 8 0.3
Fiber (%) 2.2 1.6 0.7 1.3 0.3
Fat (%) 4.2 0.1 0.5 3 0.4
2.3.1
Maize and Waxy Maize
As starch has been produced industrially from yellow maize for more than a
century, the process is technically well developed. Typically maize starch fac-
tories have a daily processing capacity of 500–2000 tons of maize. The produc-
tion of maize starch (Fig. 2.6) starts with steeping purified maize kernels. This
means that maize is not just swelling in water, which has a temperature of
about 50 8C and a pH of 3.8–4.2, but these conditions cause a controlled lactic
acid fermentation and loosen the protein matrix. Steeping is an important step,
because the maize kernels absorb up to 45% (wet basis) of water, release solu-
bles, absorb sulfur dioxide (0.2–0.4 g per kg) and become soft enough to be suit-
able for separation of its components.
After steeping, the grains are coarsely ground in an attrition mill, and then
the germs are separated in special hydrocyclones. The grinding and separation
processes must be performed twice for complete removal of the germs. This is
followed by fine grinding in a pin mill or impact mill in order to completely
disrupt the cells. At this stage the starch is freely in suspension and is led over
bend screen cascades for separation from fiber and other maize components.
The separated residues are dehydrated and dried for use as an animal feed com-
ponent referred to as maize feed.
The crude starch milk still contains all the dissolved proteins. This fraction is
called gluten, and most of it is separated off by means of two successive nozzle
type continuous centrifugal separators. The gluten is dehydrated by means of a
rotary vacuum filter, then dried and used as a feed additive similar to maize
feed. The starch milk, which still contains approximately 2% of protein after
separation, is then refined in a multi-step cyclone plant. The refined starch
milk, having a residual protein content of approximately 0.3%, is dehydrated in
peeler centrifuges and dried by means of a flash dryer. The residual water con-
tent of the starch at this stage is less than 14%, and the product is storable.
In some cases the starch production plant is connected with saccharification
and ethanol production plants.
Waxy maize is a special form of yellow maize. Its starch contains more than
99% amylopectin. The production of waxy maize starch is similar to normal yel-
low maize starch.
2.3.2
Wheat
Wheat starch can be produced either from wheat flour or by wet milling of
wheat grain. Industrial production of wheat starch is almost exclusively from
wheat flour. There are a large number of processes which are basically similar
but differ in the means of producing and further processing the wheat dough.
In the first step the wheat flour is stirred with water in special mixing machines
to form a dough or a suspension (Fig. 2.7). Then the wheat paste is separated
Fig. 2.6 Maize starch production.

Fig. 2.7 Wheat starch production.
in extractors, decanters, or in a hydro-cyclone plant. Fiber and gluten residues

are subsequently removed by sieving through centrisieves. Wheat starch is char-
acterized in that it is comprised of two fractions of different granule size; these
are obtained separately while producing the starch.
The bigger wheat starch granules of 20–50 lm in diameter are called prime
starch and comprise 90% of the starch. This starch is refined by means of sep-
arators or a multi cyclone plant; normally the product obtained contains less
than 0.3% of residual protein. The second-grade starch, which comprises the
smaller granules of 2–15 lm in diameter, is purified by means of separators. It
has a higher protein content and is contaminated with pentosans and hemicel-
luloses. Dehydration and drying of prime starch is similar to the process in the
production of maize starch. The paste fraction is usually dried with a ring dryer.
2.3.3
Potato
Potato is the most important tuber used for industrial isolation of starch. It has the
advantage that the extraction process is simpler than that used for cereals, because
it is not necessary to swell or mill them or prepare a dough. The disadvantages are
the lower starch content of tubers and the large quantity of fruit water produced,
which is the main cause of sewage problems arising from potato starch plants.
Processing (Fig. 2.8) starts after purification of the tubers by rasping by
means of a rotary saw blade rasp, in which potatoes are converted into a mash.
This process results in a nearly complete disruption of the potato cells, which
therefore release the starch. Because of this disruption technique, starch yields
in modern potato starch plants are in excess of 97%. Sulfur dioxide is added to
the rasped potatoes to avoid discoloration, which is a result of the action of phe-
nol oxidases.
Fig. 2.8 Potato starch production.

In modern plants the high protein fruit water is separated off by means of de-
canters before the starch is extracted. This reduces the problem of sewage, and
the valuable potato protein may be separated. The soluble proteins are coagu-
lated by treatment with acid and heat and then separated in decanters. The
starch in the rasped potatoes is isolated by means of centrisieves, yielding 99%
of pure starch. The remaining pulp is drained or pressed off and used directly
as feed while still damp or dried in flash dryers. The dilute starch milk had to
be concentrated in separators or multi cyclone units prior to the next step of
processing. Potato starch is refined in several steps in hydro cyclone plants. The
starch is dewatered by rotary vacuum filtration and subsequently dried in a
flash dryer.
2.3.4
Tapioca
Tapioca starch is a root starch which is produced from the roots of manioc or
cassava plants. Its starch content is about as low as that of potato. One disad-
Fig. 2.9 Tapioca starch production.

vantage is that the roots are not storable. Therefore they have to be processed
within 24 h of harvesting. Another problem is that the roots contain the glyco-
side linamarin, from which prussic acid may emerge during decomposition.
Most of the steps of industrial tapioca starch production are similar to those
of potato starch production (Fig. 2.9). After purification the roots are first cut
into potato-size pieces with a crusher and then rubbed with a rotary saw blade
rasp, which is the same as that used for potato rasping. The starch is extracted
in centrisieves. The steps of concentration and refining correspond to those in
potato-starch production. The only difference is that finer sieves are used be-
cause the starch granules are smaller in size. Like maize starch, tapioca starch
is dewatered with peeler centrifuges and subsequently dried.
2.3.5
Other Starches
Apart from the main starch sources described above, there are a number of
starch containing plants of less importance for industrial application. They are,
however, worth mentioning because of their often very interesting properties.
Sweet potatoes, for example, are used for starch production in Japan and are
processed in the same way as normal potatoes.
In the field of cereal starches, rice and millet are used regionally for industrial
starch production, whereas rye and barley are rarely utilized because of their
lower availability, their low starch content and their difficult production. Rice
starch is usually produced from broken rice. Rice is first steeped under alkaline
conditions and then ground and washed through a sieve. The separated fiber
fraction is used as feed. The starch milk is separated from the paste by means
of separators, dewatered in centrifuges and subsequently dried. Millet starch
production is similar to the maize starch process. Therefore, it can be produced
in maize starch plants, although an adaptation of the plant is necessary, because
the millet starch granules are smaller and therefore require the use of smaller
sieves and some changes in the milling process.
The starch from different kinds of peas, beans and tropical plants such as ba-
nana, shoti roots and curcuma has been investigated because of its scientifically
interesting properties. The production of so-called “tropical starch” is still in the
experimental stage although it has been ascribed great potential in the future.
2.4
Properties of Commercial Starches
As starch is a polymer synthesized in plants, it has different physical and chem-

ical properties depending on its origin. All starches are synthesized in the plant
from glucose molecules and stored as water-insoluble granules in several differ-
ent parts of the plant. During the starch synthesis two types of polysaccharide,
namely amylose and amylopectin, are generated at a ratio of approximately 1 : 3
Fig. 2.10 Molecular weight distribution of different starches (determined by SEC).
in most kinds of starches. Amylose is a largely linear molecule built from 1,4
linked a-D-glucopyranosyl units having an average molecular mass of 1 ´ 105 to
1 ´ 106. Amylopectin is different from amylose in that it has both a-1,4 and a-
1,6 bonds and a higher molecular mass of 1 ´ 107 to 1 ´ 108. The molecular mass
varies substantially, depending on the method of isolation and determination. A
method for the determination of the molecular mass of the polymers is a liquid
chromatographic method called size-exclusion chromatography (SEC), in which
the molecules are separated according to their hydrodynamic volume. Figure
2.10 shows the molecular mass distributions of six different types of starch.
Because of these different structures, the two fractions of starch have different
specific properties. Amylopectin is responsible for the partial crystallinity of
starch, with the crystalline properties resulting from clusters of layers of the
amylopectin molecules. The crystalline units are separated by amorphous layers.
When studied by light microscopy with polarized light, these layers appear as
Maltese crosses because of the optical properties of the starch granules
(Fig. 2.11). These optical properties enable assessment of whether or not the
starch granules are intact, because they are lost after e.g. thermal treatment.
The shape and size of starch granules can be measured microscopically,
which allows identification of the origin of the starch. The shape of the gran-
ules is difficult to describe, because it is never uniform. The shapes often range
from round to oval, oblong, kidney-shaped, edged, polyhedral, and rosette-
shaped. The size of the granules is between 1 lm and approximately 100 lm,
and each type of starch usually contains a broad distribution (Table 2.2). Ama-
Fig. 2.11 Maltese crosses of potato starch in polarized light.
ranth and rice starch are examples of starches having small granules, potato
starch is an example of a large granule starch.
Diffraction patterns obtained by X-ray irradiation of starch allow identification
of different types of starch and further classification of the material. Cereal
starches have been assigned to type A, root and tuber starches to type B, and
mixed forms (e.g. legume starch) to type C. The diffraction patterns result from
different degrees of polymerization of the amylopectin side chains, resulting in
double helix chains of different pack density. In type B, the double helices are
less closely packed than in type A, in which more water can be stored, giving a
different diffraction pattern.
Commercially produced starch contains several contaminants, the amounts
and types of which are generally dependent on the type of starch plant but also
on the quality of the production process. Because the properties and quality of
the starch can be severely affected by these substances, attempts are being made
to reduce the total amount to less than 1%. This is usually achieved in a mod-
ern production plant. Typical accompanying substances are proteins, lipids, and
salts. Phosphorus is especially important, because it is bonded to starch in the
form of a salt of phosphoric acid or, in potato starch, an ester. This bonding to
the hydroxyl groups of starch strongly affects the properties of the starch, partic-
ularly potato starch. Potato starch has greater swelling properties, stronger an-
ionic character and forms a paste of clearly higher viscosity stability. The main
minerals in starch are sodium, potassium, calcium, and magnesium.
74
Table 2.2 Composition and properties of commercial starches.
MS WMS PS TS WS RS
Source Maize Waxy maize Potato Manioc Wheat Rice

Moisture [%] <14 <14 13–21 <17 <14 <14
Amylose [%] d.s. 20–31 <1 16–24 16–18 27–31 15–25
Amylopectin [%] d.s. 69–80 >99 70–80 84 69–73 75–80
Crystal structure A A B C A A
Particle shape Polyhedrons, Polyhedrons, round, oval Round, hemi- round, elliptical Polyhedrons,
sharp edged, sharp edged, spheric sharp edged,
round round fragmented
Particle size range [lm] 5–25 2–25 15–100 (40) 3–25 2–35 a) 2–10
(median value) (15) (15) (15) (5)
Protein content [%] d.s. 0.2–0.4 0.2–0.3 0.05–0.1 0.1–0.2 0.2–0.5 0.2–0.5
Phosphorus [%] d.s. 0.015–0.02 b) 0.015–0.02 b) 0.04–0.12 – 0.06–0.12 c) 0.01–0.04 b)
Ash [%] d.s. 0.05–0.1 0.1 0.2–0.4 0.1–0.2 0.1–0.4 0.3–0.6
DH (DSC) [J/g] 1–4 1–7 2–0 1–5 1–0 1–3
Brabender peak viscosity [BU] 870 2800 2010 540 300 680
(concentration) (6.25%) (6.25%) (4%) (5%) (6.25%) (6.25%)
Gelatinization temperature [8C] 62–80 63–72 56–66 52–65 52–85 61–78
Swelling power 24 64 >1000 71 21 20–30
a) Bimodal distribution
b) Resulting mainly from salt
2 Industrial Starch Platform – Status quo of Production, Modification and Application
c) Resulting mainly from phospholipids

Viscosity is an important criterion for assessing starch quality. Native starches

only dissolve if heated in an aqueous suspension above a certain temperature,
the so-called gelatinization temperature. This process of gelatinization is accompa-
nied by irreversible swelling of the starch granules, because due to the disruption
of hydrogen bonds the starch helices can store water. The volume of the starch
thus increases twenty-fold to thousand-fold (Table 2.2). This reshaping of the
starch granules results in changes of rheological behavior, which may be mea-
sured. Appropriate instruments have been developed for this purpose which, un-
der defined conditions, may be used to measure the viscosity of starch pastes.
These instruments, for example the Brabender Viscograph and the Brookfield
Viscometer, are based on the principle of rotation viscosity and determine the ro-
tation moment necessary for maintaining a given rotation number. These instru-
ments allow recording of a temperature profile of viscosity during heating and
cooling (Fig. 2.12). These measurements are an important criterion for assessing
the behavior of starch for later practical applications, for example as a thickener or
a rheology enhancer. Other instruments, such as the falling ball viscometer and
the capillary viscometer, are based on different principles and are frequently ap-
plied for very specific measurements required in industrial applications.
Another specific property of starch paste is the phenomenon of retrogradation.
This is a kind of crystallization process caused by the formation of hydrogen bonds
between parallel starch molecules, particularly the amylose part of the starch. The
formation of these bonds reduces the ability to bind water and enhances viscosity,
Fig. 2.12 Brabender curves of various starches at different concentrations.

which can lead to the formation of gels. Retrogradation may be substantially re-
duced by different methods, for example by chemical modification.
2.5
Modification of Starch
2.5.1
Modification Technology
Native starch has a granular structure and is used in this form in only a few specific
applications. For normal applications starch is converted into a hydrated or gela-
tinized form, either by the user or the producer. In any case, any change of the
original starch means a modification. Natural starch can be modified by chemical,
physical, enzymatic treatment or by a combination of these methods (Fig. 2.13).
2.5.1.1 Slurry Process (Heterogeneous Conditions)

In slurry technology, the grain structure of starch remains substantially un-
changed. In this process starches are usually suspended in aqueous solution with
permanent circulation and are chemically, physically or enzymatically modified.
The maximum slurry concentration depends on the extent of swelling, which de-
pends on the raw materials and the reaction conditions, e.g. temperature and al-
kalinity, or other factors which cause thickening of the slurries. Protective salts
Fig. 2.13 Starch modification technology.

2.5 Modification of Starch 77
(e.g. sodium sulfate) are often used, which allow the application of more intensive
and therefore more efficient reaction conditions. The slurry concentration is
usually 30–40% of dry matter, and the reaction temperature is usually from 25
to 50 8C. Under these conditions starch is often modified by treatment with dilute
acids or alkalis (pH 1– pH 12). This treatment results in only minor substitution
and limited degradation. More substitution leads to undesired swelling of the
starch, which makes further processing more complicated.
These modified starches are usually washed free from salt, drained and dried
in a flash dryer. The advantages of this process are that it yields almost pure
starch derivatives without any side-products, and that energy costs are low.
These starches are normally sold as products that are insoluble in cold water
(cook up starches). In special cases the starches may be modified by extrusion,
drum drying, or spray cooking to make them cold-water soluble according to
consumers’ needs.
2.5.1.2 Dry Reactions

Another process which only slightly changes the grain structure is the dry reac-
tion. This process is limited to a few modification processes, mainly dextriniza-
tion processes. The process is also used, to a limited extent, for chemical modi-
fications, such as phosphatation and production of carbamate starches. In dry
reactions the starches are dried under vacuum, then subjected to thermal treat-
ment at temperatures of up to 180 8C with or without the addition of chemicals.
Semi dry technology, such as heat–moisture treatment or dry cationization, is
an intermediate case. Such modification is performed under wet but not fluid
conditions. Storable products are obtained by subsequent drying.
2.5.1.3 Paste Reactions (Homogeneous Conditions)

Conversion paste reactions are performed under homogeneous reaction condi-
tions, often in autoclaves, to obtain a high degree of substitution. This allows
the use of more severe reaction condition than in slurry processes. In these pro-
cesses starches are converted into an amorphous structure by steam or alkali ge-
latinization and subsequently subjected to high temperatures over a relatively
short period of time. This technology is applied to concentrations of up to 40%
dry weight, but because of the high viscosity of such mixtures, they require vig-
orous mixing. The type of modification depends on alkalinity and reaction tem-
perature, which ranges from 808 (carboxymethylation) to 110 8C (hydroxypropy-
lation). The advantage of this process is that it is possible to achieve a high de-
gree of substitution and a high reaction yield in a short reaction time. The vis-
cosity of the final products may be substantially affected by mechanical stress
during processing. The paste-like products obtained are usually dried in a drum
at high energy costs. This type of dryer produces a product film that can be
ground to flakes or powder which is very soluble in cold water. The disadvan-
tage of this process is that the final products contain salts and side-products.
2.5.1.4 Extrusion Cooking

Extrusion cooking also yields products that are soluble in cold water. Starches
having a water content of *25% are digested by a mechanical thermal melting
process and rapidly expanded through a die head, whereupon the molten paste
solidifies. In this process the energy input is mechanical shearing with a screw
or external heating elements. Mechanical degradation may be regulated by
choosing an appropriate screw configuration from a wide variety. Because the
starch remains in the system for only a short period of time, this energetically
highly favorable and continuous process allows only limited chemical modifica-
tions and low degrees of substitution. The granules produced are poorly soluble
compared to the flakes obtained by drum drying. Extrusion cooking is predomi-
nantly used for physical modification of starch; only few types of chemical deri-
vatizations are possible, and they are rarely implemented on a large scale.
2.5.2
Types of Starch Modification
Starch can be changed by physical, chemical, and enzymatic treatments or by

combinations thereof. Derivatization of native starch is achieved by means of a
variety of technologies, e.g. extrusion, the slurry/paste approach, or dry/semi-
dry processes (Section 2.5). In these processes starch molecules are degraded,
chemically modified by the introduction of functional groups, or changed by hy-
drothermal technology. Widely different combinations of these processes are
possible and are already being realized on a large scale. The aim of derivatiza-
tion is to change the natural properties of starch and to introduce new proper-
ties to allow application of the starch in different areas. Derivatization of starch
may be used to
· change gelatinization or cooking properties
· decrease the retrogradation tendency for gel formation
· increase hydrophilic properties
· confer hydrophobic properties
· introduce ionic substituents
· adjust viscosity (molecular weight).
2.5.2.1 Physical Modification

Hydrothermal technology is the simplest way to change the properties of native
starch. Annealing and heat–moisture treatment are two hydrothermal treat-
ments that modify the physicochemical properties of starch without destroying
the granular structure. These treatments can change the gelatinization tempera-
ture and gelatinization enthalpy. Annealing of starch slurries involves condition-
ing at below the gelatinization temperature with excess water. Heat–moisture
treatment uses far higher temperatures and a limited amount of water.
Another special physical modification of starch is the production of cold-water
soluble granular starches by hydrothermal conditioning. As starch is a polymer,
it does not form a real solution but a colloid dispersed gel. The addition of
chemicals such as alkalis influences the swelling properties as well as the solu-
tion conditions. Swelling starches for non-food applications are mostly produced
by drum drying, extrusion, or spray cooking.
2.5.2.2 Degraded Starches

Degraded starches are produced by mechanical, thermal, acid, oxidative and enzy-
matic degradation or combinations thereof. Starches having different degrees of
reduced polymerization are obtained, depending on the chosen process and its pa-
rameters. Usually the amylopectin is degraded and the amylose only slightly split.
Acid modification is usually performed with hydrochloric, sulfuric, or phosphoric
acid. The products obtained are described as “thin cooking starches”, because heat-
ing of large concentrations in water yields a low viscosity material.
The effect of oxidation depends on the oxidizing agent, e.g. chromic acid, hy-
drogen peroxide, hypochlorite, periodate, or peroxosulfate, and the catalyst used.
The starch may be degraded to different degrees, resulting in different amounts
of carbonyl and carboxyl groups; the products have different properties, for ex-
ample paste stability and color, and different applications.
Dry processes may also be used to produce products of different levels of deg-
radation, depending on the reaction temperature, reaction time, and type and
amount of acid. The starch derivatives obtained are called white dextrins and
yellow dextrins (roast dextrins). Roasting technology without the addition of
acids produces dextrins known as British gums. Of the techniques used, the
Noredux process is the most well known. In this process the starch is dried un-
der vacuum and then reacted with gaseous hydrogen chloride and heated. This
yields products of different viscosities and cold-water solubilities (up to > 98%),
depending on the reaction time and temperature (up to 200 8C). Apart from the
reduction of viscosity, the transglycosylation reaction is also important in the
production of roast dextrins. Dextrins are used in the paper industry, the adhe-
sive industry and the textile industry.
Other important methods for the degradation of starch are enzymatic pro-
cesses. Enzymes capable of catalyzing the hydrolysis of starch are widely avail-
able in nature. The enzymes may be classified according to their action: a-amy-
lases, b-amylases, glucoamylases, and oligosaccharide hydrolases. a-Amylases
cleave the a-d-1,4-linkages converting starch to reduced sugars. b-Amylases pro-
mote hydrolysis of starch to maltose; these enzymes cannot cut a-d-1,6-linkages,
therefore a high-molecular-weight limit dextrin remains. Glucoamylases and
oligosaccharide hydrolases hydrolyze starch directly to d-glucose. Enzymatically
degraded starch products are mainly used in food applications and, to some
extent, for adhesives and paper surface treatment.
2.5.2.3 Chemical Modification

Dry reactions may be used to produce acetylated, phosphated, phosphate cross-
linked and carbamate starch, the latter by esterification of starch with urea.
Usually, a dry reaction of starch under the action of heat and vacuum is carried
out with urea and phosphoric and sulfuric acids. Because of their good solubili-
ty properties, such carbamate starches are used in textile applications.
Modification of starch by reaction of the hydroxyl groups in the C2, C3, and
C6 positions of the anhydro glucose units with acids, acid anhydrides or alco-
hols yields starch ethers and starch esters.
Reaction with bifunctional or polyfunctional compounds results in intra-
molecular and intermolecular bonds or crosslinking of the starch. Compared to
cellulose, even relatively low degrees of substitution of starch (degree of substi-
tution 0.01–0.1) will result in major changes in the properties. Starch may be
derivatized both in the granules and in the paste.
Esterification of starch may be performed by reaction with both inorganic and
organic acids or derivatives thereof. Inorganic acids and their derivatives are pre-
dominantly esters. For example, ortho- or metaphosphate usually leads to starch
monophosphate. Starch diphosphate is produced by conversion with phosphorus
oxychloride (phosphoryl chloride). The monophosphate and diphosphate esters of
starch have a wide range of application, for example in the pharmaceutical and
textile industries. Other starch esters based on inorganic acids, e.g. starch nitrate,
starch sulfate, or starch xanthogenate, are of only limited importance. The most
important starch esters obtained from organic acids are products based on acetic
acid. Acetylated starches are produced by reaction not only with acetic acid anhy-
dride but also with vinyl acetate. Acetylated starches and native starch hardly differ
under the microscope, but in the form of pastes the derivatives have obvious ad-
vantages, namely low gelatinization temperature, very low gel formation, and
clearer pastes. Apart from acetylation, starch may be modified with citric, succinic,
and higher organic acids. An important reaction is with octenylsuccinic acid anhy-
dride (OSA). It yields an extremely hydrophobic starch ester which has good emul-
sifier properties and is used in the cosmetics and pharmaceutical industries. Be-
sides OSA, higher aliphatic fatty acids may also be used.
The etherification of starch is the most important kind of modification. The hy-
droxyl groups of the starch are reacted with nucleophilic reagents, usually in a
highly alkaline medium, forming very stable ether bonds. Typical reactions are:
· the Williamson ether synthesis (with aliphatic halogen or sulfate groups)
· ring-opening of epoxides
· Michael addition (addition of starch to unsaturated C-C bonds).
Hydroxyalkylation of starch, performed by reaction with propylene oxide, is cer-

tainly the most important reaction. Products of this type are often produced in
combination with further modification steps and are broadly applied. Because
of their thickening effect, highly substituted starch ethers are used in the con-
struction industry, and degraded starch ethers are used for sizing. Hydroxyethyl
starches, produced by reaction with oxirane, are used as blood plasma substi-
tutes. Ionic groups may also be introduced into starch by etherification. Starch
is carboxymethylated by reaction with chloroacetic acid, chloroacetic acid salts or
esters. Anionic starches are used in construction, sizing and textile printing.
The solubility of carboxymethylated starch in non-aqueous solvents increases
with increasing substitution. Therefore, the most highly substituted products
are optimally produced by reaction in alcohol solutions. Cationic starch ethers,
i.e. polysaccharides which become positively charged in acid as a result of proto-
nation, are as industrially important as anionic starches, the most important
ones being the tertiary and quaternary ammonium alkyl ethers. Quaternary am-
monium alkyl ethers are produced by reacting starch with appropriate epoxides
or the corresponding chlorohydroxy compounds. Cationic starches have a spe-
cial affinity to negatively charged celluloses, fibers, sludges, etc. They are pro-
duced by slurry reaction, paste reaction, extrusion, and by semi dry technology.
They are predominantly applied in the paper industry and as flocculants. One
example of a Michael addition is the preparation of cyanoethylated starch by re-
action with acrylonitrile. Depending on the strength of the base, the product
may be further hydrolyzed to the amide or the carboxylic acid. Carbamoyl ethyl
starch ethers are used in the cement and textile industries. Another important
modification of starch is cross-linking. Reaction with a variety of polyfunctional
reagents, e.g. phosphorus oxychloride, epichlorhydrin, adipic acid, vinylsulfones,
or aldehydes, e.g. propionaldehyde, allows construction of a three-dimensional
network in the starch. Cross-linking may proceed both on the intramolecular
and on the intermolecular levels. Pure cross-linking may be used to reduce the
extent of swelling of the starch. Usually, however, cross-linking is carried out in
combination with further modification steps, resulting in increased shear stabil-
Fig. 2.14 Types of starch modification.

ity and better thickening properties. Such materials are used as textile print
thickeners.
The types of modification described in this section lead to different classes of
products summed up in Fig. 2.14. This figure is not just an overview of these
products, but it also shows how modification of starch varies depending on the
chosen application.
2.6
Application of Starch and Starch Derivatives
Starch and modified starches or starch derivatives have a broad range of applica-
tions both in the food and non-food sectors. A comparison of the compositions
of the two largest markets, the US and the EU, shows how starches are used
differently (Table 2.3). While in the US production of isoglucose, high-fructose
corn syrup (HFCS), and ethanol are the most important uses of starch, these
are of secondary importance in the European Union.
Table 2.3 Starch use by product in EU and US in 2000 (million tons).
EU US
Ethanol 0.1 10.1

Isoglucose/HFCS 0.3 7.9
Other syrup-based products 3.6 3.2
Native and modified starches 4.5 3.6
Fig. 2.15 Applications of starch in the EU in 2002.

In Europe in 2002, the total consumption of starch and starch derivatives was
approximately 7.9 million tons, of which 54% or 4.3 million tons were used in
food applications and 46% in non-food applications. By far the largest user of
starch and starch derivatives in the EU (29%) is in the paper, board, and corru-
gating industries. Other important fields are textiles, adhesives, cosmetics, phar-
maceuticals, construction, paints, and agrochemicals. The use of starches in the
manufacture of detergents and biodegradable plastics has become of increasing
interest in recent years (Fig. 2.15).
2.6.1
The Paper and Corrugating Industries
2.6.1.1 Use of Starch in the Paper Industry

In terms of quantity (2.3 million tons in 2002), the paper industry is the most
important user of starch products in non-food applications. After fiber and fill-
ing materials, starch is the most important material for the production of paper.
In Germany, in 1998, of the 600 000 tons of starch products used in the non-
food sector, 345 000 tons (i.e. about 60%) were used in paper production, and
90 000 tons (about 15%) in corrugated cardboard production (Fig. 2.16). Because
of permanently rising quality requirements and the increasing use of waste pa-
per, the need for starch continues to increase; it is currently about 21 kg of
starch per ton of paper (in 1975: 12 kg per ton).
Most of the starch in paper production is used for paper sizing (about 80% in
Germany); other applications are paper coating (8%), wet end (7%), and spray
starch (5%). The fundamental characteristic that starch confers to the paper is
dry strength. It depends on the quantity and the properties of the starch used.
The starch is partly used as starch derivatives during wet processing. Mainly cat-
ionic starches are used, because they are readily adsorbed and bind well to the
mostly anionic surface of the fibers and filling materials, and they have a desir-
able flocculation effect. Starch improves not only the strength of the paper but
Fig. 2.16 Starch consumption in the paper industry in Germany.

also dewatering and retention of fiber and filling materials. The industry also
uses amphoteric starch and, in special applications, anionic starches. Combina-
tions of native starches and cationic polymers, e.g. polyvinylamines, are only
used to a limited extent.
Addition and retention of starch in the paper pulp by electrostatic interaction
between starch and fibers is quantitatively limited to 1–2% of starch per paper
and depends on the type of fiber and starch. Higher additions of starch to paper
to obtain greater strengths are achieved by sizing paper with starch solutions in
a size press. Usually, degraded starches are used in this application. The degra-
dation is usually a depolymerization carried out either in the starch factory (oxi-
dized starches, dextrin) or in the paper factory directly before use (thermochem-
ical or enzymatic degradation). The method of production and the type of starch
allows control of the strengths of the paper (dry strength, bursting strength, sur-
face strength) as well as other quality features, for example smoothness, print-
ability, and stiffness. In addition to degraded native starches, degraded slightly
modified starches (cationized or acetylated) may be used as well.
A fundamental disadvantage of paper sizing with starch solutions is that the
dampening of the paper means that the machine speed must be reduced, which
affects the output. Therefore, size presses have been mostly replaced by film
presses, which allow less penetration of the paper. In addition to this global trend,
efforts are being made to replace paper sizing with starch by increasing the addi-
tion of starch to the pulp. This requires dual technologies that permit an increased
use of cationic starch (>1.5%) through the addition of an anionic component (e.g.
silicic acid, bentonite, polyaluminum oxide, alum, anionic starch). Many systems
are now being tested in an experimental stage wherein the starch is not in a sol-
uble state but in a more or less particulate state and is retained mechanically in the
paper sheet. Hopefully, this will include significantly more starch (> 2%) in the pa-
per. As of now, however, these new systems are still not economical.
In paper coating, starch is mainly employed as a binder or as a co-binder in coat-
ing colors. Degraded starch products are usually used to bind the pigments to the
paper fibers. The starch also controls water retention and rheology of the coating
colors. Starch as a co-binder is in widespread use for precoating, usually in com-
bination with latex as the binder in the top coat. Coating starches are usually oxi-
datively degraded starches, dextrin, or enzymatically degraded starches, which are
used in highly concentrated solutions. Modified, e.g. acetylated, ethoxylated, and
propoxylated starches are also commonly used in this field.
Spray starch is mainly used in carton production. The different paper layers
are stuck together by spraying starch on them. The starch becomes gelatinized
in the drying section of the paper machine, and thus the paper layers stick
together. Usually, native starches are used in this application, but modified
starches having a reduced gelatinization temperature, and some cold-water solu-
ble starches may be used as well.
2.6.1.2 Use of Starch in the Corrugating Industry

The flutes in corrugated cardboard are usually glued together by means of
starch. In this process the starch is used as a two-component system: The first
component is completely or partly gelatinized starch, and the second compo-
nent is native starch. To produce the glue for corrugated cardboard, the primary
starch is either completely or partly gelatinized in aqueous suspension with so-
dium hydroxide. The processes differ according to the degree of gelatinization:
Stein Hall, Minocar, or Super Glue. After gelatinization, or to stop the gelatini-
zation of the primary starch, the secondary starch and the remaining water are
added, borax is also added, and the glue is sheared until the desired viscosity is
reached. During this procedure the secondary starch remains in the glue in its
non-gelatinized state. The primary starch gives the glue viscosity, water reten-
tion, rheology, and the so-called green bond. The secondary starch gives the
glue its adhesive strength by gelatinization in the dry section of the corrugated
cardboard machine. Wheat and maize starch are usually used for gluing corru-
gated cardboard, although potato and tapioca starches may also be used. In
many cases a mixture of different types of starch is used.
So-called “one bag products”, also referred to as “Readymix”, are widely avail-
able. This means that all the components (cold-water soluble primary starches,
secondary cooking starches, alkali, borax) are already mixed, and the user just
has to add the required amount of water and to shear the glue to achieve the
desired viscosity (Stein Hall, Lory).
Modified starches are also used in this field, for example crosslinked starches,
which have an especially high and shear-stable viscosity under alkaline condi-
tions, degraded starches, which allow greater penetration of the glue into the
paper, and other modifications which improve the tack.
2.6.2
The Textile Industry
Large quantities of starch are used in the textile industry. Usually, the products
are used to facilitate textile treatment and are removed from the product after
the process. Here it is an advantage that starch degrades naturally. The starches
used are native, modified or even highly modified, predominantly starch ethers.
In this application starch has to compete against synthetic polymers.
2.6.2.1 Sizing Agents

Before weaving, sizing agents give yarn closeness, smoothness, suppleness and
strength, making it more resistant to the enormous mechanical stress of the
weaving machine. The yarns are coated in a hot sizing bath, squeezed and dried
on dry drums. After the weaving process the sizing agent is washed out with
aqueous enzymatic or chemical (alkaline, oxidizing) solutions. This process is
necessary to produce soft and easily colorable material. The demands on the siz-
ing agent are, therefore, high solubility, good adhesion to the fiber, formation of
a tough, abrasion-resistant and elastic film and good desizing. With the develop-
ment of high-performance weaving machines achieving high weft insertion
rates, it has become necessary to modify the starches accordingly. Originally, na-
tive products were used for sizing; nowadays, degraded modified starch esters
and starch ethers are used. The starch may be degraded under oxidizing or ther-
mal conditions by use of an extruder. Modifications include propoxylation, car-
boxymethylation, acetylation and carbamate production by the Noredux process.
These products have to compete against synthetic polymers, for example poly-
acrylates and polyvinyl alcohols, as well as against semi-natural carboxymethyl
celluloses. Mixtures of starch and synthetic sizing agents are used for fine cot-
ton products and semi-synthetic materials (e.g. mixtures of cotton and poly-
ester), again competing with synthetic products.
In countries where potato and tapioca starches are available, these are usually
favored as raw materials. If such starches are not available, maize and wheat
starches are used, but mostly in combination with synthetic products. Cooking
starches and cold-water soluble starches are used as well. The great advantage
of starch products is their still low costs and their sustainability because they
are biodegradable.
2.6.2.2 Textile printing Thickeners

In textile printing, large amounts of starch are used as thickening agents, espe-
cially for acid-disperse printing and reactive printing. They are used in the pro-
duction of both clothing and home textiles like curtains, upholstery cloth, and
carpets. The starch products give the print pastes a printable, paste-like consis-
tency, preferably shear thinning. The thickening agent should liquefy under
pressure, penetrate the fabric well, and then stiffen immediately for good sharp-
ness of print. The print paste should not penetrate too well, though, because
this will result in bad color yield. It is important that the products are very pure
to avoid gaze blocking, and that the starch-based print-thickening agent is well
washable to obtain a soft and supple fabric. As opposed to sizing, in textile
printing only highly modified starches are used, usually cross-linked carboxy-
methylated maize starches, and sometimes analogous modified tapioca starches.
Potato starches are unsuitable because of their grain size, which will block the
gauze. The products compete with synthetic polyacrylates and polyvinyl alcohols
as well as with semi-synthetic celluloses and natural polymers, e.g. guar, tamar-
ind, and alginates.
2.6.2.3 Finishing Agents

In addition to the classical uses as auxiliaries in textile production, where the
products are washed out from the fabrics after the process, in some applications
the starches remain on the textiles. For example, starches are used as adhesives
on non-woven textiles, or as a glaze for protection, or for stiffening the fabrics
(e.g. shirt collars). In laundries, spray starches are used as ironing auxiliaries.
2.6.3
Adhesives
Starch is a very widespread starting material for adhesives. Adhesive is a generic

term encompassing other common terms, such as glue, paste, dispersion adhe-
sive, contact adhesive etc., all having different physical and chemical properties.
Applications of starch-based adhesives range from industrial applications to ad-
hesives for every-day use. Usually, amylopectin-containing starches are used in
most of these applications because they have a similar adhesive power, but bet-
ter long-term viscosity than starches containing amylose. There are, however,
many exceptions where amylose starches have been found to be superior to
amylopectin starches, depending on the type of adhesive and whether degraded
and/or derivatized starch is used. Different additives may be used in different
fields of application, e.g. thickening agents or rheology modifiers, such as modi-
fied celluloses or casein, bond agents for wet paper, or preservatives.
Starches and starch products are mainly used for the adhesion of paper and
paper-like raw materials. For paper adhesives, cross-linked clear cold soluble
starches are usually used. Products are characterized by good tack and short-
structured performance. This short-structured performance allows low splatter
application of the glue. Surfactants may be added to reduce surface tension, re-
sulting in better penetration of the paper and therefore better adhesion.
Almost the same process is used for the preparation of cigarettes, in book-
binding, and for spiral covers, but in these applications degraded starches or
dextrin-like products having a high solids contents are used. Another interesting
application is adhesives that may be re-moistened. They are important in the
production of envelopes, postage stamps and adhesive tape. A clear paste having
highly stable viscosity and good re-moistening properties is required for these
products. Usually, waxy maize starches are used. In addition to the use on pa-
per, starch is also used in the production of labels and all around adhesives. For
these applications the starch must not only have strong and fast adhesive prop-
erties for attaching labels to glass, aluminum, or plastic, but it must also be suf-
ficiently stable and durable to resist condensed water, e.g. in cold rooms.
Another field of starch-containing adhesives is the application of posters and
wallpaper to walls. This adhesion is achieved by using cold water soluble
carboxymethylated and propoxylated starches. Essential qualities are good wet
adhesion and good slip resistance.
2.6.4
Building Chemistry
In the construction industry, starch and starch derivatives based on a variety of

raw materials are used as additives to both hydraulic and organopolymer bin-
ders in aqueous systems. Starch or starch derivatives may have a strong effect
on the rheology of aqueous systems. In particular, they act as efficient thicken-
ing agents, as a means of improving water retention, and as processing addi-
tives. The systems in which starches are used range from a wide variety of
structural materials, e.g. mortars and concretes, to surface-applied plaster, etc.
Cold-water soluble starch derivatives, i.e. highly modified so-called starch ethers
of substantially modified starch grains, are usually produced by drum drying.
The extent of modification is very important for special applications. In the field
of plastering, highly propoxylated starch ethers based on potato starch are used.
Such starch ethers are used for both hand plasters and machine plasters based
on gypsum and cement. In these plasters the starch acts as a synergistic addi-
tive to highly modified cellulose, which is also responsible for water retention.
The starch improves the stability of the plaster, making it easier to handle. The
use of starch increases productivity and stand-up quality. Starch ethers are only
applied in very small quantities of up to 0.1%, depending on the formulation.
In fillers, the amount of modified celluloses is higher. Starch ethers are used
to reduce the stickiness and to slow down the formation of a film on material
which may be exposed to the atmosphere for a long time.
Another important field of application is tile cement, which also exploits the
synergistic effects of highly modified celluloses and starch ethers, like plaster.
For tile cement, however, the important properties are workable time, ease of
application, and setting time.
Another application of starch products which has come up in recent years is
self-levelling concrete, a self-compacting concrete that gives a very flat surface
without requiring vibration. Starch ethers are used as new viscosity-modifying
agents that help reduce bleed and improve the stability of the concrete mixtures.
A new field of application for starch was recently found in the dry-jet process for
conventional tunnelling. Tunnelling not only requires economic efficiency, but
ecological considerations and working conditions are important, too. Dust forma-
tion may be reduced, thus improving work hygiene and mitigating the effect of
dust on the environment. A clear reduction of rebound may be achieved by using
a highly hydroxypropylated cold-water soluble starch ether based on maize starch.
If it is possible to pre-moisturize dry spray concrete, starch ether will have even
more favorable properties. Depending on the starch ether used, less than 0.20%
is usually required in the cement to give the spray concrete the desired properties.
The starch must not negatively affect the early strength of the fresh sprayed con-
crete, but the required concrete strength must still be achieved with aging.
Hydrolyzed wheat or maize starches are used in the manufacturing of plaster-
boards. The starches are responsible for binding the gypsum core to the card-
board. The starch is believed to migrate into the surface of the gypsum core
and act as a glue between gypsum and cardboard.
2.6.5
Pharmaceuticals and Cosmetics
Many pharmaceutical products such as tablets etc. are taken orally, but not as
nutrients, so they are not classified as food. Starches act as excipients and must
fulfill special requirements. Specially modified products are used as binding
agents to give tablets high strength, low abrasion loss, and stable consistency.
Starch is also used in products which are solid when dry but swell when wet
(oral administration), and it is also used to accelerate the breakdown of the ta-
blet to release the drug. Such products are based either on swelled, spray-cooked
starches or, if the grain structure has to be preserved on in organic solvent, on
highly modified starches, which can develop an enormous swelling capacity.
The opposite effect is required for tablet coatings. In this case, the starch has to
make the tablets stable enough for handling and trading, and it has to prevent
the release of the drug before the moment of oral administration.
A new application of starches is hard or soft capsules. So far, gelatin products
have usually been used, but these are now out of favor because of BSE (bovine
spongiform encephalopathy). In the production of capsules, gelatin may be suc-
cessfully substituted by specially modified starches.
Hydroxyethylated, highly degraded starches which may be obtained in high
purity and toxin-free may be used as blood plasma substitutes.
Starch products are also used in cosmetics, for example in toothpastes, pow-
ders, and shampoos. They often contain crosslinked waxy maize and rice
starches which support the active ingredient and are responsible for the soft,
velvety feeling of the pastes on the skin. Such starch derivatives are also found
in baby powder, eye shadow, and face powder. Highly crosslinked sterilisation
stable cooking starches are used as separating agents and lubricants in the pro-
duction of condoms and surgical gloves. Acylated products are used as emulsi-
fiers in pastes, and propoxylated phosphate ester starches are used in shampoos
to give them a soft feeling on the skin. An essential requirement for all these
products is high purity and non-toxicity.
2.6.6
Laundry Starches
Native starch is widely used for washing bed sheets and table cloths to extend
the lifetime of the fabric and to impart stiffness and finish to the material. Mod-
ern laundries often use soluble starches packaged with a suitable propellant in
aerosol cans for starching garments during steam ironing.
As an alternative to non-biodegradable co-builders, graft copolymers of starch
having a high carboxylate content are often used as ion-exchange materials in
water softeners.
2.6.7
Bioconversion of Starch
Industrial biotechnology and the application of starch as a source of carbon in

the fermentation industry are becoming increasingly important and, indeed, es-
sential, because fossil energy resources are becoming scarce. Due to the current
basic conditions of energy availability and political will, recently the production
of “bioethanol” (ethanol prepared from renewable resources) from starch-con-
taining raw materials, e.g. wheat, maize, and rye, is booming. Usually, starch is
saccharified enzymatically (by a-amylase and amyloglucosidase), and the organic
hydrolysate is fermented to alcohol by means of yeast. “Bioethanol” thus pro-
duced is mostly used as an admixture to mineral fuels, but it is also used as a
raw material for the synthesis of ethyl tertiary butyl ether (ETBE). The residues
from fermentation may be made into highly nutritious animal feed.
In addition to the production of ethanol, starch hydrolysate also serves as a
carbon source in many other fermentations. For example, many organic acids
such as citric acid, acetic acid, gluconic acid, 2-ketogluconic acid (the first step
in the production of erythorbic acid), and itaconic acid are produced from starch
syrups. Other organic acids, e.g. maleic acid, fumaric acid, and l-tartaric acid,
are produced biotechnically from glucose syrup in an economical process. As-
corbic acid (vitamin C) may also be produced from starch syrup.
Enzymes are widely used in food and non-food applications. Most of the en-
zymes are produced in bio-technical processes using glucose of starch as the
source of carbon. The production of many hydrolytic enzymes, e.g. amylases,
proteases, and cellulases, is catabolically inhibited by glucose, and therefore it is
advisable to use only slowly metabolized substrates such as starch or partly hy-
drolyzed starch. a-Amylases and amyloglucosidases are produced on an indus-
trial scale by fermenting hydrolyzed maize starches (with bacillus and aspergil-
lum). Many amino acids, e.g. arginine, lysine, glutamic acid, glutamine, histi-
dine, threonine, and valine, may be produced biotechnically from starch glu-
cose. Antibiotics such as penicillin G and V, cephalosporin, and oxytetracycline
are also obtained by fermentation of substrates containing hydrolyzed starches
as essential nutrient components.
Many other industrial chemicals are produced by fermentation of glucose
syrups, and because of the permanent shortage and increase in price of fossil
resources there is a growing interest in alternative economical processes for pro-
ducing organic chemicals such as propanediol, glycerin, butanediol, 2-propanol,
polyalkonates, polyhydroxybutanoic acid, and polylactic acid. Other products that
may be obtained by fermentation of glucose or glucose syrups include alginate,
dextran, and xanthan.
Starch hydrolysates and starch syrups are used as starting materials for the
industrial production of iso-syrups on a large scale. Fructose syrups are pro-
duced by enzymatic transformation from glucose to fructose (iso-glucose) using
so-called glucose isomerase. Iso-syrups are often used as sweetening agents and
sugar substitutes in pharmaceuticals and dietary products.
Cyclodextrins a, b and c are water-soluble cyclic non-reducing oligosaccharides
produced by liquefaction of starch with special enzymes, the so-called CGT-ases
(cyclomaltodextrin glycosyl transferases). The special complex forming proper-
ties of the cyclodextrins make them successfully applicable in many fields in-
cluding cosmetics, pharmaceuticals, analytical chemistry, textile production, etc.
2.6.8
Other Applications of Starch
A new field of application of starches is the emulsion (co)polymerization process.

A (co)polymerization process is a process wherein liquid monomers are present
in an aqueous emulsion and are subjected to polymerization in this form. The
monomers are usually alkenes. In these processes starch acts mainly as a pro-
tective colloid but also as an emulsifier for the monomers and thus serves as a
balancer for the monomer emulsions and for the polymer dispersions produced.
Various patents cover the application of differently modified starches, including
dextrins, decomposed, carboxymethylated, cationized, and acylated starches, in
different applications. Succinylated starches have good emulsifying properties.
Starch derivatives are also used as flocculants. Their polymeric structure in
combination with special substitutions allows formation of complexes which
promote coagulation in aqueous solutions and thus effect sedimentation and
precipitation. A classical application of this is in paper production, where starch
is used to achieve precipitation of a cellulose–starch complex. Such starches are
also used in the cleaning of waste water and the production of drinking water,
where they are used in the additives such as colloidal silica and sodium silicate.
Again, the objective is to bind the components causing turbidity and to precipi-
tate them. Other, very specialized applications of flocculants are the cleaning of
protein suspensions, the dehydrogenation of sludge suspensions in chemical
processes, and sludge sedimentation in the treatment of iron ore. Anionic, cat-
ionic, amphoteric, or non-ionic starches based on maize, wheat, and potato
starch are used, depending on the chemical composition of the agent causing
the turbidity. Flocculants and binders based on starch have been widely used in
the ceramics industry. This is of particular importance, because flocculants and
binders based on starch are of natural origin and hence more environmentally
friendly. Thus, no hazardous substances that might have an impact on the envi-
ronment are released during carbonization. Ceramic products, especially refrac-
tories, are manufactured by a flocculation process usually using cationic
starches to precipitate inorganic materials, followed by drying/forming and fir-
ing processes. Cationic starches are used as temporary binders with special
properties because they form of a stable porous matrix.
Starch derivatives are also found in the formulations of drilling fluids used to
drill wells into subterranean formations containing oil, gas, or minerals, for the
purpose of extraction and production of the minerals. Drilling fluids are usually
composed of water and a wide array of additives to give them the characteristics
required for the specific purpose. They are used to clean and cool the drill bit
and to flush to the surface the rock cuttings, stones, gravel, clay, or sand that
are broken loose by the drill bit. Pre-gelatinized, carboxymethylated, drum dried
or extruded starches are added to improve the coating capacity and rheological
properties of the fluid.
Another field of application of starches is the foundry. Here starches are used
as binders and/or rheology enhancers in foundry glaze. They are used to coat
sand grains with a fine-grained, fire-proof filler and act as a separating agent al-
lowing ready detachment of the cast from the mold. They also have a positive
effect on the surface of the casts so that they may be used without much addi-
tional treatment. In this field, starch derivatives compete with celluloses, poly-
saccharides, acrylate dispersions, and alkyd emulsions.
A growing field of application of starches is the textile glass-fiber industry.
Glass-fiber weavings are coated with a complex dressing and dried to produce
glass-fiber wallpaper. Such dressings usually contain dispersing and crosslink-
ing agents, a paraffin emulsion, and a number of starch derivatives, which are
responsible for the good wet strength and elasticity of the wallpaper. Usually,
phosphorized carbamate starches or propoxylated oxidized cold-water soluble
starches based on potato starch are used.
2.7
Future Trends and Developments
In recent years, the demand for starch has grown constantly, and this develop-
ment is expected to continue during the next decade. If growth continues ac-
cording to recent trends, starch production in 2010 is estimated to be more than
70 million tons. This growth scenario will be boosted by the development of
new application areas, new technologies in starch modification resulting in new
properties, and tailor-made starches produced mainly by applying biotechnologi-
cal processes.
The increasing need for crude oil a basis for petrochemicals leading to high
prices will trigger starch use in new application areas. New product properties,
e.g. biodegradability and compatibility with the “bio–energy” market, will be
based on starch-producing plants. In recent years increasing efforts have been
made to investigate new modification technologies, for example high-pressure
treatment, low-temperature modification, pulsed high electric field treatment,
and ultrasonic treatment, resulting in starches having new properties. The in-
corporation of new functionalities by chemoenzymatic derivatization or biotrans-
formation will also open new markets for starch-based products. Transgenic
plants produced by new biotechnological tools are expected to produce new tai-
lor-made starches.
2.7.1
Tailor-made Starches made by using Biotechnological Tools
The property profiles of starch products for a variety of food and non-food appli-
cations require complex physical, chemical, or enzymatic modifications of the
starches. Therefore, growers try to breed varieties of starch-producing plants of
higher agricultural performance, e.g. higher yield per hectare, better resistance
against diseases and pests etc., as well as plants that grow to produce starch
having the required polymer characteristics. These efforts have not only been
2.7 Future Trends and Developments 93
made by conventional breeding methods, but also, for some 20 years, by genetic
modification of the plants. A classical example from the past is the development
of mutants of yellow maize, so-called waxy maize species, that produce a starch
made up of pure amylopectin. This provided a qualitatively and economically
superior alternative to the complicated fractionation of conventional starch for
the production of pure amylopectin. Similar procedures for obtaining amylopec-
tin starches have been applied to other starch-producing plants. Mutants or
genetically modified potatoes, tapioca, wheat, sweet potato and rice, etc., which
are now available and have, to a certain extent, already found their ways into in-
dustrial uses. Plant species which produce starches having a high amylose con-
tent have been bred in a conventional way. Especially famous are, e.g., mutants
of maize (amylomaize) and different types of rice, rye, and pea species. Cur-
rently, potatoes are being developed which produce high-amylose starch.
Beside the required amylose or amylopectin content, further attempts have
been made to develop plants which grow to produce new starches having specif-
ic properties. Examples already developed or under development include
starches having an increased phosphor content, altered particle size distribution,
different branching degree or amylopectin-sidechain length, higher viscosity,
special rheology and retrogradation properties, or combinations thereof. It is to
be expected that in the very near future an increasing number of starches hav-
ing special functions, obtained from new plant species, will be introduced to
the market.
2.7.2
New Modification Technologies for New Properties
The permanent need for starch products having better or different properties
leads to the development and use of new technologies for the production and
modification of starch products. For example, pulsed high electric field or ultra-
sonic treatment have been shown to be useful for extracting starch from e.g. po-
tatoes or maize. The use of these new technologies reduces the amount of en-
ergy needed to extract the starch, resulting not only in higher yields but also in
starch and starch derivatives of higher purity.
New ways of modifying starch, e.g. high-pressure or cryo treatment, are the
subject of very intensive research. These new treatment methods furnish
starches e.g. with special rheology characteristics. Wide-spread application of
these new technologies will depend on whether the higher costs of the pro-
cesses can be justified by better performance of the modified starches. These
new technologies may become very important in the production of organic
starches having improved properties. Another important topic of high interest
is the further development or combination of current technologies. For example,
the combination of spray-drying technology and gelatinization of starch using
steam directly in the spray drier (spray cooking) has led to many new starches
having special viscosity characteristics. Similar developments are now being in-
vestigated e.g. in the field of reactive extrusion processing.
2.7.3
New Fields of Application
While in the 2nd half of the 20th century petrochemical products boomed and
put some applications of starch under pressure (e.g. in the field of textiles and
adhesives), this trend is expected to reverse in the 21st century. A number of ap-
plications of classical petrochemical products are criticized for their lack of sus-
tainability, and the demand for sustainable alternatives is increasing. A typical
example is biodegradable packaging material. Highly intensive research has
been done in recent years in the field of thermoplastic starches, and it is just a
matter of time until new products enter this huge market.
In this general trend, in many fields of application new solutions are being
investigated that are based on starch. Intensive work is done in the field of
application of specially modified starches for inks, coatings, and paints, and
new products are expected to be launched very soon.
The type-dependant, particular granular structure of starches allows use of
these starches in special applications, e.g. as a pigment in paper coatings.
Recently, hydrophobized starch derivatives have been described for use in de-
tergents in a number of applications. Considering the polyalkyl glycosides,
which are already known, a wide range of new starch based detergents is ex-
pected to hit the market in the near future.
The use of special starch derivatives in construction applications is gaining
importance. While application in plasters, mortars, and tile cements have long
been known, new products for new applications have been developed in recent
years, particularly in the field of conventional concrete and spray concrete.
These new products are starch-based anti-blocking agents and so-called rebound
minimizers. It is to be expected that further new applications of special starches
will be developed in the future.
Bibliography
Sections 2.1 and 2.2 Potato Science and Technology, G. Lisinska

(Edt.), Elsevier (1989)
http://europa.eu.int/comm/agriculture/eval/ http://www.cassava.org/
reports/amidon/index_en.htm http://www.inaro.de/Deutsch/ROHSTOFF/
http://www.aac-eu.org industrie/STAERKE/starke.htm
http://www.starch.dk/
http://www.biosicherheit.de/kartoffeln/
Sections 2.3, 2.4, and 2.5 14.doku.html
Starch: Chemistry and Technology, R. L.
Starch in food: Structure, function and Whistler, J. N. Bemiller, E. F. Paschall
applications, Ann-Charlotte Eliasson (ed.), (Eds.), Academic Press, Inc., 2nd Edition
Woodhead Publishing (2004) (1984)
Technology of Corn Wet Milling, P. H. Blan- Stärke und Stärkederivate, G. Tegge (ed.),
chard (Edt.), Elsevier (1992) Behr’s Verlag GmbH and Co. KG Ham-
burg, 3rd Edition (2004)
Bibliography 95
H. Röper: Renewable Raw Materials in Eu- “Corn: Chemistry and technology”, Stanley
rope – Industrial Utilisation of Starch and A. Watson, Paul E. Ramstad (eds.), Ameri-
Sugar [1], Starch/Stärke 54 (2002) 89–99 can Association of Cereal Chemists, Inc.,
H. Röper: Starch: present use and future St. Paul, Minnesota, USA
utilisation, in Carbohydrates as Organic Otto B. Würzburg (ed.), Modified Starches:
Raw Materials III (Eds. H. van Bekkum, Properties and Uses, CRC Press Boca Ra-
H. Röper, F. Voragen), VCH Verlagsge- ton, 1986
sellschaft Weinheim, 1996, 17–35 “Starch and its Modifications”, M. V. Ruten-
Otto B. Würzburg (ed., Modified Starches: berg
Properties and Uses, CRC Press Boca Ra- “Handbook of Water-Soluble Gums and
ton, 1986 Resins”, R. L. Davidson (ed.), McGraw-Hill
Book Co (1980)
Section 2.6 Lorenz, A., Beiträge zur Anwendung von
Stärkeveredelungsprodukten auf dem Ge-
biet der Tablettierung in der pharmazeu-
“Applications of Wet-End Paper Chemistry”,
tischen Technologie. Dissertation, Martin-
Che On Au, Ian Thorn (eds.), Blackie Aca-
Luther-Universität Halle-Wittenberg, 1986
demic and Professional, 1995
Colloidal silica/polyelectrolyte blends for
“Paper Chemistry”, J. C. Roberts (ed.),
natural water clarification, Kristine S.
Blackie Academic and Professional, 1996
Slmen, Pek Lee Choo, David A. Picco, Mi-
“Stärkeeinsatz in der Papierindustrie”; PTS-
chio Kobayashi, Nalco Chemical Company,
IZP-Seminar, SE-SE 358 HEI, 1993
(1997); Naperville (US)
“An overview of developments in starch ad-
“Industrial Starches”, Roland W. James,
ditives and starch kitchens for corrugated
Noyes Data Corporation (1974); New Jer-
board”, Bob Boades, International Paper
sey (US)
Board Industry, January 1996, 59–66
Ullmanns: Encyklopädie der technischen
Chemie, 4., neubearb u erw Aufl. Verlag Section 2.7
Chemie, Weinheim (D)
“Handbuch der Textilhilfsmittel”, A. Chwa- “Gentechnik in der Stärkeforschung”,
la, V. Anger, C. Chwala, Verlag Chemie, J. Koßmann, M. Emmermann, R. Lorberth,
Weinheim, 1977 F. Springer, L. Willmitzer, G. Abel, V.
“Grundlagen der Textilveredelung”, M. Pe- Büttcher, E. Duwenig, T. Welsh, Getreide
ter, H. K. Rouette; 13. überarb Aufl. Mehl und Brot 51 (1997) 5, 259–262
Deutscher Fachverlag, Frankfurt am Main, “Approaches to influence starch quantity
1989 and starch quality in transgenic plants”,
EP 1232189 Emulsionspolymerisationsver- B. Müller-Röber, J. Koßmann, Plant, Cell
fahren; M. Pfalz, M. Wastyn, D. Grüll and Environment (1994) 17, 601–613
(2003); Südzucker AG Mannheim (D) “Towards modifying plants for altered starch
EP 1075839 Plasmaexpander, Blutverdün- content and composition” R. G. F. Visser,
nungsmittel und Kryoprotektor, hergestellt E. Jacobsen, TIBTECH February 1993
unter Verwendung von Amylopektin-Kar- (Vol 11), 63–68
toffelstärke; D. Grüll, U. Stifter (2001); Mutation: Modified Starch Metabolism in
Südzucker AG Mannheim Mutant and Transgenic Plants, P. Sathish,
EP 1313682 Flockungs- bzw. Bindemittel C. Sun, A. Lönneberg, C. Jansson in Pro-
für den keramischen Bereich; D. Grüll, M. gress in Botany, Vol. 56, Springer, Berlin,
Wastyn, M. Kozich (2003); Südzucker AG (1995), 301–318.
Mannheim (D)
97
3
Lignocellulose-based Chemical Products
and Product Family Trees
Birgit Kamm, Michael Kamm, Matthias Schmidt, Thomas Hirth, and Margit Schulze
3.1
Introduction
Lignocellulose biomass, for example wood or straw, is an almost inexhaustible,

consistently renewable source of energy and chemical diversity. Lignocellulose is
a complex combination of the two polymeric carbohydrates hemicellulose and
cellulose, and lignin, a phenylpropane-based polymer. Together, they form the
scaffold of the plant cell wall. Lignocellulose is part of almost all higher plants,
mostly found in vessels and roots. So, lignocellulose-rich hardwood consists of
30 to 45% cellulose, 20 to 30% hemicellulose, and 20 to 25% lignin; cereal
straw contains approximately 38 to 40% cellulose, 20 to 30% hemicellulose, and
6 to 20% lignin.
Of all continental sources of biomass, lignocellulose is the most abundant
natural source characterized by many advantages compared with field crops
such as corn grain, cereals, or sugar beet.
Lignocellulose biomass, for example straw, grass, reeds, or fast-growing lum-
ber, is significantly less expensive than cereals. It contains up to 80% cellulose
and hemicellulose that can be converted into sugars. Because of modern bio-
technical and chemical methods, those conversions are expected to be of high
economic efficiency. In addition, improved enzymatic methods of conversion of
cellulose to glucose enable economically efficient production of basic chemicals
such as ethanol, which at the same time is a fuel. Conversions of hemicellulose
open up economic routes to furfural and furan chemistry and a variety of poly-
meric materials, e.g. Nylon, polyalcohols, polyesters, and furan resins. There is
a very good chance that catalytic oxidation and hydrogenation reactions and al-
kaline hydrolysis of lignin could establish a biomass-based chemical lignin fami-
ly tree.
Complex LCF biorefineries with cellulose, hemicellulose, and lignin product
lines and family trees of the corresponding chemically and biotechnically avail-
able derivatives open the route for biobased industrial production of high added
value, economy, and efficiency.
ISBN: 3-527-31027-4
98 3 Lignocellulose-based Chemical Products and Product Family Trees
3.2
Historical Outline of Chemical and Technical Aspects of Utilization Lignocellulose
in the 19th and 20th Century
3.2.1
From the Beginnings of Lignocellulose Chemistry Until 1800
The history of utilization of lignocellulose and cellulose is closely related to the

production of paper and dates back to the original old-Egyptian pressing of stipe
pith of the papyrus plant (Cyprus papyrus) to a substance “to write with” (“Be-
schreibstoff”), (third millennium before Christ) until the first attempt to prepare
paper in the year 105 AD. Tsai Lun from Gue Yang in the Province Hunan re-
ported to the emperor of China Ho Ti about his discovery of paper produced
from bark, cannabis, rags and fishing nets.
In 1546, the German physician and pharmacist Georg Pawer, better known as
Georgius Agricola (1494–1555) investigated succinic acid [HOOC–CH2CH2–
COOH] [1] obtained by dry distillation of amber (succinite). Today, succinic acid
is available via fermentation of sugar or cellulose hydrolyzates.
In the middle of the 18th century, only four organic acids were known. The
German-Swedish pharmacist Carl Wilhelm Scheele (1742–1786) increased this
number to 13: 1767–1770 wine acid [HOOC(CHOH)2COOH] together with Ret-
zius, 1768–1782 hydrocyanic acid [HCN], 1769–1786 gallic acid (3,4,5-trihydroxy-
benzoic acid), and pyrogallic acid (1,2,3-trihydroxybenzene), 1776 oxalic acid
[HOOC–COOH] together with Torbern Olof Bergman (1735–1784) (in 1769 also
explored by Johann Christian Wiegleb “Kleesäure” [2]), 1780 lactic acid [H3C–
C(OH)–COOH], mucic acid and furandicarboylic acid (Fig. 3.1) and 1784/85
malic acid [HOOCCH(OH)CH2COOH] [3, 4, 5]. Today, tartaric acid and the last
four acids mentioned above today play an important role in the concept of “Bio-
based Products and Biobased Building Blocks” [6, 7, 8]. For example, mucic acid,
available by oxidation of several carbohydrates is part of furan chemistry
(Fig. 3.1).
Lignocellulose and cellulose chemistry itself is only approximately 200 years
old.
disaccharide
Fig. 3.1 Furan derivatives by dry distillation.

3.2 Historical Outline of Chemical and Technical Aspects of Utilization Lignocellulose in the 19th 99
3.2.2
Lignocellulose Chemistry in the Nineteenth Century
3.2.2.1 Cellulose Saccharification

In 1819, the French savant and plant chemist Henri Braconnot (1780–1855) dis-
covered that canvas is dissolved by vitriol oil (old-fashioned term for sulfuric
acid). Boiling a dilute solution Braconnot found a fermentable sugar, identical
with the sugar from grapes (glucose) or starch [9]. So, Braconnot for the first
time described a reaction that later, in a changed form, would achieve technical
importance as cellulose saccharification (“Holzverzuckerung”).
3.2.2.2 Oxalic Acid

In 1829, Joseph Louis Gay-Lussac developed a technically interesting synthesis
of oxalic acid (ethane diacid, clover acid) by heating sawdust and similar raw
materials with potassium hydroxide [10]. Oxalic acid, which is one of the most
frequent natural plant acids [sour clover, Oxalis acetosella, sorrel, turnip-top, pie-
plant], was first prepared in 1776 by Carl Wilhelm Scheele (1742–1786) by oxi-
dation of saccharose (cane or beet sugar) with nitric acid, and in 1824 by Frie-
drich Wöhler (1800–1882; the father of the synthesis of urea) by saponification
of dicyan (oxalonitrile) (Fig. 3.2) [11].
3.2.2.3 Xyloidin and Nitrocellulose

In 1833, Braconnot observed the formation of a flammable compound after
treatment of wood fiber or starch with nitric acid which he called xyloidin [12];
even today this name is sometimes used for water-soluble starch nitrate. In
1846, nitrocellulose (so-called gun cotton) was explored by Christian Friedrich
Schönbein (1799–1868), Professor of Chemistry at Basel (Switzerland), who also
reported its use in shot. At the same time Julius Otto and Rudolf Christian
Böttger (1806–1881, Professor of Chemistry in Halle/Saale, Germany) also dis-
covered this special cellulose derivative [3 b].
The observations of G. S. C. Kirchhoff on the hydrolysis of starch (1812 [13])
and those of Braconnot [9] on the chemical relationship of cellulose, starch, and
sugar, and the corresponding analyses performed by Gay-Lussac (1778–1850)
and Louis Jacques Baron Thénard (1777–1857) had been the basis of a further
discovery – in 1827 the British physician and pharmacist William Prout (1786–
1850) reported that a variety of different sugars, starches, rubbers, or wood com-
pounds could be regarded as combination of carbon-with-water – “carbohy-
Fig. 3.2 Oxalic acid synthesis by Scheele 1776. *Today catalyzed with HNO3 and V2O5.
drates” [14]. Prout also classified organic food for the first time into the three
groups – proteins, fats, and carbohydrates, a classification still used today. Even
so, he called the carbohydrates the “saccharine class”. The current name carbo-
hydrates instead of “saccharine class” was introduced in 1844 by Carl Schmidt
(1822–1894), Professor of Medical Chemistry at the University of Dorpat/Tartu,
formerly Ostpreußen, today Estonia [15].
3.2.2.4 Cellulose
In 1839, Anselme Payen, a French manager of a sugar refinery, later an univer-
sity lecturer, reported the generation of a substance called les cellules (France
Cellule = cell) [16] formed after treatment of wood with nitric acid and subse-
quently with sodium hydroxide solution. The name was recommended by his
scientific friend Jean-Baptiste André Dumas who developed the hydrogen–halo-
gen substitution theory and discovered trichloroacetic acid, propionic acid, fatty
acids, and anthracene from tar [17 a].
In 1839, cellulose was converted into dextrose (d-glucose) for the first time by
Payen, by treatment with concentrated sulfuric acid. Payen discovered that cellu-
lose, starch, and sugar all have the same chemical composition. He also found
cellulose in cotton and recognized that wood is not a uniform substance. By
treatment of wood with nitric acid he obtained a carbon-like substance he, how-
ever, believed to be a mixture of different compounds. He called it “incorporated
substance”; today it is known as lignin [17 a/18].

In 1840, the Dutch professor of chemistry Gerardus Johannes Mulder (1802–
1880, who also introduced the word “proteins” in 1839) reported the synthesis
of levulinic acid (4-oxopentanoic acid, c-ketovaleric acid) by boiling of fructose
with hydrochloric acid. The old name levulose for fructose also gave the name
for the corresponding acid [19]. Chemically, levulinic acid has the properties of
a ketone and an acid. Degradation of hexoses to levulinic and formic acids is
not yet clarified. Today, HMF (5-hydroxymethylfurfural) is assumed to be inter-
mediate [20] (Fig. 3.3).
Fig. 3.3 Levulinic acid process with HMF as intermediate.

3.2 Historical Outline of Chemical and Technical Aspects of Utilization Lignocellulose in the 19th 101
3.2.2.6 Lignin
In 1856, Franz Ferdinand Schulze (1815–1873), chemist at the agricultural faculty
of the University of Greifswald, reported the isolation of cellulose by treatment of
wood with a mixture of nitric acid and potassium chlorate (KClO3) (see also Payen,
1839 [16]). For the first time, F. F. Schulze called the solved part lignin (Latin lig-
num, wood) [21, 22]. Schulz’s “Contributions to the knowledge of lignins” [23]
was the beginning of intensive investigation of lignin [17 b, 24 ].
It was still many years, however, before the chemical structure of lignin was
described. F. Bente (1868) [25] reported the aromatic character of alkaline lignin
melts. In 1897 the Swedish scientist P. Klasen described the “non-cellulosic”
aromatic character of lignin. Klason assumed coniferyl alcohol (4-hydroxy-3-
methoxycinnamyl alcohol) to be the central building block of lignin cellulose
biosynthesis [26, 27]; in 1919, W. Fuchs described a phenol nucleus [28].
Karl Freudenberg (1886–1983), Professor of Wood Chemistry at Karlsruhe
and Heidelberg, discovered in 1927 that lignin is built of phenylpropane deriva-
tives [29]. In addition, lignin can be described as a product of a continuous
etherification of those building blocks [30–33]. In the 1940s, systematic investi-
gation of a variety of lignin-containing plants and wood species started on an in-
ternational scale. The results were published in a review in 1960 by Friedrich
and Dorothy Brauns [34].
3.2.2.7 Hemicellulose (Polyoses) and Furfural

In 1891, the German-Swiss chemist E. Schulze for the first time called the carbo-
hydrates of wood hemicellulose [35]. Difference from cellulose they are soluble in
dilute alkaline solution. Schulze still assumed, however, that hemicellulose was
a precursor of cellulose [36 a]. For the first time, the Nobel Prize winner Hermann
Staudinger in 1952 introduced and specified the terms polyoses and hemicellulose as
an integrated complex of macromolecular carbohydrates that are found with cellu-
lose in wood and other lignocellulose-containing resources [37, 38].
Another milestone of lignocellulose chemistry was the discovery of furfural
(2-furfuryl aldehyde), obtained from the pentoses of hemicellulose. In 1831 the
founder of Technical Chemistry, J. W. Döbereiner, obtained furfural (originally
called furfurol) by distillation of bran [bran (old) = furfur] with dilute sulfuric
acid. Döbereiner obtained the same result by treating sugar with sulfuric acid
in the presence of manganese dioxide. Especially useful was the treatment of
hemicellulose-containing raw materials with dry steam from hydrochloric acid
(HCl) [39]. Several raw materials were treated by this process between 1912 and
1927 (Table 3.1).
Because of the possibility of furfural production from straw pulp, during
World War I, the German “War Commission for Feed Substitutes” (“Kriegsaus-
schuss für Ersatzfutter”) held a contest to find new applications for furfural
[40]. Technical furfural synthesis was mainly promoted by the Quaker Oats com-
pany which, since 1922, produced furfural from waste obtained during produc-
tion of oat flakes [41].
Table 3.1 Yields of furfural obtained by application of the

HCl–steam process to different raw materials (from 1912 till
1927 [39, 40]).
Raw material Furfural yield (%)
Laboratory attempts Technical attempts
Straw 9.48 5.60–8.31

Flax pares 10.11
Oat hulls 6.72
Corncobs 7.76, 9.74 a)
Coniferous wood 3.72
Wood 3.5
Rice chaff 4.00
Wheat bran a) 3.00
a) H2SO4
3.2.2.8 Lignocellulose
The term lignocellulose was introduced by Edward John Bevan (1856–1921) and
Charles Frederick Cross (1855–1935), the two British discoverers of the “Cellu-
lose–Xanthogenate Process” (also called the Viskose process) [42]. In 1903 they
described lignocellulose as one of five natural cellulose types, assuming a chem-
ical bond between lignin and cellulose [43]. There is some controversy about the
first use of the term lignocellulose in the current meaning as a type of biogenic
feedstock. It is assumed that the “lignocellulose feedstock” term was first intro-
duced at the end of the first third of the 20th century. Until that time, raw ma-
terials such as cotton or wood were mainly evaluated with regard to the techni-
cal utilizability of the corresponding cellulose. In the American literature, the
term lignocellulose has been used since 1942, for example by Raphael Katzen
and Donald F. Othmer (co-editor of the Kirk-Othmer Encyclopedia for Technical
Chemistry) [44].
3.2.3
Industrial Lignocellulose Utilization in the 19th and Beginning of the 20th Century
In the 19th century and the first third of the 20th, technical and industrial utili-
zation of lignocellulose-containing raw materials was mainly focused on:
· paper and pulp production from wood,
· soluble cellulose derivatives, viscose and other cellulose-based synthetic fibers,
· wood-based sugar production and wood liquification,
· vanillin production from lignin,
· nitration of cellulose (guncotton and nitro rayon),
· furfural and nylon.
This topic will not be discussed more in detail at this point because the histori-
cal and technological aspects are discussed several times from different points
of view in different chapters of this monograph [45–47].
3.3
Lignocellulosic Raw Material
3.3.1
Definition
Lignocellulosic feedstock is biomass mainly consisting of a complex composite of

the two structural carbohydrates cellulose and hemicellulose, and phenolic lig-
nin. Depending on the corresponding chemical–technical or pulping processes,
lignocellulose feedstocks can be divided into the subgroups cellulose, hemicellu-
lose (polyoses), lignin, extractive substances, and ash (Fig. 3.4) [48].
Cellulose, hemicellulose and lignin are biopolymers and act as precursors in LC-
biomass. Depending on type and concentration they are accompanied by other
biopolymers – polyhydroxy fatty acid esters such as cutin (a wax-like heteropoly-
mer with ester bonds between the monomers, e.g. C16 and C18 fatty acids and cor-
responding x-hydroxy fatty acids and epoxy fatty acids), polyisopropenoids, for ex-
ample terpenes and steroids, and high-molecular-weight glycosidic pectins. In ad-
dition, so-called storage carbohydrates, for example several sugars and starch, can
be found in minor amounts, as also can dyes, pigments, flavors, alkaloids, inor-
ganic compounds and many more [48–53].
Cellulose, the main part of plant cell walls, and hemicellulose together act as sup-
porting and scaffold substances. Cellulose is a non-branched water-insoluble poly-
Fig. 3.4 Chemical–technical major groups of lignocellulosic feedstock [48].

saccharide consisting from several hundred up to tens of thousands of b-glucose

molecules of formal composition (C6H10O5)n; more specifically, cellulose is an iso-
tactic b-1,4-polyacetal of cellobiose (4-O-b-d-glucopyranosyl-d-glucose). Cellobiose
(C12H22O11) thus consists of two molecules of glucose (Fig. 3.5). The disaccharide
cellobiose can be obtained by enzymatic degradation of cellulose with cellulase, a
cellulose-degrading enzyme complex, or by mild hydrolysis [54].
Cellulose is the most abundant biopolymer synthesized by Nature, approxi-
mately 1011 tons per annum. A tree, for example, produces approximately 14 g
cellulose per day. A chain formed from all the cellulose molecules produced per
day would reach 175 times from the earth to sun (2.6 ´ 1010 km).
Hemicelluloses (Greek: hemisys = half) or pseudocellulose, in the German litera-
ture also called polyoses, are part of the plant cell membranes and serve, together
with cellulose, as scaffolds and supporting substances. The name hemicellulose
encompasses all polysaccharides based on polymeric hexosans generated from a
variety of monomers (e.g. glucose, mannose, galactose) and/or polymeric pento-
sans based on monomers like arabinose or xylose. Figure 3.6 shows a selection
of hemicelluloses.
Hemicelluloses, with cellulose and pectins, belong to the so-called “structural
carbohydrates”, whereas carbohydrates like saccharose (cane and beet sugar)
and starch are so-called storage compounds. So, wheat and barley contain hexo-
sans; rye and oat mainly pentosans; and bran contains up to 40% hemicellulose
[49, 50, 54].
Lignin (Latin lignum wood) is an amorphous, thermoplastic, three-dimensional
polymer network based on phenylpropane subunits (aromatic monomers), which
is incorporated in the cell wall causing so-called lignification of the cells. Approxi-
mately 20 to 30% of the solids content of lignified plants consists of lignin.
Fig. 3.5 Section of the chemical structure of cellulose.
Fig. 3.6 Selection of arabino-4-O-methyl-glucuronoxylan hemicellulose [54].

Fig. 3.7 Lignin structural units and an example of coupling.
The chemical structure of lignin consists of the aromatic phenylpropane subunits

trans-p-coumaryl alcohol, trans-p-coniferyl alcohol, and trans-p-sinapyl alcohol con-
nected in different ways – b-O-4 coupling, b–b coupling, b-5 coupling, b-1 coupling,
and 5-5-coupling – of which b-O-4-coupling is the most common one (Fig. 3.7).
Lignin from different hard and soft woods is characterized by different per-
centages of the corresponding alcohols. The subunits are connected in different
ways to form the final network (O–O ether and C–C bonds). The phenylpropane
subunits of lignin and hemicellulose are connected by ether bridges. After cellu-
lose, lignin is the most common organic substance in the world [24, 34, 55].
3.3.2
Sources and Composition
3.3.2.1 Sources
After green biomass, lignocellulose feedstock is the most common raw material
for continental biorefinery processes. In addition, LCF are least regulated by fed-
eral law and exclusively reserved for food applications. The best known re-
sources are wood, fast-growing lumber, old forest and timber, recovered paper,
Table 3.2 Placement of lignocellulosic feedstock (LCF).
No. Source Examples
Group 1 Existing landscape species Softwood, hardwood, reed, reed grass, switch
grass, dry grasses
Group 2 Fast-growing plantations Poplar, willow, wood grass, eucalyptus, Sudan
grass
Group 3 Landscape conservation Old forest, residual wood and under-wood from
forestry, switch grass, dry grasses, hay, straw
Group 4 Process lignocelluloses Straw, corn stover, press cake from crop drying
plant, ethanol plants and oil mills, by-products
from cereal mills, whole crop refineries, paper
mill and pulp industry
Group 5 Used materials and waste Timber, used wood, recovered paper, cellulosic
municipal solid wastes
and straw. In Table 3.2, lignocellulosic sources are summarized and placed into
five different groups.
All five source groups will play an important role in the supply of LCF,
although there will be significant regional differences. Groups 2 (fast-growing
plantations) and 4 (process lignocelluloses) will grow the fastest, because of the
significant resources (e.g. fast-growing woods and straw). In Europe, Group 3
will attract more attention because of substantial changes in agricultural poli-
tics.
3.3.2.2 Chemical Composition of Lignocelluloses

All the raw materials listed in Tables 3.2 to 3.5 contain the precursors cellulose,
hemicellulose and lignin in different amounts. The chemical composition varies
from raw material to raw material, and from plant to plant, and also in between
species. As shown in Fig. 3.8, the compositions of hardwoods and softwoods are
substantially different. The figure shows that:
1. the lignin content of softwoods is usually higher than that of hardwoods;
2. the hemicellulose content of hardwoods is similar to that of softwoods; and
3. the cellulose content of hardwoods is usually higher than that of softwoods.
Fig. 3.8 Comparison of the compositions of hardwood and softwood [56].

Table 3.3 Chemical composition of different lignocellulosic feedstock a) [52].
Name (raw material) Latin name Lignin (%) Alpha- Hemicellulose (%) Extractives c) Ash
cellulose b) (%) (%)
% Pentosans Other
Spruce Picea 29 43 12 14 1.7 0.3

Popular Populus 23 43 18 13 1.5 0.7
Beech Fagus silvatica 25 38 21 14 2.3 0.4
Ailanthus Ailanthus 23 42 22 19 2.6 0.8
Hornbeam Carpinus betulus 21 38 27 12 1.6 0.8
Alder Alnus 26 42 20 9 2.2 0.5
Willow Salix 23 43 21 9 3.3 0.4
Oak Quercus 26 35 24 8 2.0 0.5
Turkey oak Quercus cerris 28 38 21 10 2.3 0.8
Wheat straw Triticum aestivum 18 32 23 14 5.0 8.2
a) As a percentage of dry matter

b) Alpha-cellulose in 17.5% NaOH or 24% KOH solution, insoluble part with de-
gree of polymerization > 200
c) Extractives: alcohol–benzene extract
Table 3.3 presents the compositions of different types of straw. Straw species are
more uniform in composition than wood species. Straw usually has a lower cel-
lulose content than wood, but despite this it has a total carbohydrate fraction
(holocellulose) approximately equal to that of wood. This is because of its high
hemicellulose and low lignin content compared with wood. The ash content of
straw is higher than that of wood [57].
Differences also arise as a result of different investigation and pulping meth-
ods [58, 59]; because of this complexity, it is still difficult to determine specific
group composition. The data shown in Tables 3.3 to 3.6 are collected from dif-
ferent sources.
Table 3.4 Chemical composition of different types of American straw a) [57].
Name Latin name Lignin Alpha- Hemicellulose Extractives c) Ash

(raw material) of plant (%) cellulose b) pentosans only (%) (%)
(%) (%)
Rice straw Oryza 11.9 36.2 24.5 4.6 16.1

Barley straw Hordeum vulgare 14.5 33.8 24.7 4.7 6.4
Wheat straw Triticum aestivum 16.7 39.9 28.2 3.7 6.6
Rye straw Secale cereale 19.0 37.6 30.5 3.2 4.3
Oat straw Avena sativa 17.5 39.4 27.1 4.4 7.2
Flax shives Linum 22.3 34.9 23.6 4.1 3.5
Soybean stalks Glycine max 19.8 34.5 24.8 3.9 2.3

b) Alpha-cellulose in 17.5% NaOH or 24% KOH solution, insoluble part with de-
gree of polymerization > 200
c) Extractives: alcohol–benzene extract
Table 3.5 Ranges of variation of the chemical composition of

different lignocellulosic feedstock [50].
Feedstock Cellulose (%) Hemicellulose (polyoses) Lignin (%)
Hexoses (%) Pentoses (%)
Softwood 40–48 12–15 7–10 26–31

Hardwood 30–43 2–5 17–25 20–25
Cereal straw 38–40 2–5 17–21 6–21
Maize straw 35–41 2 15–28 10–17
Rape straw 38–41 – 17–22 19–22
Recovered paper 50–70 – 6–15 15–25
Table 3.6 Composition of selected biomass materials a) [60].
Feedstock Cellulose (%) Hemicellulose (%) Lignin (%)
Tarnbark oak 44.8 19.6 24.8

Corn stover 36.5 28.1 10.4
Red clover hay 26.7 20.6 15.1
Bagasse 41.3 20.4 14.9
Oat hulls 33.7 20.5 13.5
Newspaper 61.0 16.0 21.0
Processed MSW b) 47.0 25.0 12.0

b) Municipal solid wastes
3.3.2.3 Carbohydrates in Lignocelluloses

Lignocellulosic feedstocks (LCF) consist of 65 to 85% carbohydrates, mainly
polysaccharides. Linear and branched polymers of glucose are summarized as
so-called glucan (polyglucan); cellulose also belongs to group of polysaccharides,
as also do amylose, laminatin, and lichenin. Hemicelluloses mainly consist of
polysaccharides such as mannan, xylan, and galactan. Arabinan, polymeric acet-
ates, acetate derivatives of hemicellulose, and polyuronoic acid (polymeric alde-
hyde acids) are also included as carbohydrate building blocks (Fig. 3.9 [61, 62]).
Mannan and xylan are the most common polysaccharides of polyoses. Man-
nanes are polyoses (hemicelluloses) built from mannose instead of glucose sub-
units. The chains consist of 1,4-linked mannose monomers forming a b-pyra-
nose. Xylanes (C5H8O4) are polyoses (hemicelluloses) that are part of many
trees, cereals, straw, glumes, bran, pectin, tragant, and, in common continental
plants, mostly as heteroxylanes. Xylanases are special hemicellulases (enzymes)
that can hydrolyze xylane into xylose and other pentoses. Such xylanases are
generated by fungi. Genotechnologically modified Zymomonas mobilis bacteria
can ferment xylane into ethanol [63]. Galactanes are polysaccharides (glycanes)
that are mainly composed of galactoses. Arabinanes (C5H8O4)n are polyoses of
Fig. 3.9 Carbohydrates in lignocelluloses.
Table 3.7 Carbohydrate composition of different lignocellulosic feedstock a) [50].
Component Glucan Mannan Xylan Galactan Arabinan Total carbo- Lignin

plant name (%) (%) (%) (%) (%) hydrates (%) (%)
White wood fir, 46.5 11.6 6.8 1.2 1.6 67.70 26.7
Abies alba
Douglas fir, 43.46 10.76 2.77 4.66 2.67 64.32 31.30
Pseudotsuga
menziesiicaesia
Oak, Quercus 40.63 1.97 19.19 1.22 0.36 63.37 23.91
Aspen, Populus 45.97 2.10 17.74 7.9 1.23 67.83 20.30
tremula
Maize cob 34.0 0.5 14.0 1.0 1.7 51.20 13.1
Wheat straw 37.0 0.3 18.9 6.5 5.6 62.30 13.6
linear a-(1,5)-glycosidic linked l-arabinofuranoses with single l-arabinose units

connected via a-(1,3)-glycosidic bonds to the main chain [62]. The amount of
the different carbohydrates in lignocelluloses varies very much depending on
wood type and age (one-year-old plants compared with older trees) (Fig. 3.8 and
Table 3.7).
3.4
Lignocelluloses in Biorefineries
3.4.1
Background
An important aspect of utilization of biomass as a chemical raw material is the cost

of corresponding raw materials. In 2002, approximate raw material prices were:
US $ 30 t–1 for corn stover or straw (LCF); US $ 3 bushel–1 or US $ 110 t–1 for corn
or grain [64]. The objective of all hitherto introduced processing methods based on
lignocellulosic feedstocks is to produce fibers, paper, pulp, or sugar. Because cur-
rently only the fraction is cellulose used, however, most of the valuable biomass raw
material is lost. The following two examples should help explain this in more detail.
3.4.1.1 Example 1
Is an entire tree (from roots to treetop) as biomass is processed into pulp a sig-
nificant weight loss is observed. After pre-treatment and pulping more than
70% of the original mass is lost. Before arrival at the plant the “biomass tree”
will already have lost 40% of its original mass; a further 30% will be lost during
the pulping process [65].
3.4.1.2 Example 2
Table 3.3 shows the composition of wheat straw [52]; none of the three main
components cellulose, hemicellulose, or lignin is dominant. So, economically all
three components must be processed to be efficient.
The following conclusions could therefore be drawn.
· Every plant, every feedstock, is a raw material that must be converted entirely.
· All components of such a raw material are of special value and therefore
must be treated within a complex integrated process.
There are still many problems to be solved, e.g. with regard to conversion of LCF
into precursors. It is still the problem that one or more components may be de-
stroyed or changed in structure during conversion. Another unsolved problem
is that lignin still is not really used but regarded as waste. To solve the problem
completely it will be necessary to learn from more than 100 years experience with
and efficiency of petroleum-based chemistry. Petroleum refineries deliver well-de-
fined, chemically-pure basic chemicals (building blocks) that are easily handled.
On the basis of these simple building blocks, more complex intermediates can
be generated in a controlled manner by chemical reactions. Because of an almost
countless number of combinations, those intermediates then can be converted into
a broad variety of even more complex intermediates and/or final products.
The principles of the highly efficient fossil-based chemical industry – specific
product lines, building blocks, product family trees, commodity chemicals, so-
called coupling production processes and backwards integrated production –

should be transferred and adapted appropriately to bio-based chemistry. By fol-
lowing this approach one ends up with the concept of biorefineries [7, 66–73],
integrated biobased “platforms” and building block structures [74, 76]. The term
biorefinery may, in general, be defined as follows [72]: A biorefinery is an entire
integrated complex concept of a processing plant in which biomass feedstocks are ex-
tracted and converted into a broad spectrum of valuable products analogous to petro-
chemical refineries.
3.4.2
LCF Biorefinery
A lignocellulosic biorefinery (LCF biorefinery) converts lignocelluloses into

fuels, chemicals, polymers, and many other materials. A general scheme of a
biorefinery is shown in Fig. 3.10. Here, all thermo-chemical processes, for exam-
ple combustion, gasification, and pyrolysis (syngas platform) are considered
solely as utilization of waste or residual materials.
An LCF biorefinery is, analogous to most biorefineries, a rather complex and
integrated system of conventionally used technology and more recent, modern
processes. Complex technology resources are (with others):
· the processing of cereal waste into furfural [39, 40], e.g. the well-known pro-
duction of flakes and furfural from oats [English Patent (E.P.) 203,691 (1923),
French Patent (F.P.) 570 531 (1923)] an industrial-scale process of Quaker
Oats, Illinois, USA [77, 78]
· commercial levulinic acid production using hexoses of low-cost cellulose prod-
ucts; in 1956 levulinic acid was regarded for the first time as a “platform
chemical substance” of high synthetic potential [79–81]; and
Fig. 3.10 General scheme of a lignocellulosic biorefinery.

Fig. 3.11 The general equation of the conversion in the LCF-biorefinery.
· the complex technological approaches of wood processing according to Timell

1961 [82], James 1969 [83], or Prink and Pohlmann 1972 [84], the straw pro-
cessing methods according to Puls and Dietrich 1980 [85], or the studies of
Shen 1982–88 in the field of wood grass processing [86].
Lignocellulose materials consist of three primary chemical fractions or precur-
sors:
1. hemicellulose/polyoses, sugar polymer of predominantly pentoses,
2. cellulose, a glucose polymer, and
3. lignin, a polymer based on phenol subunits.
A general equation for the conversion of LCF precursors into “intermediate plat-
forms” sugar (xylose, glucose) and lignin is given in Fig. 3.11 [7, 68, 71, 87] and an
overview of the potential products of an LCF biorefinery is shown in Fig. 3.12.
Fig. 3.12 Products of lignocellulosic feedstock biorefinery

(LCF-Biorefinery, Phase III) [7, 71, 87].
Furfural and hydroxymethylfurfural are particularly interesting products. Fur-

fural is the starting material for the production of Nylon 6,6 and Nylon 6. The
original process for production of Nylon 6,6 was based on furfural. The last of
these production plants was closed in 1961 in the United States for economic
reasons (low price of petroleum). Nevertheless, the market for Nylon 6 is huge.
There are, however, still some problems to be solved in the LCF process, e.g.
utilization of lignin as a fuel, adhesive, or binder. This still is unsatisfactory, be-
cause the lignin scaffold contains substantial amounts of monoaromatic hydro-
carbons which, if isolated economically and efficiently, could add significant val-
ue to the primary process. It should be noted that no natural enzymes have yet
been found that can split naturally formed lignin into basic monomers as easily
as can occur for carbohydrates or proteins.
3.4.3
LCF Conversion Methods
3.4.3.1 Pretreatment Methods

Several physical and/or chemical methods can be used for pre-treatment of lig-
nocelluloses, especially to separate cellulose from its protective sheath of lignin
and increase the surface area of the cellulose crystallite by size reduction and
swelling [88]:
1. Physical pre-treatment [89]
a) Milling and grinding
b)High-pressure steaming and steam explosion
c) Extrusion and expansion
d)High-energy radiation
e) Pyrolysis
2. Chemical methods [57, 90, 93]
a) Alkali treatment (e.g. NH3, NH4SO3, NaOH)
b)Acid treatment (e.g. H2SO4, HCl, H3PO4)
c) Gas treatment (e.g. ClO2, NO2, SO2)
d)Oxidizing agents (e.g. H2O2, O3) [92]
e) Cellulose solvents (e.g. Cadoxen, CMCS) [93]
f) Solvent extraction of lignin (e.g. ethanol–water, benzene–ethanol, ethylene
glycol, butanol–water)
g) Swelling agents [94].
3. Biological methods [90 b, 95–97]
a) Lignin-consuming microorganisms (fungi, e.g. Phanerochaete chrysosporium;
bacteria, e.g. Nocardica sp.)
b)Cellulose-attacking microorganisms (fungi, e.g. brown rot)
c) Lignin and cellulose-attacking microorganisms (fungi, e.g. white and red
rot)
d)Lignin and/or cellulose attacking insects (e.g. termites).
3.4.3.2 Chemical Pulping Methods

Chemical and chemical–thermal pulping processes of lignocelluloses can be di-
vided into two main groups [50, 57 b, 90 c, 91]:
1. Processes with lignin and cellolignin as residues
a) Steam/pressure processes (autohydrolysis)
b)Hydrolysis with weak acids
c) Hydrolysis with concentrated acids or water-free acids
2. Processes with cellulose and cellulose/hemicellulose as residues
a) Alkaline pulping
b)Sulfate pulping
c) Sulfite pulping
d)Organosolv process
e) Phenol process (Battelle)
f) Extraction with H2O (pressure, high temperature).
Figure 3.13 shows combined steam alkali pulping of lignocellulose.

All lignocellulose feedstock contains the three components lignin, hemicellu-
lose, and cellulose. There are, however, differences between the way and the ex-
tent these compounds are chemically and physically connected. There are also
differences between the reactivity of the components, depending on the pulping
reagents used. There is, nevertheless, a demand for enhanced selectivity of the
process which, in the end, means reducing yields and often also changing the
characteristics of the target product (e.g. degree of polymerization, mechanical
Fig. 3.13 Combined steam-alkaline pulping of lignocelluloses.

strength, etc.). Therefore, all processes are a compromise focused on the final
product.
3.4.3.3 Enzymatic Methods

Biodegradation of untreated native lignocellulose is a very slow process, giving
rise to little degradation, often below 20% [85, 98–100]. Thus, the low rate and
extent of conversion inhibit the development of an economically feasible bio-
technological process for hydrolysis of lignocelluloses. To increase the suscepti-
bility of cellulosic material, structural modification by means of various pre-
treatment processes is essential (see above, “Pretreatment methods”). Enzymatic
hydrolysis of pre-treated lignin-free cellulose (obtained from lignocellulose) is
technically feasible. Enzymatic hydrolysis of cellulose accomplishes degradation
of cellulose to glucose. Glucose is an important sugar building block for chemi-
cals and fuels [8]. Enzymatic hydrolysis as a heterogeneous catalytic reaction is
typically characterized by an insoluble reactant (cellulose) and a soluble catalyst
(enzymes) [57 b]. Several comprehensive reviews have been published on the
mode of attack by cellulases on crystalline cellulose. The rate of this reaction is
affected by both structural features of the cellulose and the mode of enzyme
action [90 e, 101–105]. Enzymatic hydrolysis processes are of interest because en-
zymes exclusively catalyze specific reactions (stereospecific and/or stereoselec-
tive reactions). Therefore, different from acidic hydrolysis, there are no side re-
actions or by-products and the hydrolysis can potentially be performed with
yields approaching 100%. All enzymatic hydrolysis processes consist of four ma-
jor steps that may be combined in a different ways – pretreatment, enzyme pro-
duction, hydrolysis, and fermentation. During the last few years enzymatic hy-
drolysis processes have gained importance, particularly for ethanol production
based on lignocellulose [87, 90, 91, 106–108] (Fig. 14).
Fig. 3.14 Enzymatic transformation of lignocelluloses into ethanol.

3.5
Lignin-based Product Lines
3.5.1
Isolation and Application Areas
Current and future application of lignin are a broad field of increasing impor-
tance, as demonstrated during the last 20 years by an increasing number of
worldwide scientific publications and patents on basic scientific knowledge and
technological aspects. One reason for this development is an increasing appre-
ciation of lignocelluloses as a renewable resource. Lignin as a raw material is
still very far from intensive utilization, however, despite its high potential for
several applications with regard to its chemistry, properties, and the large
amounts available from pulp production or LCF biorefinery processes.
During lignin isolation, there are mainly two problems to be solved – main-
taining the natural lignin structure and achieving high yields. Usually, however,
the chemical structure of the isolated lignin is changed to some extent. The
principal isolation methods are shown Fig. 3.15 [34, 48, 54, 109 , 110–114].
Today, approximately 50 Mio tons alkalignin- and lignosulfonic acids are pro-
duced per year during pulp production, although only a very few percent of these
acids are used. Combustion combined with recycling of chemicals is still the most
efficient process (economically). Both sulfate and (increasingly) sulfite pulping
processes include combustion (in combination with recycling). The heat gener-
ated covers 80 to 100% of the energy demand of the pulping factories (except
bleaching processes). Although, there are several application areas for lignin
Fig. 3.15 Processes for isolation of lignin [109 a].

3.5 Lignin-based Product Lines 117
Fig. 3.16 Areas of application of different insulated and modified lignins [48].
and modified lignins; their market potentials would significantly increase in a bio-
based economy [34, 48, 50, 54, 110–118] (Fig. 3.16).
3.5.2
A Lignin-based Product Family Tree
Processes for substantial utilization of lignin can be divided into four groups:
· utilization in polymeric forms: e.g. as adhesives for wood materials, cement
additive for enhanced low-temperature durability and low-temperature resis-
tance etc.;
· utilization as polymer component: co-reactant for polymers and resins;
· fractionation into low-molecular-mass particles and monomers: e.g. genera-
tion of vanillin from softwood lignosulfonates; production of dimethyl sulfox-
ide (an important solvent); and
· complete degradation to gas, oil and coal by pyrolysis.
Figure 3.17 shows a potential chemical lignin-based product family tree. Alka-
line hydrolysis/oxidation is already technically established, e.g. vanillin produc-
tion; residues are used as dispersants. Alkaline demethylation via sulfide is used
for DMS production, which later could be used for DMSO solvent production
[50, 119]. Up to 33% phenolic substances can be obtained by addition of NaS
and NaOH at temperatures of 250 to 290 8C [120].
Fig. 3.17 A lignin-based product family tree [48].
An interesting recently developed process is the catalytic pulping of wood in

sodium hydroxide and water. By this method syringaldehyde, 3,4,5-trimethoxy-
benzaldehyde, vanillin, and levulinic acid are synthesized within one integrated
biorefinery regime [121, 122].
It is also possible to obtain well-defined products such as phenols and cresols
by catalytic hydrogenation/hydrocracking. Best known is the so-called Noguchi
process [123]. Reports of investigations in the US reveal production of up to
38% monophenols and 7–8% higher phenols [50]. This demonstrates the poten-
tial of lignin as raw material particularly for synthesis of cresols more efficiently
and less expensive than from coal or crude oil. Pyrolytic reactions and gasifica-
tion, respectively, are analogous for lignin and coal processing. A major part of
lignin hydrolysis and gasification is comparable with the corresponding wood-
processing steps. In addition, partial oxidation with oxygen can be performed to
produce syngas (here, it must be considered that this process might lead to
large amounts of ash). In very fast oxidation at 1200 8C with subsequent
quenching up to 23% w/w acetylene is obtained [124].
In addition, lignin has an important potential as a co-reactant for synthesis of
polymers and resins (phenolic compounds, furans, epoxides, urethanes, urea–
formaldehydes, and others). Although well-known, many new developments
and novel potential applications have been discussed for lignin-based polymers
and resins [114, 116, 118, 125].
3.6
Hemicellulose-based Product Lines
3.6.1
Isolation and Application Areas
The building blocks of hemicellulose (polyoses) are glucose, xylose, mannose,

galactose, arabinose, and rhamnose. Polymeric polyoses consist of a specific
combination of these building blocks. Hardwood and straw, for example, con-
tain more xylanes (pentosans, degradation to pentoses); soft wood contains
mainly glucomannanes [hexosans, degradation to hexoses] (see also Tables 3.5
and 3.7).
Industrial or technical supply of pure hemicellulose fractions is still a very
laborious process. Current wood pulping (sulfite pulping and corresponding
pre-hydrolyzates) generate fractions of hemicellulose that are still contaminated
with cellulose and/or lignin residues. Therefore, many applications are limited
to a so-called “low-tech” sector, such as energy regeneration, production of feed
yeast, adhesives for chipboards or fermentation to alcohol [50]. The latter is be-
coming increasingly important because improved processes for fermentation of
hemicellulose pentoses have been developed in which ethanol yields are 30%
higher than for conventional fermentation [104, 126].
Potential sources for almost pure hemicellulose fractions are:
· pre-hydrolysis (acid hydrolysis),
· precipitation component/solution component (Organosolv process, Batelle
process),
· steam-/pressure-processes in water,
· aqueous extraction at higher temperature.
To use the rich chemical and biotechnological potential of hemicelluloses, the

hydrolysis of hemicellulose to xylose (pentose) and mannose (hexose) is prefer-
entially used (Fig. 3.9). The resulting xylose, isolated and purified, could be used
as a building block for subsequent reactions, for example oxidation, reduction,
or esterification, to generate a wide array of technical products (Section 3.6.2,
Fig. 3.18).
3.6.2
A Hemicellulose-based Product Family Tree
3.6.2.1 Mannan/Mannose Product Lines

Figure 3.18 shows a technically feasible chemical/biotechnical hemicellulose-
based product family tree in which chemical reactions are preferred. In the fol-
lowing text the hexoses of polyoses mannane/glucomannane are discussed only
briefly because the LCF concepts are mainly based on utilization of pentose-rich
plants (annual plants, grass, straw, seed vessels, grain etc.). In addition, it must
be considered that chemical conversions of mannose always compete with those
Table 3.8 Potentially pure chemicals derived from glucoman-

nan [127].
Product Source Process Possible application
Mannose Softwood a) Acid hydrolysis Food products, chemicals

Mannitol Mannose b) Reduction Sweetening agents, carrier for
(Manna sugar, Mannit) medicine, resins, drying oils,
plasticizers, emulsifiers
Methyl mannoside Mannose b) Methanolysis Polyurethane, polyester,
polyethers
Sodium mannose Mannose b) Bisulfite addition Synthesis of medicinals,
bisulfide germicides, dyes, carrier for
sulfite reagents
Mannoheptonic acid Mannose b) Kiliani synthesis Additives in alkaline washing
compounds, detergent scale
inhibitors, additive to cement
a) Spent liquors from pulping softwood

b) Residues from softwoods or softwood pulping liquors
of glucose; however, glucose is available from lignocellulose (cellulose fraction)

in a more efficient way. Furthermore, the reaction of mannitol (C6 sugar alco-
hol) to arabitol (C5 sugar alcohol) is of growing interest. Production of arabitol
from sugar beet pulp via reduction of arabinose is, anyway, economically more
efficient. LCF-mannan will gain a special importance as a “hexose supplier” for
fermentation to ethanol or yeast products, because of waste liquor can be used
without mannose isolation. Table 3.8 shows some technically feasible conversion
processes for glucomannan [127].
If mannose could be produced from polyoses of lignocelluloses in an econom-
ically advantageous way, however [128], its importance would be focused on hy-
drogenation to the sugar alcohol mannitol and subsequent catalytic hydrogena-
tion via C–C bond cleavage to glycols and polyalcohols for polymer synthesis
(e.g. polyurethane) [129] or the use of mannitol derivatives as simple or even
chiral building blocks [130–132].
3.6.2.2 Xylan/Xylose Product Line

Figure 3.18 shows a product line preferentially focused on xylane. Isolation of
xylan (e.g. by precipitation from alkaline lignocellulose extract using alcohol)
and subsequent hydrolysis to xylose is currently not profitable, however. More
efficient is isolation of xylose from the hydrolyzate. So, different technical hy-
drolysis processes have been developed for production of xylose from lignocellu-
loses, e.g. acid processes with dilute sulfuric acid at higher temperatures [109 b,
133], alkaline processes and mixed alkaline–acid processes [134, 135], processes
based on ion exchange [136, 137], and the use of nitrogen oxides as pretreat-
ment agents [138, 139].
Fig. 3.18 A hemicellulose-based chemical product family tree.
Xylose (d-xylose) is the representative pentose of lignocelluloses. Pentoses are

reducing sugars, built up of five carbon atoms. Xylose is also the cheapest pen-
tose, readily accessible from wood- or straw-derived xylans. In 2004 approxi-
mately 25 000 tons of d-xylose were produced worldwide [128]. Xylose crystal-
lizes much more rapidly than glucose and forms crystals of uniform grain size.
The crystals can easily be separated from the mother liquid. Xylose can be used
as a sweetener in the form of crystalline powder or large crystals. Table 3.9 sum-
marizes the most important processes and applications of xylan and xylose (see
also Fig. 3.18).
The principal derivatives of xylose are xylitol and furfural. Xylitol is obtained by
catalytic-high pressure hydrogenation of xylose and was first synthesized in 1891 by
Fisher and Strobel by reduction of xylose using sodium amalgam. The reaction con-
ditions of the hydrogenation are the same as those for sorbitol manufacture (below).
Reduction catalysts are nickel fixed to different supports or Raney Nickel. Xylitol, as
sorbitol, is sweet and used for food sweetening and as a moisture-retaining agent in
cosmetics (e.g. in tooth pastes). Use of xylitol as a raw material in the chemical
industry is analogous to sorbitol. In the 1960s large amounts of xylitol produced
in the former USSR were used as starting material for production of vitamin C
[50 b, 127]; today, sorbitol is preferentially used for this process. Xylitol can also
be used as raw material for alkyd resins, surfactants, and plasticizers [109 b, 128].
Furfural is produced when three molecules of water are removed from xylose
under ring-closure. Furfural chemistry is discussed more in detail in Section 3.6.3.
Table 3.9 Potentially pure chemicals derived from xylan [140].
Product Source Process Possible application
d-Xylose Xylan Acid hydrolysis Secondary products, food

additives, detergents after
esterification, polyurethane
from methyl ether
Xylitol Xylose a, b) Reduction Sweetener, humectant, plastic
plasticizer
Xylonic acid, Xylose Oxidation of spent Binders, sequestering agents
tartaric acid, sulfite liquors
triox glutaric acid
Furfural Xylan c), Xylose Acidic dehydration Plastics, solvents, chemical
products
a) Wood and agricultural wastes

b) Spent pulping liquors
c) Vegetable materials
3.6.3
Furfural and Furfural-based Products
3.6.3.1 Furfural
Furfural (the structural formula is given in Fig. 3.18) is the oldest and most im-
portant substance industrially produced from hemicellulose [141, 142]. In 1998,
approximately 142,000 t a1 furfural were been produced worldwide, mainly in
China, the Dominican Republic, South Africa, and the USA [41]. Industrially
used raw materials are: corn cobs (23.4%), oat hulls (22.3%), cottonseed hull
bran (18.6%), cane trash (17.4%), and rice hulls (11.4%) – the numbers given
in parentheses are potential furfural yields [143]. Synthesis of furfural on the
basis of fossil raw materials (e.g. catalytic oxidation of 1,3-dienes) is economical-
ly not competitive. Because of the acidic hydrolysis, the biobased synthesis of
furfural can be combined with alcohol production based on lignocellulose. Fur-
fural is a liquid with a boiling point of 161.7 8C that is almost infinitely miscible
with all solvents except saturated aliphatic compounds. Freshly distilled, furfural
is a colorless, temperature-stable liquid with a very high durability under anae-
robic conditions. All technically established processes start with pentosan-con-
taining raw materials. The primary reaction is acidic hydrolysis (1). Kinetically,
this reaction depends on proton concentration and temperature [144]. The sec-
ond step is a dehydration of pentoses to furfural (2) (Fig. 3.19). Finally, furfural
is obtained by steam distillation, another time-determining reaction step [145].
The following processes are known for preparation of furfural; except for (9) and
(10) all are used industrially applied [147] (1–4, 6, 8, 12 [41]; 5, 7, 9, [50 c]; 10 [146,
147]; 11 [143]).
Fig. 3.19 Furfural formation starting with pentosan.
1. Quaker Oats batch process (dilute H2SO4, T 153 8C)

2. Chinese process (dilute H2SO4, T 160 8C)
3. Agrifurane process (1% H2SO4, rector cascade, 177 8C to 161 8C)
4. Continuous Quaker Oats process (dilute H2SO4)
5. Roni and Sebara process (dilute and conc. H2SO4)
6. Rosenlew process (catalytic hydrolysis with acetic and formic acid as cata-
lyst)
7. Duipopetrovski process (dilute HCl)
8. Escher Wyss process (cat. hydrolysis with H2SO4, acetic and formic acid)
9. Steug-Sevo process (steam 18 bar)
10. Supratherm process/Staketech process (high temperature, 200 8C)
11. Natta process (HCl, normal pressure)
12. Process based on spent sulfite
Fig. 3.20 Furfural-based Nylon process.

The main applications of furfural can be summarized as [41 128, 148]:

1. Starting material for the production of derivatives such as furfuryl alcohol,
furan, methylfuran, tetrahydrofuran, and furoic acid. Approximately 60% of
all furfural produced is used to make furfuryl alcohol. Even adiponitrile for
Nylon was produced from furfural from 1946 to 1961 (Fig. 3.20, see also
Fig. 3.21).
2. As an extractant in the refining of lubricating oils, diesel fuels, and vegetable
oils.
3. As a fungicide.
4. As a nematocide.
"
anhydride from furfural: oxidation [172].

Fig. 3.21 A furfural-based chemical product (17) maleic anhydride from furan: oxidation
family tree: (1) furfural from biomass and/or in gas phase [173]. (18) maleic acid from
hemicellulose: acid hydrolysis [40]. (2) furan furfural: cat oxidation, modif. V2O5-cat,
from furfural: (a) cat. decarbonylation, -CO, Ni/Al-Fe-Tubes [172]. (19) fumaric acid from
cat: Zn-, Fe(II)-, or Cr2O3/ZnO or KOH/ furfural: cat. oxidat. with NaClO3 in pres. of
NaOH [149–152]; (b) thermal decarbonyla- V2O5-cat. [174]. (20) furyliden acrolein from
tion with steam over Ca(OH)2 and slaked furfural: + acetaldehyde (cond.) [40]. (21)
lime at 350 8C, y: 90% [153]. (3) furoic acid furyliden ketone from furfural: aldol conden-
from furfural: (a) by Cannizarro reaction; (b) sation [175, 176]. (22) 2-furanacrylic acid
by oxidation with O2 [150, 154] (b) by oxida- from furfural: (a) + malonic acid in pres. of
tion with O2 [155, 156]. (4) furan from furoic pyridine (Perkin reaction) [177, 178];
acid: -CO2, decarboxylation [154]. (5) tetrahy- (b) + acetic anhydride, by 150 8C in the
drofuranyl alcohol from furfural: cat. vapor- presence of potassium acetate [179]. (23)
phase Hydrogenation, Ni-, or Ru(IV)-oxide, furanacrylonitrile from furfural: + cyanoacetic
or CuCrO3 cat. [157–159]. (6) furfuryl alcohol acid in pres. of ammonium acetate and
from furfural: red., Ni- and CuCrO-cat. [160, pyridine [180]. (24) 2-furyl-2-nitroethylene
161]. (7) methyl furan from furfural: from furfural: aldol cond./nitroacetic acid or
(a) cat. red., H2 + Ni-cat. (+ furfuryl alcohol) esters and base [109 c, 179, 181]. (25) tetra-
[162, 163] (b) cat. hydrogenation, CU-Cr-cat. hydrofuran (THF) from furan: cat. hydro-
[164]. (8) fufuryl amine from furfural: reduc- genation, cat: paladous oxide [182–185].
tive amination [165]. (9) 5-nitro furfural from (26) 1,4-butanediol from furan: cat. hydroge-
furfural: nitration with HNO3 in (CH3CO)2O nation by-product of THF-process [186].
[109 c, 166]. (10) tetrahydrofuranyl alcohol (27) thiofuran from furan: reaction of H2S in
from furfuryl alcohol: cat. hydrogenation presence of alumina by heating [187, 188].
[152, 167–169]. (11) levulinic acid from fur- (28) pyrrole from furan: reaction with NH3
furyl alcohol: by heating in hydrochloric over Al2O3-cat by heating [189, 190].
acidic ethyl methyl ketone, y: 90–93% [170]. (29) 1,4-butanediol from succinic acid:
(12) methyl furan from furfuryl alcohol: cat. reduction of succinic diethyl ester with so-
hydrogenation, Cu-Fe-cat., y: 80% [162]. (13) dium in ethanol [191]. (30) 1,4-butanediol
methyltetrahydrofuran (MTHF) from methyl- from THF: hydrogenation and hydration
furan: Cat. hydrogenation, cat: Raney Ni, [192 a, 193]. (31) tetrahydrofuran (THF) from
200 8C [161]. (14) methyltetrahydrofuran 1,4-butanediol: cat. dehydration [193, 194].
(MTHF) from tetrahydrofuranyl alcohol: cat. (32) succinic acid from THF: oxidation with
hydrogenation [109 c]. (15) succinic acid HNO3 or NO3 [195]. (33) maleic anhydride
from furfural: (a) electrolytic oxidation from succinic acid: dehydration [196]. (34)
(by product maleic acid) [109 c]; (b) cat. maleic
oxidation with O2 [109 c, 171]. (16) maleic
Legend Fig. 3.21 (continued) anhydride from (56) c-valerolactone from d-valerolactone:
THF: cat. airborne oxidation [196]. (35) isomerization, a) H2SO4-cat. in H2O [215],
maleic acid from THF: cat. airborne oxida- b) with J2 [216]. (57) d-cyanovaleric acid from
tion in gas phase [197]. (36) maleic acid d-valerolactone: NaCN, H2O [192 c, 215].
from maleic anhydride: [198]. (37) fumaric (58) e-caprolactam from d-cyanovaleric acid:
acid from maleic acid: (a) cis-trans isomer- hydrogenation, (+ 4H, – H2O) [192 c, 215].
ism by heating with HCl solution [199], (59) 1,5-dichloropentane from THP: acidoly-
(b) cis-trans isomerism with H2O2, thiourea, sis with 2 HCl [208]. (60) 1,5-dicyanopentane
and ammonium persulfate by 100 8C [198]. from 1,5-dichloropentane: + KCN in Metha-
(38) DL-malic acid from fumaric acid: cat. or nol, 130 8C [217]. (61) 1,7-diaminoheptane
thermal hydration [54, 200]. (39) DL-malic from 1,5-dicyanopentane: cat. hydrogenation,
acid from maleic acid: thermal hydration Ni-cat. [218]. (62) 1,5-dicyanopentane from
[201]. (40) DL-tartaric acid from maleic acid: 2-cyano-THP: acidolysis [192 b]. (63) pimelic
Oxidation with H2O2 or KClO4 [202]. (41) acid from 1,5-dicyanopentane: hydration
maleic acid from furoic acid: oxidation with [219, 220]. (64) butanol from THF: cat. or
ClO–2 (by-product 2-furancarboic acid) [203, acid hydration [221, 222 a]. (65) d-valerolac-
204]. (42) tetrahydrofuroic acid from furoic tone from THF: + CO, cat. carbonylation
acid: cat. Hydrogenation [150]. (43) 2,5-fur- [223, 224]. (66) c-valerolactone from THF:
andicarboxylic acid (FDCA) from furoic acid: cat. methylation [216, 225]. (67) 1,3-buta-
react. of potassium salt of furoic acid over diene from THF: -H2O, cat. Dehydration
CdI2-cat [205]. (44) 5-nitrofurancarboxylic [222 b]. (68) c-butyrolactone from THF:
acid from furoic acid: Nitration with HNO3 a) oxidation with O2 [223]; b) oxidation by
[109 c, 166]. (45) a-ketoglutaric acid from bromate and lactonization [226]. (69) c-valer-
furoic acid: oxidation under esterification olactone from c-butyrolactone: cat. oxidation
and ether forming, then oxid. ring open./ and methylation [227]. (70) pyrrolidone from
hydrolysis [109 c, 206]. (46) 1,2,5-trihydroxy- c-butyrolactone: reaction with NH3 [228].
butane from tetrahydrofuranyl alcohol: with (71) N-vinylpyrrolidone from pyrrolidone:
(CH3CO)2O/ZnCl2 to 1,2,5-triacetyl-butane N-alkylation [228]. (72) adipic acid from
and then KOH/CH3OH [192 a]. (47) 2,3-dihy- THF: 2 CO + 2 H2O, cat. (Ni(CO)4 and
dropyran (DHP) from tetrahydrofuranyl NiCl2 [229]. (73) 1,4-dichlorobutane from
alcohol: cat, (alumina-cont. cat.) dehydration THF: (a) with HCl and higher pressure
and ring expansion at 200–500 8C [152 b, [152 a, 222 c] (b) in presence of water re-
207]. (48) acrolein + ethylene from DHP: by moval agents H2SO4/ZnCl2/SOCl2 [230].
thermolysis [41, 192 b]. (49) 2-hydroxytetrahy- (74) poly(THF) from THF: polymeris. in
dropyran (R=H) and tetrahydropyran (THP) pres. of oxonium salts, chlorosulfonic acid,
ethers from DHP: ethers by addition of ROH or phosphor(V)-chloride [231]. (75) 4,4'-di-
[41, 208]. (50) 2-cyano-THP from DHP: addi- chlorodibutyl ether from THF: + HCl, cat.
tion of cyanide [208]. (51) tetrahydropyran [222 b]. (76) poly(THF) from 4,4'-dichlorodi-
(THP) from DHP: cat. hydrogenation [152 b, butyl ether: + KOH, 1,4-butanediol [222 b].
207]. (52) 1,5-pentanediol] from DHP: cat. (77) adiponitrile from 4,4-dichlorodibutyl
Hydrogenation in presence of Raney-Ni [209, ether: KCN and normal pressure, or NaCN
210]. (53) d-valerolactone from DHP: cat. and higher pressure [222 d, 232]. (78) 1,6-
Hydrogenation [41, 208]. (54) d-valerolactone diaminohexane from adiponitrile: hydrogena-
from 1,5-pentanediol: select. oxidation and tion under higher pressure over Ni- or Co-
lactonization [211]. (55) glutaric acid from cat. [222 d, 233]. (79) adipic acid from adipo-
d-valerolactone: cat. oxidation [212–214]. nitrile: + 4 H2O, hydrolysis [234].
3.6.3.2 A Furfural-based Family Tree

Furfural is a very interesting chemical compound. Fig. 3.21 shows a furfural-
based family tree with technically important products. Some are commercial
products, e.g. furfuryl alcohol, others, for example furan, tetrahydrofuran, adipo-
nitrile, were of economic interest until they were replaced by fossil-based prod-
ucts. Products such as methylfuran and methyltetrahydrofuran will gain in im-
portance in the future, for example as fuel additives. In general, in a biobased
economy, all products of a furfural family tree will gain more importance be-
cause of improved economy and a modified market demand.
3.7
Cellulose-based Product Lines
3.7.1
Isolation, Fractionation and Application Areas
Cellulose is by far the most frequent organic compound. Cotton and other cellu-
lose-rich fibers (ramie, flax, cannabis) release their cellulose rather easily; in cel-
lulose production from lignocellulose (wood, reed, straw, cane strash, cornstalks,
or sunflower stalks, and others) special pulping processes must be performed to
separate lignin and other polyoses and to obtain cellulose of uniform molecular
weight. The most important chemical pulping processes are discussed in Sec-
tion 3.4.3 (see also Fig. 3.13), the generated cellulose is called pulp. The corre-
sponding pulping process can often be identified by the prefix of the pulp, e.g.
sulfite-pulp, sulfate-pulp, sodium hydroxide-pulp etc.
Cellulose is one of the most important raw materials for a variety of branches of
industry. By far the largest amounts of cellulose are used in the paper and textile
industries. In addition, cellulose is used in medicine and pharmacy. Cellulose is
also a raw material for many plastics, fibers, rayon (viscose silk), vulcanized fiber,
and cellophane. Further cellulose derivatives are cellulose wadding (batting), gun
cotton, cellulose-based varnishes, and adsorbent materials for chromatographic
use and other applications. Several cellulose derivatives are used in ion-exchange
chromatography. Microcrystalline cellulose and cellulose powder are used as fillers
or binders in tablets, as sedimentation delayers in tooth pastes and creams, in ci-
garettes as tobacco substitutes, and as filter materials, emulsifiers, dispersants, and
filtration additives in the food industry, etc. Another application is bacterial trans-
formation of cellulose into so-called single-cell protein (SCP) [54].
Use of cellulose while maintaining its original structure is discussed in detail
in Ref. [48]. In the following text “cellulose chemistry” describes the chemistry
of the corresponding cellulose conversion products, e.g. glucose, the major hex-
ose of lignocellulose sugars, and its derivatives, for example fructose, polyalco-
hol, methylglucoside, hydroxymethylfurfural (HMF), and levulinic acid.
When using cellulose prepared from lignocellulose feedstocks for conversion
processes, it should always be checked in advance if the pure isolated and thus ex-
pensive cellulose pulp is necessary as raw material for further conversion, or if it is

possible to convert the desired hexoses by appropriate processes using the ligno-
cellulose itself (instead of isolated cellulose pulp). Thus, occasionally, e.g. ethanol
production, even hexoses from hemicellulose can be used. Looking for appropriate
conversion methods, it is also reasonable to consider biorefinery principles [45].
3.7.2
Cellulose-based Key Chemicals
3.7.2.1 Glucose
Glucose, a hexose (d-glucose, dextrose, grape-sugar, IUPAC/IUB abbreviation d-
Glc, acyclic aldose, monosaccharide) is the most important cellulose derivative
of lignocellulose. The importance of glucose is primarily a result of the fre-
quency of the molecule (Section 3.3.1). Glucose also plays an outstanding role
as so-called “intermediate platform” or “key chemical” within the concept of bio-
based products [75]. For biotechnical conversion, in particular, glucose is of spe-
cial significance [128].
d-Glucose or dextrose occurs as a pure crystalline material; for medical appli-
cations, glucose is available as 5 to 50% aqueous solution. Most glucose is cur-
rently produced by hydrolysis of starch [235]. Most is used as glucose syrup for
candy production; for this application an increasing amount of enzymatically
isomerized glucose syrup, so-called “isosyrup”, is used. In the chemical industry,
large amounts of glucose are used for chemical synthesis, e.g. of sorbit, glucon-
ic acid, ascorbinic acid, glutaminic acid, and glutamates or methylglycoside and
for technical application, for example fermentation into ethanol, acetic acid and
lactic acid [236]. In recent years production of d-glucose and glucose syrups, re-
spectively, starting from cellulose and lignocellulose, has attrached increasing
interest, especially ethanol production from lignocellulose [90 d]. Industrial
chemical processes of lignocellulose saccharification are mainly based on acid
hydrolysis in so-called wood saccharification by the Bergius (Bergius-Rheinau
process) [237] and Scholler (Scholler-Tornisch process) [238, 239], processes that
were industrially used between 1930 and 1960 [240]. After World War II crystal-
line glucose was also successfully manufactured in the so-called New Rheinau
process [241].
In recent times, enzymatic methods have been increasingly used for extrac-
tion of cellulose degradation products, e.g. saccharification of cellulose (see also
Fig. 3.14). Although this degradation works successfully when applied to pure
cellulose, it is still a problem to use lignocelluloses, particularly non-uniform
lignocelluloses. Here, pretreatment and prepulping of lignocellulose are neces-
sary to end up with economically efficient saccharification. Nevertheless, the
first success has been obtained in enzymatic saccharification [90 e, 91].
Fig. 3.22 shows a chemical industrial cellulose-derived product family tree
based on glucose as so-called “intermediate platform”. This does not mean, how-
ever, that crystalline cellulose or cellulose syrup is necessary as starting material
for all the processes and product lines listed.
Fig. 3.22 Chemically industrial cellulose-based product family tree.
Remarkable recent publications have described glucose oxidation via heteroge-

neous catalysis, enzymes, or fermentation to products such as glucosone, 6-alde-
hyde-d-glucose, gluconic acid, glucuronic acid, glucaric acid, 2-keto-d-gluconic
acid, 5-keto-d-gluconic acid, 2,5-di-keto-gluconic acid, l-ascorbic acid (vitamin C,
by fermentation), and Koji acid [242].
3.7.2.2 Sorbitol
d-Sorbitol (d-glucitol according to IUPAC/IUB; glucit, C6H14O6) is a crystalline su-
gar with approximately 50% of the sweetening efficiency of saccharose. d-Sorbitol
belongs to the hexite group; it is a hexa-alcohol (sugar alcohol, polyalcohol, polyol)
which can intramolecularly split off one or two molecules water and can form cyclic
ethers (e.g. sorbitan and sorbit) (Fig. 3.23) [243]. Sorbitol is obtained when a glucose
Fig. 3.23 Synthesis of cyclic ether based on sorbitol and glycose [243].
solution, e.g. the hydrolysis solution is hydrogenated. Manufacture of sorbitol from

glucose has already been industrialized and the technique is firmly established.
The first technical production of d-sorbitol was electrolytic reduction of glu-
cose solution in caustic soda and sodium sulfate as electrolyte, with amalga-
mated lead as the cathode and an anode chamber filled with dilute sulfuric acid
[244, 245]. Today, catalytic hydrogenation, especially high-pressure hydrogena-
tion (Raney Ni catalyst, hydrogen pressure 100–150 atm, temperature 100–
150 8C) of glucose or starch and saccharose is preferred [54].
The chemical relationship of sorbitol and glycerin with the glycols on the one
hand side and the carbohydrates on the other is decisive for its multi-purpose
application. Sorbitol is widely used as an additive for paper, fiber, tobacco, cos-
metics, leather, and pharmaceuticals, because it is suitably hygroscopic and
maintains a fixed moisture content despite changes in atmospheric humidity.
Crystalline sorbitol can be used as a sweetener in the form of a powder or as
large crystals (rock sugar), and as dietary sugar.
Sorbitol is also widely used as a raw material in the chemical industry. d-Sor-
bitol is used commercially as a raw material for vitamin C (l-ascorbic acid) pro-
duction and was used as raw material for explosives during the war. Its most
important current application is its use as a raw material for alkyd resins and
surfactants [245–247] (Fig. 3.22).
3.7.2.3 Glucosides
Glucosides are the glycosides of glucose. “Glycosides” is a collective term for an
extensive group of plant substances and synthetic compounds that – by boiling
in water or dilute acids or by treatment with glycosidases – can be fractionated
into one or more carbohydrates (mono- or oligosaccharides) or into one or more
aglycones (or non-sugars). The chemistry and biochemistry of glycosides is
therefore very extensive [248]. In the following text glucosides are discussed as
compounds in which the reducing group of sugar is condensed with an alcohol
or phenol to form acetals. Among different types of glucosides the methygluco-
sides (formula in Fig. 3.24) are commercially important. A simple methylgluco-
Fig. 3.24 Synthesis of methylglucoside.
side is synthesized by treatment of glucose with methanol in presence of 1%

hydrochloric acid as catalyst (Fischer reaction; Fig. 24).
A variety of acids have been proposed as catalyst [249–252], but cation ex-
change resins of the sulfate type are most commonly used. The yield is 88% of
the theoretical value [253]. Methylglucoside is used to manufacture alkyd resins.
The alkyd resin obtained by esterification of linseed oil and soybean oil has ex-
cellent properties. Drying oil obtained from fatty acid and methylglucoside
forms a strong membrane, with satisfactory waterproof and adhesive properties,
and is used as raw material for paints [109 c]. Application is also being devel-
oped in the surfactant field [249, 250].
3.7.2.4 Fructose
d-Fructose (levulose, fruit sugar) is the sweetest of the sugars. On a relative scale
with sucrose taken as 100 the sweetness of fructose is 173. The molecular weight
and the elemental composition of glucose and fructose are exactly the same. The
only difference is that glucose is of the aldehyde type whereas fructose is of the
keto type (ketohexoses). The most important natural sources are saccharose and
inulin. A variety of methods have been proposed for converting glucose into fruc-
tose by isomerization. In general, these can be divided into chemical and enzy-
matic methods. Until 1970 chemical isomerization in alkaline solution with alka-
line catalysts such as ammonia, slaked lime, lime water, sodium carbonate, potas-
sium carbonate, or caustic soda via d-glucose-1,2-enediol was preferred [254–256];
today enzymatic and mixed enzymatic/chemical catalysis methods are of advan-
tage. Under optimum conditions the enzyme glucose-2-oxidase converts > 99%
of the glucose into glucosone, which can subsequently be transformed into d-fruc-
tose by use of a metal catalyst, e.g. Pd or Raney Ni, and hydrogen at elevated tem-
perature and pressure, or by homogenous NaBH4 reduction [242, 257] (Fig. 3.25).
d-Fructose is used as sugar substitute in the pharmaceutical and dietetics in-
dustries, particularly in cases of diabetes. For the biobased chemical industry,
fructose is an outstanding starting material for manufacture of 5-hydroxy-
methylfurfural (HMF) and levulinic acid [175]. Nevertheless, both these impor-
tant building blocks are also available from non-specific hexoses (Section 3.7.2).
Utilization of natural fructose raw material, for example inulin (polyfructosan)
from special cultures such as artichoke and topinambur (Jerusalem artichoke)
or saccharose (sucrose) from sugar cane and sugar beet, and utilization of semi-
Fig. 3.25 Synthesis of d-fructose from d-glucose [242, 257].
synthetic d-fructose for HMF and levulinic acid, will both be exposed to signifi-
cant economic pressure.
3.7.2.5 Ethanol
The significance of ethanol as an important bulk product from lignocellulose con-
version and from the different processes of utilization of lignocellulose-based
hexoses and pentoses has already been discussed. In this section the importance
of ethanol among biobased products as a C2 building-block chemical is reviewed.
Most currently produced ethanol is used in beverage industry. Technically, ethanol
is a valuable solvent for fats, oils, and resins, particularly in lacquer and varnish
manufacture and also for production of essences. Ethanol is the most important
solvent for scents and perfumes and cosmetics (aftershaves, hair tonic, etc.). Be-
cause of its germicidal effect, ethanol is used as a preservative and a disinfectant.
Fig. 3.26 An ethanol-based chemical product family tree.

As substrate for protein generation (SCP), ethanol can replace petroleum. Because
of its high calorific value (29 kJ g–1 or 7 kcal g–1), ethanol in the form of denatured
alcohol or so-called solid alcohol is used for combustion or (mixed with gasoline)
as a fuel (gasohol, E10- or E85-biofuel etc.).
Use of ethanol as raw material for synthesis of important industrial chemicals
is most promising for those countries that decided to use ethanol for fuel pro-
duction. Changing the raw material basis from cost-intensive agricultural prod-
ucts (e.g. grain, corn) to lignocelluloses (e.g. straw) and introducing energy-sav-
ing biotechnological processing steps could further improve the competitiveness
of bio-based versus fossil-based ethanol. Ethanol is the starting material for
many chemicals, for example diethyl ether, chloroform, ethyl chloride, dyes, and
pharmaceutical compounds [258]. Fig. 3.26 shows an industrial chemical prod-
uct family tree. The product lines for ethyl lactate and ethylene, and the corre-
sponding derivatives are of particular interest in chemical industry [259].
3.7.2.6 Hydroxymethylfurfural
Two very important biobased building blocks, 5-hydroxymethylfurfural (HMF)
and levulinic acid, are available via acid-catalyzed dehydration of hexoses. For-
mally, elimination of one molecule of water leads to a mixture of levulinic acid
and formic acid. It is assumed that during degradation HMF is formed as an
intermediate which is not stable in acids and degrades into levulinic and formic
acids. This means that three molecules water are eliminated to form HMF,
which then reacts with two molecules of water in a kind of “re-hydration” to
form levulinic acid and formic acid [260, 261] (Fig. 3.3). To achieve high yields
in the isolation of HMF, it is essential to suppress HMF hydrolysis, which is
most successful in non-aqueous systems [20].
5-Hydroxymethylfurfural (HMF) is a versatile sugar derivative which can be
regarded as a key intermediate between bio-based carbohydrate chemistry and
mineral oil-based industrial organic chemistry. HMF is a common dehydration
product of all hexoses and thus is an aldehyde, an alcohol, an aromatic com-
pound, a cis diene, and a difunctional diene. HMF is available from biomass
in a simple dehydration reaction and is convertible into di- and tetrahydro-
furan derivatives and also benzene, pyridazine, pyridine, and other derivatives
[262. 263]. Some possible reactions and applications are shown in Figs. 3.27 and
3.29.
Starting with HMF, furan-2,5-dicarboxylic acid (FDCA) is available in an one-
step reaction [264, 265]. Terephthalic acid or isophthalic acid are bulk products
which could be replaced by furan-2,5-dicarboxylic acid (FDCA) (Fig. 3.27). Cur-
rently, fossil-based terephthalic acid is the most important dicarbonic acid, pro-
duced on the million ton scale and mainly used for polyester production, e.g.
poly(ethylene terephthalate) (PET) and polycondensation products of terephthal-
ic acid (TPA) and ethylene glycol (EG) for packaging, beverage bottles, foil, etc.
[266]. Polymers, especially polyesters and polyamides have been prepared using
FDCA and other HMF derivatives instead of terephthalic acid [267, 268].
Fig. 3.27 Comparison of the syntheses of TPA and FDCA [269].
HMF can be produced by three different acid catalyzed dehydration processes

in which the ketohexose fructose is mainly used as starting material [262, 269,
270]:
1. reaction in aqueous solution with H2SO4 as catalyst, a simple process, but
HMF yields are low (50–60%) (Fig. 3.27);
2. elimination of water in DMSO as solvent leads to high HMF yields, but sub-
sequent isolation of HMF is rather difficult; and
3. elimination of water in multi-phase reaction mixtures, thereby continuously
collecting the generated HMF by use of an appropriate solvent.

Levulinic acid, or 4-oxopentanoic acid (m.p. 33.5 8C, b.p. 245 8C), is the simplest
member of the comparatively rare class of organic compounds known as c-keto
acids. Having both a ketone carbonyl group and an acidic carboxyl group it
reacts as a ketone and as a fatty acid. The chemical structure of levulinic acid
can be represented as CH3COCH2CH2COOH [271]. Levulinic acid is formed by
acid hydrolysis of hexoses and hexosic materials (Fig. 3.3, Section 3.7.2).
Levulinic acid is easily converted into chemical derivatives [80, 81, 272]
(Fig. 3.29) and, because of its high stability, can be used as a liquid fuel ex-
tender [273]. Fig. 3.28 gives a summary of possible applications.
Levulinic acid is a starting product for preparation of organic chemicals, dye-
stuffs, polymers, pharmaceutically active compounds, and flavor substances. Le-
vulinic acid is also an inhibitor of chlorophyll synthesis. If levulinic acid is used
in food products, rigorous purity, color, and stability requirements must be met.
Esters of levulinic acid are known to be useful as plasticizers and solvents,
and have been suggested as fuel additives. Levulinic acid is useful as a solvent,
as a food-flavoring agent, and as a starting material for preparation of a variety
of industrial and pharmaceutical compounds such as diphenolic acid (useful as
a component of protective and decorative finishes) and calcium levulinate (a par-
ticularly suitable form of calcium for intravenous injection used for calcium re-
plenishment and to treat hypocalcemic states). Levulinic acid is also useful for
preparing a glass-like synthetic resin, as a constituent of hydraulic brake fluids,
Fig. 3.28 Levulinic acid – overview of production and application.
and in the manufacture of nylon and rubber. Use of the sodium salt of levulinic
acid as a replacement for ethylene glycols as an antifreeze has also been pro-
posed [81].
3.7.3
An HMF and Levulinic Acid-based Family Tree
HMF and levulinic acid both are multifunctional compounds with a very broad
reaction and application potential. Figure 3.29 shows a chemical family tree
with HMF- and levulinic-based products that already have or might achieve
technical importance. Today, some of these products are as economically effi-
cient as levulinic acid itself, others were of economic significance years ago and
were then replaced by fossil-based products, e.g. several hydroxymethylfuranyl
derivatives. Several others could gain importance in the future, for example 2,5-
furandicarboxylic acid (FDCA) as a monomer for polymer synthesis and methyl-
tetrahydrofuran (MTHF) as a fuel additive. In a biobased economy, market de-
mand and economic efficiency of all the products discussed will develop ex-
tremely positively.
Fig. 3.29 An HMF and levulinic acid-based acetic acid: electrochemical oxidation at a
chemical product family tree: (1) 5-hydroxy- Ni-oxide-hydroxide anode [298]. (24) 2,5-
methylfurfural (HMF) from hexosic material: bis(hydroxymethyl)tetrahydrofuran from
acid hydrolysis [20, 274]. (2) levulinic acid HMF: cat. hydrogenation, Raney Ni, 90%
(LEVA) direct from biomass or over HMF: [283, 290, 292]. (25) 1,2,5-trihydroxyhexane
acid hydrolysis [81, 272, 275, 276]. (3) levu- from HMF: cat. hydrogenation, cat: Ru/C,
linic acid (LEVA) from HMF: cleavage in 96% [290]. (26) 1,2,5-trihydroxyhex-3-ene
acidic medium [277]. (4) levulinate esters from HMF: cat. hydrogenation, cat: Pt or Ru
from cellulose: acid cat. + alcohol, + higher [290]. (27) 2,5-bis(hydroxymethyl)tetrahydro-
temp. [278]. (5) levulinate esters from HMF: furan from FDCA: cat. reduction, cat: Raney-
acid. cat. + ROH [278]. (6) levulinate esters Ni [292]. (28) 2,5-bis(hydroxymethyl)-
from LEVA: + ROH [279, 280]. (7) aldaric tetrahydrofuran from BHMF: cat. hydrogena-
acids from hexoses: oxidation of glucose (or tion, cat: Ru/C, T. neutral med. [283, 290].
hexoses): (a) acid oxid. HNO3; (b) cat. oxid. (29) 2,5-bis(aminomethyl) tetrahydrofuran
O2, Pt on C; (c) biotechn. by Aspergillus niger from FDCA: cat. hydrogen, NH3 [290]. (30)
[242]. (8) 2,5-furandicarboxylic acid (FDCA) succinic acid from FDCA: (a) Potassium Salt
from aldaric acids: (a) with dehydrating of FDCA and Brom to dibromosuccinic acid
agents, e.g. HBr [281, 282]; (b) by cyclodehy- and then hydrogenation [299 a]; (b) FDCA
dration of mucic acid with p-TsOH at 140 8C and Brom in heat water to fumaric acid and
[283]; (c) esters, from d-glucaric acid, alcs. then hydrogenation [299 b]. (31) succinic
and acids by using microwave radiation acid from LEVA: (a) cat. oxidation, O2/V2O5
[284]. (9) 2,5-furandicarboxylic acid (FDCA) [272, 300]; (b) H2O2 on Cu-cat. [81].
from hydroxymethyl furoic acid: by cat. oxyd. (32) 2-oxoglutaric acid from LEVA: cat. oxi-
with charcoal-on-Pt-cat. [285]. (10) 2,5-furan- dation (Riley reaction) SeO2 [272, 301].
dicarboxylic acid (FDCA) from HMF: cat. (33) 5-methyl-2-pyrrolidone deriv. from LEVA:
oxydat. different cat. methods [286–289]. + R-NH2, reductive amination over Co-,
(11) 2,5-bis(hydroxymethyl)furan (BHMF) Raney Ni, Pt or Pd-catalysts [302–305].
from HMF: (a) by cat. hydrogenation [290]; (34) 5-furfurylidenelevulinic acid from LEVA:
(b) by Cannizzaro [291]. (12) 2,5-bis(hydroxy- + furfural [306–308]. (35) dilevulinic acid
methyl)furan (BHMF) from FDCA: cat. hy- from 5-furfurylidenelevulinic acid: acids treat-
drogenation [292]. (13) 2,5-furandicarbalde- ment [272, 309]. (36) sebacic acid from dile-
hyde (FDC) from HMF: cat. oxyd. BaMnO4, vulinic acid: cat. hydrogenation, H2/Ni [272,
93% [289, 293]. (14) 2,5-furandicarbaldehyde 309]. (37) 4,4-diaryl subst. valeric acids (di-
(FDC) from BHMF: cat. hydrogenation [292]. phenolic acid) from LEVA: acid-cat. conden-
(15) 2,5-furandicarbaldehyde (FDC) from sation with phenols or naphthols [310–312].
FDCA: cat. hydrogenation [292]. (16) 2,5-fur- (38) 1-keto-non-6-en from LEVA, + hex-3-
andicarboxylic acid (FDCA) from FDC: cat. enoic acid [313]. (39) acrylic acid from LEVA:
oxid. AgO2, 80% [289]. (17) 5-hydroxymethyl- condensat. with aldehydes and ketone split-
furoic acid from HMF: (a) cat. with Ag2O, ting [80]. (40) b-acetylacrylic acid from LEVA-
100 8C, 75% [289] ; (b) by Cannizzaro [291]. esters: oxidation of levulinic acid esters with
(18) methyl malonate from HMF: electro-oxi- SeO2 [314]. (41) a-angelica lactone from
dation on platinum anode in methanol, LEVA, cat. dehydration, cat e.g. phosphoric
LiClO4 electrolyte [294]. (19) bis(5-methylfur- acid, [81, 315]. (42) b-angelica lactone from
furyl)ether from HMF: TsOH, 89% [295, a-angelica lactone: base cat. (cat. e.g. tert.
296]. (20) 2-aminomethyl-5-hydroxymethylfur- Amine) isomerization [81]. (43) c-valerolac-
an from HMF: reductive amination, Ni/H2, tone from a-angelica lactone: reduction
NH3, 72% [297]. (21) 2,5-bis(aminomethyl)- [316]. (44) c-subst.-b-acetyl-c-butyrolactones
furan from HMF: + NH2OH, then Ni/H2 from a-angelica lactone: rect. with aldehydes
[289]. (22) 5-hydroxymethyl-furylideneacetic in presence of BF3-O(C2H5) [317].
acid from HMF: + malonic acid, cat. react in (45) 4-hydroxypentanoic acid from LEVA: cat.
pyridine [298]. (23) 5-carboxy-2-furylideneace- hydrogenation or reduction with diluted HCl
tic acid from 5-hydroxmethyl-furylidene- [318].
Legend 3.29 (continued) (46) c-valerolac- tenol (oxymethyl thiophene) from LEVA,
tone from hydroxypentanoic acid: acidic de- heating with P4S10 [80, 324]. (56) [5-methyl-
hydration [318, 319]. (47) c-valerolactone 5-fluoro-c-butyrolactone] from LEVA, fluori-
from LEVA: (a) cat. hydrogenation, cat: Ra- nating with SF4 (over-5-hydroxy-c-valerolac-
ney-Ni on Pt [273, 320]; (b) Platinum-Group tone) [326]. (57) dihydropyridazinones from
metal catalyst (e.g. Rh/C) in the presence of LEVA: + hydrazine or derivatives [327, 328].
hydrogen [321]. (48) 1,4-pentanediol from (58) pyridazinones from dihydropyridazi-
c-valerolactone: cat. hydrogenation [316]. nones: oxidation with Br2 or SeO2 [327, 328].
(49) 1,4-pentanediol from LEVA: cat. hydro- (59) dihydropyridazinone-3-carboxylic acid
genation, cat: Raney-Ni on Pt [320]. from dihydropyridazinones: oxid. w. 10%
(50) 2-methyl-THF (MTHF) from 1,4-penta- HNO3 [329]. (60) glutamic acid from dihy-
nediol: dehydration [316]. (51) 1,4-penta- dropyridazinone-3-carboxylic acid: cat. hydro-
diene from 1,4-pentanediol: cat. dehydration genation, H2/Ni [329]. (61) brominating le-
[322]. (52) 2-methyl-THF (MTHF) from vulinic acids from LEVA: + Br2 in different
LEVA: cat. hydrogenation, 240 8C, 100 atm solv. [272, 330]. (62) d-aminolevulinic acid,
(ca. 101 bar) [47, 316, 323]. (53) 5-cyano-4- (5-aminolevulinic acid) from LEVA or LEVA-
methylpent-4-enoic acid from LEVA: Knoeve- esters: (a) regioselect. Bromination to 5-bro-
nagel condensation with cyanoacetic acid molevulinic acid and then react. w. alkali
[324]. (54) 3-methyladipic acid from 5-cyano- metal diformylamide [331]; (b) fermentation
4-methylpent-4-enoic acid: reduction of dou- by photosynthetic bacterium (Rhodobacter
ble bond and acid hydrolysis [324]. (55) thio- sphaeroides IFO 12203) [332].
3.8
In a biobased economy, lignocelluloses will most probably be the main source

of raw materials.
First, there are a variety of sources of lignocellulose (e.g. wood, straw, reeds,
grass, etc.) and lignocellulose is the abundant continental biomass. There are
also other sources, for example cellulose-containing waste materials from public
life (recovered paper, hospital waste, municipal waste etc.) and industrial waste
products (e.g. pulp and paper industry).
Second, lignocelluloses, with their main components cellulose, hemicellulose,
and lignin, contain organic structures that serve as source for a variety of deriva-
tives and conversion products. There are almost inexhaustible possibilities in
chemistry and biotechnology to use lignocellulose and corresponding deriva-
tives. In addition, industrially established processes and products have already
been developed in the past (e.g. saccharification, furfural-based nylon produc-
tion); unfortunately they could not compete with extremely inexpensive petro-
leum. Those processes and corresponding experience can be used for further
development.
Third, lignocelluloses are, to a large extent, independent of economic policy
(in contrast with agricultural products such as corn, grain, sugar beet, availabil-
ity of hemicellulose raw materials is not state-controlled); this, together with
their reasonable raw material prices, makes lignocelluloses very interesting for
industrial use. (Prices for lignocellulose corn stover or straw are approximately
30% of those of corn and grain.)
References 139
Fourth, lignocelluloses can be produced even in environmentally sound less

intensive agriculture and forestry which is another positive effect, in addition to
the general CO2-neutrality of biomass.
The main requirement for economic success of lignocelluloses, its technolo-
gies and its products seems to be an integrated approach of lignocellulose pro-
cessing and utilization. By analogy with the extremely successful petrochemis-
try, it is absolutely essential to improve biorefinery technologies and to develop
sustainable and marketable product lines, multiproduct systems, and competi-
tive biobased products.
References
1 Agricola, G.; De natura fossilium libri 12 Braconnot, H.; Annales de chimie et de

X, Basel 1546 [Reprint: Dover Publ.; physique (Ann. chim. phis/A. ch.), 52
Mineola, N.Y., 2004, ISBN 0-486-49591- (1833) 290
4] 13 Kirchhoff, G. S. C.; Schweigers Journal
2 Hufbauer, K. The Formation of the für Chemie und Physik, 4 (1812) 108
German Chemical Community (1720– 14 Prout, W.; On the ultimate composition
1795); [University of California Press, of simple alimentary substances. Philo-
1982] p 190–191 sophical Transaction (Phil. T.), (1827) 355
3 Graebe, C.; History of Organic Chemis- 15 Schmidt, C.; Annalen der Chemie (A.),
try. Bd 1 [Verlag Julius Springer, Ber- 51 (1844) 30
lin, 1920, germ.] a) 28, b) 122 f 16 Payen, A.; Comptes rendus de l’Académie
4 Pötsch, W.R; Lexicon of famous Chem- des Sciences (C.r.), 8 (1939) 51
ists. [Bibliographisches Institut, Leip- 17 Walden, P.; Geschichte der Organi-
zig, 1988, ISBN 3-323-00185-0, germ.] schen Chemie seit 1880. Bd 2 [Graebe,
a) 305 C. (Ed.), Verlag Julius Springer, Berlin,
5 Dictionary of Scientific Biography 1941] a) 656, b) 686, c) 690
[Charles Scribner’s Sons, 1970–1990] 18 Beneke, K.; Anselme Payen (1795–
6 Ohara, H.; Biorefinery. Appl. Microb. 1871) In: Beiträge zur Geschichte der
Biotechnol. (AMB), 62 (2003) 474–477 Kolloidwissenschaften [VII. Mitteilun-
7 Kamm, B.; Kamm, M.; Principles of gen der Kolloid-Gesellschaft, 1998] 18–
Biorefineries. Appl. Microbiol, Biotech- 20
nol., (AMB), 64 (2004) 137–145 19 Mulder, G. J.; J. Prakt. Chem., 21 (1840)
8 Werpy, T., Petersen, G. (eds.); Top Val- 219
ue Added Chemicals from biomass 20 Kuster, B. F. M.; 5-Hydroxymethylfur-
(ed) [U.S. Department of Energy, Office fural (HMF). A Review Focussing on its
of scientific and technical information, Manufacture. Starch, 42 (1990) 314–321
2004, No.: DOE/GO-102004-1992, 21 Boeck, G.; Franz Ferdinand Schulze.
www.osti.gov/bridge] In: Liebigs Spuren in Mecklenburg.
9 Braconnot, H.; Annales de chimie et de Fachgruppe Geschichte der Chemie
physique (Ann. chim. phis/A. ch.), 12 Nr. 17 [Gesellschaft Deutscher Chemi-
(1819) 172 ker (ed.), Frankfurt am Main, 2004]
10 Crosland, M. P.; Gay-Lussac: Scientist 22 Hägglund, E.; Chemistry of Wood.
and Bourgeois. [Cambridge University [Academic press Inc., New York, 1952]
Press, Cambridge, England, 1978] 30 ff 23 Schulze, F. F.; Beiträge zur Kenntnis
11 Partington, J. R.; A Short History of des Lignin. Chem. Zent., NF2 (1857)
Chemistry [Dover, N.Y., 1989] 226 321–325
24 Brunow, G.; Methods to Reveal the 41 McKillip, W. J.; Furan and Derivatives.
Structure of Lignin. In: Biopolymers In: Ullmann’s Encyclopedia of Indus-
Vol. 1 [M. Hofrichter, A. Steinbüchel trial Chemistry. 6th Ed., Vol. 15 [Wiley-
(eds.), Wiley–VCH, Weinheim, New VCH, Weinheim, 2003] 187–206
York et al, 2001, ISBN 3-527-30220-4] 42 Cross, C. F., Bevan, E. J., Beadle, C.
90 (Viscose Syndicate); British Patent
25 Bente F.; Über die Konstitution des 8,700, (1892/1893)
Tannen- und Pappelholzes. Chem. Ber., 43 Bevan, J. E; Cross, C. F.; Cellulose (an
8 (1868) 476–479 Outline of the Chemistry of the Struc-
26 Klason, P.; Ber. dt. chem. Ges. (B.), 53 tural Elements of Plants), 2. Ed. [Lon-
(1920) 1864 don, 1903]
27 Klason, P.; Berichte der Deutschen Che- 44 Katzen, R.; Othmer, D. F.; Ind. Eng.
mischen Gesellschaft (B.), 69 (1936) 676 Chem., 34 (1942) 314.
28 Fuchs, W.; Berichte der Deutschen Che- 45 Kamm, B.; Kamm, M.; Gruber, P. R.;
mischen Gesellschaft (B.), 54 (1921) 484 Kromus, S.: Biorefinery Systems – An
29 Freudenberg, K.; Zur Konstitution des Overview, In this book, 2005
Lignins. II. Mittl., B., 60 (1927) 581 46 Katzen, R.; Schell, D. J.; Lignocellulosic
30 Freudenberg, K.; Polysaccharides and Feedstock Biorefinery: History and
Lignin Annual Rev. Biochem., 8 (1943) Plant Development for Biomass Hydro-
81 lysis. In this book, 2005
31 Freudenberg, K.; Neish, A. C. (eds.); 47 Hayes, Daniel, J.; Fitzpatrick, Steve;
Constitution and Biosynthesis of Lig- Hayes, M. H. B.; Ross, J. R. H.; The Bio-
nin [Springer, Berlin, 1968] fine Process: Production of Levulinic
32 Freudenberg, K.; Ploetz, Th., Dumpert, Acid, Furfural and Formic Acid from
G.; Stufenweise Hydrolyse von Fichten- Lignocellulosic Feedstocks. In this
holz. Cellulosechem., 19 (1941) 89. book, 2005
33 Freudenberg, K.; Lautsch, W.; Engler, 48 Fengel, D.; Wegener, G.; Wood, Chem-
K.; Zur Konstitution des Lignins. istry, Ultrastructure, Reactions [Walter
XXXIV. Mittl., B., 73 (1940), 167 de Gruyter, Berlin, New York, 1984]
34 Brauns, F. E., Brauns D. A.; The Chem- 49 Bikales, N.M.; Segal, L.; Cellulose and
istry of Lignin. Supplement Volume cellulose derivatives [Wiley, New York,
[Academic press, New York and Lon- 1971]
don, 1960, LCCCN: 60-9065] 50 Schliephake, D. et al.; Nachwachsende
35 Schulze, E.; Zur Kenntnis der chemi- Rohstoffe, Teil I Holz und Stroh [Land-
schen Zusammensetzung der pflanzli- wirtschaft-Industrie e.V. (ed.), Verlag J.
chen Zellmembranen. Ber. Deutsch. Kordt, Bochum, 1986] a) I3–I226,
Chem. Gesell., (B.), XXIV (1891) 2277 b) I–160, c) I–166
36 Sandermann, W.; Grundlagen der Che- 51 Gruber, E.; Krause, Th.; Schurz, J.: Cel-
mie und chemischen Technologie des lulose. In: Ullmann, Enzyklopädie der
Holzes [Akademische Verlagsge- technischen Chemie, 4. Aufl. Bd. 9
sellschaft, Leipzig, 1956] a) 97 ff b) 147 [Bartolomé, E. et al (Hrsg), Verlag Che-
37 Staudinger, H.; Reinecke, F.; Holz, mie, Weinheim, 1975] 184 ff
Roh- und Werkstoff, 2 (1939) 321 52 Lengyel, P.; Morvay, S.; Chemie und
38 Staudinger, H.; Makromolekulare Che- Technologie der Cellulose-Herstellung
mie und Biologie [Wepf & Co., Basel, [Akadémia Kiadó, Budapest, 1973]
1947] 53 Hägglund, E.; Chemistry of Wood.
39 Pringsheim, H.; Cellulosechemie, 2 [Academic press Inc., New York, 1952]
(1921) 123 54 Römpp Chemie Lexikon – CD Version
40 Ullmann, F.; Enzyklopädie der Tech- 2.0 [Georg Thieme Verlag, Stuttgart/
nischen Chemie. 2. Aufl., 5. Bd. New York, 1999]
[Urban & Schwarzenberg, Berlin und 55 Brauns, F. E., The Chemistry of Lignin.
Wien, 1930] 442 ff [Academic press, New York and Lon-
don, 1952]
References 141
56 Browning, B. L.; Handbook of pulp and refineries. In: Perspectives on new

paper technology, 2nd edn. [K.W. Britt crops and new uses. [J. Janeck (ed),
(ed.), Van Nostrand Reinhold, New ASHS Press, Alexandria, Va., 1999]
York, 1970] p. 7 pp 114–123
57 Fan, L. T.; Gharpuray, M. M., Lee, Y.-H.; 69 Kamm, B.; Kamm, M.; The Green
Cellulose Hydrolysis. Biotechnology Biorefinery – Principles, Technologies
Monographs [Springer, Berlin Heidel- and Products. In: Proceedings 2nd In-
berg New York, 1987] a) 13 b) 21 tern. Symp. Green Biorefinery, Feld-
58 Lengerken, J. v.; Zimmermann, K.; bach, Austria 1999, [SUSTAIN (ed.),
Handbuch – Futtermittelprüfung. 1. TU Graz, 1999] 46–69
Aufl. [Deutscher Landwirtschaftsverlag 70 National Research Council USA (ed.);
Berlin GmbH, Berlin, 1991, ISBN 3- Biobased Industrial products. [Nat.
331-00293-3] Acad. Press, Washington DC, 2000,
59 Jewoch, H.; Flachkowsky, G.; Weis- ISBN 0-309-05392-7]
bach, F.; Futtermittelkunde. [Gustav 71 Kamm, B.; Kamm, M.; Biorefinery-Sys-
Fischer Verlag, Jena, 1993] ISBN 3-334- tems. Chem. Biochem. Eng. Q., 18,
00384-1, p. 20 ff (2004) 1–6
60 Ackerson, M. D.; 16th IGT Conf. En- 72 U.S. Department of Energy (DEO); Na-
ergy from Biomass and Wastes [Wash- tional Biomass Initiative and Energy,
ington D.C., 1991] 725 Environmental and Economics (E3)
61 Pigman, W., et al (eds.); The Carbohy- Handbook [www.bioproducts-bioener-
drates, Bd. 2A [Academic Press, New gy.gov]
York, 1970] 400 ff. 73 Biomass R&D Technical Advisory Com-
62 Aspinal G. O.; In: The Carbohydrates, mittee (ed.); Vision for bioenergy and
Chemistry and Biochemistry Bd. 2B, biobased products in the United States.
[Pigman, W. et al (eds.), Academic [http://www.bioproducts-bioenergy.gov/
Press, New York, 1970] S. 517, 500 ff. pdfs/BioVision_03_Web.pdf, 2002]
See also: Aspinal, G. O.; The Polysac- 74 Biomass R&D Technical Advisory Com-
charides, 1 [Academic Press, New York, mittee (ed.); Roadmap for biomass
London, et al, 1982] technologies in the United States.
63 Buchholz, S. E.; Eveleigh, D. E.; Genetic [http://www.bioproducts-bioenergy.gov/
modification of Zymomonas mobilis. pdfs/FinalBiomass-Roadmap.pdf, 2002]
Biotechnol. Adv., 8 (1990) 547–581 75 Werpy, T.; Petersen, G.; and US-Depart-
64 Dale, B.; Encyclopedia of physical ment of Energy (eds.); Top Value
science and technology. Vol 2, 3rd edn. Added Chemicals from Biomass [U.S.
[McGraw-Hill, New York, 2002] pp DEO, Office of Scientific and Technical
141–157 Information, 2004, GO102004-1992]
65 Frank, G.; Das Papier, 30 (1976) 120 76 Kamm, B., Kamm, M.; Hempel, M.
66 Soyez, K.; Kamm, B., Kamm M.; (eds.) (eds); Biorefinica-2004. Proceedings In-
The Green Biorefinery, Proceedings of ternational Symposium Biobased Prod-
1st International Green Biorefinery ucts and Biorefineries [Verlag biopos,
Conference, Neuruppin, Germany, Teltow, 2004, ISBN: 3-00-015166-4]
1997 [Verlag GÖT, Berlin, 1998, ISBN 77 Hitchcock, L.; Duffey, H.; Chem. Eng.
3-929672-06-5, germ.] Prog., 44 (1948) 669
67 Gravitis, J.; Suzuki, M.; Biomass refin- 78 Dunlop, A. P.; Furfural. In: Kirk-Oth-
ery. In: Proceedings of 99 Intern. Conf. mer 2nd edn, Vol. 10 (1966) 237–251
on Agriculture Engineering, Beijing, 79 A. E. Staley, Mfg. Co. A. E. (Decatur Ill.);
China, 1999 [The United Nations Uni- Levulinic Acid 1942 [C.A. 36, 1612]
versity Press, Tokyo, 1999] III-9–III-23 80 Kitano, M.; Tanimoto, F.; Okabayashi,
68 Van Dyne, D. L.; Blasé, M. G.; Clem- M.; Levulinic Acid, A New Chemical
ents, L. D.; A strategy for returning Raw Material – Its Chemistry and Use.
agriculture and rural America to long- Chemical Economy & Engineering Review,
term full employment using biomass 7 (85)(1975) 25–29
81 Leonard, R. H.; Levulinic acid as a Ba- 95 Ander, P.; Ericksson, K.E.; Progress in
sic Chemical Raw Material. Ind. Eng. ind. Microbial. Vol. 14 [M. J. Bull (ed.),
Chem., 48 (1956) 1331–1341 Elsevier, New York, 1978] 1
82 Timell, T. E.; Tappi, 44 (1961) 99 96 Schurz, J.; Bioconversion of Cellulosic
83 James, R. L.; In: The pulping of wood, Substances into Energy, Chemicals and
2nd edn., vol. 1 [McGraw-Hill, New Microbiol. Protein. Symp. Proc. BERC
York, 1969] p 34 IIT Delhi [T.K. Ghose (ed.), New Delhi,
84 Brink, D. L.; Pohlmann, A. A.; Tappi, 55 1978]
(1972) 381 ff. 97 Scott, G. M.; Akhtar, M.; Biotechnologi-
85 Puls, J.; Dietrichs, H. H.; Energy from cal Applications of Lignin-Degrading
Biomass. Proceed. EC-Conference Fungi. In: Biopolymers Vol. 1 [M. Hof-
Brighton 1980 [1980] 348 richter, A. Steinbüchel (eds.), Wiley-
86 Shen, S.; Biological Engineering for VCH, Weinheim, New York et al, 2001,
Sustainable Biomass Production. ISBN 3-527-30220-4] 181
In: Biodiversity [E. O. Wilson, Harvard 98 Dunlap, C. E. et al; AIChE Symp. Ser.
University, ed.; National Academy of 72/158 (1976) 58
Sciences/Smithsonian Institution, 99 Fan, L. T. et al; In: Biotechnol., Bioeng.
Washington DC, 1988, German Symp. no. 11 [C.D. Scott (ed.), Wiley,
Edition: “Ende der biologischen Viel- New York, 1981]
falt?”, 1992, ISBN 3-89330-661-7] 404– 100 Gharpuray, M. M.; Biotechnol. Bioeng.,
416 25 (1983) 157
87 Van Dyne, D. L. et al.; Estimating the 101 Reese, E. T.; Sinn, R. G. H.; Levinson,
Economic Feasibility of Converting Lig- H. S.; J. Bacterial., 59 (1950) 485
no-Cellulosic Feedstocks to Ethanol 102 Reese, E. T.; Recent Adv. Phytochem., 11
and Higher Value Chemicals under the (1977) 311
refinery concept: A Phase II Study 103 Ghose, T. K.; Ghosh, P.; J. Appl. Chem.
[University of Missouri, 1999, Biotechnol., 28 (1978) 1489
OR22072-58] 104 Wood, T. M.; 6th Int. Ferment. Symp.
88 Fan, L. T.; Adv. Biochem. Eng. Vol. 23 London, Canada, 1980, p 87
[A. Fiechter (ed.), Springer, Berlin-Hei- 105 Chang, M. M.; Chou, T. Y. C.; Tsao,
delberg New York, 1982] G. T.; Adv. Biochem. Eng., 20 (1981) 15
89 Fan, L. T. et al.; Biotechnol. Bioeng., 23 106 Keller, F. A.; Integrated bioprocess de-
(1981) 419 velopment for bioethanol production.
90 Kosaric, N.; Vardar-Sukan, F.; Potential In: Handbook of bioethanol: production
Sources of Energy and Chemical Prod- and utilization. [C. E. Wyman (ed), Tay-
ucts. In: The Biotechnology of Ethanol lor & Francis, Bristol, Pa. 1996] 351–379
[M. Roehr (ed.), Wiley-VCH, Weinheim 107 Wolley, B. R.; Ruth, M.; Sheehan, J.;
et al, 2001, ISBN 3-527-30199-2] a) 89 ff Ibsen, K.; Majdeski, H.; Galvez, A.;
b) 131, c) 132 d) 125 e) 137 Lignocellulosic Biomass to Ethanol
91 Kamm, B.; Kamm, M.; Schmidt, M.; Process Design and Economic Utilizing
Starke, I.; Kleinpeter, E.; Chemical and Co-Current Dilute Acid Prehydrolysis
biochemical generation of carbohy- and Enzymatic Hydrolysis Current and
drates from lignocellulose-feedstock Futuristic Scenarios. [National Renew-
(Lupinus nootkatensis), Quantification of able Energy Laboratory (NERL), Golden,
glucose, Chemosphere, (in press), DOI Co, USA, 1999, NREL/TP-580-26157]
information: 10.1016/j.chemosphere. 108 Galbe, M.; Zacci, G.; A review of the
2005.03.073 production of ethanol from softwood.
92 Han, Y. W.; Callihan, C. D.; Appl. Micro- Appl. Microbiol. Biotechnol., 59 (2002)
biol., 27 (1974) 159 618–628
93 Turbak, A. F. et al.; Chemtech, Jan. 1980 109 Oshima, M.; Wood Chemistry – Pro-
94 Warwicker, J. O.; In: Shirley Institute cess Engineering Aspects [Noyes Devel-
Pamp No. 93 (1966) opment Corp. New York, 1965, No.
10965] a) 121 b) 88 ff c) 109–119
References 143
110 Freudenberg, K.; Vorträge über Bio- 123 Groheen, D. W.; Lignin, Structures and
chemie. Vergleichende Untersuchung Reactions. [American Chemical Society,
des natürlichen und biosynthetischen Washington DC, 1966] 205
Lignins. J. Prakt. Chemie, 10 (1960) 124 Goheen, D. W.; Henderson, J.; The
220–238 Preparation of Unsaturated Hydrocar-
111 Sarkanen, K. V.; Ludwig, C. H. (eds.); bons from Lignocelluloses Materials.
Lignins: Occurrence, Formation, Struc- Cellul. Chem. Tech., 12 (1978) 363
ture and Reactions [Wiley-Interscience, 125 Lora, J. H.; C. F. Wu; Pye, E. K.; Balati-
New York, 1971] necz, J. J.; In: Lignin: Properties and
112 Adler, E.; Lignin chemistry – Past, Materials. American Chemical Society
Present and Future, Wood Sci. Technol., Symposium Series 397 [W.G. Glasser
11 (1977) 169–218 and S. Sarkanen (eds.)]. Washington,
113 Hofrichter, M.; Steinbüchel, A. (eds.); DC, USA 1989, p. 312–324
Lignin, Humic Substances and Coal. 126 Lawford, H. G.; Rosseau, J.D.; 16th IGT
Biopolymers Vol. 1 [Wiley-VCH, Wein- Conf. Energy from Biomass and Wastes,
heim, New York et al, 2001, [IGT, Washington DC, 1991] 583
ISBN 3-527-30220-4] 127 Goldstein, I. S.; Organic Chemicals
114 Brunow, G.; Lignin chemistry and the from Biomass [CRC press, Florida,
role in biomass conversion. In this USA, 1981].
book, 2005 128 Lichtenthaler, F. W.; The Key Sugars of
115 Northey, R. A.; Low-Cost Uses of Lig- Biomass: Availability, Present Non-
nin, Emerging Technology if Materials Food Uses and Potential Industrial De-
and Chemicals from Biomass, ACS velopment Lines. In this book, 2005
Symposium Series 476, Washington, 129 Schuster, L.; Saccharide Selektive Hy-
D.C., 1992 drogenolyse zu Polyalkoholen. In:
116 Lin, S. Y.; Lebo, S. E. Jr., Lignin, Kirk- Nachwachsende Rohstoffe – Perspekti-
Othmer Encyclopedia of Chemical ven für die Chemie [M. Eggersdorfer,
Technology, Fourth Edition, Volume S. Warwel, G. Wullf (eds.), VCH, Wein-
15, 268–289, 1995 heim New York Basel Cambridge
117 Lignin Institute; http://www.lignin.info Tokyo, 1993, ISBN 3-527-29019-2] 211
118 Pye, E. K.; Industrial Lignin Production 130 Van Bekkum, H.; Verrast, D. L.; New
and Applications. In this book, 2005 Starch and Inulin Derived Products
119 Hearon, W.; Macgregor, W. et al; Sulfur with potential Applications. In: Per-
Chemicals from Lignin. Tappi, 45 (1962) spektiven nachwachsender Rohstoffe in
120 Enquist, F. et al; Tappi, 45 (1962) 128 der Chemie [E. Eierdanz (ed.) VCH,
121 Taraban’ko, V. E.; Ivancehneko, N. M., Weinheim New York Basel Cambridge
Koropatchinskaya, N. V., Kuznetsov, Tokyo, 1996, ISBN 3-527-29417-1] 200
B. N.; Study of wood and lignosulfonate 131 Van Bekkum, H.; Studies on Selective
processing into fine chemicals. Chem. Carbohydrate Oxidation IN: Carbohy-
for sustainable development, 4–5 (1996) drates as Organic Raw Materials [F. W.
391–402 Lichtenthaler (ed.), VCH, Weinheim
122 Taraban’ko, V. E.; Kuznetsov, B. N.; New York Basel Cambridge Tokyo,
Chernyak, M.; Yu., Pervyshina, E. P., 1991, ISBN 3-527-28280-7] 289
Swistek, M.; The production of fine 132 Defay, J., Pedersen, C.; Hydrogen
chemicals from hard wood. In: Catalytic Fluoride, Solvents and Reagent for car-
and Thermochemical Conversion of bohydrate Conversion Technology. In:
Natural Organic Polymers. Proc. Fourth Carbohydrates as Organic Raw Materi-
Intern. Sym., 2000, Krasnoyarsk, Russia als [F.W. Lichtenthaler (ed.), VCH,
[B. N. Kuznetsov; R. Gruber (eds.), Rus- Weinheim New York Basel Cambridge
sian Academy of Science, Russia and Tokyo, 1991, ISBN 3-527-28280-7] 47
University of Metz, France, 2000] 77 133 Hosaka, H.; Suzuki, H.; In: Wood Hy-
drolysis, FAO Technical panel on Wood
Chemistry [FAO, FAO/WC/60/WH-5, 150 Hurd, C. D.; Garrett, J. W.; Osborne,

Tokyo, 1960] E.N.; Furan Reactions. IV. Furoic acid
134 Fahn, R.; Bernd, B.; US Pat. 4.072.538 from Furfural, J. Am. Chem. Soc., 55
(1978) (1933) 1082–1084
135 Friese, H.; US Pat. 3.907.120 (1978) 151 Gilman, H.; Organic Syntheses, Collec-
136 Steiner, K.; US Pat 3.586.537 (1971) tive Vol. 1 [John Wiley & Sons, New
137 Kohno, S.; US Pat. 3.558.765 (1971) York, London, 1953] 274
138 Borrevik, R. K. et al.; In: Effect of nitro- 152 Cass, O. W., Ind. Eng. Chem., 40 (1948)
gen oxide pretreatment on enzymatic a) 216, b) 219
hydrolysis of cellulose [Lawrence 153 Noyori, G.; Takijiri, S.; Kakehashi, S.;
Berkeley Lab., Univers. of Calif., Berke- Japanese Patent JP 202.746
ley, 1978, LBL 7879] 154 Wilson; Org. Syn. Coll. Vol. I, 2nd ed.
139 Wilke, C. R.; IN: Process development (1941), 276
studies of the bioconversion of cellu- 155 Quaker Oats; U.S. Patent 2,041.184
lose and production of ethanol [Lawr- (1936) [C.A. 30, 4515]
ence Berkeley Lab., Univers. Of Calif., 156 Dunlop, A. P.; Furfural Formation and
Berkeley, 1978, LBL 6881] Behavior. Ind. Eng. Chem., 40 (1948) 204
140 Naughton, G. G.; Geyer, W. A.; Econom- 157 Kotake, M.; Fujita, Y.; J. Chem. Soc.
ic Analysis of Energy from Biomass. Jap., 51 (1930) 354
In: Energy from Biomass [A. Strub, P. 158 Dunlop and Schegulla; U.S. Pat.
Chartier, G. Schleser (eds.); EC-Confer- 2.838.523 (1958 to Quaker Oats)
ences, Brüssel, 1983] 159 Deutsche Hydrierwerke AG; France
141 Monroe, K. P.; The Preparation and Patent (F.P.), 829 113 (1937)
technical uses of furfural. J. Ind. Eng. 160 Peters; U. S. Patent 1.906.873 (1933 to
Chem., 13 (1921) 133 Quaker Oats)
142 Dunlop, A. P.; Peters, F. N.; The Furans 161 Wojcik, B. H.; Catalytic hydrogenation
[Reinhold Publ. Corp., New York, 1953] of furan compounds. Ind. Eng. Chem.,
143 Dunlop, A. P.; McKillip, W. J.; Furan 40 (1948) 210
and Derivative. In: Ullmanns Encyklo- 162 Lukes, R. M.; Wilson, C. L.; J. Am. Soc.,
pedia der technischen Chemie. 73 (1950) 4790
4. neub. Aufl., Bd 12 [Verlag Chemie, 163 Soc. Anon des Distelleries des Deux
Weinheim New York, 1976] Sévres (France). France patent: F. P.
144 Schoenemann, K.; Chem. Eng. Sci., 8 639 756 (1927)
(1957) 161–176 164 Burnette, L. W.; Johns, I. B., Holdren,
145 Root, D. F.; Saeman, J. F.; Harris, J. F.; R. F.; Hixon, R. M.; Production of 2-
Neill, W. K.; Forest Prod. J., 9 (1959) Methylfuran by Vapor-Phase Hydroge-
158–165 nation of Furfural. Ind. Eng. Chem., 40
146 Zeitsch, K. J.; Patent: US 4.912.237 (1948) 502
(1990); DE 38 42 825 C2 (1991) 165 Lichtenthaler, F. W.; Acc. Chem. Res., 35
147 Zeitsch, K. J.; The chemistry and Tech- (2002) 729 ff
nology of Furfural and its many By- 166 Gilman, H.; Wright, G.; J. Am. Chem.
Products [Elsevier, Amsterdam, 2000] Soc., 52 (1930), 1170, 2550, 4165
148 Lichtenthaler, F. W. et al; Practical 167 Brenner, G. M.; McNeil, D.; English
Routes from Mono- and Disaccharides pat. E.P. 547 334 (1942) [C.A. 37 (1943)
to Building Blocks with Industrial Ap- 5988]
plication Profiles [F. W. Lichtenthaler 168 Paul, R.; Bull. Soc. Chim. France, 53
(ed.), VCH, Weinheim New York Basel (1933) 1489
Cambridge Tokyo, 1991, ISBN 3-527- 169 Lukes, R. M.; Nelson L. S; The Concur-
28280-7] 208 rent Hydrogenation and Hydrolysis of
149 Hurd C. D.; Goldsby, A. R.; Osborne, Furfuryl Alcohol. J. Org. Chem., 21
E. N.; Furan Reactions. II. Furan from (1956) 1096
Furfural, J. Am. Chem. Soc., 54 (1932) 170 Timokin, B. V.; Bransky, V. A.; Eliseeva,
2532 G. D.; Levulinic acid in organic synthe-
References 145
sis. Russ. Chem. Rev., 68 (1999) 73–84, lin, 1965, ger.] a) 1402 ff, b) 1542 ff, c)
75 [see there also Ref. [33] US Patent 3 1535
752 849 and [34] Jpn. Patent 5 139 205 193 Reppe, W.; Chemie und Technik der
171 Kenkyujo, Z. H. R.; English patent, E.P. Acetylen-Druck-Reaktionen [Verlag
253 877 (1926) Chemie, 1952] 118 (Beilstein XVII, 27
172 Nielson, R. E.; Vapor Phase Oxidation and XVII(2), 15),
of Furfural. Ind. Eng. Chem., 41 (1949) 194 Schmoyer and Case; Nature, 187 (1960)
365 592
173 Milas, N. A.; Walsh, W. L.; J. Am. Chem. 195 Reppe, W., et al; Bernsteinsäure aus
Soc., 57 (1935) 1389 Tetrahydrofuran. In: Äthinylierung V.
174 Milas, N. A.; Fumaric Acid. Org. Syn., Liebigs Ann. Chem., 596 (1955) 156
Coll. Vol. II (1943) 302 196 Reppe, W., et al; Oxydation von Tetra-
175 Lichtenthaler, F. W., Peters, S.; C.R. hydrofuran IN: Äthinylierung V. Liebigs
Chimie, 7 (2004) a) 80, b) 65–90 Ann. Chem., 596 (1955) 107
176 McKillip, W. J.; Furan and Derivatives. 197 Reppe, W., et al; Maleinsäure aus Tetra-
In: Kirk-Othmer Encyclopedia Chem. hydrofuran. In: Äthinylierung V. Liebigs
Technol. 11, 1981, 501–527 Ann. Chem., 596 (1955) 157
177 Rajagopalan, S.; Raman, P. V. A.; Org. 198 Weissermel, K., Arpe, H.-J.; Maleic An-
Syn., 25 (1945) 51 hydride. In: Industrial Organic Chem-
178 Rice, C. C.; Dudley, E. A.; J. Am. Chem. istry. Fourth, Rev. Ed. [Wiley-VCH
Soc., 78 (1956) 68 GmbH Co. KGaA, 2003] 367 ff
179 Johnson, J. R.; 2-Furanacrylic acid. Org. 199 Dobratz, E. H. (Monsanto); Patent AP.
Synth., 20 (1940) 55 2.704.296 (1951)
180 Patterson, J. M.; 2-Furanacrylonitrile. 200 Saito, K. et al (Nippon Chem. Ind.);
Org. Synth., 40 (1960) 46 Patent JP 4360(50), 1950
181 Hinz, A., Meyer, G.; Schlucking, G.; 201 Takashi, Y.; Hibino, O.; Morita, S.; Syn-
Ber. (Chem. Ber.), 76 B (1943) 676 thesis of malic acid from maleic acid.
182 Starr, D.; Hixon, R. M.; Org. Syn., Coll. J. Chem. Soc. Japan, Ind. Chem. Sec., 53
Vol. II (1943) 566 (1950) 361
183 DuPont; Deutsches Bundespatent 202 Milas, N. A.; Tery, E. M.; J. Am. Chem.
(DBP), 106872 (1958) Soc., 47 (1925) 1412
184 Bandford & Manes; U.S. pat. 2.846.449 203 Schmidt, E.; Haag, W., Sperling, L.;
(1959 to DuPont), Ber. Dtsch. Chem. Ges. (B.), 58 (1925)
185 Manly; U.S. pat. 3.021.342 (1962 to 1400
Quaker Oats) 204 Session, W. V.; J. Am. Chem. Soc., 50
186 Pinkos, R.; Fischer, R. (BASF AG); Pro- (1928) 1696
cess for preparation of 1,4-butanediol 205 Andrisano, R.; Angelori, A. S.; Prepara-
and tetrahydrofuran from furan. Ger. tion of 2,5-furandicarboxylic acid from
Patent; Ger. Offen., DE 19510438 A1 furoic acid. Ann. Chim. (Rome), 53
(1996) (1963) 1658–1664
187 Yur’ev, Y. K.; Tranova, V. A.; J. Gen. 206 Bottorff, Moore; Org. Syn. Coll. vol. V,
Chem., USSR Engl. Tranl., 18 (1940) 31 (1973) 687
[CA: 34, 4733] 207 Paul, R.; Bull. Soc. Chim. France, 53
188 Yur’ev, Y. K.; Org. Khim., 175 (1956) (1933) 1489
159 [CA: 51, 10470b] 208 Houben–Weyl, Methods in Organic
189 Cass, O. W.; Chemurgic Digest, 8 (1949) Chemistry. Bd. 6/4 [E. Müller (ed.),
3 Georg Thieme Verlag Stuttgart, 1965,
190 E. I. DuPont; Patent US 2.600.289 ger.] a)12–70, b) 286–300
(1952) 209 Sharples Chem Inc.; AP 2.516.337
191 Boesken, M. J.; Receuil Trav. chim. Pays- (1946)
Bas., 24 (1915) 100 210 Ritter, H.; Dihydropyran. In: Ullmann’s
192 Fodor, G.; Organic Chemistry Enzyklopädie der technischen Chemie.
[Deutscher Verlag Wissenschaften, Ber- Bd. 14, 1966, 456
211 Kaufmann, D.; Reeve, W.; Org. Syn. 231 Meerwein, H.; Delfs, D.; Morschel, H.;
Coll. vol. III, (1955) 693 Die Polymerisation des Tetrahydrofu-
212 Paris et al.; Org. Syn. Coll. vol. IV, rans; Angew Chem., 72 (1960) 927–1006
(1963) 496 232 Scott, L. T.; Roelofs, N. H.; Tsang, T.-H.;
213 English, Dayan; Org. Syn. Coll. vol. IV, Automerization of Benzene. J. Am.
(1963) 499 Chem. Soc., 109 (1987) 5456–5461
214 Gordon, D.; Kline, M.; Modern Plas- 233 DuPont; Patent AP. 2.284.525 (1942)
tics, 23 (1946) 174 234 Danly, D. E.; Campell, C. R.; In: Kirk-
215 Hopff, H.; Caprolactam. In: Ullmann’s Othmer Encyclopedia of Chemical
Encyklopedia Chemie. 3. Aufl., Bd 5, Technology vol. 1, 3rd. [Wiley-Inter-
1954, 68 ff science, New York, 1978] 510–531
216 Jenner, G., Kheradman, H., Kienne- 235 Grüll, D.; Jetzinger, F.; Kozich, M.;
mann, A.; Journal of Organometallic Wastyn, M.M.; Wittenberger, R.; Starch
Chemistry, 277 (1984) 427–438 Product family trees Starch / Starch
217 Beilstein; Handbook of Organic Chem- Platform, Products. In this book, 2005
istry; 4. Aufl., Vol. I. 131, Vol. I1 43, 236 Schenk, F.W.; Hebeda, R.W. (eds.);
Vol. I2 95 Starch Hydrolysis Products, Worldwide
218 Houben-Weyl Methods in Organic technology, production and Applica-
Chemistry. Polyamide, Vol. E20/2 tions [VCH Publishers, Inc., NY/Wein-
[E. Müller (ed.), Georg Thieme Verlag heim/Cambridge, 1992]
Stuttgart, 1965, ger.]1497–1560 237 Bergius, F.; Trans. Inst. Chem. Eng.
219 Beilstein, Handbook of Organic Chem- (London), 11 (1933) 162
istry; Beilstein EIV 2, 2003 238 Scholler, H.; Dissertation, Technical
220 Cornils, B.; Lappe, P.; Dicarboxylic University of Munich, Germany 1923
acid, Aliphatic. In: Ullmann’s Encyklo- 239 Scholler, H.; French Patent, F.P.
pedia, 5. Ed., Vol A (1987) 531 777,824 (1935)
221 Grimm, A.; Schimpf, H.U.; Patent 240 Conrad, T. F.; Holzzuckerbrennerei. In:
DWP 7259 (1953) Die Hefen, Vol. II 8 F. Reiff, R. Kautz-
222 Reppe, W., et al; Äthinylierung V. Lie- mann, H. Lüers, M. Lindemann (eds.)
bigs Ann. Chem., 596 (1955) a) 89 b) [Verlag Hans Carl, Nürnberg, 1962]
80 ff c) 120 d) 127 437–444
223 Flickinger, E.; Tetrahydrofuran. In: Ull- 241 Gläzer, H.; Flügge, F.; Scholler, H.;
mann’s 3. Aufl. Bd. 17 (1966) 72 Praetorius; Chemische Technologie des
224 Ullmann’s Encyclopedia Chemistry, Holzes [Carl Hanser Verlag, 1954]
Bd. 13, p. 82 242 Röper, H.; Selective Oxidation of d-
225 Patai, S. (ed); The chemistry of acid de- Glucose; Chiral Intermediates for In-
rivatives (2 vol.). In: Chemistry of func- dustrial Utilization. In: Carbohydrates
tional groups series [Chichester, Wiley as Organic Raw Materials [F.W. Lich-
& Sons, 1979] tenthaler (ed.), VCH, Weinheim New
226 Metsger, L.; Bittner, S.; Autocatalytic York Basel Cambridge Tokyo, 1991,
Oxidation of Ethers with Sodium Bro- ISBN 3-527-28280-7] 267–288
mate. Tetrahedron, 56 (2000) 1905–1910 243 Wulff. G.; Übersicht über neue poly-
227 Ullmann’s Encyclopedia Chemistry; Bu- mere Materialien auf Basis nachwach-
tyrolactone, Bd. 4, p. 802 sender Rohstoffe IN: Nachwachsende
228 Ullmanns Encyclopedia Chemistry, Pyr- Rohstoffe – Perspektiven für die Che-
role and Derivatives, Bd. 14 (1966) mie [M. Eggersdorfer, S. Warwel, G.
507 ff Wulff (eds.), VCH, Weinheim New
229 Reppe, W.: Neue Entwicklungen auf York Basel Cambridge Tokyo, 1993,
dem Gebiet des Acetylens und Kohlen- ISBN 3-527-29019-2] 291
oxyds [Springer, Heidelberg, 1949] 47 ff 244 Kirk-Othmer Encyclopedia of Chemical
230 Treischmann, H. G.; Manchen, F. Technology. Vol. 1 [R. E. Kirk, D. F. Oth-
(BASF); DBP 859 734 (1939) mer (eds.) The Interscience Encyclope-
dia Inc., 1947] 329
References 147
245 Creighton, H. J.; US Patent 1.990.582 sellschaft mbH, Weinheim/Basel/Cam-

(1935) bridge/NY/Toronto, since 1985] 587
246 Rappaille, A.; Starch/Stärke 40 (1988) 259 Thoen, J., Busch, R.; Industrial Chemi-
356–359 cals from Biomass – Industrial con-
247 Ullmann’s Encyclopedia of Industrial cepts. In this book, 2005
Chemistry. A25, 5. ed. [VCH Verlagsge- 260 Moye, C. J.; Rev. Pure App. Chem., 14
sellschaft mbH, Weinheim/Basel/Cam- (1964) 161–170
bridge/NY/Toronto, since 1985] 413– 261 Van Dam, H. E.; Kieboom, A. P. G.; van
437 Bekkum, H.; Starch/Stärke, 38 (1986)
248 Schmidt, R. R.; Displacement by Sub- 95
stitutions Processes: Synthesis of Gly- 262 Schiweck, H.; Munir, M.; Rapp, K. M.;
cosides. In: Comprehensive Organic Scheider, B.; Vogel, M.; New Develop-
Synthesis. Vol. 6 Heteroatom Manipu- ments in the Use of Sucrose as an In-
lation (B. M. Trost, Fleming, I.; Winter- dustrial Bulk Chemical. 5-hydroxy-
feld, E. (eds.), Pergamon Press, Oxford, methylfurfural (HMF). In: Carbohy-
1991] drates as Organic Raw Materials [F. W.
249 Klotz, W.; Schmidt, C.; Schmidt, R. R.; Lichtenthaler (ed.), VCH, Weinheim
Tenside durch direkt anomere O-Alky- New York Basel Cambridge Tokyo,
lierung von ungeschützten Zuckern. 1991, ISBN 3-527-28280-7] 78–94
In: Perspektiven nachwachsender Roh- 263 Lichtenthaler, F. W.; Carbohydrates. In:
stoffe in der Chemie [E. Eierdanz (ed.) Ullmann’s Encyclopedia Ind. Chem.,
VCH, Weinheim New York Basel Cam- Vol. 6, 6th. Ed. [Wiley-VCH, Wein-
bridge Tokyo, 1996, ISBN 3-527-29417- heim/NY, 2002] 263 ff
1] 280 264 Hoechst AG; Verfahren zur Oxydation
250 Schmidt, R. R.; Kohlenhydrate – von 5-hydroxmethylfurfural. Patent
Synthesebausteine für Spezialchemika- 38.26.073
lien. In: Nachwachsende Rohstoffe – 265 Vinke, P.; van Dam, H. E.; van Bek-
Perspektiven für die Chemie [M. Eg- kum, H.; Platinum Catalyzed Oxidation
gersdorfer, S. Warwel, G. Wulff (eds.), of 5-hydroxmethylfurfural. In: New De-
VCH, Weinheim New York Basel Cam- velop. In Selective Oxide. [G. Centi,
bridge Tokyo, 1993, ISBN 3-527-29019- F. Trifiro (eds.), Elsevier, B.V., Amster-
2] 225 dam, 1990] 147–158
251 Schmidt, R. R.; Recent developments in 266 Franck, H.-G.; Stadelhofer, J. W.; Indus-
the Synthesis of Glucoconjugates. Pure trielle Aromatenchemie [Springer Ver-
Appl. Chem., 61 (1989) 1257–1270 lag, Berlin, Heidelberg, 1987]
252 Wulff, G.; Röhle; Angew. Chemie, 86 267 Gandini, A.; Furan Polymers. In: En-
(1974) 173–187 cycl. Polym. Sci. Engineer., 2nd ed. Vol.
253 Dean, G. R.; Pyle, R. E.; British Patent 7 [Interscience Publ., New York, 1984]
670.480 (1952) 268 Mitiakoudis, A.; Gandini, A.; Chera-
254 de Bruyne, D. C. A.; von Eckstein, dame, H.; Polyamides Containing
W. A.; Rec. Trav. Chim., 41 (1895) 156, Furanic Moieties. Polym. Commun., 26
203 (1985) 246–249
255 Wolfrom, M. L.; J. Am. Chem. Soc., 50 269 Rapp, K. M.; Daub, J.; Herstellung und
(1928) 842 Derivatisierung von 5-Hydroxymethyl-
256 Bamford, C. H.; Collins, J. R.; Proc. furfural. In: Nachwachsende Rohstoffe
Roy. Soc. A., 228 (1955) 100 – Perspektiven für die Chemie [M. Eg-
257 Neidlemann, S. L.; Amon, W. F.; Gei- gersdorfer, S. Warwel, G. Wulff (eds.),
gert, J.; Process for the Production of VCH, Weinheim New York Basel Cam-
d-Glucosone and d-fructose. US Patent bridge Tokyo, 1993, ISBN 3-527-29019-
4.246.347 (1981) [C.A.: 94 (1981) 2] 183
207379s] 270 Rapp, K. M. (Südzucker AG); Process
258 Ullmann’s Encyclopedia of Industrial for Preparing Pure 5-Hydroxymethyl-
Chemistry. A9, 5. ed. [VCH Verlagsge- furfural. US Patent 4.740.605 (1987)
271 Zoebelin, H. (ed.); Dictionary of Re- 2,5-furandicarboxylic acid by in situ

newable Resources [VCH Verlagsge- oxidation of 5-hydroxymethyl-furfural
sellschaft mbH, Weinheim/NY/Basel/ starting from fructose. Topics in Cataly-
Cambridge/Tokyo, 1997] a) 186 sis, 13 (2000) 237–242.
272 Timokhin, B. V.; Baransky, V. A.; Elisee- 288 Leupold, E. I.; Wiesner, M.; Schling-
va, G.D.; Levulinic acid in organic syn- mann, M.; Rapp, K. (Hoechst AG, Ger-
thesis. Russ. Chem. Rev., 68 (1999) 73– many). Catalytic oxidation of 5-(hy-
84 droxymethyl)furfural. Ger. Offen, DE
273 Ghorpade, V.; Hanna, M.; Industrial 3826073 (1990)
applications for levulinic acid. In: Cer- 289 El Hajj, T.; Masroua, A.; Martin, J. C.;
eals – Novel Uses and Processes [G. M. Descotes, G.; Synthese de l’hydroxy-
Campbell, C. Webb, S. L. Mckee (eds.) methyl-5 furanne carboxaldehyde-2 de
Plenum press, New York London, ses derives par traitment acide de
1997, ISBN 0-306-45583-8] 49–55 sucres sur resine echangeuses d’ions,
274 Wiggins, L. F.; Adv. Carbohydr. Chem., 4 Bull. Soc. Chim. Fr., 5 (1987), 855–860
(1949) 306 290 Schiavo, V.; Descotes, G.; Mentech, J.;
275 Beilsteins Handbuch der Organischen Hydrogenation catalytique du 5-hydro-
Chemie. Lävulinsäure. Bd. 3, 4. Aufl. xymethyl-furfural en milieu aqueux,
[Verlag Julius Springer, Berlin, 1921] Bull. Soc. Fr., 128 (1991) 704–711
671 291 Lewkowski, J.; Synthesis, Chemistry
276 Dahlmann, J.; Notiz über die Darstel- and Applications of 5-Hydroxymethyl-
lung von Lävulinsäure. Chem. Ber., 101 furfural And Its Derivatives, University
(1968) 4251–4223 of Lodz, Poland (ISSN 1424-6376)
277 Choudury, P. K.; J. Sci. Ind. Res., 16B 292 Cope, A.C.; Baxter, W. N.; J. Am. Chem.
(1957) 289–294 Soc., 77 (1955) 394.
278 Olson, E. S.; Kjelden, M. R.; Schlag, 293 Firouzabadi, H.; Ghadari, E.; Barium
A. J.; Sharma, R. K.; ACS symposium Manganate. An Efficient Oxidizing Re-
series 784 (2001) 51–63 agents for Oxydation of Primary and
279 Bart, H. J.; Reidetschlager, J.; Schatka, Secondary Alcohols to Carbonyl Com-
K.; Lehmann, A.; Ind. Eng. Chem. Res., pounds. Tetrahedron Lett., 1978, 839–842
31 (1994) 21 294 Kawana, O.; Nakamura, Y.; Yoshihiro,
280 Shu, C.-K.; Lawrence, B. M.; J. Agric. Y.; Nippon Kogaku Zaishi, 12 (1983)
Food Chem., 43 (1995) 782 1747–1752 [ref. C.A. 100, 15644 f
281 Fittig, R.; Heinzelmann, H.; Chem. (1984).
Ber., 9 (1876) 1198 295 Moye, C. J.; Goldsack, R. J.; J. Appl.
282 Tollens, B.; Yoder, P. A.; Chem. Ber., 34 Chem., 16 (1966) 206–208
(1901) 3446 296 Larousse, C.; Rigal, L.; Gaset, A.; Ther-
283 Haworth, W. N.; Jones, W. G. M.; Wig- mal degradation of 5-hydroxymethylfur-
gins, L. F.; The Conversion of Sucrose fural. J. Chem. Techn. Biotechnol., 53
into Furan Compounds. Part II. J. (1992) 111–116
Chem. Soc., (1945) 2 297 Elming, N.; Clauson-Kaas, N.; Acta
284 Bratulescu, G.; Novel technique for Chem. Scand., 10 (1956) 1603–1605
one-step synthesis of 2,5-bis(alkoxycar- 298 Skowronski, R.; Grabowski, G.; Lew-
bonyl)furans. J. Soc. Algerienne Chim., kowski, J.; Descotes, G.; Cottier, L.;
10 (2000) 135–137 (fr.) Neyret, C.; Org. Prep. Proced. Int., 25
285 Lew, B. W.; (Atlas Chem. Ind.), US Pat- (1993) 321–323
ent: US P. 3326944 (1967) [ref. CA 68, 299 Beilstein – Handbuch der Organischen
P4943 (1968)] Chemie; Furandicarbonsäure. 4. Aufl.
286 Blanksma, J. J.; Über die Konstitution [Deutsch. Chem. Gesell. (ed.), Verlag
des Oxymethylfurfurols, Chemisches Julius Springer, Berlin, 1934] a) 329
Zentralblatt I (1910) 539 [see also Phelps, Hale; J. Am. Chem.
287 Kroger, M.; Prusse, U.; Vorlop, K.-D.; Soc., 25, 440 ff; b) 329 [see also Klink-
A new approach for the production of hardt; J. Prakt. Chem., 25, 41 ff ]
References 149
300 US patent: USP 2 676 186 [ref. Zh. 317 Mukaiyama, T.; Hanna, J.; Inoue, T.;
Khim. (20) 47161P (1955)] Sato, T.; A Convenient method for the
301 Japan patent: Jpn. P. 4513 [ref. Zh. synthesis of c-substituted-b-acetyl-c-bu-
Khim. 16N 34P (1961)] tyrolactones. Chem. Lett. (1974), 381–
302 Frank, R. L.; Schmitz, W. R.; 1,5-Di- 382
methyl-2-Pyrrolidone. Org. Synth. Coll. 318 Erdmann, H.; Ueber Abkömmlinge
Vol. 3 (1954) 328 und Umwandlungen der Benzallävu-
303 US patent: US P. 4 389 531 [ref. C.A. linsäure. Ann. d. Chem. (Annalen), 254
99, 89 638 (1983)] (1889) 213 [see also Wolff, L.; Annalen
304 Moffett, R. B.; Pyrrolidyl Alkanols. 208 (1881) 106]
J. Org. Chem., 14 (1949) 862 319 Rosenmund, K. W.; Zymalkowski, F. et
305 Manzer, L. E. (E. I. Dupont); PCT Int. al; Chem. Ber., 84 (1951) 711
Appl: WO 2004084888, A1 20041007 320 Delpine, M.; Horena, A.; Bull. Soc.
(2004) Chim, Fr., Hem [5], 4 (1937) 32
306 Kumashiro, G.; Hatikava, E.; J. Chem. 321 Manzer, L. E. (E. I. DuPont); PCT Int.
Soc. Jpn., Chem. Ind. Chem., 57 (1954) Appl.: WO 2002074760, A1 20020926
299 (2002)
307 Joda, N.; Macromol. Chem., 55 (1962) 174 322 Houben-Weyl – Methods in Organic
308 Fetizon, M.; Montanfirer, M.; Rens, J.; Chemistry; Bd 5/1c [E. Müller (ed.),
J. Chem. Res. (S), 9 (1982) Georg Thieme Verlag Stuttgart, 1965,
309 US patent: US P. 2 815 375 [ref. Zh. ger.] 1–975
Khim. (12) 2072P (1960)] 323 Elliott, D. C., U.S. Patent 5,883,266
310 Bader, A. A.; Kontowic, A. D.; J. Am. (1999)
Chem. Soc., 76 (1954) 4465 324 Stevens, R.; Laevulinic acid. Part I.
311 Japan patent: Jpn. P. 6 270 338 [ref. J. Chem. Soc. (1960) 1118
Chem. Abstr., 107, 154 076 (1987)] 325 Kues, W.; Paal, C.; Synthese des Thio-
312 Zhang, R.; Moore, J. A.; Polymer Pre- tenols und des Thiolens. Ber. Deutsch.
prints, 43 (2002) 1007–1008 Chem. Ges. (Ber.), 19 (1886) 555
313 Knole, J.; Schaefer, H.; Angew. Chem., 326 Larsson, V.; Carlson, R.; Leroy, J.; Acta
87 (1975) 777 Chem. Scand., Ser. A, 47 (1993) 380
314 Raymond, S.; J. Am. Chem. Soc., 72 327 Stevens, F. J.; Griffin, T. D.; Fields, T. L.;
(1950) 3296 J. Am. Chem. Soc., 77 (1955) 42
315 Leonard, R. H.; Conversion of levulinic 328 Lepangol, A.; Deprey, J.; Bull. Soc.
acid into alpha-angelicalactone. US pat- Chim. Fr., 606 (1961)
ent: US P. 2 809 203 [ref., Chem. 329 Fudzise, M.; Nacamura, F.; J. Chem.
Abstr. 52 2819b] Soc. Jpn., Pure Chem., 75 (1954) 348
316 Bozell, J. J., Moens, L., Elliott, D. C., 330 Ha, H.-J.; Lee, S.-K., Ha, J.-L.; Synth.
Wang, Y., Neuenschwander, G. G., Fitz- Commun., 24 (1994) 10945
patrick, S. W., Bilski, R. J. and Jarnefeld, 331 Moens; Luc (Midwest Research Insti-
J. L. (2000), Production of levulinic acid tute) US patent: US P. 5,907,058, May
and use as a platform chemical for de- 25, 1999
rived products. Resources, Conservation 332 Matsuda, S.; Yunoue, M.; Appl. Microb.
and Recycling, 28 (2000) 227–239. Eng. Div., 24 (1998) 197–202
151
4
Lignin Chemistry and its Role in Biomass Conversion
Gösta Brunow
4.1
Introduction
Lignin is among the most abundant biopolymers on earth and, being renew-
able, it has attracted much effort to make use of lignin as feedstock in biomass
conversion processes. Lignins are an essential component of the woody stems
of arborescent gymnosperms and angiosperms in which amounts range from
15 to 36%. Lignins are not restricted to arborescent plants, they are found as in-
tegral cell wall constituents in all vascular plants including the herbaceous vari-
eties. The lignin in the cell walls is intimately mixed with the carbohydrate
components. The structure of the polymer is complex and irregular and isola-
tion of lignin from other plant constituents is not easy. Lignin is an essential
component of higher plants, giving them rigidity, water-impermeability, and re-
sistance against microbial decay. In the pulp and paper industry, lignin is re-
moved chemically and residual lignin in pulp is removed or degraded using
bleaching agents, e.g. chlorine dioxide, oxygen, or ozone. In mechanical pulping
much energy is needed to eliminate the cementing effect of lignin. Vast
amounts of lignin derivatives from pulp and paper industry are created and
these compounds are a threat to the environment if not detoxified or otherwise
treated in effluent treatment plants. White-rot fungi are the only organisms able
to mineralize lignin efficiently to carbon dioxide and water by processes initially
catalyzed by extracellular enzymes. Biotechnological applications of these fungi
and their lignin-modifying enzymes are being developed as alternative methods
for pulping and bleaching and for bioremediation, i.e. removal of toxic pollu-
tants from soil, groundwater, and effluents.
ISBN: 3-527-31027-4
152 4 Lignin Chemistry and its Role in Biomass Conversion
4.2
Historical Overview
Lignin is encountered industrially during the process of papermaking from

wood. This involves the chemical and mechanical separation of the cellulosic fi-
bers from woody or other lignified plant material. The chemical separation of
lignin from cellulose has been termed “delignification” and it is one of the com-
plex processes of the pulp and paper industry. The lignin products resulting
from the delignification processes may vary widely in properties depending on
which delignification process has been employed and at which stage of deligni-
fication the lignin was isolated. The sulfate or Kraft process produces the largest
amount of pulp. The sulfate or kraft lignin has consequently been the most
plentiful lignin available in spent pulping liquors. The kraft lignin can be recov-
ered in reasonably high yields by acidifying and filtering the precipitated lignin.
Very few kraft mills do process kraft lignin for sale; the bulk of the spent liquor
lignin is burned for the production of energy. The fuel value of the organic mat-
ter in the black liquor from the kraft process is substantial. The value of any
chemical to be produced from black liquor must be high enough to compensate
for the cost of the capital installation and of the additional fuel used to replace
the lignin removed.
4.3
The Structure of Lignin
4.3.1
Definition
The term “lignin” is often loosely used both for the lignin in the intact wood
and for preparations obtained from diverse procedures with the objective of sep-
arating the lignin from other cell-wall constituents. To be precise, the lignin in
the cell wall should be termed “protolignin”, and all other preparations “lignin
products” with a clear declaration of what procedure was used to obtain the lig-
nin. A published description (Brunow et al. 1998) gives an useful overview of
our present knowledge about the chemical composition of protolignin:
This description is not a chemical definition of protolignins, but summarizes
the main structural features based on knowledge available today. Protolignins
are biopolymers consisting of phenylpropane units with an oxygen atom at the
p-position (as OH or O–C) and with none, one, or two methoxy groups in the
positions ortho to this oxygen atom. These ortho positions may alternatively be
C-substituted or O-substituted with substituents other than methoxy. Only a few
of the aromatic units are substituted in other ring positions. A few percent of
the building blocks in protolignins are not phenylpropane units. The side chain
is missing or shortened, or the unit is replaced by a quinoid group. The phenyl-
propane units are attached to one another by a series of characteristic linkages
(b-O-4, b-5, b-b, etc.) or, alternatively, exist as members of a series of characteris-
tic end groups (e.g. cinnamaldehyde units). Practically all the types of structural
element detected in protolignins have been demonstrated to be formed on oxi-
dation of the p-hydroxycinnamyl alcohols in vitro (Freudenberg 1968; Adler
1977). The structural elements in protolignins are not linked to one another in
any particular order. Protolignins are not optically active. The polymer is
branched and cross-linking occurs. In addition, the following facts should be
noted: (1) there are strong indications of the occurrence of linkages between
protolignin and carbohydrates, (2) some types of protolignin are esterified with
phenolic acids (grass lignins with p-coumaric acid and other lignins, for exam-
ple aspen lignin, with p-hydroxybenzoic acid), and (3) scattered observations
suggest that there are some units, for example, dihydroconiferyl alcohol units,
that cannot be thought to have been produced by oxidation of p-hydroxycinna-
myl alcohols.
4.3.2
The Bonding of the Phenylpropane Units
Phenylpropane units of types 1 (guaiacylpropane), 2 (syringylpropane), and 3 (p-

hydroxyphenylpropane) are the main building blocks in lignins. The propor-
tions of 1–3 differ with the botanical origin of the lignin. The biosynthesis of
lignins is regarded as proceeding via oxidative polymerization of three primary
precursors – the p-hydroxycinnamyl alcohols 4–6. It has been shown that practi-
cally all the types of structural element detected in lignins are formed by in
vitro enzymic oxidation of the p-hydroxycinnamyl alcohols 4–6 and phenolic
lignin model compounds.
The growth of the lignin polymer is visualized as an oxidative coupling be-

tween a phenoxy radical formed from a monolignol (4–6) with a radical formed
from a phenolic center in the growing polymer. The isolation of numerous di-
meric lignin degradation products derived from lignin structures consisting of
different types of unit show that cross-coupling of units of types 1–3 occurs dur-
ing the biosynthesis.
It follows from the types of reaction that the structural elements in lignins
are not linked to each other in any particular order and that lignins do not have
optical activity. That this is the case has recently been demonstrated (Ralph et
al. 1999; Matsumoto et al. 1999). The reaction sequences do not rule out the
possibility that certain sequences of units are more probable than others.
There are strong indications of the occurrence of linkages between lignin and
carbohydrates. Certain types of lignin are esterified with phenolic acids (grass
lignins with p-coumaric acid and other lignins (e.g. aspen lignin) with p-hy-
droxybenzoic acid). It has been found that there are some types of units in lig-
nins, for example dihydroconiferyl alcohol units, that cannot be thought to be
produced by oxidation of p-hydroxycinnamyl alcohols.
The proportions of structural units of types 1–3 provides a basis for the clas-
sification of lignins. The composition of some important classes of lignins
based on this criterion are listed in Table 4.1.
According to tracer studies (Tomimura et al. 1980) roughly 50% of the 5-posi-
tions and a small percentage of the 2- and 6-positions in the guaiacyl units (1)
in softwood and hardwood lignins carry a C or O-substituent (“condensed
units”).
The so called nucleus exchange method gives similar results (Funaoka et al.
1992). As judged from a recent reassessment of the nucleus exchange method,
however, the number of condensed units determined by this method can be ex-
pected to be too low (Chan et al. 1995). Results based on studies of degradation
products obtained on permanganate oxidation of lignins point to a smaller pro-
portion of condensed guaiacyl units (condensed guaiacyl units/total number of
guaiacyl units * 0.4) (Erickson et al. 1973; Larsson and Miksche 1971). A combi-
nation of degradation (thioacidolysis) and subsequent examination by gel per-
Table 4.1 Approximate composition (%) of some important classes of lignins.
1 2 3
Softwood lignin 95% 1% 4%

Hardwood lignin a) 50% 50% 2%
Grass lignin b) 70% 25% 5%
Compression wood lignin 70% 0% 30%
a) Most hardwood lignins are composed of approximately equal

amounts of 1 and 2 but quite a few exceptions are known.
Some hardwood lignins are esterified with p-hydroxybenzoic acid.
b) p-Coumaric acid attached by ester linkages not included.
meation chromatography is an alternative procedure for determination of the

extent of condensation (Suckling et al. 1994).
The lignin polymer is expected to be branched and cross-linking may occur to
some extent. If the lignin were a linear polymer the number of interconnections
per unit in a molecule consisting of n units would be (n–1)/n and cannot exceed
1. This is true even if branching occurs. Cross-linking leads to the formation of
rings of units. A consequence of this is that the number of interconnections per
unit increases (values larger than 1 are possible). Available lignin data suggest
that the number of rings is small and it can therefore be assumed that the
number of interconnections per unit is close to 1.
In this chapter we have tried to amalgamate the results from studies of lignin
in situ with those obtained in studies of isolated lignins. Most of the data given
emerge from examinations of milled wood lignin (MWL), however (Björkman
1956; Lundquist 1992). Comparative studies of wood and MWL (Erickson et al.
1973; Lapierre et al. 1991; Rolando et al. 1992; Lapierre and Lundquist 1999)
suggest that MWL is, in most respects, representative of the lignin in wood.
The nature of the functional groups and bonding patterns in lignins are nowa-
days well established, although many results regarding the quantitative contri-
bution of different types of structural element are contradictory and controver-
sial. This is not surprising, because it is very difficult to obtain reliable quantita-
Scheme 4.1 Important structural features in lignins.

tive lignin data. An example of the types of difficulty encountered in quantita-

tive lignin analysis occurs in calculation of the abundance of condensed units
in lignins on the basis of 1H NMR spectroscopy (Ludwig et al. 1964; Lundquist
1980). Calculation of condensed units is based on integration of the signal from
aromatic protons (corrected for the contribution of some vinyl protons) together
with determination of the number of phenylpropane units and consideration of
the distribution of units 1–3 in the sample. A rather optimistic estimate of the
experimental error in a determination of the number of aromatic protons/unit
by this method would be ± 5%. If, for instance, the number of aromatic pro-
tons/unit is found to be 2.5 in a softwood lignin sample (composition, see Ta-
ble 4.1) a 5% error makes determination of the extent of condensation uncertain
(50% ± 12.5%). In this and in many other instances quantification of functional
groups and bonding patterns in lignins leads to uncertain results despite basi-
cally sound approaches. The problems related to lignin analysis are outlined in
Brunow et al. (1998).
4.3.3
Bonding Patterns and Functional Groups
4.3.3.1 General
Biosynthetic considerations are often used among the arguments for the exis-
tence of particular structural units in lignins. Biosynthetic arguments are only
pointed out occasionally in the discussion below. Each one of the types of unit
discussed below can be expected to be present in all the different classes of lig-
nin listed in Table 4.1. For simplicity the lignin units are usually depicted as non-
condensed guaiacylpropane units (1) but when applicable the formulas also represent
the other types of phenylpropane unit. Data given for hardwood lignins refer to such
lignins composed of similar amounts of units of types 1 and 2.
4.3.3.2 Survey of Different Types of Lignin Unit
Arylglycerol Units Attached to an Adjacent Unit by a b-O-4 Linkage Evidence for

the occurrence of arylglycerol units attached to an adjacent unit by a b-O-4 link-
age (linkages between units A and B, and D and E, see Scheme 4.1) as an im-
portant lignin structure was obtained by synthesis of appropriate model com-
pounds and comparison of their reactions with lignin reactions. Both models
and lignins gave, for instance, so called Hibbert ketones on acid degradation
(Adler et al. 1957). The existence of lignin structures of this type was later con-
firmed in numerous studies. It has been shown that all the three types of phe-
nylpropane unit (1–3) participate in structures of the arylglycerol b-aryl ether
type. Degradation products obtained from the three types of unit are, for exam-
ple, obtained on acidolysis (Lundquist 1992), thioacidolysis (Rolando et al. 1992)
and DRFC degradation (Lu and Ralph 1998). Distribution of the diastereomeric
forms in lignins has been studied by NMR spectral methods. The results are in
agreement to the extent that approximately equal amounts of the diastereomeric

forms are found in softwood lignins and there is a predominance of the erythro
form in hardwood lignins (Lundquist 1980; Nimz et al. 1984; Hauteville et al.
1986; Ede and Ralph 1996; Saake et al. 1996). Studies of the stereochemistry
based on the formation of threonic acid and erythronic acid on ozonation (Mat-
sumoto et al. 1986; Taneda et al. 1989; Sarkanen et al. 1992) are largely in accor-
dance with the NMR spectroscopic results. The distribution of diastereomeric
forms of arylglycerol units attached to guaiacylpropane (1) units and syringyl-
propane (2) units in a hardwood lignin has been studied using the 2D INADE-
QUATE experiment (Bardet et al. 1998). The results show that the predomi-
nance of erythro forms is because of the presence of large amounts of erythro
forms attached to syringylpropane units. NMR studies suggest there are 30–
40% units of this type in softwood lignins (Robert and Brunow 1984; Lundquist
1991; Jiang and Argyropoulos 1994) and 40–50% such units in hardwood lig-
nins (Lundquist 1991; Argyropoulos 1994). Most of the b-O-4 units in hard-
woods are linked to syringylpropane units (Bardet et al. 1998).
Phenylpropane Units Attached to an Adjacent Unit by a b-5 Linkage Units linked

to an adjacent unit by a b-5 linkage are present in phenylcoumaran structures
(B–C in Scheme 4.1). Freudenberg and co-workers (1968) have shown that per-
manganate oxidation of methylated lignin gives isohemipinic acid. This acid
can be expected to originate from b-5-linked units, and labeling studies have
shown that this is true to some extent (Freudenberg 1968). Acidolysis studies
provided evidence of the occurrence of a significant number of b-5 units in soft-
wood lignin (Adler and Lundquist 1963). Ozonolysis similarly confirms the oc-
currence of units of this type and indicates primarily the trans stereochemistry
(Habu et al. 1990). Attempts to detect the cis form in spruce lignin by 1H NMR
spectroscopy based on model compound data (Iliefski et al. 1997) have failed
(Lundquist 1999). The occurrence of b-5 units in lignins has been confirmed in
a large number of studies of degradation products (Lai and Sarkanen 1971) and
in several 1H NMR and 13C NMR spectroscopic studies. On the basis of UV
spectroscopic acidolysis studies (Li and Lundquist 1997) it was concluded there
are approximately 10% b-5 units in spruce lignin. This figure should be ad-
justed somewhat downwards because of interference of diguaiacylstilbene
formed from b-1 structures on acid treatment (Lundquist 1992; Lai and Sarka-
nen 1971). Recent acidolysis studies point to a frequency of 6–9% units in
spruce lignin (Li and Lundquist 1999). Permanganate studies suggest 12% b-5-
linked units (Erickson et al. 1973) and this agrees fairly well with results ob-
tained from ozonolysis studies (Habu et al. 1990). The number of units of this
type in hardwood lignins is comparatively small (Larsson and Miksche 1971;
Landucci et al. 1992).
Phenylpropane Units Attached to an Adjacent Unit by a b–b Linkage Units linked

to an adjacent unit by a b-b linkage are present in pinoresinol structures and in
analogous structures. Solvolytic (Lapierre et al. 1991; Wallis 1971) and reductive
(Lapierre et al. 1991; Nimz 1974; Sakakibara 1992) degradation studies provide
evidence for the occurrence of different types of pinoresinol structure in lignins.
Both pinoresinol and syringaresinol structures have been detected in lignins by
a variety of NMR spectroscopic techniques (Lundquist 1991; Kilpeläinen et al.
1994). Ogiyama and Kondo determined the abundance of b-b units in a soft-
wood lignin to be 5–10% (Ogiyama and Kondo 1968). NMR spectral studies
suggest somewhat lower values (Lundquist and Stomberg 1988).
The formation of 3,4-divanillyltetrahydrofuran on acidolysis (Lundquist and
Stomberg 1988; see also references cited therein dealing with the formation of
2,3-divanillyl-1,4-butanediol from lignin) or thioacidolysis of softwood lignin (La-
pierre and Lundquist 1999) suggests the occurrence of b-b-linked units with re-
duced side-chains. These structures cannot be formed by oxidation of coniferyl
alcohol.
Biphenyl Units, Dibenzodioxocin Structures and Diaryl Ether Structures The oc-
currence of biphenyl units in lignins has been demonstrated in studies of lignin
degradation products. Methylation then permanganate oxidation gives dehydro-
diveratric acid (Erickson et al. 1973). Degradation by reductive methods gives
dehydrodicoerulignol (Lapierre et al. 1991; Nimz 1974; Sakakibara 1992). This
provides unambiguous proof of the occurrence of phenylpropane units in lig-
nins attached to each other by a biphenyl linkage. Pew (1963) attempted to esti-
mate the number of biphenyl units in softwood lignin by a UV spectrometric
method. He concluded that “coniferous lignin may well contain 25% or more
biphenyl-linked units.” Permanganate oxidation studies suggested that 19% of
the lignin units are of the biphenyl type in softwood lignin (Erickson et al.
1973). This estimate is based on results obtained by permanganate oxidation of
lignin pre-treated by cupric oxide oxidation. Model compound studies (Pearl
and Beyer 1954; Bose et al. 1998) show that biphenyl coupling occurs to some
extent on cupric oxide oxidation. This indicates that the value derived from per-
manganate oxidation studies is slightly too high. On the basis 13C NMR exami-
nations Drumond et al. (1989) arrived at a higher value for the frequency of bi-
phenyl units (24–26%). The accuracy of this estimate can be questioned, be-
cause no separate signals from biphenyl units can be discerned in the spectra.
In view of the many sources of error the results obtained by different methods
are not directly incompatible. Estimation of biphenyl units is complicated by
the recent finding (Karhunen et al. 1995) that there are significant amounts of
dibenzodioxocin structures in lignins. Conclusive evidence of the occurrence of
units of this type (Scheme 4.1, units C–D) has been obtained by NMR spectral
studies (Karhunen et al. 1995). Tentative estimates suggest there are approxi-
mately 6% such units in softwood lignin (Sipilä and Brunow, unpublished re-
sults). This implies that approximately 12% biphenyl units may be present in
dibenzodioxocin structures. From model compound studies (Karhunen et al.
1999) it can be concluded that the biphenyl units in dibenzodioxocin structures
are included in the estimates of biphenyl units by NMR spectroscopy and per-
manganate oxidation. The number of biphenyl units is smaller in hardwood lig-
4.4 Role of Lignin in Biomass Conversion 159
nins. Permanganate oxidation suggests there are 9% such units in birch lignin
(Larsson and Miksche 1971).
The occurrence of diaryl ether structures in lignin was concluded on the basis
of permanganate oxidation (Freudenberg 1968) and was later confirmed in stud-
ies of lignin degradation by reductive methods (Lapierre et al. 1991; Nimz 1974;
Sakakibara 1992).
Permanganate oxidation studies suggest 3.5% of such units in softwood lig-
nin (Erickson et al. 1973) and significantly larger amounts (6.5%) in hardwood
lignin (Larsson and Miksche 1971).
Phenylpropane Units Attached to an Adjacent Unit by a b-1 Linkage The occur-

rence of b-1 structures of the 1,2-diaryl-1,3-propanediol type in lignins was dis-
covered by studying degradation products (Lundquist and Miksche 1965; Nimz
1965). Almost all types of solvolytic and reductive degradation of lignins yield
comparatively large amounts of dimeric products that can be envisaged as origi-
nating from structures of this type. Nevertheless, it has not, until recently, been
possible to obtain spectroscopic evidence of the occurrence of b-1 structures in
lignins (Kilpeläinen et al. 1994; Lundquist 1987). Degradation studies suggest,
however, much larger amounts of such structures than do the spectroscopic
studies. Two possible explanations of the discrepancy have been suggested – un-
even distribution of the b-1 structures in lignins (Lapierre et al. 1991; Ede et al.
1996) or formation of b-1 structures during lignin degradation from cyclohexa-
dienone precursors (Brunow and Lundquist 1991; Lundquist 1987). 1H NMR
spectroscopy suggest some 1–2% of such units in softwood lignins and perhaps
as much as 5% in hardwood lignins (Lundquist 1987). The number of cyclohex-
adienone units has been estimated to about 1% in softwood lignin in a recent
study (Zhang and Gellerstedt 1999).
4.4
Role of Lignin in Biomass Conversion
4.4.1
Introduction
Because of the very nature of this complex organic polymer and its derivatives,
there has been rather slow development towards an increasing number of uses
and products. Improved understanding of the structure of lignins and their de-
rivatives will help researchers to relate their experimentation to a fundamentally
sound framework of ideas.
4.4.2
Low-molecular-weight Chemicals from Lignin
At present only a few simple chemicals (such as vanillin, dimethylsulfoxide) are

produced in commercial quantities from lignin. The main reason is the absence
of competitive production methods. An important factor is the cost of separat-
ing the lignin from large volumes of aqueous industrial pulping liquors. Even
after drying the product is not pure lignin but contains the ash, carbohydrates,
and extraneous material of the spent liquor. In the long run lignin is a renew-
able resource but it may well be that it will become important only when petro-
leum becomes an expensive raw material.
4.4.3
Polymeric Products
The uses to which polymeric lignin products can be put may be broadly sepa-
rated into three classes: (1) combustion, (2) utilization of the surface active
properties of salts of a lignin derivative, and (3) condensation of lignin so that it
becomes an integral part of the product. A useful review of the utilization of
polymeric lignin products is found in Hoyt and Goheen (1971).
4.4.4
Biodegradation
White-rot fungi have been shown to degrade lignin in an essentially oxidative

process. Degradation of the lignin polymer occurs in the side chains, which are
oxidized with formation of carbonyl and carboxyl groups, and in the aromatic
nuclei, which are oxidatively cleaved after demethylation and introduction of hy-
droxyl groups in phenolic units to give 2,3- and/or 3,4-dihydroxyphenyl moi-
eties. It is assumed (Higuchi 1990) that various lignin structural units are de-
graded by the mediation of extracellular enzymes which attack both low-molecu-
lar-weight and polymeric substrates via many intermediate products along sev-
eral different pathways. In practice it has proved difficult to imitate with
individual enzymes the degradation of lignin achieved by basidiomycetes. Oxida-
tive enzymes (peroxidases and laccases) tend to polymerize the lignin via cou-
pling of radical intermediates. Further research is needed to clarify the mecha-
nism that prevents the polymerization when lignin is degraded in vivo.
References
Adler, E., JM Pepper, E Eriksoo. (1957) Ac- Adler, E., K Lundquist. (1963) Spectrochemi-
tion of mineral acid on lignin and model cal estimation of phenylcoumaran ele-
substances of guaiacylglycerol-beta-aryl ments in lignin. Acta Chem Scand 17:13–
ether type. Ind Eng Chem 49:1391–1392, 26, 1963.
1957.
References 161
Adler, E. (1977) Lignin chemistry – past, Freudenberg, K. (1968) The constitution and
present and future. Wood Sci Technol biosynthesis of lignin. In: K Freudenberg,
11:169–218, 1977. AC Neish, eds. Constitution and Biosyn-
Argyropoulos, DS. (1994) Quantitative phos- thesis of Lignin. Berlin–Heidelberg:
phorus-31 NMR analysis of six soluble lig- Springer, 1968, pp 47–122.
nins. J Wood Chem Technol 14:65–82, Funaoka, M., I Abe, VI Chiang. (1992) Nu-
1994. cleus exchange reaction. In: SY Lin, CW
Bardet, M., D Robert, K Lundquist, S von Dence, eds. Methods in Lignin Chemistry.
Unge. (1998) Distribution of erythro and Berlin: Springer, 1992, pp 369–386.
threo forms of different types of b-O-4 Habu, N., Y Matsumoto, A Ishizu, J Naka-
structures in aspen lignin by 13C NMR no. (1990) The role of the diarylpropane
using the 2D INADEQUATE experiment. structure as a minor constituent in spruce
Magn Reson Chem 36:597–600, 1998. lignin. Holzforschung 44:67–71, 1990.
Björkman, A. (1956) Studies on finely di- Hauteville, M., K Lundquist, S von Unge.
vided wood. Part I. Extraction of lignin (1986) NMR studies of lignins. 7. 1H
with neutral solvents. Sven Papperstidn NMR spectroscopic investigation of the
59:477–485, 1956. distribution of erythro and threo forms of
Bose, SK., KL Wilson, RC Francis, M Aoya- b-O-4 structures in lignins. Acta Chem
ma. (1998) Lignin analysis by permanga- Scand B40:31–35, 1986.
nate oxidation. I. Native spruce lignin. Higuchi, T. (1990) Lignin biochemistry: Bio-
Holzforschung 52:297–303, 1998. synthesis and biodegradation, Wood Sci.
Brunow, G., K Lundquist, G Gellerstedt. and Technol. 24, 23–63.
(1998) Lignin. In: E Sjöström, R Alén, eds. Hoyt, CH., DW Goheen. (1971) Polymeric
Analytical Methods in Wood Chemistry, Products. In: KV Sarkanen, CH Ludwig,
Pulping and Papermaking. Berlin: Sprin- eds. Lignins – Occurrence, Formation,
ger, 1998, pp 77–124. Structure and Reactions. New York: Wiley-
Brunow, G., K Lundquist. (1991) On the Interscience, 1971, pp 833–865.
acid-catalysed alkylation of lignins. Holz- Jiang, Z-H., DS Argyropoulos. (1994) The
forschung 45:37–40. stereoselective degradation of arylglycerol-
Chan, FD., KL Nguyen, AFA Wallis. (1995) beta-aryl ethers during kraft pulping.
Estimation of the aromatic units in lignin J Pulp Pap Sci 20:J183–J188, 1994.
by nucleus exchange – a reassessment of Karhunen, P., J Mikkola, A Pajunen, G Bru-
the method. J Wood Chem Technol now. (1999) The behaviour of dibenzodiox-
15:473–491, 1995. ocin structures in lignin during alkaline
Drumond, M., M Aoyama, C-L Chen, D Ro- pulping processes. Nord Pulp Pap Res J
bert. (1989) Substituent effects on C-13 14:123–128, 1999.
chemical shifts of aromatic carbons in Karhunen, P., P Rummakko, A Pajunen,
biphenyl type lignin model compounds. G Brunow. (1996) Synthesis and crystal
J Wood Chem Technol 9:421–441, 1989. structure determination of model com-
Ede, RM., J Ralph, KM Torr, BSW Dawson. pounds for the dibenzodioxocine structure
(1996) A 2D NMR Investigation of the het- occurring in wood lignins. J Chem Soc,
erogeneity of distribution of diarylpropane Perkin Trans 1 1996:2303–2308, 1996.
structures in extracted Pinus radiata lig- Karhunen, P., P Rummakko, J Sipilä, Bru-
nins. Holzforschung 50:161–164. now G. (1995) Dibenzodioxocins; a novel
Ede, RM., J Ralph. (1996) Assignment of type of linkage in softwood lignins. Tetra-
2D TOCSY spectra of lignins: the role of hedron Lett 36:169–170, 1995.
lignin model compounds. Magn Reson Kilpeläinen, I., E Ämmälahti, G Brunow, D
Chem 34:261–268, 1996. Robert. (1994) Application of three-dimen-
Erickson, M., S Larsson, GE Miksche. sional HMQC–HOHAHA NMR spectros-
(1973) Gaschromatographische Analyse copy to wood lignin, a natural polymer.
von Ligninoxydationsprodukten. VIII. Zur Tetrahedron Lett 35:9267–9270, 1994.
Struktur des Lignins der Fichte. Acta Kilpeläinen, I., J Sipilä, G Brunow, K Lund-
Chem Scand 27:903–914, 1973. quist, RM Ede. (1994) Application of two-
dimensional NMR spectroscopy to wood Lundquist, K., GE Miksche. (1965) Nach-

lignin structure determination and identif- weis eines neuen Verknüpfungsprinzips
ication of some minor structural units of von Guaiacylpropaneinheiten im Fichten-
hard- and softwood lignins. J Agric Food lignin. Tetrahedron Lett: 2131–2136, 1965.
Chem 42:2790–2794, 1994. Lundquist, K., R Stomberg. (1988) On the
Lai, YZ., KV Sarkanen. (1971) Isolation and occurrence of structural elements of the
structural studies. In: KV Sarkanen, CH lignan type (b–b structures) in lignins.
Ludwig, eds. Lignins – Occurrence, For- The crystal structures of (+)-pinoresinol
mation, Structure and Reactions. New and (±)-trans-divanillyltetrahydrofuran.
York: Wiley-Interscience, 1971, pp 165– Holzforschung 42:375–384, 1988.
240. Lundquist, K., S Li. (1999) Structural analy-
Landucci, LL., GC Deka, DN Roy. (1992) A sis of lignin and lignin degradation prod-
13
C NMR study of milled wood lignins ucts. Proceedings of 10th International
from hybrid Salix clones. Holzforschung Symposium on Wood and Pulping Chem-
46:505–511, 1992. istry Vol 1, Yokohama, 1999, pp 2–10.
Lapierre, C., B Pollet, B Monties, C Rolan- Lundquist, K. (1980) NMR studies of lig-
do. (1991) Thioacidolysis of spruce lignin: nins. 4. Investigation of spruce lignin by
1
GC–MS analysis of the main dimers re- H NMR spectroscopy. Acta Chem Scand
covered after Raney nickel desulphuration. B34:21–26, 1980.
Holzforschung 45:61–68, 1991. Lundquist, K. (1987) On the occurrence of
Lapierre, C., B Pollet, B Monties. (1991) b-1 structures in lignins. J Wood Chem
Heterogeneous distribution of diarylpro- Technol 7:179–185.
pane structures in spruce lignin. Phyto- Lundquist, K. (1991) 1H NMR spectral stud-
chemistry 30:659–662. ies of lignins. Quantitative estimates of
Lapierre, C., K Lundquist. (1999) Investiga- some types of structural elements. Nord
tions of low molecular weight and high Pulp Pap Res J 6:140–146, 1991.
molecular weight lignin fractions. Nord Lundquist, K. (1992) Acidolysis. In: SY Lin,
Pulp Pap Res J 14:158–162, 170, 1999. CW Dence, eds. Methods in Lignin Chem-
Larsson, S., GE Miksche. (1971) Gaschroma- istry. Berlin: Springer, 1992, pp 289–300.
tographische Analyse von Ligninoxy- Lundquist, K. (1992) Wood. In: SY Lin, CW
dationsprodukten. IV. Zur Struktur des Dence, eds. Methods in Lignin Chemistry.
Lignins der Birke. Acta Chem Scand Berlin: Springer, 1992, pp 65–70.
25:647–662, 1971. Matsumoto, Y., A Ishizu, J Nakano. (1986)
Li, S., K Lundquist. (1997) Synthesis of lig- Studies on chemical structure of lignin by
nin models of b-5 type. Acta Chem Scand ozonation. Holzforschung 40 (Suppl):81–
51:1224–1228, 1997. 85, 1986.
Li, S., K Lundquist. (1999) Acid reactions of Matsumoto, Y., T Akiyama, A Ishizu, G Me-
lignin models of b-5 type. Holzforschung shitsuka, K Lundquist. (1999) Proof of the
53:39–42, 1999. presence of racemic forms of arylglycerol-
Li, S., T Iliefski, K Lundquist, AFA Wallis. b-aryl ether structures in lignin – studies
(1997) Reassignment of relative stereo- on stereo structure of lignin by ozonation.
chemistry at C-7 and C-8 in arylcoumaran Proceedings of 10th International Sympo-
neolignans. Phytochemistry 46:929–934, sium on Wood and Pulping Chemistry Vol
1997. 1, Yokohama, 1999, pp 126–129.
Lu, F., J Ralph. (1998) The DFRC method Nimz, H. (1965) Über die milde Hydrolyse
for lignin analysis. 2. Monomers from iso- des Buchenlignins, II. Isolierung eines
lated lignins. J Agric Food Chem 46:547– 1.2-Diaryl-propan-Derivates und seine
552, 1998. Überführung in ein Hydroxystilben. Chem
Ludwig, CH., BJ Nist, JL McCarthy. (1964) Ber 98:3160–3164, 1965.
Lignin. XIII. The high resolution nuclear Nimz, H. (1974) Beech lignin – proposal of
magnetic resonance spectroscopy of pro- a constitutional scheme. Angew Chem Int
tons in acetylated lignins. J Am Chem Soc Ed 13:313–321, 1974.
86:1196–1202, 1964.
References 163
Nimz, H. H., U Tschirner, M Stähle, R Leh- NMR spectroscopy. Phytochemistry

mann, M Schlosser. (1984) Carbon-13 43:499–507, 1996.
NMR spectra of lignins, 10. Comparison Sakakibara, A. (1992) Hydrogenolysis. In:
of structural units in spruce and beech lig- SY Lin, CW Dence, eds. Methods in Lig-
nin. J Wood Chem Technol 4:265–284, nin Chemistry. Berlin: Springer, 1992,
1984. pp 350–368.
Ogiyama, K., T Kondo. (1968) On the Sarkanen, KV., A Islam, CD Anderson.
pinoresinol type of structural units in lig- (1992) Ozonation. In: SY Lin, C.W. Dence,
nin molecule. IV. The changes of dilac- eds. Methods in Lignin Chemistry. Berlin:
tone-yield during enzymic dehydrogena- Springer, 1992, pp 387–406.
tion. Mokuzai Gakkaishi 14:416–420, Suckling, ID., MF Pasco, B Hortling, J
1968. Sundquist. (1994) Assessment of lignin
Pearl, IA., DL Beyer. (1954) Studies on lig- condensation by GPC analysis of lignin
nin and related products. IX. Cupric oxide thioacidolysis products. Holzforschung
oxidation of lignin model substances. J 48:501–503, 1994.
Am Chem Soc 76:2224–2226, 1954. Taneda, H., N Habu, J Nakano. (1989) Char-
Pew, JC. (1963) Evidence of a biphenyl acterization of the side chain steric struc-
group in lignin. J Org Chem 28:1048– tures in the various lignins. Holzfor-
1054, 1963. schung 43:187–190, 1989.
Ralph, J., J Peng, F Lu, RD Hatfield, RF Tomimura, Y., T Yokoi, N Terashima. (1980)
Helm. (1999) Are lignins optically active? Heterogeneity in formation of lignin. V.
J Agric Food Chem 47:2991–2996, 1999. Degree of condensation in guaiacyl nu-
Robert, DR., G Brunow. (1984) Quantitative cleus. Mokuzai Gakkaishi 26:37–42, 1980.
estimation of hydroxyl groups in milled Wallis, AFA. (1971) Solvolysis by acids and
wood lignin from spruce and in a dehy- bases. In: KV Sarkanen, CH Ludwig, eds.
drogenation polymer from coniferyl alco- Lignins – Occurrence, Formation, Struc-
hol using 13C NMR spectroscopy. Holz- ture and Reactions. New York: Wiley-Inter-
forschung 38:85–90, 1984. science, 1971, pp 345–372.
Rolando, C., B Monties, C Lapierre. (1992) Zhang, L., G Gellerstedt. (1999) Detection
Thioacidolysis. In: SY Lin, CW Dence, and determination of carbonyls and qui-
eds. Methods in Lignin Chemistry. Berlin: nones by modern NMR techniques. Pro-
Springer, 1992, pp 334–349. ceedings of 10th International Symposium
Saake, B., DS Argyropoulos, O Beinhoff, O on Wood and Pulping Chemistry Vol 2,
Faix. (1996) A comparison of lignin poly- Yokohama, pp 164–170.
mer models (DHPs) and lignins by 31P
165
5
Industrial Lignin Production and Applications
E. Kendall Pye
5.1
Introduction
As one of the three major polymers always present in woody biomass, lignin
represents a considerable proportion of the structural components of plants. It
can account for approximately 10–12% of the aerial portion of some short an-
nual plants and up to 30% or more for some coniferous trees [1]. As such it is
claimed to be the second most abundant organic chemical on earth [2] and is
therefore a major renewable chemical that should not be overlooked, either in
volume or value. Significant commercial markets, totaling over one million tons
per year, already exist for lignins that have been recovered from chemical pulp
mills [3]. The value of lignin in these markets is generally an order of magni-
tude higher than its fuel value. With the anticipated future construction of bio-
refineries processing lignocellulosic feedstocks, the amount of lignin potentially
available for marketing for its chemical value, rather than its fuel value, is likely
to be enormous. Furthermore, lignin from some types of biorefineries will most
probably have better performance characteristics and be of greater commercial
value for its chemical properties than lignins from existing chemical pulping
operations. The availability of these “new” lignins with enhanced physical and
chemical properties will no doubt stimulate major new markets for this renew-
able material. However, it is likely that these materials will find their initial mar-
kets in the same sectors as the current lignin products. A review of the manu-
facturing, properties and present markets for lignins from pulping operations is
therefore instructive in assessing the potential economic value of lignin that
might be recovered from biorefineries processing lignocellulosic materials.
Presently lignin is separated on an industrial scale from wood and from some
annual plants, such as straw, bagasse and flax, by the chemical pulping indus-
try, using primarily the kraft, sulfite and soda pulping processes [4]. The vast
majority of this lignin is not isolated and recovered but is burned in chemical
recovery boilers as components of the concentrated black liquor feed. As such it
provides low value fuel to the pulp mill for the production of steam and power
ISBN: 3-527-31027-4
166 5 Industrial Lignin Production and Applications
[4]. A small number of companies have, on the other hand, introduced systems
for the partial recovery and purification of lignin and have developed significant
businesses that market these materials to a large number of different industries
for diverse applications [3].
In future, biorefineries processing lignocellulosic materials with the primary pur-
pose of producing fermentable sugars from cellulose and hemicellulose, will gen-
erate lignin in very substantial quantities. The lignin produced by these operations
will almost certainly be chemically different from the presently available industrial
lignins and they will most probably be closer to the chemical structure of the native
material. Consequently, new applications will arise for these more versatile prod-
ucts, but they will also be capable of competing in the existing lignin markets, re-
presenting strong competition to the presently available lignin products.
The lignins that are currently produced and marketed today are recovered
from the cooking liquors of the chemical pulping industry. They are, however,
chemically modified to a greater or lesser extent by the chemistry of the specific
chemical pulping process from which they are derived [1]. In the sulfite pulping
process, for example, sulfonic acid groups are introduced into partially hydro-
lyzed lignin in the wood converting it into a fully water-soluble lignosulfonate.
This becomes a major component, together with the hemicellulose sugars, of
the spent sulfite liquors. In the kraft pulping process the native lignin is con-
verted into lower molecular weight fragments of thiolignin by the introduction
of thiol groups through the action of the sodium sulfide employed in the pro-
cess. This thiolignin is soluble in the strong alkali present in the cooking liquor,
but can be precipitated and recovered by acidification of the kraft black liquor
[1]. Soda pulping causes a hydrolytic cleavage of the native lignin into smaller
fragments that are soluble in the strongly alkaline cooking liquors, but the re-
sultant lignin is otherwise relatively unmodified chemically.
The markets and the applications of the various forms of lignin presently re-
covered from commercial chemical pulping operations are therefore determined
by the nature of the lignin that is produced by each process. As mentioned pre-
viously, when compared with the total amounts that are separated from wood
and woody fibers by the chemical pulping industry, the amounts of the various
forms of lignin that are presently recovered and sold commercially are very
small, representing a few percent of all that is extracted by that industry. World-
wide, almost all of the lignin separated from woody feedstocks by the pulping
industry is burned in chemical recovery furnaces because of the need to recover
and recycle the inorganic cooking chemicals. An exception to this is in some
lesser developed countries where many small soda pulp mills that process an-
nual fibers cannot afford chemical recovery boilers and therefore dispose of
their cook liquors into the environment, either directly or following minimal
treatment. This latter activity is now being actively discouraged by almost all
governments, with one result being the closure over the last decade of many
small chemical pulp mills, especially in China [5]. In the larger chemical pulp
mills, where chemical recovery boilers are an economic necessity, the lignin is
part of a low value fuel that provides steam and power to the pulp mill.
Several lignocellulosic biorefinery technologies that are now under develop-

ment produce lignin as a wet, solid residue that remains after the saccharifica-
tion and/or fermentation stages, often in a form contaminated with residual cel-
lulose. As such, these particular biorefinery processes find it most convenient to
use this lignin residue as a low value boiler fuel to provide power and steam for
the process [6]. On the other hand, pure lignin that can be readily produced by
some biorefinery technologies, such as organosolv technology, could be an excel-
lent source of industrial specialty and commodity chemicals, displacing materi-
als now produced from crude oil and natural gas [7]. Lignin, with its dominant
aromatic chemical structure, represents a potential new source of aromatic
chemicals and other products that are used extensively in industry. Simply
using this versatile material as a fuel would represent its lowest value applica-
tion.
In principle, using known technology, higher value, purified lignin can be re-
covered equally effectively from either the original woody biomass feedstock
prior to saccharification, or from the residues of the saccharification and/or fer-
mentation stages. However, it is now becoming well recognized that the pres-
ence of lignin and also hemicellulose to some extent, can significantly inhibit
the velocity and efficiency of cellulose saccharification by cellulolytic enzymes,
and even saccharification by acid hydrolysis [8]. Feedstock pretreatment by
either mechanical or chemical methods, which partially removes or modifies
these two materials, is therefore a universal feature of almost all presently pro-
posed biorefinery processes. Most of these pretreatments, such as steam explo-
sion [9], dilute acid hydrolysis [10], and ammonia fiber explosion (AFEX) [11],
serve to disrupt the physical and possibly the chemical relationships between
the cellulose, hemicellulose and lignin, which in native woody biomass is highly
integrated. Such pretreatments can influence the chemical structure and the
physical properties of the lignin making it more or less valuable in potential
commercial applications. Most of the proposed biorefinery pretreatments will
produce a lignin that has been partially depolymerized by hydrolysis, mostly at
the aryl–alkyl ether linkages [12], creating a lignin product that, unlike the ma-
jority of presently available lignins, has no sulfur-containing substituents. Pro-
vided that the conditions of the pretreatment and the lignin recovery do not in-
troduce substantial recondensation of the lignin fragments the number average
and the weight average molecular weight of these lignin products should be
small (1000–2000) and the polydispersity should be low [7], leading to a very
uniform product. This is different from the situation that exists for currently
available commercial lignins. Lignosulfonates from the sulfite pulping process,
for example, have a very high degree of sulfonic acid substitution and a very
high polydispersity [13].
Over the past fifty years or so, numerous research papers and conferences
have been devoted to the development of specialty and commodity applications
for pure, unmodified lignin [3, 14, 21], but today few of these exciting applica-
tions have been commercialized. The reason is clear. Until very recently, the
only lignins available commercially and in large quantities were thiolignins
from the kraft pulping process and lignosulfonates from the sulfite pulping pro-
cess. These chemically-modified lignins are presently being marketed primarily
by two major suppliers with roots in the pulp and paper industry, the Specialty
Chemicals division of MeadWestvaco Corporation and Borregaard Lignotech di-
vision of Borregaard Industries [3]. Chemically nonderivatized lignins, of the
type used in many investigative studies and of the type likely to be produced by
biorefineries, have not been available in the quantities required to support a sig-
nificant commercial market.
With the anticipated commercial introduction of biorefineries processing lig-
nocellulosics, this situation is about to change. These biorefineries could pro-
duce large quantities of lignin in a relatively uniform and purified form. Such
lignins can either be used for low value fuel, or they can be used for much
higher value industrial chemical applications, which would substantially im-
prove the economics of the biorefineries producing them. The lignin output
from these future biorefineries could become the raw material for a major new
industry making products that either substitute for chemicals presently made
from crude oil or natural gas, or products that are new to the market place.
Some current and future market and product opportunities are presented in
this chapter.
5.2
Historical Outline of Lignin Production and Applications
An excellent and detailed account of the discovery and history of lignin, as well
as the history of the chemical pulping industry, has recently been provided by
J. L. McCarthy and A. Islam [15]. Starting with the discovery of lignin in 1838
by Payen in France [16], there has been a substantial continuing interest in lig-
nin both at the industrial and the chemical research levels. This interest acceler-
ated with the introduction of a process in 1866 to produce pulp for papermak-
ing from woody materials by chemical pulping (in which the bulk of the lignin
is removed in order to release intact individual fibers) using sulfurous acid [17].
Before this time high quality printing and writing paper had been produced al-
most exclusively from old rags.
5.2.1
Lignosulfonates from the Sulfite Pulping Industry
The first chemical wood pulp mill, using a calcium based sulfite liquor, was
built in Sweden in 1874 followed by a mill in the US in 1882 and a mill in Ca-
nada in 1888 [15]. From that time on the sulfite pulping process became the
dominant chemical pulping process for wood until the kraft process started to
expand in the 1930s following the invention of the Tomlinson recovery furnace.
Prior to this time, the lignin-containing “spent liquors” from the sulfite pulp
mills were discharged into the environment, usually into the nearest waterway.
As this practice became more socially and environmentally unacceptable alterna-

tive disposal mechanisms were introduced including the spraying of these mate-
rials onto road surfaces for dust suppression and their inclusion into animal
feeds. These spent sulfite liquors from the sulfite pulping industry were erro-
neously referred to as “lignin” in the trade, a misnomer that still is problematic
for many attempts to convince individuals in the pulp and paper industry of the
high value of purified lignin. In fact, the solids composition of this sulfite spent
liquor, known as “lignin”, includes a substantial quantity of inorganic materials
and even the organic fraction contains approximately 50% sugars, which ac-
counts for its value as an animal feed additive. Lignosulfonate therefore repre-
sents considerably less than 50% by weight of the total solids of the so-called
“lignin” from a sulfite mill.
In the early part of the twentieth century a small number of sulfite pulp mills
began to consider the potential commercial value of purified lignosulfonates
and initiated investigations on applications and commercial development. Per-
haps the earliest of these, in 1909, was the Marathon Corporation at their
Rothschild mill near Wausau, Wisconsin, in the US [18]. This led, starting in
1927, to the development of commercial products from the organic components
of the spent sulfite liquor and was a milestone in the commercial development
of lignin-based products. Full-scale commercial production of lignosulfonate
products started at Marathon with the construction of a production facility in
1936. Marathon, after passing through several corporate hands that included
American Can, Reed Lignin and Daishowa Chemicals, is now the North Ameri-
can flagship site of Borregaard LignoTech, Inc., the major supplier of special-
ized products from lignosulfonates that are produced by the sulfite pulping in-
dustry [18].
With the successful introduction of chemical recovery furnaces for the kraft
process in the 1930s, many sulfite mills changed from the classical calcium-
based sulfite liquors to using pulping liquors containing cations such as so-
dium, ammonium and magnesium [15]. These systems provided not only tech-
nical advantages for the pulping operations, but also allowed the use of recovery
furnace systems that recovered both the cation and the SO2. In these systems
the lignosulfonate is burned and it is therefore no longer available for industrial
applications. Today, purified lignosulfonates are produced from those sulfite
pulp mills that do not practice chemical recovery, or that have spent liquor in
excess of their capacity to burn it.
5.2.2
Lignin from the Kraft Pulping Industry
With the invention of the Tomlinson chemical recovery furnace in the early
1930s the kraft process started to displace the sulfite process as the primary
chemical wood pulping technology, except for the production of some specialty
pulps such as dissolving pulps. Even though a number of specialty sulfite pulp
mills have been constructed in the intervening period, the sulfite pulping indus-
try has been a steadily declining fraction of the overall chemical pulping indus-
try since that time; a trend that continues today.
In 1942, at Charleston, South Carolina, the Westvaco Company started to pro-
duce lignin products from the black liquors obtained from the kraft pulping of
softwoods and hardwoods. That company, now MeadWestvaco Corporation,
through its Specialty Chemicals division, continues as the dominant supplier of
lignin products obtained from kraft black liquor. While this business appears to
be quite profitable, MeadWestvaco has attracted few competitors for its kraft lig-
nin business, probably because the recovery of kraft lignin from black liquors is
neither simple nor inexpensive. Furthermore, the manufacture and marketing
of lignin products appears as a complicating divergence from their principle
business for most pulp and paper companies. Today, vast quantities of lignin
are produced in the kraft industry worldwide, probably greater than 70 million
tonnes per year, but of this amount more than 99% is burned in chemical re-
covery furnaces and is not recovered for industrial applications. Separation and
recovery of significant quantities of lignin from kraft black liquors changes the
organic to inorganic ratio in the recovery furnace feed and will potentially dis-
rupt operations of those critical pieces of equipment. With the huge scale of
present day chemical pulp mills, any possibility for an expensive disruption of
normal operations is carefully avoided by most company managers.
It is likely that no more than 100 000 tonnes per year of kraft lignin is mar-
keted for its chemical value, worldwide. However, a significant fraction of this
amount is chemically modified to convert it into a water-soluble sulfonated lig-
nin that competes in some applications against lignosulfonates from the sulfite
pulping industry [19].
5.2.3
Lignin from the Soda Pulping Industry
While it is the oldest of the chemical pulping processes, the soda process [4], in
which chemical pulp is produced by the delignifying action of sodium hydrox-
ide, is now almost exclusively used for the production of chemical pulps from
annual plants, such as sugar cane bagasse, flax and cereal straws. Soda pulping
is not as effective a pulping process as kraft or sulfite when applied to either
hardwoods or softwoods. Because of the relatively high ash content of the raw
materials, annual fiber pulp mills usually face the serious problem of a high in-
organic content, particularly silica, in the soda black liquors, which restricts
their ability to use chemical recovery methods similar to the kraft process.
Furthermore, because of the seasonal nature of the availability of the annual fi-
ber feedstock most of these mills are very small and are mostly located in devel-
oping countries. In the past, the standard treatment of the black liquors from
these pulp mills has been to discharge them directly into the environment. Lig-
nin has not been recovered from such sources in the past primarily because of
the relatively unsophisticated nature of the industry in the regions that practice
soda pulping.
However, the lignin produced in the soda pulping of annual fibers is poten-
tially a very useful material. Unlike kraft and sulfite lignins, soda lignins have
not had sulfur introduced into their chemical structure. In addition, they have
interesting thermal and solubility properties that make them especially valuable
in certain commercial applications. One company, Granit S.A., of Lausanne,
Switzerland, is now selling sulfur-free lignin from the soda pulping of annual
fibers for higher value applications [20]. As noted above, most annual fiber pulp
mills are small and located in developing countries, so the availability of this lig-
nin is a concern as the markets for it expand. Granit has addressed this prob-
lem in an innovative way. Recognizing that small annual fiber soda pulp mills
have severe difficulties dealing with their silica-containing pulping liquors,
Granit has developed a technology that allows these mills to solve their liquor
disposal problems while at the same time providing Granit with the lignin prod-
uct it requires for its lignin marketing business [22].
The lignin recovery process developed by Granit, called the Lignin Precipita-
tion System (the LPS process), recovers lignin from the soda black liquor [23].
In this process, the black liquor is first filtered to remove any contaminating
pulp fibers. The filtered liquor is then acidified to create a lignin slurry, which
is conditioned in a maturation step and then filtered to remove the lignin sol-
ids. Following washing, the lignin cake is dried to a powder of high purity lig-
nin containing about 5% moisture. The removal of lignin reduces the COD of
the original black liquor by about 50%, which can then be treated either by
anaerobic digestion or by wet oxidation. The capital cost of the combined sys-
tem would appear to much lower than that of a typical alkali recovery system
used in conventional pulp mills, which in any case would be difficult to scale
down to a size appropriate for the small annual fiber mills. The interesting fea-
ture of this business arrangement is that Granit will purchase the high purity
dried lignin powder from the mill [24]. Thus, installation of the LPS system in-
creases revenues to the mill, solves an environment problem and generates lig-
nin product that is required by the lignin marketing arm of Granit. The first
commercial installation of the LPS technology was undertaken at the flax pulp-
ing mill of Papeteries du Léman in Thonon, France, in 2000 [20]. In this opera-
tion the entire black liquor generated from the soda pulping of flax is fed into
the relatively small LPS lignin recovery unit. After the lignin is filtered from the
slurry and dried, the filtrate is sent presently to a municipal treatment plant,
but soon it will be treated in a Granit wet oxidation reactor that will be part of a
new on-site effluent treatment plant. A second LPS system is under construc-
tion at a mill in India and is anticipated to be operational in 2005. The potential
lignin production is over 10 000 tonne per year of dried high purity lignin pow-
der ready for the market. Interesting alternative uses of the LPS technology are
for the debottlenecking of an existing conventional alkali recovery system, or for
the expansion of pulping capacity in an existing mill.
5.3
Existing Industrial Lignin Products
5.3.1
Lignosulfonates
As previously stated, the sulfite pulping industry, in its earlier days, simply dis-
charged its spent liquor into the environment. As this practice became less ac-
ceptable and also for economic reasons, some in the industry started to look for
alternative uses for the spent sulfite liquor. Initially, the simplest approach was
to partially evaporate the liquor to reduce its volume and its transportation cost
and then to use the concentrated material as road dust suppressant, or animal
feed supplement [3]. One of the processes used to upgrade the spent liquors in-
volves the fermentation of the sugars in the liquor, which represents close to
50% of the organic material, to produce food and fodder torula yeasts. The re-
maining partially purified lignosulfonates can then be concentrated and modi-
fied to produce a series of industrial products suitable for commercial applica-
tion. Such a plant is operated by Wausau Paper Mills at the Rhinelander sulfite
mill in Wisconsin, but other similar plants have been constructed in the wes-
tern US but are not presently operating. As the sulfite pulp mills that supplied
the spent liquors were closed the fermentation and lignosulfonate plants were
also mostly closed.
5.3.1.1 Chemical Characteristics of Lignosulfonates

The sulfite pulping process can be operated either at acid, neutral or alkaline
pH. In all cases the native lignin in the wood is cleaved into smaller fragments
and the bulky sulfonic acid group is introduced at the a and/or the c carbons of
the phenylpropane unit of the lignin. The reactions responsible for this modifi-
cation include sulfonation (the introduction of the sulfonic acid group mostly at
the a-position), hydrolysis (the hydrolytic cleavage of the aryl–alkyl ether bonds
between the phenylpropane groups, mostly the b-O-4 linkage), sulfitolysis (simi-
lar to hydrolysis, but leads to the formation of lignosulfonic acid, and condensa-
tion (reactions that produce new linkages between the lignin fragments, result-
ing in higher molecular weight components). The resulting lignosulfonate can
have a broad range of molecular weights that might range from several hundred
up to ninety thousand daltons, or greater. Unless deliberately fractionated, the
same lignosulfonate product will span this entire molecular weight range, lead-
ing to a relatively nonuniformly functioning product in some applications. Be-
cause of the introduction of the bulky, polar sulfonic acid groups into the mole-
cule, lignosulfonates are water soluble over essentially the entire useful pH
range. Consequently, they find their greatest utility in applications that exploit
their surfactant and dispersant properties.
The sulfonic acid group cannot be readily or economically removed from the mol-
ecule. Therefore lignosulfonates, especially with their high polydispersity, do not
represent a useful source of underivatized sulfur-free lignin. Lignosulfonates are

considered to be non-toxic at typical usage levels [3] and are regularly used in animal
feeds and agricultural and horticultural applications.
5.3.1.2 Lignosulfonate Producers

With the steady decline in the number of operating sulfite pulp mills worldwide,
the number of lignosulfonate producers has declined and these have been conso-
lidated into just a few suppliers [3]. Over the last thirty years or so, previously well-
known suppliers of lignosulfonates, such as Crown Zellerbach, Rayonier Silvi-
chemicals, American Can, Georgia-Pacific and Reed Lignin have gone out of the
business. Almost always this was because of the sale, or more frequently the per-
manent closure, of the sulfite pulp mill that supplied the raw material for the busi-
ness. A classic example is that of Georgia-Pacific which had a major lignosulfo-
nates business, producing almost 200 000 tonnes per year of solids, until the com-
pany closed its Bellingham, Washington, sulfite mill in March 2001 for reasons
unrelated to the ligosulfonate business. At this point, without a supply of raw ma-
terial Georgia-Pacific exited the lignosulfonates business.
Today the worldwide lignosulfonates business is dominated by Borregaard
LignoTech, which makes a number of products and grades. LignoTech has op-
erations worldwide and markets about 400 000 tonnes of lignosulfonate solids
per year [3]. There are several smaller sellers of lignosulfonate products, includ-
ing Tembec, Fraser Papers and Nippon Papers in Japan that have total sales just
less than those of Borregaard. Even as recently as twenty years ago it could be
calculated that the worldwide production of commercial lignosulfonate chemi-
cals was well in excess of 1.2 million tonnes per year. But, with the precipitous
closure in 2001 of the Bellingham sulfite mill that was a major production
source, the available supply of lignosulfonates dropped to below 800 000 tonnes
per year, which caused a considerable short term market disruption. Earlier, in
1997, Borregaard assured itself of an additional significant supply of lignosulfo-
nates when it signed an agreement with Sappi/Saiccor to construct a 55 000 ton
per year lignosulfonates plant at its Umkomass sulfite mill in Natal, S. Africa
that started up in 1998. This plant was expanded to 155 000 tonnes per year in
2003 partly in response to the capacity shortfall caused by the closure of the
Georgia-Pacific Bellingham mill [18]. The Umkomass mill has two pulping
lines: one a calcium sulfite line of approximately 450 tonnes per day of pulp,
while the other is a magnesium sulfite line of approximately 550 tonnes per
day of pulp that has its own recovery boiler. At the time, this calcium sulfite
line represented one of the larger non-utilized lignosulfonate sources available
for lignosulfonate recovery anywhere in the world. The project also benefited
Sappi by removing one of its environmental problems, since in the past the mill
had discharged the spent liquors into the ocean [18].
An example of a typical smaller lignosulfonate producer is Fraser Papers Inc.
that produces about 170 tons of liquid lignosulfonate products daily at its Park
Falls, Wisconsin, mill. This pulp mill produces 170 tons of calcium bisulfite
birch hardwood pulp per day. These lignosulfonate products are sold in liquid
form having a solids content of between 52% and 62% and a pH as low as 2–3
and as high as pH 7–8, depending on the grade. The more purified grades,
which are used primarily as binders and dispersants, have approximately 5% su-
gars, 4–5% calcium, and 6% sulfur as a percent of dry matter, with up to 80%
of the solids being lignin. The less pure grades would have between 30% and
35% of the solids as sugars and about 55% as lignin.
5.3.1.3 Markets for Lignosulfonates

Many smaller lignosulfonate producers still rely on the traditional markets for
spent sulfite liquors such as road dust suppression, feed pellet binders and ani-
mal feed additives. But the larger producers exploit newer, more specialized
markets with products that are relatively more purified and refined. A partial list
of applications and markets for lignosulfonates is given in Table 5.1.
In many of these applications lignosulfonates act as dispersants, emulsifiers
and surfactants. For example, in concrete admixtures, the lignosulfonate per-
forms as a plasticizer, reducing the amount of water required to produce a
workable concrete mixture and thereby creating a final concrete having greater
compressive strength, durability, density and better uniformity. They act in a
similar manner in the production of bricks, ceramics and refractories by dis-
persing the clay particles and reducing the amount of water required in the
early stages of manufacture. This reduces drying costs and increases the “green”
strength of the products, which holds the shape better before the formed bodies
are sent to the kiln for firing.
As binders in feed pellet production and for agricultural fertilizers and miner-
als, the lignosulfonates coat and bind the small particles together providing
greater granule strength, less dusting and better handling. Because of the water
solubility of the lignosulfonates, the products break down easier in the environ-
Table 5.1 Major applications of lignosulfonate products.
Applications of crude spent liquor lignosulfonates Applications of refined lignosulfonates
Feed and pellet binders Oil well drilling fluids

Feed molasses extenders Dye and pigment dispersants
Dust suppression and road stabilization Protein precipitants
Granulation and agglomeration Tanning agent
Plant micronutrients and horticulture Gypsum board manufacture
Agricultural dispersants and emulsifiers Cement manufacture
Grinding aids Concrete admixtures
Metal ore processing Refractory clays and ceramics
Carbon black
Phenolic resins
Lead acid storage battery plates
ment or, in the case of feed pellets, in the animals digestive tract. An added ad-
vantage in pelletizing operations is that the lignosulfonates provide lubrication
for the die of the pellet forming machine, which reduces the energy consump-
tion and increases productivity of the pelletizing equipment.
5.3.2
Kraft Pulping and Kraft Lignin Recovery
The kraft pulping process has now become the dominant chemical pulping process
for making pulp from wood. It uses a combination of sodium hydroxide and sodium
sulfide to delignify the wood by causing thiolysis mostly of the aryl–alkyl ether lin-
kages, which occur predominantly at the b carbon of the phenylpropane unit. This
thiolysis introduces a thiol group into the partially depolymerized lignin, which can
account for about 2–3% by weight of the lignin product. This lignin becomes a
component of the kraft black liquor, which in almost all kraft pulp mills is evapo-
rated to greater than 60% by weight of solids and then burned in an on-site kraft
chemical recovery boiler [4]. The purpose of the recovery boiler is to allow the recover
and recycle of the inorganic cooking chemicals. The burning of the concentrated
black liquor produces steam, which can be used for power production and also pro-
cess steam. To recover kraft lignin it is necessary to remove some lignin from the
black liquor prior to its entry into the recovery boiler. This can be done by acidifying
the black liquor, initially with carbon dioxide from flue gas, and then by addition of
sulfuric acid. A precipitate of sodium lignate is formed, which is coagulated and
filtered and in most circumstances dried. Only a small portion of the lignin in
the black liquor can be recovered otherwise the organic content of the concentrated
liquor becomes too low to support combustion in the recovery boiler.
5.3.2.1 Producers of Kraft Lignin

At this time the one major producer of kraft lignin is the MeadWestvaco Cor-
poration at Charleston Heights, South Carolina, USA. Kraft lignin is also sold
by Borregaard LignoTech. MeadWestvaco uses some of its production of kraft
lignin internally to make sulfonated derivatives for many of the lignosulfonate
markets and other products.
5.3.2.2 Markets for Kraft Lignin

Kraft lignins find applications [19] mostly as:
· Rubber reinforcers
· Activated carbon
· Carbon black substitutes
· Phenolic resin components
· Raw materials for production of methylsulfonates, which find applications in
mostly the same markets as lignosulfonates, but in some cases with superior
performance.
5.3.3
Lignins Produced from the Soda Process
As stated previously, the Swiss company Granit is now producing and market-
ing annual fiber lignin from the soda pulping process [20]. The markets for this
product are developing rapidly and it is finding applications in many of the
markets currently being exploited by kraft lignin producers. However, with its
particular advantageous properties it is likely to find new markets, especially in
animal nutrition and health, which are not presently being served by other
forms of lignin.
5.3.4
Lignin from Other Biomass Processing Operations
Furfural is made by treatment of pentose-rich biomass, such as bagasse and oat

hulls, with strong mineral acid at high temperature. These conditions favor the
hydrolysis of hemicellulose and the dehydration of the resultant pentose sugars
to furfural. Following recovery of the furfural a solid residue consisting mostly
of lignin and cellulose remains in the digester. This residue can be extracted
with solvent or alkali to dissolve and recover a substantial fraction of the lignin.
One commercial form of this lignin, derived from bagasse residues from a fur-
fural plant, is known as Sucrolin. Because it is derived from an annual fiber,
the methoxy content of this lignin is extremely low, which could imply greater
reactivity than lignins from wood. The solubility characteristics of this lignin,
however, are similar to kraft lignin and it might be anticipated that the lignin is
more condensed than lignins produced under less intense conditions, such as
the soda process.
5.3.5
Comparisons of the Physical and Chemical Properties of Commercially Available
Lignins
A general comparison of the chemical and physical properties of the three com-
mercially available lignin is given in Table 5.2. It can be seen that lignin from
the soda process is distinguished from the other two since it contains no mea-
surable sulfur and has a very low water solubility.
Table 5.2 Comparative properties of commercial lignins.
Property Softwood kraft lignin Softwood Soda lignin from straw

lignosulfonate
Carbon, % 66 53 56
Hydrogen, % 5.9 5.4 7.5
Methoxy, % 14 12.5 N/A
Ash, % 3 2.5 < 2.5
Wood sugars, % Low Up to 50% 2.5–3.5
Sulfur, % 1.6 6–7.9 N/A
Water solubility Low Very high Very low
Tg, 8C 140 Not detected 150
Softening pt. 8C Not detected Not detected N/A
Mol. wt., MN 2000 400–150 000 2300–2900
5.4
Lignin from Biorefineries
5.4.1
Advantages of Lignin and Hemicellulose Removal on Saccharification
and Fermentation of Cellulose
Strong mineral acid can be employed to saccharify cellulose, as was done in the
Scholler process and other processes reviewed by Wenzl [25], to produce ethanol
from wood. However, acid hydrolysis creates a number of significant difficulties.
Strong mineral acid will readily penetrate the walls of the woody material and
quickly initiate both cellulose and hemicellulose saccharification but, being a
rather indiscriminate catalyst, it also induces other less desirable reactions in
wood hydrolysis. These side reactions include the dehydration of pentose and
hexose sugars to form furfural and 5-hydroxymethylfurfural, two potent inhibi-
tors of fermentation organisms. This not only makes it difficult to ferment the
resultant sugars directly, but it also leads to a significant reduction of glucose
yield below the theoretical maximum. Additionally, strong mineral acid, at the
elevated temperatures required for cellulose hydrolysis, induces potentially un-
desirable condensation reactions in the lignin, thus reducing its versatility and
value as a chemical intermediate and product. Consequently, lignin recovered
from acid hydrolysis processes is usually utilized only as a boiler fuel.
Cellulase enzymes are much more specific catalysts that operate closer to am-
bient temperature than do acid hydrolysis systems. They do not catalyze reac-
tions in lignin, although they may have secondary hydrolytic activity on hemi-
cellulose. Neither do they create fermentation inhibitors. As a consequence, they
have the ability to carry out almost complete saccharification of relatively clean
cellulose, when the enzyme profile is appropriately adjusted. The glucose yields
from cellulose using enzymatic hydrolysis can be greater than 90% of theoreti-
cal [26], compared with acid-catalyzed hydrolysis which may be little better than
70% of theoretical. For these and other reasons the technology focus for future
lignocellulosic biorefineries has now mostly shifted towards enzymatic saccharif-
ication of cellulose, especially with the recent major reductions in the price of
cellulase enzymes [27]. Starch saccharification has moved almost exclusively to-
wards enzymatic systems, for similar reasons. However, enzymes are large
bulky catalysts that cannot readily diffuse through the encrusting hemicellulose
and lignin that surrounds the cellulose in woody materials. Cellulase enzymes
react very slowly with native lignocellulosic materials. For this and other practi-
cal reasons biorefinery technology for lignocellulosic conversion has now found
it essential to include some type of feedstock pretreatment stage.
Such pretreatments must accomplish the physical disruption of the native lig-
nocellulosic biomass sufficiently to allow the diffusion and penetration of the
enzymes into the biomass structure and give ready access to the cellulose mole-
cules in the primary and secondary walls of the woody fiber. Woody biomass
pretreatments that are under investigation include (1) one stage steam explo-
sion, (2) two stage steam explosion in the presence of SO2 or sulfuric acid, (3)
ammonia fiber explosion (AFEX), (4) dilute mineral acid, and (5) organosolv de-
lignification. More exotic pretreatments, such as treatment with super critical
water, are also being investigated. The various fiber explosion processes and di-
lute mineral acid pretreatments have the effect of hydrolyzing and solubilizing
hemicellulose and also of opening up the tight structure of the fiber structure
to expose cellulose more for enzymatic attack. They do not remove lignin from
the pretreated material unless specific extraction steps, usually with hot ethanol
or hot alkali, are included after the initial explosion stage. Recent studies have
indicated that the presence of lignin and even hemicellulose in the pretreated
biomass can reduce the speed and possibly the extent of enzymatic saccharifica-
tion of the cellulose [28]. Partly this is because of nonspecific, and possibly irre-
versible, binding of the cellulase enzymes to the lignin. The effect of this phe-
nomenon, which can be overcome by adding excess enzymes, is to increase the
enzyme requirement and consequently the operating costs of the biorefinery.
Organosolv pretreatment, which involves treating the raw lignocellulosic ma-
terial with an aqueous organic solvent (frequently ethanol) at temperatures in
the range of 180–200 8C, specifically hydrolyzes most of the hemicellulose and
lignin and causes them to dissolve in the liquor [29]. Following washing of the
remaining solid fiber, the residual lignin is a minor part of the finely disrupted
material. Studies have shown that organosolv-pretreated woody biomass is
highly susceptible to cellulase hydrolysis, with the extent of cellulose saccharifi-
cation being greater than 90% of theoretical [26]. Even though dehydration prod-
ucts of the sugars are formed in organosolv processes, they do not contaminate
the saccharification and fermentation stages because the solid fiber is washed
extensively to remove and recover lignin that is dissolved in the liquor retained
in the fibers following the pretreatment. The other advantage of organosolv pre-
treatment is that a relatively pure, partially hydrolyzed lignin product is easily
recovered from the liquor as well as various components of the hydrolyzed

hemicellulose [29]. This lignin has excellent properties for use in various com-
mercial applications [7].
It should also be possible to recover a useful lignin from the solid residuals
that remain following saccharification and fermentation of woody biomass that
has been pretreated by steam explosion and possibly dilute acid hydrolysis. This
could be accomplished by subjecting these lignin-rich residuals, which would
be contaminated with undissolved cellulose and hemicellulose to an organosolv
extraction. However, this approach would lose the advantage of removing the
lignin prior to enzymatic saccharification. Alternatively, the lignin-rich residuals
of the saccharification stage, despite their contamination with polysaccharides,
might find a valuable use in industry.
5.4.2
Lignin from an Organosolv Biorefinery
Recent interest in environmentally friendly chemical pulping has encouraged

the investigation and development of organosolv pulping. Various organosolv
processes have been proposed and investigated over the last twenty-five years or
so. These include the Alcell process [29], using ethanol; Acetosolv, using acetic
acid; Formacell, using formic acid; pulping with phenol; and the Organocell
process using methanol. The Alcell process, developed by Repap Enterprises
Inc, during 1987–1997, is perhaps the most commercially advanced of the etha-
nol-based organosolv pulping processes. This technology employs an aqueous
solution of ethanol as the pulping liquor. Cooking at relatively high tempera-
tures, approximately 1958C (and consequently relatively high pressures, about
28 bars) allows the production of a bleachable pulp from hardwoods and from
most annual fibers. A key characteristic of this process is the required recovery
of a natural “organosolv” lignin as one of a series of co-products that also in-
clude bleachable pulp, furfural, acetic acid and xylose [29]. The Alcell pulping
process was operated in a pre-commercial demonstration plant at Repap’s Mira-
michi pulp and paper mill in New Brunswick, Canada. This demonstration
plant is shown in Fig. 5.1. This plant, with a design capacity of 30 tonnes of
pulp per day, operated intermittently from 1989 to 1996 and produced more
than 3700 tonnes of organosolv lignin. This lignin was in the form of a dry
powder and was sold in supersacks containing approximately 800 kg each. All
of the lignin produced at the plant was sold commercially during the time the
plant operated, except for some minor portion that was used for internal study
and development purposes. Unfortunately, with the financial collapse and con-
sequent breakup of Repap in 1997, this plant was closed, but the technology
has since been acquired by Lignol Innovations Corporation of Vancouver, Cana-
da, and is now being commercialized as a biorefinery technology with the cellu-
lose fraction being used for ethanol production instead of pulp [30].
The availability of tonnage quantities of this new form of lignin ignited a veri-
table explosion of interest in identifying new applications for this product. Large
Fig. 5.1 View of the organosolv-based Alcell Demonstration

Plant at Miramichi, New Brunswick, Canada, showing the dis-
tillation column for ethanol recovery (left) and the lignin dryer
building (right, with loading door).
samples were distributed to universities and to companies for applications test-

ing in many different market areas. Commercial sales started as early as 1990.
In 1995, when Repap was preparing to construct a 450 tonne per day Alcell
pulp mill at Atholville, New Brunswick, several studies were commissioned to
identify marketing strategies for the 56 000 tonnes of organosolv lignin output
from this mill. These studies clearly showed that several hundred thousand
tonnes of lignin per year could be easily sold at that time for an average price
close to the price of PF resins.
A different form of organosolv pulping technology was commercialized in
Germany in 1992 under the name of Organocell. Initially, it was the intention
of Organocell to recover lignin and market it as a co-product. Before mill con-
struction a significant amount of successful development work was undertaken
to identify markets in Europe for this lignin product. Unfortunately, process
changes introduced during mill construction eliminated the ability to recover
lignin [31].
5.5
Applications and Markets for Lignin
5.5.1
Phenol–Formaldehyde Resin Applications
One of the earliest types of thermoset synthetic resins is phenol–formaldehyde

(PF) resin produced by reacting phenol and formaldehyde in the presence of an
acid or alkaline catalyst. PF resins now have a wide range of industrial applica-
tions [32]. These resins display a useful range of hardness, physical strength,
glossy finish, electrical properties, heat resistance and chemical stability. They
are one of the most widely used groups of plastics in the world, with total an-
nual market size that can now be estimated to be greater than 2.5 million
tonnes. The estimated worldwide capacities, markets and major applications are
excellently reviewed by others [33].
Phenolic resins are normally divided into resols or novolacs. Resols are so-
called one-stage resins that are most usually used as binders in the production
of exterior grade wood panels (such as plywood, particleboard and OSB), glass
fiber insulation, friction materials, foundry sand molds and resin-saturated la-
minated products. Novolacs are so-called two-stage phenolic resins that require
the presence of a crosslinking agent to cure. They are used principally in abra-
sives, protective coatings, friction materials, heat-resistant and general purpose
molding compounds, foundry sand mold binders and as specialized binders.
Since they contain a higher proportion of phenol to formaldehyde, novolacs
usually are more costly than resols.
The worldwide demand for phenolic resins is generally increasing in line
with world economic activity, since it is used in many broad-based industrial ac-
tivities and in housing. The highest prices are for specialized phenolic resins
used in the manufacture of heat-resistant and high electrical resistant molded
products, as well as in the manufacture of abrasives. Since phenol is now pro-
duced almost exclusively from benzene instead of from coking operations, sharp
increases in crude oil prices that have occurred in 2004 have caused a signifi-
cant rise in the cost of raw materials and a consequent increase in the prices
for phenolic resins.
5.5.2
The Potential Use of Biorefinery Lignin in Phenolic Resins
A simple examination of the chemical structure of native lignin [15] shows

some interesting similarities to the structure of phenol–formaldehyde resins.
Lignin consists of a three-dimensional amorphous matrix of crosslinked phenyl-
propanoid units. These residues, depending on their botanical source, may
have, to a greater or lesser extent, substitution of methoxy groups on the ortho
positions to the hydroxyl group on the aromatic ring. Lignin from annual
plants, with a high proportion of p-hydroxy phenylpropane residues, will have a
significantly lesser degree of ortho substitution, while lignins from hardwoods,

with a high content of syringyl moieties, will have a high proportion of ortho
substituents on the aromatic ring. Softwood lignin with a high content of guaia-
cyl groups (more than 90%) and some p-hydroxy phenylpropane moieties will
have an intermediate degree of methoxy substitution.
Both native lignin and phenol–formaldehyde resins are polyphenolic materials
consisting of phenolic rings linked together by short aliphatic chains. However,
the form of these linkages in lignin are much more diverse and different from
the linkages in PF resins. In lignin many of the crosslinking groups are aryl–
alkyl ethers, but there are numerous carbon–carbon links also. Furthermore, lig-
nin has a significant number of functional groups, such as aliphatic hydroxyls,
on the propane residues, a feature that is not shared with phenolic resins.
Nevertheless, the structural similarities are close enough to suggest a strong
compatibility between these two materials and even opportunities for chemical
interaction between them, with or without added crosslinking agents.
Lignosulfonates and thiolignins have been marketed for incorporation into
phenolic resins for some time, but they do not appear to have met with signifi-
cant acceptance by the resin industry.
Some of the problems experienced by current lignins in this market may be
overcome with biorefinery lignins, but not by all forms of biorefinery lignins.
Organosolv processing of woody biomass can readily yield a nonderivatized lig-
nin having high purity, significant chemical reactivity, low polydispersity and
low molecular weight. Organosolv lignins from hardwood produced in tonnage
quantities by the Alcell process in Miramichi, Canada, in the 1990s has been
readily substituted into phenolic resins [7] and used successfully on a commer-
cial basis. Commercial applications in which it was used included in phenolic
resins used in OSB manufacture and the production of friction materials. Nu-
merous other applications where organosolv lignin was being substituted in
part or in whole for phenolic resins were also under development at that time,
such as in saturating resins for laminates production, stiffening agents for con-
tainer boards and in rubber processing additives. Figure 5.2 shows samples of
several commercial and pre-commercial products manufactured using Alcell or-
ganosolv lignin.
The manner of substituting lignin for phenol–formaldehyde resins can vary
from a simple blending of dry powder lignin with dry powder phenolic resin to
the use of organosolv lignin as a primary phenolic component during the man-
ufacture of the resin. In both cases the lignin represents the substitution of a
renewable material for nonrenewable chemicals produced from fossil carbon.
The advantages to the users and suppliers of phenolic resins of partial or
complete substitution of biorefinery lignin are numerous. They include the po-
tential of lower cost raw material and the opportunity to receive valuable carbon
credits since phenolic resins are produced from phenol, now almost totally pro-
duced from crude oil, and formaldehyde, which is a product of natural gas. In
addition, recent studies have shown that the incorporation of organosolv lignin
into PF resins that are used in the manufacture of OSB will reduce the emis-
Fig. 5.2 Some commercial and pre-commer- sections of oriented strand board (OSB) and
cial products made with organosolv lignin other panel boards, while on the right is a
from hardwoods. On the left is an automo- section of rubber belting. In the middle is a
bile brake pad, next to a pot handle made sample of the original organosolv lignin
from a molding compound. To the rear are powder, with a granulated version to its left.
sions of formaldehyde from the press during manufacture [34]. This is a major
advantage to manufacturers because these emissions of a known carcinogen are
being closely monitored and regulated by various governments. An added bene-
fit to OSB manufacturers is that it has been shown that the substitution of up
to 35% of PF resin with an equal weight of organosolv lignin will provide sub-
stantial improvements to the final board properties, especially in reduced swell
of the wet board and a higher modulus of rupture following the boil test [7].
5.5.3
Panelboard Adhesives
Resin binders for exterior grade wood panel production is the largest part of the
market for PF resins, representing over half of the PF resin used today. The
market is divided mostly between plywood (a declining market), waferboard and
oriented strandboard (OSB), and exterior grade granulated wood panels, such as
particleboard. Plywood uses only liquid resins, while waferboard and OSB resins
are divided between dry powder and liquid forms. For a number of technical
and production reasons, isocyanate resins, despite their higher cost, are becom-
ing the favored resins for the core layer of OSB, while PF resins remain the re-
sins of choice for the two face layers. The estimated North American demand
for this market segment is over 550 000 tonnes annually on a solids basis, while
total world demand for this purpose is probably more than 1.1 million tonnes
annually.
In work conducted in the 1970s it was shown that lignin could replace up to
50% or more of the expensive MDI on an equal weight basis in an isocyanate resin
used in wood binder applications [35]. The properties of the finished board were in
many cases superior to the control board with 100% isocyanate resins. Isocyanate
resins sell for about double the price of PF resins on a solids basis.
5.5.4
Thermoset Resins for Molded Products
One of the oldest applications for PF resins is in the production of phenolic

molding compounds that are used to make a variety of products in the automo-
tive, electrical and appliance fields, such as electrical circuit boxes and circuit
breaker components, heat resistant pot holders and handles (Fig. 5.2). They nor-
mally consist of 50% PF resin and 50% fillers, but their exact composition is de-
termined by their application.
In the US the phenolic molding compound market is approximately 100 000
tonnes per year, which equates to more than 55 000 tonnes per year of PF resin,
while the total world usage of PF resin in this application is about 200 000
tonnes per year.
5.5.5
Friction Materials
During the manufacture of friction products, such as brake pads, brake shoes
and clutch facings, PF resins are added to the initial mix of components in the
range of 2–5% by weight. They serve as green strength binders to hold the
shape of the final product during molding and while in the bake oven. In the
bake cycle the PF resins are carbonized and form a matrix that supports the
other components. Approximately 27 000 tonnes per year of PF resins are used
in the manufacture of friction materials in the US, while world consumption is
probably in excess of 120 000 tonnes per year. Lignin can substitute for a signifi-
cant amount of this PF resin in friction materials [36].
Before the closing of the Alcell plant in 1996, Repap was regularly selling or-
ganosolv lignin to a commercial brake lining manufacturer who was substitut-
ing lignin for PF resin at a 20% level, but a higher level of substitution was
anticipated following these successful introductory levels (Fig. 5.2). Organosolv
lignin was found to provide some technical advantages to the final product, in
addition to cost advantages.
5.5.6
Foundry Resins
PF resins are used to bind the sand in metal casting molds and core. Mostly
the molds are made of green sand without binders, but the more delicate sand
cores are bonded with PF and other types of resins, such as furan resins. These
resins have the advantage that they are burned out of the sand, which can then
be recycled. The incorporation of lignin into these resins would have the same
advantage. Phenolics account for slightly more than 50% of the resins used in
the foundry market. In North America approximately 55 000 to 60 000 tonnes of
phenolic resins are used in the foundry mold binder market each year. World-
wide the usage is estimated to be about 150 000 tonnes per year. Alcell organo-
solv lignin was found to effectively displace more than 20% of furan and
phenolic foundry resins without loss of performance.
5.5.7
Insulation Materials
Glass fiber in insulation batting is bonded together using mostly liquid phenolic
resins but some powder resins, about 15–20% of the total, are used in certain
specialty applications. PF resins are used because they are temperature stable
and flame resistant. They are also used in phenolic foams, mineral wool and
waste fiber insulation. In the latter case, waste fiber such as cotton, polyester
and wool is shredded and bonded together with powder resin and molded.
Much of this material is used for acoustical insulation and applications. In
North America over 110 000 tonnes of phenolic resins are used by the insulation
materials market sector each year. The prices are similar to those for resins
used in other applications.
5.5.8
Decorative Laminates
A major market for PF resins is in the manufacture of decorative laminates.

Decorative laminates are made up of numerous layers of kraft paper that are
saturated generally with alcohol-based liquid PF resin. It is then partly cured
and dried before a photographic melamine surface layer is attached and the en-
tire system is placed in a press to be bonded. The amount of PF resin usage in
the US is approximately 100 000 tonnes per year, while the total world demand
for this application is probably close to 300 000 tonnes per year. Prices in this
application are similar to those in other PF resin applications.
5.5.9
Panel and Door Binders
There is a growing market for PF resins in the production of laminated and ve-
neered doors and certain types of molded fibrous structural panels. While this
is a fast growing business it is relatively small compared to the production of
structural wood panels for roofing, siding and underlaying, such as OSB and
plywood.
5.5.10
Rubber Processing
Several applications for lignin have been identified in the rubber industry.
These are:
· phenolic tackifiers
· antioxidants
· reinforcers.
Tack is the term given to the resistance to separation of two materials after they
have been brought in contact with each other, under light pressure, for a short
time. One type of tack is classified as autoadhesive tack, where the two materi-
als have the same chemical composition. The other type, adhesive tack, is used
where the two materials are of different composition.
In the production of multi-layered rubber products, such as tires and belting,
tack is needed to hold the components together during manufacture. Various
additives, known as tackifiers, are added to rubber formulations to increase tack
in the compound. Three types of tackifying resins are mostly used – hydrocar-
bon resins, rosin and its derivatives, and phenol–formaldehyde resins. Interest-
ingly, the molecular weight of the typical tackifying resins is around 2000 or
less, a number consistent with the number average weight of several soda and
organosolv lignins.
As a result of recognizing these similarities Repap entered into a collaborative
development program in the early 1990s with two major rubber products manu-
facturers for the evaluation of Alcell lignin as a tackifier in rubber processing.
This program demonstrated the capacity of dry powder Alcell lignin to function
as an effective tackifier in SBR compounds. Initially, lignin was used as a 50%
replacement of PF resin. Because tackifiers also affect the cured physical proper-
ties of rubber, it was necessary to not only test for tack improvement, but also
to test the hardness and tensile strength of the cured rubber as well as other
properties such as cut growth resistance and resistance to oils and solvents. A
patent covering this application was awarded in the US and several other coun-
tries [37].
Reinforcers are additives employed to obtain certain performance properties
in rubber products, such as tires, where abrasion resistance, in particular, is im-
portant. Reinforcing agents are normally carbon blacks, inorganic fillers, or re-
inforcing resins. Kraft lignin powders are also used in this application and it
has been suggested that, because of the specific gravity of organosolv lignin
powders, they should also be effective reinforcers.
A major advantage in this application was that the lignin also acted as an
antioxidant. In the manufacture of rubber products, antioxidants are also added
to the compound. Therefore, lignin acted as a multi-functional additive; a major
advantage, since a single additive could be used in place of the two additives
normally employed.
5.6 Lignin as an Antioxidant 187
5.5.11
The Opportunity for Lignin in Phenol–Formaldehyde Resin Markets
As phenolic resin substitutes, natural lignins have the following characteristics

and advantages, which should provide inducements for their acceptance by ex-
isting users.
· They are highly compatible with phenolic resins, which allows for effective
partial substitution and resin mixing. This accommodates applications where
blending to achieve specific performance properties is necessary.
· Lignin condenses under heat without the need for inorganic acid or base cata-
lysts. This, together with its very low ash content, is useful for resin use in
the electronics industry.
· It is a natural product that does not release formaldehyde during condensa-
tion. This is important in the wood panel industry, which is under tight regu-
lation of formaldehyde release during the hot pressing stage of panel manu-
facture.
· Lignin is a renewable natural product that should easily qualify for carbon
credits when it is used to displace phenol–formaldehyde resins.
· Lignin also brings additional properties, such as being a polymeric hindered
phenol antioxidant, which makes it multifunctional in many applications.
5.6
Lignin as an Antioxidant
Antioxidants are a group of chemicals that inhibit atmospheric oxidation and its
degradative effects on polymer systems, lubricants, foodstuffs and animal feed
additives. They minimize degradation during fabrication, storage and use.
In polymers and long chain molecules, such as fatty acids and polyalkanes,
chemical bonds are broken under the influence of heat, ionizing radiation, me-
chanical stress and chemical reactions to form free radicals. Oxygenation of
these free radicals forms peroxy radicals that initiate a chain reaction, which will
eventually badly degrade the polymer or long chain molecule, frequently intro-
ducing colored compounds into the product.
In order to inhibit oxidation in this chain reaction and to slow polymer degra-
dation, free radical scavengers are introduced into the polymer or foodstuff.
These are known as primary antioxidants, which include hindered phenols and
secondary arylamines. Lignin is a polymeric hindered phenol, a class of com-
pounds known to be effective primary antioxidants. Alcell organosolv lignin was
tested for its antioxidant properties and found to be an effective antioxidant in
grease, rubber and animal vitamin supplements.
Presently, the major antioxidants used in industry include butylated hydroxy-
toluene (BHT), butylated hydroxyanisole (BHA), propyl gallate (PG), natural to-
copherols and polymeric hindered phenols. These are all expensive materials
($ 3.00 per kg) and many of them are petroleum-based. Particularly in food and
feed applications, they are highly regulated. Some synthetic antioxidants con-
tinue to be under suspicion for adverse health effects. Lignin is a natural prod-
uct, which is ingested daily by both humans and animals. It might therefore be
expected to be a preferred form of natural antioxidant especially in food and
feed since there is a high likelihood that lignin is physiologically benign in hu-
mans.
5.6.1
Antioxidants in Animal Feed Supplements
In 1996 a leading feed vitamin supplier tested Alcell lignin to assess its poten-
tial as a natural antioxidant able to protect the potency of the antioxidant vita-
mins, such as vitamin E in animal feed vitamin supplements. It was reported
that the lignin functioned well in this application. Interestingly, the lignin also
served the added function as a pellet binder.
5.6.2
Antioxidants in the Rubber Industry
Antioxidants are also used in the rubber industry to suppress deterioration in

rubber products subject to long term exposure of air and sunlight, such as the
sidewalls of automobile tires. A number of different antioxidants are used by
the industry that can cost upwards of $ 3.00 per kg. Since lignin is a polymeric
hindered phenol, with hardwood lignin being especially hindered because of its
dominant syringyl structure, lignin will compete in the middle price range of
antioxidants.
5.6.3
Antioxidants in the Lubricants Industry
An area that is very attractive for lignin as an antioxidant is in the field of lubri-
cating greases. In this application the dark color of lignin is not usually impor-
tant and it is not necessary for it to be soluble in the grease. Addition levels to
greases is between 1% and 10% by weight, depending on product and applica-
tion.
Alcell organosolv lignin was marketed in 1996 as a multifunctional grease ad-
ditive (antioxidant and extreme pressure/antiwear additive) through a specialty
lubricants additives company for a price competitive with existing antioxidants.
Lubricating greases represent only 2% of all lubricant sales. Sales of antioxi-
dant/antiwear additives for greases in the US in the late 1990s were approxi-
mately 3.0 million kg with a value in excess of $10 million. Many customers are
looking for “green additives” that are nonmetallic, nonchlorinated, nonsulfur ad-
ditives and also are looking for natural products with low cost relative to the
conventional additives.
Approximately 300 000 tonnes of antioxidant/antiwear additives for lubricating

oils are being sold annually in the US, with a total value of approximately $ 520
million. Globally, the market was twice this size, with a value of over $ 1 billion
per year.
5.7
Applications for Water-soluble, Derivatized Lignins
As discussed earlier, lignosulfonates from the sulfite pulping industry and delib-
erately sulfonated kraft lignins represent the largest single form of lignin used
in industrial applications. Together these products account for approximately 1
million tonnes per year, worldwide. They function mostly as dispersants,
emulsifiers and surfactants in numerous commercial applications. Hydrolysis
lignins obtained from future biorefineries will also be capable of being modified
to produce equivalent or, because of their greater purity, improved materials for
these markets.
As the sulfite pulping process has moved from being used to produce com-
modity papermaking pulps to being the source for the manufacture of specialty
dissolving pulps, more sulfite mills have closed over the last fifty years or so,
and continue to close. Most of the recovered lignosulfonates from the remain-
ing mills is going into the surfactants, dispersants and emulsifiers markets.
With the increase in demand for these products and the need for improved per-
formance, some market share has been lost to synthetic products. Sulfonated
kraft lignins are also sold into this market by MeadWestvaco. Methylsulfonated
biorefinery lignins should easily enter this market.
5.7.1
Concrete Admixtures
Water-reducing concrete admixtures are surface-active chemicals that function

to break down cement particle agglomerates, thereby enhancing the fluidity and
workability of the fresh concrete. The less water that is present in the fresh con-
crete mix, the higher the strength of the final set concrete. Water reducing ad-
mixtures can therefore be used to reduce the total amount of concrete and
therefore the weight of concrete needed to achieve a desired strength. Ideally,
water-reducing admixtures should have no negative effects on the strength and
durability of the hardened concrete. Also, they should have no detrimental side
effects such as excessive air entrainment or retardation of set time. The addition
rate and therefore the effectiveness of many admixtures currently in use is lim-
ited by these undesirable side effects.
Current commercial water-reducing admixtures fall into four broad categories:
1. modified and unmodified lignosulfonates and sulfonated kraft lignins
2. sulfonated melamine formaldehyde condensates
3. sulfonated naphthalene formaldehyde condensates
Fig. 5.3 Concrete test cylinder made with methyl-sulfonated

organosolv lignin, shown next to a brake pad for size
comparison.
4. others, including polycarboxylic acids, and mixtures of acids, amines and

polysaccharides.
The first three categories rely upon sulfonic acid groups to impart a negative
charge on the cement particle. The positioning of sulfonic acid groups on ligno-
sulfonates is a direct result of the pulping process chemistry. This may not be
ideal for this application and deliberately sulfonated natural lignins may yield
higher performing products than lignosulfonates. Methyl-sulfonated Alcell lig-
nin was tested as a water reducer in concrete and found to be quite effective.
Figure 5.3 shows a concrete cylinder produced with methyl-sulfonated Alcell lig-
nin that is used for testing of compressive strength.
5.7.2
Dye Dispersants
Before the advent of synthetic fibers, fabrics were made from naturally occur-
ring materials such as wool, cotton, linen etc. These natural fibers were dyed
from solution, in a dye bath. The dissolved, dyestuff molecule chemically
bonded to reactive sites on the fiber surface. Solutions exhibited uniform con-
centration so that the fabric was evenly-colored.
Synthetic fibers have now become equally as important as natural materials
in clothing, furniture coverings, drapes, and carpeting. Since many of the newly
discovered fibers were synthetic polymers, they were chemically unreactive.
Often it proved impossible to color them using traditional dyestuffs or dyeing

techniques because they would not react with the dyestuff molecule. A new
technique became necessary and, by the 1960s, one had been developed, known
as disperse dyeing. In this process, the dye is applied to the fabric in the form
of a fine, uniform suspension. The insoluble particles of dye, uniformly dis-
persed, are physically entrapped within the polymer structure. In effect, they
are in a solid solution.
Disperse dyes account for about 100 000 tonnes per year. Of the different
classes of dyestuff, only disperse dyes (by far the most important), sulfur dyes
in some applications and, to a lesser extent, vat dyes, are in the insoluble form
during the dyeing process.
All types of dye must be applied in a manner that produces uniformly colored
fabric, free of specks or color shadings. In the case of disperse dyes, this means
that the dispersion must be extremely uniform and contain particles of dyestuff
in the 1-lm diameter size range. The only way to meet these criteria is to use
dispersing agents.
Dispersing agents are incorporated into the dyestuff during its manufacture
(both before and after the milling process) and may amount to as much as 50%
of the finished product. In addition to providing excellent dispersion, the
dispersing agent must also offer other properties, including:
· low staining
· ability to maintain stable dispersions at high temperatures (above 100 8C)
· nonfoaming
· contain no sludge (fabric is an excellent filter)
· nonreactive with the dye molecule
· be readily spray-dried
· assist in grinding.
It is not easy to meet these demanding requirements. Over the past 30 years,
the dyestuffs industry has found only three dispersants that will perform ade-
quately:
1. Sulfonated kraft lignin
2. Lignosulfonate
3. Condensation products of naphthalene sulfonate.
Sulfonated kraft lignin and lignosulfonates together account for about 90% of
the dispersant market. While they are the best products available, it is generally
admitted that they are far from ideal. The third class of products, the naphtha-
lene sulfonates, account for 5 to 10% of the dispersant market. They exhibit
questionable high-temperature stability – a vital property – and are expensive.
They are seldom used in formulations in North America, but they are often
used in vat dyes for cotton in other countries. The total amount of lignin-based
dye dispersants used worldwide is about 25 000 tonnes annually.
5.7.3
Asphalt Emulsifiers
The strong move to water-based asphalt emulsions and away from organic sol-
vent-based systems has been driven by environmental concerns about organic
solvent releases into the atmosphere. Asphalt emulsions are now used exten-
sively in a variety of applications including road building, road and driveway
sealing, soil stabilization, surface coating of asphalt pavement and built-up roof-
ing. In these applications the emulsions are formed and stabilized with emulsi-
fiers that are generally used at a level of about 1 to 2% by weight of the emul-
sion.
Pinova, a division of Hercules, of Wilmington, DE, markets a series of asphalt
emulsifiers based on polymerized rosin, Vinsol resin (a “bottoms” fraction from
the refining of rosin) and rosin acids. These materials are obtained from rosins
extracted from Southern Pine stumpwood. For a number of reasons the avail-
ability of these rosins is declining and Hercules has been searching for new
raw materials to support its asphalt emulsifier business.
During discussions with Repap in the mid-1990s Hercules became aware of
the new organosolv lignin product from the Alcell process and tested its perfor-
mance in asphalt emulsifiers. These trials were successful and two US patents
[38, 39] was awarded and assigned to Hercules covering the formulation of lig-
nin-containing asphalt emulsifiers and the asphalt emulsions containing such
emulsifiers. These new emulsifiers were prepared by combining and melting
lignin and polymerized rosin to obtain a homogeneous molten blend, then cool-
ing the blend until it solidified. In a preferred embodiment, the ingredients
further comprise added rosin acids and Vinsol resin, and the lignin is an orga-
nosolv lignin.
The total world output of asphalt is well in excess of one billion tons. It is
stated that 90% of all road networks are asphalt. Lignin-based asphalt emulsi-
fiers should be well received by the industry. There is a continuing public con-
cern about leaching of asphalt components into the surrounding soils and into
ground and surface water. The leaching of lignin would have no negative envi-
ronmental consequences, and may even have positive effects.
5.7.4
Agricultural Applications
Lignin products from conventional chemical pulping operations have for a long
time been used in agriculture. Lignosulfonates from the sulfite pulping industry
have found applications as feed pellet binders, dispersants for insecticides, fun-
gicides and herbicides, and even as dust suppressants on farm roads. Initially
these applications were driven by a need of the sulfite pulping industry to find
uses for its spent sulfite pulping liquors. In these applications there was little
need to purify the spent liquors and so the product was frequently a dried pow-
der of the spent liquor, or even the liquor itself.
Worldwide, agriculture has several emerging needs that can be satisfied by hy-
drolysis lignins, such as organosolv and biorefinery lignins. Among these needs are
· improved environmental performance
· improved animal health without using human applicable antibiotics
· greater efficiency in use of resources
· lower costs of operation
· improved productivity
· insulation from rising energy costs
· lower usage of fossil carbon-based chemicals.
Natural hydrolysis lignins have been shown to be multifunctional and have

properties that help satisfy these needs of agriculture. They include:
· biodegradability, with the degradation products having a positive environmen-
tal effect
· photodegradability
· physiologically active
· specific antimicrobial effects
· natural antioxidant
· relatively immunity to rising energy prices
· natural product not derived from fossil carbon sources.
These properties have been exploited recently in several new applications in

agriculture. They include:
· enhanced feed efficiency in calves
· antidiarrheic in cattle
· antioxidant component in vitamin supplement formulas for animals
· slow release matrix for fertilizer and pesticide applications, with positive envi-
ronmental effects
· beneficial effects on rumen metabolism
· reduced ammonia concentrations in broiler operations.
5.7.5
Dispersants for Herbicides, Pesticides and Fungicides
The active ingredient in herbicide formulations is generally an organic chemical

that is insoluble or immiscible in water. In this regard, herbicide formulations
are closely related to disperse dyes. Many of the manufacturers are the same,
particularly the giant Swiss and German chemical companies.
With the advent of micro-emulsion technology, post-emergent herbicides, and
the greater use of dry granular compositions, herbicide manufacturers and for-
mulators now require that the dispersant system do more than merely emulsify
or disperse the active ingredient in water. Now, the additive must also be com-
patible with fertilizers and other pesticides, insecticides or fungicides, to allow
combined-field application. In addition, the additive should increase the effi-
ciency of the application by improving penetration of the leaf or insect.
A range of surfactants provide these properties to some degree or another. In

the United States, the annual surfactant market in herbicide applications totals
about 45 000 tonnes and is divided between several product classes, principally
lignin-based surfactants and synthetic surfactants, such as alkylphenol ethoxy-
lates. The total market in the US for herbicide surfactants is about 50 000
tonnes annually, of which only about 5% is currently lignin based. The total
world market for lignin-based herbicide surfactants is approximately 18 000 to
20 000 tonnes per year.
5.8
New and Emerging Markets for Lignin
As can be readily seen from the above descriptions of the present markets for
lignin and its water-soluble derivatives, there is an existing major market oppor-
tunity for hydrolysis lignins that might be obtained from new biorefinery opera-
tions. Not only will the new lignins promise superior performance in many ap-
plications that currently are filled by the lignosulfonates and kraft lignins, but,
because of their anticipated superior purity and functionality, they will open ma-
jor new markets that presently do not exist. Already there are indications of
such future market opportunities that were being developed from the very lim-
ited supply of organosolv lignins generated from Repap and from the limited
work undertaken by Organocell. Some of these exciting opportunities are pre-
sented below.
5.8.1
Printed Circuit Board Resins
A report from the IBM company in 1996 [40] identified a novel application for
lignin in the manufacture of printed circuit boards (PCBs) for the electronics in-
dustry. In the manufacture of PCBs the glass fabric, which is the largest part of
the PCB, is dip coated with a resin. This is usually an epoxy resin, dissolved in
a low boiling solvent, such as methyl ethyl ketone or acetone. The release of
these solvents during resin curing is an undesirable health and environmental
issue.
At that time, IBM was in the process of examining its various production pro-
cesses and operations in order to substitute, where possible, “green” technology.
One area of interest was the use of renewable biopolymers to replace the oil-
based epoxy resins typically used in the industry. Lignin became of interest be-
cause its molecular structure could provide the thermal stability and chemical
resistance that was required for PCBs. It was reported that in initial research,
PCBs produced with an epoxy resin containing 50% lignin showed better ther-
mal and electrical performance than current high volume PCBs and that the re-
sin cost was significantly lower than standard resins. Furthermore, this material
reduced the dependence on fossil fuels.
5.8.2
Animal Health Applications
In the mid 1980s, Organocell, of Munich, developed a methanol-based organo-

solv pulping process for wood from which they obtained an organosolv lignin.
This lignin was manufactured in sub-tonnage quantities in their pilot plant and
was distributed to various academic institutions in Germany for applications re-
search. Some of this lignin was sent to Professor J. Gropp, of the Veterinary De-
partment of the Ludwig-Maximilians University of Munich. He and his students
tested various applications in animal feeds and several doctoral theses were pro-
duced from this work [41–43]. The conclusions from this work can be summa-
rized as follows.
Prophylactic use of 1.5 to 4.5% organosolv lignin in the feed of piglets inocu-
lated with pathogenic E. coli led to a significant decrease in morbidity and inten-
sity of diarrhea.
· The reduction of feed efficiency caused by diarrhea could be avoided by feed-
ing 1.5% lignin.
· The therapeutic administration of 3.0% and 6.0% lignin after appearance of
the diarrhea resulted in an improved fecal consistency and a reduced water
excretion.
· Nevertheless, the beneficial effects of feed efficiency and average daily weight
gain showed a maximum at 4.5%, with 6.0% lignin proving to be deleterious
to these criteria.
· The egg laying performance of hens was negatively affected by the presence
of lignin in the diet.
The antidiarrheic effects of dietary lignin in piglets were also demonstrated in

calves. The addition level having optimal effects was very similar for both ani-
mal models, with both showing negative effects at 6% and above.
Support for these observations comes from US Patent #4, 473 556 awarded in
1984 that describes the use of a feed formulation containing acid-hydrolysis lig-
nin (Scholler lignin), used in the treatment of gastrointestinal disturbances in
farm animals.
The significance of these observations is that there is often substantial mor-
bidity, especially in young farm animals, as a direct result of acute gastrointesti-
nal disturbances. In the past sub-therapeutic levels of antibiotics have been used
to reduce the incidence of this problem and the economic losses associated with
it. The same antibiotics are used to treat human infections and there is wide-
spread concern over the appearance of antibiotic-resistant bacterial strains that
result from this practice. Many European countries have now banned the broad
use of human therapeutic antibiotics in farm animal husbandry. An alternative,
nonantibiotic treatment for this problem would be of great importance.
5.8.3
Animal Feed Supplement
As a result of the above observations on the positive effects of lignin on animal

gastrointestinal disturbances, a major well controlled feeding trial and related
research program at the experimental farm of McGill University was underta-
ken in 1995–96. The feeding trials involved groups of veal calves that were fed
rations containing no or different levels of organosolv lignin from the Alcell
process. The microbial studies involved in vitro and in vivo experiments on the
responses of various bacteria to lignin. Some of the results of these studies have
been published and reported at scientific conferences [44, 45]. Among the most
interesting results was the observation that 1.25% of added organosolv lignin to
the diet of calves increased weight gain by 12% and reduced ammonia concen-
trations in the feces. In addition it was noted that enteric bacteria were found to
adhere to the lignin particles and that at certain levels lignin destroyed Gram-
positive and Gram-negative bacteria. It was concluded that lignin has the poten-
tial to alter feed utilization by animals through its effect on the physiology and
microbiology of the gut.
The importance of the finding on weight gain and feed efficiency cannot be
overstated. If these observations are carried through to the feeding and fattening
of mature cattle, lignin could significantly enhance the economics of feedlot op-
erations, together with a resultant benefit to the environment. This one applica-
tion could become a major market for natural lignins.
5.8.4
Carbon Fibers for Mass-produced Vehicles
A fascinating and potentially very large new market for lignin is for the produc-
tion of low cost carbon fibers for use in automobile and light truck body compo-
nents.
It is well recognized that major transportation fuel savings, and an equivalent
reduction in the demand for oil, would be achieved if the weight of vehicles
could be reduced. There would also be a beneficial concomitant reduction in
emissions. Today, despite a rising use of lower weight materials, between half
and two thirds of the weight of most vehicles is contributed by ferrous metals.
Much of these could be replaced by components made from lighter weight car-
bon fiber composites, which could reduce the weight of the vehicle by about
one third. Such carbon fiber composite materials are used extensively in the
aerospace industry where they have proven their value and performance. Carbon
fibers for these applications are made mostly from oil-derived pitch and poly-
acrylonitrile (PAN) and in their final form cost in excess of $ 25 per kg.
In order for auto body parts made from carbon fiber composites to be price-
competitive in today’s mass-produced vehicles, it has been determined that the
carbon fibers would need to cost less than half this amount. This price cannot
be achieved using existing feedstocks, especially as oil prices continue to climb.
What is needed to achieve this goal is a high carbon content, relatively low
priced, preferably renewable feedstock that can be produced on a large scale,
the price of which would be relatively independent of the price of oil.
Researchers at the Oak Ridge National Laboratory in Tennessee, USA, are in-
vestigating the use of lignin for this application [46, 47]. Most of their work has
been with kraft lignin, but they have also successfully used organosolv lignin in
this application. This work has produced industrial grade carbon fibers suitable
for use in vehicles from blends of lignin and post-consumer recycled polyester
and other polymers. The commercial kraft lignin that was used had to be
washed free of contaminants and desalted prior to use to avoid the presence of
voids in the fibers, which would significantly reduce their strength and value.
Purer lignins produced from biorefinery processes could eliminate the need for
this processing step. Figure 5.4 shows spools of the melt spun lignin/polymer
fibers containing different amounts of lignin. Figure 5.5 displays microscopic
images of lignin/polyethylene oxide fibers at different stages in the manufacture
of carbon fibers [47].
Once this application becomes fully developed it has been estimated that if
each newly manufactured vehicle in the US used only 10 kg of carbon fiber,
there would be an annual demand for 125 000 tonnes of carbon fiber. With a lig-
nin carbon content of 0.68 and an expected carbon yield of 0.45–0.55, just this
limited consumption could require up to approximately 250 000 tonnes of lignin
annually and could support a lignin price greater than US $ 1.20 per kg. Each
new vehicle could ultimately use many times the 10 kg suggested in this illus-
tration. In fact, it is conceivable that each vehicle could ultimately use up to
500 kg of carbon fiber making the carbon fiber demand for US manufactured
vehicles alone at least 4 million tonnes per year, which in turn would require
up to 8 million tonnes of lignin per year. Today the worldwide demand for car-
bon fiber is approximately 28 000 tonnes per year. Therefore, capacity would
have to increase by close to five times just to satisfy this application at a level of
10 kg per new vehicle in the US alone and a further fifty times to meet the ulti-
mate potential demand.
Fig. 5.4 Spools of melt spun fibers made from blends of kraft lignin and polymer.
Fig. 5.5 Microscopic images of fibers made from blends of

kraft lignin and polyethylene oxide during the manufacture of
carbon fibers.
5.9
Conclusions and Perspectives
Lignin that is now being recovered from conventional chemical pulping opera-
tions has established a significant commercial market in numerous industries
and applications. These markets basically employ two different forms of lignin.
The first of these are the water-soluble lignosulfonates recovered from the sulfite
pulping process and the deliberately sulfonated lignins derived from the kraft pro-
cess. The second form of commercial lignin is the thiolignin obtained from the
kraft process. Because of their markedly different physical and chemical proper-
ties, each of these has established its own range of applications, with very little
overlap between them. The combined worldwide market that presently exists
for these two product forms appears to be about one million tonnes per year.
With the increasing cost of crude oil and natural gas and the developing trend
and incentives towards the use of renewable chemicals to replace fossil-carbon-de-
rived materials it can only be anticipated that these traditional markets for lignin
products will expand. At the same time, the number of pulp mills that practice the
sulfite process are slowly declining, which raises the question as to where the sup-
ply to meet this increased demand will come from. An additional trend in the
pulping industry is the ever increasing scale of new kraft mills and the frequent
closure of small kraft mills now regarded as too small to be competitive. It is un-
likely that recovery of lignin will be an attractive option for the very large kraft
mills because of the process integration with large recovery boilers.
This situation can benefit future biorefineries that will be processing lignocel-
lulosic feedstocks. Certain biorefinery pretreatment technologies, such as orga-
nosolv processes, will have the capability to produce a superior form of pure lig-
nin ideal for chemical applications. The marketing of this lignin as a co-product
will add substantial revenues and vastly improve the economics of the biorefin-
ery. The new biorefineries will more than likely be on a considerably smaller
scale than world-scale kraft pulp mills that are now processing 3 000 to 4 000
tonnes of wood on a dry weight basis per day. Consequently biorefineries will
be more flexible and better suited to produce and market some of the special-
ized lignins that will emerge from them.
References 199
The past experience from the production of large volumes of Alcell lignin
shows that the availability of commercial quantities of these improved lignins
will stimulate sizeable new market opportunities that do not presently exist for
lignins from the chemical pulping industry. Some of these new markets are al-
ready clearly visible. What is needed now is the commercial-scale production of
the lignin to service these opportunities.
References
1 R. Alén, Forest Products Chemistry, Fapet 12 R. Alén, Forest Products Chemistry, Fapet
Oy, Helsinki, Finland, 2000. Oy, Helsinki, Finland, 2000, p. 66.
2 W. G. Glasser, R. A. Northey, T. P. 13 W. G. Glasser, Forest Prod. J. 1981, 31,
Schultz, Lignin: Historical, Biological and 24–29.
Materials Perspectives, ACS Symposium 14 Lignin: Properties and Materials, eds.
Series 742, American Chemical Society, W. G. Glasser, S. Sarkanen, American
Washington, DC, 2000. Chemical Society Symposium Series
3 J. D. Gargulak, S. E. Lebo. In Lignin: His- 397. Washington, DC, USA, 1989.
torical, Biological and Materials Perspec- 15 J. L. McCarthy and A. Islam, Lignin: His-
tives, ACS Symposium Series 742, Amer- torical, Biological and Materials Perspec-
ican Chemical Society, Washington, DC, tives, ACS Symposium Series 742, Amer-
2000, p. 304. ican Chemical society, Washington, DC,
4 G. A. Smook, Handbook for Pulp and Pa- 2000, p. 2–99.
per Technologists, 2nd edn., Joint Textbook 16 A. Payen, Compt. Rend. 1838, 7, 1052.
Committee of the Paper Industry, TAP- 17 B. C. Tilghman, British Patent No. 2924,
PI, Atlanta, USA, and CPPA, Montreal, 1866.
Canada. 18 Borregaard LignoTech website. http://
5 X.-J. Zhong, A. Gan, Profit Through Inno- www.ltus.com/about us/history.html.
vation, Pira International, London, UK, 19 Meadwestvaco website, http://
1997, p. 222–224. www.meadwestvaco.com/specialtychem-
6 F. Zimbardi, E. Ricci, G. Braccio. Applied icalshome.nsf
Biochem. Biotechnol. 2002, 18/20, 89–99. 20 Granit S. A. website, http://www.granit.-
7 J. H. Lora, C. F. Wu, E. K. Pye, and J. J. net
Balatinecz, In Lignin: Properties and Ma- 21 Chemical modification, Properties, and
terials. W.G. Glasser and S. Sarkanen Usage of Lignin, 2002, ed. T. Q. Hu,
(Eds.). American Chemical Society Sym- Kluwer Academic/Plenum Publishers,
posium Series 397. Washington, DC, New York.
USA 1989, p. 312–324. 22 J. H. Lora, A. Abächerli, F. Doppenberg,
8 X. Pan, X. Zhang, D. J. Gregg, J. N. Sad- 2000, Tappi Pulping Conference Proceed-
dler. Applied Biochem. Biotechnol. 2004, ings, Tappi Press, Atlanta, USA.
113/116, 1103–1114. 23 J. H. Lora, Proceedings of the 6th Interna-
9 A. Boussaid, A. R. Esteghlalian, D. J. tional Conference of Pulp and Paper Indus-
Gregg, K. H. Lee, J. N. Saddler, Applied try – Paerex, December 5–7, 2003, New
Biochem. Biotechnol. 2000, 84/86, 693– Delhi, India.
705. 24 J. H. Lora, personal communication, 2004.
10 G. P. van Walsum, Applied Biochem. Bio- 25 H. F. J. Wenzl, The Chemical Technology of
technol. 2001, 91/93, 317–328. Wood, 1970, Academic Press, New York.
11 F. Teymouri, L. Laureano-Perez, H. Ali- 26 X. Pan, C. Arato, N. Gilkes, D. Gregg,
zadeh, B. E. Dale, Applied Biochem. Bio- W. Mabee, K. Pye, Z. Xiao, X. Zhang, J.
technol. 2004, 113/116, 951–963. Saddler, “Biorefining of Softwoods Using
Ethanol Organosolv Pulping – Prelimin-
ary Evaluation of Process Streams for 38 R. R. Suchanec, US Patent #5 683 497,

Manufacture of Fuel-Grade Ethanol and 1997, “Asphalt emulsion with lignin-con-
Co-products”, 2005, Biotechnol. Bioeng., taining emulsifiers”.
90, 473–480. 39 R. R. Suchanec, US Patent #5 656 733,
27 C. Mitchinson, “Improved Cellulases for 1997, “Lignin-containing resinous com-
Biomass Conversion”, 26th Symposium positions”.
on Biotechnology for Fuels and Chemicals, 40 J. M. Shaw, S. L. Buchwalter, J. C. He-
May 9–12, 2004, Chattanooga, Tennes- drick, S. K. Kang, L. L. Kosbar, J. D. Ge-
see, USA. lorme, D. A. Lewis, S. Purushothaman,
28 H. Palonen, “Role of Lignin in the Enzy- R. Saraf and A. Viehbeck, Printed Circuit
matic Hydrolysis of Lignocellulose”, Ph. Fabrication, 1996, 19, 38–44.
D. Dissertation, 2004, VTT Technical Re- 41 R. Stich, Doctoral Thesis, Ludwig-Maximi-
search Centre of Finland. lians University of Munich, 1984, “Tests
29 E. K. Pye, J. H. Lora, 1991, Tappi J., 74, for Different Lignin Effects in Veal
113–118. Calves, Rats, Laying Hens and Quails”.
30 C. Arato, E. K. Pye and G. Gjennestad, 42 A. J. Kloner, Doctoral Thesis, Ludwig-Maxi-
2005, Applied Biochem. Biotechnol. milians University of Munich, 1985, “Re-
121/124, 871. duction in Infectious Piglet Diarrhea by
31 W. Zitzelsberger, “Production of Lignin Lignin”.
from the Organocell Pulping Process”, 43 A. Ott, Doctoral Thesis, Ludwig-Maximi-
1st European Workshop on Lignocellulosics lians University of Munich, 1983, “Effect
and Pulp (EWLP); Utilization and Analy- of Dietary Lignin on Nutrient Utilization
sis of Lignin. 1991, Sept 18–20, Ham- of Piglets”.
burg-Bergedorf, Germany. 44 L. E. Phillip, E. S. Idziak, Proc. of the
32 G. S. Brady, H. R. Clauser, Materials XVIII International Grassland Congress,
Handbook, Twelfth Edition, McGraw-Hill, June 1997, Winnipeg, Canada.
New York, USA, 1986, pp. 599–601. 45 L. Phillip, E. Idziak, S. Kubow, Proc. of
33 E. Camara, “Phenolic Resins”, Chemical the Eastern Nutrition Conference, Animal
Economics Handbook, 1999, SRI Interna- Nutrition Conference of Canada, May 25,
tional, Menlo Park, USA. 2000.
34 W. C. Senyo, A. W. Creamer, C. H. Wu, 46 J. F. Kadla, S. Kubo, R. A. Venditti, R. D.
J. H. Lora, Forest Products J. 1996, 46, 73– Gilbert, A. L. Compere, W. Griffith. 2002.
77. Carbon, 40, 2913.
35 A. L. Lambuth, US Patent No. 4 279 788, 47 W. L. Griffith, A. L. Compere, J. T. Shaf-
1981. fer, C. F. Leitten, R. D. Gilbert, J. F. Ka-
36 M. G. Jacko, R. F. Gager, US Patent No. dia, S. Kubo, R. A. Venditti, 2000, Proc.
4 239 666, 1980. Midwest Advanced Materials and Proces-
37 J. H. Lora, M. J. Trojen, W. H. Klingen- sing Conference, Sept 12–24, Dearborn,
smith, US Patent #5 196 460, 1990, “Rub- MI, USA. Society for the Advancement
ber Compositions Containing High Pur- of Material and Process Engineering.
ity Lignin Derivatives”.
201
6
Towards Integration of Biorefinery and Microbial Amino Acid
Production
6.1
Introduction
In the chemical industry the utilization of feed stocks produced in biorefineries

is an emerging field of interest, because a wide range of low cost raw materials
is potentially available. On the other hand amino acid production is a large-vol-
ume, steadily growing business already with several decades of experience with
the utilization of renewable resources such as sucrose, molasses and starch hy-
drolyzates. Integrating both concepts opens up further opportunities for a sus-
tainable production of amino acids. In our contribution we discuss environmen-
tal, commercial and technical aspects of microbial amino acid production
(MAAP) integrated in a biorefinery.
The optimization of MAAP has made tremendous progress during the last
four decades. In this article we restrict our review to biotechnologically pro-
duced amino acids with a world-wide yearly production capacity of more than
thousand tons per year. Because excellent reviews are already available [1–4] we
do not cover details of metabolic engineering, strain development, alternative
fermentation processes, and downstream processing. Instead the major focus of
our contribution is to illustrate synergies of developments at the interface be-
tween emerging biorefinery and the well established amino acid industry and to
outline how to integrate both.
ISBN: 3-527-31027-4
202 6 Towards Integration of Biorefinery and Microbial Amino Acid Production
6.2
Present State of the Industry
6.2.1
Microbial Amino Acid Production
Amino acids are produced by extraction from protein-hydrolyzates, by chemical

synthesis, by enzymatic synthesis, or by fermentation. Because of tremendous
progress in strain development during the last two decades, the fermentation
industry has grown rapidly with a steady increase of process efficiency. Esti-
mated world-wide production by fermentation in 1996 was 1000, 250, 4, 1.3,
and 1.2 thousand tons per year for mono sodium glutamate, l-lysine HCl, l-
threonine, l-glutamine, and l-arginine, respectively [2]. The fermentative pro-
duction of l-tryptophan, l-valine, l-leucine, l-isoleucine, l-histidine, l-proline,
and l-serine accounts for world-wide production capacities below 600 tons per
year.
For the bulk amino acids used for food and feed applications, the carbon
source is the major variable cost followed by variable energy cost and the cost of
the nitrogen source. Other raw material costs are less than 5% of the variable
cost. Fix costs have been significantly reduced by economy of scale, so that vari-
able cost dominates the overall manufacturing cost.
Rational strain development, the application of genome information, and in-
formation about metabolic fluxes, metabolite concentrations, the transcriptome,
and the proteome have led to the development of high-performance production
strains with high productivity and high conversion of the carbon source. Re-
cently, success stories for application of metabolic engineering for improving
lysine production by Corynebacterium glutamicum have been reviewed [3].
6.2.2
Biorefinery and the Building-block Concept
Biorefineries are defined as processing facilities that extract carbohydrates, oils,

lignin, and other materials from biomass and convert them into multiple prod-
ucts including fuels and high-value chemicals and materials [5]. Many technical
concepts are discussed, mainly focusing on the pretreatment and hydrolysis of
lignocellulose-based feedstocks [6]. Processing of green leaf material toward
high amino acid-containing grass juice as a raw material for fermentation has
also been used for commercial amino acid production [7]. The biorefinery defi-
nition is derived from the petrochemical definition but biorefineries can poten-
tially yield many high-value organic products that oil refineries cannot.
Recently, a comprehensive overview on the variety of biobased products which
are already produced in biorefineries or may originate from biorefineries in the
future has been presented [5]. Biobased products fall into three categories: com-
modity chemicals (including fuels), specialty chemicals, and materials. Either di-
rect physical or chemical processing of biomass such as cellulose, starch, oils,
6.2 Present State of the Industry 203
protein, or lignin results in biobased products or indirect processing from car-

bohydrates by biotechnological methods such as microbial (e.g., fermentation)
and enzymatic processing.
Competitive microbial fermentation of large-volume amino acids is already
closely integrated with large-scale starch-based (corn milling) or sucrose-based
(sugar cane) biorefinery operations. But the question arises if there are more fa-
vorable process options, if MAAP is integrated in a lignocellulose-based opera-
tion. The fermentation of amino acids might benefit from experience of ligno-
cellulose-based ethanol production. Requirements of feedstock quality are, how-
ever, substantially higher, because the MAAP process is more sophisticated than
the ethanol process and a consistent fermentation performance is of high im-
portance. The downstream process for feed-grade amino acids will, on the other
hand, tolerate the use of more impure carbon sources, compared with the very
sensitive downstream processes for biobased polymers. Production of high vol-
ume, high value feed additives like lysine and threonine will therefore have low-
er technological hurdles to overcome for successful integration into a lignocellu-
lose-based biorefinery concept than other large-scale fermentation processes
(e.g. poly(lactic acid), 1,3-propanediol).
The motivation for the integration of fermentation processes into a lignocellu-
lose-based biorefinery has been the availability of low-cost carbon sources and
the reduction of energy cost, because of the utilization of waste material for
generation of steam and electricity. Despite the 20- to 30-fold reduction of en-
zyme costs for cellulose hydrolysis [6, 8], however, significant disadvantages of
the lignocellulose-base biorefinery still exist:
· the use of dilute, acidic carbohydrate solution from hydrolysis and the slower
hydrolysis of cellulose at higher temperatures compared with hydrolysis of
starch increase the specific capital investment [9]
· the lower protein content of lignocellulose material compared with corn or
wheat and the absence of an established commercial application for co-prod-
ucts results in no additional benefit from co-products [9]
· despite progress in improving the utilization of xylose (C5 sugar from hemi-
cellulose) by yeast and other industrial important production organisms, utili-
zation of xylose is still not as efficient as the use of dextrose or sucrose.
Therefore, despite recent technical improvements, cellulose-based ethanol still

has significantly higher manufacturing cost compared to starch-based ethanol.
This cost disadvantage will only increase when cellulose-based production of fer-
mentation products with a greater need for consistent raw material quality and
more stringent downstream requirements are compared with the starch-based
or sucrose-based technologies. For a starch-based operation the state-of-the-art
technology converts 97% of the starch into rather pure and well-defined
fermentable sugars with 95% dextrose content. For the sucrose-based operation
from sugar cane, no hydrolysis is necessary in the first place, to yield a well de-
fined carbon source. Lignocellulose-based technology has a long way to go to
achieve this with the same level of process stability.
Table 6.1 Building-block concept in biorefinery. Twelve sugar-

derived building blocks which serve as a platform for the syn-
thesis of derivatives or secondary chemicals [5].
1,4-Succinic, fumaric and malic acids

2,5-Furandicarboxylic acid
3-Hydroxypropionic acid
Aspartic acid
Glucaric acid
Glutamic acid
Itaconic acid
Levulinic acid
3-Hydroxybutyrolactone
Glycerol
Sorbitol
Xylitol/arabinitol
For all renewable resources processed in a biorefinery, irrespective of the

main carbon source, the so called building block concept, which has been illus-
trated in detail [5], enables potential generation of additional value from co-prod-
ucts. The building block concept introduces twelve building blocks that can be
produced from sugar by biological or chemical conversion and can be subse-
quently converted to a number of high-value biobased chemicals or materials.
Building blocks, as considered in the biorefinery concept, are chemicals such as
succinic acid with multiple functional groups with the potential to be trans-
ferred into new families of useful molecules (Table 6.1). This will eventually also
benefit MAAP if it actually leads to reduced cost for carbon sources and energy
supply. For example, it has recently been reported that succinic acid can be pro-
duced economically and efficiently by fermentation with Mannheimia succinici-
producens at a product yield of 55% and productivity of 3.19 g L–1 h–1 [10] from
an inexpensive biomass-based wood hydrolyzate.
In an integrated biorefinery it should be also possible to use a variety of co-
products and intermediates for MAAP; there will, however, also be limitations
to this concept that will be discussed in more detail in Section 6.3.
6.2.3
Metabolic Engineering and the Building-block Concept
Because, in multi-product operations, flexibility is mandatory to adapt to ever-

changing market situations for raw materials and products, it will be important
that the production organisms and technical constraints of fermentation pro-
cesses integrated in a biorefinery enable the effective conversion of a wide vari-
ety of substrates. This can be achieved by application of metabolic engineering,
and with intelligent design of bioreactors and flexible utility operation. Meta-
bolic engineering has been defined as the improvement of enzymatic, transport,
and regulatory functions of the cell with the application of recombinant DNA
6.3 Environmental and Commercial Consideration of Microbial Amino Acid 205
technologies [11]. Developments in metabolic engineering have been reviewed

extensively [12–14]. Analytical tools have been developed to investigate intracel-
lular metabolic fluxes [3, 14, 15]. For metabolic flux analysis the building block
concept introduced by Neidhardt et al. [16] is of central importance. According
to this concept in microbial intermediary metabolism twelve building blocks are
derived from carbon sources which subsequently are required for biosynthesis
of biomass and products formed by a microorganism. Interestingly, a similar
concept has been defined in biorefinery (Table 6.1). In Table 6.2 the building
block concept of cellular metabolism is illustrated for C. glutamicum. Here only
eleven central intermediates of metabolism were considered to calculate the
molecule demand to generate one gram of dry cell weight [17].
For the predominant amino acid production species C. glutamicum this con-
cept has been extensively applied [17–19] so that nowadays the metabolic net-
work is well understood. With this knowledge of metabolic flux distributions for
a large number of physiological states it is feasible to determine the complex re-
adjustment of metabolic networks in response to changes in environmental con-
ditions or to changes in the metabolic network structure. Large differences in
intracellular building block demand and metabolic fluxes were observed in C.
glutamicum when the production of glutamate and lysine were compared with
physiological state of growth [20]. Another example for alterations in metabolic
network structure is the modification of cofactor dependency of important en-
zymes. In a lysine-producing C. glutamicum strain a glutamate dehydrogenase
was introduced which is dependent on NADH instead of NADPH. The building
block demand and metabolic fluxes changed dramatically [21].
Similar redirections of metabolic fluxes might occur with use of alternative
carbon sources from biorefinery side streams which differ in their degree of re-
duction. During the last two decades a platform was established which enables
us to quantify metabolic effects rather than just to evaluate them qualitatively.
This way it will be possible to quantify the effects of new carbon source input
from emerging biorefinery. Subsequently, metabolic engineering strategies can
be designed for rational metabolic engineering or for traditional strain breeding.
Also, models and control schemes for a cost-effective operation and control of a
biorefinery considering metabolic constraints and substrate supply of the differ-
ent fermentation operations can be developed.
6.3
Environmental and Commercial Consideration of Microbial Amino Acid Production
Integrated in a Biorefinery
The role of amino acids in world-wide ecology and in relation to the future de-
velopment of the world population has been evaluated recently [22]. Feeding an-
imals in a well balanced manner leads to reduced release of ammonia into the
environment. When methionine instead of soybean meal is given to poultry the
amount of ammonia released to the environment is reduced more than tenfold
Table 6.2 Building block concept in metabolic engineering of microbial amino

acid production. Eleven building blocks are produced from carbon sources in mi-
crobial metabolism and subsequently are used as intermediates for biosynthetic
pathways. The typical demand in lmol to build one gram of dry cell weight of C.
glutamicum is shown. These data are relevant for a leucine auxotroph strain. For
a leucine prototroph strain the demand for pyr and accoa is 2685 and 2939 lmol
per gram of dry cell weight, respectively. The three letter code for amino acids is
indicated.
Precursor Amount Stoichiometry
g6p f6p ri5p e4p gap pga pep pyr accoa oaa akg
Ala 606 1
Arg 189 1
Asx 399 1
Cys 87 1
Glx 806 1
Gly 361 1
His 71 1
Ile 202 1 1
Leu 0 2 1
Lys 202 1 1
Met 146 1
Phe 133 1 2
Pro 170 1
Ser 225 1
Thr 275 1
Trp 54 1 1 1
Tyr 81 1 2
Val 284 2
Dmp 146 1 1
Protein 0 0 125 268 0 673 482 1724 0 1370 1165
RNA 630 368 262
DNA 100 50 50
Lipids 129 129 2116
LPS 51 16 24 24 24 329
Peptidoglycan 55 28 83 55 28 28
Glycogen 154
CI units 49
Polyamines 59
Precursor g6p f6p ri5p e4p gap pga pep pyr accoa oaa akg
Total 205 71 879 268 129 1293 534 1807 2500 1710 1252
Abbreviations: Dmp, diaminopimelate; g6p, glucose 6-phosphate; f6p, fructose 6-

phosphate; ri5p, ribose 5-phosphate; e4p, erythrose 4-phosphate; gap, glyceraldehyde
3-phosphate; pga, 3-phosphoglycerate; pep, phosphoenolpyruvate; accoa, acetyl coen-
zyme A; oaa, oxaloacetate; akg, a-ketoglutarate [17]
6.3 Environmental and Commercial Consideration of Microbial Amino Acid 207
[22]. In this way surplus nitrogen and especially nitrate can be avoided so that
ground-water quality is improved. The same is true for substitution of soybean
meal by lysine and threonine in low-protein diets.
An ecological balance for the fermentative production of polyhydroxyalkano-
ates revealed that the major burden on the environment was caused upstream
of the fermentation in the supply chain which provides the substrate for the fer-
mentation [23]. Similar results were obtained for the fermentative production of
lysine and threonine [24]. This means that a re-engineering in the sugar-provid-
ing industry could further improve the ecological impact of MAAP. Because the
lignocellulose-based process is significantly less efficient for the preparation of
the monomer carbon source, however, compared with the well established
starch or sucrose-based process there might not be a significant improvement.
Considering the ecological balance the most efficient process will be the prepa-
ration of a sucrose stream from sugar cane and utilization of sugar cane ba-
gasse for energy generation. The utilization of bagasse for energy supply or sup-
ply of lignocellulose-based carbon sources has a significant advantage over corn
stover, because it is already available at the processing site and no costs arise for
collection and transport.
The markets for the amino acids glutamate, lysine, and threonine are charac-
terized by dynamic growth and they are highly competitive. Monosodium gluta-
mate is used in food as a taste enhancer which is added in prepared food at 0.1
to 0.8% or even more in East Asian dishes. Lysine is used as a feed additive
and the addition of 1 kg lysine · HCl increases poultry feed quality to the same
extent as addition of 35 kg soybean meal [22]. Also threonine is used as feed ad-
ditive especially in diets for pig and poultry. Addition of up to 0.75% of threo-
nine to sorghum-peanut meal increases breast meat deposition by more than
15%.
Lysine and monosodium glutamate are the largest products in the category of
MAAP, and the total market value for amino acids including threonine and
tryptophan was estimated to be in the range of 2.5 billion 1 in 2004. A publica-
tion by Ajinomoto illustrates the decrease of manufacturing cost for lysine and
threonine during the last two decades [25]. It becomes obvious that in this
highly competitive market the product price stabilizes on a quite narrow low
price level. Innovative concepts for reduction of production cost are of tremen-
dous importance to stay competitive in MAAP.
The size and location of a MAAP site is affected by the following considera-
tions:
· availability and price of carbon sources as major single cost
· availability and price of electricity, gas, and steam supply
· availability and price of nitrogen source
· equipment prices and prices for engineering and construction driving capital
investment
· proximity to the market and large-scale customers to achieve low transport
and distribution cost
· currency effects in a global market.
Table 6.3 Commercially available lysine preparations for feed

use based on different downstream technologies [3].
Product Lysine-base Downstreaming Wastes Advantages

preparation content (%) steps
Lysine HCl 78.8 Biomass separation, Biomass, organic No crystallization

[26] ion exchange, drying acids, salts,
ammonia, water
Liquid lysine Approx. 50 Biomass separation, Biomass, salts No drying step
[27] evaporation, filtration
Granulated 40–50 Evaporation, Almost none No wastes, no
lysine sulfate [28] spray drying separation step
The same considerations are valid if MAAP is evaluated in the context of any
biorefinery operation. MAAP processes consist of four major steps: receiving,
storage and sterile preparation of carbon sources and other raw materials; culti-
vation of the production strain in an aerobic process; downstream of fermenta-
tion broth; and purification of amino acids and waste treatment. Table 6.3 illus-
trates, as an example, that depending on the downstream process the overall
process yield and thus the quantity of waste materials differs significantly [3, 4].
Even though the process flow in the downstream part of the production of a
special lysine sulfate product which contains the whole culture broth was sim-
plified to a minimum number of unit operations, great challenges had to be
overcome to enable good handling properties and variations of lysine content in
the final product. Because, in the feed-additive market, customers require only
a guaranteed minimum content of the active substance and handling properties
suitable for large scale operations (low dustiness, low caking tendency, good
flow ability, high bulk density) there is room for the development of further
low-purity product forms if the savings in manufacturing cost are substantially
higher than the costs of product registration and marketing efforts for a new
product form. Even if a feed mill is integrated into a biorefinery operation and
a liquid, amino acid-enriched product [29] is used directly for the feed prepara-
tion, large variation of the amino acid content will be very problematic for high-
quality feed preparation. This puts significant higher constraints on process
control of MAAP within a biorefinery operation compared with production of
ethanol.
6.4 Technical Constraints for Integration of Microbial Amino Acid Fermentation into a Biorefinery 209
6.4
Technical Constraints for Integration of Microbial Amino Acid Fermentation
into a Biorefinery
6.4.1
Mono-septic Operation
Because all amino acid fermentation processes operate between 30 8C and 40 8C

at approximately neutral pH conditions they are highly susceptible to contami-
nation. Contaminants usually have higher growth rates than the production or-
ganisms. They also might degrade amino acids during the cultivation or add en-
zymatic activities that lead to increased degradation during further processing
of the fermentation broth. Contaminants also may express toxins that are not
desired for food and feed applications. This means that much effort must de-
voted to ensuring sufficient sterilization of all incoming raw materials, mono-
septic operation of the fermentation process, and sufficient sanitation of all
downstream equipment. Sterilization of the raw material is especially difficult if
large amounts of suspended solids or precipitation result in fouling of heat-ex-
changer surfaces. Increased viscosity also negatively affects turbulent flow in
continuous sterilizers and thus reduces sterilization efficiency. This means that
flexible utilization of different raw material streams for fermentation will most
probably result in increased investment and an increased maintenance effort to
ensure sterile operation. This must be taken into account if the benefit to over-
all production cost is evaluated.
6.4.2
Carbon Sources
The broadening of substrate utilization spectrum for MAAP will be of central

importance for a competitive production process. During the last two decades
metabolic engineering strategies have mainly focused on the final terminal bio-
synthetic pathways. For lysine biosynthesis this resulted in extremely high prod-
uct yields of up to 0.5 g lysine · HCl g–1 sucrose or glucose [3]. Threonine pro-
duction strains which are able to grow on ethanol [30] or methanol [31] are
available. Production of lysine [32, 33] and glutamate [34] from methanol has
also been described.
A future challenge will be the broadening of the substrate-utilization spec-
trum of production strains for MAAP. Because Saccharomyces cerevisiae is of
great industrial importance and is thoroughly characterized in its genetics and
in its physiology, different substrate utilization systems have been established
for this yeast [35]. Similar strategies must be used to broaden the substrate utili-
zation spectrum of production strains in MAAP. In particular, to confer poly-
mer-degrading capabilities on hosts, access to the substrate must be ensured by
efficient secretion of the enzymes from the host. Additional requirements arise
for sugar transport and for a new set of metabolic activities. The design of these
new sets of metabolic activities is a challenge which must be addressed by a ra-

tional design of the metabolic network coping with the optimization of a large
variety of cellular regulation systems.
Lignocellulose is the most abundant biomass in the biosphere and enzymes
for utilization of lignocellulose must attack in a sequential concerted and syner-
gistic manner. Hydrolyzates of lignocellulose contain compounds that are inhib-
itory to most microorganisms, so strain development is required to facilitate the
development of robust “platform” biocatalysts that can ferment biomass sugars
into either ethanol or other desired biobased products with economically viable
rates and yields on industrial scales. Tolerance to harsh environments, including
elevated temperatures, high salt, and low pH, will be essential.
Currently, available strains are severely limited in pentose utilization and ex-
hibit poor hydrolyzate tolerance. Zymomonas mobilis has been successfully engi-
neered to utilize xylose for ethanol production by introducing a xylose assimila-
tion pathway and two genes encoding transketolase and transaldolase resulting
in a product yield which is 85% of the theoretical maximum [36].
Acetate has not been considered as a building block in the biorefinery concept
because of its lower potential compared with acetone, which is already a petro-
chemical byproduct. Nevertheless, it is worthwhile discussing acetate as an alter-
native or additional carbon source to sucrose or glucose because acetate metabo-
lism of C. glutamicum has been investigated in detail by using modern analyti-
cal tools of metabolic engineering [37–39]. Metabolic flux analysis revealed how
the carbon flow inside the bacterial cell is readjusted and how intermediate
building block metabolism is affected. For example, in vivo citric acid cycle ac-
tivity was increased almost twofold when acetate was fed compared with growth
on glucose plus acetate [37]. Furthermore, the synthesis of building blocks was
superior during growth on glucose plus acetate. Interestingly, a recent publica-
tion [38] has revealed that even small amounts of acetate, injected with a glu-
cose acetate pulse into a steady-state continuous culture, significantly increased
the experimental lysine yield from C. glutamicum. The use of carbon sources
such as palmitate and propionate has been evaluated [39]. On palmitate and
propionate very low growth rates and biomass yields have been observed. This
kind of information might help in the design of optimum carbon source cock-
tails at the interface between biorefinery and MAAP.
A similar study has been performed for other carbon sources [40]. This kind
of information will, in the future, help to redesign metabolism of C. glutamicum
for the utilization of new carbon sources from the biorefinery. In this context it
is important to know where redox equivalents from carbon sources can be chan-
neled into the electron transport chain and how efficient energy generation will
be for different carbon sources. For C. glutamicum this topic has been reviewed
by Bott and Niebisch [41].
Although lactose is very dilute in whey it has been addressed as carbon
source for C. glutamicum. The introduction of the lac operon of Escherichia coli
resulted in growth of C. glutamicum on lactose and with the use of heterologous
expression of the lac and gal genes from Lactococcus lactus, and Lactobacillus del-
6.4 Technical Constraints for Integration of Microbial Amino Acid Fermentation into a Biorefinery 211
brueckii lysine was formed at a concentration of up to 2 g L–1 [42, 43] which is

far from target values which would be regarded as economical. So, there are
ways of utilizing a wide range of carbon sources for MAAP, but for a biorefin-
ery operation they must all be available at consistent qualities and quantities at
a price significantly lower than starch hydrolyzate or sucrose. The use of MAAP
as a sink for undesired co-product stream with a highly variable composition
will not be competitive.
6.4.3
Nitrogen Source
Complex nitrogen sources such as corn steep liquor or yeast extract are already
added to MAAP processes to increase productivity and stability of the microbial
fermentations. They do not contribute significantly to the supply of nitrogen
sources. This is achieved with low-cost alternatives, for example ammonium sul-
fate or liquid ammonia. Corn steep liquor is a by-product in the production of
corn starch and contains amino acids, nucleic acids, vitamins, minerals, and a
significant amount of phosphorus. The natural fluctuation in quality of complex
nitrogen sources results in fluctuations of MAAP process performance. Inten-
sive quality monitoring for the raw materials is required. This must be consi-
dered when new nitrogen sources from a biorefinery are to be introduced in
MAAP. The use of high-value amino acid or peptide fractions as nitrogen
source in amino acid fermentation could be an outlet for these components,
however, only if other applications of those amino acids are not successful. This
application would force a biorefinery operation to price those “high-value” nitro-
gen sources at the same level as “low-value” ammonium sulfate or ammonia.
6.4.4
Phosphorus Source
Sufficient supply of phosphorus is essential for oxidative metabolism. Fermenta-

tion media contain synthetic and/or complex phosphorus sources. Interestingly,
corn steep liquor as a complex source of phosphorus is a traditional product of
corn processing and, depending on the steeping process, the major fraction of
phosphorus is available in the inorganic form or bound as inositol phosphate.
Media must be carefully designed with regard to phosphorus input because ex-
cess input leads to extensive oxidative metabolism and low biomass and product
yields. On the other hand, low input results in slow biomass formation, weak
intermediate energy supply, weak export capacity, and low productivity. Phos-
phate limitation elicits a specific gene expression response in C. glutamicum
[44]. By the introduction of simultaneous and concerted carbon and phosphate
limitation continuous fermentation for lysine production has been optimized
[45]. All mass flows in a biorefinery must certainly be evaluated for their phos-
phorus content before they can be integrated in MAAP.
6.4.5
Mixing and Oxygen Supply
Depending on the extent of reduction of the carbon source and on the energetic
efficiency of MAAP processes more or less oxygen is required to produce bio-
mass and amino acids. The fermentation equipment for mixing, oxygen supply,
and heat removal will have to handle a rather large range of requirements, espe-
cially when flexible utilization of carbon sources with different physical proper-
ties (i.e. viscosity) and different reduction state of the carbon atoms are used. If
the carbon sources are less oxidized than sugars, the fermentation will use sig-
nificantly more oxygen and produce more CO2. For E. coli this effect seems to
be uncritical up to a concentration of 30% of carbon dioxide in the exhaust gas
[46].
6.4.6
Toxicity
A major problem of the usage of raw materials from biorefinery in MAAP may
be the toxicity of components which are contained in the raw material. This top-
ic has already been addressed in Section 6.4.2 when the use of lignocellulose
was discussed. Advanced monitoring technologies such as cytometry [47, 48]
and DNA microarray analysis of gene expression [49, 50] might be used to
monitor toxicity effects, to reveal the nature of toxicity, and to search for meta-
bolic engineering or strain breeding strategies to overcome the hurdles caused
by toxic compounds. Recently, metabolic flux analysis has been applied to acid-
tolerant yeast Candida milleri which would be ideal to convert industrial pulp
mill xylose containing waste streams into xylitol [51]. This kind of investigation
is a first step in the direction of a comprehensive understanding of metabolic
network responses to stimuli from substrate streams which originate from bio-
refineries. It is crucial to select robust host strains such as, in this case, C. mill-
eri in advance and afterwards to optimize the metabolic pathways for maximum
product formation. In the field of MAAP this approach is in its infancy, because
metabolic engineering efforts have been focused on well established hosts. Al-
ternatively, before proceeding with further optimization of terminal pathways
for amino acid biosynthesis, metabolic engineering and classical strain breeding
should be initiated for improvement of resistance against toxic effects. A re-
cently described example is the development of a raffinate (ammonium sulfate
rich eluent from ion exchange)-resistant lysine production strain that enables
re-utilization of the ammonium sulfate generated as a waste stream during pur-
ification [52].
6.4.7
Cultivation Temperature
A significant part of production costs is caused by cooling demand. To reduce

costs of cooling utilities C. glutamicum strains have been selected which are
characterized by a growth optimum at 40 8C instead of 30 8C [53]. This strategy
might be useful to integrate mass flows from biorefinery into MAAP processes
so that costly cooling operations are avoided.
6.5
Biorefineries are defined as processing facilities that extract carbohydrates, oils,

lignin, and other materials from biomass, and convert them into multiple prod-
ucts including fuels and high-value chemicals and materials [5]. In our contri-
bution we have illustrated the major hurdles which must be overcome to gener-
ate value in industrial production of amino acids. Sucrose, molasses, and starch
hydrolyzates have been used as renewable carbon sources for decades. Existing
starch processing already has the advantage that co-product markets as for glu-
ten and corn oil contribute to overall plant economics. In industrial amino acid
production experience in handling these product streams is available. Based on
this experience it is obvious that potential new product streams which might
enter MAAP must be of low cost and characterized by low concentrations of im-
purities and by low fluctuations in raw material composition.
It will be a prerequisite for the success of the biorefinery concept to yield new
well defined carbon sources of high purity and lower cost compared with starch
or sugar-based feedstock. If these characteristics can be ensured in future the
integration of biorefineries and MAAP will be successful. Especially, with regard
to downstream processing and product formulation, stable product characteris-
tics and guaranteed purity must be delivered by a MAAP process which is inte-
grated into a biorefinery.
Not only will the large part of variable cost attributable to the carbon source
in MAAP be reduced substantially, but also the fixed cost will be reduced when
different co-products can be produced in one biorefinery. We have illustrated
the example of generation of energy or even an additional carbon source from
sugar cane bagasse.
The processing of natural compounds requires extensive equipment size and
energy consumption for water handling. This problem must be addressed to
improve cost structure. The availability and price of carbon and nitrogen
sources, electricity, gas, and steam supply, and equipment prices and prices for
engineering and construction must be considered together, to define the opti-
mum for economic operation of MAAP as part of a biorefinery. Proximity to the
market and to large-scale customers guarantees low transport and distribution
cost which means that sophisticated optimization of the supply chain must be
performed. An additional option might be to combine biorefinery locations with

animal feed-production facilities, to make use of synergies in logistic planning
and transportation. If all prerequisites for the establishment of this kind of bio-
refinery concept are fulfilled, lignocellulose-based processes will be beneficial
for the economics and ecology of MAAP.
Taken together, the biorefinery concept promises to provide cheap carbon
sources with unlimited availability, environmentally friendly processes, and valu-
able co-products. Glycerol and ethanol might in future be inexpensive and
might be used for the biotechnological MAAP as an alternative to sugar sub-
strates, depending on public regulation. Even for chemical synthesis of amino
acids and other specialty chemicals glycerol and ethanol might be of importance
in the future.
Until now we have considered topics related to the business model of MAAP.
The basis for a successful operation of this business process is a well elaborated
technology platform. To establish this platform a systematic approach must be
designed which guarantees that future product streams are compatible within a
biorefinery so that they can be combined and integrated. The so called building
block concept in biorefineries, which has been illustrated in detail [5], has been
defined to support the systematic design of biorefineries. If technical solutions
for this concept become available, large opportunities will arise for MAAP. We
have illustrated in our contribution that also in metabolic engineering a similar
building block concept has been defined. Using this concept metabolic engi-
neering has contributed to tremendous progress in MAAP during the last two
decades. Now it will be the challenge to redirect metabolic fluxes in microorgan-
isms depending on changes in carbon sources, phosphorus source, mixing and
oxygen supply, toxicity, and cultivation temperature. The building block concept
will help to systematically develop platform microbes with the use of metabolic
engineering.
The question arises whether to build technology platforms in homologous
systems or to intensify the establishment of heterologous pathways for broaden-
ing of the substrate utilization spectrum for MAAP in selected host organisms.
The answer must be found individually, because we just have started the jour-
ney toward developing robust high-performance strains which can efficiently
use new carbon sources and can resist harmful effects in their microenviron-
ment.
If the major hurdles described in our contribution are overcome the concept
of biorefinery is beneficial for MAAP. The major benefit will originate from the
additional value of co-products for which a clear marketing and sales concept
must be demonstrated.
Acknowledgment
We acknowledge the support of Dr Klaus Huthmacher, Professor Dr Wolfgang

Leuchtenberger, and Dr Michael Binder, Degussa AG, Hanau, Germany.
References 215
References
1 L. Eggeling, H. Sahm, Metabolic engineer- 18 W. Wiechert, C. Siefke, A. Marx, A. A. de

ing, Marcel Dekker, New York, 1999, Graaf, Biotechnol. Bioeng. 1997, 55, 118–
153–175. 135.
2 M. Ikeda, Advances in Biochemical Engi- 19 K. Schmidt, A. Marx, A. A. de Graaf, W.
neering/Biotechnology 2003, vol. 79, 1–35. Wiechert, H. Sahm, J. Nielsen, J. Villad-
3 W. Pfefferle, B. Möckel, B. Bathe, A. sen, Biotechnol. Bioeng. 1998, 58, 254–
Marx, Advances in Biochemical Engineer- 257.
ing/Biotechnology 2003, vol. 79, 59–112. 20 A. Marx, K. Striegel, A. A. de Graaf, H.
4 R. Kelle, T. Hermann, B. Bathe, Hand- Sahm, L. Eggeling, Biotechnol. Bioeng.
book of Corynebacterium glutamicum, 1997, 56, 168–180.
CRC Press, Boca Raton, 2005, 465–488. 21 A. Marx, B. J. Eikmanns, H. Sahm, A. A.
5 T. Werpy, G. Petersen, Top Value Added de Graaf, L. Eggeling, Metab. Eng. 1999,
Chemicals From Biomass, 2004, vol. I, 1, 35–48.
http://www.nrel.gov/docs/fy04osti/ 22 H. Wennemer, G. Flachowsky, G. Hoff-
35523.pdf, accession 07. 04. 2005. mann, Protein, Population, Politik, Plexus
6 J. R. Cherry, K. Wenger, BioWorld Europe, Verlag, Frankfurt, 2005, 86–87.
2005, 01, 10–13. 23 I. Renner, W. Klöpffer, Untersuchung der
7 Food production daily, http://www.fodpro- Anpassung von Ökobilanzen an spezifische
ductiondaily.com/new/new-ng.asp?id= Erfordernisse biotechnischer Prozesse und
30072-agroferm-ups-lysine, accession Produkte. Umweltbundesamt, 2005.
15. 04. 2005. 24 M. Binder, Personal communication,
8 M. Knauf, M. Moniruzzaman, Int. Sugar Degussa AG, 2005.
J. 2004, vol. 106, 147–150. 25 Ajinomoto, http://www.ajinomoto.com/ar/
9 A. McAloon, F. Taylor, W. Yee, K. Ibsen, i_r/pdf/fact/feeduse_amino.pdf, accession
R. Wooley, Determining the cost of produc- 07. 04. 2005.
ing ethanol from corn starch and lignocellu- 26 W. Fechter, J. H. Dienst, J. F. Le Patourel,
losic feedstocks. A joint study sponsored by ZA 9409059, 1995.
U.S. Department of Agriculture and U.S. 27 P. Lucq, C. Domont, EP534865, 1993.
Department of Energy, National Renew- 28 A. Höfler, H. C. Alt, C. J. Klasen, H. Frie-
able Energy Laboratory, Golden drich, U. Hertz, L. Mörl, R. Schütte,
Colorado, 2000. EP809940, 1997.
10 D. Y. Kim, S. C. Yim, P. C. Lee, W. G. Lee, 29 M. Binder, K. E. Uffmann, US 6 465 025,
S. Y. Lee, H. N. Chang, Enzyme Microbial 2002.
Technol. 2004, 35, 648–653. 30 H. Yukawa, T. Nara, Y. Takayama, US
11 J. E. Bailey, Science 1991, 252, 1668–1677. 4 427 774, 1984.
12 W. R. Farmer, J. C. Liao, Curr. Opin. Bio- 31 H. Anazawa, H. Motoyama, S. Teshiba,
technol. 1996, 7, 198–204. US 5 217 883, 1993.
13 D. C. Cameron, F. W. R. Chaplen, Curr. 32 F. J. Schendel, C. E. Bremmon, M. C.
Opin. Biotechnol. 1997, 8, 175–180. Flickinger, M. Guettler, R. S. Hanson,
14 E. T. Papoutsakis, S. Y. Lee, Metabolic en- Appl. Environ. Microbiol. 1990, 56, 963–
gineering, Marcel Dekker, New York, 970.
1999, 1–49. 33 H. Motoyama, H. Yano, Y. Terasaki, H.
15 U. Sauer, Curr. Opin. Biotechnol. 2004, Anazawa, Appl. Environ. Microbiol. 2001,
15, 58–63. 67, 3064–3070.
16 F. C. Neidhardt, J. L. Ingraham, M. 34 T. Brautaset, M. D. Williams, R. D. Dil-
Schaechter, Physiology of the bacterial cell, lingham, C. Kaufmann, A. Bennaars, E.
Sinauer, Sunderland, MA, 1990, 133– Crabbe, M. C. Flickinger, Appl. Environ.
173. Microbiol. 2003, 69 , 3986–3995.
17 A. Marx, A. A. de Graaf, W. Wiechert, L. 35 E.-S. Choi, S.-K. Rhee, Metabolic engineer-
Eggeling, H. Sahm, Biotechnol. Bioeng. ing, Marcel Dekker, New York, 1999,
1996, 49, 111–129. 281–307.
36 M. Zhang, C. Eddy, K. Deanda, M. Fin- 45 J. A. de Hollander, F. R. Eswilder, J. A. C.

kelstein, S. Picataggio, Science 1995, 267, Noordover, EP 0796916 A1 970924, 1997.
240–243. 46 R. Fass, T. R. Clem, J. Shiloach, Appl. En-
37 V. F. Wendisch, A. A. de Graaf, H. Sahm, viron. Microbiol. 1989, 55, 1305–1307.
B. J. Eikmanns, J. Bacteriol. 2000, 182, 47 A. Marx, C. J. Hewitt, R. Grewal, S.
3088–3096. Scheer, K. Vandre, W. Pfefferle, B. Koss-
38 L. Paegle, M. Ruklisha, J. Biotechnol. mann, P. Ottersbach, C. Beimfohr, J.
2003, 104, 123–128. Snaidr, C. Auge, M. Reuss, CIT 2003,
39 R. Gerstmeir, V. F. Wendisch, S. 75, 608–614.
Schnicke, H. Ruan, M. Farwick, D. Rein- 48 C. J. Hewitt, R. Franke, A. Marx, B. Koss-
scheid, B. J. Eikmanns, J. Biotechnol. mann, P. Ottersbach, Biotechnol. Lett.
2003, 104, 99–122. 2004, 26, 549–557.
40 D. Molenaar, M. E. van der Rest, A. 49 V. F. Wendisch, J. Biotechnol. 2003, 104,
Drysch, R. Yücel, J. Bacteriol. 2000, 182, 273–285.
6884–6891. 50 T. Polen, V. F. Wendisch, Appl. Biochem.
41 M. Bott, A. Niebisch, J. Biotechnol. 2003, Biotechnol. 2004, 118, 215–232.
104, 129–153. 51 T. B. Granström, A. A. Aristidou, J. Joke-
42 W. Brabetz, W. Liebl, K. H. Schleifer, la, M. Leisola. Biotechnol. Bioeng. 2000,
Arch. Microbiol. 1992, 155, 607–612. 70, 197–207.
43 E. Barrett, C. Stanton, O. Zelder, G. Fitz- 52 H. J. Laiw, J. M. Eddington, Y. Yang,
gerald, R. P. Ross, Appl. Environ. Micro- R. Dancey, S. Swisher, W. Mao, WO01/
biol. 2004, 70, 2861–2866. 09306 A2, 1999.
44 T. Ishige, M. Krause, M. Bott, V. F. Wen- 53 Y. Murakami, H. Miwa, S. Nakamori,
disch, H. Sahm, J. Bacteriol. 2003, 185, JP 4 004 887 A, 1992.
4519–4529.
217
7
Protein-based Polymers:
Mechanistic Foundations for Bioproduction and Engineering
Dan W. Urry
7.1
Introduction
7.1.1
Definitions
7.1.1.1 Proteins and Protein-based Polymers

A protein is a polypeptide, (–NH–CHR–CO–)n, where R is the side chain of any
of the twenty different amino acid residues available to biological protein syn-
thesis, or in practice the R group can also be a chemical or enzymatic modifica-
tion thereof. In the living organism the molecular weight can range from a few
thousand Daltons, for example, some tens of residues, to two million Daltons,
that is, twenty thousand residues. Protein-based polymers are polymers of re-
peating peptide sequences, The repeating unit may be as small as a dipeptide,
(–NH–CHR–CO–NH–CHR'–CO–)n, or as large as hundreds of peptide residues
and n can be as large as several hundred, or more. Protein-based polymers hold
promise of low-cost production by means of recombinant DNA technology. By
this process genes are constructed that encode for the desired peptide sequence,
and a biological organism is transformed to produce (express) the designed pro-
tein-based polymer sequence that could even be a thermoplastic. Thus, proteins
and protein-based polymers constitute renewable resources with the potential,
at least in part, to replace petroleum-based polymers.
7.1.1.2 Two Basic Principles for Protein-based Polymer Engineering

As introduced here, two basic principles, referred to as the hydrophobic and
elastic consilient mechanisms, for protein-based polymer engineering bear on
controlling hydrophobic association and utilizing near ideal elasticity [1]. More-
over, an understanding of hydrophobic association is key to bioproduction, puri-
fication and function, and the nature of elasticity relates to efficient function
and to an enabling of a remarkable biocompatibility for medical applications.
ISBN: 3-527-31027-4
218 7 Protein-based Polymers: Mechanistic Foundations for Bioproduction and Engineering
7.1.2
Proteins in Aqueous Media
Proteins in an aqueous milieu provide the ultimate in amphiphilic polymers.

There is absolute control of sequence where in each position can occur any one
of twenty disparate amino acid residues, ranging from very hydrophobic to very
polar (for example, charged), and this occurs with strict maintenance of stereo-
chemistry. The result is a structural and functional diversity that is unmatched
by any other polymer.
7.1.3
Thermodynamics of Proteins in Water
7.1.3.1 Exothermic Hydration of Apolar Groups

In 1937 Butler reported the fundamental finding that dissolution of CH2 groups
of an alcohol series in water is an exothermic reaction [2]. Thinking within the
framework of the Gibbs free energy for solubility, DG(solubility) = DH–TDS. For
the methanol to n-pentanol series, the heat released (DH) on hydration is
1.3 kcal mol–1 CH2 with the (–TDS) entropic term limiting solubility by a
+1.7 kcal mol–1 CH2. This requires that there be structured (low entropy) water
surrounding dissolved hydrophobic groups, and that too much hydrophobic
hydration means insolubility (Section 7.1.4 below).
7.1.3.2 The Change in Gibbs Free Energy of Hydrophobic Association

The most fundamental property deciding aqueous protein-based polymer struc-
ture formation and function resides in DG8HA, the change in Gibbs free energy
for hydrophobic association, that is, for loss of solubility of hydrophobic groups
[3]. Insight into the primary operative component of DG8HA gains from the old
adage, “oil and vinegar do not mix.” In proteins and protein-based polymers,
however, oil-like and vinegar-like groups are forced by sequence to coexist along
a chain molecule; they are forced to interact. As one might expect from the old
adage, they display their reluctance to intermix by a water-mediated repulsion.
Oil-like (apolar) and vinegar-like (polar) groups of proteins, forced by primary, sec-
ondary, tertiary and quaternary structure to coexist, repulse each other as each seeks
hydration unperturbed by the other. Apolar and polar groups compete for hydration!
7.1.3.3 The Apolar–Polar Repulsive Free Energy of Hydration, DG8ap

The operative component of DG8HA is the apolar–polar repulsive free energy of
hydration, DGap; it measures the competition for hydration [4]. These funda-
mental thermodynamic quantities, DG8HA and DGap, are quantifiable by a num-
ber of experimental methods. The value of DGap can be determined using differ-
ential scanning calorimetry as the change in DG8HA attending ionization of a
functional R group, and it can also be measured using acid–base titrations from
the shift that occurs in pKa of an ionizable side-chain on changing the hydro-
phobicity of the protein-based polymer. From the standpoint of bioproduction,
ionization can give solubility and neutralization can effect selective phase sepa-
ration for purification.
7.1.4
The Inverse Temperature Transition for Hydrophobic Association
By the Second Law of Thermodynamics, an increase in temperature results in

an overall decrease in order of the total system, which in our case is water plus
protein-based polymer. With the correct balance of oil-like and vinegar-like
groups, raising the temperature through the transition range results in a phase
separation in which the protein-based polymer goes from being randomly dis-
persed in solution to being more ordered, and even crystalline, on separation
[4]. This is called an inverse temperature transition. Structured water surround-
ing the dissolved polymer, which becomes less ordered bulk water as the poly-
mer associates, makes the inverse temperature transition congruent with the
Second Law. Such water does exist surrounding oil-like (hydrophobic) groups
and is called hydrophobic hydration. Thus, the inverse temperature transition
represents a transition from a state of hydrophobic groups being hydrated to
the loss of hydrophobic hydration as the hydrophobic groups associate. Develop-
ment of too much hydrophobic hydration for a given temperature results in the
insolubility due to hydrophobic association. On the other hand, in the process
of achieving their own hydration, charged species disrupt hydrophobic hydra-
tion, even the hydrophobic hydration that forms during a transient hydrophobic
dissociation. In this way charged species decrease the amount of hydrophobic
hydration, and thereby disrupt hydrophobic association. As considered above,
the driving force for loss of solubility (hydrophobic association) derives from the
development of too much hydrophobic hydration for a given temperature [2].
Since DG(solubility) = DH–TDS, insolubility occurs as the term (–TDS) becomes
too positive and dominates the inherently negative DH term.
7.1.5
The Role of Elasticity in the Engineering of Protein-based Polymers
7.1.5.1 Near Ideal Elasticity Provides for Efficient Energy Conversion

In general, an energy input changes hydrophobic association, and as the means
of achieving function, the input energy is transiently stored in chain deforma-
tion. Efficient energy conversion requires that most or all of the energy ex-
pended during elastic deformation be recovered on relaxation, as can only occur
with near ideal elasticity. This is most apparent in the performance of mechani-
cal work. During an isometric contraction, for example, the input energy con-
verts to an increase in elastic force. If the force–relaxation curve falls below the
force–extension curve, the result is called hysteresis, and the result is that only
a part of the input energy spent on extension is recovered on relaxation. There-
fore, without near ideal elasticity, too little energy of deformation becomes avail-
able for the function of producing motion
7.1.5.2 Mechanism of Near Ideal Elasticity

A mechanism of elasticity that allows for near complete recovery of the defor-
mation energy on relaxation is central to the design of an efficient protein-based
machine that is to perform mechanical work. The structure and mechanism
should be such as to yield the experimental result of a near perfectly reversible
stress–strain curve. In our view, such near ideal elasticity arises, in general,
from a decrease in chain mobility within the extended chain segment. Specifi-
cally, the decrease in chain mobility on extension is well described as a decrease
in amplitude of torsion angle oscillations within the load-bearing chain. This
allows that very little energy is lost from the chain to its environment, such as
to associated chains that bear no load.
7.1.6
Many of the Advantages of Protein-based Polymeric Materials
One of the most important advantages of protein-based polymers is that they

constitute perhaps the most favorable model proteins with which to determine
the sought-after engineering principles. This, of course, becomes possible due
to the absolute control of amino acid sequence and due to the availability of a
diverse set of residues from which to choose. In addition to the model proteins
on which they are based, these principles provide a consilience, “a common
groundwork of explanation” [5] for the hydrophobic mechanism that is relevant
to all amphiphilic polymers and for an entropic elastic mechanism for all poly-
mers of whatever composition so long as the polymer exhibits a chain mobility
that becomes damped on deformation. For these reasons the derived engineer-
ing principles are referred to as the hydrophobic and elastic consilient mecha-
nisms.
Table 7.1 lists eighteen advantages of protein-based polymers for their utiliza-
tion as advanced materials for the future. Each item on the list warrants a sub-
stantial paragraph, but length limitations require that the listing suffice. A
number of the advantages, however, become apparent in the following sections
more specifically focused on the mechanistic foundations of bioproduction and
engineering.
Table 7.1 Many of the advantages of protein-based polymeric materials.
1 Two modes of synthesis: chemical and recombinant DNA technology

2 Diversity of monomers
3 Precise control of the sequence of amino acid residues
4 Exact control of stereochemistry
5 Precise chain lengths
6 Capacity to introduce natural bioactive peptide sequences
7 Circumstances of protein function to guide analyses and approach
8 Properties and uses beyond those of known proteins, e.g. thermoplastics (polymers
that melt)
9 Low cost of bioproduction
10 Produced from renewable resources
11 Axioms for protein-based polymer engineering
12 Perform pair-wise energy conversions using intensive variables of mechanical force,
pressure, chemical potential, temperature, electrochemical potential, and electromag-
netic radiation
13 Quantitative principles available for effective design of protein-based polymer ma-
chines
14 Allows for most efficient mechanism for function in an aqueous environment
15 Remarkable biocompatibility of elastic protein-based polymers
16 In vivo breakdown products simply amino acids
17 A near infinite number of polymer compositions
18 Permits de novo design of specific composition for each intended application
7.2
Historical Outline
7.2.1
Historical Beginnings of (Elastic) Protein-based Polymer Development
As the result of a collaboration in 1969 with S. M. Partridge of Bristol, England, we

reported that a temperature-induced phase separation of a-elastin, the Partridge
70 kDa chemical fragmentation product of purified ligament elastic fiber, resulted
in increased intramolecular order of this protein fragment [6]. Subsequent observa-
tion, by means of transmission electron microscopy with negative staining, showed
that on phase separation a-elastin increased its intermolecular order as represented
by formation of filaments and fibrils. These heat-induced increases in intramole-
cular and intermolecular order gave rise to the term inverse temperature transition [7].
In 1971, based on amino acid composition and on in vivo properties of the elastic
fiber, the proposal was made that glycine containing repeating tetra-, penta-, and
hexapeptide sequences would be probable in the elastic fiber [8]. Shortly thereafter,
L. B. Sandberg, then at the University of Utah, called by telephone to report that his
research group had found a repeating tetrapeptide in elastin [9]. Subsequently, they
found that the dominant repeating peptide sequence was a pentapeptide [10]. Our
response was to synthesize repeating tetra-, penta-, hexa- and nonapeptide se-
quences, to carry out many physical characterizations [11], to crosslinked high mo-
lecular weight polypentapeptides, and to find the latter material to be highly elastic
[12]. Then followed the proposal of a new mechanism of elasticity in 1982 [13],
which was supported by test syntheses, by many more physical characterizations
[14, 15], and by calculations using the ECEPP molecular mechanics approach of
Scheraga [13] and the CHARMM molecular dynamics program of Karplus [16].
The development of elastic-contractile model proteins capable of diverse en-
ergy conversions, based on inverse temperature transitions, rapidly ensued for-
mation of crosslinked elastic matrices that contracted either on raising the tem-
perature above Tt, the onset of the transition, or on lowering Tt from above to
below the operating temperature. Five phenomenological Axioms summarize
the use of Tt for design of Tt-type polymers capable of eighteen classes of pair-
wise energy conversions [4]. Development of the mechanistic foundations fol-
lowed enumeration of the Axioms.
7.2.2
Mechanistic Foundations: Fundamental Engineering Principles
Development of the mechanistic foundations proceeded on two distinct tracks that

then couple to achieve function. The two tracks, referred to as the hydrophobic and
elastic consilient mechanisms, couple hydrophobic association or apolar–polar re-
pulsion to elastic deformation to produce motion or even chemical work.
7.2.2.1 The Hydrophobic Consilient Mechanism

The change in the Gibbs free energy of hydrophobic association, DGHA, under-
girds the hydrophobic consilient mechanism [1, 3, 4]. Initial insights began with
expectation (based on the Second Law of Thermodynamics) that the increase in
order (structure formation) of elastin peptides on raising the temperature was
due to hydrophobic association [7]. Again many physical characterizations were
utilized, including direct observation of hydrophobic association and the accom-
panying decrease in water structure as ordered hydrophobic hydration became
disordered bulk water [4, 17].
The apolar–polar repulsive free energy of hydration, DGap, represents the op-
erative component of DGHA. DGap derives most effectively from changes in the
presence of charged states associated with protein, such as the ionization of car-
boxyls and the binding of phosphates. Stretch-induced [18] and hydrophobic-in-
duced [19] pKa shifts demonstrate competition for hydration and allow evalua-
tion of DGap by the entirely independent experimental means of acid–base titra-
tions. The identification of hydrophobic hydration and the direct observation
that charge formation destroys hydrophobic hydration provide confirmation of
the competition for hydration [20]. The change in DGHA resulting from charge
formation, as measured from differential scanning calorimetry, gave the same
value of DGap as obtained from acid–base titration data of hydrophobic-induced
pKa shifts [1, 3, 21]. Such are these highlights in the historical development of
the hydrophobic consilient mechanism.
7.2.2.2 The Elastic Consilient Mechanism

The history of the development of the elastic consilient mechanism has been
one of controversy. Our view noted above that the damping of internal chain dy-
namics on extension of even a single chain gives rise to an entropic elastic force
[16] has been decried by adherents to the established classical (random chain
network) theory of rubber elasticity [22, 23] and even by those currently sub-
scribing to the view that solvent entropy changes attending changes in hydro-
phobic association give rise to an entropic elastic force [24].
In your author’s view, the issue is clear. First, the proposed contribution of
solvent entropy changes resolves on consideration of the development of elastic
force under isometric conditions. Elastic force development at fixed extension
results in the development of entropic elastic force due to a negative change in
entropy, whether due to thermally or chemically driven hydrophobic association
[1, 21, 25]. Under these conditions for hydrophobic association, the entropy
change is positive for solvent, but negative for developed elastic force. Second,
in AFM single-chain force–extension studies a single chain, fixed at both ends,
develops entropic elastic force [26, 27]. It would appear that extension of a ran-
dom chain network comprised of a Gaussian distribution of end-to-end chain
lengths is not required for entropic elasticity. Having eliminated the solvent-
based mechanism and also invoking an elementary statistical mechanics analy-
sis [26, 27], entropic elastic force derives from decreased amplitude of chain tor-
sional angle oscillations on extension [15, 16].
Analyses of crystal data for the Rieske Iron Protein support this understand-
ing of elastic force development. Hydrophobic association stretches a single
chain that on relaxation moves a protein domain to transfer an electron in
Complex III of the electron transport chain. Also our crystal data analyses for
the myosin II motor of muscle contraction argues that the power stroke due to
ATP hydrolysis and loss of phosphate causes two hydrophobic associations that
stretch interconnecting single chains to produce elastic force under isometric
conditions of a near rigor state [1, 21].
7.2.3
Highlights of Bioproduction
The history of protein-based polymer bioproduction essentially began in 1986

with an Accelerated Research Initiative of the Office of Naval Research (ONR)
with the purpose of developing biotechnology, specifically recombinant DNA
technology, for the production of protein-based adhesives and elastomers. This
five-year initiative, administered by Michael Marron, rightly receives the credit
as the critical starting point for the production of the hundreds of transformed
E. coli of Table 7.5 (below) and the approximately one hundred and fifty (150)
expressed protein-based polymers.
Of the many highlights of this striking development, several may be illus-
trated (Figs. 7.1–7.3).
Fig. 7.1 A. E. coli transformed to express the of the volume of the transformed E. coli. B.
phase-separated protein-based polymer, The same strain of E. coli before transforma-
(GVGVP)121, with the expression product tion. Reproduced with permission from Ref.
seen as large phase-separated inclusion [28].
bodies which comprise approximately 80%
Fig. 7.2 Two-hundred and thirty-sev-

en-gram yield of a single fermenta-
tion in 1997 of transformed E. coli
that produced (GVGIP)260. Now the
yields can be several times greater.
7.3
Bioproduction
Bioproduction of protein-based polymers may be considered in three stages:

(1) gene construction using recombinant DNA technology, (2) transformation of
the cell to be used for expression, and (3) fermentation of the transformed
microbial cell. This process has been reported in detail for the production of
(GVGVP)10 ´ n, where the monomer gene encodes for ten pentameric repeats
without DNA repeats [29].
7.3 Bioproduction 225
Fig. 7.3 Three thermoplastics (polymer melts isms but of growing interest to society.
pulled into plastic fibers). Two are plastics Furthermore, protein-based polymer thermo-
of our daily experience (B, polypropylene; plastics can be programmed to biodegrade
C, polystyrene) whereas the third, A, is a in a sufficiently wet environment with half-
protein-based plastic that melts at 160 8C lives that could be varied from days to de-
and does not decompose until 250 8C. Thus, cades. It might be said that these protein-
using living organisms, the potential is to based thermoplastics portend food rather
prepare protein-based polymeric materials than death for marine life. From Ref. [1].
with properties inaccessible to living organ-
7.3.1
Gene Construction using Recombinant DNA Technology
7.3.1.1 Preparation of Monomer Genes and the PCR Technique [29]

A protein sequence of no more than about 50 residues is chosen. Such a length
makes possible chemical synthesis of two nucleotide sequences, each of about
80 to 90 bases, one from the 3' end and the other from the 5' end. These two
base sequences associate by base pairing with an overlap of some 10 to 20 base
pairs. The polymerase chain reaction (PCR) is then used to complete the double
stranded DNA sequence. The appropriate restriction enzyme is used to cut the
ends of the double stranded DNA to produce the monomer gene for insertion
into the chosen plasmid. Commonly, the BamH 1 restriction site is used for in-
sertion into pUC-118.
7.3.1.2 Transformation, Monomer Gene Production and Sequence Verification

The prepared plasmid, in our case pUC-118 containing the monomer gene, is
introduced into the chosen strain of E. coli, which is grown up to prepare many
copies of the monomer gene. The purified large-scale monomer gene prepara-
tion is sequenced to verify that the chemical synthesis did not introduce se-
quence errors, and prepared for concatenation (polymerization) into multimer
genes.
7.3.1.3 Monomer Gene Concatenation Produces Multimer Genes of Monomer [30]

After excision exposes the PflM 1 restriction site, which allows only head-to-tail
alignment, the ligase reaction polymerizes the monomer gene into multimer
genes. In Fig. 7.4 the gene ladder of lane 2 shows each multimer gene. Lane 1
is the monomeric gene fragment. Lanes 3, 4, and 5 contain pUC-118 and genes
encoding (GVGVP)10 ´ n+1 released on treatment with restriction endonuclease,
Bam H 1. Lane 3 gives (GVGVP)10 ´ 4+1, which is (GVGVP)41. Lane 4 gives
(GVGVP)121, and Lane 5 gives (GVGVP)251. As seen in Lane 5, the capacity to
make genes is so effective, that the gene is larger than the plasmid into which
it is placed. In fact, it has been possible to prepare and to express genes that en-
code for the equivalent of more than 400 repeats of the pentamer. In terms of
Fig. 7.4 Gene-ladder. From Ref.

[30].
molecular weights that would be some 200 kD, whereas the original maximum
size was thought to be 50 kD in E. coli.
7.3.2
E. coli Transformation for Protein-based Polymer Expression
The multimer gene, prepared in pUC-118, is excised and inserted into an expres-
sion vector, such as pET-23d, which is specialized for expression of the designed
protein-based polymer and inserted into a strain of E. coli, selected for the expres-
sion [29]. Figure 7.1 B, above, shows the non-transformed E. coli, whereas Fig. 7.1A
shows the strain of E. coli, transformed to produce (GVGVP)121. This demon-
strates the high production capacity of the chosen expression system, for example,
the BL21(DE3) strain of E. coli, to produce the elastic protein-based polymer.
7.3.3
Fermentation using Transformed E. coli
A portion of a glycerol stock of the transformed E. coli is utilized to grow an in-

oculum for addition to a large fermenter. In addition to the choice of carbon
source, nitrogen source, salts, and cofactors, the medium contains an antibiotic
to which the chosen expression vector confers resistance and an inducer that
turns on the expression vector after the cell density has reached an optimal lev-
el. In the example given above the antibiotic was antimycin and the induction
system was designed for IPTG.
Figure 7.2, above, is an example of an early single fermentation using a work-
ing volume of 400 L. The yield of a single fermentation for this product,
(GVGIP)260, was 237 grams after purification, by phase separation, to less than
1 ppm impurities.
7.4
Purification of Protein-based Polymers
The purification of protein-based polymers that exhibit inverse temperature

(phase) transitions is addressed in three sections: (1) The essential properties of
the inverse temperature transition are discussed as a very effective method of pur-
ification. It should be realized that all of the means of changing Tt and DGHA in
Tables 7.3 and 7.4, below, can be useful means for selective phase separation of the
designed protein-based polymer from the cell lysate. (2) A number of physical
methods, such as multi-dimensional nuclear magnetic resonance spectroscopy
and mass spectrometry are shown to be particularly effective in the verification
of product. And (3) after achieving adequate purification, subcutaneous injection
in the guinea-pig is shown to provide effective demonstration of the remarkable
biocompatibility of elastic protein-based polymers, where large quantities of bioma-
terial disperse within days without eliciting any evidence of having been present.
7.4.1
Use of the Inverse Temperature Transition as a Method of Purification
7.4.1.1 Purification by Phase Separation as Demonstrated by SDS–PAGE [30]

Figure 7.5, below, uses sodium dodecyl sulfate polyacrylamide gel electropho-
resis (SDS–PAGE) to demonstrate the usefulness of the inverse temperature
transition exhibited by the elastic protein-based polymer, (GVGVP)141 as a meth-
od of purification. Using the negative CuCl2 stain, the expressed product is seen
as the bulge in Lane 1. Lane 4 shows how the effective utilization of the inverse
temperature transition purifies these protein-based polymers.
7.4.1.2 Purification by Phase Separation Shown by Carbon-14-labeled E. coli [31]

Without showing the data, the effectiveness of the phase separation was fol-
lowed by C-14 labeling of all of the components of E. coli except the protein-
based polymer [1]. With each phase separation the purification improved by a
factor of ten or more.
Fig. 7.5 SDS–PAGE with CuCl2 stain of ex- tion leaving polymer in solution. Lane 3:
pressed (GVGVP)141 demonstrates use of Supernatant of cold spin after heating to
phase transition to separate from E. coli pro- phase separate (GVGVP)141. Lane 4: Phase
teins. Lane 1: Crude lysate of transformed separated elastic protein-based polymer on
E. coli. Lane 2: Precipitate of cold centrifuga- heating. From [30].
7.4.2
Physical Characterization and Verification of Product Integrity
7.4.2.1 Gross Visualization of the Phase Separated Product

In Fig. 7.6 direct visualization of the phase separated product provides graphic
demonstration of both the physical nature of the product and the effectiveness
of the inverse temperature transition as a means of purification.
7.4.2.2 Sequence Integrity and Purity Evaluated by Nuclear Magnetic Resonance

Routinely, one-dimensional nuclear magnetic resonance (NMR) verifies the ab-
sence of impurities and other molecular groups. Two-dimensional NMR, by pro-
ton–proton through bond couplings, gives proton assignments, and, using pro-
ton–proton through space nuclear Overhauser interactions, allows determina-
tion of the sequence integrity of the repeating unit [32]. In this way the com-
plete sequence of the repeat monomer can be verified. Now, verification of
number of repeats follows below.
7.4.2.3 Mass Spectra Reaffirm Size of Expressed Polymer [32]

The agarose gel gene ladder allows determination of gene size, as seen in
Fig. 7.4, where the integral number of monomer repeats of the designed gene
can be discerned from the number of monomer gene repeats plus one penta-
mer. Thus, one has an expected molecular weight for the expressed protein-
based polymer. Using matrix assisted laser desorption time-of-flight (MALDI–
Fig. 7.6 A. Phase separated, taffy-like mass cake, which in C. is pulled into a tough band
of (GVGVP)251 produced by a single fermen- of a meter in length. The addition of a single
tation. The mass readily pulls into long, fine CH2 group per pentamer causes this
viscoelastic strands [1]. B. A small amount dramatic change in physical property. Cross-
of the protein-based polymer, (GVGIP)320, linked (GVGVP)251 exhibits reversible stress–
phase separated into the bottom of a strain curves, whereas (GVGIP)320 exhibits a
250 mL centrifugation bottle to form a pan- marked hysteresis. From Ref. [1].
TOF) mass spectrometry, the whole-molecule (polymer) peak can be determined

plus or minus a number of sodium ions, if neutral or negatively charged, or the
number of chloride ions if positively charged. Such efforts allow confirmation
of polymer size [32]. Accordingly, a combination of physical methods achieves
verification of structure.
7.4.3
Biocompatibility
7.4.3.1 The Challenge of Using E. coli-produced Protein as a Biomaterial

Required purification levels pose a challenge for protein biomaterials produced by
E. coli fermentation. The target level for purification of protein pharmaceuticals
has been to achieve impurities of less than 10 ppm (parts per million). Since bio-
materials may be used in amounts one thousand to one million times greater than
a pharmaceutical, purification requires an equivalent increase in level of purity.
The most challenging test situation, that is, a gold standard, would be for the in-
troduced biomaterial to have released all of its impurities in a relatively short time,
say in a few days, without an adverse reaction from the host tissues. This chal-
lenge has been met. When injected subcutaneously in the guinea-pig, purified
(GVGVP GVGVP GEGVP GVGVP GVGVP GVGVP)36(GVGVP) does disperse
in a short time without a trace of ever having been present (Fig. 7.7 A below).
7.4.3.2 Removal of Endotoxins and Determination of Levels

After phase separation, a pre-filtration step followed by an ultrafiltration
approach is used to remove endotoxins. Determination of endotoxin levels uti-
lizes a 20 mg mL–1 protein-based polymer solution in the Pyrotell Limulus
Amebocyte Lysate test (Associates of Cape Cod, Inc). The values obtained for
the endotoxin units per mg (EU mg–1) can be routinely less than 0.01 and often
less than 0.003 [32].
7.4.3.3 Western Immunoblot Technique to Demonstrate Level of Purity

In the standard Western blotting technique, 5 lg per well of positive control
(lyzed E. coli supernatant) and of test E. coli-produced protein-based polymer(s)
are run in parallel lanes of a 12.5% SDS–PAGE gel. The separated protein
bands, as in Lane 3 of Fig. 7.5, are then transferred, by contact, to a nitrocellu-
lose membrane. After suitable treatment, the membranes are incubated with
polyclonal rabbit anti-E. coli immunoglobulin G (IgG) as the primary antibody.
The membranes are then incubated with biotinylated anti-rabbit IgG. The detec-
tion procedure utilizes an alkaline phosphatase procedure to detect antigens to
E. coli proteins with a limit of 5 pg. When no bands are detected from an appli-
cation of 5 lg, the E. coli protein impurity is considered to be below 1 ppm (one
part per million). Phase separations by means of elicited inverse temperature
transitions are sufficient to achieve this level of purification using any of the
means of Tables 7.3 and 7.4 and Section 7.5 below. It is important to realize,
however, that this level of purification is insufficient when using the protein-
based polymer in vivo, as a biomaterial that can release all of its impurities in a
short time without leaving any trace of its ever having been present.
7.4.3.4 Western Immunodotblot Technique to Demonstrate Medical Grade Purity

Based on the subcutaneous injection test, discussed below, an impurity level of
less than 1 ppm is not satisfactory. Accordingly, a more sensitive technique is
required to assess impurity level. The next step is to use the Western Immuno-
dotblot technique, where 1 mg of test sample is placed in a single spot and the
above technique is again employed. With a detection limit of 5 pg, the absence
of detection (absence of a spot) indicates an impurity level of less than 5 ppb
(parts per billion). This level of purification is required for protein-based poly-
mer to be a candidate for medical use. The ultimate test uses subcutaneous in-
jection, as discussed immediately below.
7.4.3.5 Subcutaneous Injection in the Guinea-pig

Protein-based polymer (30 mg) at phase separation concentration in normal sa-
line is injected subcutaneously in the guinea-pig into the region of the fatty
layer. The injection site is followed externally as a bump that results from the
volume of injected sample. In the case of the protein-based polymer, (GVGVP
GVGVP GEGVP GVGVP GVGVP GVGVP)36(GVGVP), the bump lasted for no
more than several days. At two weeks the injection site was examined histologi-
cally. As seen in Fig. 7.7A, no evidence is found of the polymer’s ever having
been injected. When (GVGVP)251 is injected, the bump is retained, and the in-
Fig. 7.7 Histological sections of subcuta- GVGVP)36(GVGVP), in B. (GVGVP)251 and in

neous injections in the guinea-pig using C. {(GVGVP)10–GVGVPGRGDSP–
three different protein-based polymer com- (GVGVP)10}16(GVGVP). The results of A and
positions. The injected polymers are in C will be discussed below in Section 7.6.1.
A. (GVGVP GVGVP GEGVP GVGVP GVGVP From Ref. [32].
jected polymer is seen in Fig. 7.7 B to be intact and surrounded by a fine fibrous
capsule [32].
7.4.3.6 ASTM Tests

It should be noted in addition to the tests routinely run above, three composi-
tions, (GVGVP)n [33], (GGAP)n [30], and (AVGVP)n [34], have been examined
using the complete set of eleven tests recommended by the American Society
for the Testing of Materials (ASTM) for materials to be in contact with tissues,
tissue fluids and blood. As this set of tests had been run on chemically synthe-
sized polymers, they were continued with microbially synthesized polymers to
achieve an adequate understanding of the impurities derived from E. coli. Elas-
tic protein-based polymers, (GVGVP)n and (GGAP)n, exhibit truly remarkable
biocompatibility. The plastic protein-based polymer, (AVGVP)n, exhibits good
biocompatibility, but being inelastic and an unnatural sequence, its biocompat-
ibility is not as extraordinary.
7.5
Mechanistic Foundations for Engineering Protein-based Polymers
The mechanistic foundations derive from determination of the physical basis

for a set of Axioms that enumerate observations arising out of elastic–contractile
model proteins designed de novo for energy conversions. As yet unexplained by
popular descriptions of protein function, the Tt-based Axioms argue that new
mechanistic understandings be sought. Therefore, this section begins with a
table that succinctly states these practical Axioms [4] in terms of changes in Tt
that function as simple on–off switches for contraction/relaxation by hydropho-
bic association/dissociation.
7.5.1
Phenomenological Axioms (see Table 7.2)
7.5.2
The Change in Gibbs Free Energy for Hydrophobic Association, DGHA
The experimentally developed phenomenological Axioms, listed below in Ta-

ble 7.2, do not derive from present treatments of protein structure and function.
Therefore, the Axioms require that physical bases, i.e., mechanistic foundations,
be sought for these observations. Over the last two decades, our focus has been
to design and characterize a particular class of protein-based polymers called
elastic–contractile model proteins with which to develop the mechanistic foun-
dations. In short, virtually every experimental variable that effects a change in
protein structure and function changes the Gibbs free energy of hydrophobic
7.5 Mechanistic Foundations for Engineering Protein-based Polymers 233
Table 7.2 Phenomenological AXIOMS for protein-based polymer engineering.
AXIOM 1: The change in temperature interval, over which occurs the hydrophobic associa-
tion transition of a host protein-based polymer on introduction of different guest substitu-
ents, becomes a practical measure of relative hydrophobic character of the substituents, and
it approximates the change in free energy of the resulting hydrophobically associated state.
This provides the phenomenological data-base for design of protein-based machines.
AXIOM 2: Heating to raise the temperature from below to above the temperature interval
for hydrophobic association of crosslinked elastic protein-based polymers drives contraction
with the performance of mechanical work.
This is thermally driven contraction inherent in inverse temperature transitions.
Example: Thermo « mechanical transduction
AXIOM 3: At constant temperature, any energy input that changes the temperature interval
for hydrophobic association in a protein-based polymer can drive contraction with the perfor-
mance of mechanical work
Examples: Chemo « mechanical transduction Electro « mechanical transduction
Baro « mechanical transduction Photo « mechanical transduction
AXIOM 4: Two or more different functional groups of an amphiphilic macromolecule, each
of which can be acted upon by a different energy input that changes the temperature inter-
val for hydrophobic association, become coupled one to the other by acting upon the same
hydrophobic association domain. In other words, an energy input acts on one functional
constituent to change its hydrophobicity and that change in hydrophobicity becomes an en-
ergy output that changes the other functional constituent due to its dependency on hydro-
phobicity.
This axiom is the fundamental energy-coupling axiom; it utilizes the hydrophobic association
transition common to all energy conversions by the consilient mechanism. Importantly, in
doing so, it involves the performance of kinds of work in addition to mechanical work.
Examples: Electro « chemical transduction Electro « thermal transduction
Baro « electrical transduction Photo « voltaic transduction
Thermo « chemical transduction Photo « thermal transduction
Baro « thermal transduction Baro « chemical transduction
Photo « baric transduction Photo « chemical transduction
Chemo « chemical transduction Electro « electrical transduction
Electromagnetic radiation (1) « electromagnetic radiation (2) transduction
AXIOM 5: More hydrophobic domains make more efficient the energy conversions involving
polar constituents undergoing conversion between more and less hydrophobic functional
states.
This is the efficiency axiom. (Poising or Biasing)
association, DGHA. Because of this and all that has been demonstrated by sim-
ply controlling hydrophobic association, your author believes DGHA to be the
underlying thermodynamic quantity that dictates protein function. Accordingly,
a proper derivation of the quantity, an analysis of its component parts, and reali-
zation of the range of experimental variables contributing to DGHA are central

to understanding bioproduction, purification and engineering of protein-based
polymers. By way of example, every change in DGHA of Tables 7.3 and 7.4, be-
low, provides a unique opportunity for purification and engineering.
The treatment begins with the derived statement of the change in Gibbs free
energy attending a phase transition, rewrites the statement with respect to in-
verse temperature transitions, and uses differential scanning calorimetry to ob-
tain the necessary heats and temperatures of the transition for calculation of
DGHA. The first useful result of the treatment is the DG8HA-based hydrophobi-
city scale for the amino acid residues and where relevant in each of their un-
charged and ionized states. The next step brings in prosthetic groups and chem-
ical modifications of side chains relevant to biological systems. Finally, the ef-
fects of solvent-based variables on DGHA are noted. All of these results guide
bioproduction and engineering.
7.5.2.1 The Change in Gibbs Free Energy Attending a Phase Transition, dDGt(v) [3]
The relationship that the chemical potential for molecules undergoing a phase
transition is the same in solution as in the phase separated state means that
the heat of the transition, DHt, equals TtDSt, which is then written for both a
reference state, ref, and a state resulting from the change in a variable, v. Suit-
able identification of the change in the heat, dDHt, and entropy, dDSt, of the
transition as the result of v allows the statement [3] that,
dDGt v dDHt v Tt vdDSt v 1
7.5.2.2 The DGHA-based Hydrophobicity Scale for Amino Acid Residues [3]
Rewriting Eq. (1) for the endothermic inverse temperature transition allows that
dDGt(v) = DGHA(v). When the variable is the amino acid residue, X, within the
pentamer, dDGt(v) = DGHA(GXGVP) where DGHA is written per mole GXGVP. If
the glycine residue (Gly, G) is taken as the zero reference, DG8HA(GGGVP) = 0.
Thus, at the temperature of the inverse temperature transition, Tt(GXGVP), we
can write:
DGHA GXGVP DH t GGGVP DH t GXGVP 2
Using the basis set of protein-based polymers, poly[ fX(GXGVP),fV(GVGVP)],

where fX and fV are mole fractions with fX + fV = 1, the experimental values of Tt
and DHt are determined for values of fX and linearly extrapolated to fX = 1. At
fX = 1, these reference values are indicated by the bold faced quantities, Tt and
DHt. The resulting DG8HA-based hydrophobicity scale for the naturally occurring
amino acid residues is given in Table 7.3 for the experimental conditions of
40 mg mL–1, mw & 100 kDa in 0.15 m NaCl, 0.01 m phosphate.
Table 7.3 Hydrophobicity Scale in terms of DG8HA , the

change in Gibbs free energy of hydrophobic association for
amino acid residues.
b Residue X Tt (8C) DG8HA(GXGVP) kcal mol–1 pentamer
W Trp –105 –7.00

F Phe –45 –6.15
Y Tyr –75 –5.85
H8 His8 –10 (Tt) –4.80 (from graph)
L Leu 5 –4.05
I Ile 10 –3.65
V Val 26 –2.50
M Met 15 –1.50
H+ His+ 30 (Tt) –1.90 (from graph)
C Cys 30 (Tt) –1.90 (from graph)
E8 Glu(COOH) 20 (2) –1.30 (–1.50)
P Pro 40 –1.10
A Ala 50 –0.75
T Thr 60 –0.60
D8 Asp(COOH) 40 –0.40
K8 Lys(NH2) 40 (38) –0.05 (–0.60)
N Asn 50 –0.05
G Gly 55 0.00
S Ser 60 +0.55
R Arg 60 (Tt) +0.80 (from graph)
Q Gln 70 +0.75
Y Tyr(}-O–) 140 +1.95
D Asp(COO–) 170 (Tt) *+3.4 (from graph)
K+ Lys(NH+3 ) (104) (+2.94)
E Glu(COO–) (218) (+3.72)
S-Pi Ser(PO2–
4 ) 860 (Tt) *+8.0 (from graph)
Data in parentheses utilized microbial preparations of poly(30-mers), e.g.

(GVGVP GVGVP GXGVP GVGVP GVGVP GVGVP)n, with n & 40. The notation
(from graph) indicates that the value of Tt was used to obtain DG8HA(GXGVP)
from the experimental sigmoid curve of Tt versus DG8HA of Ref. [3]. Table adapted
from Ref. [3].
7.5.2.3 DG8HA-based Hydrophobicity Scale of Prosthetic Groups, etc.

For this set of comparative data, the modification is carried out by addition
of the new chemical group to the side chain of a functional amino acid
residue and indicated by enclosure in brackets. Using the example of a
functional glutamic acid residue (Glu, E), DG8HA may be defined for addition of
reduced nicotinamide adenine dinucleotide (NADH) by amide linkage as
DG8HA(GK{NADH}GVP). In this way the basis is exactly the same as for the
DG8HA-based hydrophobicity scale for amino acid residues of Table 7.3.
Included in Table 7.4 are reduced and different oxidative states of N-methylni-
cotinamide (NMeN), oxidized and reduced nicotinamide adenine dinucleotide
(NAD and NADH), oxidized and reduced flavin adenine dinucleotide (FAD and
FADH2), adenosine monophosphate (AMP), sulfated serine, threonine, and
tyrosine (Ser{–O–SO3H}, Thr{–O–SO3H}, and Tyr{–O–SO3H}, respectively), ni-
trated tyrosine (Tyr{–O–NO–2}), and finally phosphorylated serine (Ser{PO2–4 }).
Table 7.4 shows large effects of changing the oxidative states of the most com-
mon redox groups of biology and of changing the state of phosphorylation. The
DGHA on reduction of FAD is about two-thirds that of NAD as might be ex-
pected for their roles in oxidative phosphorylation. On inclusion of the effects
of salt, solvent and ligands on DGHA , one gains a comprehensive understand-
Table 7.4 DG8HA -based Hydrophobicity Scale (preliminary Tt

and DG8HA values) to include chemical modifications and
prosthetic groups of proteinsa).
Residue X DGHA (kcal mol1) [g

Tt at fX = 1 (8C)
Lys{dihydro NmeN}b, d) 7.0 130

Glu{NADH}c) 5.5 30
Lys{6-OH tetrahydro NMeN}b, d) 3.0 15
Glu{FADH2} 2.5 25
Glu{AMP} +1.0 70
Ser{–O–SO3H} +1.5 80
Thr{–O–SO3H} +2.0 100
Glu{NAD}c) +2.0 120
Lys{NMeN,oxidized}b, d) +2.0 120
Glu{FAD} +2.0 120
Tyr{–O– SO3H}e) +2.5 140
Tyr{–O–NO2–}f) +3.5 220
Ser{PO2–
4 }
g)
+8.0 860
a) The usual conditions are for 40 mg mL–1 polymer, 0.15 m

NaCl and 0.01 m phosphate at pH 7.4
b) NMeN is for N-methylnicotinamide pendant on a lysyl side
chain, i.e. N-methylnicotinate attached by amide linkage to
the –NH2 of Lys and the most hydrophobic reduced state is
N-methyl-1,6-dihydronicotinamide (dihydro NMeN), and the
second reduced state is N-methyl-6-OH-1,4,5,6-tetrahydroni-
cotinamide or (6-OH tetrahydro-NMeN)
c) For the oxidized and reduced nicotinamide adenine dinu-
cleotides, the conditions were 2.5 mg mL–1 polymer, 0.2 m
sodium bicarbonate buffer at pH 9.2
d) For the oxidized and reduced N-methylnicotinamide, the
conditions were 5.0 mg mL–1 polymer, 0.1 m potassium bi-
carbonate buffer at pH 9.5, 0.1 m potassium chloride
e) The pKa of polymer bound –O–SO3H is 8.2
f) The pKa of Tyr{–O–NO2} is 7.2
g) Gross estimates of DG8HA using the Tt values in the right
column in combination with the Tt versus DG8HA values from
sigmoid curve of Tt versus DG8HA. Adapted from Ref. [1].
ing of the hydrophobic effect in biology, a universal foundation for protein and
protein-based polymer function.
7.5.2.4 Comprehensive Hydrophobic Effect: DGHA Responds to all Variables

Tables of the effect of salts, solvents and additional ligands on DGHA are avail-
able [1], but space limitations do not permit their inclusion here. Even so, it is
important to make the fundamental point here. The significance and general re-
levance of DGHA gains from consideration of the following perspective of Gre-
gorio Weber [35]: “A complete description of the energetics of hemoglobin, or of
any other oligomeric protein of similar size and complexity, is well nigh impos-
sible. It would involve not only the determination of the energetic couplings of
any number of ligands with each other and with the subunit interactions but
also the variations of these quantities with pH, temperature, and pressure.” In-
deed, the unifying result of the comprehensive hydrophobic effect comes from
finding that virtually every variable of interest in protein and protein-based poly-
mer function brings about, and is responsive to, a change in DGHA [1]. Thus,
the solution to the problem is no longer “well nigh impossible,” but rather the
solution simplifies due to the common denominator of the hydrophobic consili-
ent mechanism.
7.5.2.5 The Apolar–Polar Repulsive Free Energy of Hydration, DGap

The apolar–polar repulsive free energy of hydration can be seen in Table 7.3 in
terms of the change in DGHA on ionization of the Glu carboxylate of 5.22 kcal
mol–1 (GEGVP). The DGap repulsion is also measured from the shift in pKa on
increasing hydrophobicity where DGap = 2.3RTDpKa [4]. Whether determined
from the heat and temperature of the transition or from a titration curve, DGap
is the same. The repulsion that arises out of a competition for hydration be-
tween apolar and polar groups provides the basis for function.
7.5.3
The Coupling of Hydrophobic and Elastic Mechanisms
A brief description of the elastic mechanism occurs above in Sections 7.5.1

and 7.5.2. The coupling of hydrophobic and elastic mechanisms can occur in sev-
eral ways. In the elastic contractile model proteins for energy conversion and in
the myosin II motor of muscle contraction an energy input causes hydrophobic
associations that stretch interconnecting chain segments [1, 21]. The stretched
chains retract (i.e., contract) to perform mechanical work in a smooth, efficient
conversion of the input energy into the work output of producing motion. In
the ATP synthase example, proton chemical energy is transduced into the chem-
ical energy of ADP phosphorylation, but it occurs in two steps. Again, chemically
driven hydrophobic association produces (rotary) motion with elastic deformation
that in turn uses apolar–polar repulsion to produce ATP. Thus, motion involving
elastic deformation is central to this energy conversion even though motion is

neither the initial input energy nor the final output energy [1].
7.6
Examples of Applications
Examples of applications are literally endless. Many are addressed in Chapter 9

of Ref. [1]. Here, mention will be made of applications in each of the wide-rang-
ing medical areas of soft tissue restoration (3) and drug delivery (2). In areas
with both medical and nonmedical utility are biodegradable thermoplastics that
can be programmed for rate of degradation, fibers with improved elastic moduli
and strength and sound absorption.
7.6.1
Soft Tissue Restoration
Soft tissue restoration can be considered in terms of three categories: (1) pre-
vention of post-surgical adhesions to restore the tissues to a near normal state,
(2) soft tissue augmentation, for example, to support the urinary bladder and
improve body contours, and (3) soft tissue reconstruction where elastic–contrac-
tile materials function as temporary functional scaffoldings that induce normal
cells of the tissue to remodel into a natural tissue.
7.6.1.1 Prevention of Post-surgical Adhesions

The application for which the most work has been completed is the prevention
of post-surgical adhesions. This work has been performed in three separate ani-
mal model studies: (1) the bloodied and feces-contaminated abdominal cavity of
the rat [36], (2) the strabismus surgery model of the rabbit [37], and (3) the
spinal surgery (post-laminectomy) model in the rabbit [38] and goat. In each
case emplacement of crosslinked matrices and gels of (GVGVP)251 or of
(GVGVP GVGVP GEGVP GVGVP GVGVP GVGVP)36(GVGVP) prevented ad-
hesions, and when purified to the highest level, the host exhibited no reaction
whatever to the protein-based polymers. Once large-scale purification of medical
grade polymers is in place, this application should move quickly to commerciali-
zation.
7.6.1.2 Soft Tissue Augmentation

One approach to soft tissue augmentation is to introduce an inert material that
would occupy the desired volume and stay in place. This could be achieved as
shown above in Fig. 7.7 B. An approach uniquely possible with protein-based
polymers is to include in the sequence a bioactive peptide such as GRGDSP.
This peptide acts both as a chemoattractant [39] and a cell attachment sequence
[40]. When injected subcutaneously in the guinea-pig, it causes a generation of

natural tissue with angiogenesis, as shown in Fig. 7.7 C [32].
This shows the diversity of responses to simple changes in protein-based
polymer composition. With just one Glu residue in six GVGVPs, the injected
sample disperses in a few days without a trace (Fig. 7.7 A). In the absence of
the Glu residue, (GVGVP)251 forms a large inclusion body the size of the in-
jected bolus (Fig. 7.7 B). On replacing the Glu used in Fig. 7.7 A with GRGDSP,
the generation of natural tissue of Fig. 7.7 C occurs. These polymers exhibit re-
markable versatility.
7.6.1.3 Soft Tissue Reconstruction:

The Concept of Temporary Functional Scaffoldings
The concept of a temporary functional scaffolding derives from two principal
factors. The initial enabling feature is the capacity to prepare biocompatible
elastic matrices of protein-based polymers that match the compliance of the tis-
sue to be replaced and contain attachment sequences for the natural cells of the
tissue. Second is the important feature that tissue cells function as mechano-
chemical transducers. They attach to the extracellular matrix, sense the chang-
ing tensional forces, and remodel the tissue continually in order to sustain the
sensed forces. Important tissues that have been considered in this context are
vascular wall, intervertebral discs and urinary bladder. Consideration of the lat-
ter is given in Fig. 7.8 where the dynamic changes chosen to simulate the filling
and emptying of the urinary bladder did indeed stimulate cellular proliferation
and extracellular matrix elaboration. Elastic protein-based polymers make a tem-
porary functional scaffolding approach possible.
Fig. 7.8 Outgrowth of human uroepithelial designed to simulate bladder filling and
cells from a ureteral explant (dark area) onto emptying. A. The static case where good
a crosslinked elastic matrix of {(GVGIP)10– outgrowth is seen. B. The dynamic case of
GVGVPGRGDSP–(GVGVP)10}12(GVGVP). filling in three hours and emptying in 25 s
The elastic matrix, with an elastic modulus shows enhanced cellular proliferation and
that closely matches that of the human much greater development of extracellular
urinary bladder, is placed within a chamber matrix. Reproduced from reference [41].
7.6.2
Controlled Release Devices for Amphiphilic Drugs and Therapeutics
The capacity to use elastic protein-based polymers as controlled release devices is as

diverse as the energy conversions of Table 7.2 and as expansive as the many advan-
tages of Table 7.1. The extraordinary biocompatibility of elastic protein-based poly-
mers amplifies the opportunity, for example, due to the possibility of no fibrous
capsule formation in vivo that can defeat the competence of the drug release device.
Here, we note two examples of the uniqueness of elastic protein-based poly-
mers in light of the mechanistic foundations. One is the utilization of the apo-
lar–polar repulsive free energy, DGap, in the design of families of polymers that
can give a predetermined set of near constant release rates. Second is the advan-
tage of an elastic cushion for controlled release patches to prevent pressure ul-
cer formation.
7.6.2.1 The Use of DGap in the Design of Controlled-release Devices

Protein-based polymer designs utilize charged side chains with methodical in-
creases in hydrophobicity, commonly by replacing, in each six pentamer repeat,
2, 3, 4, and then 5 valine (Val, V) residues by much more hydrophobic phenyl-
alanine (Phe, F) residues. The result is a systematic shift in pKa values that are
quantifiable by means of DGap = 2.3RTDpKa. Generally, the polymer, with one io-
nized functional group per 30-mer (5 ´ 6 pentamers), is soluble in solution. Ad-
dition of an amphiphilic pharmaceutical of the opposite charge lowers the value
of Tt below the operating temperature, and the pharmaceutical effects phase
separation to form the loaded drug-delivery vehicle. The level of pharmaceutical
release depends on the DGap value of the polymer’s functional group. For a con-
stant surface area zero order release occurs, and the drug delivery vehicle dis-
perses at the same rate as the drug.
The loading and zero-order release of positively charged drugs and therapeu-
tics, using negatively charged protein-based polymers, has been demonstrated
for Leu-enkephalin amide [41] and Naltrexone (a narcotic antagonist) [1]. All cat-
ionic anesthetics, analgesics, and protein could be released in this remarkably
controlled manner using the DGap approach. Analogously, using positively
charged protein-based polymers, the loading and controlled release of negatively
charged drugs and therapeutics, such as dexamethasone phosphate and beta-
methasone phosphate (anti-inflammatory glucocorticoids) [42], anti-sense oligo-
nucleotides [43], including genes and proteins with net negative charge could
be released in this manner [1].
7.6.2.2 Prevention of Pressure Ulcers by Means of Elastic Patches for Drug Delivery
Pressure ulcers appear over bony prominences where the pressure leads to skin
necrosis and ulcers. In the Swaim coaptation cast model for the dog model, even
the elastic patch (Fig. 7.9) made of crosslinked (GVGIP)260 placed over the bony
Fig. 7.9 A. Disk-shaped patches of cross- 350%. C. The two polymers, (GVGVPGVG
linked (GVGIP)260 contain Dazmegrel from FPGEGFPGVGV PGVGVPGVGV P)40–
left to right of 0, 0.1, 1.0, and 10 mg cm–2. (GVGVP) and (GVGVP GVGFP GKGFP
B. The two polymers, (GVGVP GVGVP GVGVP GVGVP GVGVP)22(GVGVP), were
GEGVP GVGVP GVGVP GVGVP)36–(GVGVP) similarly treated to obtain fibers with an
and (GVGVP GVGVP GKGVP GVGVP elastic modulus of 6.1 ´ 106 Pa, a break
GVGVP GVGVP)22(GVGVP), were cross- stress of 6.9 ´ 106 Pa and a break strain of
linked by amide linkage to form a fiber with 280%. The bar indicates 100 lm. B and C re-
an elastic modulus of 3.86 ´ 106 Pa, a break produced from Ref. [45].
stress of 1.3 ´ 106 Pa and a break strain of
prominence pressure point without drug, appeared to have efficacy in preventing

pressure ulcer formation. When loaded with dazmegrel (a thromboxane synthe-
tase inhibitor), efficacy appeared to be complete as long as the patch did not split
or tear [44]. The patches split 25% of the time at shear pressure points. Tougher
patches have since been made exhibiting twice the essential work of fracture [45].
7.6.3
Fibers of Improved Elastic Moduli and Break Stresses and Strains
The carboxyl and amine functions in the polymers of Fig. 7.9 exhibit hydropho-
bic-induced pKa shifts. Formation of the –COO–···+H3N ion pairs on mixing of
the polymers reduces Tt and DGHA from the values of the individual polymers.
This aligns the polymers for chemical crosslinking and results in improved elas-
tic moduli and break stress values by one to two orders of magnitude (see leg-
end of Fig. 7.9).
7.6.4
Programmably Biodegradable Thermoplastics
Thermoplastics derive from the designed repeat pentamer, (AVGVP)n, that ex-
hibits an inverse temperature transition, but to a plastic rather than elastic-like
state [46]. When the polymer is made more hydrophobic, the dry polymer melts
and can be pulled into the fibers of Fig. 7.3 A. Also, introduction of asparagine
residues allows for timed breakdown to the carboxylate of aspartic acid. This
raises the value of Tt, and in water the plastic surface swells to become bio-
degradable. Since life cannot exist at the 160 8C required to melt the plastic, we
have designed protein-based polymers for functions that go beyond what nature
has had reason to evolve. Nonetheless, society can find uses for such environ-
mentally friendly plastics.
7.6.5
Acoustic Absorption
To the best of our knowledge, elastic protein-based polymer (GVGIP)320 has an

intense mechanical resonance in the acoustic absorption range with an absorp-
tion intensity that is an order of magnitude greater than the usually considered
sound-absorbing petroleum-based elastomers. Once the relevant “idol of the
past” [47] abates, this material can be expected in numerous sound absorption
applications.
7.7
7.7.1
List of Gene Constructions and Expressed Protein-based Polymers
Appropriate in considering the list of Table 7.5 is a quotation due to Jacob Bro-
nowski “In effect, the modern problem is no longer to design a structure from
the materials (available) but to design materials for a structure” [48]. Each of the
gene constructs of Table 7.5 was made for a specific purpose, either for elucidat-
ing an understanding of mechanism or for a specific application or for both. In-
deed, no longer is it required to coerce, by physical manipulation, many struc-
ture/function uses out of a single composition of matter. Now, one can design a
specific polymer sequence to be near optimal for the particular application and
then use physical manipulation in the role of fine-tuning the product for its de-
signed application. This is the power of protein-based polymeric materials. Each
transformed E. coli, below, becomes the factory for producing, with extraordi-
nary fidelity, the specific designed protein-based polymer.
7.7.2
Efforts Toward Low-cost Production in other Microbes and in Plants
Bioproduction of protein-based polymers has been under consideration using to-

bacco [49, 50], mushrooms [51], yeast [52], and the seeds of arabidopsis, canola,
and soy [53]. Early optimism that cost of production by E. coli could reach levels
of less than $ 10.00 kg–1 has yet to be realized. Production by yeast, as proposed
by Casal [52], gains from the capacity for protein-based polymer to be excreted
from the yeast cell, where harvesting could proceed without cell destruction,
and purification would not be as demanding as described above for E. coli [52].
The promise of truly low-cost production appears to reside with plants. One
attractive approach, considered by Somers [53], would be to express protein-
based polymer in seed as a value-added product under circumstances where the
cost of production would be the cost of purification. For example, production in
the canola seed would occur with retention of the canola oil product, and the
Table 7.5 List of gene constructions and expressed protein-

based polymers of Bioelastics Research.
1 {(GVGVP)10}n(GVGVP): n = 25*, 14*, 12*, 4*

2 (GVGVP GVGFP GHGFP GVGVP GVGFP GFGFP)n(GVGVP): n = 25*, 15, 12, 9, 8, 7,
5, 3
3 (GVGVP GVGFP GKGFP GVGVP GVGFP GFGFP)n(GVGVP): n = 75*, 41*, 33*, 22*,
15, 12, 6*, 5, 2
4 (GVGVP GVGFP GEGFP GVGVP GVGFP GFGFP)n(GVGVP): n = 42*, 32*, 17*, 14*,
8*, 6*, 4*, 3*, 2*
5 (GVGVP GVGFP GDGFP GVGVP GVGFP GFGFP)n(GVGVP): n = 12*, 8*, 7, 6, 4
6 (GVGVP GVGFP GEGFP GVGVP GVGFP GKGVP)n(GVGVP): n = 40, 21*, 20, 11*, 10,
8, 7
7 (GVGVP GVGKP GEGFP GVGVP GVGFP GFGVP)n(GVGVP): n = 48, 39*, 26, 22*, 21,
20, 17, 15, 12, 9, 7
8 (GVGVP GVGFP GEGFP GVGVP GVGVP GKGVP)n(GVGVP): n = 22*, 20, 18, 15, 13,
11, 10, 9, 8, 7, 6, 5, 4
9 (GVGVP GVGKP GEGFP GVGVP GVGVP GFGVP)n(GVGVP): n = 38, 22*, 15, 6
10 {(AVGVP)10}n(GVGVP): n = 29*, 7*, 4*, 2
11 {(GVGIP)10}n(GVGVP): n = 26*, 15*, 13*, 7*, 4*
12 (GVGIP GFGEP GEGFP GVGVP GFGFP GFGIP GVGIP GFGEP GEGFP GVGVP
GFGFP GFGIP)n(GVGVP): n = 30*, 24, 20*, 13*, 11, 10, 6, 5
13 (GVGIP GFGEP GEGFP GVGVP GFGFP GFGIP GVGIP GFGEP GEGFP GVGVP
GFGFP GFGIP GVGVP GVGRGYSLG VP)n(GVGVP): n = 20*
14 (GVGVP GVGFP GEGFP GVGVP GVGFP GVGFP)n(GVGVP): n = 41*, 29, 15*, 12, 9,
7, 6, 3
15 (GVGVP GVGVP GEGVP GVGVP GVGFP GFGFP)n(GVGVP): n = 60*, 39*, 24*, 15
16 (GVGVP GVGFP GEGFP GVGVP GVGVP GVGVP)n(GVGVP): n = 40*, 25, 16, 14, 10,
8, 7, 6, 5, 4, 3, 2
17 (GVGVP GVGFP GKGFP GVGVP GVGFP GVGFP)n(GVGVP): n = 21*, 12, 6
18 (GVGVP GVGVP GKGVP GVGVP GVGFP GFGFP)n(GVGVP): n = 45*, 22*, 20, 15,
14*, 12*, 7
19 (GVGVP GVGFP GKGFP GVGVP GVGVP GVGVP)n(GVGVP): n = 26*, 22*, 17*, 16,
14*, 13, 11, 10, 9
20 {(GVGIP)10–GVGVPGRGDSP–(GVGIP)10}n(GVGVP): n = 21, 18*, 11*
21 {(GVGVP)10–GVGVPGRGDSP–(GVGVP)10}n(GVGVP): n = 18*, 15*, 8*
22 {(GVGIP)10–GVGVPGRGDSP–(GVGVP)10}n(GVGVP): n = 12*, 8*
23 {(GVGIP)10–GVGRGYSLGIP–(GVGIP)10}n: n = 10*, 4*
24 [{(GGVP)3(GGFP)}3]n: n = 27, 22
25 GKGKAPGK–{(GVGVP)10}n: n = 10*
26 GKGKAPGK–{(AVGVP)10}n: n = 20*, 5*
27 {(GVGVP)10(GVGVAP)8(GVGVP)10}n: n = 8*, 5*, 2, 1
28 {(AFGFPAEGFP)5}n(GVGVP): n = 53, 22, 18, 16*, 14, 6
29 {(GVGFPGEGFPGFGVP)3}n(GVGVP): n = 30*, 14*, 12
30 {(GVGVP)10–GVGVPGNGVP(GVGVP)10}n(GVGVP): n = 10*, 5*
31 {(GVGIP)10–GVGIPGNGIP}n(GVGVP): n = 30*, 40*
32 {GVGVP GVGVP GEGVP GVGVP GVGVP GVGVP}n(GVGVP)YGSEFELRRQACGR-
TRAPP–PPPLPSGC: n = 44*, 36*
33 {GVGVP GVGVP GKGVP GVGVP GVGVP GVGVP}n(GVGVP): n = 55*, 36*, 22*
34 {(FEGFPAEGFP)5}n(GVGVP): n = 19*
35 {(GVGVP)10–GVGRGYSLGIP–(GVGVP)10)}n: n = 12*, 7*
36 {(GVGVP)10–GVGRGYSLGIP–(GVGIP)10)}n: n = 7*
37 {(GVGIP)10–FPGVGQKRPSKRSKYLIP–(GVGIP)10)}n: n = 11*, 6*
38 {(GVGVP)10–FPGVGQKRPSKRSKYLIP–(GVGVP)10}n(GVGVP): n = 9*, 6*
39 {(GVGVP)10–FPGVGQKRPSKRSKYLIP–(GVGIP)10}n(GVGVP): n = 9*, 7*, 5*
40 GKGKAPGK–(GVGVP GVGVP GEGVP GVGVPGVGVP GVGVP}n(GVGVP): n = 1
41 {(FEGVP)10}n: n = 28*, 15*, 11, 4
42 {(GVGVP)10–GVGVPGRGDSP–(GVGVP GVGVP GKGVP GVGVP GVGFP
GFGFP)2}n(GVGVP): n = 8*, 5
43 GKGKAPGK–{(GVGVP)10–GVGVPGRGDSP–(GVGVP)10}n: n = 7*
44 (FEGFP AFGFP)5: n = 33, 29, 26, 25, 23, 19, 18, 16, 14, 11, 6
45 {(GVGVP GVGVP GKGVP GVGVP GVGFP GFGFP)2–GVGVPGRGDSP–(GVGVP)10
(GVGVAP)8}n: n = 11*, 8*, 4*, 3*
46 {(GVGVP GVGVP GKGVP GVGVP GVGFP GFGFP)2– (GVGVP)10(GVGVAP)8}n:
n = 12*, 8*, 4*
47 {(GVGIP)11–GYGIP–(GVGIP)10}2(GVGIP):Rea): n = 11*, 10, 9, 8, 7, 6*, 5
48 {(FVGEP FVGFP)5}n: n = 18, 17, 12, 2, 1
49 (GIGVP GAGVP GIGVP GIGVP GAGVP GIGVP)n: n =22*, 11*
50 {(GVGIP)11–GYGIP}n(GVGIP):Rea): n = 18*, 17, 16, 15, 14, 13, 12, 11*, 6, 3
51 {(GVGVP)10}n(GVGVP): Rea): n = 28, 26, 18, 17*, 16, 15, 14,13*, 12, 11, 6, 4
52 {(GVGIP)10}n(GVGVP): Rea): n = 32*, 25*, 21*, 15*, 9*, 6*, 4*, 2*
53 [{(GVGIP GEGIP GVGIP)2}2]n: n = 20*, 9*
54 [{GVGIP GEGIP (GVGIP)4}2]n: n = 23*, 10*
55 [(GVGIP GEGIP GVGIP GEGIP GVGIP GVGIP)2]n: n = 26*, 17*, 15*
56 [{(GVGIP GKGIP GVGIP)2}2]n: n = 19, 18, 17, 12
57 [{GVGIP GKGIP (GVGIP)4}2]n: n = 21*, 12*
58 [(GVGIP GKGIP GVGIP GKGIP GVGIP GVGIP)2]n: n = 73, 41, 21, 17, 14, 11
59 [(GVGIP)8(GYGIP]n: n = 32*, 24*, 18*
60 [{(GVGIP)5GYGIP}2]n: n = 20*, 12*, 11*
61 [{(GVGIP)2GYGIP}3]n: n = 26, 15*, 14
62 {(GVGIP)10}n1–c)Fn3–{(GVGIP)10}n2: n1 = 16 and n2 = 6
63 {GVGVP GVGVP GEGVP GVGVP GVGVP GVGVP}n(GVGVP): n = 42*, 23*, 22*, 18*,
16*
64 AKKKKKKG–{(GVGIP)10}n–VCCC: n = 15*
65 AKKKKKKG–{(GVGIP)10}n–VCCC: n = 18*
66 GKGKAPGK–{(GGAP)12}n(GVGVP): n = 1
67 { (GGAP)12}n: n = 1
68 {(FVGVP FEGVP)5}n: n = 1
69 {[(GVGVP)2–GFGVP]3}n: n = 1
70 {(GVGIP)11–GSGIP–(GVGIP)10}n(GVGIP):Rea): n = 1
71 {(GVGIP)11–GTGIP–(GVGIP)10}n(GVGIP):Rea): n = 1
72 {(FFGEP)10}n: n = 1
73 {(GVGIP)11–GSGIP}n(GVGIP):Rea): n = 1
74 {(GVGIP)11–GTGIP}n(GVGIP):Rea): n = 1
75 {(GVGVP GVGVP GKGVP GVGVP GVGFP GFGFP)2–GVGVPGRGDSP–(GVGVP
GVGVP GKGVP GVGVP GVGFP GFGFP)2}n: n = 1
76 (GIGVP GAGVP GKGVP GIGVP GAGVP GIGVP)n: n = 1
77 (GIGVP GAGFP GKGFP GIGVP GAGVP GIGVP)n: n = 1
78 (GIGVP GAGFP GKGFP GIGVP GAGFP GVGFP)n: n = 1
7.8 Patents 245
79 {ACPGCGGVGIPCPGCG–[(GVGIP)26–b)Fn3(RGYSLG)–(GVGIP)6]
CPGCGGVGIPCPGCG}n: n = 1
*) Indicates that the protein-based polymer was expressed

a) Re: Indicates that the gene was made explicitly using the
same codon for a repeating residue in the sequence and
then a different codon was used in the next repeat
b) Fn3(RGYSLG) stands for the tenth type III domain of fibro-
nectin in which the cell attachment sequence, GRGDSP,
was replaced by the kinase site, RGYSLG, as a site for phos-
phorylation
c) Fn3 stands for the tenth type III domain of fibronectin.
Adapted from Ref. [1]
protein-based polymer would be in the protein meal byproduct, currently used

for cattle feed at costs of less than a dollar per pound. Water-based purification,
using the inverse temperature transition, would allow selected removal of the
protein-based polymer and return the remaining protein for cattle feed. In
short, the time can be anticipated when the cost of production of protein-based
polymers will compete favorably with that of petroleum-based polymers. Accord-
ingly, the outlook is promising for protein-based polymers to make a significant impact
in the marketplace of the near future.
7.8
Patents
7.8.1
Patents of D. W. Urry on Protein-based Polymers
A list of your author’s patents exclusively on uses of protein-based polymers is

given in Table 7.6. There are many additional patent drafts and concepts for
patents that have yet to be drafted. These will be pursued as finances allow. In
addition, there are enabling trade secrets that have been maintained.
7.8.2
Result of Ex Parte Patent Reexamination Request to the USPTO
Our routine utilization of GVGVP, or any of the five permutations of this se-
quence, in development of the mechanistic foundations and the applications
considered above is apparent. As noted above in Section 7.2, the efforts, specifi-
cally using GVGVP and VPGVG, date back more than three decades. Nonethe-
less, a patent issued in 1993 that claimed the use of recombinant DNA technol-
ogy to produce GVGVP and VPGVG. Once the patent appeared collaboration/li-
cense agreements between Bioelastics Research (BRL), the entity that held the
Table 7.6 D. W. Urry patents on protein-based polymers.
Country Patent no. Date filed Date issued
1 Inventors: D. W. Urry and K. Okamoto

Title: Synthetic Elastomeric Insoluble Crosslinked Polypentapeptide
USA 4,132,746 07/09/76 01/02/79
Canada 1,103,238 02/26/79 06/16/81
2 Inventors: D. W. Urry and K. Okamoto
Title: Synthetic Elastomeric Insoluble Crosslinked Polypentapeptide
USA 4,187,852 08/14/78 02/12/80
3 Inventor: D. W. Urry
Title: Elastomeric Composite Material Comprising a Polypeptide
USA 4,474,851 10/02/81 10/02/84
Title: Elastomeric Material Comprising a Polypentapeptide of Opposite Chirality in Posi-
tion Three
USA 4,500,700 12/23/82 02/19/85
Title: Enzymatically Crosslinked Bioelastomers
USA 4,589,882 09/19/83 05/20/86
6 Inventors: D. W. Urry and R. M. Senior
Title: Stimulation of Chemotaxis by Chemotactic Peptides (Hexapeptide)
USA 4,605,413 09/19/83 08/12/86
7 Inventors: D. W. Urry and M. M. Long
Title: Stimulation of Chemotaxis by Chemotactic Peptides (Nonapeptide)
USA 4,693,718 10/31/85 09/15/87
8 Inventors: D. W. Urry and K. U. M. Prasad
Title: Temperature Correlated Force and Structure Development of Elastin Polytetra and
penta
USA 4,783,523 08/27/86 11/08/88
Europe EP 0,321,496 03/30/94
Japan 2,726,420 08/27/87 12/05/97
9 Inventors: D. W. Urry and K. U. M. Prasad
Title: Segmented Polypeptide Bioelastomers to Modulate Elastic Modulus
USA 4,870,055 04/08/88 09/26/89
Japan 2,085,090 04/17/87 08/23/96
Title: Bioelastomer Containing Tetra/Pentapeptide Units
USA 4,898,926 06/15/87 02/06/90
Europe 06/13/88
Title: Reversible Mechanochemical Engines Comprised of Bioelastomers Capable of Inter-
conversion of Chemical and Mechanical Work
USA 5,032,271 06/15/87 07/16/91
Europe EP 0,425,491 06/13/88 07/20/94
USA 5,085,055 04/30/91 02/04/92
7.8 Patents 247
Table 7.6 (continued).
USA 5,255,518 12/24/91 10/26/93
14 Inventors: D. W. Urry and M. M. Long
Title: Stimulation of Chemotaxis by Chemotactic Peptides
USA 4,976,734 09/19/87 12/11/90
Europe EP 0,366,777 06/13/88 07/20/94
Title: Bioelastomeric Materials Suitable for burn areas or the Protection from Adhesions
USA 5,250,516 04/21/88 10/05/93
Europe EP 0,365,655 09/21/94
Japan 2,820,750 04/14/89 08/28/98
Title: Elastomeric Polypeptides as Vascular Prosthetic Materials
USA 5,336,256 04/22/88 08/09/94
Europe EP 0,365,654 01/12/94
Japan 2,115,962 04/14/89 12/06/96
Title: Polynonapeptide Bioelastomers Having an Increased Elastic Modulus
USA 5,064,430 02/23/89 11/12/91
Title: Polymers Capable of Baromechanical and Barochemical Transduction
USA 5,226,292 04/22/91 07/13/93
Title: Superabsorbent Materials and Uses Thereof
USA 5,393,602 04/19/91 02/28/95
Europe EP 0,580,811 03/10/93 08/04/99
Japan 4,510,189 03/10/92 07/26/02
Title: Superabsorbent Materials and Uses Thereof
USA 5,520,672 02/06/95 05/28/96
Title: Elastomeric Polypeptide Matrices for Preventing Adhesion of Biological Materials
USA 5,527,610 05/20/94 06/18/96
Japan 05/20/95
Title: Elastomeric Polypeptide Matrices for Preventing Adhesion of Biological Materials
USA 5,519,004 06/07/95 05/21/96
Title: Bioelastomeric Drug Delivery System
USA 6,328,996 B1 10/03/94 12/11/01
Europe EP 0,449,592 11/30/94
Japan 1,994,740 11/22/95
Table 7.6 (continued).
24 Inventors: D. W. Urry, Peter Shewry and U. Prasad Kari

Title: Bioelastomers Suitable as Food Product Additives
USA 5,972,406 10/13/95 10/26/99
Europe 96,912,815.6 04/15/96 Pending
Japan 8-531,270 04/23/2002 Pending
25 Inventors: D. W. Urry, H. Daniell, D. McPherson and J. Xu
Title: Hyperexpression of Bioelastomeric Polypeptides
USA 6,004,782 10/13/95 12/21/99
Title: Bioelastomers Suitable as Food Product Additives
USA 5,900,405 06/07/95 05/04/99
Europe 0,830,509 06/07/96 03/25/98
Japan 9-519,202 08/06/97 Pending
27 Inventors: D. W. Urry, D. McPherson and J. Xu
Title: A Simple Method for the Purification of a Bioelastic Polymer
USA 5,854,387 10/13/95 12/29/98
Europe 96,912,768.7 04/15/96 Pending
Japan 8-531261 04/16/2002 Pending
Title: Acoustic Absorption Polymers and Their Methods of Use
USA 09,746,371 12/20/00 Pending
Europe PCT/US 00/ 7/22/02 Pending
34,658
Title: Bioelastomer Nanomachines and Biosensors (NEMS)
USA 09/888,260 06/21/01 Pending
Europe PCT/US01/20045 06/21/01 Pending
Title: Injectable Implants for Tissue Augmentation And Generation
USA 09/837,969 04/18/01 In Allowance
Canada 2,319,558 02/26/99 Pending
Title: Injectable Implants for Tissue Augmentation And Generation
USA 09/841,321 04/23/01 Pending
Europe 99,908,590.5 02/26/99 Pending
Canada 2,319,558 02/26/99 Pending
Japan 533,072/2000 02/26/99 Pending
32 Inventors: D. W. Urry, P. Glazer and T. M. Parker
Title: Injectable Implants for Tissue Augmentation And Generation (Disk Repair)
USA 6,533,819 B1 04/23/01 03/18/03
Table adapted from Ref. [1].
Note added in Proof: Since the completing of the manuscript for this book, I have learned,
following my resignation as General Partner of Bioeleaxtec Research Ltd. that the prosecu-
tion and maintenance of this patent portfolio did not continue.
References 249
rights to the patents of Table 6, and major US companies as well as an impor-

tant Option Agreement with a major foreign entity terminated. In my view, this
was largely due to the perceived lack of BRL’s access to bioproduction of this
pivotal repeating unit. It was thought that only an interested party, prepared to
initiate litigation, would allow BRL access to GVGVP and VPGVG for commer-
cialization. Fortunately, the legal firm for BRL suggested that an Ex Parte Patent
Reexamination Request to the US Patent and Trademark Office (USPTO) would
provide the opportunity, at relatively low cost, to remove the impairing claims.
The submitted Request, which presented the BRL prior art, resulted in the rela-
tively recent rejection of the claims by the Examiner, and the entity that owned
the patent containing the offending claims made no effort to overcome the re-
jections. Accordingly, this major impediment for moving forward with commer-
cialization has been removed.
Note added in Proof: On August 2005, just prior to receiving these page proofs quite inex-
plicably, your author has learned that the Examiner has allowed amended claims to read on
the other three permutation of the polypentapeptide, namely VGVPG, GVPGV, and PGVGV.
These should be rejected on the same basis as were GVGVP and VPGVG. For example, one
can group the pentameric repeats of figures 7.8 and 7.9 to reach as repeats of any of this five
permutations.
Acknowledgment
The author gratefully acknowledges support of the Office of Naval Research

(ONR) and Program Officers, Michael Marron and Keith Ward, by means of
Grant No. N00014-98-1-0656 and Contract No. N00014-00-C-0178.
References
1 D. W. Urry, What Sustains Life? Consilient 8 D. W. Urry, Proc. Natl. Acad. Sci. USA,
Mechanisms for Protein-based Machines 1971, 68, 810–814.
and Materials. Springer, LLC, New York, 9 W. R. Gray, L. B. Sandberg and J. A. Fos-
2006 (in press), ISBN: 0817639101. ter, Nature, 1973, 246, 461–466.
2 J. A. V. Butler, Trans. Faraday Soc., 1937, 10 L. B. Sandberg, N. T. Soskel and J. G.
33, 229–238. Leslie, N. Engl. J. Med., 1981, 304, 561–566.
3 D. W. Urry, Chem. Phys. Letters, 2004, 11 D. W. Urry and M. M. Long, CRC Crit.
399, 177–183. Rev., Biochemistry, 1976, 4, 1–45.
4 D. W. Urry, J. Phys. Chem. B, 1997, 101, 12 D. W. Urry, K. Okamoto, R. D. Harris,
11007–11028. C. F. Hendrix and M.M. Long, Biochemis-
5 E. O. Wilson, Consilience, The Unity of try, 1976, 15, 4083–4089.
Knowledge, A. E. Knopf, New York, 1998, 13 D. W. Urry, C. M. Venkatachalam, M. M.
p. 8. Long and K. U. Prasad, In Conformation
6 D. W. Urry, B. Starcher and S. M. Par- in Biology, (R. Srinivasan and R H. Sar-
tridge, Nature, 1969, 222, 795–796. ma, Eds.) Adenine Press, USA, 1982,
7 B. A. Cox, B. C. Starcher and D. W. Urry, pp. 11–27.
Biochim. Biophys. Acta, 1973, 317, 209– 14 D. W. Urry, Methods in Enzymology, 1982,
213. 82, 673–716, (L.W. Cunningham and
D. W. Frederiksen, Eds.) Academic Press, 29 D. T. McPherson, J. Xu, and D. W. Urry,

Inc., New York, New York. Protein Expression and Purification, 1996,
15 D. W. Urry, In Molecular Conformation and 7, 51–57.
Biological Interactions, (P. Balaram and S. 30 D. W. Urry, A. Nicol, D. T. McPherson,
Ramaseshan, Eds.) Indian Acad. of Sci., C. M. Harris, T. M. Parker, J. Xu, D. C.
Bangalore, India, 1991, pp. 555–583. Gowda and P. R. Shewry, In Encyclopedic
16 D. K. Chang and D. W. Urry, J. Computa- Handbook of Biomaterials and Bioengineer-
tional Chem., 1989, 10, 850–855. ing – Part A – Materials, Vol. 2, (D. L.
17 D. W. Urry, Prog. Biophys. Mol. Biol., Wise, D. J. Trantolo, D. E. Altobelli, M. J.
1992, 57, 23–57. Yazemski, J. D. Gresser and E. R.
18 D. W. Urry, S.-Q. Peng, L. Hayes, J. Jag- Schwartz, Eds.) Marcel Dekker, Inc.,
gard and R. D. Harris, Biopolymers, 1990, New York, 1995, pp. 1619–1673,
30, 215–218. 31 A. Pattanaik, T. M. Parker and D. W.
19 D. W. Urry, D. C. Gowda, S.-Q. Peng and Urry, unpublished results.
T. M. Parker, Chem. Phys. Letters, 1995, 32 D. W. Urry, A. Pattanaik, J. Xu, T. C.
239, 67–74. Woods, D. T. McPherson and T. M. Par-
20 D. W. Urry, S.-Q. Peng, J. Xu and D. T. ker, J. Biomater. Sci. Polymer Edn., 1998,
McPherson, J. Amer. Chem. Soc., 1997, 9, 1015–1048; Reproduced in Polymers
119, 1161–1162. for Tissue Engineering, (M. S. Shoichet
21 D. W. Urry, In Protein-Based Nanotechnol- and J. A. Hubbell, Eds,) VSP BV, The
ogy, (V. Renugopalakrishnan and R. Netherlands, 1998, pp. 19–52.
Lewis, Eds.), Kluwer Academic Publish- 33 D. W. Urry, T. M. Parker, M. C. Reid, and
ers, in press. D. C. Gowda, J. Bioactive Compatible
22 P. S. Flory, Rubber Chem. and Tech., 1968, Polym., 1991, 6, 263–282.
41, G41–G48. 34 D. W. Urry, D. T. McPherson, J. Xu, D. C.
23 C. A. J. Hoeve and P. J. Flory, Biopolymers, Gowda, and T. M. Parker, In Industrial
1974, 13, 677–686. Biotechnological Polymers, (C. Gebelein
24 L. B. Alonso, B. J. Bennion and V. Dag- and C. E. Carraher, Jr., Eds,), Technomic,
gett, J. Am. Chem. Soc., 2001, 123, Lancaster, PA, 1995, pp. 259–281.
11991–11998. 35 G. Weber, Protein Interactions, Chapman
25 D. W. Urry and T. M. Parker, J. Muscle & Hall, Inc. 1992, p. 104.
Res. Cell Motility, 2002, 23, 541–547; Spe- 36 L. D. Hoban, M. Pierce, J. Quance, I.
cial Issue: Mechanics of Elastic Biomole- Hayward, A. McKee, D. C. Gowda, D. W.
cules. (H. Granzier, M. Kellermayer Jr., Urry and T. Williams, J. Surgical Res.,
W. Linke, Eds.) 1994, 56, 179–183.
26 D. W. Urry, T. Hugel, M. Seitz, H. Gaub, 37 F. J. Elsas, D. C. Gowda and D. W. Urry,
L. Sheiba, J. Dea, J. Xu and T. Parker, J. Pediatr. Ophthalmol. Strabismus, 1992,
Phil. Trans. R. Soc. Lond. B, 2002, 357, 29, 284–286.
169–184. 38 R. N. Alkalay, D. H. Kim, D. W. Urry,
27 D. W. Urry, T. Hugel, M. Seitz, H. Gaub, J. Xu, T. M. Parker, and P. A. Glazer,
L. Sheiba, J. Dea, J. Xu, L. Hayes, F. Pro- Spine, 2003, 28, 1659–1665.
chazka and T. Parker, In Elastomeric Pro- 39 M. M. Long, V. J. King, K. U. Prasad and
teins: Structures, Biomechanical Properties D. W. Urry, Biochim. Biophys. Acta, 1987,
and Biological Roles, (P. R. Shewry, A. S. 928, 114–118.
Tatham and A. J. Bailey, Eds.) University 40 M. D. Pierschbacher and E. Ruoslahti,
Press, The Royal Society; Chapter 4, Nature, 1984, 309, 30–33.
2003, pp. 54–93. 41 D. W. Urry, A. Pattanaik, M. A. Accavitti,
28 D. W. Urry, D. T. McPherson, J. Xu, D. C. C.-X. Luan, D. T. McPherson, J. Xu, D.
Gowda, N. Jing, T. M. Parker, H. Daniell, C. Gowda, T. M. Parker, C. M. Harris,
C. Guda, In The Polymeric Materials Ency- and N. Jing, In Handbook of Biodegrad-
clopedia: Synthesis, Properties and Applica- able Polymers, (A. J. Domb, J. Kost and
tions, CRC Press, Boca Raton, 1996, D. M. Wiseman, Eds.) Harwood Aca-
pp. 7263–7279.
References 251
demic Publishers, Chur, Switzerland, Pro2–Ala3–Val4–Gly5): A Reversible, In-

1997, pp. 367–386. verse Thermoplastic,” In Biotechnology
42 D. W. Urry, T. C. Woods, L. C. Hayes, J. and Polymers, (C. G. Gebelein, Ed.), Ple-
Xu, D. T. McPherson, M. Iwama, M. Fu- num Press, New York, 1991, 265–274.
ruta, T. Hayashi, M. Murata, and S. M. 47 P. Zagorin, Francis Bacon, 1998, p. 82.
Parker, In: Tissue Engineering and Novel 48 J. Bronowski, “The Ascent of Man,” Little,
Delivery Systems, Marcel Dekker, Inc., Brown and Company, Boston, 1973,
New York, 2004, pp. 31–54. p. 110.
43 T. C. Woods, Ph.D. Dissertation of The 49 X. Zhang, C. Guda, R. Datta, R. Dute,
University of Alabama at Birmingham, D. W. Urry, and H. Daniell, Letters, 1995,
1998. 17, 1279–1284.
44 B. Kemppainen, N.-Z. Wang, S. Swaim, 50 X. Zhang, D. W. Urry, and H. Daniell,
D. W. Urry, C.-X. Luan, J. Xu, E. Sartin, Plant Cell Reports, 1996, 16, 174–179.
R. Gillette, S. Hinkle and S. Coolman, 51 R. W. Herzog, N. K. Singh, D. W. Urry
Wound Repair and Regeneration, 2004, 12, and H. Daniell, Applied Microbiol. & Bio-
453–460. tech., 1997, 47, 368–372.
45 D. W. Urry, J. Xu, W. Wang, L. Hayes, F. 52 Collaboration with Prof. Margarida Ca-
Prochazka, and T. M. Parker, Mat. Res. sal, Dept. Biology, University of Minho,
Soc. Symp. Proc.: Materials Inspired by Braga, Portugal.
Biology, 2003, 774, 81–92. 53 Collaboration with Prof. D. A. Somers,
46 D. W. Urry, J. Jaggard, K. U. Prasad, T. Dept. Agronomy and Plant Genetics,
Parker, and R. D. Harris, “Poly(Val1– University of Minnesota.
253
8
New Syntheses with Oils and Fats as Renewable
Raw Materials for the Chemical Industry
Ursula Biermann, Wolfgang Friedt, Siegmund Lang, Wilfried Lühs, Guido Machmüller,
Jürgen O. Metzger, Mark Rüsch gen. Klaas, Hans J. Schäfer, Manfred P. Schneider
This work is dedicated to Professor Siegfried Warwel, Münster, for his impor-
tant contributions to the development of oleochemistry.
8.1
Introduction
Sustainable development had become the key ideal of the 20th century [1]. In
the search for sustainable chemistry, considerable importance is being attached
to renewable raw materials which exploit the synthetic capabilities of nature [2,
3]. Oils and fats of vegetable and animal origin make up the greatest proportion
of the current consumption of renewable raw materials in the chemical indus-
try, since they offer to chemistry a large number of possibilities for applications
which can be rarely met by petrochemistry. The extent of the use of natural oils
and fats in chemistry was summarized in 1988 [4]. It stated that “more than
90% of oleochemical reactions have been those occurring at the fatty acid car-
boxy group, while less than 10% have involved transformations of the alkyl
chain. However, future progress will be along the lines of these latter types of
reactions with their potential for considerably extending the range of com-
pounds obtainable from oils and fats. Such progress is essential for a growth in
the use of oils and fats as renewable raw materials”. For the future, this means
that “oils and fats of vegetable and animal origin offer possibilities for providing
chemistry with a wealth of reaction products which will be of great value in the
future. The chemical possibilities of renewable oils and fats are still very far
from being fully exploited. Interdisciplinary collaboration involving chemistry,
biochemistry, plant breeding, and agriculture is necessary to extend the success-
ful applications of this technology.” A good example of this is the alkyl polygly-
cosides, the use and properties of which have been recently reviewed [5].
ISBN: 3-527-31027-4
254 8 New Syntheses with Oils and Fats as Renewable Raw Materials for the Chemical Industry
We report here the advances made in the chemistry and biotechnology of fatty
materials over the last ten years and include the improvements in natural oils
and fats by plant breeding.
8.2
Reactions of Unsaturated Fatty Compounds
By means of simple industrial reactions, fatty materials are available from vege-
table oils in such purity that they may be used for further chemical conversions
and for the synthesis of chemically pure compounds. Predominantly, oleic acid
1 a and elaidic acid (E)-1 a, petroselinic acid 2 a, erucic acid 3 a, linoleic acid 4 a,
and linolenic acid 5 a have been used in the syntheses described below
(Fig. 8.1). Ricinoleic acid 6 a carries an additional hydroxyl group which is useful
in stereo- and regioselective syntheses. By pyrolysis of 6 b and subsequent hy-
drolysis, 10-undecenoic acid 7 a, a x-unsaturated carboxylic acid, is obtained [4],
which is very useful for selective reactions. Both 9-decenoic acid 8 a and 13-tetra-
decenoic acid 9 a are accessible by the metathesis reaction of ethylene with oleic
acid 1 a and erucic acid 3 a, respectively, thus extending the range of x-unsatu-
rated fatty compounds available (Section 8.2.3). Conjuenic acid 10 a, with conju-
gated double bonds, is obtained as a regio- and stereoisomeric mixture by the
isomerization of linoleic acid 4 a [4]. The alkyne fatty compounds 11–13, with
internal or terminal triple bonds, are readily available on a laboratory scale [6].
The epoxides 14–16, the synthesis of which has been greatly improved re-
cently, are also available as reactive fatty compounds (see Section 8.2.1). Methyl
ricinoleate 6 b may be oxidized to methyl 12-oxooleate 17 which, in turn, may
be readily isomerized to the enone 18 [7]. Similarly, the allyl alcohol 19, ob-
tained by selenium oxidation of methyl 10-undecenoate 7 b (see Section 8.3.2.2),
can be dehydrogenated to the x-unsaturated enone 20 [7 b]. The fatty com-
pounds 17–20 are suitable substrates for interesting follow-up reactions.
8.2.1
Oxidations
8.2.1.1 New Methods for the Epoxidation of Unsaturated Fatty Acids

Unsaturated fatty compounds are preferably epoxidized on an industrial scale by
the in situ performic acid procedure [4]. Numerous new methods have been used,
particularly with oleic acid, such as epoxidation with aldehydes and molecular oxy-
gen [8], dioxiranes [9–11], H2O2/tungsten heteropolyacids [12, 13], and H2O2/
methyl trioxorhenium [14–17]. Epoxidation by the Halcon process [18]. with alkyl
hydroperoxides also succeeds with unsaturated fatty compounds [19, 20]. As yet,
however, none of these methods has achieved industrial significance.
Chemo-enzymatic epoxidation is of considerable interest because this method
totally suppresses undesirable ring opening of the epoxide. Initially, the unsatu-
rated fatty acid [21] or ester [22] is converted into an unsaturated percarboxylic
Fig. 8.1 Starting materials for the synthesis 9,10-epoxyoctadecanoic acid 14 a, cis-9,10;cis-
of novel fatty acids: Oleic acid 1 a, elaidic 12,13-bisepoxyoctadecanoic acid 15 a, cis-
acid (E)-1 a, petroselinic acid 2 a, erucic acid 9,10;cis-12,13;cis-15,16-trisepoxyoctadecanoic
3 a, linoleic acid 4 a, linolenic acid 5 a, ricino- acid 16 a, 12-oxooleic acid 17 a, 12-oxoocta-
leic acid 6 a, 10-undecenoic acid 7 a, 9-dece- dec-10-enoic acid 18 a, 9-hydroxy-10-undece-
noic acid 8 a, 13-tetradecenoic acid 9 a, con- noic acid 19 a, 9-oxo-10-undecenoic acid
juenoic acid 10 a (regio- and stereoisomeric 20 a, and the respective methyl esters
mixture), stearoleic acid 11 a, 17-octadecy- 1 b–20 b and alcohols 1 c–20 c.
noic acid 12 a, 10-undecynoic acid 13 a, cis-
Scheme 8.1 Reaction principle of the chemo-enzymatic “self”

epoxidation of unsaturated fatty acids: intermediate enzymatic
formation of peroxyoleic acid 21 from oleic acid 1 a [23].
acid, such as 21, by a lipase-catalyzed reaction with H2O2 and is then self-epoxi-
dized in an essentially intermolecular reaction (Scheme 8.1) [23]. The second re-
action step occurs without involvement of the enzyme, following the rules of
the Prileshajev epoxidation.
Excellent stability and activity is shown by Novozym 435, a Candida antarctica
lipase B immobilized on polyacryl. This remarkable, readily separable, heteroge-
neous biocatalyst can be used several times without loss of activity; a turnover
of more than 200 000 moles of product per mole of catalyst has been achieved.
If vegetable oils are subjected to perhydrolysis, they are likewise epoxidized by
the peroxy fatty acid formed (Scheme 8.2) [24]. The formation of mono- and di-
glycerides can be fully suppressed by the addition of 5 mol% free fatty acid.
Soybean and other vegetable oils have been oxidized by these methods with con-
versions and selectivities of 90% and above. Even with the highly unsaturated
linseed oil, the selectivity of this reaction is maintained.
Industrially, vegetable oil epoxides are currently used mainly as PVC stabiliz-
ers. New applications have been opened by the possibility of photochemically in-
itiated cationic curing [25].
Follow-Up Reactions of Epoxides to Aziridines and Episulfides Epoxidized fats,

such as 14–16, are reactive reactants for a number of interesting follow-up pro-
Scheme 8.2 Chemo-enzymatic epoxi-

dation of vegetable oils [24].
Scheme 8.3 Synthesis of methyl epiminooctadecanoate 22 a

from methyl epoxyoctadecanoate 14 b: a) NaN3, NH4Cl,
EtOH, H2O; b) Ph3P, THF [26]. Methyl di- and triepiminoocta-
decanoates 22 b and 22 c can be synthesized in a similar man-
ner from the methyl di- and triepoxyoctadecanoates 15 b and
16 b [27].
Scheme 8.4 Synthesis of methyl epithiooctadecanoate 23 a

from methyl epoxyoctadecanoate 14 b: a) HC(=S)N(CH3)2,
CF3COOH, ClCH2CH2Cl. Methyl diepithiooctadecanoate 23 b
can be synthesized in a similar manner from methyl diepoxy-
octadecanoate 15 b [28].
cesses [4]. The corresponding methyl epiminooctadecanoates 22 a–c have been

synthesized as potentially bioactive compounds (Scheme 8.3) [26, 27].
The aziridines 22 and the episulfides 23 [28], the latter being accessible from
the epoxides 14 b and 15 b (Scheme 8.4), are interesting intermediates in the
synthesis of heterocyclic and highly functionalized fatty compounds.
8.2.1.2 Oxidation to vic-Dihydroxy Fatty Acids

Vicinal diols of unsaturated fatty compounds – polyols for polyurethanes based
on renewable raw materials – may be prepared by epoxidation and subsequent
nucleophilic ring opening of the epoxide. Since harsh reaction conditions are
technically necessary for ring opening of fatty epoxides [29], the direct synthesis
of vicinal dihydroxy fatty acids is an interesting alternative.
Scheme 8.5 Enantioselective oxidation of methyl elaidate (E)-

1 b with AD-mix-a to methyl (–)-(9S,10S)- and with AD-mix-b
to methyl (+)-(9R,10R)-dihydroxyoctadecanoate 24 [35a].
a) AD-mix, MeSO2NH2, H2O, tBuOH, 0 8C.
The hydroxylation of oleic acid 1 a by H2O2 and a molybdenum [30], tungsten

[31], or rhenium-based catalysts [32, 33]. affords syn diols via the epoxide inter-
mediate.
The enantioselective oxidation of methyl elaidate (E)-1 b to the chiral syn-dihy-
droxyoctadecanoate (S,S)-24 and its enantiomer with AD-mix-a and AD-mix-b
[34], respectively, in a yield of 97% and an enantiomeric excess of 95% ee is
especially noteworthy (Scheme 8.5) [35 a]. With methyl oleate 1 b, the enantio-
meric excess was very low using this method. The enantiomerically enriched
carboxylates derived from 24 associate to gels in carbon tetrachloride. Like the
corresponding gels from enantiomerically pure ricinoleic acid salts [36], they
form helical fibers which can be visualized with atomic force microscopy [35 b].
8.2.1.3 Oxidative Cleavage

The cleavage of oleic acid 1 a to pelargonic 25 and azelaic acids 26 a with ozone
as oxidant is the most important industrial application of ozonolysis [4, 37]. A
catalytic alternative, which uses a more suitable and safe oxidant is of consider-
able interest [4].
The direct oxidative cleavage of internal C–C double bonds with peracetic acid
and ruthenium catalysts or with H2O2 and Mo, W, or Re-based catalysts leads to
yields of only 50–60% (Scheme 8.6) [38]. In contrast, terminal C–C double
bonds can be cleaved in yields of 80% with Ru(acac)3/CH3CO3H [39] or Re2O7/
H2O2 [40] (acac = acetylacetonato). This gives rise to the possibility of initially
converting natural, internally unsaturated fatty acids into x-unsaturated fatty
acid methyl esters such as 8 b and 9 b by means of metathesis, followed by oxi-
dative cleavage. The advantage here is that the production of azelaic acid 26 and
pelargonic acid 25 can be uncoupled, independent of the oxidation method.
Scheme 8.6 Transition metal-catalyzed oxidative cleavage of

methyl oleate 1 b to pelargonic acid 25 and azelaic acid half
ester 26 with peracetic acid [39] or hydrogen peroxide [40].
a) [Ru(acac)3]/CH3CO3H or Re2O7/H2O2.
8.2.2
Transition Metal-Catalyzed Syntheses of Aromatic Compounds
The route to aromatic compounds from renewable raw materials is of impor-

tance [4]. The transition metal catalyzed trimerization of the alkyne fatty com-
pounds 11 and 13 gives the highly functionalized aromatic species 27 and 28,
respectively (Scheme 8.7), and co-trimerization with nitrile moieties affords the
highly varied and functionalized pyridine derivatives 29 (Scheme 8.8) [42].
Scheme 8.7 Cyclotrimerization of the internal alkyne 11 c and

the terminal alkyne 13 b to the regioisomeric benzene deriva-
tives 27 and 28 [42]. TMS=trimethylsilyl, Cp=cyclopentadienyl,
cod = cycloocta-1,5-diene.
Scheme 8.8 Cyclization of methyl 17-octadecynoate 12 b with

nitrile species to the pyridine derivatives 29 [42]
8.2.3
Olefin Metathesis
Transition metal metathesis of olefins, which is used in the industrial petro-

chemistry and polymer chemistry for the production of special olefins and un-
saturated polymers, is also applicable to unsaturated fatty acid esters. However,
the low loading of the expensive catalysts has, until now, stood in the way of
the technical utilization of this interesting reaction in oleochemistry [4].
Over the past few years, Warwel et al. have developed significantly more active
catalysts in the form of Re2O7 · B2O3/Al2O3 · SiO2 + SnBu4 and CH3ReO3
Scheme 8.9 Co-metathesis of methyl oleate 1 b and ethylene

to methyl 9-decenoate 8 b and 1-decene. The ester 1 b used
(new sunflower) was 87% pure, the conversions and selectiv-
ities each > 90%, and the yields of 8 b were > 80% [43].
+ B2O3 · Al2O3 · SiO2 and successfully tested them in a series of metathesis

transformations [43]. The industrial application of olefin metathesis to unsatu-
rated fatty compounds thus moves realistically nearer. Scheme 8.9 illustrates the
co-metathesis of methyl oleate 1 b and ethylene to form methyl 9-decenoate 8 b
and 1-decene. Similarly, methyl 13-tetradecenoate 9 b and 1-decene are obtained
from methyl erucate 3 b and ethylene [43]. Methyltrioxorhenium is also a suit-
able catalyst for the metathesis of unsaturated fatty compounds [44].
8.2.4
Pericyclic Reactions
The thermal Diels-Alder reaction of methyl conjuenate 10 b with electron-

deficient dienophiles has been thoroughly investigated [4]. and is carried out
industrially with maleic anhydride. With a Lewis acid, such as boron trichloride
or tin tetrachloride, and catalytic amounts of iodine it was possible to obtain
the cycloadducts 31 with the dienophiles 30, even at room temperature
(Scheme 8.10) [45]. Building on the cycloaddition of dimethyl acetylenedicarbox-
ylate 32 to 10 b, a further aromatic synthesis has been developed. Adduct 33 was
dehydrogenated with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) to the di-
methyl phthalate 34 into which the fatty acid side chain is incorporated
(Scheme 8.11).
Scheme 8.10 Diels-Alder reactions of methyl conjuenates 10 b

with the dienophiles 30 a–d to the regioisomeric addition
products 31 [45].
Scheme 8.11 Diels-Alder addition reactions of methyl conjue-

nates 10 b and dimethyl acetylenedicarboxylate 32 to the cy-
cloaddition product 33 which was dehydrogenated to the di-
methyl phthalates 34 with DDQ [45]. BHT = 4-methyl-2,6-
bis[(1,1-dimethyl)ethyl]phenol.
In addition, Diels-Alder reactions with the enones 18 and 20 as dienophiles

[45, 46], ene reactions [47, 48], [2+2] cycloadditions of ketenes [49], and isocya-
nates [50], to unsaturated fatty compounds, as well as sigmatropic [3,3] rear-
rangements of allylvinyl ethers derived from fats [49], have been investigated,
which lead to a number of interesting and novel fatty compounds.
8.2.5
Radical Additions
With the development of modern preparative radical chemistry, radical additions

to unsaturated fatty compounds with the formation of new C–C bonds have
been investigated systematically. Normal tin hydride radical chemistry [51] can-
not be applied to the sterically constrained, internal, and electron-rich double
bonds, as in 1 b. In contrast, enolizable compounds such as acetic acid, malonic
acid, monomethyl malonate, and cyanoacetic acid were added to the fatty acid
Scheme 8.12 Manganese(III) acetate-

induced radical addition of malonic
acid to methyl oleate 1 b with forma-
tion of the regioisomeric c-lactones 35
[53–55].
esters 1 b and 7 b by initiation with manganese(III) acetate [52] to give the re-
spective c-lactones such as 35 (Scheme 8.12) [53–55]. Unfortunately, higher car-
boxylic acids cannot be oxidized to radicals with manganese(III) acetate and
added to alkenes [54, 55]. For this purpose, a new method was developed.
8.2.5.1 Solvent-Free, Copper-Initiated Additions of 2-Halocarboxylates

Higher carboxylic acids can be added to alkenes, such as unsaturated fatty com-
pounds, as their a-haloesters, a process initiated by electron transfer from cop-
per [56–58]. The addition of 2-iodocarboxylates, for example, methyl 2-iodopro-
panoate 36 a, to 7 b gave the c-lactone 37 in high yields (Scheme 8.13). The reac-
tion procedure is very simple: The unsaturated fatty compound, the 2-halocar-
boxylate, and commercial copper powder are mixed without further pretreat-
ment and heated at 100–130 8C under an inert atmosphere. After a simple
work-up, analytically pure products are obtained in good yields. Esters of 2-iodo-
carboxylic acids can be obtained in situ from the readily available bromo com-
pounds by the addition of a stoichiometric amount of sodium iodide.
Methyl 2-bromopropanoate 36 b was also added to methyl oleate 1 b with cop-
per by the addition of stoichiometric amounts of sodium iodide. The regioiso-
meric addition products 38 a were isolated in 58% yield. The addition–elimina-
tion product 38 b was isolated as a byproduct (Scheme 8.14). Comparable results
were achieved with methyl petroselinate 2 b and methyl erucate 3 b [57, 58]. This
generally applicable addition reaction was also carried out with bromomalo-
Scheme 8.13 Copper-initiated addition of methyl 2-iodopro-

panoate 36 a to methyl 10-undecenoate 7 b [56–58].
Scheme 8.14 Copper-initiated addition of methyl 2-bromopro-

panoate 36 b to methyl oleate 1 b in the presence of sodium
iodide yields the regioisomeric c-lactones 38 a and the addi-
tion–elimination product 38 b [56–58].
Scheme 8.15 Radical cyclization of methyl 2-iodopetroselinate

39 induced by AgOAc/SnCl2 (a-40 : b-40 : trans-40 = 35 : 31 : 34)
[59].
nates, 2-bromo-3-alkylsuccinates, and a,a'-diiododicarboxylates, among others, in

good to very good yields [57, 58]. In an analogous manner, 2-haloalkane nitriles
have also undergone addition [56–58].
The reaction can also be used for intramolecular cyclization. The cyclization
of methyl 2-iodopetroselinate 39 to the cyclopentane derivatives 40 was best car-
ried out with the initiator system AgOAc/SnCl2 (Scheme 8.15) [59].
8.2.5.2 Addition of Perfluoroalkyl Iodides

Radical additions of perfluoroalkyl iodides to terminally unsaturated carboxylic
acids such as 10-undecenoic acid 7 a with 2,2'-azobisisobutylnitrile (AIBN) as
initiator give perfluoroalkylated products in good yields [60]. In contrast, for rad-
ical additions to alkenes with internal double bonds, such as methyl oleate 1 b,
the addition products are only obtained in very low yields by this method [61].
Scheme 8.16 Synthesis of 9- and

10-perfluoroalkyloctadecanoic
acids 42 as a regioisomeric mix-
ture: Addition of perfluoroalkyl
iodides to 1 b give the regioiso-
meric perfluoroalkylated iodo-
esters 41, which were then re-
duced to iodine-free esters and
hydrolyzed to free perfluoroalky-
lated fatty acids 42 [61, 62].
Perfluoroalkyl iodides 41 can be added to both methyl 10-undecenoate 7 b and

methyl oleate 1 b with good to very good yields if the reaction is initiated by
electron transfer from metals such as finely divided silver [61], copper powder
[62], or lead with a catalytic amount of copper(II) acetate [62] (Scheme 8.16).
The best yields of addition product 41 are obtained with copper powder or with
lead/Cu(OAc)2 [62].
Perfluoroalkylated fatty compounds such as 42 are of interest because of their
surfactant properties [63].
8.2.5.3 Thermal Addition of Alkanes

Alkylated fatty acids have interesting properties [64] and an effective synthesis
of these products is important [4]. The ane reaction is the thermally initiated
radical addition of alkanes to alkenes at elevated temperatures (200–450 8C) and
pressures (200–250 bar) [65]. The addition of cyclohexane to methyl 10-unde-
cenoate 7 b gave methyl 11-cyclohexylundecanoate 43 (Scheme 8.17) [66]; 11-cy-
clohexylundecanoic acid is the main lipid of thermophilic archaebacteria [67].
Scheme 8.17 Thermally initiated addition of cyclohexane to

methyl 10-undecenoate 7 b. The reaction was carried out in a
high pressure–high temperature flow reactor [66].
8.2.6
Lewis Acid-Induced Cationic Addition
x-Hydroxycarboxylic acids, including alkyl-branched acids such as 44, which are

of interest as polyester components, are obtained with high selectivity by the
ene addition of formaldehyde to unsaturated fatty acids (Scheme 8.18) [68].
However, stoichiometric amounts of dimethylaluminum chloride or ethylalumi-
num dichloride are used as reagents [69, 70]. A catalytic variant would be highly
significant. The acid (Z)-45 (Scheme 8.18), obtained by the addition of formalde-
hyde to 10-undecenoic acid 7 a, induces wound healing of tissue damage in soy-
beans by stimulation of callus formation at the damaged site [71].
Ene additions of formaldehyde to natural oils proceed with formation of the
respective di- and trifunctionalized triglycerides [72], and jojoba oil gives mix-
tures of 1 : 1 and 1 : 2 adducts [73]. Homoallyl ethers are obtained in an analo-
gous reaction with acetals [74].
Formaldehyde and higher aldehydes react with unsaturated fatty compounds
in the presence of aluminum chloride to form the corresponding alkyl-substi-
tuted 4-chlorotetrahydropyrans in good yields and with high selectivity [75]. The
reaction of two equivalents of formaldehyde with, for example, methyl oleate
Scheme 8.18 Me2AlCl-induced addition of paraformaldehyde

to oleic acid 1 a to give the regioisomeric homoallyl alcohols
44. The corresponding addition to 10-undecenoic acid 7 a
gives the homoallyl alcohol 45 [(E):(Z) = 4 : 1] [68].
Scheme 8.19 AlCl3-induced addition of two equivalents of

paraformaldehyde to methyl oleate 1 b to give the 4-chloro-
tetrahydropyrans 46 (mixture of two regioisomers) [75].
1 b, gave the 3,5-dialkyl-substituted 4-chlorotetrahydropyran 46 (Scheme 8.19).

Variation of the alkene, on the one hand, and the carbonyl component, on the
other, leads to a broad range of alkyl-substituted chlorotetrahydropyrans.
The Friedel-Crafts acylation is an interesting and versatile method for the
functionalization of unsaturated fatty compounds [76]. The EtAlCl2-induced acy-
lation of oleic acid 1 a, among others, with acyl chlorides 47 gave the (E)-config-
ured b,c-unsaturated oxocarboxylic acids 48 with high selectivity (Scheme 8.20).
Cyclic anhydrides, such as succinic anhydride, gave oxo diacids in a similar
manner [76]. The acylation products 48 are substrates for a number of interest-
ing follow-up reactions, for example, 48 g for Nazarov cyclizations [77].
8.2.7
Nucleophilic Addition to Reversed-Polarity Unsaturated Fatty Acids
Addition to the double bond of unsaturated fatty acids mainly occurs with electro-
philes (Section 8.2.6), radicals (Section 8.2.5), or in pericyclic reactions (Section
8.2.4). Totally new coupling possibilities arise when the polarity of the electron-
Scheme 8.20 EtAlCl2-induced Friedel-Crafts acylations of oleic

acid 1 a with the acyl chlorides 47 a–g give the unsaturated
regioisomeric oxocarboxylic acids 48 a–g [76].
rich double bond is reversed, as in the enone fatty acids 18 and 20. In this way, a
number of nucleophiles may be coupled to the double bond by Michael additions
[78]. Interesting and novel fatty compounds have also been obtained from the en-
ones 18 and 20 in Stetter [7, 78] and Mukaiyama additions [79]. Similarly, methyl
conjuenate 10 b has been treated anodically with numerous alcohols to afford
methyl dialkoxyoctadecanoates, some of which had interesting surfactant proper-
ties [80]. Numerous carbon, oxygen, and nitrogen-based nucleophiles may be in-
serted into fatty allyl carbonates by palladium catalysis (from the corresponding
allyl alcohols; Section 8.3.2.2) in very good yields [79 b, 79 d].
8.3
Reactions of Saturated Fatty Compounds
8.3.1
Radical C–C Coupling
8.3.1.1 Oxidative Coupling of C2 Anions of Fatty Acids

The C–C coupling, with the concurrent formation of symmetrical products, may
be achieved by the dimerization of two radicals. Radicals may be formed selec-
tively under mild conditions in high concentrations by the oxidation of anionic
precursors. Unsaturated fatty acids possess several sites with comparably high
C–H acidities, which are suitable for anionization and subsequent reactions,
Scheme 8.21 Radical a,a' dimeriza-

tion of the fatty acid methyl esters
49. Ozonolysis of the dimer 50 c
gives the dimethyl tetracarboxylate
51 [81]. LDA = lithium diisopropyla-
mide.
particularly the a-positioned C–H bond of the ester group. The fatty acid methyl
esters 49 were anionized and treated oxidatively with 0.9 equivalents CuBr2. In
this way, the dimers 50 were formed with a (d,l) : meso ratio of about 1.2 : 1
(Scheme 8.21) [81 a]. The dimethyl ester of the tetracarboxylic acid 51 was ob-
tained from 50 c by ozonolysis in 90% yield.
8.3.1.2 Anodic Homo- and Heterocoupling of Fatty Acids (Kolbe Electrolysis)

The anodic decarboxylation of aliphatic carboxylic acids gives a rapid, and also
technically useful, path to radicals for dimerization and coupling (Kolbe electroly-
sis) [82]. This efficient synthetic method was used extensively in homocouplings
with natural and modified fatty acids, such as in the preparation of specifically
functionalized alkanes [83] or in the preparation of long chain diesters. Isostearic
acid may be dimerized in 63% yield to a methyl-branched C34 hydrocarbon whose
cosmetic property profile resembles that of squalane. As the half ester with the
currently greatest number of carbon atoms, the methyl ester of the C36 dimeric
fatty acid was coupled in 38% yield to a C70 dimethyl dicarboxylate [83, 84].
In addition, the dimeric fatty acids 53 may be obtained from Diels-Alder ad-
ducts of fatty acids such as 52 (Section 8.2.4) by homocoupling (Scheme 8.22 a)
[47], or dicarboxylic acids with four alkyl chains, such as 55, are obtained from
2,2'-coupled diacids like 54 (Section 8.3.1.1, Scheme 8.22 b) [78 b].
Through heterocoupling, that is, the electrolysis of two different carboxylic acids,
new unsaturated fatty acids are formed [82 a, 84, 85 a], such as methyl 17-octade-
cenoate 56 (Scheme 8.23 a) [84], partially perfluoronated fatty acids like 57 (Sche-
Scheme 8.22 Homocoupling of the half esters 52 and 54 by

Kolbe electrolysis to give the dimers 53 [45] and 55 [78 b]
respectively.
Scheme 8.23 Kolbe electrolysis of two different fatty acids:

Synthesis a) of new x-unsaturated fatty acids such as 56 [84],
b) of perfluoroalkylated fatty acids such as 57 [84], and
c) of C-glycosides such as 59 [86 a].
me 8.23 b) [84], pheromones [85 b], C-glycosides 59 formed by co-electrolysis with

carbohydrate carboxylic acids 58 (Scheme 8.23 c) [86], or long chain diesters [85 b].
8.3.2
Functionalization of C–H Bonds
Selective reactions at the alkyl chain of fatty acids are still rare but are of great
interest [4].
8.3.2.1 Oxidation of Nonactivated C–H Bonds

Nonactivated C–H bonds may be functionalized chemically [87] or enzymatically
[88] (Section 8.4.2.5). Particularly important, but yet to be solved satisfactorily, is
the regioselectivity of C–H functionalization.
Notable advances have been achieved by photochemical gas phase chlorination
of fatty acids that are adsorbed onto aluminum oxide with chlorine or tBuOCl. For
this reaction, selectivities increase with increasing chain length of the fatty acid.
Stearic acid 60 reacts with chlorine or tBuOCl at –35 8C in the x-(x-2) position
to form the chlorostearic acids 61 in yields of 96% and 93%, respectively
(Scheme 8.24) [89]. The selectivity is significantly better than with the more estab-
lished radical chlorination with dialkylchloramines in an acidic medium [90, 91].
The methyl esters of shorter chain fatty acids and fatty alcohols may be hydroxy-
lated with amine oxides with good conversions and (x-1)-(x-2) selectivities [7 a].
Scheme 8.24 Photochemical gas-phase chlorination of stearic

acid 60 adsorbed on aluminum oxide to chlorostearic acids
61 with chlorine (relative product distribution (rpd): x-2
14.2%; x-1 38.4%; x 43.7%) and tBuOCl (rpd: x-2 11.6%;
x-1 51.5%;x 30.1%) [89].
8.3.2.2 Oxidation of Allylic C–H Bonds

The allylic C–H bonds of unsaturated fatty acids are activated C–H bonds,
which, in principle, may be functionalized with a number of oxidizing agents.
For the allylic oxidation of methyl 10-undecenoate 7 b and methyl oleate 1 b,
SeO2/tBuOOH has been shown to be suitable [7].
The reaction with singlet oxygen has proved to be considerably more suitable
for the preparation of the allyl alcohol 62. For this purpose, 1 b was photooxyge-
nated with oxygen by means of a high pressure sodium-vapor lamp and tetra-
phenylporphin as sensitizer, and the resulting hydroperoxide 63 was reduced
Scheme 8.25 Photooxygenation of methyl oleate 1 b with

singlet oxygen and tetraphenylporphin as sensitizer to give
a) allyl alcohols 62 and b) a,b-unsaturated ketones 18
(both regioisomeric mixtures form) [79 b, 79 c].
with triphenylphosphine (Scheme 8.25) [79 b, 79 c]. In the presence of acetic an-
hydride, pyridine, and catalytic amounts of 4-dimethylaminopyridine (DMAP),
the hydroperoxide may converted directly into the regioisomeric mixture of the
enone fatty acids 18 [79 b, 79 c]. Since the photooxidation occurs even in sun-
light, unsaturated fatty acids oxidized in the allylic position are available by a
route which is particularly favorable from both an economical and an ecological
viewpoint.
8.4
Enzymatic Reactions
8.4.1
Lipase-Catalyzed Transformations
Lipases and their applications have been reviewed in this journal in 1998 [92].
We will limit ourselves therefore to a few examples of selective syntheses of
fatty compounds.
8.4.1.1 Lipase-Catalyzed Syntheses of Monoglycerides and Diglycerides

Mono- and diglycerides (partial glycerides) are among the most important non-
ionic surfactants emerging from oleochemistry. They are used widely as emulsi-
fiers in the preparation of foodstuff. “Monoglycerides” are prepared on a large
scale by the glycerolysis of natural fats and oils in the presence of inorganic cat-
alysts and must be subsequently purified by molecular distillation [92]. Biocata-
lytic transformations offer a more gentle alternative to this process [4]. Consider-
able advances have been made here in the last few years but no breakthrough
could yet be considered economic.
Glycerol, protected as the isopropylidene [93] or phenylboroester [94, 95] deri-
vatives, can be converted into pure monoglycerides 68 with interesting surfac-
tant properties in the presence of a lipase from Rhizomucor miehei (lipozyme)
and free fatty acids as acyl donors (Scheme 8.26).
If glycerol is immobilized on silica gel, the esterification runs surprisingly
smoothly in aprotic solvents such as n-hexane or tert-butylmethyl ether in the
presence of different lipases and acyl donors (free fatty acids, fatty acid methyl
esters, vinyl esters, triglycerides, and so forth) [96–98]. Thus, for example, iso-
merically pure (> 98%) 1,3-sn-dilaurin 70 is readily obtained with vinyl laurate
69 in the presence of a “1,3-selective” lipase (Scheme 8.27) [96, 99]. Because of
their ready accessibility, up to the kilogram scale, and their high purity and sta-
bility, these 1,3-sn-diglycerides represent interesting building blocks for further
surfactant compounds (for example, by coupling with amino acids) and for the
preparation of reagents for lipid modification of natural products [100].
If the 1(3)-sn-monoglycerides 68 are prepared by this method, they must be
selectively separated from the reaction mixture, since they are very readily esteri-
fied by the lipases to 1,3-sn-diglycerides. This separation is achieved by exploit-
ing the poor solubility of the monoglycerides 68 at low temperatures in appro-
priate solvent mixtures [97, 98]. The method has proved to be exceptionally suit-
Scheme 8.26 Lipase-catalyzed synthesis of 1(3)-sn-monogly-

cerides 68 by acylation of glyceryl phenylborates 66 with fatty
acids to give 67 once the protecting group is cleaved [94, 95].
Scheme 8.27 Lipase-catalyzed synthesis of 1,3-sn-diglycerides,

such as 1,3-sn-dilaurin 70, by the vinyl lauroate (69) acylation
of glycerol immobilized on silica gel [96, 99].
able for the conversion of natural fats and oils from palm kernels, coconuts,
soybeans, sunflowers, and rapeseeds into the respective monoglyceride mix-
tures. Such highest-quality products are particularly well suited for uses in the
cosmetic and pharmaceutical industries.
8.4.1.2 Lipase-Catalyzed Syntheses of Carbohydrate Esters

Alkylpolyglucosides (APGs) and (polyethoxylated) sorbitan esters (Span, Tween)
– both directly or indirectly derivatives of glucose – are already used extensively
as nonionic surfactants or emulsifiers [5]. The lipase-catalyzed synthesis of car-
bohydrate esters has been recently reviewed [92].
It is particularly noteworthy, that in aprotic solvents such as tetrahydrofuran,
dioxan, monoglyme, or diglyme, monosaccharides such as d-glucose, d-galac-
tose, d-mannose, and d-fructose can be transformed regioselectively and in
high yields into the respective 6-O-acyl derivatives in the presence of the lipase
from Candida antarctica B (Novozym SP 435). Thus, for example 6-O-lauroyl-
d-glucose 72 was obtained directly from glucose 71 and lauric acid 49 d
(Scheme 8.28). The method may also be applied to the esterification of l-ascor-
bic acid and even, if only to a limited extent, to saccharose. The fatty acids
caprylic, capric, lauric, myristic, palmitic, stearic, oleic, 12-hydroxystearic, and
erucic acids have been used as acyl donors [101].
Scheme 8.28 Lipase-catalyzed selective esterification of glu-

cose 71 with lauric acid 49 d in aprotic solvents to give 6-O-
lauroyl-D-glucose 72 [101].
8.4.2
Microbial Transformations
8.4.2.1 Microbial Hydration of Unsaturated Fatty Acids

The chemical addition of water to unsaturated fatty compounds such as 1 a is
neither regioselective nor stereoselective [102]. In contrast, microbial hydration
is frequently both regio- and stereoselective.
Microbial water attachment to an unsaturated fatty acid was first reported by
Wallen et al. in 1962 [103]. The authors observed that a Pseudomonas species,
isolated from fat-containing materials, hydrated oleic acid 1 a to (R)-10-hydroxy-
stearic acid 73 in 14% yield (Scheme 8.29 a). This water attachment was also
observed with the bacterial genera Nocardia, Rhodococcus, Corynebacterium, and
Micrococcus [104–106]. Compound 73 was obtained in 45% yield with the yeast
Scheme 8.29 Microbial enantioselective and regioselective ad-

dition of water to a) oleic acid 1 a [103–106]; b) linoleic acid
4 a [108]; and c) linolenic acid 5 a [108]. to give the 10-hydroxy
fatty acids 73, 75, and 76; further, d) 73 can be enzymatically
dehydrogenated to 10-oxooctadecanoic acid 74 [107].
Saccharomyces cerevisiae [107]. The hydration product 73 can be oxidized to 10-

oxooctadecanoic acid 74 in a subsequent enzymatic dehydrogenation reaction
(Scheme 8.29 d) [107].
Microbes Lactobacillus plantarum and Nocardia cholesteriolicum assisted the for-
mation of (Z)-10-hydroxyoctadec-12-enoic acid 75 in 71% yield from linoleic acid
4 a (Scheme 8.29 b) [108]. a-Linolenic acid 5 a was converted into (12Z,15Z)-10-
hydroxyoctadeca-12,15-dienoic acid 76 in 77% yield (Scheme 8.29 c) [108]. The
hydratase was not active towards trans-unsaturated fatty acids, such as elaidic
acid (E)-1 a, and unsaturated fatty acids without a double bond in the 9 position,
such as erucic acid 3 a. Enzymatic hydration activity occurred less readily with a
greater number of double bonds in the substrate. Since hydratases from a num-
ber of bacteria and yeasts convert (9Z)-fatty acids into 10-hydroxy fatty acids, it
can be assumed that the C10 specificity is universal in nature [109].
In the future, it is expected that protein structure elucidation in algae, higher
plants, and marine lifeforms will advance and these enzymes will cloned in mi-
croorganisms so that biocatalysts will be available for synthetic use in ever
larger amounts [110].
8.4.2.2 Microbial x- and b-Oxidation of Fatty Acids

Microbial x-oxidation of fatty acids, which leads to dicarboxylic acids, is of great
interest [4]. Advances have been made here in recent years: Yi and Rehm [111]
were able to convert oleic acid 1 a into the corresponding unsaturated dicar-
boxylic acids 77 with yeast of the genus Candida tropicalis (Scheme 8.30 a). With
alkaline fermentation procedures, it was possible to increase the yields of 77
from 23 to 50% and also to oxidize solid fatty acids, such as palmitic acid, stea-
ric acid, and erucic acid, to the respective dicarboxylic acids [112 a]. In a batch
Scheme 8.30 Microbial x-oxidation of oleic acid 1 a to a) (Z)-

9-octadecendioic acid 77 with Candida tropicalis [111, 112 a]
and b) (Z)-3-hydroxy-9-octadecendioic acid 78 with the yeast
mutant Candida tropicalis M25 [114].
fermentation with 70 g L–1 palmitic acid, hexadecanedioic acid was obtained in

a yield of 36% and a concentration of 28.1 g L–1. In comparison with reported
yields [113], this value is among the highest values that have been obtained with
genetically unaltered microorganisms. This reaction is therefore not subject to
the gene technology regulations and is therefore of additional industrial interest.
The yields are significantly lower with linoleic acid 4 a and ricinoleic acid 6 a
[112 b].
Oleic acid 1 a can be transformed into the unsaturated hydroxy diacid 78 with
76% ee with the yeast mutant Candida tropicalis DSM 3152. The productivity of
the strain is improved by N-methyl-N'-nitro-N-nitrosoguanidine (NMG) muta-
genesis, and in a Fed batch fermentation with the mutant Candida tropicalis
M25 19.4 g L–1 78 were obtained from oleic acid (Scheme 8.30 b) [114]. Under
similar conditions, 6.1 g L–1 hydroxydiene diacid were obtained with 30 mL L–1
linoleic acid [115].
8.4.3
Microbial Conversion of Oils/Fats and Glucose into Glycolipids
The broad structural palette of biosurfactants, along with their numerous appli-
cations and biosynthetic pathways, were comprehensively reviewed by Kosaric
[116], Ratledge [117], Desai and Banat [118], Banat [119], and Lang and Wagner
[120]. Of particular interest, because of the remarkably high yield, 300–400 g L–
1
, is the sophorose lipid formation with Candida bombolica from glucose and ra-
peseed oil as substrates [121, 122]. Moreover, natural oils, fatty acid methyl es-
ters, and free fatty acids have been converted into glycolipids in the presence of
Ustilago maydis DSM 4500. In comparison with natural oils, the use of free fatty
acids has brought about an increase in yield to 30 g L–1 glycolipid containing 90
wt% of mannosyl erythritolipids [123].
8.5
Improvement in Natural Oils and Fats by Plant Breeding
In recent years, knowledge of the biochemical relationships of plant metabolism –

in particular, of the biosynthesis of the storage fats in commercial use – has in-
creased considerably [124]. Breeding has always been aimed mainly at an improve-
ment in the yield performance of useful plants. Efforts are also now being made to
meet the demands of industry for tailor-made oils and fats. The potential for fu-
ture growth in this area is mainly expected where suitability for chemical proces-
sing already exists due to the natural structure or purity of the vegetable raw ma-
terial. Possibilities for the genetic modification of oil plants exist primarily in re-
spect of the composition of the storage lipids, since, where chain length, number,
and position of double bonds and functional groups are concerned, nature has al-
ready generated an enormous variety of fatty acids. Even drastic variations in the
fatty acid pattern of seeds are tolerated by the plants and the seedlings.
8.5.1
Gene Technology as an Extension of the Methodological Repertoire
of Plant Breeding
Concurrent with the increasing demand for renewable raw materials, modern
biotechniques, including gene technology, have made enormous steps in extend-
ing the methodological repertoire of plant breeding, so that today’s breeding
procedures are even more efficient and selective. Although classical plant breed-
ing, when combined with experimental mutagenesis (“mutation breeding”) and
modern in vitro cell- and tissue-culture methods, has frequently proved to be
successful in oil plants such as, for example, soybean, rapeseed, sunflower, or
linseed [125, 126], gene technology offers an additional, universal approach for
changing the amount and composition of the stored oil [127, 128].
This development was recently made possible by a series of methodological
improvements. This is illustrated by continued progress both in natural vector-
transformation systems based upon natural infection by the soil-borne bacteri-
um Agrobacterium tumefaciens (or A. rhizogenes) and in a series of vector-free
transformation systems where, for example, the foreign recombinant gene is in-
tegrated into cells lacking a cell wall (protoplasts) or by bombardment of regen-
erable meristems with DNA-loaded particles [129, 130]. In molecular-biological
experiment, the necessary regulatory “gene switches” (promoters) as expression
signals are normally combined (cloned) with structural parts of previously iso-
lated genes (structural genes) to form a new functional unit, a chimeric gene
[131]. Moreover, selection markers, namely, genes which confer antibiotic or her-
bicide resistance and thus permit a selection of successfully transformed plant
cells, are also frequently inserted [129].
A widely used method for the genetic modification of useful plants, which
are used particularly for the prevention of the undesired expression of species-
specific genes, is the “antisense RNA” approach. Although the mode of action
in transgenic plants is not yet fully understood, the simplest explanation is that
the species-specific sense RNA binds to the transferred, complementary anti-
sense RNA and, thus, the translation and biosynthesis of the relevant protein is
inhibited. The resulting double-stranded RNA hybrid appears to be degraded
very rapidly by nucleic-acid digesting enzymes (the nucleases, in this case
RNAse H) [132]. The extent of the gene expression achieved is dependent to a
significant extent upon the transcriptionally active promoter used. Thus, in the
preparation of the gene constructs that are to be transferred, bacterial or viral
promoters are frequently used. Meanwhile, however, a series of seed-specific
promoter sequences such as those of napin, phaseolin, or oleosin genes have
also been successfully used [131, 133]. Since at present, almost exclusive reli-
ance is placed upon cell cultures or embryonic tissue in vitro for genetic trans-
formation, further improvements are also indispensable for the regeneration to
intact, transgenic plants; this has also been achieved [129].
8.5.2
New Oil Qualities by Oil Designed with Available Agricultural Varieties
New genetic variation is fundamental to every commercial breeding activity, that

is, selection is only successful if the characteristic to be changed varies in the
starting material. Because of the intensive breeding to which they have been
subject, the variability for new, desirable quality properties in agriculturally culti-
vated plants is, in practise, highly restricted. In contrast, a very rich reservoir of
genetic resources for industrially interesting raw material for fatty compound in
high purity is present in wild plants. These are, for example, medium to very
long chain fatty acids as well as fatty acids with unusual functionality resulting
from the number and position of double bonds or the presence of hydroxyl,
oxo, or epoxy groups [134, 135]. Plant breeding efforts to domesticate wild
plants such as species of the genera Cuphea, Calendula, Euphorbia, Vernonia, Les-
querella, Crambe, or Limnanthes, in order to develop useful plants that may be
more productive, are not lacking [136].
Where the genetic distance between the wild and the cultivated species is not
too large, it is possible, in principle, to transfer the desired, quality improving
property into the cultivated form by conventional methods of interspecific and
intergeneric hybridization or with the support of biotechniques (for example,
“embryo rescue”). Such breeding programs are, among others, very laborious,
since many adverse properties of the “gene donor”, such as the low yield cap-
ability, late ripening, or low shattering resistance of a wild species, is also trans-
ferred. These unfavorable properties must once more be eliminated with diffi-
culty through repeated backcrossing and subsequent selection (often even with-
out success). This clarifies why transferring a novel oil quality into a high yield-
ing, agronomically adapted plant species by conventional methods is indeed
possible, but is fraught with difficulty.
In this scenario, gene technology is well suited to accelerate breeding progress
or, in many cases, to make it even possible. In practice, this grants the ability to
implant a specific and desired quality property from distantly related plant species,
from microorganisms (such as bacterial or yeasts), or even from mammals with-
out detrimental effects to the genetic background or yield capabilities of the pro-
ductive species. Numerous genes (cDNA clones) exist for the biosynthesis of un-
usual fatty acids such as ricinoleic, petroselinic, linoleic, vernolic, or crepenynic
acids, which may be cloned and transformed in cultivated plants. With the help
of this material, it should become more possible to optimize genotypes (such as
species) for the production of oleochemical raw materials [127, 137–139].
8.5.3
Overview of Renewable Raw Materials Optimized by Breeding
A series of oil plants of world-wide significance is suitable for the production of

renewable raw materials, namely, for the extraction of oils and fats with a specif-
ic fatty acid composition. Thus, commercially exploited oil seeds such as soy-
bean (Glycine max), rapeseed (Brassica napus), sunflower (Helianthus annuus),
peanut (Arachis hypogaea), or linseed (Linum usitatissimum) now exhibit a con-
siderable variation in their fatty acid pattern, both in nature and as modified by
breeding (Table 8.1) [126, 140–143]. Where “nonfood” uses are concerned, genet-
ic engineering approaches can make a special contribution to the expansion in
the wealth of raw materials available to oleochemistry, such as increasing the
content of individual fatty acids or drastically changing the oil quality by the in-
troduction of a new fatty acid. Within this context, variants of important oil
seeds, which have become available by plant breeding with different methods,
will be discussed in the following on the basis of selected examples (Table 8.2).
8.5.3.1 Soybean
As a result of intensive quality breeding, the fatty acid pattern of the soya bean
is remarkably variable. In addition to the low linolenic acid varieties, which
should contribute considerably to the improvement in oxidative stability (mainly
in the food oil area), there are further varieties with modified proportions of in-
dividual saturated fatty acids (Table 8.2) [144–146]. “High oleic” (HO) soybeans
have been produced by routes based on genetic engineering. It has been esti-
mated that 40 000 ha of this variety was planted in the USA in 1998 [138, 147].
8.5.3.2 Rapeseed
For a number of reasons, the intensive, ongoing work inducing alterations in
the oil quality of cultivated plants is currently concentrated on rapeseed (B. na-
pus). Since both summer and winter forms of this species are available, they
can be planted as oil plant in climatically different regions of the world. A
further advantage of rapeseed over other cultivated species is in its accessibility
to biotechnological methods and, in particular, in its capability for transforma-
tion and regeneration [141, 148].
278
Table 8.1 Commercially available fatty acid variants of important oil seeds.
Type Variant Origin 12 : 0 a) 14 : 0 16 : 0 18 : 0 18 : 1 18 : 2 18 : 3 20 : 1 22 : 1 Others Source
soybean conventional – – 11 4 23 54 8 – – – [126]

rapeseed high erucic acid conventional – – 3 1 11 12 9 8 52 4 [126]
0 or 00 (canola) natural mutation – – 4 2 60 21 10 1 1 1 [126]
low linolenic acid mutagenesis – – 4 2 61 28 3 1 – 1 [140]
high lauric acid gene technology 37 4 3 1 33 12 7 – – 3 [141]
sunflower conventional – – 7 5 19 68 – – – 1 [126]
high oleic acid mutagenesis – – 3 4 83 10 – – – – [126]
(HO)
peanut conventional – – 12 4 47 31 – – – 6 [142]
high oleic acid natural mutation – – 6 2 81 3 – – – 8 [142]
(HO)
linseed conventional – – 6 4 18 14 58 – – – [126]
low linolenic acid mutagenesis – – 6 3 15 73 3 – – – [143]
(linola)
a) 12 : 0 = lauric acid, 14 : 0 = myristic acid, 16 : 0 = palmitic acid, 18 : 0 = stearic acid 60, 18 : 1 = oleic acid 1 a, 18 : 2 = linoleic acid 4 a,
18 : 3 = linolenic acid 5 a, 20 : 1 = eicosenoic acid, 22 : 1 = erucic acid 3 a.
8 New Syntheses with Oils and Fats as Renewable Raw Materials for the Chemical Industry
Table 8.2 Extreme fatty acid variants in breeding material from important oil seeds.
Type Variant Method 14 : 0 a) 16 : 0 18 : 0 18 : 1 18 : 2 18 : 3 20 : 1 22 : 1 Others Source
soybean low linolenic acid mutagenesis – 10.5 4.6 23.2 59.6 2.0 – – – [144]
low palmitic acid mutagenesis – 3.7 3.7 24.1 58.9 8.9 – – 0.7 [145]
high palmitic acid mutagenesis – 17.3 2.9 16.8 54.5 8.3 – – 0.2 [146]
high stearic acid mutagenesis – 8.4 28.1 19.8 35.5 6.6 – – 1.6 [146]
high oleic acid gene technology – 6.6 3.6 84.9 0.6 1.9 – – 2.4 [147]
(HO)
rapeseed high myristic acid gene technology 17.7 23.1 2.4 33.7 14.8 3.8 – – 4.5 [155]
high stearin gene technology – 4 29 15 19 22 1 – 10 b) [153]
high oleic acid mutagenesis – 4.2 2.2 80.2 4.5 5.2 1.8 – 1.9 [151]
(HO)
high oleic acid gene technology – 4.3 1.4 84.1 5.2 2.9 0.9 – 1.2 [133]
(HO)
low linolen gene technology – 3.8 1.5 68.5 22.1 1.2 1.1 – 1.8 [133]
sunflower high palmitin mutagenesis – 25.2 3.5 11.4 55.1 – – – 4.8 c) [158]
high stearin mutagenesis – 5.1 26.0 13.8 55.1 – – – – [158]
high oleic acid com- mutagenesis – 3.2 2.4 92.1 2.3 – – – – [144]
bined with low satu-
rated fatty acids
linseed high palmitin mutagenesis – 27.8 1.8 17.5 6.0 42.0 – – 4.8 d) [164]
a) 14 : 0 = myristic acid, 16 : 0 = palmitic acid, 18 : 0 = stearic acid 60, 18 : 1 = oleic acid 1 a, 18 : 2 = linoleic acid 4 a, 18 : 3 = linolenic acid
5 a, 20 : 1 = eicosenoic acid, 22 : 1 = erucic acid 3 a;
b) includes 6% arachidic acid (20 : 0), 2% behenic acid (22 : 0), 1% lignoceric acid (24 : 0);
c) includes 3.7% palmitoleic acid (16 : 1);
d) palmitoleic acid (16 : 1).
8.5 Improvement in Natural Oils and Fats by Plant Breeding
279
Rapeseed oil is very rich in erucic acid (3 a), a widely sought raw material for
many nonfood uses [149, 150]. In the context of improvement in nutritional oil
quality, the low erucic acid varieties – named zero and double zero or canola
types, which exhibit about 60% oleic acid (1 a) – were developed by classical
breeding methods. The breeding of linolenic acid deficient (< 3% 5 a) or high
oleic acid (> 80% 1 a) rapeseed forms has been achieved both by induced muta-
tion [151, 152] and genetically by inhibition of the inherent 12- or 15-desaturase
genes [133] (Table 8.2). Anti-sense inhibition of the desaturation step in Brassica
rapa (a close relative of B. napus) yields up to 40% stearic acid 60 in the seed oil
and has been already tested under field conditions [153]. In approaches which
promise success, special fatty acid variants, which previously could not be real-
ized in rapeseed, have been developed by gene technology. In this way, success
was obtained in establishing the synthesis of short- and medium-chain saturat-
ed fatty acids (chain lengths of 8–14 carbon atoms), which are of special interest
for oleochemistry and which could only be obtained previously from imported
tropical fats (coconut, palm kernels) [154, 155]. Most advanced is the develop-
ment of “high lauric acid rapeseed” with about 40–50% lauric acid by Calgene
(CA, USA), which depends on the transfer of a thioesterase gene from the Cali-
fornian bay (Umbellularia californica) and which has been already commercially
planted [141, 154].
There is a constant demand for high erucic acid rapeseed oil for industrial
use. Here, breeding is devoted to increasing the fraction of this very long chain
fatty acid well above the current maximum of 55–60% 3 a. It has been known
for a long time that, because of the nonoccupation of the middle of the three
triacylglycerol positions by erucic acid 3 a, a (theoretical) maximum of 67% can-
not be exceeded [149]. However, partial success was achieved recently when
transgenic rape forms were developed with varying contents of trierucin (trieru-
coylglycerol) in the seed oil by transfer of the gene for sn-2-acyltransferase (lyso-
phosphatidic acid acyltransferase, LPAAT) from different Limnanthes species
(meadowfoam) and by inhibition of the inherent LPAAT of rapeseed [141, 156].
8.5.3.3 Sunflower
In addition to the conventional sunflower oils, which exhibit a high content of
linoleic acid (4 a), HO types were developed experimentally some time ago by
mutagenesis [126, 157]. Furthermore, forms with increased proportions of satu-
rated fatty acids, which could provide advantages for margarine production (Ta-
ble 8.2), have been produced by mutagenic treatment [158]. However, the indus-
trial use of HO sunflower oil requires that the content of saturated fatty acids
should be as low as possible. Breeding has already reduced the content of stea-
ric acid 60 to 1.5%, which adversely affects the solidification temperature and
the cloud point. Under favorable climatic and cultivation conditions, a stable
proportion of 90% 1 a with a concurrently reduced content of stearic acid 60
could be achieved from current HO sunflower lines or hybrids developed in this
way [144]. In contrast for nonfood purposes in Germany, economic reasons de-
mand at least 83% oleic acid 1 a in the product in order to avoid additional
purification steps and thus increase the advantage to this production, against 1 a
derived from the competing raw material, beef tallow [159].
8.5.3.4 Peanut
In the case of the peanut, an HO mutant (breeding line F435 from the Univer-
sity of Florida) was found in the available varieties which was then used to
breed varieties which provide an oil with high oxidative stability [142, 160].
8.5.3.5 Linseed
If the possibilities for the use of linseed oil in the nonfood areas are considered, its
main uses are in the production of dyes, coatings, and linoleum [135]. On the
other hand, in the oleochemical area, the high reactivity of the polyene structure
of linseed fatty acids results in a pronounced sensitivity towards autoxidation of
products based on linseed oil as well as complex reaction pathways which lead
to poorly defined products [161]. From this point of view, breeding efforts are
being made to improve the variability of linseed oil in respect of its fatty acid
and triglyceride composition in order to provide new, specific oil qualities [162].
Here too, the classical approach of mutagenesis has also been used to breed
new linseed varieties with a linolenic acid 5 a content of less than 5% (linola qual-
ity) [143, 163] or with an increased palmitic acid content in the oil [164].
8.5.4
Concluding Remarks on the Use of Gene Technology
The preceding presentation clearly illustrates that gene technology is a very suit-
able breeding instrument in order to induce new genetic variation. In the ideal
situation, it allows the breeder to introduce totally new qualities into cultivated
plant varieties with greater precision without impairing the performance cap-
abilities of the respective genotype. There are certain barriers to the realization
and use of gene technology because of the poor acceptance by some end users;
this applies especially to the nutritional and feed area (novel food, novel feed).
However, since the novel products do not enter the human food chain directly,
it is not surprising that the initial applications of modern bio- and gene technol-
ogy methods are found in the technical and chemical area, where they provide
vegetable raw materials of improved quality and yield. In this way, important
but limited raw material resources can be saved for future generations. To what
extent these “new” plant types achieve practical relevance depends on economic
factors. Thus, from the viewpoint of industry, demand will only be generated if
new raw materials of vegetable origin are available in sufficient quantities at
competitive prices, economically viable isolation of the relevant components is
possible, and they are held in a higher esteem and preference to alternatives
which come, for example, from the petrochemical industry.
8.6
Future Prospects
After years of relative stagnation, the synthesis of novel fatty compounds based
on oils and fats has made important advances. With the breeding of new oil
plants – including the use of gene technology – numerous fatty compounds of
adequate purity are now available which makes them attractive for synthesis.
The use of modern synthetic methods together with enzymatic and microbiolog-
ical methods has lead to an extraordinary expansion in the potential for the syn-
thesis of novel fatty compounds, which are selectively modified in the alkyl
chain. These are now being investigated for their action, properties, and possi-
bilities for new applications.
However, numerous synthetic problems remain unsolved and solutions must
be found in the coming years. Chemists, biotechnologists, and plant breeders
are all challenged to continue development of the advances made in recent
years in an integrated, interdisciplinary approach and thus prepare the way for
oils and fats to be increasingly used as renewable raw materials in the chemical
industry.
Acknowledgments
We thank the Departments of Bildung und Forschung, and Ernährung, Land-

wirtschaft und Forsten of the German government, as well as the companies
Bayer AG, Henkel KGaA, Harburger Fettchemie, Brinckmann & Mergel GmbH,
Hoechst, BASF AG, Condea Chemie GmbH, Süd-Chemie AG, and Wella AG
for inspirational and material support of our work.
References
1 a) Konzept Nachhaltigkeit, vom Leitbild 3 Perspektiven nachwachsender Rohstoffe

zur Umsetzung, Concluding report of in der Chemie (Ed.: H. Eierdanz), VCH,
the Symposium “Schutz des Menschen Weinheim, 1996.
und der Umwelt – Ziele und Rahmenbe- 4 a) H. Baumann, M. Bühler, H. Fochem,
dingungen einer nachhaltig zukunftsver- F. Hirsinger, H. Zoebelein, J. Falbe, An-
träglichen Entwicklung” of the 13th Ger- gew. Chem. 1988, 100, 42–62; Angew.
man Parliament (Ed.: Deutscher Bundes- Chem. Int. Ed. Engl. 1988, 27, 41–62;
tag, Referat Öffentlichkeitsarbeit), Bonn, b) U. Biermann, J. O. Metzger, Topics in
1998; b) J. O. Metzger, M. Eissen, C. R. Catalysis, 2004, 38, 3675–3677; c) J. O.
Chimie, 2004, 7, 569–581; c) M. Eissen, Metzger, Chemosphere 2001, 43, 83–87;
J. O. Metzger, E. Schmidt, U. Schneide- d) U. Biermann, S. Fürmeier, J. O. Metz-
wind, Angew. Chem. 2002, 114, 402–425; ger, “New Chemistry of Oils and Fats”,
Angew. Chem. Int. Ed. 2002, 41, 414–436. Oleochemical Manufacture and Applica-
2 Nachwachsende Rohstoffe, Perspektiven tions (F. D. Gunstone, R. J. Hamilton),
für die Chemie (Eds.: M. Eggersdorfer, S. Sheffield Academic Press and CRC
Warwel, G. Wulff), VCH, Weinheim, 1993. Press, 2001, ISBN 1-84127-219-1, 266–
References 283
299; e) U. Biermann, W. Friedt, S. Lang, 1709; Angew. Chem. Int. Ed. Engl. 1991,
W. Lühs, G. Machmüller, J. O. Metzger, 30, 1638–1641.
M. Rüsch, gen. Klass, H. J. Schäfer, M. P. 16 M. Gerle, PhD thesis, RWTH Aachen
Schneider, Angew. Chem., 2000, 112, (Germany), 1997.
2292–2310; Angew. Chem. Int. Ed. 2000, 17 W. A. Herrmann, J. D. G. Correia, F. E.
39, 2206–2224; f) J. O. Metzger, R. Mah- Kühn, G. R. J. Artus, C. C. Romao, Chem.
ler, G. Francke, A. Hayen, “Synthesis of Eur. J. 1996, 2, 168–173.
New Oleochemicals: Functionalization of 18 R. Landau, G. A. Sullivan, D. Brown,
Fatty Compounds Using Radical Reac- CHEMTECH 1979, 602–607.
tions”, Recent Developments in the Synthe- 19 P. J. Martinez de la Cuesta, E. Martinez,
sis of Fatty Acid Derivatives L. M. Cotoluero Minguez, Grasas Aceites
(G. Knothe, J. T. P. Derksen), OCS Press, (Seville) 1985, 36, 181–185, and refer-
1999, ISBN 1-893997-00-6, 90–99; ences therein.
g) U. Biermann, J. O. Metzger, “Synthe- 20 A. Debal, G. Rafaralahitsimba, E. Uccia-
sis of New Oleochemicals: Products of ni, Fat Sci. Technol. 1993, 95, 236–239.
Friedel-Crafts Reactions of Unsaturated 21 a) F. Björkling, S. E. Godtfredsen, O.
Fatty Compounds”, Recent Developments Kirk, J. Chem. Soc. Chem. Commun.
in the Synthesis of Fatty Acid Derivatives 1990, 1302–1303; b) F. Björkling, H.
(G. Knothe, J. T. P. Derksen), AOCS Frykman, S. E. Godtfredsen, O. Kirk, Tet-
Press, 1999, ISBN 1-893997-00-6, 80–89. rahedron 1992, 48, 4587–4592; c) O. Kirk,
5 W. von Rybinski, K. Hill, Angew. Chem. F. Björkling, T. Damhus, S. E. Godtfred-
1998, 110, 1394–1412; Angew. Chem. Int. sen, Biocatalysis 1994, 11, 65–77.
Ed. 1998, 37, 1328–1345. 22 M. Rüsch gen. Klaas, S. Warwel, J. Mol.
6 K. E. Augustin, H. J. Schäfer, Liebigs Ann. Catal. A 1997, 117, 311–319.
Chem. 1991, 1037–1040. 23 S. Warwel, M. Rüsch gen. Klaas, J. Mol.
7 a) L. Hinkamp, PhD thesis, Universität Catal. B 1995, 1, 29–35.
Münster (Germany), 1993; b) H. J. Schä- 24 a) M. Rüsch gen. Klaas, S. Warwel, J.
fer, M. aus dem Kahmen, L. Hinkamp, Am. Oil Chem. Soc. 1996, 73, 1453–1457;
R. Maletz in 3. Symposium Nachwach- b) M. Rüsch gen. Klaas, S. Warwel, Ind.
sende Rohstoffe, Perspektiven für die Che- Crops Prod. 1999, 9, 125–132.
mie (Ed.: Bundesministerium für Ernäh- 25 J. V. Crivello, R. Narayan, Chem. Mater.
rung, Landwirtschaft und Forsten), Land- 1992, 4, 692–699.
wirtschaftsverlag, Münster, 1994, pp. 26 M. S. F. Lie Ken Jie, M. S. K. Syed-Rahma-
217–234. tullah, J. Am. Oil Chem. Soc. 1992, 69,
8 M. C. Kuo, C. T. Chou, Ind. Eng. Chem. 359–362.
Res. 1987, 26, 277–284. 27 a) J. O. Metzger, S. Fürmeier, Eur. J. Org.
9 W. Adam, J. Bialas, L. Hadjiarapoglou, Chem. 1999, 661–664; b) S. Fürmeier,
Chem. Ber. 1991, 124, 2377. J. O. Metzger, Eur. J. Org. Chem. 2003,
10 P. E. Sonnet, M. E. Lankin, G. P. Lankin, 649–659.
J. Am. Oil Chem. Soc. 1995, 72, 199–204. 28 M. S. F. Lie Ken Jie, Y. F. Zheng, Chem.
11 M. S. F. Lie Ken Jie, M. K. Pasha, Lipids Phys. Lipids 1988, 49, 167–178. For
1998, 33, 633–637. further heterocyclic fatty compounds see:
12 Y. Ishii, K. Yamawaki, T. Ura, H. Yama- a) S. Fürmeier, J. O. Metzger, Eur. J. Org.
da, T. Yoshida, M. Ogawa, J. Org. Chem. Chem. 2003, 885–893; b) S. Fürmeier,
1988, 53, 3587–3593. M. M. L. Lau, M. S. F. Lie Ken Jie, A. Lüt-
13 P. Bavaj, PhD thesis, RWTH Aachen zen; c) J. O. Metzger, Eur. J. Org. Chem.
(Germany), 1995. 2003, 4874–4878.
14 W. A. Herrmann, R. W. Fischer, M. U. 29 B. Dahlke, S. Hellbardt, M. Paetow,
Rauch, W. Scherer, J. Mol. Catal. 1994, W. H. Zech, J. Am. Oil Chem. Soc. 1995,
86, 245–266. 72, 349–353.
15 W. A. Herrmann, R. W. Fischer, D. W. 30 “Stets geforscht . . .Chemieforschung im
Matz, Angew. Chem. 1991, 103, 1706– Degussa-Forschungszentrum Wolfgang”:
M. Dankowski, G. Goor, G. Prescher,
Degussa AG company publication, Vol. 2, 43 a) B. Wolff, PhD thesis, RWTH Aachen

Frankfurt, 1988, p. 63. (Germany), 1994; b) S. Warwel, P. Bavaj,
31 T. M. Luong, H. Schriftmann, D. Swern, M. Rüsch gen. Klaas, B. Wolff in Perspek-
J. Am. Oil Chem. Soc. 1967, 44, 316–320. tiven nachwachsender Rohstoffe in der Che-
32 W. A. Herrmann, D. Marz, J. G. Kuchler, mie (Ed.: H. Eierdanz), VCH, Weinheim,
G. Weichselbaumer, R. W. Fischer, DE-A 1996, pp. 119–135.
3902357 A1, 1989; Chem. Abstr. 1991, 44 W. A. Herrmann, W. Wagner, U. N. Fless-
114, 143714. ner, U. Volkhardt, H. Komter, Angew.
33 S. Warwel, M. Rüsch gen. Klaas, M. Chem. 1991, 102, 1704–1706; Angew.
Sojka, Chem. Commun. 1991, 1578–1579. Chem. Int. Ed. Engl. 1991, 30, 1641–
34 K. B. Sharpless, H. Kolb, M. S. van Nieu- 1643.
wenhuze, Chem. Rev. 1994, 94, 2483– 45 M. aus dem Kahmen, H. J. Schäfer, Fett/
2547. Lipid 1998, 100, 227–235.
35 a) M. Plate, M. Overs, H. J. Schäfer, Syn- 46 M. aus dem Kahmen, PhD thesis, Uni-
thesis 1998, 1255–1258; Applications of versität Münster (Germany), 1993.
enantioenriched vic-dihydroxy fatty acids: 47 J. O. Metzger, K. F. Leisinger, Fat Sci.
M. Overs, M. Fix, S. Jacobi, C. Löbbe, Technol. 1988, 90, 1–5.
L. F. Chi, M. Sieber, H. J. Schäfer, H. 48 J. O. Metzger, U. Biermann, Fat Sci. Tech-
Fuchs, H.-J. Galla; Langmuir 2000, 16, nol. 1994, 96, 321–323.
1141–1148; M. Overs, F. Hoffmann, H. J. 49 E. M. Zobel, PhD thesis, Universität
Schäfer, H. Hühnerfuss; Langmuir 2000, Münster (Germany), 1997. C. Kalk, PhD-
16, 6995–6998; M. Fix, M. Sieber, M. thesis, Universität Münster (Germany)
Overs, H. J. Schäfer, H.-J. Galla, Physical 2001.
Chemistry Chemical Physics (Phys. Chem. 50 C. Kalk, Diploma thesis, Universität
Chem. Phys.) 2000, 2, 4515–4520; K. Münster (Germany), 1998.
Dreger, PhD-thesis, Universität Münster 51 “Carbon radicals”: Methoden Org. Chem.
(Germany) 2004; b) Jacobi, L. Chi, M. (Houben-Weyl), 4th ed. 1952–, Vol. E19a,
Plate, M. Overs, H. J. Schäfer, H. Fuchs, 1989.
Thin Solid Films 1998, 327–329, 180–184. 52 G. G. Melikyan, Synthesis 1993, 833–850.
36 T. Tachibana, T. Mori, K. Hori, Bull. 53 J. O. Metzger, U. Riedner, Fat Sci. Tech-
Chem. Soc. Jpn. 1980, 53, 1714–1719. nol. 1989, 91, 18–23.
37 G. Fayter in Perspektiven nachwachsender 54 J. O. Metzger, U. Linker, Fat Sci. Technol.
Rohstoffe in der Chemie (Ed.: H. Eier- 1991, 93, 244–249.
danz), VCH, Weinheim, 1996, pp. 107– 55 U. Linker, PhD thesis, Universität
118. Oldenburg (Germany), 1991.
38 a) M. Rüsch gen. Klaas, P. Bavaj, S. War- 56 J. O. Metzger, R. Mahler, Angew. Chem.
wel, Fat Sci. Technol. 1995, 97, 359–367; 1995, 107, 1012–1016; Angew. Chem. Int.
b) S. Warwel, M. Rüsch gen. Klaas, Lipid Ed. Engl. 1995, 34, 902–906.
Technol. 1997, 10–14. 57 J. O. Metzger, R. Mahler, G. Francke,
39 S. Warwel, M. Sojka, M. Rüsch gen. Liebigs Ann. 1997, 2303–2313.
Klaas, Top. Curr. Chem. 1993, 164, 79–89. 58 R. Mahler, PhD thesis, Universität
40 S. Warwel, M. Rüsch gen. Klaas, US-A Oldenburg (Germany), 1994.
5321158, 1994; Chem. Abstr. 1996, 125, 59 J. O. Metzger, R. Mahler, Liebigs Ann.
136578. 1993, 203–205.
41 K. M. Draths, J. W. Frost in Green Chemis- 60 N. O. Brace, J. Org. Chem. 1962, 27,
try, Frontiers in Benign Chemical Syntheses 4491–4498.
and Processes (Eds.: P. T. Anastas, T. C. 61 J. O. Metzger, U. Linker, Liebigs Ann.
Williamson), Oxford University Press, 1992, 209–216.
Oxford, 1998, pp. 150–165. 62 J. O. Metzger, R. Mahler, A. Schmidt,
42 K. Augustin, PhD thesis, Universität Liebigs Ann. 1996, 693–696.
Münster (Germany), 1991. C. Rüdiger, 63 J. Greiner, A. Manfredi, J. G. Riess, New
PhD-thesis, Universität Münster (Ger- J. Chem. 1989, 13, 247–254.
many) 1999. 64
References 285
D. V. Kinsman in Fatty Acids in Industry teriums für Ernährung, Landwirtschaft

(Eds.: R. W. Johnson, E. Fritz), Marcel und Forsten, 3. Symposium „Nachwach-
Dekker, New York, NY, 1989, pp. 233– sende Rohstoffe – Perspektiven für die
276. Chemie“, Landwirtschaftsverlag Münster,
65 J. Hartmanns, K. Klenke, J. O. Metzger, 1994, 217–234; d) M. Zobel, H. J. Schä-
Chem. Ber. 1986, 119, 488–499. fer, Tagungsband 5. Symposium Nachw.
66 J. O. Metzger, F. Bangert, Fat Sci. Tech- Rohstoffe 1997, Berlin, 186–190.
nol. 1995, 97, 7–9. 79 a) M. Zobel, Diploma thesis, Universität
67 a) H. G. Floss, Nat. Prod. Rep. 1997, 14, Münster (Germany), 1994; b) M. Zobel,
433–452; b) S. Handa, H. G. Floss, Chem. PhD thesis, Universität Münster (Ger-
Commun. 1997, 153–154. many), 1997; c) M. Zobel, H. J. Schäfer,
68 a) U. Biermann, J. O. Metzger, Fat Sci. Schriftenreihe „Nachwachsende Rohstoffe
Technol 1991, 93, 282–284; b) J. O. Metz- Bd. 12“, Chemische Nutzung heimischer
ger, U. Biermann, Synthesis 1992, 463– Pflanzenöle – Abschlusskolloquium des
465. BML, Landwirtschaftsverlag, Münster,
69 B. B. Snider, D. J. Rodini, T. C. Kirk, 1998, p. 75–110; d) H. J. Schäfer, M. Zo-
R. Cordova, J. Am. Chem. Soc. 1982, 104, bel in Recent Developments in the Synthe-
555–563. sis of Fatty Acid Derivatives (Eds.: G.
70 B. B. Snider, G. B. Phillips, J. Org. Chem. Knothe, J. T. P. Derksen), AOCS Press,
1983, 48, 464–469. Champaign, IL, 1999, pp. 59–79.
71 E. Blée, INFORM 1995, 6, 852–861. 80 a) M. Plate, PhD thesis, Universität
72 J. O. Metzger, U. Biermann in Chemische Münster (Germany), 1997; b) M. Plate,
Nutzung heimischer Pflanzenöle (Ed.: H. J. Schäfer in 5. Symposium Nachwach-
Fachagentur Nachwachsende Rohstoffe), sende Rohstoffe (Ed.: Fachagentur Nach-
Landwirtschaftsverlag, Münster, 1998, wachsende Rohstoffe), Landwirtschafts-
pp. 144–176, see p. 145. verlag Münster, 1997, pp. 195–198; c) M.
73 J. F. McLellan, R. M. Mortier, S. T. Orszu- Plate, H. J. Schäfer, M. aus dem Kahmen
lik, R. M. Paton, J. Am. Oil Chem. Soc. in Nachwachsende Rohstoffe, Band 12,
1994, 71, 231–232. Chemische Nutzung heimischer Pflan-
74 J. O. Metzger, U. Biermann, Liebigs Ann. zenöle, Landwirtschaftsverlag, Münster,
1996, 1851–1854. 1998, p. 50.
75 J. O. Metzger, U. Biermann, Bull. Soc. 81 a) R. Quermann, R. Maletz, H. J. Schä-
Chim. Belg. 1994, 103, 393–397. fer, Liebigs Ann. Chem. 1993, 1219–1223;
76 a) U. Biermann, J. O. Metzger, Fat Sci. b) R. Quermann, PhD thesis, Universität
Technol. 1992, 94, 329–332; b) J. O. Metz- Münster (Germany), 1991; c) C. Rüdiger,
ger, U. Biermann, Liebigs Ann. 1993, PhD-thesis, Universität Münster (Ger-
645–650; c) U. Biermann, A. Lützen, many) 1999.
M. S. F. Lie Ken Jie, J. O. Metzger, Eur. J. 82 a) H. J. Schäfer, Top. Curr. Chem. 1990,
Org. Chem. 2000, 3069–3973. For hydro- 152, 91–151; b) D. Degner, Top. Curr.
alkyl-additions to unsaturated fatty com- Chem. 1988, 148, 1–95, see p. 24.
pounds see: a) U. Biermann, J. O. Metz- 83 A. Weiper-Idelmann, M. aus dem Kah-
ger, J. Am. Chem. Soc. 2004, 126, 10319– men, H. J. Schäfer, M. Gockeln, Acta
10330; b) U. Biermann, J. O. Metzger, Chem. Scand. 1998, 52, 672–682.
Angew. Chem. 1999, 111, 3874–3876; An- 84 H. J. Schäfer, A. Weiper, M. aus dem
gew. Chem. Int. Ed. 1999, 38, 3675–3677. Kahmen, A. Matzeit in Nachwachsende
77 J. O. Metzger, U. Biermann, Fett/Lipid Rohstoffe (Eds.: M. Eggersdorfer, S. War-
1998, 100, 2–6. wel, G. Wulff), VCH, Weinheim, 1993,
78 a) R. Maletz, H. J. Schäfer, R. Quer- p. 97.
mann, Fett/Lipid 1996, 98, 370–379; b) 85 a) B. C. L. Weedon, Adv. Org. Chem. 1960,
R. Maletz, PhD thesis, Universität Müns- 1, 1–60; b) H. J. Schäfer, Chem. Phys.
ter (Germany), 1994. c) H. J. Schäfer, M. Lipids 1979, 24, 321–333.
aus dem Kahmen, L. Hinkamp, R. 86 a) A. Weiper, H. J. Schäfer, Angew. Chem.
Maletz, Schriftenreihe des Bundesminis- 1990, 102, 228; Angew. Chem. Int. Ed.
Engl. 1990, 29, 195–197; b) A. Matzeit, 96 M. Berger, M. P. Schneider, J. Am. Oil

M. Harenbrock, H. J. Schäfer, Liebigs Chem. Soc. 1992, 69, 961–965.
Ann. 1996, 55–62. 97 M. Berger, K. Laumen, M. P. Schneider,
87 a) J. A. Davies, P. L. Watson, J. F. Lieb- J. Am. Oil Chem. Soc. 1992, 69, 955–
man, A. Greenberg, Selective Hydrocarbon 960.
Activation, Principles and Progress, VCH, 98 C. Waldinger, M. P. Schneider, J. Am.
Weinheim, 1990; b) D. Mansuy, Pure Oil Chem. Soc. 1996, 73, 1513–1519.
Appl. Chem. 1990, 62, 741–766; c) O. Rei- 99 a) H. Berger, M. P. Schneider, Biotech-
ser, Angew. Chem. 1994, 106, 73–76; An- nol. Lett. 1991, 13, 333–338; b) P. Ville-
gew. Chem. Int. Ed. Engl. 1994, 33, 69–72; neuve, T. A. Foglia, INFORM 1997, 8,
d) H. J. Schäfer in Chemistry of Alkanes 640–650.
and Cycloalkanes (Eds.: S. Patai, Z. Rapo- 100 M. Berger, M. P. Schneider, Fat Sci.-
port), Wiley, New York, NY, 1992, pp. Technol. 1993, 95, 169–175.
781–808. 101 a) B. Haase, G. Machmüller, M. P.
88 a) M. Bühler, J. Schindler, in Biotechnol- Schneider in Biokonversion nachwach-
ogy, Vol. 6a (Eds.: J. Rehm, G. Reed), sender Rohstoffe (Ed.: Fachagentur Nach-
VCH, Weinheim, 1984, pp. 329–385; wachsende Rohstoffe), Landwirtschafts-
b) K. Kieslich, Microbial Transformations verlag, Münster, 1998, pp. 218–224;
of Non-Steroid Cyclic Compounds, Thieme, b) B. Haase, G. Machmüller, M. P.
Stuttgart, 1976. Schneider, DE-B 19 62 6943.1, 1996;
89 L. Hinkamp, H. J. Schäfer, B. Wippich, Chem. Abstr. 1998, 128, 127145t.
H. Luftmann, Liebigs Ann. Chem. 1992, 102 T. Lucas, PhD thesis, Universität
559–563. Münster (Germany), 1991.
90 N. C. Deno, W. E. Billups, R. Fishbein, 103 L. L. Wallen, R. G. Benedict, R. W. Jack-
C. Person, R. Whalen, J. Wyckoff, J. Am. son, Arch. Biochem. Biophys. 1962, 99,
Chem. Soc. 1971, 93, 438–440. 249–253.
91 a) E. Cramer, H. J. Schäfer, Fat Sci. Tech- 104 S. Koritala, L. Hosie, C. T. Hou, C. W.
nol. 1988, 90, 351–357; b) J. R. L. Smith, Hesseltine, M. O. Bagby, Appl. Micro-
R. O. C. Norman, A. G. Rowley, J. Chem. biol. Biotechnol. 1989, 32, 299–304.
Soc. Perkin Trans. 1 1973, 566–571; 105 C. W. Seo, Y. Yamada, N. Takada, H.
c) N. C. Deno, D. G. Pohl, J. Am. Chem. Okada, Agric. Biol. Chem. 1981, 45,
Soc. 1974, 96, 6680–6682. 2025–2030.
92 R. D. Schmid, R. Verger, Angew. Chem. 106 W. Blank, H. Takayanagi, T. Kido, F.
1998, 110, 1694–1720; Angew. Chem. Int. Meussdoerfer, N. Esaki, K. Soda, Agric.
Ed. 1998, 37, 1608–1633. Biol. Chem. 1991, 55, 2651–2652.
93 a) C. C. Akoh, Biotechnol. Lett. 1993, 15, 107 S. H. El-Sharkawy, W. Yang, L. Dostal,
949–954; b) S. Pecnik, Z. Knez, J. Am. J. P. N. Rosazza, Appl. Environ. Micro-
Oil Chem. Soc. 1992, 62, 261–265; c) Y. F. biol. 1992, 58, 2116–2122.
Wang, J. J. Lalonde, M. Momongan, D. E. 108 a) Y. Yamada, H. Uemura, H. Nakaya,
Bergbreiter, C. H. Wong, J. Am. Chem. K. Sakata, T. Takatori, M. Nagao, H.
Soc. 1988, 110, 7200–7205; d) W. A. Sza- Iwase, K. Iwadate, Biochem. Biophys.
rek, A. Zamojeski, K. N. Tiwari, E. R. Res. Commun. 1996, 226, 391–395;
Ison, Tetrahedron Lett. 1986, 27, 3827– b) S. Koritala, M. O. Bagby, J. Am. Oil
3830; e) V. Partali, A. G. Melbye, T. Alvik, Chem. Soc. 1992, 69, 575–578.
T. Anthonsen, Tetrahedron: Asymmetry 109 C. T. Hou in Advances in Applied Micro-
1992, 3, 65–72. biology, Vol. 41 (Eds.: S. L. Neidleman,
94 a) O. Papendorf, B. Steffen, S. Lang, A. I. Laskin), Academic Press, San Die-
F. Wagner, Chim. Oggi, 1995, 10, 17–20; go, 1995, pp. 1–23.
b) B. Steffen, A. Ziemann, S. Lang, 110 a) I. Gill, R. Valivety, Trends Biotechnol.
F. Wagner, Biotechnol. Lett. 1992, 14, 1997, 15, 470–478; b) A. Nuñez, G. St.
773–778. Armand, T. A. Foglia, G. J. Piazza, Bio-
95 B. Steffen, S. Lang, D. Hamann, P. technol. Appl. Biochem. 1997, 25, 75–80.
Schneider, H. K. Cammenga, F. Wagner, 111 Z.-H. Yi, H.-J. Rehm, Appl. Microbiol.
Fat Sci. Technol. 1995, 97, 132–136. Biotechnol. 1988, 30, 327–331.
References 287
112 a) D. Fabritius, Diploma thesis, Univer- C. Syldatk, Biotechnol. Lett. 1998, 20,
sität Münster (Germany), 1993; b) D. 1153–1156; e) U. Rau, S. Hammen, R.
Fabritius, PhD thesis, Universität Heckmann, V. Wray, S. Lang, Ind.
Münster (Germany), 1996. Crops Products 1999, in press; f) Y. Hu,
113 a) J. Schindler, F. Meussdorfer, H. G. L. K. Ju, Enzyme Microbial Technol.
Bühler, Forum-Biotechnol. 1990, 274– 2001, 29, 593–601; g) V. Guilmanov, A.
281; b) N. Uemura, A. Taoka, M. Taka- Ballistreri, G. Impallomeni, R. A.
gi, J. Am. Oil Chem. Soc. 1988, 65, Gross, Biotechnol. Bioeng. 2002, 77,
148–152. 489–494; h) D. A. Cavalero, D. G. Coop-
114 D. Fabritius, H. J. Schäfer, A. Steinbü- er, J. Biotechnol. 2003, 103, 31–41.
chel, Appl. Microbiol. Biotechnol. 1996, 123 S. Lang, U. Rau, D. Rasch, S. Spöck-
45, 432–438. ner, E. Vollbrecht in Biokonversion nach-
115 a) D. Fabritius, H. J. Schäfer, A. Stein- wachsender Rohstoffe (Ed.: Fachagentur
büchel, Appl. Microbiol. Biotechnol. Nachwachsende Rohstoffe), Land-
1996, 45, 432–438; b) D. Fabritius, H. J. wirtschaftsverlag, Münster, 1998, pp.
Schäfer, A. Steinbüchel, Appl. Microbiol. 154–164.
Biotechnol. 1998, 50, 573–578; 124 a) T. Voelker, A. J. Kinney, Annu. Rev.
c) D. Fabritius, H. J. Schäfer, Schriften- Plant Physiol. Plant Mol. Biol. 2001, 52,
reihe „Nachwachsende Rohstoffe Bd. 12“, 335–361; b) H. Drexler, P. Spieker-
Chemische Nutzung heimischer Pflan- mann, A. Meyer, F. Domergue, T.
zenöle – Abschlusskolloquium des Zank, P. Sperling, A. Abbadi, E. Heinz,
BML, Landwirtschaftsverlag Münster, J. Plant Physiol. 2003, 160, 779–802.
1998, p. 111–140. 125 A. Thierfelder, W. Lühs, W. Friedt, Ind.
116 N. Kosaric in Biotechnology, Vol. 6 Crops Prod. 1993, 1, 261–271.
(Eds.: H. J. Rehm, G. Reed, A. Pühler, 126 W. Lühs, W. Friedt in Designer Oil
P. Stadler), VCH, Weinheim, 1996, pp. Crops (Ed.: D. J. Murphy), VCH, Wein-
659–717. heim, 1994, pp. 5–71.
117 C. Ratledge in Biotechnology, Vol. 7 127 D. J. Murphy, Lipid Technol. 1994, 6(4),
(Eds.: H. J. Rehm, G. Reed, A. Pühler, 84–91.
P. Stadler), VCH, Weinheim, 1997, pp. 128 a) R. Töpfer, M. Martini, J. Schell,
133–197. Science 1995, 268, 681–686;
118 J. D. Desai, I. M. Banat, Microbiol. Mol. b) W. Friedt, W. Lühs in Perspektiven
Biol. Rev. 1997, 61, 47–64. nachwachsender Rohstoffe in der Chemie
119 I. M. Banat, Acta Biotechnol. 1995, 15, (Ed.: H. Eierdanz), VCH, Weinheim,
251–267. 1996, pp. 11–20; c) S. Weber, M. K.
120 a) S. Lang, F. Wagner, Fat Sci. Technol. Zarhloul, W. Friedt, Progress in Botany
1995, 97, 69–77; b) S. Lang, Curr. Opin. 2000, 62, 140–174; d) W. Friedt, W.
Colloid Interface Sci. 2002, 7, 12–20; c) Lühs in 7. Symp. Nachwachsende Roh-
S. Lang, In Novel Surfactants (Ed.: K. stoffe für die Chemie, Landwirtschafts-
Holmberg), Marcel Dekker, Inc., New verlag Münster, 2001, Schriftenreihe
York, Basel, 2003, pp. 279–315. Nachwachsende Rohstoffe, Bd. 18,
121 A. Albrecht, U. Rau, F. Wagner, Appl. S. 45–75; e) W. Lühs, M. K. Zarhloul,
Microbiol. Biotechnol. 1996, 46, 67–73. S. Weber, W. Friedt in 7. Symp. Nach-
122 a) A.-M. Davila, R. Marchal, J.-P. Van- wachsende Rohstoffe für die Chemie,
decasteele, Appl. Microbiol. Biotechnol. Landwirtschaftsverlag, Münster, 2001,
1992, 38, 6–11; b) Q. H. Zhou, N. Kosa- Schriftenreihe Nachwachsende Roh-
ric, J. Am. Oil Chem. Soc. 1995, 72, 67– stoffe, Bd. 18, S. 750–762; f) W. Friedt,
71; c) L. Fischer, A. Boger, S. Lang, C. W. Lühs, Biol. Unserer Zeit 1999, 29,
Manzke, S.-H. Park, U. Rau, A. Schlot- 142–150.
terbeck, F. Wagner in Perspektiven nach- 129 a) M. De Block, Euphytica 1993, 71,
wachsender Rohstoffe der Chemie (Ed.: 1–14; b) H. J. Fisk, A. M. Dandekar,
H. Eierdanz), VCH, Weinheim, 1996, Sci. Horti. (Amsterdam) 1993, 55,
pp. 250–254; d) H.-J. Daniel, M. Reuss, 5–36.
130 R. Luthra, Varsha, R. K. Dubey, A. K. New Crops (Ed.: J. Smartt, N. Haq),

Srivastava, S. Kumar, Euphytica 1997, International Centre for Underutilized
95, 269–294. Crops, Southampton University (UK),
131 a) J. C. Kridl, V. C. Knauf, G. A. Thomp- 1997.
son in Control of Plant Gene Expression 137 a) E. B. Cahoon, J. B. Ohlrogge, Plant
(Ed.: D. P. S. Verma), CRC, Boca Raton, Physiol. 1994, 104, 827–837; b) A. S.
FL, 1993, pp. 481–498; b) T. J. Guilfoyle Reddy, T. L. Thomas, Nat. Biotechnol.
in Genetic Engineering, Vol. 19 (Ed.: J. K. 1996, 14, 639–642; c) P. Broun, P. C.
Setlow), Plenum, New York, NY, 1997, Somerville, Plant Physiol. 1997, 113,
pp. 15–47. 933–942.
132 a) J. Green, O. Pines, M. Inouye, Annu. 138 A. J. Kinney, Fett/Lipid 1998, 100, 173–
Rev. Biochem. 1986, 55, 569–597; 176.
b) G. M. Fader, A. J. Kinney, W. D. Hitz, 139 M. Lee, M. Lenman, A. Banas, M. Ba-
INFORM 1995, 6, 167–169; c) B. Le- for, S. Singh, M. Schweizer, R. Nilsson,
win, Genes, 6th ed., Oxford University C. Liljenberg, A. Dahlqvist, P. O. Gum-
Press, Oxford, 1997. meson, S. Sjoedahl, A. Green, S.
133 a) W. D. Hitz, C. J. Mauvis, K. G. Ripp, Stymne, Science 1998, 280, 915–918.
R. J. Reiter, Rapeseed Today and Tomor- 140 R. Scarth, P. B. E. McVetty, S. R. Rim-
row, Vol. 2, Dorset, Dorchester, 1995, mer, Can. J. Plant Sci. 1997, 77, 125–
pp. 470–472 (Proc. 9th Int. Rapeseed 126.
Congr. (GCIRC)); b) W. D. Hitz, N. S. 141 W. Friedt, W. Lühs, Fett/Lipid 1998,
Yadav, R. J. Reiter, C. J. Mauvis, A. J. 100, 219–226.
Kinney in Plant Lipid Metabolism (Eds.: 142 J. B. Mugendi, C. A. Sims, D. W. Gor-
J.-C. Kader, P. Mazliak), Kluwer, Dor- bet, S. F. O’Keefe, J. Am. Oil Chem.
drecht, 1995, pp. 506–508. Soc. 1998, 75, 21–25.
134 a) T. P. Hilditch, P. N. Williams, The 143 J. C. P. Dribnenki, A. G. Green, G. N.
Chemical Constitution of Natural Fats, Atlin, Can. J. Plant Sci. 1996, 76, 329–
3rd ed., Chapman & Hall, London, 331.
1964; b) C. R. Smith, Jr. in Progress in 144 G. Cole, G. S. Coughlan, N. Frey, J. Ha-
the Chemistry of Fats and other Lipids, zebroek, C. Jennings, Fett/Lipid 1998,
Vol. 11 (Ed.: R. T. Holman), Pergamon, 100, 177–181.
Oxford, 1970, pp. 137–177; c) R. C. Ba- 145 X. Ndzana, W. R. Fehr, G. A. Welke,
dami, K. B. Patil, Progr. Lipid Res. 1981, E. G. Hammond, D. N. Duvick, S. R.
19, 119–153; d) B. G. Muuse, F. P. Cu- Cianzio, Crop Sci. 1994, 34, 646–649.
perus, J. T. P. Derksen, Ind. Crops Prod. 146 R. F. Wilson, INFORM 1993, 4, 193–
1992, 1, 57–65; e) H. K. Mangold, Fat 200.
Sci. Technol. 1994, 96, 23–27; f) K. Ait- 147 a) E. P. Heppard, A. J. Kinney, K. L.
zetmüller in Perspektiven nachwachsen- Stecca, G. H. Miao, Plant Physiol. 1996,
der Rohstoffe in der Chemie (Ed.: H. 110, 311–319; b) A. J. Kinney, S. Knowl-
Eierdanz), VCH, Weinheim, 1996, pp. ton in Genetic Modification in the Food
209–217; g) V. Spitzer, Fett/Lipid 1999, Industry (Eds.: S. Roller, S. Harlander),
101, 2–19. Blackie, London, 1998, pp. 193–213.
135 W. Lühs, W. Friedt in Designer Oil 148 a) D. J. Murphy, Trends Biotechnol.
Crops (Ed.: D. J. Murphy), VCH, Wein- 1996, 14, 206–213; b) G. B. Poulsen,
heim, 1994, pp. 73–130. Plant Breed. 1996, 115, 209–225;
136 a) F. Hirsinger in Oil Crops of the World c) B. Fitch Haumann, INFORM 1997,
(Eds.: G. Röbbelen, R. K. Downey, A. 8, 804–811.
Ashri), McGraw-Hill, New York, NY, 149 W. Lühs, W. Friedt, Fat Sci. Technol.
1989, pp 518–532; b) Progress in New 1994, 96, 137–146.
Crops, Proc. 3rd Nat. Symp. New 150 a) N. O. V. Sonntag, INFORM 1991, 2,
Crops (Ed.: J. Janick), ASHS Press, 449–463; b) N. O. V. Sonntag in Brassica
Alexandria, VA, 1996; c) Domestica- Oilseeds – Production and Utilization
tion, Production and Utilization of (Eds.: D. S. Kimber, D. I. McGregor),
References 289
CAB International, Wallingford, 1995, 157 a) K. I. Soldatov in Proc. 7th Int. Sun-
pp. 339–352; c) K. Coupland, K. B. T. flower Conf., Krasnodar, USSR, Intern.
Hatton in Synthesis in Lipid Chemistry Sunflower Assoc., 1976, pp. 352–357;
(Ed.: J. H. P. Tyman), The Royal Society b) W. Lühs, K. J. Dehmer, R. Berg-
of Chemistry, Cambridge, 1996, pp. mann, W. Friedt in Perspektiven nach-
57–66. wachsender Rohstoffe in der Chemie (Ed.:
151 D. L. Auld, M. K. Heikkinen, D. A. H. Eierdanz), VCH, Weinheim, 1996,
Erickson, J. L. Sernyk, J. E. Romero, pp. 232–238; c) J. M. Martínez-Rivas,
Crop Sci. 1992, 32, 657–662. P. Sperling, W. Lühs, E. Heinz, Mol.
152 B. Rücker, G. Röbbelen in Rapeseed To- Breed. 2001, 8, 159–168.
day and Tomorrow, Vol. 2, Dorset Press, 158 J. Osorio, J. Fernández-Martínez, M.
Dorchester, 1995, pp. 389–391 (Proc. Mancha, R. Garés, Crop Sci. 1995, 35,
9th Int. Rapeseed Congr. (GCIRC)). 739–742.
153 D. S. Knutzon, G. A. Thompson, S. E. 159 a) W. Lühs, W. Friedt, Anbauempfehlun-
Radke, W. B. Johnson, V. C. Knauf, J. C. gen für hochölsäurehaltige Sonnenblumen
Kridl, Proc. Natl. Acad. Sci. USA 1992, (HO-Sonnenblumen) in Deutschland,
89, 2624–2628. 3rd ed., Universität Giessen (Ger-
154 a) T. A. Voelker, A. C. Worrell, L. Ander- many), 1998; b) W. Lühs, W. Friedt in
son, J. Bleibaum, C. Fan, D. J. Haw- Proc. 6th Symp. Renewable Resources
kins, S. E. Radke, H. M. Davies, Science and 4th Eur. Symp. on Ind. Crops Prod.
1992, 257, 72–74; b) A. J. Del Vecchio, (Ed.: Fachagentur Nachwachsende Roh-
INFORM 1996, 7, 230–243; c) D. S. stoffe), Landwirtschaftsverlag Münster,
Knutzon, T. R. Hayes, A. Wyrick, H. 1999, pp. 401–406.
Xiong, H. M. Davies, T. A. Voelker, 160 a) A. J. Norden, D. W. Gorbet, D. A.
Plant Physiol. 1999, 120, 739–746. Knauft, C. T. Young, Peanut Sci. 1987,
155 C. Stoll, W. Lühs, M. K. Zarhloul, W. 14, 7–11; b) T. G. Isleib, C. T. Young,
Friedt, Eur. J. Lipid Sci. Technol. 2005, D. A. Knauft, Crop Sci. 1996, 36, 556–
107, 244–248. 558.
156 a) D. Weier, C. Hanke, A. Eickelkamp, 161 R. Höfer, W. Knörr, A. Westfechtel in
W. Lühs, J. Dettendorfer, E. Schaffert, 3. Symposium Nachwachsende Rohstoffe,
C. Möllers, W. Friedt, F. P. Wolter, M. Perspektiven für die Chemie (Ed.: Bun-
Frentzen, Fett/Lipid 1997, 99, 160–165; desministerium für Ernährung, Land-
b) M. Frentzen, Fett/Lipid 1998, 100, wirtschaft und Forsten), Land-
161–166; c) A. Gräfin zu Münster, W. wirtschaftsverlag, Münster, 1994, pp.
Lühs, D. S. Borchardt, F. P. Wolter, M. 235–256.
Frentzen in Advances in Plant Lipid Re- 162 a) C. Bickert, W. Lühs, W. Friedt, Ind.
search (Eds.: J. Sánchez, E. Cerdá-Ol- Crops Prod. 1994, 2, 229–237; C. Bick-
medo, E. Martinez-Force), Secretariado ert, W. Lühs, W. Friedt, Ind. Crops Prod.
de Publicaciones de la Universidad de 1994, 2, 317; b) R. Steiss, A. Schuster,
Sevilla (Spain), 1998, pp. 671–674; W. Friedt, Ind. Crops Prod. 1998, 7,
d) W. Lühs, A. Voss, J. Han, A. Gräfin 303–309.
zu Münster, D. Weier, F. P. Wolter, 163 a) A. G. Green, Can. J. Plant Sci. 1986,
M. Frentzen, W. Friedt in Genetics and 66, 499–503; b) A. G. Green, Theor.
Breeding for Crop Quality and Resistance Appl. Genet. 1986, 72, 654–661; c) G. G.
(Eds.: G. T. S. Mugnozza, E. Porceddu, Rowland, Can. J. Plant Sci. 1991, 71,
M. A. Pagnotta), Kluwer, Dordrecht, 393–396.
1999, pp. 323–330; e) J. Han, W. Lühs, 164 C. Ntiamoah, G. G. Rowland, D. C. Tay-
K. Sonntag, U. Zähringer, D. S. Bor- lor, Crop Sci. 1995, 35, 148–152.
chardt, F. P. Wolter, E. Heinz, M. Frent-
zen, Plant Mol. Biol. 2001, 46, 229–239.
291
9
Industrial Development and Application
of Biobased Oleochemicals
Karlheinz Hill
9.1
Introduction
Vegetable oils and fats are important constituents of human and animal food-
stuffs. Some grades are used industrially and, together with carbohydrates and
proteins, are numbered among renewable resources compared with fossil and
mineral raw materials, whose occurrence is limited and finite. For new products
price, performance, and product safety are equally important criteria and have a
correspondingly high importance at the start of product development. To ensure
high product safety for consumers and the environment renewable resources
have often been shown to have advantages compared with petrochemical raw
materials and can, therefore, be regarded as being the ideal raw material. Re-
sults from oleochemistry show that the use of vegetable fats and oils enables
the development of competitive, powerful products which are both consumer-
friendly and environmentally-friendly. Products from recent developments fit
this requirement profile.
In polymer applications derivatives of oils and fats, for example epoxides,
polyols, and dimerization products based on unsaturated fatty acids, are used as
plastic additives or components for composites or for polymers such as poly-
amides and polyurethanes. In the lubricant sector oleochemically-based fatty
acid esters have proved to be powerful alternatives to conventional mineral oil
products. For home and personal care applications a wide range of products, for
example surfactants, emulsifiers, emollients, and waxes based on vegetable oil
derivatives has proved to provide extraordinary performance benefits to the end-
customer. Selected products, for example the anionic surfactant fatty alcohol sul-
fate, have been investigated thoroughly with regard to their environmental im-
pact compared with petrochemical based products, by life-cycle-analysis. Other
product examples include carbohydrate-based surfactants and oleochemical
based emulsifiers, waxes, and emollients.
ISBN: 3-527-31027-4
292 9 Industrial Development and Application of Biobased Oleochemicals
9.2
The Raw Materials [1–3]
The sources of oils and fats are a variety of vegetable and animal raw materials
(e.g. tallow, lard), with the vegetable raw materials soybean, palm, rapeseed, and
sunflower oil being the most important in terms of the amounts involved
(Fig. 9.1). Of approximately 125 million tonnes of fats and oils produced world-
wide in 2003, by far the largest share was used in human foodstuffs. For oleo-
chemistry 17.4 million tonnes were available. The composition of the fatty acids
contained in the oil (fatty acid spectrum) determines the further use of the oils.
Special attention must be given to coconut oil and palm kernel oil (lauric oils)
because they contain a high proportion of fatty acids of short or medium chain
length (mainly 12 and 14 carbon atoms – C12, C14). For example, these are par-
ticularly suitable for further processing to surfactants, for washing and cleans-
ing agents, and to cosmetics. Palm, soybean, rapeseed, and sunflower, and ani-
mal fats such as tallow contain mainly long-chain fatty acids (C18, saturated and
unsaturated) and are used as raw materials for polymer applications, surfac-
tants, and lubricants. Figure 9.2 shows the composition of a typical lauric oil
(coconut oil) compared with that of sunflower oil.
Fats and oils are available in large amounts. In recent years the amounts pro-
duced have increased continuously by approximately 3% per year. Price develop-
ment – similar to crude oil – has not been constant, but has sometimes under-
gone drastic changes. In parallel with the price increases in crude oil in the
seventies caused by shortage of supply (oil crisis), price jumps in the fats and
oils sector have also occurred; this has been particularly noticeable for coconut
oil which in those days was the most important source of short-chain fatty
acids. In this market palm kernel oil, a second source of short-chain fatty acids,
Fig. 9.1 World production of oils and fats in 2003 (in million
tonnes) and main uses (source: Oil World).
9.3 Ecological Compatibility 293
Fig. 9.2 Composition of coconut and sunflower oil.
has had a stabilizing effect on raw material availability and price. In the medi-
um and long term it must be assumed that fats and oils will be offered at com-
petitive prices. The increasing demand will be met by the amounts produced,
which are also increasing [3, 4].
9.3
Ecological Compatibility
On the basis of on results from cycle analyses (see Chapter 9.4.3.1) and selected
ecological and toxicological studies one can assume that products based on
renewable resources are usually more ecologically compatible than petrochem-
ical-based substances – an important criterion in the development of a new
product, just as price and performance are [5]. This general assumption must
be proven for each new product, however, so ecological compatibility plays a de-
cisive role in all research and development projects. Basically, it covers two dif-
ferent aspects – remaining in the environment and the effects on the environ-
ment (Table 9.1). A variety of criteria are used to evaluate these two aspects. By
exposure analysis the expected environmental concentration of a particular sub-
stance (in wastewater, in exhaust gases, or in a sewage treatment plant) is esti-
mated, taking into consideration the amount of the substance produced and its
biodegradation behavior. The effect on the environment, e.g. toxicity to organ-
isms such as fish, algae, or microorganisms, is determined by a series of stan-
dardized testing methods. The two results are compared with each other. If the
expected environmental concentration of a particular substance is less than the
amount at which negative effects can no longer be determined then the product
Table 9.1 Evaluation of environmental compatibility of chemicals.
Method Result
Basic information
– Environmental fate Biodegradation tests
– Environmental effect Ecotoxicity tests
Criteria
– Environmental concentration Exposure analysis, PEC: Predicted environmental concentration
– Ecotoxicological concentration Biodegradation PNEC: Predicted no effect concentration
Analysis of effects
Evaluation Comparison PEC vs. PNEC PEC < PNEC: environmentally compatible
is ecologically compatible [6]. Apart from ecological investigations, toxicological

tests and microbiological and dermatological investigations are also carried out.
In the framework of a successful marketing strategy all data relevant to product
safety for the environment and the consumer are evaluated at each stage of
product development (selection of raw materials, development of test formula-
tions and application tests, process development, development of packaging,
testing consumer satisfaction in test markets).
9.4
Examples of Products
Oils and fats are triglycerides which typically consist of glycerin and saturated
and unsaturated fatty acids. There are a few exceptions to this rule, for example
castor oil, a glycerol triester of 12-hydroxyoleic acid (ricinoleic acid). Triglycer-
ides have two chemically reactive sites – the double bond in the unsaturated
fatty acid chain and the acid group of the fatty acid chain. In product develop-
ment based on triglycerides most derivatization reactions are performed out at
the carboxyl group (> 90%) whereas oleochemical reactions involving the alkyl
chain or double bond amount to less than 10% (Fig. 9.3).
For most other uses oils and fats must be split into the so-called oleochemical
base materials – fatty acid methyl esters, fatty acids, glycerol, and, as hydrogena-
tion products of the fatty acid methyl esters, fatty alcohols (Fig. 9.4) [1]. In the
Fig. 9.3 Reactive sites in triglycerides.

Fig. 9.4 Industrial processing of natural oils and fats and se-
lected product derivatives.
text below innovative products derived from glycerides, fatty acids, or fatty alco-
hols are discussed – oleochemicals for polymers, esters for lubricants, and sur-
factants, emulsifiers, and emollients for home and personal care applications.
9.4.1
Oleochemicals for Polymer Applications
The use of oleochemicals in polymers has a long tradition. One can differentiate
between their use as polymer materials, for example linseed oil and soybean oil
as drying oils, polymer stabilizers and additives, for example epoxidized soybean
oil as plasticizer, and building blocks for polymers, for example dicarboxylic
acids for polyesters or polyamides (Table 9.2) [7]. Considering the total market
for polymers of approximately 150 million tonnes in 1997 the share of oleo-
chemical based products is relatively small – or, in other terms, the potential for
these products is very high. Without doubt there is still a trend in the use of
naturally derived materials for polymer applications, especially in niche mar-
kets. As an example, the demand for linseed oil for production of linoleum has
increased from 10,000 tonnes in 1975 to 50,000 tonnes in 1998 (coming from
120,000 tonnes in 1960!) [8 a]. Epoxidized soybean oil (ESO) as a plastic additive
has a relatively stable market of approximately 100,000 tonnes/year [8 b].
A few years ago research was undertaken to use oleochemicals to build up
matrices for natural fiber reinforced plastics [9]. The use of natural fibers, for
example flax, hemp, sisal, and yuca is of increasing interest for a variety of ap-
plications, among them the automotive and public transportation industries, in
which the composites could be used in door pockets, covers, and instrument
panels, and for sound insulation [9 a]. Other applications could be in the manu-
Table 9.2 Oleochemicals for polymers – selected examples [7].
Product/use Source
Polymer materials
– polymerized soybean oil, castor oil Drying oils Soybean oil, castor oil
– polymerized linseed oil Linoleum Linseed oil
Polymer additives
– epoxides Stabilizers, plasticizers Soybean oil
– soaps (Ba/Cd, Ca/Zn) Stabilizers Stearic acid
– fatty acid esters, – amides, waxes Lubricants Rapeseed oil
Building blocks for polymers
– dicarboxylic acids Polyamides, polyesters, Tall oil, soybean oil, castor
– ether-/ester polyols alkyd resins, polyur- oil, sun-flower Oil, linseed
ethanes oil, oleic acid
facture of furniture. In this field Cognis was coordinating a research project,

funded by the Federal Ministry of Food, Agriculture and Forestry (BML) and the
National Agency for Renewable Resources (FNR) – project partners being the
German Aerospace Center (DLR) and Wilkhahn. The objective was the develop-
ment of a matrix-system with a high content of renewable raw materials (70–
75%) and comparable or better performance compared with purely petrochem-
ical based matrices. Various oleochemical based monomers, for example epoxi-
dized oils, maleinated oils, polyols and amidated fats were investigated and
tested, including manufacture of prototype products based on epoxidized oil
(Tribest) [9 b]. In the meantime Alstom has used this technology in the manu-
facture of urban public transport vehicles (Hamburger Hochbahn) [9 c].
Oleochemical-based dicarboxylic acids – azelaic, sebacic, and dimer acids
(Figs. 9.5 and 9.6) – amount to approximately 100,000 tonnes/year as compo-
Fig. 9.5 Building blocks for polymers based on natural oils.

Fig. 9.6 Dimerization of unsaturated fatty acids.
nents for polymers. This is approximately 0.5% of the total dicarboxylic acid
market for this application, in which phthalic and terephthalic acids represent
87%. The chemical nature of these oleochemical derived dicarboxylic acids can
alter or modify condensation polymers, and therefore will remain a special
niche market area. Some of these special properties are elasticity, flexibility, high
impact strength, hydrolytic stability, hydrophobicity, lower glass transition tem-
peratures, and flexibility [10]. The crucial reactions in the development of build-
ing blocks for polymers based on oils and fats are caustic oxidation, ozonolysis,
dimerization, (aut)oxidation, epoxidation, and epoxy ring opening. All of these
reactions except biooxidation [10 b] are performed on the double bond of an un-
saturated fatty acid or glyceride. Figure 9.5 summarizes the end products and
areas of application derived from these reactions. In the following text recent
developments in the field of diols and polyols for polyurethanes will be pre-
sented in more detail [11].
9.4.1.1 Dimerdiols Based on Dimer Acid [11, 12]

Dimerization of vegetable oleic acid or tall oil fatty acid (TOFA) yields dimer
acids, originally introduced in the 1950s by General Mills Chemicals and Emery
(both now Cognis Corporation). The reaction is very complex resulting in a mix-
ture of aliphatic branched and cyclic C36-diacids (dimer acid) as the main prod-
ucts, besides trimer acids and higher condensed polymer acids and a mixture of
isostearic acid and unreacted oleic and stearic acid. Hydrogenation of dimer
acid methyl ester or dimerization of oleyl alcohol leads to dimer alcohols (dimer
diols) (Fig. 9.6). Oligomers based on dimer diol are industrially manufactured
by acid-catalyzed dehydration of dimer diol. The reaction can be easily moni-
tored by determination of the amount of water produced. Oligomers in the
molecular weight range 1000–2000 are commercially available by this route
Table 9.3 Specificaitons of high molecular weight aliphatic diols [11].
Dimer diol a) Dimer diol a) 12-Hydroxystearyl 1,10-Decane diol

alcohol
Outer appearance Yellow liquid Colorless liquid White flakes White flakes
Hydroxyl value 180–200 180–210 345–360 625–645
Viscosity (25 8C, mPas) 3500–4300 1800–2800 Solid Solid
Melting Point (8C) – – 61–65 68–73
Composition
– monomer (%) 13 2 – –
– dimer (%) 68 >96 – >98
– trimer (%) 19 2 – –
Trademark Sovermol 650NS Sovermol 908 Sovermol 912 Sovermol 110
a) Molecular weight = 1000–2000 by oligomerization of dimer diol
(Table 9.3). Another method used to produce oligomers is the transesterification

of dimer diol with dimethyl carbonate. The resulting dimer diol polycarbonate
has a average molecular weight of 2000. Both types of oligomer, ethers and car-
bonates, are more chemically stable than dimer diol polyesters.
Because of their improved stability toward hydrolysis and oxidation dimer diol
polyethers (and dimer diol polycarbonates) are used as soft segments in the
preparation of thermoplastic polyurethanes. Polyurethanes prepared from these
oleochemical building blocks are very hydrophobic and have the expected stabili-
ty. The products were almost unaffected when stored either in 60% sulfuric acid
or 20% sodium hydroxide solution at 60 8C for 7 weeks. There was no signifi-
cant change in the weight of the testing sticks. For comparison, ester-based
polyurethanes used as standards were destroyed completely under these testing
conditions after one week (sulfuric acid) and two weeks (sodium hydroxide), re-
spectively. Soft segments based on dimer diol ethers are used to prepare saponi-
fication-resistant TPU-seals, which withstand contact with aggressive aqueous
media at elevated temperatures. A typical field of application is in nutrition
technology [13].
9.4.1.2 Polyols Based on Epoxides [13, 14]

Low-molecular-weight liquid epoxy polyol esters or ethers which can be used as
polyols for polyurethane systems are obtained by reaction of epoxidized oils
with low-molecular-weight mono- or polyfunctional alcohols or acids. Depend-
ing on the reaction conditions either polyols with high OH-functionality (com-
plete reaction) or epoxy polyol esters with remaining epoxy groups (partial con-
version) are obtained (Fig. 9.7). Although hydroxyl-functional triglyceride oils
have found applications in casting resins and adhesives the carboxyl groups of
the triglyceride backbone are not fully resistant to hydrolytic attack, particularly
by alkali. To overcome this specific behavior fatty acids are used as starting oleo-
Fig. 9.7 Oleochemical polyols for polyurethanes (brand name: Sovermol).
chemicals in the preparation of (epoxy) polyols. In principal three categories of

product are obtained by this route – fatty acid derivatives with reactive OH-
groups in the ester position only, e.g. glycerol monostearate, fatty acid deriva-
tives with reactive OH-groups in the ester position and in the hydrocarbon
chain, e.g. glycerol monoricinoleate, and fatty acid derivatives with reactive OH-
groups in the hydrocarbon chain only. These new polyols are of low molecular
weight and relatively low viscosity. They have outstanding hydrolytic stability
against both alkali and acids and very high chemical resistance toward corrosive
solvents such as super fuel. In addition they have significantly improved me-
chanical properties compared with hydroxyl functional oils after reaction with
aliphatic isocyanates [11].
Oleochemical polyols have an average molecular weight of 250 to 2500. Because
of their relatively low viscosity and compatibility with methylene di(phenylisocya-
nate) (MDI) they are particularly suitable for solvent-free, two pack, full solids
polyurethane systems, to be applied as thin decorative or protective coatings by
brush, roller or spraying. They can also be applied in thick coatings, bearing even
high filler loads. In industrial flooring applications, self leveling polyurethane or
epoxy/polyurethane multilayer systems have good chemical and mechanical prop-
erties and benefits such as minimum shrinkage, high mechanical strength and
durability, and favorable cost of installation. They are widely used for wear and
crack-resistant floorings on parking decks, for concrete protection in assembly
areas, and in large kitchens, slaughterhouses, and grocers, because of the ease
of cleaning. Oleochemical polyols can also be used to bind porous filler materials,
for example perlite, and rubber particles for applications of composites in con-
struction, soil protection, sport tracks, and playing fields [8, 11].
9.4.2
Biodegradable Fatty Acid Esters for Lubricants [15]
Apart from being used as “bio-diesel”, fatty acid esters, which are obtained from
fatty acids and alcohols, are becoming increasingly interesting as biodegradable
replacements for mineral oils. In some application areas, for example chain saw
oil, gearbox oils, hydraulic oils, and lubricants for crude oil production these
oleochemical products have already proved themselves.
Esters for lubricant applications are divided into five groups: monocarboxylic
acid esters (monoesters), dicarboxylic acid esters (diesters), glycerol esters, polyol
esters, and complex esters.
Monoesters are obtained by reacting carboxylic acids with alkyl chain lengths
from C8 to C22 and branched or linear alcohols. Typical diesters are obtained,
for example, from adipic, sebacic, azelaic, or dimer fatty acids by reaction with
butanol, ethyl hexanol, isodecanol, isotridecanol, or Guerbet alcohol (see Chap-
ter 9.4.4.3). Using renewable resources as a base, sebacic acid is obtained from
castor oil by oxidation with lead(II) oxide as catalyst. Azelaic acid and dimer
fatty acids are obtained from oleic acid by technical processes – the first by
ozone cleavage, with pelargonic acid being formed as a coupling product, the
latter by thermal dimerization (see also previous section). Although the diesters
already have excellent lubricating properties, their thermal stability is surpassed
by the polyol esters. These products are based on polyols with a quaternary car-
bon atom – for this reason glycerol esters form a separate class of products
(Fig. 9.8). Complex esters are formed by esterification of polyols with mixtures
of mono, di, and tricarboxylic acids and are oligomer mixtures; from a technical
application viewpoint they are characterized by their high shear stability [15 a].
The decisive factor is that the specially designed fatty acid esters which are
used as replacements for mineral oil products not only have ecologically com-
patible properties but also comparable or even better performance than that of
conventional products. That this is possible can be demonstrated very clearly by
an example from crude oil production. In coastal drilling (e.g. in the North Sea)
the demands placed on the lubricants (drilling fluids) are particularly high. The
drilling fluid is pumped to the surface together with the drill cuttings and after
coarse separation disposed of directly into the sea. In addition to the good lubri-
cating effect the biodegradability assumes particular importance in this applica-
tion. A specially developed fatty acid ester (Petrofree) not only fulfils the re-
Fig. 9.8 Polyols used for the manufacture of complex esters.

Table 9.4 European potential market for biodegradable lubricants (1000 tonnes year–1) a).
Application Total Biodegradable

lubricants
Automotive oils 2305 250

Hydraulic oils 750 200
Turbine oils 200 20
Compressor oils 65 25
Industrial gear oils 200 10
Metal working oils 500 10
Demolding oils 110 110
Chainsaw oils 60 60
Process oils 600 200
Lubricating greases 100 100
a) According to Reference [15b].
quirement of biodegradability but also has a better lubricating effect than prod-
ucts based on mineral oils [16].
Current developments include the use of specially designed fatty acid esters
in a wide range of applications as biodegradable lubricants, and environmentally
friendly alternatives are available for almost all mineral oil-based products. In
Europe, the long-term potential is estimated to be 10–20% of the total market
(500,000–1,000,000 tonnes/year; Table 9.4) [15 c]. In 1997 40,000 tonnes of biode-
gradable lubricants were sold in Germany alone (4.5% of the total market)
[15 c]. An increase of this share is the objective of a variety of measures taken
by government and other authorities. The success of these efforts will finally
also depend on statutory regulations which should govern the use of environ-
mentally-friendly products [17].
9.4.3
Surfactants and Emulsifiers Derived from Vegetable Oil
The basic manner in which surfactants act is determined by their structure.

With their hydrophilic head and hydrophobic tail, surfactant molecules inter-
pose themselves between water and water-insoluble substances. By enriching
themselves at the boundaries which water forms with air or oil they lower its
surface tension; as ingredients in soaps and washing agents they make contact
with soiled material in this way. When dissolved in water at high concentrations
these molecules group themselves together to form spherical structures (mi-
celles); their inwards-pointing hydrophobic groups surround soil particles and
keep these in solution. Surfactants are usually classified as anionic, cationic,
nonionic, or amphoteric, depending on the type and charge of the hydrophilic
groups (Fig. 9.9) [18 a].
Fig. 9.9 Production of surfactants and examples of products.
Surfactants are used in a wide range of fields. By far the most important
fields of application are the washing and cleansing sectors and in textile treat-
ment and cosmetics; these use more than 50% of the total amount of surfac-
tants. Surfactants are also used in the food sector, in crop protection, in mining,
and in the production of paints, coatings, inks, and adhesives. The basic manu-
facturing routes to important surfactants are listed out in Fig. 9.9. It is true that
the most important surfactant on the basis of the amount produced, apart from
soap, is still the petrochemical-based alkyl benzene sulfonate; in recent years,
however, a continuous trend toward surfactants based on renewable resources
has become apparent. The total worldwide market amounts to approximately
19.2 million tonnes (2000, including soap). The amounts involved, broken down
Table 9.5 Global surfactant consumption in 2000 a).
Surfactant class 1000 tonnes
Soap 8800
Anionic surfactants
– Alkyl benzene sulfonates 3400
– Fatty alcohol ether sulfates 1000
– Fatty alcohol sulfate 500
Nonionic surfactants
– Alcohol ethoxylates 800
– Alkylphenol ethoxylates 700
Cationic surfactants 810
Amphoteric surfactants 180
Others (including carbohydrate-based surfactants) 2900
a) Soap, Perf., Cosmet., November 2000, p. 51; Colin A. Hous-

ton and Associates
Fig. 9.10 Worldwide surfactant market.
into the individual surfactant classes, are summarized in Table 9.5; Fig. 9.10
shows the regional distribution [18 b].
9.4.3.1 Fatty Alcohol Sulfate (FAS) [5 a, b]

Fatty alcohol sulfate has been known for a long time and has already been used
as surfactant in a variety of products, for example detergents. It is produced
directly from fatty alcohol by reaction with sulfur trioxide (SO3) gas (1–8% v/v
in air or nitrogen) in a falling-film reactor. The crude product is then neutral-
ized with aqueous sodium hydroxide, using a buffer if necessary (Fig. 9.11).
Some forms of the product, for example aqueous solutions or granulates, are
typically produced according to the requirements of the market. Fatty alcohol
sulfates are readily biodegradable (no metabolites from the degradation) under
both aerobic and anaerobic conditions.
Because FAS can be produced either from vegetable oil-based or petrochem-
ical-based fatty alcohol (Fig. 9.9), both types have been evaluated in a life-cycle
analysis with a positive overall result for the natural product. For vegetable
Fig. 9.11 Synthesis of fatty alcohol sulfate (FAS).

based fatty alcohol sulfate the analysis starts with harvesting of the oil fruits
(palm kernels or coconuts) and their processing to isolate the desired plant oil.
Subsequent transesterification and hydrogenation of the methyl ester intermedi-
ates leads to the fatty alcohols, which are finally sulfated to produce the desired
product. On the basis of on this analysis the environmental impact of vegetable
oil-based fatty alcohol sulfate compared with the petrochemical based product
is:
· 70% less use of fossil resources
· 50% less emission to the atmosphere
· 15% less waste
· 50% more emission to water (low toxic waste water from small, decentralized
oil plants)
9.4.3.2 Acylated Proteins and Amino Acids (Protein–Fatty Acid Condensates) [19]
In the development of protein–fatty acid condensates it was possible to combine
the renewable resources fatty acids (from vegetable oil) and protein, which can
be obtained both from animal waste (leather) and from many plants, to con-
struct a surfactant structure with a hydrophobic (fatty acid) and a hydrophilic
(protein) part (Fig. 9.12). This was achieved by reacting protein hydrolyzate with
fatty acid chloride under Schotten–Baumann conditions using water as solvent.
The products obtained have an excellent skin compatibility and also a good
cleaning effect (particularly on the skin) and, in combination with other surfac-
tants, lead to an increase in performance. For instance, even small additions of
the acylated protein hydrolyzate improve the skin compatibility of other prod-
ucts. This protective effect could be because of the amphoteric nature of the
product. There is an interaction between the protein–fatty acid condensate and
skin collagen. This could lead to the formation of a protective layer, which re-
duces excessive attack of surfactants on the upper layers of the skin, their
strong degreasing effect, and the direct interaction of anionic surfactants with
the skin. Comparable reaction conditions have been used to develop acylated
amino acids, and acyl glutamates, in particular, have found broad uses in recent
years (Fig. 9.13).
In the personal care market, fatty acid derivatives of proteins and amino acids
(glutamic acid) are mainly used in mild shower and bath products, mild sham-
poos, surfactant-based face cleansers, cold-wave preparations and fixatives, baby
wash formulations, and special emulsifiers for “leave-on” products.
Fig. 9.12 Structure of protein hydrolyzate fatty acid condensates.

Fig. 9.13 Synthesis of acyl glutamate.
9.4.3.3 Carbohydrate-based Surfactants – Alkyl Polyglycosides [20]

The development of surfactants based on carbohydrates and oils is the result of
a product concept which is based on the exclusive use of renewable resources.
In industry saccharose, glucose, and sorbitol, which are available in large
amounts and at attractive prices, are used as the preferred carbohydrate raw ma-
terials.
The selective functionalization of saccharose and sorbitol with fatty acids for
construction of a perfect amphiphilic structure cannot be realized by simple
technical processes, because of the polyfunctionality of the molecule. This is
why the products offered on the market contain different amounts of mono-,
di-, and tri-esters and are, therefore, only suitable for particular applications,
e.g. as emulsifiers for foodstuffs and cosmetics or, for the sorbitan esters, also
in technical branches such as explosives and in emulsion polymerization.
The ideal raw material for selective derivatization is glucose. Reaction with
fatty alcohol produces alkyl glucosides; N-methylglucamides are prepared by re-
ductive amination with methylamine and subsequent acylation. Both products
have proved to be highly effective surfactants in washing and cleansing agents.
The alkyl glucosides have also established themselves in the cosmetic products
sector, as auxiliaries in crop protection formulations, and as surfactants in in-
dustrial cleansing agents. On the basis of yearly production they can already be
regarded as the most important sugar surfactants.
Alkyl polyglycosides have been known for a long time but only in the 1990s,
after several years’ research work, reaction conditions were developed, which en-
abled manufacture on a commercial scale. The structure on which these com-
pounds are based corresponds exactly to the surfactant model described above.
The hydrophobic (or lipophilic) hydrocarbon chain is formed by a fatty alcohol
(dodecanol/tetradecanol) obtained from palm kernel oil or coconut oil. The hy-
drophilic part of the molecule is based on glucose (dextrose) obtained from
starch (Fig. 9.14).
Fig. 9.14 Synthesis of alkyl polyglycosides.
The chemical challenge to process technology was to find reaction conditions

which enabled the fatty alcohol to react directly with glucose on a commercial
scale and at acceptable cost. To develop a method that was as environmentally
friendly as possible the use of solvents was rejected. Method development was
successfully completed and Cognis was the first company to offer alkyl polygly-
cosides on an industrial scale of the required quality. Currently Cognis has an
annual capacity of approx. 50,000 tonnes available for manufacture of this class
of compound (other manufacturers include Kao, Seppic, ICI, and LG). By com-
bining vegetable oil and sugar as raw materials it has for the first time become
possible to offer commercially important amounts of nonionic surfactants
which are completely based on renewable resources (Fig. 9.15).
Fig. 9.15 Manufacturing processes for alkyl polyglycosides.

Unique properties had previously been observed for alkyl polyglycosides, par-
ticularly in combination with other surfactants. For example, use of alkyl poly-
glycosides in light-duty detergent or shampoo formulation means that the total
amount of surfactant can be reduced without sacrificing performance. In other
combinations a particularly stable and fine foam can be produced which pro-
tects sensitive textiles during the washing process. Toxicological and ecological
laboratory investigations have also produced favorable results. Alkyl polyglyco-
sides have a good compatibility with the eyes, skin, and mucous membranes
and even reduce the irritant effects of surfactant combinations. On top of this
they are completely biodegradable, both aerobically and anaerobically. The rela-
tively favorable classification (for surfactants) as class I under the German water
hazard classification (WGK I) is a consequence of this.
9.4.3.4 Alkyl Polyglycoside Carboxylate [21]

Alkyl polyglucoside carboxylate (INCI name sodium lauryl glucose carboxylate
(and) lauryl glucoside, Plantapon LGC Sorb), is a new anionic surfactant with
an excellent performance in personal care cleansing applications. In shampoo
and shower bath formulations the anionic surfactant has good foaming behav-
ior. In body wash applications it improves sensorial effects. These properties
make Plantapon LGC Sorb suitable for several cosmetic applications, e.g. mild
facial wash gel, mild baby shampoo, mild body wash for sensitive skin, wet
wipes, and special sulfate-free shampoo applications.
A new industrial process based on reaction of sodium monochloroacetate
with aqueous alkyl polyglycoside (without additional solvents) enables economic-
al and ecologically favorable manufacture of this product (Fig. 9.16).
9.4.3.5 Polyol Esters [22]
Polyglycerol Esters Emulsifiers based on glycerol or polyglycerol are a class of

product well known commercially and used particularly in products for personal
care and in food production. Further development will focus on the design and
Fig. 9.16 Synthesis of alkyl polyglycoside carboxylate.

Table 9.6 Properties of selected polyglycerol esters as emulsifiers for personal

care and food technology.
INCI name Trade name Properties
Polyglyceryl-3-diisostearate Lameform TGI W/O – emulsifier

Polyglyceryl-2-dipolyhydroxystearate Dehymuls PGPH MW = 725; pale yellow liquid
PEG-4-polyglyceryl-2-stearate Lamecreme DGE 18 W/O – emulsifier for lotions and creams
Pentaerythritol distearate Cutina PES MW >3000; yellow, cloudy, viscous
Polyglycerol stearate (E 475) Polymuls 4G (ca. 15 000 mPas, Brookfield, 23 8C)
Polyglycerol polyricinoleate (E 476) Polymuls PGPR O/W – emulsifier
Consistency wax with sensorial benefits
Food – emulsifier
Food – emulsifier
optimization of specific emulsifier formulations. The combination of different

types of emulsifier can lead to new uses for mono- and diglycerides. Polyglycer-
ol esters are obtained by esterification of polyglycerol, which is produced by oli-
gomerization of glycerol under basic conditions, with fatty acids. The properties
of the different products can be adjusted by selection of the type of polyglycerol
used and the chain lengths and chain type of the fatty acids used. Selected types
of product of different molecular weight are shown as an example in Table 9.6.
For example, Dehymuls PGPH is recommended for use in body lotions. It
leaves the skin with a smooth, non greasy, and well cared for feeling, spreads
easily, and is absorbed quickly.
Pentaerythritol Esters As for glycerol esters, these esters are produced by esteri-
fication of pentaerythritol with the desired fatty acids. For example, under de-
fined reaction conditions and use of stearic acid at a defined concentration, pen-
taerythritol distearate has been recently developed as an off-white wax with a
very weak odor (Cutina PES). This type of product is offered as co-emulsifier
and consistency factor for cosmetic products with high sensorial elegance and
can be applied in a variety of formulations (Fig. 9.17).
Emulsifier compound based on polyglycerol ester and alkyl polyglycoside Re-

quirements for modern emulsifiers not only include outstanding performance
Fig. 9.17 Chemical structure of pentaerythritol distearate (main component in Cutina PES).
Fig. 9.18 Capacity increase in cold emulsification processes

using Eumulgin VL 75 as emulsifier.
but also compatibility with modern emulsification techniques and balanced sen-
sory feeling. One product which fulfils these requirements is a compound based
on glycerin, alkyl polyglycoside and polyglyceryl-2-dipolyhydroxystearate (Eumul-
gin VL 75). In combination with selected emollients it enables the preparation
of O/W emulsions of high quality and stability (small droplet sizes). In addition,
because of the liquid appearance of the product, and, as a consequence, the pos-
sibility of cold processing, manufacturing time and cost of preparation of emul-
sions are significantly reduced (Fig. 9.18).
9.4.3.6 Multifunctional Care Additives for Skin and Hair [23]

Intelligent combination of alkyl polyglycoside and glyceryl oleate resulted in a
new product which combines emulsifying and cleansing properties with out-
standing care effects, such as enhancing of the skin lipid layer. This effect is
proven in a standardized test by washing the forearm, rinsing, drying, and ex-
tracting the lipid layer on the skin with ethanol pads. The lipid content is mea-
sured by quantitative analysis of glycerol oleate in the extract (Fig. 9.19). Techni-
cal data for the product (Lamesoft PO 65) are summarized in Table 9.7.
For application in hair-care products it was found that a compound based on
dioctyl ether from coconut or palm kernel oil-based octanol (Cetiol LDO) en-
ables the formulation to be silicone oil-free. Cetiol LDO is a highly efficient
hair-care additive and is particularly suitable for the use in hair-cleansing prep-
arations to improve the tactile hair feel and hair gloss. Cetiol LDO, in combina-
Table 9.7 Technical data for Lamesoft PO 65.
Application Multifunctional care additive for clear and pearlescent cleans-

ing preparations
Composition Glyceryl Oleate (lipid), Coco Glucoside (surfactant)
Dry residue 65–70%
Lipid ca. 30%
pH value (5%) 3.0–3.5
Viscosity (Brookfield, 23 8C) max. 12 000 mPas
Fig. 9.19 Evaluation of lipid layer enhancing effect.
tion with wax esters and cationic polymers also has the benefits of silicon-free
body-cleansing preparations with regard to improved sensorial skin properties.
9.4.4
Emollients [24]
The physicochemical nature of the oil-phase components in a cosmetic emul-
sion, the emollients, determines their skin-care effects, such as smoothing,
spreading, sensorial appearance. Test methods have been developed to character-
ize and classify the numerous emollients available on the market, for example
Table 9.8 List of selected emollients.
Structure Emollient INCI name
Ester Cetiol A Hexyl laurate

Cetiol LC Coco-caprylate
Eutanol G16 S Hexyldecyl stearate
Cetiol V Decyl oleate
Cetiol J 600 Oleyl erucate
Myritol 318 Caprylic/capric triglyceride
Guerbet Alcohols Eutanol G 16 Hexyl decanol
Eutanol G Octyl dodecanol
Hydrocarbons Cetiol S Diethylhexyl-cyclohexane
Ethers Cetiol OE Dicaprylyl ether
Carbonates Cetiol CC Dicaprylyl carbonate
silicones, paraffins, and oleochemical-based products. The latter include glycer-

ides, esters, alcohols, ethers, and carbonates with tailor-made structures depend-
ing on the performance needed (Table 9.8). With regard to additional effects,
however, there is still a demand for new products with unique performance
properties.
9.4.4.2 Dialkyl Carbonate

One example of a new class of compound in this field is dioctyl carbonate. The
product is synthesized by transesterification of octanol and dimethyl carbonate
in the presence of alkali catalyst (Fig. 9.20). Dioctyl carbonate (Cetiol CC) is a
dry emollient with excellent dermatological compatibility and a comprehensive
and convincing performance profile for various applications in the personal care
segment. Properties reported are good emulsifiability, outstanding behavior in
Deo/AP formulations, solubilizing and dispersing properties for sun care, and,
last but not least, the special sensorial feeling given to the final formulation (Ta-
ble 9.9).
9.4.4.3 Guerbet Alcohols [25]

R. C. Guerbet in 1899 discovered the self condensation reaction of alcohols,
which, via the aldehyde as an intermediate, lead to branched structures (2-alkyl
alcohols) as shown in Fig. 9.21. Starting from fatty alcohols from vegetable
sources, for example octanol and decanol, the corresponding C16 and C20 alco-
hols are produced, 2-hexyldecanol and 2-octyldecanol. The reaction is conducted
under alkali catalysis and at high temperatures (>200 8C). Over the years, both
Fig. 9.20 Synthesis of dialkyl carbonates.
Table 9.9 Main properties of dioctyl carbonate.
Chemical structure
INCI Dicaprylyl carbonate

Spreading value 1600 mm2/10 min
Sensory feeling Dry, volatile silicone-oil like
Skin compatibility Excellent
Stability Hydrolysis stable
Origin Vegetable oil (fatty alcohol)
Biodegrability Yes
Fig. 9.21 Synthesis of Guerbet alcohol 2-hexyldecanol (Eutanol G 16).
products have proven to be efficient emollients, but are also used for other ap-
plications, for example as plasticizers or as components of lubricants (Fig. 9.21).
9.5
Perspectives
Examples of recent product innovations from oleochemistry illustrate the suc-

cessful sustainable development of environmentally compatible and powerful
products. It can be assumed that in the future further possibilities for using
renewable resources will be intensively investigated. Here the combination of a
variety of vegetable raw materials to form new products and intelligent product
concepts in order to meet market and consumer needs will be a challenge for
research and development. However, this will depend on the scope of imple-
mentation of the Bioenergy and Biofuel Strategy as part of the Kyoto protocol,
too. The subsidized use of vegetable oils for bioenergy and biofuel production is
completely contradictory to their established use in nutrition and to future in-
dustrial developments of high value added, oleochemical-based products. There-
fore edible oils and fats should not be part of this biomass regulation and sub-
sidies for biofuel and bioenergy in general should be more flexible [26].
9.6
Trademarks
APG, Cetiol, Cutina, Dehymuls, Emulgade, Eumulgin, Eutanol, Lamecreme,

Lameform, Lamesoft, Myritol, Polymuls, Sovermol, and Tribest are registered
trademarks of the Cognis group. Petrofree is a registered trademark of Baroid
Drilling.
References
1 (a) H. Baumann, M. Bühler, H. Fochem, 2 H. Eierdanz (Ed.), Perspektiven nachwach-

F. Hirsinger, H. Zoebelein, J. Falbe, An- sender Rohstoffe in der Chemie, VCH,
gew. Chem. Int. Ed. Engl. 1988, 27, 41; (b) Weinheim, 1996.
A. Behler, M. Biermann, N. Huebner, L. 3 (a) B. Brackmann, C. Hager, The statisti-
Zander, A. Westfechtel, 95th AOCS cal world of raw materials, fatty alcohols
Meeting, Cincinnati, USA, 2004. and surfactants, Proceedings 6th World
References 313
Surfactant Congress, Cesio 2004, Berlin, Automobilindustrie, in Gülzower Fach-

June 20–23, 2004; (b) P. Renaud, Natu- gespräche: Nachwachsende Rohstoffe –
ral-based fatty alcohols: Completely in line Von der Forschung zum Markt, Work-
with the future?, Proceedings 6th World shop 25./26. 05. 1998, Fachagentur Nach-
Surfactant Congress, Cesio 2004, Berlin, wachsende Rohstoffe e.V. (Ed.), 1998,
June 20–23, 2004; (c) G. Kreienfeld, G. pp. 27–47; (b) B. Dahlke, H. Larbig, H.
Stoll, Surfactants in Consumer Products D. Scherzer, R. Poltrock, J. Cell. Plastics,
and Raw Material Situation – A Brief Sur- 1998, 34, 361; (c) J. Neubauer, Der Schritt
vey. In: K. Hill, W. von Rybinski, G. Stoll in die Industrialisierung – Nachwachsende
(Eds.), Alkyl Polyglycosides – Technology, Rohstoffe im Schienenfahrzeugbau, Confer-
Properties and Applications, VCH, Wein- ence presentation at Konstruktionswerk-
heim, 1997, p. 225. stoffe aus nachwachsenden Rohstoffen
4 (a) G. Röbbelen, Pflanzliche Öle als Roh- 2002, September 2002, Braunschweig.
stoffbasis – Potential und Veränderungen in 10 (a) R. Fayter, Technical Reactions for
der Verfügbarkeit. In: Tagungsband 3. Production of Oleochemical Monomers,
Symposium Nachwachsende Rohstoffe – In: H. Eierdanz (Ed.), Perspektiven nach-
Perspektiven für die Chemie, Schriften- wachsender Rohstoffe in der Chemie, VCH,
reihe des Bundesministeriums für Er- Weinheim, 1996, p. 107–117;
nährung, Landwitschaft und Forsten, (b) D. L. Craft, R. C. Wilson, D. Eirich,
Landwirtschaftsverlag, Münster, 1994, Y. Zhang, Candida CYP52A2A promoter
p. 115; (b) K. Hill, Agro–Food Industry and uses in increasing gene expression in
Hi-Tech, 1998, 9[5], 9. yeast for dicarboxylic acid production,
5 (a) F. Hirsinger, F. Bunzel, Ökobilanz PCT Int. Appl. WO2002008412, 2002,
von Fettalkoholsulfat – Petrochemische ver- (Cognis).
sus oleochemische Rohstoffe. In: H. Eier- 11 R. Höfer, Oleochemical polyols – New
danz (Ed.), Perspektiven nachwachsender Raw Materials for Polyurethane Applica-
Rohstoffe in der Chemie, VCH, Weinheim, tions, Proceedings European Coatings
1996, p. 228; (b) M. Stalmans, M. et al., Conference, Berlin, 1999.
Tenside Surf. Det., 1995, 32, 84; (c) M. K. 12 A. Heidbreder, R. Höfer, R. Grützma-
Patel, A. Theiß, E. Worrell, E., Resources, cher, A. Westfechtel, C. W. Blewett,
Conservation and Recycling, 1999, 25, 61. Fett/Lipid, 1999, 101, 418.
6 J. Steber, Textilveredlung 1991, 26, 348. 13 J. Möschel, Thermoplastische Polyurethane
7 R. Höfer, Anwendungstechnische Aspekte sowie ihre Verwendung, DE-PS 19512310,
der Verwendung natürlicher Öle und ihrer 1995, (Parker-Prädifa).
Derivate in der Polymer – Synthese und – 14 (a) B. Gruber, R. Höfer, H. Kluth, A.
Verarbeitung, In: H. Eierdanz (Ed.), Per- Meffert, Fat Sci. Technol., 1987, 89, 147;
spektiven nachwachsender Rohstoffe in der (b) P. Daute, R. Gruetzmacher, R. Höfer,
Chemie, VCH, Weinheim, 1996, pp. 91– A. Westfechtel, Fat Sci. Technol., 1993,
106. 95, 91.
8 (a) B. Schulte, B. Schneider, Linoleum: 15 (a) F. Bongardt, A. Willing, J. Synthetic
Traditionelle und moderne Problemlösung Lubrication, 2003, 20-1 (April), 53;
für den Fußboden auf Basis nachwachsen- (b) J. Legrand, K. Dürr, Agro–Food Indus-
der Rohstoffe, In: H. Eierdanz (Ed.), Per- try Hi-Tech, 1998, 9[5], 16; (c) Th. Mang,
spektiven nachwachsender Rohstoffe in der Fett/Lipid, 1998, 100, 524.
Chemie, VCH, Weinheim, 1996, p. 338– 16 C.-P. Herold (Cognis Deutschland
344; (b) M. W. Formo, Industrial Use of GmbH & Co.KG), personal communica-
Soybean Oil, in Proceedings of the 21st tion.
World Congress of the International So- 17 Bundesministerium für Ernährung,
ciety of Fat Research (ISF), The Hague, Landwirtschaft und Forsten (Ed.), Bericht
1995, P. J. Barnes & Associates (Eds.), über den Einsatz biologisch schnell abbau-
pp. 519–527. barer Schmierstoffe und Hydraulikflüssig-
9 (a) D. Schäfer, Einsatz und Potential keiten und Maßnahmen der Bundesregie-
naturfaserverstärkter Kunststoffe in der rung, 1996, Germany.
18 (a) J. Falbe (Ed.), Surfactants in Consumer 23 (a) T. Morris, M. Hansberry, W. Seipel,

Products: Theory, Technology, Applications, C. Nieendick, Cosmetics & Toiletries, 2004,
Springer, Heidelberg, 1987; (b) W. Dolke- 119 (5), 79; (b) W. Seipel, N. Boyxen,
meyer, Surfactants on the Eve of the Third Moderne Formulierungskonzepte mit Care-
Millennium, 5th World Surfactant Con- Effekten, in Conference Proceedings 51st
gress, Cesio 2000, Florence May 29–June SEPAWA Kongress, Würzburg, 2004,
02, 2000. pp. 70.
19 (a) A. Sander, E. Eilers, A. Heilemann, 24 (a) H. Tesmann, Nachwachsende Rohstoffe
E. von Kries, Fett/Lipid, 1997, 99, 115. in der Kosmetik, in Ref. [2], pp. 31–39;
20 (a) M. Biermann, K. Schmid, P. Schulz, (b) R. Kawa, A. Ansmann, B. Jackwerth,
Starch/Stärke, 1993, 45, 281; (b) J. Knaut, M. Leonard, Parfüm. Kosmet., 1999, 80,
G. Kreienfeld, Chimica Oggi 1993, 41; 17; (c) Th. Förster, U. Issberner, H. Hen-
(c) K. Hill, W. von Rybinski, G. Stoll sen, J. Surfactants and Detergents, 2000,
(Eds.), Alkyl Polyglycosides – Technology, 3, 345; (d) B. Jackwerth, Cetiol CC –
Properties and Applications, VCH, Wein- The New Benchmark for Dry Emollients,
heim, 1997; (d) W. von Rybinski, K. Hill, In-Cosmetics 2000, Barcelona, April
Angew. Chem. Int. Ed., 1998, 37, 1328. 2000.
21 A. Behler, H. Hensen, W. Seipel, Alkyl 25 (a) R. C. Guerbet, C. R. Hebd, Seances
Polyglycoside Carboxylate – A New Anionic Acad. Sci., 1899, 128, 5118;
Surfactant, Proceedings 6th World Sur- (b) K. S. Markley, Fatty Acids (Vol. 2),
factant Congress, Cesio 2004, Berlin, Interscience Publishers, 1961, New York,
June 20–23, 2004. p. 1353.
22 C. Mitchell, A. Ansmann, S. Bruening, 26 H. Sauthoff, Bioenergy and Biofuels – Op-
U. Issberner, S. Nefkens, Formulation portunity or Threat for Oils and Fats Trad-
Design by Texture Modifications, in Con- ing?, Handout and Lecture, FOSFA
ference Proceedings 51st SEPAWA Kon- “Contact Day”, London, September 8,
gress, Würzburg, 2004, pp. 283. 2005.
315
10
Phytochemicals, Dyes, and Pigments
in the Biorefinery Context
George A. Kraus
10.1
Introduction
The idea of a biorefinery is modeled after the highly successful oil refinery
wherein petroleum is converted into gasoline, oil, and monomers such as ethyl-
ene and propylene. As a result of several decades of improvements, these petro-
leum refineries are flexible and efficient [1–3]. They produce high-volume chem-
icals plus a large number of low volume, high-value materials.
For the biorefinery to be successful, it must produce high-volume fuels such
as ethanol or biodiesel plus monomers and a portfolio of high-value chemicals
for niche markets. An outline of a biorefinery is depicted in Fig. 10.1.
The monomers produced by the biorefinery will probably be diols and acids,
because the feedstocks are more highly oxygenated than petroleum. Among the
high-value products produced by the biorefinery will be phytochemicals, dyes,
and pigments. Occasionally these phytochemicals are foods or drugs. Often (e.g.
Fig. 10.1 The biorefinery.
ISBN: 3-527-31027-4
316 10 Phytochemicals, Dyes, and Pigments in the Biorefinery Context
the saponins, phytoestrogens, and the protease inhibitors) the pharmacological

potential has only recently begun to be realized.
10.2
Historical Outline
The fundamentals of a biorefinery already exist in the corn and soybean indus-
tries. The corn wet milling industry converts approximately two billion bushels
of corn per year into corn oil, starch, corn gluten feed, corn gluten meal, and
corn germ meal plus a variety of sugars and other chemicals [4–6]. The basic
outline of a corn wet milling plant is shown in Fig. 10.2.
In the wet milling process, corn is first soaked in water containing sulfur di-
oxide. It is then ground and the components are physically separated using a
series of centrifuges, screens, and washes. Some of the separated fractions then
undergo additional refining steps.
In a soybean-processing plant, the soy protein is separated from the oil [7–9]
and the crude soybean oil is then purified by degumming to remove lecithin,
by refining to remove fatty acids, by bleaching with clay to remove chlorophylls,
and by deodorization (steam distillation) to remove phytosterols and tocopher-
ols. This process is outlined in Fig. 10.3.
Fig. 10.2 Corn wet milling.
Fig. 10.3 Soybean processing.

The deodorizer distillate contains phytochemicals such as phytosterols and to-

copherols. The soy protein contains phytochemicals such as saponins and pro-
tease inhibitors.
Some cooperatives then convert the soybean oil into biodiesel by treating the
oil with methanol or ethanol and a catalyst [10, 11]. Biodiesel is formed by a
transesterification reaction which is catalyzed by acid or, more commonly, by
base. It is the methyl or ethyl ester of a fatty acid. The byproduct of this reac-
tion is glycerin, a hygroscopic triol. Glycerin can be converted into higher-value
products by catalysis or biocatalysis [12, 13]. Glycerin can also be converted into
1,3-propanediol which is used in the synthesis of Sorona [14].
10.3
Phytochemicals from Corn and Soybeans
Corn and soybeans are, at least initially, the most likely feedstocks for a biore-
finery. There is an enormous knowledge base about the fundamental plant
science of corn and soybeans [15]. Additionally, the means of production and
harvesting of these two crops on a million-acre scale is well documented and is
improved each year. I have therefore focused this chapter on describing the phy-
tochemicals, dyes, and pigments from these two crops. Phytochemicals are
chemicals from plants. We will focus on chemicals that are reported to have
beneficial health effects but are not essential for life.
10.3.1
Phytosterols
Phytosterols are present in both corn and soybeans. They are separated from
soybean oil by a steam distillation process and are isolated in the deodorizer dis-
tillate with other organic chemicals. The structures of the major phytosterols
found in corn and soybeans are depicted in Fig. 10.4.
The mixture obtained from soybeans consists of sitosterol (60%), sigmasterol
(20%), and campesterol (20%). These sterols are converted into commercially
important steroids by microbial fermentation [16]. Biotransformation procedures
for the efficient production of androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-
dione, and testosterone have been reported [17, 18].
Sitosterol has recently been shown to reduce plasma VLDL and LDL (“bad”)
cholesterol and increases plasma HDL (“good”) cholesterol [19, 20]. Because ap-
proximately 25% of American adults have high cholesterol, the market for this
high-value phytochemical may soon increase dramatically.
Corn contained dimethyl sterols and monomethyl sterols which amount to
approximately 6% of the total sterol content. In corn and soybeans, sterols are
also present as esters and as glycosides. In Illinois high oil corn, 53% of the
sterols are free sterols, 46% of the sterols are acylated, and 1% of the sterols are
present as glycosides [21]. Most of the acylated sterols are esters of linoleic acid.
Fig. 10.4 Phytosterols from corn and soybeans.
10.3.2
Lecithin
Lecithin, also known as phosphatidylcholine, is available from both corn and

soybeans. In soybean oil processing it is separated from crude soybean oil at
the degumming step. The structure of lecithin is depicted in Fig. 10.5.
Although the structure in Fig. 10.5 shows the phosphatidylcholine unit, com-
mercial lecithin is a mixture of phospholipids in which R1CO2 represents a mix-
ture of saturated fatty acids and R2CO2 represents a mixture of, mostly, unsatu-
rated fatty acids. The major soy phospholipids have been separated by means of
liquid chromatography with use of a flame ionization detector [22].
Lecithin is used commercially as an emulsifier. Lecithin also has several bene-
ficial effects at the cellular level. It is involved in cell-membrane function, fat
transport, and cholinergic neurotransmission [23]. Lecithin reduces plasma cho-
lesterol levels [24] and provides a source of choline which is beneficial for cogni-
tive development [25].
Fig. 10.5 The structure of lecithin.

10.3.3
Tocopherols
Tocopherols are antioxidants that are present in both corn and soybeans. They
are separated from soybean oil in the deodorizer distillate fraction. The struc-
tures of the tocopherols are shown in Fig. 10.6. In soybeans the major constitu-
ent is c-tocopherol (62%), followed by d-tocopherol (25%), a-tocopherol (12%),
and b-tocopherol (1%) [26]. In corn the major component is c-tocopherol, with
a-tocopherol second. The total tocopherol content of corn varies as a function of
genetics and geography, with a range of 500–1000 mg tocopherols per kilogram
of corn oil [27].
The tocopherols are a source of vitamin E. Natural vitamin E is a-tocopherol
in which each of the three stereogenic centers has the R configuration. The b, c,
Fig. 10.6 Structures of the tocopherols.

and d isomers can be converted into vitamin E or used as antioxidants in the

animal feed industry [28]. Vitamin E has cardioprotective activity [29].
10.3.4
Carotenoids
The main pigments in yellow corn are carotenes and xanthophylls. Depending
on the growing conditions, corn contains from 20–80 mg kg–1 carotenes and
xanthophylls [30]. The main carotenes are b-carotene and b-cryptoxanthin. Their
structures are shown in Fig. 10.7. Because of the conjugated polyene structure
of carotenes, they are not as stable as other phytochemicals present in corn.
They can be readily degraded by oxygen in the air, by a combination of oxygen
and sunlight, and also by heat. The time of storage and storage conditions are,
therefore, factors in the yield of carotenoids.
Carotenoids are a significant dietary source of vitamin A for animals. The effi-
ciency of conversion of carotenes into vitamin A varies with the type of animal.
Carotenes are also good antioxidants [31].
Fig. 10.7 Structures of the carotenoids.

Xanthophylls are pigments responsible for color but do not function as pre-
cursors to vitamin A. The principal xanthophylls in corn are lutein and zea-
xanthin. Their structures also are illustrated in Fig. 10.7. Like carotenes, the
xanthophylls are labile in light, heat, and air. They are also good antioxidants.
Chromatography-based detection methods have been developed for the caro-
tenes and xanthophylls [32].
Lutein has recently been recognized as an effective drug for treatment of
macular degeneration, a disease that affects eyesight [33]. Although marigolds
are currently the primary source of lutein, genetic modification of corn may
lead to enhanced levels of the compound.
10.3.5
Phytoestrogens
Phytoestrogens are found in soybean protein. Levels of these phytochemicals

are approximately 400 mg kg–1 soy protein [34]. Soybeans, particularly tofu, are
the major dietary source of isoflavanoids. Isoflavone levels can vary as a func-
tion of genetics, growing conditions, and processing techniques. The main phy-
toestrogens in soybeans are isoflavanoids such as genistein and daidzein. In
non-fermented foods these compounds are present mainly as the b-glycosides
[35]. The structures of genistein and daidzein are depicted in Fig. 10.8.
These phytochemicals are associated with reduced incidence of breast cancer
and prostate cancer [36]. Genistein is reported to reduce levels of LDL (“bad”)
cholesterol [37].
10.3.6
Saponins
Saponins are glycosylated terpenes found in several crops, including soybeans.

Structures of representative soybean saponins aglycones are illustrated in
Fig. 10.9. Soybeans have a saponin content of approximately 6% by weight in
whole soybeans. A procedure for producing a purified saponin fraction on an
industrial scale was recently patented [38].
Soy saponins have several health benefits. Soy saponins may reduce cholester-
ol levels in humans by binding to cholesterol, thus preventing its re-adsorption
into the blood stream [39]. Saponins have been reported to inhibit accumulation
Fig. 10.8 Structures of genistein and daidzein.

Fig. 10.9 Structures of saponin aglycones.
of visceral fat, providing a mechanism for controlling obesity [40]. Saponins also
bind tightly to bile acids, thereby reducing the risk of colon cancer [41]. Sapo-
nins have also been reported to remove toxins and hardened matter from the
intestinal tract [42]. Researchers have recently reported an in-vitro effect of sapo-
nins on the infectivity of the human immunodeficiency virus [43]. Saponins
have also been reported to protect against aflatoxin-induced mutagenicity. They
were ranked as more effective than tocopherol or l-ascorbic acid [44].
10.3.7
Protease Inhibitors
Over the past decade, protease inhibitors, particularly those derived from soy-
beans, have emerged as potential cancer chemopreventive agents. Soybeans con-
tain significant amounts of protease inhibitors – approximately 6% by weight of
total soybean protein is protease inhibitors [45]. Protease inhibitors have also
been found in crops such as corn and rice.
The inhibitor known as the Bowman–Birk inhibitor, more commonly referred
to as BBI, has received the most attention [46]. This inhibitor makes up a signif-
icant fraction of the inhibitors in soy protein and is readily available in commer-
cial tofu. The molecular structure of BBI, a polypeptide of molecular weight
8000, has recently been determined [47]. Extensive experimentation has shown
that BBI is a potent chymotrypsin and trypsin inhibitor.
Although BBI has been effective in animal trials, results of human trials are
not yet known. On the basis of public health records in Asia and the United
States, there certainly seems to be a connection between soybean consumption
and the incidence of breast, colon, and prostate cancers [48]. BBI has been re-
commended as a radioprotector for normal tissue to enhance radiotherapy [49].
The soybean protease inhibitor SBTI has also been characterized.
References 323
Corn gluten contains inhibitors with powerful angiotensin I enzyme-convert-

ing enzyme inhibitory activity [50]. A 12 kD protease/a-amylase inhibitor was re-
cently isolated from corn seed. CI-4a, a cysteine protease inhibitor in corn endo-
sperm has been purified and characterized [51]. Inhibitors of corn endosperm
sulfhydryl protease P-Ia, the main factor for zein degradation during germina-
tion, have been isolated and characterized [52].
10.4
Phytochemicals are high-value chemicals for niche markets. As methods for

their isolation become better defined and as their biological activity in humans
is better understood, demand for these chemicals is very likely to increase sub-
stantially. There are, moreover, other, less well understood, phytochemicals such
as conjugated linoleic acid and other protease inhibitors that could eventually
add more value to the biorefinery.
References
1 Strachan, J. F. The Petroleum Refinery En- 10 Encinar, J. M.; Gonzalez, J. F.; Rodriguez,
gineer’s Handbook, Philosophical Library, J. J.; Tejedor, A. Energy & Fuels 2002, 16,
New York, 1955. 443–450.
2 Nelson, W. L. Petroleum Refinery Engineer- 11 Zhou, W.; Konar, S. K.; Boocock, D. G. B.
ing. 4th ed. McGraw–Hill, New York J. Am. Oil Chem. Soc. 2003, 80, 367–371.
1958. 12 Schlaf, M.; Ghosh, P.; Fagan, P. J.;
3 Peterson, D. J. New Forces at Work in Re- Hauptman, E.; Bullock, R. M. Ange-
fining: Industry views of Critical Business wandte Chemie, International Ed. 2001,
and Operation Trends, Rand, Santa Moni- 40, 3887–3890.
ca, CA 2003. 13 Antal Jr, M. J.; Mok, W. S. L.; Richards,
4 Johnson, L. A.; May, J. B. Corn: Chemistry G. N. Carbohydrate Research, 1990, 199,
and Technology, Chapter 12, American 111–115.
Association of Cereal Chemists, USA, 14 Schuster, L.; Eggersdorfer, M. Eur. Pat.
2003. Appl. 1996. EP 713849
5 Blanchard, P. H. Technology of Corn Wet 15 For an excellent overview, see:
Milling and Associated Processes, Elsevier P. J. White, L. A. Johnson Corn: Chemistry
Publishing, Amsterdam, 1992. and Technology, American Association of
6 Brekke, O. L. Corn Culture, Processing, Cereal Chemists, USA, 2003.
Products, AVI Publishing, Westport, CT, 16 Shah, K.; Mehdi, I.; Khan, A. W.; Vora,
1970. V C. European J. Applied Microbiol. Bio-
7 Erickson, D. R. Practical Handbook of technol. 1980, 10, 167–169.
Soybean Processing and Utilization, AOCS 17 Liu, W.-H.; Pan, C.-P.; Lo, C.-K. Food
Press, St. Louis, 1995. Science and Agricultural Chemistry 2001,
8 Friedrich, W. Fette, Seifen, Anstrichmittel 3, 139–142.
1983, 85, 346–355. 18 Lo, C.-K.; Pan, C.-P.; Liu, W.-H. Journal
9 Cowan, J. C. Agronomy 1973, 16, 619– of Industrial Microbiology & Biotechnology
664. 2002, 28, 280–283.
19 Gylling, H., Puska, P., Vartiainen, E. and 36 Lee, H. P., Gourley, L., Duffy, S. W.,
Miettinen, T. A. J Lipid Res 1999, 40, Esteve, J., Lee, J., Day, N. E. Lancet, 1991,
593–600. 337,1197–1200.
20 Gylling, H. and Miettinen, T. A. Diabeto- 37 MerzDemlow, B. E., Duncan, A. M.,
logia 1994, 37, 773–780. Wangen, K. E., Xu, X., Carr, T. P., Phipps,
21 Davis, D. L.; Poneleit, C. G. Phytochemis- W. R., Kurzer, M. S. Am. J. Clin. Nutr.
try, 1975, 14, 1201–1203. 2000, 71, 1462–1469.
22 Grieser, M. D.; Geske, J. N. J. Am. Oil 38 Wanezaki, S.; Tsuzaki, S.; Araki, H. PCT
Chem. Soc. 1989, 66, 1484–1487. Int. Appl. 2003, WO 2003075939 A1
23 Miller, D. L. Cereal Foods World 2002, 47, 20030918 Application: WO 2003-JP2881
178–184 20030311.
24 O’Mullane, J. E.; Hawthorne, J. N. Athero- 39 Arditi, T.; Meredith, T.; Flowerman, P.
sclerosis (Shannon, Ireland) 1982, 45, 81– Cereal Foods World 2000, 45, 414–417.
90. 40 Wanezaki, S.; Fukui, K.; Takamatsu, K.;
25 Ladd, S. L.; Sommer, S. A.; LaBerge, S.; Araki, H. Jpn. Kokai Tokkyo Koho 2003.
Toscano, W. Clin. Neuropharmacol. 1993, 41 Lacaille-Dubois; Wagner Phytomedicine,
16, 540. 1996, 2, 363–386.
26 Clark, J. P. Nutraceuticals: Designer Foods 42 Lipkin, R. Science News, 1995, December
III: Garlic, Soy and Licorice, [Course on 9.
Designer Foods, Proceedings], 3rd, 43 Nakashima, H. AIDS 3, 1989, 655–658.
Washington, D. C., May 23–25, 1994, 44 Jun, H.-S.; Kim, S.-E.; Sung, M.-K.
237–242. Food & Nutrition Press J. Med. Food 2002, 5, 235–240.
27 Weber, E. J. J. Am. Oil Chem. Soc. 1984, 45 Hwang, D. L. R., Davis-Lin, K. T., Yang,
61, 1231–1234. W. K. and Foard, D. T. Biochem. Biophys.
28 Pratt, D. E. AOCS Monograph 1985, 15 Acta 1977, 495, 369–382.
(Flavor Chem. Fats Oils), 145–153. 46 Kennedy, A. R Pharmacol. & Therapeutics
29 Stampfer, M. J.; Hennekens, C. H.; 1998, 78, 167–209.
Manson, J. E.; Colditz, G. A.; Rosner, B.; 47 Odani, S. and Ikenaka, T. J. J. Biochem.
Willett, W. C. N. Engl. J. Med. 1993, 328, (Tokyo) 1973, 74, 857–860.
1444–1449. 48 Dunn, J. E. Cancer Res. 1975, 35, 3240–
30 Weber, E. J. J. Am. Oil Chem Soc. 1987, 3245.
64, 1129–1134. 49 Dittmann, K. H.; Mayer, C.; Rodemann,
31 Burton, G. W.; Ingold, K. U. Science, H. P. Current Med. Chem.: Anti-Cancer
1984, 224, 569–573. Agents 2003, 3, 360–363.
32 Kurilich, A. C.; Juvik, J. A. J. Liquid Chro- 50 Suh, H. J.; Whang, J. H.; Kim, Y. S.; Bae,
matogr. Related Technol. 1999, 22, 2925– S. H.; Noh, D. O. Process Biochem.
2934. (Oxford, United Kingdom) 2003, 38,
33 Krinsky, N. I.; Landrum, J. T.; Bone, R. A. 1239–1244.
Ann. Rev. Nutrition 2003, 23 171–201. 51 Blanco-Labra, A.; Chagolla-Lopez, A.;
34 Dickson, R. A.; Ferreira, D. Phytochemis- Martinez-Gallardo, N.; Valdes-Rodriguez,
try, 2002, 60, 205–211. S. J. Food Biochem. 1995, 19, 27–41.
35 Wang, H.-J.; Murphy, P. A. J. Agric. Food 52 Abe, M.; Arai, S.; Kato, H.; Fujimaki, M.
Chem. 1994, 42, 1666. Agric. and Biol Chem 1980, 44, 685–686.
325
11
Adding Color to Green Chemistry?
An Overview of the Fundamentals and Potential
of Chlorophylls
11.1
Introduction
Chlorophylls, often termed the “pigments of life” are green colored macrocyclic
pigments which are the primary photosynthetic pigments. The term chlorophyll,
coined by Berzelius in 1838, is derived from Greek and indicates the green of
leaves [1]. In fact, as green pigments they are responsible for primary biochem-
ical energy generation in nature and are the only indication of life on earth visi-
ble from outer space. Current efforts to switch chemistry from a system based
on the use of nonrenewable resources (geological organic carbon deposits) to
one based on the utilization of renewable bioresources are one of the corner-
stones of efforts often labeled “green chemistry” [2]. Although “green” is taken
to imply an environmentally friendly approach, chlorophylls in a literal sense
are the green chemical components responsible for beauty in our natural envi-
ronment (vegetation).
If so, is green chemistry really green? At present, no. Even a cursory inspec-
tion reveals that all practical efforts aimed at the use of biological materials in
chemistry rely solely on the use of nongreen materials. This article aims to de-
scribe some of the fundamentals and highlights of chlorophyll chemistry and
gives a preliminary assessment whether chlorophylls might be a useful biore-
source.
11.2
Historical Outline
Chlorophylls have been studied for almost 200 years [1] and, although much of
their basic chemistry was established almost 100 years ago [3], real break-
throughs in their exact biochemical function were only made in recent decades
as a result of X-ray crystallographic analysis both of the reaction center [4] and
of light harvesting complexes [5]. Until WW II most studies were concerned
ISBN: 3-527-31027-4
326 11 Adding Color to Green Chemistry?
with their basic structural chemistry. This was followed by elucidation of the ba-
sic anabolic pathways of chlorophyll in the higher plants, photophysical studies
of their function in photosynthesis, and most recently their catabolism. Parallel
to studies on chlorophylls a and b from higher plants, recent decades have seen
many efforts to elucidate the structure, function, and biosynthesis of photosyn-
thetic pigments from unicellular organisms and photosynthetic bacteria [6].
Chlorophylls have never been used as bulk chemicals for transformation into
other compounds on a technical scale. As outlined below, since the middle of
the last century phytochlorin and rhodochlorin derivatives have found some use
as food additives and antiodor compounds, often in the form of simple plant ex-
tracts. During the last twenty years pheophorbide derivatives have been used as
starting materials for the synthesis of novel photosensitizers (vide infra) and are
under active investigation as compounds for solar energy conversion and hydro-
gen production. These efforts are still in a developmental stage, however, and
require only gram-scale amounts of material [7].
11.3
Chlorophyll Fundamentals
11.3.1
Occurrence and Basic Structures
Chlorophylls and the related bacteriochlorophylls are the ubiquitous pigments

of photosynthetic organisms. As such they share common structural principles
and functions. They are involved either in light harvesting (exciton transfer) as
antenna pigments or in charge separation (electron transfer) as reaction-center
pigments.
The best-known pigment is chlorophyll a, 1, which occurs in all organisms
with oxygenic photosynthesis (Fig. 11.1). In higher plants it is accompanied in a
3 : 1 ratio by chlorophyll b, 3, in which the 7-methyl group has been oxidized to
a formyl group. Both compounds typically consist of the tetrapyrrole moiety and
a C-20 terpenoid alcohol, phytol. Most compounds are magnesium chelates, but
the free base of chlorophyll a, pheophorbide a, 2, is also active in electron trans-
fer. Chlorophylls a and b can be obtained easily from plants or algae and are
the focus of this article.
Many other, similar photosynthetic pigments occur in nature, however [8]. Ap-
proximately one hundred related pigments have now been isolated and all share
either a phytochlorin 4 or 7,8-dihydrophytochlorin framework. For example,
such compounds include chlorophyll d, 5, from Rhodophytes, bacteriochloro-
phylls c, 6, d, and e (which are chlorins 9 and have significant variability in
their peripheral groups) [6, 9] from Chlorobiaceae and Chloroflexaceae, and bac-
teriochlorophylls a, 7, and b (true bacteriochlorins 10) found in Rhodospirillales.
Other natural pigments are chlorophyll c, bacteriochlorophyll g, and many re-
lated compounds with different esterified isoprenoid alcohols. Chemically re-
Fig. 11.1 Naturally occurring chlorophylls and related sys-

tems. Small numbers in the chlorophyll a formula indicate the
IUPAC numbering scheme. R groups may vary in different or-
ganisms or at different stages of development.
lated chlorins have also been found in many oxidoreductases, marine sponges,
tunicates, and in Bonella viridis. The deep-sea dragon fish Malacosteus niger even
uses a chlorophyll derivative as a visual pigment [10]. Most of these are believed
to be derived from chlorophyll which is processed by the plant or animal.
11.3.2
Principles of Chlorophyll Chemistry
Chlorophylls are heteroaromatic compounds and the aromatic character of the

underlying tetrapyrrole moiety and the reactivity of the functional groups in the
side chains govern their chemistry. Three different classes of tetrapyrrole, differ-
entiated by their oxidation level, occur in nature (Fig. 11.2): porphyrins (8, e.g.,
hemes), chlorins (9, e.g. chlorophylls), and bacteriochlorins (10, e.g., bacterio-
chlorophylls). The overall reactivity of chlorophyll, a cyclic tetrapyrrole with a
fused five-membered ring, is that of a standard phytochlorin, 4. Such com-
pounds are capable of coordinating almost any known metal with the core nitro-
gen atoms. Together with the conformational flexibility of the macrocycle and
the variability of its side chains, this accounts for their unique role in photo-
synthesis [11, 12].
Fig. 11.2 Basic tetrapyrrole structures and reactivity of chlorophyll.
The basic aromatic system is susceptible mainly to electrophilic reactions, e.g.

halogenations, with a preference for reaction at C20, the meso position closest
to the reduced pyrrole ring. The C7–C8 double bond in phytochlorin (next to
R1) is not part of the aromatic delocalization pathway and is thus a convenient
point for modification of the chlorophyll macrocycle by addition and oxidation
reactions. The side-chain substituents undergo standard transformations, e.g.
the C3 vinyl group undergoes addition and oxidation reactions, the ester groups
at C173 and C132 can be saponified or transesterified, and the reactivity of ring
E is usually governed by enolization and follow-up chemistry of the b-keto ester
system. The latter includes the so-called allomerization reactions, i.e. oxidative
degradation reactions involving oxidations, hydroxylation, ring-opening or decar-
boxylations of the isocyclic pentanone ring. The book by Scheer [13] remains
the most comprehensive treatise on chlorophyll chemistry and function and a
comprehensive review on side-chain transformation has recently been published
by Pavlov and Ponomarev [14].
11.3.3
Isolation of Chlorophylls
Because of their prominent biological role, many different methods have been
developed for the qualitative and quantitative extraction of chlorophyll a and b
from plants and algae [15]. All methods require extraction of the pigments with
an organic solvent or boiling water and, despite all efforts, no single solvent or
solvent mixture has been found that will extract the pigments quantitatively and
unaltered. The natural chlorophylls are exceptionally labile compounds that
readily undergo reactions in the macrocycle core, at C20, and at the doubly acti-
vated 132-position [16]. Thus, they rapidly become demetalated and/or lose the
phytyl side-chain to yield pheophorbides 12, or undergo other chemical altera-
tions (Fig. 11.3).
Fig. 11.3 Structures of selected chlorophyll a derivatives formed during extraction.
Often, this is not only the result of the chemicals added but is promoted by
the plant material itself. For example, plants may have an acidic cytoplasm re-
sulting in the formation of pheophytins, or contain oxidative enzymes that pro-
mote oxidation reactions. The most prominent and most easily formed byprod-
ucts of the extraction process are the allomerization products, notably epimer-
ized 13 or hydroxylated derivatives 14 of chlorophyll [16, 17]. Likewise, chloro-
phyllase often remains active during extraction resulting in the formation of
acidic chlorophyllides 15 or re-esterified derivatives thereof when using alcohols
for extraction. Efforts to denature the enzymes before extraction (e.g. by scald-
ing) will result in the formation of isomerization products. All the different re-
actions described, and many others, can occur concomitantly at different stages,
giving rise to a broad range of extraction artifacts. Thus it is almost impossible
to extract plant materials without concurrent formation of a variety of chloro-
phyll derivatives.
Exceptional precautions must be taken (inert gas, cell break up by liquid ni-
trogen, etc.) to isolate chlorophylls quantitatively and unaltered. This precludes
any large-scale technical use of chlorophyll and any chemical or technical use of
chlorophyll must be based on chemically more stable derivatives thereof.
In practical terms, small-scale extractions (1–5 g plant material) may be per-
formed with almost any “green” organism, usually with chilled acetone as ex-
traction solvent. A typical procedure involves disrupting the material in a blen-
der in the presence of acetone, filtration, re-extraction of plant material, and
concentration of the combined extracts by rotary evaporation. Medium-scale ex-
tractions (up to 1 kg plant material) are best performed using fresh spinach.
The material is first heated in boiling water (1–2 min), filtration followed by ex-
traction with methanol–petroleum ether mixtures then typically yields 2 to 4 g
crude pigment extract. For large-scale extractions (1–5 kg) dried alfalfa meal or
air-dried blue–green algae, notably Spirulina, are the best sources. In our hands
the best results were obtained by freezing Spirulina with liquid nitrogen and ex-
tracting the pigments with acetone to yield approximately 2–5 g chlorophyll a
kg–1 dried algae. Spirulina contains only chlorophyll a, a major benefit which
circumvents the a/b separation problem. Although a variety of chromatographic
methods have been developed to separate the “a” and “b” series, the best “large-
scale” method utilizes chemical modification of the formyl group in pheophytin
b with Girard’s reagent T [18].
Dried blue-green algae are still the best material for the “large-scale” prepara-
tion of pure chlorophyll derivatives and have served for decades as the source of
starting materials for research groups involved in chlorophyll chemistry. Except
for production of “chlorophyllin” (see below) no attempts have been made to ex-
tract chlorophyll or derivatives thereof on an industrial scale. A “dye fraction” is,
nevertheless, one of the fractions routinely obtained when green materials
(grass, lucerne, alfalfa, etc.) are processed in a green biorefinery [19] and could
serve as a source of chlorophyll derivatives.
11.4
Chlorophyll Breakdown and Chemical Transformations
Any consideration of the potential of chlorophylls as a bioresource for fine

chemicals is critically dependent on the types of compound that can be derived
from it. Thus, we must take a look at chemical transformations occurring either
in nature or in the laboratory.
11.4.1
Biological Chlorophyll Catabolism
Although more than 109 tons of chlorophyll are degraded each year on earth
(and account for the natural beauty of fall vegetation), the biological breakdown
of chlorophyll had remained an enigma until about a decade ago [20]. The only
chemical reactions known to be involved in senescence and natural breakdown
of chlorophyll were similar to those encountered during pigment extraction.
Loss of Mg, dephytylation, and some changes at the isocyclic pentanone ring
seemed to be early steps but nothing was known about the fate of the macro-
cycle and the putative ring-opening reaction.
Work by the groups of Kräutler, Matile, and Gossauer showed that the central
step is a ring-opening reaction at the 5-position [21, 22]. This is in contrast with
the situation encountered for heme, which is oxidatively cleaved at the 20-posi-
tion. As shown in Fig. 11.4, the crucial steps of chlorophyll degradation begin
by conversion of chlorophyll a into pheophorbide a, 12, followed by enzymatic
transformation into the bilinone, 16. During this step the macrocycle undergoes
oxidative C5 ring-opening, incorporates two oxygen atoms (the CHO one from
O2) and is saturated at the 10-position. This reaction is catalyzed by a monooxy-
genase and the red compound 16 is further converted to the still fluorescing
compound 17, and finally into the nonfluorescing derivative 18, along with
some changes in the side chains which increase the hydrophilicity of the break-
down products. Chlorophyll b is first converted into chlorophyll a and then sub-
jected to the same reactions.
Fig. 11.4 Outline of chlorophyll degradation in senescent plants [22].
There is currently no evidence that chlorophyll is degraded further to smaller

nontetrapyrrolic compounds. The primary function of chlorophyll catabolism
during senescence is one of detoxification. Downsizing of the photosynthetic
machinery during low light conditions and degradation of the photosystems
during senescence releases chlorophylls and the carotenoids necessary for
photoprotection in intact chlorophyll–protein complexes. Unbound chlorophylls
are potent photosensitizers and produce highly toxic singlet oxygen (see
Fig. 11.8). Prevention of this reaction, rather than recycling of nutrients, is the
primary rationale behind chlorophyll degradation. Likewise, phytol is not re-
cycled. Its intracellular level remains largely constant during senescence and it
is transesterified to phytyl acetate and found in gerontoplasts [22].
11.4.2
Geological Chlorophyll Degradation – Petroporphyrins
It is known that chlorophyll is not only degraded in living systems but also un-
dergoes degradation in sediments [23]. Up to a few percent of all annual pri-
mary organic production is accumulated in sediments and under appropriate
conditions (low thermal or oxidative stress) significant amounts of organic com-
pounds will remain identifiable in sediments as fossil molecules [24]. These
compounds can serve as geochemical biomarkers as long as they retain enough
structural resemblance to biochemical compounds. Indeed, porphyrins are
found in many deposits and their concentration in sediments can vary from 1
to 3000 ppm. They have been termed geoporphyrins, petroporphyrins, or sedi-
mentary porphyrins. Their concentration in coal is usually below that in crude
oils, and even one porphyrin-based mineral, abelsonite, has been identified [25].
Tetrapyrroles have been isolated with the organic matter from almost all sedi-
ments, and are found at geological ages ranging from the Precambrian to re-
cent. Interest in these pigments is related to two areas of research. In practical
terms, different deposits have different porphyrin compositions, both with re-
gard to type and relative content of pigments. Thus, the petroporphyrins can be
used as a fingerprint for different oil shales. Indeed, HPLC analysis of the pet-
roporphyrin fraction often serves as an important analytical tool in the oil in-
dustry.
Second, the close structural resemblance between some geoporphyrins and
biochemical pigments (e.g. chlorophyll a) might enable development of a diage-
netic scheme for their formation. Indeed, Treibs’s basic hypothesis [23] that
petroporphyrins are derived from chlorophylls (and porphyrins) has more than
stood the test of time. By now more than 90 different petroporphyrins have
been identified, some with very unusual structures. The classic example is
desoxophylloerythroporphyrin, 20 (a derivative of phytoporphyrin in modern
nomenclature), which clearly bears a close resemblance to chlorophyll a
(Fig. 11.5). Even etioporphyrin, 19, another well-known geoporphyrin, may be
envisaged as being derived from 20 by cleavage of ring V, or from heme-type
pigments [26].
Geoporphyrins are derived from almost all different types of chlorophyll (e.g.,
21, 22). Indeed, even some “petrochlorins” (e.g. phytochlorins 4 and 23) have
been found. Many of these compounds have been prepared by total synthesis,
and for many a chemical rational can be given for their chemical formation.
Their natural formation involves extreme diagenetic conditions (geological time-
scale, heat, pressure, catalysts, . . .). Most of the reactions involving geoporphyr-
in formation are easily identified – loss of magnesium, remetalation with VO,
Ni, or other metals, aromatization of ring IV, vinyl and keto group reduction,
cleavage of the methyl ester, decarboxylation – and are standard reactions of the
porphyrin chemist [24]. Fossils of phytol 26, e.g. phytane 27 and pristine 28, are
prominent constituents of sediments also [27]. Finally, elucidation of the struc-
ture of geoporphyrins with yet unknown diagenetic origin (e.g. 24) might lead
to the identification of yet unknown biochemical pigments (Fig. 11.6).
In the context of this chapter, such pigments can serve as typical examples of
exceptionally stable compounds which can be derived from natural pigments.
Besides their importance in palaeobiology, geochemistry, and as an analytical
tool, however, they are of no industrial importance.
Fig. 11.5 Treibs’s scheme for petroporphyrin formation [23].

Fig. 11.6 Selected petroporphyrins and fossil phytol derivatives [24].
11.4.3
Chemical Degradation of Chlorophylls
As outlined in Section 11.3.2, chlorophylls contain many reactive positions that

can serve as an entry for degradation reactions. A survey of all possible reac-
tions is beyond the scope of this article and the reader is referred to relevant re-
views [3, 11, 14, 28]. Broadly, chlorophyll chemistry is divided into functional
group transformations of the side chains, manipulations involving ring V, and
reactions involving the macrocyclic ring system.
The first two will be of either no importance or must be prevented during uti-
lization of chlorophyll-derived compounds from biomass (Section 11.6). The
third is another possible obstacle to the use of chlorophylls. For our purpose,
three types of macrocycle reaction are important. First, like any other porphyrin,
the meso positions or chlorophyll derivatives are highly susceptible to electrophilic
substitution. In chlorophylls the 20 position is especially reactive and readily un-
dergoes reactions with electrophiles [29], sometimes even during chromatography
on silica gel (e.g. to yield 29) [30] (Fig. 11.7). A variety of substitution reactions can
also be used to disrupt the chlorophyll system at the 20 position [22].
The second reaction is the chlorin to porphyrin conversion. Any chlorin that
has hydrogen atoms on the sp3-hybridized centers of the reduced ring can be
oxidized to the respective porphyrin. Oxidation can be achieved by use of a vari-
ety of oxidants including oxygen [28]. Likewise, reductions to hydroporphyrins
and other reactions of the macrocycle are possible. Most of these are of interest
Fig. 11.7 Selected reactions of chlorophyll involving the macrocycle.
Fig. 11.8 Chlorophyll as a photosensitizer.
only to the specialist, however. Under appropriate conditions photochemical re-

ductions, notably the Krasnovskii reduction to 30, can occur [31].
The most important and potentially most troublesome reaction is photooxy-
genation [32]. Chlorophylls are potent photosensitizers and will produce singlet
oxygen in the presence of air or triplet oxygen [33]. Thus, chlorophylls can un-
dergo self-destruction (Fig. 11.8). The chemistry of this photooxygenation is very
involved and differs somewhat for individual types of chlorophyll [28, 34]. While
being partially responsible for the low stability of chlorophylls in solution [35],
and for unwanted side reactions in foodstuffs [36], the same reaction has great
potential for future applications. Chlorophyll and its derivatives may be used as
photosensitizers to affect desired chemical transformations and they have been
used for applications in photodynamic therapy (PDT, below) [37].
11.5 Industrial Uses of Chlorophyll Derivatives 335
11.5
Industrial Uses of Chlorophyll Derivatives
A historical look at the industrial use of chlorophyll (and derivatives) in the

20th century shows that it has been mainly used as a green pigment in the nu-
trition industry (in Europe food additive E140 for chlorophyll and E 141 for
chlorophyllin in cakes, beverages, sweets, and ice-cream, etc.) and in pharma-
ceutical and cosmetic products (color no. 125 in toothpaste, as a soap pigment,
or in shampoos). The older literature also describes its use in candles [38] and
as a lipophilic oil-bleaching additive (to neutralize the yellow color of oils in
foodstuffs or to give them a greener touch) [39].
Chlorophyll is usually used in the form of chlorophyllin and its metal com-
plexes. Chlorophyllin is an inhomogeneous water-soluble material. It is pre-
pared by saponification of the phytyl side chain with NaOH and exchange of
the central magnesium atom for copper (or other metals). The harsh reaction
conditions (and the use of the natural chlorophyll a/b mixture) results in the
formation of a mixture of compounds (Fig. 11.9). Most prominent constituents
are the derivatives 31,32-didehydrorhodochlorin (notably the chlorin e6, 31, and
e4, 32, derivatives in the old nomenclature), pheophorbide salts (e.g. 33) and the
typical allomerization products [40]. Chlorophyllin is a stable pigment with an
intense light green to dark blue–green color. Related formulations are sodium
zinc chlorophyllin, chlorophyll paste, oil-soluble chlorophyll, and sodium mag-
nesium chlorophyllin [41].
Fig. 11.9 “Chlorophyllin”.

Chlorophyllin production also yields large amounts of phytol 26, a colorless

oil by-product [42]. This compound has found industrial uses in the synthesis
of vitamin K1 (phylloquinone) [43–46] and as the lipophilic part of vitamin E
[47].
Besides the traditional use of chlorophyll and its derivatives as pigments,
early investigations on the medicinal use of chlorophyllin in the 1940s led to a
first boom in chlorophyll use and initiated more serious investigation of its
medicinal properties [48]. During those times it was used in bathroom tissue,
diapers, chewing gum, bed sheets, shoe liners, toothpaste [49], and other daily
products, mostly as an antiodorant [50]. Chlorophyll preparations are still avail-
able as OTC medicine to reduce fecal odor because of incontinence or to reduce
odor from a colostomy or ileostomy. Other applications involved use in wound
healing, germ killing, and treatment of infections and inflammations (use of
bandages, antiseptic ointments, surgical dressings) [48].
A late revival of chlorophyll use started in the 1980s when consumer demand
for natural pigments in food and other products increased [51]. Although
chlorophyllin is currently used as a food coloring agent in Europe and in Asia,
chlorophyll(in) is not approved by the FDA in the USA as a food additive. It is
permitted in drugs or cosmetics at concentrations < 1% [52, 53]. Annual chloro-
phyllin production is estimated to be several thousand tons worldwide. An exact
assessment of the total amount of “industrial chlorophyll” is difficult with many
companies being located in the USA, India, China, and Japan.
Since the 1980s use of chlorophyllin has not been limited to pigmentation
but the list of putative applications is growing daily – wound healing, treatment
of ulcers, treatment of acute and chronic hepatitis, treatment and prevention of
cancer [54], recovery of liver function [41], antiodorant in the digestive system,
treatment of iron deficiency anemia, etc. [55]. As a result, there is a growing
market for chlorophyll formulations as dietary supplements. Some of these are
based on actual research, most are rather dubious [56].
There is, nevertheless, growing evidence for medicinal use of chlorophylls.
Antimutagenic effects, both in vitro and in animal models, have been proven,
notably against aflatoxins [57]. Likewise, there are indications of an anticarcino-
genic role [58, 59]. For example, an animal study revealed inhibition of dioxin
absorption and increased fecal excretion of dioxin [60]. At the very least, these
results indicate the need for further research and offer the promise of future ap-
plications of chlorophyll.
Photodynamic therapy is the one clearly established medicinal application of
chlorophyll derivatives to date [37, 61, 62]. This method relies on selective accu-
mulation of a photosensitizer in target tissue where it can be activated with
light to produce toxic singlet oxygen resulting in, e.g., tumor necrosis, as out-
lined in Fig. 11.8. Although developed in the early seventies, this concept is only
now making its potential felt in oncology, as antiviral and antibacterial PDT,
and in the treatment of diseases such as age-related macular generation and
psoriasis. Several porphyrin-based compounds have been approved for medic-
inal applications and others are in phase-2 trials. Amongst other tetrapyrroles,
11.6 A Look at “Green” Chlorophyll Chemistry 337
Fig. 11.10 Visible light-induced hydrogen production system [64, 65].
chlorophyll derivatives are currently under active investigation and show great
promise. The current status of this field has been reviewed [37].
The use of chlorophyll derivatives in technical applications is still in the early
developmental stage. Topics of current interest are both solar energy conversion
and hydrogen production [63]. A representative investigation of biohydrogen
evolution uses Zn-chlorophyll a. This is prepared from chlorophyll a derived
from Spirulina and its photostability and photosensitizing activity are superior
to those of the natural magnesium complex. The use of chlorophyll derivatives
is based on their absorption in the visible region. Besides the photosensitizer
the photo-induced hydrogen generation-systems also require an electron donor
(for example nicotinamide adenine dinucleotide (NADH) regenerated by glu-
cose dehydrogenase (GDH)), an electron relay (Fig. 11.10, MV+/MV2+) and a cat-
alyst (for example colloidal platinum) [64, 65].
Another modern example is the use of chlorin e6, 36, also derived from Spiru-
lina in bio-photovoltaics [66]. Here, the conversion device uses chlorin e6 in a
dye-sensitized solar cell (DSSC) [67] with a nanocrystalline TiO2 film. The green
pigment absorbs visible light (to yield the chromophores in an excited singlet
state). In the next step its emission is quenched by TiO2 with effective electron
injection from 1(chlorin e6)* into the TiO2 conduction band. The final aim is
the production of low-cost solid-state photovoltaic cells. Organometallic com-
pounds such as ruthenium(II) polypyridyl complexes have been used frequently
in this field [68, 69]. Chlorophyll derivatives, however, have a better near-infrared
response and the use of environmentally undesirable heavy metals can be
avoided.
11.6
A Look at “Green” Chlorophyll Chemistry
Currently, the inherent lability of the natural pigments precludes any direct
commercial use. Exceptions are dried algae or plant materials or freeze dried ex-
tracts of these. These are hardly uses in the context of a biorefinery, however
[70], and even if a suitable application would be found, regulatory problems
might arise with regard to GMP standards. Thus, any rational use of the pig-
ments, i.e. the macrocyclic part of the chlorophylls, must involve a simple and
facile extraction process and some simple chemical transformations that pro-
duce chemically defined materials of high purity and stability. These require-
ments are counteracted by the need to produce chemicals that are still suffi-
ciently reactive (e.g. carry functional groups) that enable transformations into
desirable and marketable target compounds.
A variety of extraction methods have been developed for small-scale extrac-
tion, and it should be possible to transfer these to large-scale systems. Ignoring
the chlorophyll a/b problem for now, there are several possibilities of utilizing
chlorophyll derivatives as starting materials for other fine chemicals. Almost all
start with conversion of the chlorophylls to the respective pheophorbides, i.e. de-
metalation and hydrolysis of the 172-ester. This reaction is preferably performed
in the presence of an alcohol to yield a pheophorbide ester, 34, which can be
handled in organic solvents. Often it is beneficial to hydrogenate the 3-vinyl
group to yield “meso” compounds (35). There are two means of circumventing
allomerization problems. Either the 132 ester can be removed by demethoxycar-
bonylation [18] to yield the “pyro” compounds 37, or ring V can be opened by a
variety of nucleophiles to yield chlorin e6 36 and/or various other derivatives of
rhodochlorin. Naturally, at all stages it is possible to oxidize the chlorins to the
respective porphyrins (e.g. 38). Fig. 11.11 shows a few selected reactions that
can be applied to chlorophylls in different sequences to yield relatively stable de-
rivatives. Rhodochlorin and phytochlorin derivatives, in particular, serve as very
important intermediates in contemporary chlorophyll chemistry [14, 28].
Any use of chlorophylls as fine chemicals will start with these or closely re-
lated compounds [71]. As a result of ongoing studies in PDT (vide supra) and
the photobiological relevance of these pigments, the chemistry of these com-
pounds is well developed and most of the reactions can be performed on a mul-
tigram scale. Note that oxidation of chlorins to the porphyrins typically results
in “red” chemistry, because of a blue shift of the absorption maxima.
All of the compounds outlined in Fig. 11.11 can be prepared from crude chlo-
rophyll a in a few steps with standard chemicals. Porphyrin chemists are conti-
nually adding new and exciting compounds to this field. Current efforts concen-
trate on modeling the bacteriochlorophylls c–e [72], developing new electron-
transfer compounds [73], modeling compounds for photosynthesis [74], and ap-
plications in biomedicine [37]. Thus, no significant efforts in synthetic chloro-
phyll chemistry are necessary in respect of their use from biorefineries. Rather,
the biotechnological side and the technical side of the extraction processes must
be resolved.
One of the most overlooked areas of chlorophyll chemistry is their use as
new materials. For example, significant enhancement of their photochemical
stability is possible by adsorbing them on solid phases (silica or polymer sup-
ports or smectite conjugates) [75–79]. Such silica-adsorbed materials have been
shown to be capable of generating hydrogen gas when coupled with appropriate
electron carriers [76], whereas PEG-coupled chlide could function in the coupled
generation of glutamate [80]. Despite these promising results, very few groups
Fig. 11.11 Some chlorophyll transformations and key intermediates of chlorophyll chemistry.
are active in this area. The situation is similar with regard to applications in so-
lar energy conversion. Although this has been the catchword in almost any
modern publication dealing with chlorophyll chemistry, very few recent papers
actually deal with the use of isolated chlorophyll derivatives in photovoltaic cells
[66, 81]. Both areas require major research efforts to yield useful applications. If
successful, however, these would provide the impetus necessary to further stim-
ulate this whole line of research.
11.7
There are currently no large-scale commercial uses of chemically pure chloro-

phyll-derived materials. The few specialized applications there are very narrow
in focus and require only small amounts of material (multi-gram to kg scale),
which is conveniently prepared from a dedicated source, e.g. Spirulina. On the
other hand, well-established major industrial uses of tetrapyrroles, for example

use as coloring agents in inks, paints, or plastics and rubber, are exclusively
based on nonnatural technical pigments. For example the annual production of
phthalocyanines is about 70 000 metric tons. In fact, the technically most impor-
tant green pigment is “Phthalo green”, a chlorinated copper phthalocyanine.
These compounds are derived from simple and cheap starting materials in a
few steps. It is therefore doubtful if any bioresource-derived pigments can com-
pete with bulk organic pigments from established synthetic chemistry [82].
We currently have a situation in which people interested in chlorophylls ex-
tract chlorophyll from bioresources and discard everything else, whereas people
interested in biorefinery systems utilize most materials except chlorophylls, de-
spite listing chlorophylls and pigments as one of the target fractions [83]. There
are still several misconceptions in the latter area. For example, a green “pig-
ment/dye” fraction is taken to imply that the content can be used as an indus-
trial pigment. Lack of knowledge about the heterogeneity of the relevant bio-
mass fraction and about the chemical instability of natural tetrapyrrole pig-
ments persists. Also, scientific progress in mimicking nature using artificially
generated systems (e.g., artificial photosynthesis or solar energy conversion) is
often taken as an argument for the use of natural biorenewable systems [84].
From a tetrapyrrole chemist’s viewpoint a biorefinery has three limitations
that currently negate any use of the “green fraction”. First, any mass-produced
large-scale crop that may be used as raw material will contain chlorophylls a
and b, necessitating chromatographic or chemical separation of the two pig-
ment series. Similarly, the green dye fraction contains many other pigments
that cannot be removed by a simple step (e.g. filtration) but will always involve
chromatography (with associated costs and environmental impact because of
solvent use) if pure compounds are required. Third, compared with carbohy-
drates, the green fraction is one of the smallest in terms of mass [85] but one
of the most complex in terms of content. This prevents its use as a source of
pigments as bulk chemicals.
In conclusion, no case can currently be made for industrial-scale use of the
green material from biorefineries. This is, however, partially a result of the ab-
sence of any relevant studies in the subject, the lack of appropriate extraction
methods to yield pigments in high purity, a nonexistent industrial chlorophyll
chemistry, and the lack of large-scale industrial uses for chlorophyll-derived pig-
ments.
What is needed? In the short term, first, a quantitative assessment of the con-
tent and composition of the dye fractions derived from green biorefineries.
Next, such analyses must be correlated with different extraction methods and
processing conditions. When these data are available, it might be possible to op-
timize the work-up conditions for the “green fraction” with regard to optimum
use of the tetrapyrroles. This would also open the possibility of using the green
fraction for industrial scale production of chlorophyllin, the only chlorophyll-de-
rived material immediately marketable. In parallel with this, chemists must de-
velop simple, cost-effective, and environmentally benign reactions that can be
applied to the crude chlorophyll fraction to yield homogenous and pure materi-
als for use as fine chemicals. Similarly, use of the “green biomass fraction” must
be compared economically and environmentally with dedicated use of, perhaps,
bioengineered blue green algae as a biological source of tetrapyrroles [85]. Last,
but not least, more industrial applications must be found for natural tetrapyr-
role pigments and developed to provide an economic stimulus for this area.
Only then will “green” be truly a part of green chemistry.
Acknowledgment
Writing of this article and our own research was made possible by generous
funding from the Science Foundation Ireland.
References and Notes
1 Krasnovsky, A. A. Jr. Photosynth. Res. 11 Senge, M. O.; Wiehe, A.; Ryppa, C. In

2003, 76, 389–403. Chlorophylls and Bacteriochlorophylls: Bio-
2 Besides efforts to develop nonhazardous chemistry, Biophysics and Biological Func-
chemical procedures, see: http://www. tions; Grimm, B., Porra, R. J., Rüdiger,
epa.gov/greenchemistry/ W., Scheer, H., Eds.; Kluwer Academic
3 Willstätter, R.; Stoll, A. Untersuchungen Press: Dordrecht, 2004; in press.
über Chlorophyll. Methoden und Ergebnisse; 12 Senge, M. O. J. Photochem. Photobiol. B:
J. Springer: Berlin, 1913. Biol. 1992, 16, 3–36.
4 Deisenhofer, J.; Michel, H. Angew. 13 Scheer, H. (Ed.) Chlorophylls; CRC Press:
Chem., Int. Ed. Engl. 1989, 28, 829–847. Boca Raton, 1991.
5 McDermott, G.; Prince, S. M.; Freer, 14 Pavlov, V. Y.; Ponomarev, G. V. Khim.
A. A.; Hawthornthwaite-Lawless, A. M.; Geterosikl. Soed. 2004, 483–519; Chem.
Papiz, M. Z.; Cogdell, R. J.; Isaacs, N. W. Heterocycl. Chem. 2004, 40, 393–425.
Nature 1998, 374, 517–521. 15 Svec, W. A. In Chlorophylls; Scheer, H.,
6 Senge, M. O.; Smith, K. M. In Anoxygenic Ed.; CRC Press: Boca Raton, 1991,
Photosynthetic Bacteria; Blankenship, pp. 89–143.
R. E., Madigan, M. T., Bauer, C. E., Eds.; 16 Grese, R. P.; Cerny, R. C.; Gross, M. O.;
Kluwer Academic Publ.: Dordrecht, Senge, M. J. Am. Soc. Mass Spectrom.
1995; pp. 137–151. 1990, 1, 72–84.
7 Even after 23 years in chlorophyll chem- 17 Hynninen, P. H.; Hyvarinen, K. J. Org.
istry, the largest amount of a chemically Chem. 2002, 67, 4055–4061.
pure chlorophyll derivative I have ever 18 Kenner, G. W.; McCombie, S. W.; Smith,
seen, was about 300 g. K. M. J. Chem. Soc., Perkin Trans. 1 1973,
8 Scheer, H. In Chlorophylls; Scheer, H., 2517–2523.
Ed.; CRC Press: Boca Raton, 1991, 19 Kamm, B.; Kamm, M. Chem. Biochem.
pp. 3–30. Eng. Q. 2004, 18, 1–6.
9 Senge, M. O. Chem. Unserer Zeit 1992, 20 Hendry, G. A.; Houghton, J. D.; Brown,
26, 86–93. S. B. New Phytol. 1987, 107, 25–302.
10 Douglas, R. H.; Partridge, J. C.; Dulai, 21 Gossauer, A.; Engel, N. J. Photochem.
K.; Hunt, D.; Mullineaux, C. W.; Tauber, Photobiol. B: Biol. 1996, 32, 141–151.
A. Y.; Hynninen, P. H. Nature 1998, 393, 22 Kräutler, B.; Matile, P. Acc. Chem. Res.
423–424. 1999, 32, 35–43.
23 Treibs, A. Liebigs Ann. 1935, 510, 42.
24 Callot, H. J. In Chlorophylls; Scheer, H., 46 Isler, O. Amer. Patent 1939, 2325681.

Ed.; CRC Press: Boca Raton, 1991, 47 Schmid, M.; Gerber, F.; Hirth, G. Helv.
pp. 339–364. Chim. Acta 1982, 65, 684–702.
25 Storm, C. B.; Krane, J.; Skjetne, T.; 48 See the review by R. H. Dashwood,
Telnaes, N.; Branthaver, J.; Baker, E. W. Linus Pauling Information Center 2004
Science 1984, 233, 1075–1076. at http://lpi.oregonstate.edu/infocenter/
26 However, the ratio of “green” to “red” or- phytochemicals/chlorophylls/.
ganic material contributing to sediments 49 Hein, J. W. J. Am. Dent. Assoc. 1954, 48,
is about 100000 : 1 [24]. 14–19.
27 Rontani, J. F.; Grossi, V. Org. Geochem. 50 See the online-book by R. L. Seibold
1995, 23, 355–366. 1990, chapter 4 at www.wheatgrass.com,
28 Hynninen, P. H. In Chlorophylls; Scheer, USA, Kansas.
H., Ed.; CRC Press: Boca Raton, 1991, 51 Legally, dyes derived directly from bio-
pp. 145–209. mass may not be termed a “natural
29 Woodward, R. B.; Skaric, V. J. Am. Chem. color”, although they may not require
Soc. 1961, 83, 4676–4678. certification by the FDA.
30 Senge, M.; Senger, H. Photochem. Photo- 52 Gilman, V. Chem. Eng. News 2003, 81,
biol. 1988, 48, 711–717. 34.
31 Scheer, H.; Katz, J. J. Proc. Natl. Acad. 53 In fact, the only FDA certified green
Sci. USA 1974, 71, 1626–1629. food color (Green No. 3, Fast Green) is a
32 Wasser, P. K.; Fuhrhop, J.-H. Ann. N.Y. triphenylmethane dye.
Acad. Sci. 1973, 206, 533–548. 54 Imai, K.; Aimoto, T.; Sato, M.; Watanabe,
33 Inhoffen, H. H. Pure Appl. Chem. 1968, K.; Kimura, R.; Murata, T. Chem. Pharm.
17, 443. Bull. 1986, 34, 4287–4293.
34 Llewellyn, C. A.; Mantoura, R. F. C.; Brer- 55 A critical analysis of the literature will be
eton, R. G. Photochem. Photobiol. 1990, published elsewhere.
52, 1037–1041; 1043–1047. 56 A few excerpts from web pages: “Chloro-
35 Peled, A.; Dror, Y.; Baal-Zedaka, I.; Porat, phyll is one of the greatest food sub-
A.; Michrin, N.; Lapsker, I. Syn. Metals stances for cleansing the bowel and
2000, 115, 167–171. other elimination systems, such as the
36 Min, D. B.; Boff, J. M. Compr. Rev. Food liver and the blood. – Chlorophyll is ef-
Sci. Food Saf. 2002, 1, 58–72. fective against anemia and stimulates
37 Nyman, E. S.; Hynninen, P. H. J. Photo- the production of red blood cells in the
chem. Photobiol. B.: Biol. 2004, 73, 1–23. body. It also helps carry oxygen around
38 Arpe, H.-J., Ed. Ullmann. Industrielle the body and to the brain. – Brain Food
organische Chemie; 4th ed.; Wiley–VCH: – . . .it essentially has the same effects in
Weinheim, 1988, 11, pp. 127–129. the body as iron, so it builds our own
39 Kephart, J. C. Econ. Bot. 1955, 9, 3–38. blood naturally. – It is ... a whole food ...
40 Strell, M.; Kalojanoff, A.; Zuther, F. – wonder tonic.”
Arzneimittel-Forsch. 1955, 5, 640–642; 57 Sudakin, D. L. J. Toxicol. Clin. Toxicol.
1956, 6, 8–11. 2003, 41, 195–204.
41 See http://www.fengming.com for sev- 58 Waladkhani, A. R.; Clemens, M. R. Int. J.
eral products and use. Mol. Med. 1998, 1, 747–753.
42 Fugmann, B., Ed. Römpp Lexikon Natur- 59 Chernomorsky, S.; Segelmann, A.;
stoffe. Thieme: Stuttgart, 1997, pp. 491. Poretz, R. D. Teratogen. Carcin. Mut.
43 Almquist, H. J.; Klose, A. A. J Am. Chem. 1999, 19, 313–322.
Soc. 1939, 61, 2557. 60 Morita, K.; Ogata M.; Hasegawa, T. Envi-
44 Binkley, S. B.; Cheney, L. C. ; Holcomb, ron. Health Persp. 2001, 109, 289–294.
W. H.; McKee, R. W.; Thayer, S. A.; Mac- 61 Spikes, J. D.; Bommer, J. C. In Chloro-
Corquodale, D. W.; Doisy, E. A. J. Am. phylls; Scheer, H., Ed.; CRC Press: Boca
Chem. Soc. 1939, 61, 2558. Raton, 1991, pp. 1181–1204.
45 Fieser, L. F. J. Am. Chem. Soc. 1939, 61,
2559, 2661, 3467.
62 Bonnett, R. Chemical Aspects of Photody- ninen, P. H. J. Chem. Soc., Perkin Trans.

namic Therapy; Gordon and Breach Sci. 1 1999, 2403–2408.
Publ.: Amsterdam, 2000. 74 Fajer, J. Photosynth. Res. 2004, 80, 175–
63 Himeshima, N.; Amao, Y. Energ. Fuel. 182.
2003, 1641–1644. 75 Furukawa, H.; Kuroda, K.; Watanabe, T.
64 Woodward, J.; Orr, M.; Cordray, K.; Chem. Lett. 2000, 1256–1257.
Greenbaum, E. Nature 2000, 405, 1014– 76 Itoh, T.; Yano, K.; Inada, Y.; Fukushima,
1015. Y. J. Am. Chem. Soc. 2002, 124, 13437–
65 Inoue, T.; Kumar, S. N.; Karnachi, T.; 13441.
Okura, I. Chem. Lett. 1999, 147–148. 77 Itoh, T.; Ishij, A.; Kodera, Y.; Matsushi-
66 Amao, Y.; Komori, T. Biosens. Bioelectron. ma, A.; Hiroto, M.; Nishimura, H.; Tsu-
2004, 19, 843–847. zuki, T.; Kamachi, T.; Okura, I.; Inada, Y.
67 O’Regann, B.; Grätzel, M. Nature 1991, Bioconj. Chem. 1998, 9, 409–412.
353, 737–740. 78 Ishii, A.; Itoh, T.; Kodera, Y.; Matushima,
68 Nazeeruddin, M. K.; Pechy, P.; Renouard, A.; Hiroto, M.; Nishimura, H.; Inada, Y.
T.; Zakeeruddin, S. M.; Humphry-Baker, Res. Chem. Interm. 1997, 23, 683–689.
R.; Compte, P.; Liska, P.; Cevey, L.; 79 Ishii, A.; Itoh, T.; Kageyama, H.; Mizo-
Coast, E.; Shklover, V.; Spiccia, L.; Dea- guchi, T.; Kodera, Y.; Matsushima, A.;
con, G. B.; Bignozzi, C. A.; Grätzel, M. Torii, K.; Inada, Y. Dyes Pigments 1995,
J. Amer. Chem. Soc. 2001, 123, 1613– 28, 77–82.
1624. 80 Asada, H.; Itoh, T.; Kodera, Y.; Matsushi-
69 Smestad, G.; Bignozzi, C.; Argazzi, R. ma, A.; Hiroto, M.; Nishimura, H.;
Sol. Energ. Mat. Sol. C. 1994, 32, 259– Inada, Y. Biotechn. Bioeng. 2001, 76, 86–
288. 90.
70 Kamm, B.; Kamm, M. Appl. Microbiol. 81 Komori, T.; Amao, Y. Electrochemistry
Biotechnol. 2004, 64, 137–145. 2003, 71, 174–176.
71 The preparation of chlorophyllin uses 82 Herbst, W.; Hunger, K. Industrial Organic
similar principles: exchange of the metal Pigments: Production, Properties, Applica-
to increase stability, ester saponification tions; 3rd ed.; Wiley-VCH: Weinheim,
to yield a water soluble compound. How- 2004.
ever, due to the drastic reaction condi- 83 Kromus, S.; Wachter, B.; Koschuh, W.;
tions allomerization reactions take place Mandl, M.; Krotscheck, C.; Naradoslaws-
to some extent; in addition often the a/b ky, M. Chem. Biochem. Eng. Q. 2004, 18,
mix is used. Thus, as described, chloro- 7–12.
phyllin is not really a chemically defined 84 Moser, A. Chem. Biochem. Eng. Q. 2001,
pure compound. 15, 33–41.
72 Tamiaki, H.; Omodo, M.; Saga Y.; Mor- 85 The chlorophyll content of green feed-
ishita, H. Tetrahedron 2003, 59, 4337– stocks is approximately 0.2% compared
4350. with 2–3% for blue–green algae.
73 Helaja J.; Tauber A. Y.; Tkachenko, N. V.;
Lemmetyinen, H.; Kilpelainen, I.; Hyn-
Part II
Biobased Industrial Products,
Materials and Consumer Products
ISBN: 3-527-31027-4
347
12
Industrial Chemicals from Biomass – Industrial Concepts
12.1
Introduction
The era of a chemical industry based on mineral oil, gas and coal has lasted al-
most 150 years, but will gradually come to an end in the course of the next 50
to 75 years. The two main reasons for this development are:
· The stocks of fossil resources are finite. This has generally been accepted
since the Club of Rome published its report “Limits to Growth” in 1972. First,
mineral oil stocks will be exhausted around 2050 if we continue our present
way of life. Times during which more oil was found each year than was con-
sumed are definitely over. Natural gas will last slightly longer (some 75 years),
and coal will last longest (> 200 years).
· Environmental considerations. All kinds of pollution from global warming to
acid rain, from smog to ground water pollution, can be linked to the use of
fossil fuels. Hence we are really living on the threshold of a new era in chem-
istry – and unfortunately in most chemical research and education this has
not yet been sufficiently recognized.
The hypothesis has been formulated that from 2040 onwards we will no longer
use fossil organic raw materials, as a consequence of exhaustion and environ-
mental considerations. The fundamental question, however, is: “Is this techno-
logically feasible while maintaining our present way of life in terms of food, or-
ganic materials and energy consumption?”
12.2
Historical Outline
The use of renewable resources as raw materials for technical applications is

certainly not new. Humanity already used natural materials from the first civili-
zations onward to meet their basic needs. The first industrial activity was also
ISBN: 3-527-31027-4
348 12 Industrial Chemicals from Biomass – Industrial Concepts
largely based on the use of renewable resources and this continued until the in-
dustrial revolution.
This means that until 1850, all organic consumer products and industrial raw
materials were plant-based. Within the relatively short period of 150 years so-
ciety changed from a mainly plant-based economy to an economy based on fos-
sil fuels (coal until the end of the 19th century, until 1950 mineral oil, and now,
increasingly, natural gas). Wood supplied 70% of the fuel demand in 1870, in
1920 70% came from coal, in 1970 70% from mineral oil.
The use of renewable materials declined substantially with time, mainly as a
consequence of the extremely low prices of petrochemical resources. Currently,
approximately 96% of all organic chemical substances are based on fossil re-
sources. Nevertheless, several important industries are still based on renewable
raw materials. Half of the fiber used in the textile industry is natural material
(cotton, wool, flax), the oleo-chemical industry supplies society’s daily hygienic
needs for soaps and detergents that are based on vegetable oils. The building in-
dustry continues to use natural fiber for thermal insulation purposes. Petro-
chemistry does not always offer a realistic alternative to the use of renewable
materials. Classic examples are, for example, in the production of antibiotics
and of drugs where fermentation processes play an important role. Moreover,
industrial biotechnology frequently has further significant performance benefits
compared with conventional chemical technology, for example higher reaction
rates, increased conversion efficiency, improved product purity, reduced energy
consumption, and significantly reduced chemical waste generation. This branch
of biotechnology has recently been named “White Biotechnology”.
The penetration of bio-based product today is estimated at 5% with growth po-
tential up to 10–20% by the year 2010. The Vision for Bioenergy and Biobased
Products in the United States and the related Roadmap have established far reach-
ing goals for increasing the role of biobased energy and products in this country.
The oil crisis of the 1970s gave renewed impetus to the use of renewable re-
sources. The worlds increasing dependence on fossil resources, and the finite
availability of these, created serious concern. The concern was, however, largely
channeled in the direction of energy security. Renewable materials were less of
a concern and all attention disappeared when the oil price dropped. With in-
creasing awareness and concern in the 1990s about industrial and consumer
waste, and its effect on the environment, the need arose for better biodegrad-
able intermediates and final end products. Those products can naturally degrade
into components that are absorbed back into the natural cycle. Biodegradability
was the new key property of many new products and they were often based on
renewable resources, in view of their intrinsic biodegradability. With regard to
fossil reserves, the world is now faced with the dilemma that while crude oil is
being consumed faster than ever, “proven oil reserves” have remained mostly
unchanged. The cost of exploration and exploitation of crude oil increases,
which is reflected in increasing oil prices. In contrast with this, agricultural raw
materials such as wheat, corn, sugar, and oil crops are becoming cheaper as a
fundamental consequence of increasing agricultural efficiency and yield. This
12.3 Basic Principles 349
trend will most probably continue for some time to come. This long-term trend
may be perturbed by the transitory effects of market imbalances and politics
but for a growing number of applications the economic balance is tipping to-
ward the use of renewable resources; this is also true of bulk chemicals.
Process and catalysis technology revolutionized the chemical industry in the
20th Century. Now the same thing is happening for the production of industrial
chemicals from biomass. A wave of project initiatives is under way globally; the
objective of these is to convert renewable resources into industrial chemicals.
Industrial biotechnology uses biological systems in conjunction with existing
and new thermochemistry, for production of useful chemical entities. Biotech-
nology is mainly based on biocatalysis and bioprocessing (the use of enzymes
and cells to catalyze chemical reactions) and fermentation technology (directed
use of microorganisms), in combination with recent breakthroughs in the fore-
front of molecular genetics and metabolic engineering.
12.3
Basic Principles
Biomass can be used in different ways to provide us with organic compounds

and materials:
1. Nature already produces the desired structures, and isolation of these com-
ponents usually requires only physical methods. Examples include polysac-
charides (cellulose, starch, alginate, pectin, agar, chitin, inulin), disaccharides
(sucrose and lactose) and triglycerides, lecithin, natural rubber, gelatin,
flavors and fragrances, etc. Some current production volumes are: sucrose
115 ´ 106 t a–1, triglycerides 85 ´ 106 t a–1, natural rubber 5.5 ´ 106 t a–1. Cotton,
the natural cellulose fiber, is produced in a volume of 20 ´ 106 t a–1, an
amount which equals the sum of all synthetic fibers (volume order: polyes-
ter > polyamide > acrylic). The possibilities of producing organic chemicals di-
rectly by and from plants by means of plant biotechnology will increase dra-
matically. The plant is the “plant” of the future.
2. One step (bio)chemical modification of naturally produced structures included
in (1), above. Examples include cellulose and starch derivatives, glucose and
fructose, glycerol, fatty acids; ethanol, citric acid, glutamic acid and lactic acid
by fermentation. Lactulose, lactitol, and lactobionic acid by isomerization, hy-
drogenation, and oxidation, respectively, from lactose. Nature provides a vari-
ety of fine starting materials for pharmaceuticals.
3. By several-step modifications of naturally produced structures included in (1),
above, organic chemicals and organic materials are obtained from natural
products. For example: ethanol can be converted to today’s no. 1 organic
chemical, ethylene; sorbitol and mannitol can be produced by hydrogenation
of glucose and sucrose, respectively; vitamin C can be obtained in several
steps from glucose; fatty alcohols and amines can be obtained from triglycer-
ides; and succinic acid can be obtained from glucose and CO2.
4. “Back to C1-Chemistry” by using biomass as the carbon and hydrogen source,

converting it into small fragments (synthesis gas) and building it up again to
the desired structures. In the above the focus has been on chemical structures
and less on the product areas. For some large product groups it can be stated
that the green label (renewables-based) is accepted as a selling advantage..
12.3.1
Primary Conversion Technologies of Biomass
An overview of technically feasible technology for conversion of biomass, ranked

according to water content, is given in Fig. 12.1. The three most important tech-
nologies will be dealt with in some detail.
12.3.1.1 Gasification
Biomass can be converted into power plant fuel by gasification with high yield
and in an environmentally friendly fashion. Also, in the longer term, the eco-
nomics of this process look good, especially for energy crops. Gasification takes
place with air, at temperatures of approximately 850 8C. The gas consists of 13%
H2, 17% CO, 4% CH4, 12% CO2, 13% H2O, and 40% N2, which has a calorific
value of 6 MJ. In the 2040 scenario, 80 EJ a–1 could be produced from waste
streams and 200 EJ a–1 from energy crops, on a global scale. The removal of sul-
fur-containing components, tar, char, and ash from the gas is critical for use in
gas turbines and for methanol production. The technology is promising. Many
pilot plants are in operation, large installations are in the planning phase. The
gas could presumably also be used in Fischer–Tropsch synthesis.
Fig. 12.1 Biomass conversion technology.

12.3.1.2 Hydrothermolysis
During the period 1982–1993, the Royal Dutch Shell Laboratory developed a pro-
cess to convert biomass into liquid fuel, so-called bio-crude. This process is called
HTU (hydro-thermal upgrading). First, biomass is treated in an aqueous slurry at
200 8C and 30 bar, followed by a treatment at 330 8C and 200 bar. This process re-
sults in a bio-crude, an oil with a low oxygen content which can be further upgraded
by catalytic hydrodeoxygenation to a high-quality naphtha or diesel oil with very low
oxygen, nitrogen, and sulfur content. The oil yield is approximately 40%, on the
basis of the biomass feed stock. Wood, agricultural, and domestic (green) waste
streams have been successfully used as feedstocks. According to Shell, this
HTU process is the cheapest route to liquid biofuels. Its cost price would be ap-
proximately $20–40 per barrel, compared with fossil crude oil today at approxi-
mately $12 per barrel. Hence, the process is not yet economical under the current
tax regime. This HTU process and many variants of this process lead directly to bio-
crude, from which known transport fuels and petrochemicals can be manufactured,
without the extra sulfur-removing steps, etc., which are necessary with fossil fuel.
12.3.1.3 Fermentation to Ethanol

By fermentation of biomass (sugars, grain, cellulose, etc.) with yeast a 6.5–11%
solution of ethanol in water is formed, from which 95 or 100% ethanol can be ob-
tained by distillation (or membrane-filtration, or distillation-adsorption). Depend-
ing on the feedstock, chemical or enzymatic hydrolysis is sometimes required first,
to convert the biomass into monosaccharides. Alcohol is a raw material for many
organic chemicals among which, as was already mentioned, is today’s no. 1 organic
chemical, ethylene. In India over 400 000 t a–1 of alcohol is used to make “alco-
chemicals” with acetic acid and ethylene glycol as numbers 1 and 2. In both India
and China, Moreover, aqueous alcohol is used directly for aromatic ethylation (with
ethylbenzene, 1,4-diethylbenzene, and 4-ethyltoluene). Ethanol can also be used
directly as a liquid fuel. The technology is well developed and applied on a large
scale in the USA (corn-based) and in Brazil (sugar cane-based). It is expected that,
as a result of better enzymatic hydrolysis and ethanol processing, with increasing
fossil fuel prices, bioethanol prices will become competitive with gasoline in 2010.
12.4
Current Status
12.4.1
Europe
Renewable raw materials as industrial feedstock for the manufacture of chemi-

cal substances and products have recently received attention from policy makers
in some European Union Member States. It has been recognized that use of re-
newable raw materials as industrial feedstock is already well established in the
energy and transport sectors. In contrast, manufacture of chemicals and other

products from renewable resources, for example oils from oilseed crops, starch
from cereals and potatoes, and cellulose from straw and wood has only recently
received attention from policy makers in some member states. By employing
physical, chemical and biochemical processes, these materials can be converted
into polymers, lubricants, solvents, surfactants and specialty chemicals for
which fossil fuels have traditionally been used.
At the EU level crop-derived raw materials entered the political agenda in
2000 with the establishment of a working group “Renewable Raw Materials”
within the European Climate Change Program. The objective of this working
group was to quantify the possible reduction in greenhouse gas emissions aris-
ing from wider use of RRM-based materials in manufacturing. Although its cur-
rent direct contribution to the reduction of green house gas emissions has been
shown to be rather modest (about 8 million tonnes of CO2 equivalent by 2010),
so called indirect reductions associated with product performance improvements
could be substantially higher (more than 30 Mt CO2 equivalent). Moreover, the
potential for significant reduction in GHG emissions is expected to arise in the
medium to long term as a result of use of RRM in connection with application
of biotechnology.
Beside environmental benefits there will, in the future, be other advantages to
be derived from the wider use of RRM in industry by:
· improving the economic competitiveness of EU industry and agriculture by
giving incentives to use the most advanced technologies, including bio-tech-
nology;
· providing social benefits by rejuvenating rural communities through estab-
lishment of local industries and by providing farmers with additional sources
of income, thereby securing their jobs; and
· further enhancing environmental protection by improving soil and water
quality.
Hence, by saving fossil resources the use of RRM in industry directly contrib-
utes to sustainable development, recently endorsed by heads of States and Gov-
ernments at their summit in Gothenburg as one of the Community’s main po-
litical aims for the future.
It is felt that this industrial sector needs a more detailed presentation of its
situation and requirements, with a view to assessing what kind of action could
be taken at a Community level to increase its prospects.
12.4.2
United States
In the United States, strong motivation has developed in the past decade to re-
duce the nation’s dependence on imported oil and to increase its own energy
supplies by using a more diverse mix of domestic resources. In 1999 a presiden-
tial order triggered a series of initiatives for promotion of the use of renewable
materials. The Biomass Research and Development Act of 2000 led to the estab-
lishment of the Biomass Research and Development Technical Advisory Com-
mittee, which issued the “Vision for Bioenergy and Biobased Products in the
United States” and the “Roadmap for Biomass Technologies in the United
States”.
Challenging long-term goals have been established that will dramatically
transform the role of biomass in the everyday lives of Americans. For produc-
tion of chemicals and materials from biobased products, these goals predict a
substantial increase from five percent of the current production of target US
chemical commodities in 2001 to 12 percent in 2010, 18 percent in 2020, and
25 percent in 2030. By 2030, a well-established, economically viable, bioenergy
and biobased products industry will create new economic opportunities for rural
America, protect and enhance the environment, strengthen US energy-indepen-
dence, provide economic security, and deliver improved products to consumers.
Biobased products are a major new market opportunity for domestically
grown biomass resources. It will be a new source of revenue not only for those
who produce the feedstocks but also for the farmers and others who are in-
volved in the production of biobased products. Continued research can signifi-
cantly increase opportunities for biobased products, expand existing markets,
and open entirely new markets. Current production of biobased textile fibers,
polymers, adhesives, lubricants, soy-based inks, and other products is estimated
at 12.4 billion pounds per year. Total production (biobased and non-biobased) is,
however, in the hundreds of billions of pounds. The growth opportunities for
biobased products are, therefore, enormous. As a result of advanced research,
new concepts in industrial biorefinery could become a reality. In the industrial
biorefinery, any combination of biofuels, electric power, materials, chemicals,
and other products could be produced from local biomass resources.
12.4.3
Products
Renewable biobased products are products created from plant or crop-based

resources such as agricultural crops and crop residues, forestry, pastures, and
rangelands. Many of the products that could be made from renewable biopro-
ducts are now made from petroleum (e.g. petrochemicals).
Plant resources, mostly for paper products and chemical feedstocks, now pro-
vide approximately 5% of manufacturing inputs. Plant-based chemical products
include paints, adhesives, lubricants, inks, polymers, and resins. Use of hydro-
carbon resources is often much less expensive. For some chemical products
plant inputs are already cost-competitive, and they are a significant feedstock.
Plant-based systems are the major sources for ethanol, sorbitol, cellulose, citric
acid, natural rubber, most amino acids, and all proteins.
Companies from the chemical, life sciences, forestry, and agricultural commu-
nities are involved in establishing the renewable bioproducts industry. Their ac-
tivities range from genetic engineering of new plant species to development of
new technologies and processes for converting plants into useful industrial in-
puts. For example, DuPont recently developed a biobased method that uses corn
instead of petroleum-based processes to produce a polymer platform for use in
clothing, carpets, and automobile interiors. Similarly, Cargill Dow’s biorefinery
in Blair, Nebraska is producing polylactide (PLA) polymers from corn sugar.
12.5
Industrial Concepts
12.5.1
Introduction
Total annual biomass production on our planet is estimated at 170 billion tons
and consists of approximately 75% carbohydrates (sugars), 20% lignin, and 5%
of other substances, for example oils and fats, proteins, terpenes, alkaloids, etc.
Of this biomass production, 6 billion tons (3.5%) are presently being used for
human needs, distributed as:
· 3.7 billion tons (62%) for human food use, possibly via animal breeding as an
intermediate step;
· 2 billion tons of wood (33%) for energy use, paper, and construction needs;
and
· 300 million tons (5%) to meet the human needs for technical (non food) raw
materials (clothing, detergents, chemicals, . . .).
Other biomass is used in natural ecosystems or is lost by burning or natural

mineralization processes.
Use of bio-based materials has significant ecological advantages. Agricultural
crops are the preferred starting raw materials, instead of using fossil resources
such as crude oil and gas (Table 12.1). This technology consequently has a bene-
ficial effect on greenhouse gas emissions and at the same time supports the
agricultural sector producing the raw materials. The OECD has collected and
analyzed case studies of the application of biotechnology in such diverse sectors
as chemical, plastics, food processing, textiles, pulp and paper, mining, metal
Table 12.1 Average world market prices for raw materials.
Raw material Average world market prices (2000–2003)
Petroleum 175 1 t–1

Coal 35 1 t–1
Ethylene 400 1 t–1
Corn 80 1 t–1
Straw 20 1 t–1
Sugar 180 1 t–1
refining, and energy. The case studies show that biotechnology not only reduces
costs but also reduces the environmental footprint for a given level of produc-
tion. Capital and operating costs are sometimes reduced by 10–50%. In others,
energy and water use were reduced by 10–80% and use of petrochemical sol-
vents was reduced by 90% or eliminated completely. There are several example
in which biotechnology enabled development of new products whose properties,
cost, and environmental performance could not be achieved by use of conven-
tional chemical processes or petroleum as a feedstock.
12.5.2
Biorefinery Concepts
In biorefineries, biomass is used for production of high added-value chemicals,

materials, intermediates, and fine chemicals together with the production of en-
ergy carriers, preferably in liquid phase, for its higher energy content and easier
transport. In that respect, biorefineries can be compared with existing and fully
integrated petrochemical refineries. A parallel approach is the concept of “Bio-
Cascade”, using dedicated crops in such a way that all the constituents a plant
offers (oils, proteins, fibers, cellulose, lignin, etc. . .) result in a total product mix
with the highest economic value (Fig. 12.2).
A range of different technologies can be used industrially to convert the avail-
able biomass into renewable materials or energy carriers. Industrial activity in
renewable materials is very often linked to the food sector. Many of the renew-
able raw materials that can be considered for technical industrial applications
can be made in food processing plants. For example, sugar or glucose or natural
oils for human food use are also important raw materials for industrial pro-
cesses. The industrial sectors supplying the most important renewable raw ma-
terials are (Table 12.2):
· the sugar and starch sector, which produces carbohydrates such as sugar, glu-
cose, starch and molasses from plant materials like sugar beet, sugar cane,
potatoes, wheat, corn, etc.;
· the oil and fat processing sector which produces numerous oleo-chemical in-
termediates such as triglycerides, fatty acids, fatty alcohols and glycerol from
plant materials like rape seeds, soybeans, palm oil, coconut; and
· the wood processing sector, in particular the cellulose and the paper industry,
produce mainly cellulose, cellulose derivatives, and lignin from wood.
Biorefineries are large industrial factory complexes in which agricultural feed-

stocks are processed and fractionated into intermediate basic products that are
then partially converted into final products. In several instances the intermediate
basic products find their own applicability. The products often have little in com-
mon with the nature of the original plant feedstock. Biorefineries use physical,
chemical, and biotechnological processes; fermentation technology and biocataly-
sis, are particularly important. This technology uses microorganisms and their en-
zymes to convert basic renewable raw materials. Thermochemical conversion pro-
Table 12.2 Estimated world production figures and indicative

world market prices of several renewable and petrochemical
raw materials.
Estimated world production Indicative world market price

(million tons per year) (1 per ton)
Renewable raw materials

Cellulose 320 500
Sugar 140 180
Starch 55 250
Glucose 30 300
Bio-ethanol 26 400
Glutamic acid 1 2000
Petrochemicals
Ethylene 85 400
Propylene 45 350
Benzene 23 400
Terephthalic acid 12 700
Isopropanol 2 700
Caprolactam 3 2000
cesses, although having much potential in the future, have been insufficiently con-
sidered. Thermochemical thermolysis, pyrolysis, and gasification are well known
technology for production of char, oil, and gases. Catalytic thermochemical conver-
sions technology is mostly undiscovered for production of complex molecules
from agricultural feedstocks. Fractionation technology is a preparation process in-
tended to separate agricultural materials into separate families using chemical and
physical methods. Industrial biotechnology, enzymatic technology, and thermo-
chemical conversion technology are complementary with each other for full valor-
ization of renewable feedstocks into a portfolio of value-added chemicals and of
energy. The whole chain of different process steps, each using very different tech-
nology can occur within an industrial complex. These are then referred to as “bior-
efineries” analogous to petrochemical crude oil refineries.
12.5.3
Classes of Bioproduct
The many different industrial bioproducts produced today can be divided into
four major categories.
· Sugar and starch bioproducts derived by fermentation and thermochemical
processes include alcohols, acids, starch, xanthum gum, and other products
derived from biomass sugars. Primary feedstocks include sugarcane, sugar
beet, corn, wheat, rice, potatoes, barley, grain. and wood.
· Oil and lipid-based bioproducts including fatty acids, oils, glycerin, and a vari-
ety of vegetable oils derived from soy, canola, sunflower, or other oil seeds.
Fig. 12.2 Integrated biorefinery.
· Wood chemicals include tall oil, alkyd resins, rosins, pitch, fatty acids, lignin,
turpentine, and other chemicals derived from trees.
· Cellulose derivatives, fibers, and plastics, including products derived from cel-
lulose, including cellulose acetates (cellophane) and other cellulose deriva-
tives.
12.5.4
Opportunities for Industrial Bioproducts
For bioproducts including polymers, lubricants, solvents, adhesives, herbicides,

and pharmaceuticals potential markets are wide ranging. Although bioproducts
have already penetrated most of these markets to some extent (Table 12.5), new
products and technology are emerging with the potential to further enhance
performance, cost competitiveness, and market share (Table 12.4).
Organic chemicals are the most direct and largest target for bioproducts.
Using novel chemistry, the carbon and hydrogen moieties present in biomass
can be rearranged to yields products equivalent to or better than the products
that are produced from fossil feedstocks. Many common organic building blocks
serve as monomers for the production of plastics, which is the largest opportu-
nity for renewable materials (Table 12.3). Several advances in biotechnology,
chemicals processing, engineering, chemistry, and separation technology are
opening up new avenues for biobased polymers such as poly(lactic acid).
Table 12.3 Primary global markets for polymers.
Material Volume (million tons)
Polyethylene 48
Polypropylene 23
Poly(vinyl chloride) 26
Poly(ethylene terephthalate) 13
Polystyrene 14
Butadiene/co-polymers 8
Phenolic resins 5.5
Polyamides 4
The other major organic chemicals markets for bioproducts include organic
acids, alcohols, and solvents. Biomass-based ethanol is an established industry,
with other emerging markets for other alcohols and bio-derived acids.
Lubricants and greases were originally plant based but are now mostly petro-
leum-based. Increasing energy prices and growing environmental concerns over
the impact of petroleum-based products are supporting the entrance back in the
market of vegetable oil-based lubricants and greases.
12.5.5
Product Categories Based on C6-Carbon Sugars to Bioproducts
Biomass sugars are the most abundant renewable resource available. Many of
the products used today, for example citric acid, ethanol, and lactic acid are pro-
duced by fermentation. With a vast range of microorganisms available and
being discovered and exploited, the fermentation of sugars has great potential
for the future. Two types of sugar are present in biomass – six-carbon sugars or
hexoses, of which glucose is the most common, and five-carbon sugars or pen-
toses, of which xylose is most common. The most promising glucose derivatives
include lactic acid, succinic acid, butanol, 3-hydroxypropionic acid, 1,3-propane-
diol, and polyhydroxyalkanoates. Lactic acidderivatives include poly(lactic acid)
polymer, ethyl lactate solvent, acrylic acid, propylene glycol, and pyruvic acid.
Succinic acid derivatives include tetrahydrofuran (THF), 1,4-butanediol (BDO),
c-butyrolactone (GBL), and N-methylpyrrolidone (NMP). 3-Hydroxypropionic
acid derivatives include acrylic acid, acrylonitrile, and acrylamide.
12.5.6
Product Categories Based on C5-Carbon Sugars to Bioproducts
Pentose sugars such as xylose have thus far been an untapped resource. The
microorganisms currently available prefer glucose to pentose. By developing mi-
croorganisms that convert pentose sugars alone or in combination with glucose,
the overall economics of biobased products can be improved by enabling full
Table 12.4 Industrial biobased product opportunities.
Technology Platform Chemical Application
Sugar Fermentation Lactic acid Acidulant, electroplating

additive, textile/leather
auxiliary
Poly(lactic acid) Thermoplastic polymer
Ethyl lactate Solvent, intermediate
1,3-Propanediol Specialty resins
Succinic acid Surfactant, food, pharma, anti-
biotics, amino acid and
vitamin production
Succinic acid derivatives Solvents, adhesives, paints,
Tetrahydrofuran printing inks, tapes, plasticiz-
1,4-Butanediol er, emulsifier, de-icing
c-Butyrolactone compound, herbicide
N-Methylpyrrolidone
3-Hydroxypropionic acid and Acrylates, acrylic polymers,
derivatives fibers and resins
Acrylic acid
Acrylonitrile
Acrylamide
n-Butanol Solvent, plasticizer, polymer,
resin
Itaconic acid Aluminum anodizing agent,
reactive comonomer
Sugar fermentation and Propylene glycol Solvent, chain extender in PU,
thermochemical antifreeze, plasticizer
Sugar thermochemical Levulinic acid and derivatives Oxygenates for fuels, biode-
Methyltetrahydrofuran gradable herbicide, bisphenol
d-Aminolevulinic acid A alternative, comonomer,
Diphenolic acid solvent
THF, 1,4-butanediol
Oils and lipids Lubricants and hydraulic Polyurethanes
fluids
Solvents
Polymers
Biomass gasification Fischer–Tropsch and Fuels, solvent, aerosol, oxygen-
gas-to-liquid products ate, intermediate for mono-
Methanol mers and polymers
Higher alcohols
Biomass pyrolysis Phenol–formaldehyde resins Plywood, oriented strandboard
Table 12.5 Current industrial bioproducts from biomass.
Category Technology Feedstock Chemical Application
Starch and Biochemical Carbohydrate Lactic acid, citric Polymers, sol-

sugars biomass acid, ethanol, vent, pharma,
starch, sorbitol, cleaners, coating,
levulinic acid, inks, surfactants,
itaconic acid paints, adhesives,
cosmetics
Oil/lipids Thermochemical Oils/lipids Glycerin, alkyd Pharmaceuticals,
derived from resins, poly- resins, lubri-
soybean, canola, urethane, epoxi- cants, paints,
sunflower dized oils, fatty printing inks,
alcohol, fatty foams, elasto-
acid/ester mers, coatings,
personal care,
surfactants
Forest derivatives Thermochemical Pine, black Turpentine, oil, Solvents, soaps,
liquor, soft wood rosin, tall oil, detergents,
lignin, cellulose personal care,
adhesives, inks,
phenolic resins,
plastics, textiles
utilization of all carbohydrates present in biomass. Potentially important xylose

derivatives are itanonic acid, furfural, furfuryl alcohol, and 2-hydroxymethyl tet-
rahydrofuran.
12.5.7
Thermochemical Conversion of Sugars to Bioproducts
Biomass sugars can be upgraded to value-added products by use of thermo-

chemical conversion routes in addition to fermentation routes. Some thermo-
chemical conversion routes have been used for many years, such as the produc-
tion of sorbitol from glucose.
In recent years, production of high value-added products from biomass has
been advancing rapidly, because of the development of new and improved cata-
lysis and process technologies. Much focus on sugar thermochemistry has been
on sorbitol, levulinic acid, and their derivatives. In that respect, the conversion
of sorbitol to glycols is creating new opportunities for bioproducts. Current pro-
duction of propylene glycol and ethylene glycol is petroleum-based. Glycols are
largely used in numerous polyester resins, copolymers, polyethers, and alkyd re-
sins, and in personal care products, coatings, printing inks, heat-transfer fluids,
and antifreeze. Levulinic acid has been well explored as a platform intermediate.
The current production cost of fossil feedstock-based levulinic acid ($ 4.00–6.00
per pound) is prohibitive for large-scale applications. Levulinic acid can be pro-
duced from lignocellulose material such as paper mill sludge, paper and wood
solid waste, and agricultural residues applying high temperature dilute-acid hy-
drolysis. The manufacturing cost for levulinic acid using such technology has
the potential to be reduced to below $ 0.50 per pound. Important chemicals de-
rived from levulinic acid include methyltetrahydrofuran (MTHF), d-aminolevuli-
nic acid (DALA), and diphenolic acid (DPA).
MTHF can be blended with gasoline up to 70% by volume without adverse
engine performance or reduced mileage. MTHF will have to complete with bio-
ethanol to penetrate the transportation fuel market. DALA is the active chemical
in a new group of herbicides and pesticides. DPA is an alternative to bisphenol
A which is used in polymers such as polycarbonates and as a comonomer in
phenolic resins. Other potential derivatives of levulinic acid include tetrahydro-
furan, 1,4-butanediol, c-butyrolactone and N-methylpyrrolidone.
Thermochemical xylose derivatives include ethylene glycol, propylene glycol,
and glycerol. High hemicellulose content agricultural fiber waste can be hydro-
lyzed to xylose and other C5 sugars.
12.5.8
Thermochemical Conversion of Oils and Lipid Based Bioproducts
The fatty acid methyl ester of soybean oil is an excellent biobased solvent. It is
produced by transesterification of soy oil with methanol, resulting in a mixture
of soy fatty acid methyl esters. Methyl soyate-based biobase solvents have been
introduced commercially in recent years. They match or even surpass the per-
formance of some conventional solvents while being cost-competitive. Methyl
soyate has superior solvent properties, is readily biodegradable, and has a low
toxicity compared with other common chemicals.
The diversity of structure and inherent functionality of vegetable oils make
them prime candidates for use in polymers and resins. The fatty acid chain in ve-
getable oils, which is hydrocarbon in nature, can be transformed with a spectrum
of traditional and breakthrough chemistries to yield high-performance products
with desirable properties. The chemistry being considered to modify and functio-
nalize vegetable oils includes transesterification, epoxidation, hydroformylation,
and metathesis. Transesterification and epoxidation are already being used to
modify soy oil for use in industrial products. Hydroformylation and metathesis
are well developed for use on petroleum feedstocks. The challenge will be to de-
velop similar catalyst systems that are effective and efficient on vegetable oils.
12.5.9
Bioproducts via Gasification
The gas mixture produced by gasification is synthetic gas (syn-gas), a mixture of

carbon monoxide, carbon dioxide, hydrogen, and methane. Syn-gas can serve as
a fuel to produce power and/or may serve as one source of hydrogen for hydro-
gen fuel cells. It can also be converted to valuable chemicals and liquid fuels.
Methanol can be produced from syn-gas. Methanol is a versatile and key start-
ing compound for production of several chemicals, for example acetic acid, for-
mic acid, higher alcohols, MTBE, methyl chloride, methylamines, formaldehyde,
or dimethyl ether. The methanol to olefins route (MTO) seems an interesting
future route for production of propylene and ethylene which, in turn, are used
to produce polyethylene, polypropylene, and other bulk components, for exam-
ple propylene oxide and its derivatives. Ethanol can also be produced from syn-
gas and this might become a cost-effective alternative to the sugar fermentation
route. Fischer–Tropsch chemistry is another approach for converting syn-gas to
valuable chemicals and fuels. The chemicals that can be produced include paraf-
fins, monoolefins, aromatics, alcohols, aldehydes, ketones, and fatty acids.
12.5.10
Bioproducts via Pyrolysis
The pyrolysis technology that is closest to commercialization is the pyrolysis of

high lignin-containing lignocelluloses; this yields a replacement for phenol in
phenol–formaldehyde resins. Phenol–formaldehyde resins are used in plywood,
oriented strandboard, and many other applications that serve the building and
construction industry.
12.5.11
Biocomposites
Over the past few decades, high performance composites have replaced metals
in some applications, and wood in many applications. Biobased materials have
the potential to replace one or both parts of composite systems thereby main-
taining or improving performance. Plant fibers are very ductile and do not
splinter, producing panels that are more shatter-resistant than traditional com-
posites made with wood flour or sawdust. Fibers that could be used include ke-
naf, jute, sisal, coir, flax, and straw.
12.6
Sustained economic growth depends on a secure supply of raw material inputs.

With rapid world growth and continuing changes in consumer demands, there
is a need to find additional, preferably renewable, resources for industrial pro-
duction and energy needs. The need is growing to explore the developing tech-
nology front to capture opportunities that are provided by renewable resources.
Technology advances are beginning to make an impact on reducing the cost of
production of industrial products and fuels from biomass, making them more
competitive with their equivalents produced from petroleum-based hydrocar-
bons. Developments in pyrolysis, gasification technology, separation technolo-

gies via centrifuges or membranes, and the use of enzymes and microbes as
biological factories are enabling the extraction of value-added chemicals and in-
termediates from plant-based materials at competitive cost. As a consequence,
industry is investing in the development of new bioproducts that are steadily
gaining market share.
In the chemicals and food-processing industries, companies are developing
new technology that will enable more cost-effective production of industrial
products from biomass. Approximately 5% of chemical sales currently depend
on biotechnology, but that figure could jump to 10–20% by 2010. The Vision for
Bioenergy and Biobased Products in the United States has put forward an ambi-
tious goal for bioproducts. The share of target chemicals that are biobased is set
at 25% by 2030. Ethanol is by far the largest-volume chemical made by biopro-
cessing, but several other categories, including citric and lactic acids, amino
acids, and pharmaceutical intermediates are being investigated. The big shift,
however, will be the growing importance of biotechnology processes to make
bulk chemicals, polymers, and specialty chemicals.
In the chemicals and food-processing industries, companies are developing
new technology that will enable more cost-effective production of all kinds of in-
dustrial product from renewable resources. One example is a plastic polymer
derived from corn that is being produced at a 300 million pound per year plant
in Nebraska, a joint venture between one of the world’s largest grain merchants,
Cargill, and The Dow Chemical Company, the largest chemical producer. Other
chemical producers are exploring the use of lost-cost biomass processes to pro-
duce chemicals and plastics that are now made from more expensive petro-
chemical processes. Strategic partnerships between the chemical industry, food,
textiles, and agricultural sectors are expected to become the mainstay of the
emerging bio-products industry and foster its growth over the coming decades.
Although we look forward and point to future potential, it is also recognized
that change must start today. Change itself is often continuous, whereas break-
throughs occur at infrequent intervals. Successful progress in this field of new
technology will be achieved by integrated and multidisciplinary research in a
phase approach. Many current limitations of the use of renewable materials
arise from attempts to fit carbohydrate chemistry into a hydrocarbon based
chemistry pattern. This is often difficult. Use of renewable materials requires
the development of concepts around alternative processing. In the short term,
modified processes will enable economic use of renewable resources, and long-
er-term opportunities exist via the smart combination of chemistry with ad-
vanced engineering and with recent biotechnology advances. Performing the re-
search and development sequentially will be slow and take many years. The op-
timum approach is to ensure coordinated, parallel processing of research results
and key target area. Such an approach should also encourage partnership be-
tween the public and private sectors.
Plant based renewable resources are strategic options to meet the growing
needs for industrial building blocks. There will be economic, environmental
and societal advantages from the development of this new resource base. The
appropriate mix of research and development and technology development, in
combination with market and public policies, can support the development and
the demonstration of viable chemicals, intermediates, and materials, in combi-
nation with fuels, heat, and power supply. The impetus for new bio-products
will continue to come from favorable government policies, the implementation
of bio-refineries, and the desire to reduce the need for and dependence on im-
ported oil. Perhaps the greatest factor driving the growth of bio-products will be
acceptance by the public, business enterprises, and governments that biomass
can provide a solution to some of the most pressing global resource problems.
The impact of the bio-industry on rural development and economics has not yet
been quantified, but could be impressive. Development of a bio-industry will re-
quire increased production and processing of bio-mass and could provide a
boost to rural areas. It could create new income for farmers and foresters. De-
velopment of a larger bio-industry would require new processing, distribution,
and logistics, and new service industries. This could potentially result in posi-
tive economic impacts on rural economic growth in many parts of the world.
Using plants as feedstock instead of petroleum or natural gas can potentially
reduce the amount of carbon dioxide emitted to the atmosphere. Globally, ap-
proximately 62 Gigatons of carbon are taken-up by plants annually in photo-
synthesis. Producing chemicals and industrial products from biomass directly
reduces the associated carbon released during the production of fossil-based
products.
References
Ashford, Robert D., Ashford’s Dictionary of “Current situation and future prospects of EU
Industrial Chemicals, 2nd Edition, (Wave- industry using renewable raw materials”,
length Publications Ltd., London, 2001) European Renewable Resources and
“Affordable Resins and Adhesives from Opti- Materials Association (EERMA), Brussels,
mised Soybean Varieties”, US Department 2002
of Energy, Industrial Technologies Pro- Deamin, A. L., Small bugs, big business: The
gram Fact Sheet, May 2002 economic power of the microbe, Biotechnol-
ACS Symposium Series 742: Lignin: Histor- ogy Advances 18 (2000) 499–514
ical, Biological, and Materials Perspectives, “Developing and Promoting Biobased Products
W. G. Glasser, R. A. Northey and T. P. and Bioenergy”, ordered by William J. Clin-
Schultz, 1999, ISBN 0-8412-3611-9 ton, U.S. President, August 1999 (White
Barger, Paul. Methanol to olefins (MTO) and House, 1999)
beyond. Catalytic Science Series 2002, 3 M. Sasaki, Z. Fang, Y. Fukushima, T. Ad-
(Zeolites for Cleaner Technologies), 239– schiri, K. Arai, Dissolution and Hydrolysis
260. of Cellulose in Subcritical and Supercritical
Biomass Research and Development Act of Water, Ind. Eng. Chem. Res. 2000, 39,
2000, http://www.bioproducts-bioenergy.- 2883–2890
gov/bio_act.html Eggersdorfer, M.; Meyer, J. Eckes, P.; Use of
Bioprocessing, Reaping the Benefits of Renew- renewable resources for non-food materials;
able Resources, Chemical Week, February FEMS Microbiology Reviews 103 (1992)
11, 2004 355–364.
References 365
EU FP6 Expression of Interest, http://eoi.cor- NSF Workshop on “Catalysis for Biorenew-

dis.lu/dsp_details.cfm?ID=25887, 2002 ables Conversion”, April 13–14, 2004
“Functionalized Vegetable Oils for Utilization www.egr.msu.edu/apps/nsfworkshop
as Polymer Building Blocks”, US Depart- OECD2001 The Application of Biotechnology
ment of Energy, Industrial Technologies to Industrial Sustainability (www.oecd.org/
Program Fact Sheet, May 2001, http:// sti/biotechnology)
www.oit.doe.gov/agriculture Okkerse, H; Van Bekkum, H. From fossil to
Dale, B. E.; Greening the chemical industry: re- green. Green Chemistry, April 1999, 107–
search and development priorities for biobased 114
industrial products, J Chem. Technol. Bio- Oleochemical Manufacture and Applications,
technol. 78: 1093–1103 (2003) Edited by F. D. Gunstone and R. J. Hamil-
Industrial Biotech and Sustainable Chemistry, ton, CRC-Press 2001, ISBN 1-84127-219-1
European Biotechnology News, No 1–2, Steps towards a sustainable development, A
Volume 3, 2004, pp. 28–29 White Book for R&D of energy-efficient tech-
Industrial Biotechnology and Sustainable nologies, Eberhard Jochem, March 2004, A
Chemistry, Royal Belgian Academy Council Project of Novatlantis – Sustainability at
of Applied Sciences, January 2004 the ETH-Domain (Zurich-CH)
Johnston, S., “Emissions and Reduction of D. H. Meadows, D. L. Meadows, Jorgen Ran-
Greenhouse Gases from Agriculture and Food ders, William W. Behrens III, The Limits
Manufacturing”, S.C. Johnson Associates, to Growth, A Report to The Club of Rome
Inc., for the U.S. Department of Energy, (1972), Universe Books, New York, 1972
December 1999 The Refining Process, Corn Refiners Associa-
Industrial Bioproducts; Today and Tomorrow, tion, Washington DC, August 2002
US Department of Energy, July 2003 Transforming biomass to hydrocarbon mixtures
Matsumura, Y.; Minowa, T., International in near-critical or supercritical water: USP
Journal of Hydrogen Energy 29, 2004, 701– 6,180,845 B1
707 “Trash to Treasure”, DOE Pulse, No 50, Feb.
Mangold, E. C., Munradaz, M .A., Quellette, 28 2000, http://www.ornl.gov/news/pulse
R. P., Farah, O. G., and Cheremisinoff, Vision and Roadmap for Bioenergy and Bio-
P. N., Coal Liquification and Gasification based Products in the United States, DOE,
Technologies, (Ann Arbor Science Publish- October 2002 http://www.bioproducts-
ers, Inc., Michigan) 1982 bioenergy.gov/board.html
NACHRICHTEN – Forschungszentrum White Biotechnology: Gateway to a More Sus-
Karlsruhe Jahrg. 33 1/2001, p. 59–70, A. tainable Future, www.europabio.org, 2003
Kruse, ITC H. Wiedenroth, Zuckerrüben-Magazin,
NACHRICHTEN – Forschungszentrum Nr. 32, September 2002
Karlsruhe Jahrg. 35 – 3/2003
367
13
Succinic Acid – A Model Building Block
for Chemical Production from Renewable Resources
13.1
Introduction
The U.S. currently produces about 10 billion bushels of corn annually, with
about 20%, or 2 billion bushels, processed by either wet or dry milling. Approxi-
mately 550–600 million bushels of the processed corn are used to produce etha-
nol. The remainder is processed to produce food and non-food products.
While corn wet milling has been practiced since the mid 1800s, significant
technology advancements have continued to improve processing efficiency and
bring about a substantial reduction in water usage and energy requirements.
The corn wet milling process consists of seven major unit operations: 1) corn
cleaning and inspection, 2) steeping, 3) grinding, 4) germ separation, 5) fiber
separation, 6) starch and protein separation, and 7) downstream processing.
The major components from corn wet milling include corn oil (1.5 pounds
per bushel), corn gluten meal (2.6 pounds per bushel 60% protein), corn gluten
feed (13.5 pounds per bushel – 20% protein), and starch (32 pounds per bush-
el). Corn oil is extracted from the germ, refined and used to produce finished
oil. Corn gluten feed is produced from combining the fiber fraction with the
steep water followed by drying, and is sold primarily as animal feed. Corn glu-
ten meal is derived from the protein fraction, and is also sold as animal feed.
The starch is recovered and converted by several processes to produce various
starch products. The starch is also enzymatically hydrolyzed and used as a feed-
stock to produce products such as high fructose corn syrup, ethanol, lactic acid,
lysine, citric acid, and a variety of other fermentation products. However,
through further technology advancements, additional valuable products can be
derived from corn processing operations.
Several new technologies are under development for the production of value
added chemicals from starch. This is driven by the desire to expand the capacity
at existing corn processing facilities and for improving the overall economics of
the integrated biorefinery. The integrated biorefinery is defined here as a facility
producing both fuels and chemicals. Specific products currently under develop-
ISBN: 3-527-31027-4
368 13 Succinic Acid – A Model Building Block for Chemical Production from Renewable Resources
Fig. 13.1 Starburst diagram of succinic acid as a building block chemical.
ment or in various stages of commercialization include lactic acid for the produc-
tion of polylactic acid, direct hydrogenolysis of sorbitol for the production of pro-
pylene glycol and ethylene glycol and the production of polyols for polyurethanes.
The product targets have been established because of the potential economic at-
tractiveness and synergistic fit with the existing corn wet milling infrastructure.
One of the major considerations for the development of new technologies that
can be utilized in a corn wet mill for the production of new chemical products
is the concept of platform building blocks. This concept is based on the fact that
a single building block has the potential to create a significant number of final
products. Succinic acid represents a building block that can be used as a start-
ing material for producing a large number of commodity and specialty chemi-
cals. Succinic acid itself is derived via the fermentation of both C6 and C5 su-
gars. The derivatives of succinic acid can be obtained via a variety of catalytic
processes, primarily hydrogenation. Succinic acid and potential derivatives of
succinic acid are represented in the “starburst” diagram below (Fig. 13.1).
13.2
Economics of Feedstock Supply
The major feedstock from corn wet milling to be utilized for the production of
fuels and chemicals is starch. Starch costs are estimated to be in the range of
$0.05–0.06 per pound. The cost of starch is based on the overall corn wet
milling process and the co-product value associated with the oil, corn gluten
feed, and corn gluten meal. The actual starch cost depends on both the specific
corn wet mill and the size of the mill. Corn pricing has remained essentially
constant over the past several decades. The average annual corn price over the
past 10 years has been $2.37/bushel.
The fiber fraction of corn (5.5 pounds per bushel) is another feedstock oppor-
tunity that represents a low cost source of five carbon sugars, xylose and arabi-
nose, that could be used as a fermentation feedstock. Corn fiber is currently
sold as part of corn gluten feed, and the value of the feed is on the order of
$0.05 per pound. The real value in corn gluten feed is the protein, and the fiber
is primarily used as a carrier for the protein.
The economics of the fermentation of glucose to succinic acid are driven by a
number of key factors including sugar costs, yield of succinic acid from sugar,
media costs and productivity. Within these technical challenges the two major
issues are the development of an organism that can use a minimal media and
improving the overall productivity. The minimal media requirement is that a
low cost media such as corn steep liquor can be the sole source of nutrients. It
is also essential that the productivity be high enough that the capital costs do
not become dominant in the economics. Initial economic analysis has shown
that the productivity requirements be on the order of 2–3 g L–1 h–1. The chal-
lenge in developing a competitive process for the production of succinic acid
and its derivatives is developing a fermentation process that can produce succin-
ic acid at a cost structure comparable to the cost of maleic anhydride from bu-
tane. This cost structure must allow for purification costs of succinic acid as
well as the overall cost of the fermentation. The economics of the catalytic
transformations are competitive with the catalytic conversion of maleic anhy-
dride.
13.3
Succinic Acid Fermentation
In the last decade numerous patents have issued on anaerobic production of

succinic acid, microorganisms for such fermentations, and methods for purifi-
cation, including both separations from cellular biomass and salt splitting. Two
US organizations that have worked extensively in this area are Michigan Bio-
technology Institute (MBI) [1] and Argonne National Laboratory [2]. Other nota-
ble work has taken place at University of Georgia [3] and outside the US in-
cludes laboratories in Japan [4] and Korea [5].
Succinate fermentation faces several challenges that must be overcome before
an industrial process can be realized. A few illustrative examples are given be-
low. The first challenge is robustness of the organism. Microorganisms that
naturally produce succinic acid are often not able to tolerate large concentra-
tions of the acid or its salt and either die or cease growth and active metabo-
lism, making such organisms unsuitable for industrial use [1 a, 2 a]. This results
in low final titers which adds to production and separation costs.
A second challenge is media cost. Often expensive nutrients found in yeast
extract are required. These might include tryptophan, cysteine, methionine bio-
tin, folic acid, thiamine, riboflavin, and various others [1c]. Niacin may be the
important yeast extract component as the organism must regenerate NAD rap-
idly to achieve the required productivity levels. The cost of fermentation nutri-
ents can have substantial effect on production costs.
A third challenge is production of co-products such as acetic acid or pyruvic
acid [1 c]. These co-products result in lower titers of desired products and must
be removed if additional catalytic upgrading of succinate is to be done.
A fourth challenge is the narrow pH range under which bacterial microorgan-
isms can operate. The required neutral conditions for maintaining succinate
production (usually between pH 5.8–7.2) [1 a], necessitates base neutralization.
Common bases include ammonia or sodium or potassium hydroxide. Thus the
actual fermentation product is not succinic acid but rather the ammonium, so-
dium or potassium salt. Acidification can represent of to 30% of the total cost
of organic acid production.
The patent literature provides examples of relatively high succinate concentra-
tions produced at neutral pH. Examples of three microbial variants developed
by MBI are given in Table 13.1. The data was obtained from shake flask experi-
ments using a peptone, yeast extract, glucose medium [1 b].
The final titer for succinate approached and even surpassed 90 g L–1. How-
ever, substantial amounts of either acetate or pyruvate were co-produced.
Furthermore, the nutrient concentrations, even for the “low nutrient” conditions
were substantial. It is interesting to note that the organism that gave low acetate
had an increased production of pyruvate.
Organism FZ 6 was selected for further evaluation using various nutrient
conditions. Results are shown in Table 13.2. Increasing the corn steep liquor
concentration had a dramatic effect on the final titer, whereas increasing the
yeast extract concentration had only a moderate effect. Examples with lower
yeast extract were not provided.
Table 13.1 Succinate fermentation described by MBI (US Pat-

ent 5,573,931). Vial fermentation with “low nutrient“ condi-
tions (corn steep 10 g L–1, yeast extract 5 g L–1).
Strain Concentration (g L–1) Yield a)
Succinate Acetate Formate Pyruvate Propionate
130Z 68.5 15.1 3.9 9.8 0.9 85

FZ 6 86.9 4.9 0 12.5 0.7 94
FZ 21 96.4 16.5 0 4.4 3.6 89
a) Yield = (grams succinic acid/grams dextrose) ´ 100

Table 13.2 Effect of nutrients on FZ 6 succinate yield (g L–1).

(Reproduced from US Patent 5,573,931).
Nutrient level CSL a) Yeast extract Ave succinate Yield (wt%)
“Low” 10 5 84.9 92
“Medium” 15 5 93.4 94
“High” 10 15 90.4 82
a) Corn steep liquor
The high producing succinate strain FZ 21, was also examined in a 1 L fer-
menter using 15 g L–1 yeast extract, corn steep liquor, MgCO3 (for pH control)
and glucose. The succinate yield was 78%. Results are shown in Fig. 13.2.
Other groups have also been involved in strain development. Strains devel-
oped at Argonne have been demonstrated on more complex carbohydrate
streams. For example they have used industrially produced rice straw hydroly-
sate (Arkenol, Inc.) with Difco yeast extract (5 g L–1), tryptone (10 g L–1) as well
as various buffers. Figure 13.3 shows data for the consumption of the two major
Fig. 13.2 Succinate production with time (Reproduced from US Patent 5,573,931).
Fig. 13.3 Glucose and xylose uptake during fermentation

(Reproduced from US Patent 6,743,610).
sugars7glucose and xylose7and production of major products7succinate, ace-

tate, and ethanol. In this example the final succinate titer reached 63 g L–1 in
192 h. The amount of acetate co-produced was 4.9 g L–1.
In summary, there has been substantial strain development for the purpose
of commercial succinate production. The efforts described have concentrated on
improvements in final titer, organism robustness, and to a lesser extent selectiv-
ity (less acetate). The Argonne group has also demonstrated the use of more
complex carbohydrate hydrolysate streams. In all examples, fermentations were
done at neutral pH. However, it is believed that several groups are working on
low pH fermentations.
13.4
Succinic Acid Catalytic Transformations
The chemical conversion of succinic to produce both commodity chemicals and

specialty chemicals is shown in Fig. 13.4. The major products include 1,4-butane-
diol (1,4-BDO), tetrahydrofuran (THF) and c-butyrolactone (GBL). These products
can all be produced via catalytic hydrogenation and are analogous to the products
produced from maleic anhydride via hydrogenation. One of the potential specialty
13.5 Current Petrochemical Technology 373
Fig. 13.4 Derivatives of succinic acid.
products that can be produced from succinic acid, or more appropriately from dia-
mmonium succinate (DAS) is N-methylpyrrolidinone (NMP).
13.5
Current Petrochemical Technology
13.5.1
1,4-BDO, THF, GBL, and NMP
Several catalytic technologies have been developed in the past for the production
of 1,4-BDO from petroleum based resources. Early technology development in-
cluded a two step process based on Reppe chemistry. The first step in the pro-
cess is the conversion of acetylene and formaldehyde to produce primarily 1,4-
butynediol. The second step is the catalytic hydrogenation of 1,4-butynediol to
1,4-BDO. The 1,4-BDO produced can be dehydrated to produce THF.
A second well know technology is the Lyondell propylene oxide based process.
This process utilizes propylene oxide as the starting material and converts PO
to allyl alcohol via isomerization. The allyl alcohol is converted to 4-hydroxybu-
tyraldehyde via hydroformylation with CO and H2. The final step is the hydro-
genation of the aldehyde to produce 1,4-BDO.
A third process utilizes butadiene as the starting material. In this process, buta-
diene is reacted with acetic acid and oxygen to produce the intermediate 1,4-dia-
cetoxy-2-butene and water. The 1,4-diacetoxy-2-butene is then hydrogenated over
a catalyst to form the saturated intermediate which is then hydrolyzed to pro-
duce 1,4-BDO.
The process developed by Huntsman employs a catalyst for the direct oxidation
of butane to maleic anhydride. Maleic anhydride is then reacted with methanol
to produce dimethyl maleate. The dimethyl maleate is hydrogenated to produce
1,4-BDO.
The final process is the process developed by BP Amoco and Lurgi. The process
is also based on the direct oxidation of butane to maleic anhydride. This process
differs from the Huntsman process in that the maleic anhydride is directly re-
duced over a catalyst to produce THF.
It is currently believed that this is the low cost technology for THF production.
All of these technologies are described in more detail in the open literature and
the patent literature [6].
The production of THF can be achieved from any of the technologies that
produce 1,4-BDO. In one form or another BDO or one of its precursors is dehy-
drated to yield THF. The overall yield is generally greater than 90%.
GBL is produced by the cyclic dehydrogenation of 1,4-BDO. The majority of
GBL produced is used for the production of N-methylpyrrolidinone (NMP). In
the process to produce NMP, GBL is converted in a three stage liquid phase
process. The reaction is carried out by reacting GBL with methylamine and
water. After reaction the final NMP produce is recovered via distillation with
methylamine and water being recycled back to the process.
13.6
Current Biobased Technology
13.6.1
1,4-BDO, GBL, and NMP
There is extensive patent literature regarding the conversion of maleic acid and
succinic acid to produce 1,4-BDO, THF, GBL and NMP, the bulk of this work
has been developed utilizing petrochemical feedstocks as the starting material. There
has been a relatively low level of effort on the direct aqueous phase catalytic con-
version of bio-derived succinic acid to value added products. The remainder of this
chapter will describe work at Pacific Northwest National Laboratory on the cataly-
sis of converting succinic acid and diammonium succinate and describe some of
the unique challenges associated with fermentation derived starting materials.
Pacific Northwest National Laboratory has developed a series of supported
metal catalysts for the highly selective, aqueous phase hydrogenation of succinic
acid to GBL. The basic catalysts materials consist of an active form of palladium
on a specific carbon support. Selection of the carbon support with the appropri-
ate surface chemistry and porosity is critical in achieving highly selective cata-
lysts. Pacific Northwest National Laboratory has demonstrated that an active pal-
ladium on carbon can afford yields of GBL greater than 95%. The reaction is
carried out in either a batch mode or in a continuous mode. Operating tempera-
tures are on the order of 150–175 8C with pressures ranging from 800–2000
psig. The weight percent of succinic acid can range from 5–25%.
An important aspect of this technology development is that during the fer-
mentation of glucose to produce succinic acid, acetic acid is often seen as one
of the co-products. The technology developed at Pacific Northwest National Lab-
oratory is selective in the hydrogenation of succinic acid in the presence of
acetic acid, to the point that none of the acetic acid is hydrogenated to ethanol.
This has important economic implications in that acetic acid could be recovered
as a co-product and that selective reduction of succinic acid without the reduc-
tion of acetic acid reduces the total consumption of hydrogen.
A second element of this work relates to the production of 1,4-BDO. In an at-
tempt to maximize the yields of 1,4-BDO, a process was developed in which
GBL produced by hydrogenation of succinic acid is separated via distillation.
The purified GBL is then catalytically hydrogenated over bimetallic catalysts
supported on carbon. The bimetallic include nickel and palladium coupled with
rhenium. These catalysts have afford selectivity greater than 95% to 1,4-BDO
from GBL. Operating temperatures range from 150–200 8C with hydrogen pres-
sures ranging from 1000 to 2000 psig.
13.6.2
Derivatives of Diammonium Succinate
As has been described previously succinic acid from the fermentation is an ex-
cellent building block for the production of industrially important chemical in-
termediates. One of the challenges with the fermentation is that neutralization
is required which leads to significant downstream processing to convert the suc-
cinic acid salt back to the free acid. One strategy for deriving products from suc-
cinic acid fermentations is to identify products that can be obtained directly
from the salts. One route that affords the economical production of chemicals
from diammonium succinate is the formation of 2-pyrrolidone (2P) and N-
methylpyrrolidone (NMP). In addition to 2P and NMP, derivatives such as N-vi-
nyl-2-pyrrolidone can also be produced. The reaction chemistry for these prod-
ucts is shown in Fig. 13.5.
2-Pyrrolidone can be produced by the direct hydrogenation of aqueous diam-
monium succinate with hydrogen in the presence of an active metal catalyst [7].
A mixed product of NMP and 2P can be produced from the conversion of DAS
in the presence of methanol and an active metal catalyst. Figure 13.6 shows the
results of converting aqueous DAS in the presence of methanol and hydrogen
over a supported rhodium catalyst. Conversion for this reaction is near 100%
with a yield of NMP of about 50% and a yield of 2P of about 30%. The remain-
der of the product is a polymer of 2P. It is interesting to note the formation of
N-methylsuccinimide in the early part of the reaction, which converts to NMP
as the reaction proceeds. Once 2P is formed it essentially remains during the
course of the reaction as shown in Fig. 13.6.
Fig. 13.5 Conversion pathways of diammonium succinate.

Fig. 13.6 NMP and 2P production over a rhodium catalyst.
As was described above NMP can be produced in a single-stage reaction with

DAS, hydrogen and methanol. The drawback of the single-stage process is that
a mixture of both NMP and 2P is formed. This mixture would require substan-
tial separation costs to obtain a pure NMP product. Employing a two-stage pro-
cess in which N-methylsuccinimide is formed first, prior to reduction, leads to
near-complete conversion and selectivity to NMP. Results of the hydrogenation
of N-methylsuccinimide are given in Table 13.3. The reaction was carried out at
1900 psig hydrogen for the catalyst and temperature described. It is evident
from the results that pre-forming NMS is preferred to obtain high levels of
NMP. Using the appropriate catalyst and reaction conditions, virtually 100%
NMP can be obtained when starting with NMS [8–11].
Table 13.3 Production of NMP from NMS.
Catalyst composition Feedstock Reaction NMP +2P Yield NMP:2P Ratio

temperature
2.5%Rh/2.5%Re/C NMS 265 8C 54% (5 h) 5.3

2.5%Rh/2.5%Re/C NMS 200 8C 89% (8 h) 67.3
2.5%Rh/2.5%Zr/C NMS 200 8C 81% 38.1
13.7
Conclusions
The fermentation of glucose and other sugars derived from biomass for the pro-
duction of succinic acid provides a valuable building block for the production of
industrially important chemicals. There are still several challenges before com-
mercialization will be realized. The first major hurdle is the need for further
cost reductions in the fermentation of glucose to succinic acid. The specific cost
barriers include reducing the media requirements so that a low cost nutrient
source such as corn steep liquor could be used, improving the volumetric pro-
ductivity of the organism and ultimately a low pH fermentation. From a process
perspective, reducing the cost of separation/purification would have a signifi-
cant overall economic impact. With respect to catalysis, the overall yields for the
conversion of succinic acid and diammonium succinate to commercially impor-
tant chemical intermediates needs to be demonstrated on a longer term contin-
uous basis which will prove that the catalyst lifetime is commercially viable.
In general, the potential for biobased products to replace or displace existing
petrochemical routes to value added products continues to move closer to com-
mercialization. Overcoming a broad range of technical barriers in fermentation,
separations and catalysis will be critical to the realization of economically viable
biobased products from renewals.
References
1 (a) Guettler, M. V.; Jain, M. K.; Soni, B. K. methods of obtaining the microorgan-
US Patent 5,723,322; March 3, 1998 isms.” (e) Ponnampalam E. US Patent
(Michigan Biotechnology Institute). “Pro- US Patent 6,284,904; Sep 4, 2001 (Michi-
cess for making succinic acid, microorgan Biotechnology Institute). “Purifica-
ganisms for use in the process and tion of organic acids using anion ex-
methods of obtaining the microorgan- change chromatography.” (f) Berglund,
isms.” (b) Guettler, M. V.; Jain, M. K.; K. A.; Elankovan P.; Glassner, D. A. US
Rumler, D. US Patent 5,573,931; Nov 12, Patent 5,034,105; July 23, 1991 (Michigan
1996 (Michigan Biotechnology Institute). Biotechnology Institute). “Carboxylic acid
“Method for making succinic acid bacte- purification and crystallization process.”
rial variants for use in the process, and 2 (a) Donnelly, M. I.; Sanville-Millard, C. Y.;
methods for obtaining variants.” (c) Nghiem, N. P. US Patent 6,743,610; June
Guettler, M. V.; Jain, M. K. US Patent 1, 2004 (The University of Chicago).
5,521,075; May 28, 1996 (Michigan Bio- “Method to produce succinic acid from
technology Institute). “Method for mak- raw hydrolysate.” (b) Donnelly, M. I.;
ing succinic acid, Anaerobiospirillum Millard, C. S.; Stols, L. US Patent
succiniciprodens variants for use in pro- RE37,393; Sep. 25, 2001 (The University
cess and methods for obtaining vari- of Chicago). “Mutant E. Coli strain with
ants.” (d) Guettler, M. V.; Jain, M. K.; increased succinic acid production.” (c)
Soni, B. K. US Patent 5,504,004; April 2, Donnelly, M. I.; Sanville-Millard, C.;
1996 (Michigan Biotechnology Institute). Chatterjee, R. US Patent 6,159,738; Dec.
“Process for making succinic acid, mi- 12, 2000 (The University of Chicago).
croorganisms for use in the process and “Method for construction of bacterial
References 379
strains with increased succinic acid pro- 7 Frye Jr., J. G.; Zacher, A. H.; Werpy, T. A.;
duction.” (d) Nghiem, N. P.; Donnelly, Wang, Y. “Catalytic Preparation of Pyrro-
M.; Millard, C. S.; Stols, L. US Patent lidones from Renewable Resources” in
5,869,301; Feb 9, 1999 (Lockhead Martin Catalysis of Organic Reactions, Sowa Jr.,
Energy Research Corporation). “Method J. R. Ed; Taylor and Frances, Boca Raton
for the production of dicarboxylic acids.” 8 Werpy, T. A.; Frye, Jr, J. G.; Wang, Y.; Za-
3 Gorkan, R. R.; Eiteman, E. US Patent cher, A. H. U.S. Patent 6,706,893; March
6,455,284, Sep. 24, 2002. (The University 16, 2004 (Battelle Memorial Institute)
of Georgia Research Foundation). “Meta- “Methods of Making Pyrrolidones”
bolically engineered E. Coli for enhanced 9 Werpy, T. A.; Frye, Jr. J. G,; Wang, Y.; Za-
production of oxaloacetate derived bio- cher, A. H. U.S. Patent 6,670,483; De-
chemicals.” cember 30, 2003 (Battelle Memorial In-
4 Okino, S.; Inui, M.; Yukawa, H. Poster stitute) “Methods of Making Pyrroli-
at the Biotechnology for Fuels and dones”
Chemicals Conference, 2003. 10 Werpy, T. A.; Frye, Jr, J. G.; Wang, Y.; Za-
5 Chang, H. N.; Chang, Y. K.; Kwon, S. H.; cher, A. H. U.S. Patent 6,632,951; Octo-
Lee, W. G.; Lee, P. C.; Yoo, I. K.; Lim, S. J. ber 14, 2003 (Battelle Memorial Insti-
US Patent 6,596,521; July 22, 2003 (Ko- tute) “Methods of Making Pyrrolidones”
rea Advanced Institute of Science and 11 Werpy, T. A.; Frye, Jr, J. G.; Wang, Y.; Za-
Technology). “Method for manufacturing cher, A. H. U.S. Patent 6,603,021; August
organic acid by high efficiency continu- 15, 2004 (Battelle Memorial Institute)
ous fermentation.” “Methods of Making Pyrrolidones”
6 See for example Kirk–Othmer Encyclope-
dia of Chemical Technology, 2005 on-line
edition.
381
14
Polylactic Acid from Renewable Resources
Patrick Gruber, David E. Henton, and Jack Starr
14.1
Introduction
In today’s world of green chemistry and concern for the environment, polylactic acid
(PLA) is interesting. One hundred percent of the carbon in PLA originates from
carbon dioxide in the atmosphere. PLA has very versatile properties, giving it
the ability to compete in a wide variety of markets, and it is economical. The types
of lactic acid used to make the polymer combined with the length of polymer chain,
and the extent of branching control the versatility of PLA. The stereoisomers of lactic
acid required for PLA can only be produced economically by bioprocesses. PLA also
rapidly degrades in the environment under composting conditions and the bypro-
ducts are of very low toxicity, eventually being converted to carbon dioxide and water.
PLA is an interesting combination, it performs well, is economical, and it is “green”.
Polylactic acid (PLA) is a rigid thermoplastic polymer that can be semi-crystal-
line or totally amorphous, depending on the stereochemical purity of the poly-
mer backbone. l-(–)-Lactic acid (2-hydroxypropionic acid) is the natural and
most common form of the acid, but d-(+)-lactic acid can also be produced by
microorganisms or by racemization and this “impurity” acts much like co-
monomers in other polymers such as poly(ethylene terephthalate) (PET) or poly-
ethylene (PE). In PET, diethylene glycol or isophthalic acid is copolymerized
into the backbone at low levels (1–10%) to control the rate of crystallization. In
the same way, d-lactic acid units are incorporated into l-PLA to optimize the
crystallization kinetics for specific fabrication processes and applications.
PLA is a unique polymer that in many ways looks like PET, also a polyester,
but also performs much like polypropylene (PP), a polyolefin. It may eventually
be the polymer with the broadest range of applications because of its ability to
be stress crystallized, thermally crystallized, impact modified, filled, co-polymer-
ized, and processed in most polymer processing equipment. It can be formed
into transparent films or injection molded into blow-moldable preforms for bot-
tles, similar to PET. PLA also has excellent organoleptic characteristics and is ex-
cellent for food contact and related packaging applications.
ISBN: 3-527-31027-4
382 14 Polylactic Acid from Renewable Resources
Although PLA has a unique combination of characteristics, commercial

growth has been limited by high production costs (greater than $ 2/lb), limited
availability of both the monomer and polymer, and limited performance in ap-
plications. Until now PLA has enjoyed little success in replacing petroleum-
based plastics in commodity applications, with most initial uses limited to bio-
medical applications such as sutures [1]. PLA is not a new to the world polymer
industry. Carothers [2] investigated the production of PLA from the cyclic dimer
(lactide) of lactic acid as early as 1932. Even before that, low-molecular-weight
dimers and oligomers were detected when water was removed from an aqueous
solution of lactic acid [3]. The announcement of the formation of a new com-
pany, Cargill Dow LLC, in 1997 brought two large companies together to focus
on the production and marketing of PLA with the intention of significantly re-
ducing the cost of production and broadening the performance of PLA, and de-
veloping the markets [4].
14.2
Lactic Acid
Lactic acid (2-hydroxypropionic acid) is an a-hydroxy organic acid. It is commer-

cially available as a colorless to slightly yellow solution at concentrations of typi-
cally 50%, 80% and 88%. A variety of lactate salts, for example calcium lactate
and sodium lactate, and lactate esters are also available commercially. Lactic acid
is an important food ingredient used for its acidity, flavoring, and ability to con-
trol microorganisms. Lactic acid is also used in a variety of industrial and phar-
maceutical applications.
14.2.1
Lactic Acid Production Routes
14.2.1.1 Chemical Synthesis

Lactic acid can be made as a racemic mixture using lactonitrile as starting mate-
rial. This route has diminished because many commercial applications require
the l-lactic acid isomer and various manufacturers have left the business. Musa-
shino Chemical Laboratories in Japan remains one of the few producers of race-
mic lactic acid.
A chemical synthesis route may prove to be economical if combined with
either a low-cost chiral separation or a stereoselective synthesis. These unit op-
erations are commercial for the production of low volume, high cost chemicals
such as pharmaceuticals, but have not routinely been applied into high volume,
low cost commodity chemicals.
14.2.1.2 Fermentation
The fermentation of lactic acid by humans is very old, as shown in the produc-
tion of yogurt and other foods. In the last hundred years, people have mastered
the fermentation and purification of lactic acid for food and industrial use. The
anaerobic fermentation of sugars to produce lactic acid is the main commercial
route. Fermentation can provide either l-lactic acid or d-lactic acid with a chiral
purity near 100%. The exact chiral purity if the lactic acid formed depends on
the characteristics of the microorganism used for fermentation. Current lactic
acid production by fermentation is limited to the small number of companies
listed in Table 14.1.
There are numerous other smaller lactic acid manufacturers, particularly in
China, that are not listed here. Virtually all lactic acid manufacturers have some
affiliation with a company that provides the sugar feedstock for the lactic acid
fermentation. There has been a large increase in the worldwide lactic acid ca-
pacity in the last decade as the PGLA-1, B&G, and Cargill Dow plants have all
started up production between 1998 and 2004. All three of these plants have
been the result of joint ventures. Another joint venture between Dupont and
ConAgra, named EcoChem, was established in the early 1990s to produce lactic
acid. EcoChem closed down the manufacturing plant in 1994 because of pro-
duction problems. Cargill Dow has the largest single plant at a declared capacity
of 400 million lbs per year. Virtually all the lactic acid produced at the Cargill
Dow facility is used internally for the production of lactic acid-based polymers.
Cargill Dow does sell lactide, a six-membered ring consisting of two lactic acid
moieties that can be easily converted back to lactic acid, by addition of water, or
to other chemical derivatives. For the other seven manufacturing plants listed in
Table 14.1, for which the main applications are the food and industrial markets,
the declared capacities range from 20 million to 100 million lbs per year.
Table 14.1 Lactic acid manufacturers.
Manufacturing sites Main application Affiliations
Purac The Netherlands, Food and Industrial Subsidiary of CSM

Spain, Brazil
PGLA-1 Nebraska, USA Food and Industrial Joint venture between
Purac and Cargill
Galactic Belgium Food and Industrial Owned by Finasucre
B&G China Food and Industrial Joint venture between
Galactic and BBCA
Biochemicals
Archer Daniels Illinois, USA Food and Industrial –
Midland (ADM)
Cargill Dow LLC Nebraska, USA Polymers Joint venture between
Cargill and Dow
Chemical
14.2.2
Production by Fermentation
To make a commercial grade lactic acid product by fermentation, there must be

four main processing areas. These processing areas and their purpose are listed
in Table 14.2. A single-unit operation may actually be accomplishing two pur-
poses, such as the removal of volatile impurities during a concentration step.
Successful lactic acid fermentation needs the right combination of a microor-
ganism, sugars, nutrients, and neutralizing agent to meet the productivity, lactic
acid concentration, and low impurities levels to economically produce lactic acid
of sufficient purity for PLA production.
14.2.2.1 Microorganisms
Several microorganisms are able to produce lactic acid in an economically viable
process. There is probably no perfect microorganism because each company
and geographic location has different sugar sources, nutrient availability, and
separation technology. For instance, a Lactobacillus microorganism needs low-
cost nutrients like corn steep liquor, but this system may result in higher sepa-
ration costs to produce a high purity product. Alternatively, the use of Rhizopus
with a defined medium may produce lower yields, but the lower separation
costs of a relatively clean broth could make it viable.
Although there are numerous microorganisms that can make lactic acid, Ta-
ble 14.3 lists the genus of three microorganisms that have the characteristics of
a commercially viable lactic acid-producing microorganism. The characteristics
listed for each genus are generalizations from the literature. Given the diversity
within each genus, it is likely that a strain exists that is contrary to the general-
ized characterization listed here. Producers regard their specific production
strain as part of their competitive advantage.
Table 14.2 Lactic acid processing areas.
Area Purpose
Fermentation Microbial conversion of sugars to lactic acid, addition of neutraliz-

ing agent to maintain pH in fermentor and make lactate salts
Acidification Convert lactate salts to lactic acid
Purification Separation of cells, nutrients, and residual sugars to obtain lactic
acid solution that meets needed purity requirement.
Concentration Removal of water from lactic acid solution to achieve required
concentration
Table 14.3 Commercially viable lactic acid-producing microor-

ganisms.
Positive Characteristics Negative Characteristics
Lactobacillus High productivity Susceptible to phage

Low by-products Requires complex media
Bacillus High operating temperatures Higher by-products
High chiral purity
Rhizopus Low requirement for complex nutrients Lower yields
Naturally produces enzymes for starch Need to control morphology to
saccharification enable easy cell removal
14.2.2.2 Sugar Feedstock

Depending on location, the sugar source is either sucrose from sugar cane or
sugar beet or dextrose corn syrups made from corn (maize). Because the cost of
the sugar feedstock is a large portion of the production cost of lactic acid, vir-
tually all lactic acid production is integrated with a manufacturing plant that
produces the sugar feedstock.
Other carbohydrate sources have been tried. The EcoChem plant was located
in Wisconsin, USA, and used lactose from cheese whey as a sugar source. Pro-
duction facilities integrated with corn milling could possibly use corn starch as
a cheaper sugar source. The use of sugars from lignocellulosics for lactic acid
fermentation may be the next major technology advance to lower the cost of su-
gar feedstocks for fermentation. Because pentose sugars are also derived from
lignocellulosics, biocatalysts with the ability to convert pentoses to lactic acid are
needed.
14.2.2.3 Nutrients
The choice of nutrients depends on the production microorganism. Complex
nutrients that are typically used would be corn-steep liquor and yeast extract
paste. These are available commercially from several suppliers. Most microor-
ganisms require various salts, particularly ammonium phosphate, and vitamins.
If a complex nutrient is used, it may provide much of the microorganism’s salt
and vitamin needs. Salt and vitamins may need to be supplemented into the
media as the amount of complex nutrient decreases.
14.2.2.4 Neutralizing Agent

Because the microorganisms actually produce lactic acid, without addition of
neutralizing agent or base to form a lactate salt the pH of the fermentation
would drop quickly and lactic acid production would decrease significantly. Lime
(Ca(OH)2) and chalk (Ca(CO3) are used industrially as neutralizing agents to
control the pH of the fermentor between 5.0 and 6.8, depending on the exact
production microorganism. Other neutralizing agents have been proposed, for

example ammonia (ammonium hydroxide), sodium hydroxide, and potassium
hydroxide. EcoChem reportedly formed ammonium lactate in their process.
The choice of the neutralizing agent has important implications for the acidu-
lation area of the plant. When a base is added to control pH, the cation of the
base then forms a lactate salt. The cation of the lactate salt must be bio-compati-
ble with the microorganism and easily removed during purification.
If a microorganism could produce lactic acid in a low-pH (< 3.8) environment,
most of the lactic acid produced in the fermenter would be in the protonated
form. This fermentation would have significant cost advantage, because it would
use less neutralizing agent and less acidifying agent, and produce less by-prod-
uct salt. For instance, Cargill Incorporated. has developed a microorganism that
is able to produce lactic acid at a final pH of less than 4.0 [5].
14.2.3
Acidification
The fermentation area produces a lactate salt, because the fermenters operate at
near neutral pH. The acidulation area of the plant needs to convert the lactate
salt to lactic acid. There are two main options for the acidification – strong acid
addition and salt-splitting.
14.2.3.1 Strong Acid Addition

By directly adding sulfuric acid to the lactate salt broth, lactic acid is formed
and a by-product salt is formed. Most industrial processes form calcium lactate
in the fermenters and add sulfuric acid to the broth to crystallize out calcium
sulfate dihydrate (gypsum). The resulting solids are then removed by filtration.
As the lactic acid market grows, the cost and ability to dispose of gypsum will
be a concern. Lactic acid manufacturers have worked to develop alternatives to
land filling gypsum, for example placing the material in the drywall, cement, or
agriculture industries.
Gypsum is a relatively low-value by-product salt but this route has been used
commercially because of the low cost of the calcium base and sulfuric acid, and
the potential of putting the gypsum into industrial use. Other combinations of
bases and acids have been proposed, such as ammonia for pH control and sul-
furic acid to acidify thus producing ammonium sulfate as the by-product salt.
Ammonium sulfate can be sold as a fertilizer. Because ammonia is more expen-
sive than the calcium bases, the sale of the ammonium sulfate has to more
than make up for the higher cost of raw materials. Ammonium sulfate is also
more soluble in water than calcium sulfate, which complicates the separation
process.
14.2.3.2 Salt Splitting Technology

Considerable research effort has been devoted to reforming the neutralizing
agent from the lactate salt in a process called salt splitting. A “black box” flow
sheet of the salt splitting process for sodium lactate is shown in Fig. 14.1.
Many processes have been proposed to accomplish salt splitting. At least three
salt-splitting options have been piloted in attempts to understand the process
and economics. They are water-splitting electrodialysis, thermal cracking of am-
monium lactate, and carbon dioxide/amine extraction.
In water-splitting electrodialysis, water is split using electric force in a bipolar
membrane. The lactate anion passes through an anion-selective membrane to
combine with the proton to form lactic acid. The cation passes through a cation
selective membrane to combine with the hydroxyl anion to form a cation hy-
droxide, such as sodium hydroxide. This water-splitting electrodialysis option
has been investigated by both ADM and Cargill in conjunction with Michigan
Biotechnology Institute.
Because ammonia is volatile, ammonium lactate can be salt-split by vaporiz-
ing ammonia from the broth. As ammonia is removed from solution, the solu-
tion becomes acidic, thus making it more difficult to remove more ammonia.
To drive the salt-splitting reaction toward high conversion, the acidity associated
with the lactic acid must be addressed. One method to do this would be to add
an alcohol, for example ethanol (EtOH), to the ammonium lactate solution. The
alcohol reacts with the lactic acid to form a lactate ester. The lactate ester is then
vaporized to remove the lactic acid and enable more ammonia to be vaporized.
The overall chemistry is shown below.
NH4 Lac EtOH ! NH3 " EtLac "
Carbon dioxide is a weak acid when it dissolves into water as carbonic acid. The
carbonic acid can provide a proton to acidify a small fraction of the lactate salt.
This sequence of chemical reactions is shown below.
CO2 H2 O NaLac ! H2 CO3 NaLac ! H HCO3 NaLac

! NaHCO3 H Lac
Fig. 14.1 Salt splitting of sodium lactate.

As the solution becomes acidic, the system reaches an equilibrium. The lactic
acid must be removed from the aqueous phase to achieve high conversion to
lactic acid. Cargill solved this problem by extracting the lactic acid into an
amine-based solvent [6].
14.2.4
Purification
Several different purification processes have been commercialized on an indus-

trial scale. The choice of a purification process for lactic acid depends the
amount of residual sugars, residual nutrients, and fermentation by-products in
the broth and the purity required for the application. In all cases the microor-
ganisms or cells must be removed from the broth.
14.2.4.1 Cell Removal

The options for cell removal depend on the choice of the production microor-
ganism. For a Lactobacillus or Bacillus-based fermentation, a simple filtration
step would not work well because the cells are too small and would leak by the
filter cloth. These small cells must be flocculated before filtration to achieve a
clarified broth. A clarified liquid can be obtained by decantation from the floccu-
lated cells. Crossflow filtration using a 0.1 to 1.0 lm membrane could also pro-
vide an economical method for removing the microorganisms.
For a Rhizopus-based fermentation, the cell morphology must be controlled
as a pellet to enable easy filtration. Methods are known for controlling this as-
pect of the fermentation. A belt filter or pressure filter can provide adequate
broth clarity for Rhizopus fermentation, with good pellet morphology.
14.2.4.2 Separation of Residual Sugars, Nutrients and Fermentation By-products

Three separation schemes are used on a commercial scale – solvent extraction,
direct distillation, and distillation of lactate ester. After these operations, the lac-
tic acid solution may need to be passed through activated carbon and cation
and anion-exchange resin to produce a clear to slightly yellow, deionized prod-
uct.
Solvent Extraction Solvent extraction is performed using a solvent comprising,

typically, three components – a long chain tertiary amine, a water-immiscible al-
cohol, and an aliphatic hydrocarbon. The tertiary amine, for example trioctyl-
amine, acts as a base to form an ion pair with the lactic acid in the organic
phase. The water-immiscible alcohol, for example octanol, is designed to in-
crease the distribution of lactic acid in the organic phase. Other functional
groups such as ketones and phosphates can be used to replace the alcohol. The
aliphatic hydrocarbon is used to control the viscosity and phase separation char-
acteristics. The basic nature of the amine enables good selectivity for lactic acid
Fig. 14.2 Amine solvent extraction process flow diagram.
over residual sugars and nutrients, but does not provide good separation from
acidic impurities.
The forward extraction of the lactic acid into the solvent phase is performed at
relatively low temperatures to help increase the distribution of the lactic acid into
the solvent. The lactic acid is released from the amine and the solvent phase by
back extracting the lactic acid into an aqueous solution at high temperatures. A
process flowsheet for this solvent extraction process is shown in Fig. 14.2.
Solvent exchange can be integrated with salt-splitting operations such as those
described above with carbon dioxide. The solvent extraction acts as a second
phase to help drive the salt splitting reaction to high conversion. Solvent extrac-
tion could also be used to sequester the lactic acid when vaporizing the ammo-
nia from ammonium lactate.
Direct Distillation Lactic acid can be purified by distillation. The distillation

scheme can be a single stage or multi-stage depending on the purification re-
quired. This can be an efficient method for separating low volatility compounds
like sugars and amino acids from lactic acid. A single stage distillation is less ef-
ficient for separating fermentation by-products that have a vapor pressure near
the vapor pressure of lactic acid. In a multi-stage distillation, care has to be
taken to minimize the formation of lactic acid oligomers, such as lactyllactate,
that have significantly lower vapor pressures and may result in loss of yield.
Distillation of Lactate Ester The distillation of lactic acid can be difficult, be-
cause it can form lactic acid oligomers, require high temperatures, and low
pressures. By reacting lactic acid with an alcohol, such as ethanol, to form a lac-
tate ester, many of the issues with the distillation of lactic acid can be avoided.
High-purity lactic acid can be obtained by this process as multi-stage distillation
has the potential to separate other carboxylic acids. A simplified process flow-
sheet with ethanol as the alcohol is shown in Fig. 14.3.
The alcohol must be recycled in the process which adds complexity. The water
and the alcohol have higher vapor pressures than ethyl lactate, so they preferen-
tially go into the vapor phase compared with the lactate ester. Thus ethanol
must be recycled back to the esterification reactor and water must be removed
from the system to drive the esterification reaction to high conversions. Re-
Fig. 14.3 Ethyl lactate purification process flow diagram.
searchers at Argonne National Laboratories have developed a pervaporation pro-

cess that has the potential to minimize the cycle streams around the esterifica-
tion reactor by using a selective membrane [7].
This route may have benefits for companies like Archer Daniels Midland and
Cargill, that also have ethanol production, compared with other routes. This
purification scheme may also favor companies that have significant sales of lac-
tate esters, for example ethyl lactate, because they would not have to hydrolyze
the esters to produce a final product.
Concentration Finally the lactic acid is concentrated using standard technology

and then transferred via pipeline to the PLA plant. Typically the lactic acid
would have an optical purity of >98% at about a 60–70% concentration.
14.3
PLA Production
Production of commercially useful PLA requires control of the optical composi-

tion of the polymer, control of rheology, and stabilization of the polymer, all
with high yield under simple processing conditions. Control of the optical com-
position affects both the melt behavior in polymer-converting equipment and
the end use properties. Control of the rheology enables PLA to be processed on
a wide variety of polymer processing equipment, and melt stabilization makes
the PLA stable to conditions typically seen in polymer-fabrication operations.
PLA can be prepared by both direct condensation of lactic acid and by the
ring opening polymerization of the cyclic lactide dimer, as shown in Fig. 14.4.
Because the direct condensation route is an equilibrium reaction, difficulties re-

moving trace amounts of water in the late stages of polymerization usually limit
the ultimate molecular weight achievable by this approach. Most work has fo-
cused on the ring opening polymerization of lactide, although other approaches,
for example azeotropic distillation to drive the removal of water in the direct es-
terification process, have been evaluated [8].
Cargill Dow LLC has developed a patented, low cost, continuous process for
the production of lactic acid-based polymers [9]. The process combines the sub-
stantial environmental and economic benefits of synthesizing both lactide and
PLA in the melt rather than in solution and, for the first time, provides a com-
mercially viable biodegradable commodity polymer made from renewable re-
sources. The process starts with lactic acid produced by fermentation of dex-
trose, followed by a continuous condensation reaction of aqueous lactic acid to
produce low molecular weight PLA pre-polymer (Fig. 14.5). Next, the low-molec-
ular-weight oligomers are converted into a mixture of lactide stereoisomers
using tin catalysis to enhance the rate and selectivity of the intramolecular cycli-
zation reaction. The molten lactide mixture is then purified by vacuum distilla-
tion. Finally, PLA high polymer is produced using a tin-catalyzed, ring-opening
lactide polymerization in the melt, completely eliminating the use of costly and
environmentally unfriendly solvents. When polymerization is complete any re-
maining monomer is removed under vacuum and recycled to the beginning of
the process (Fig. 14.6). This process is currently in operation in a recently con-
structed 300 million lb/yr commercial-scale PLA plant in Blair, Nebraska, USA.
As we move into the 21st century, increased utilization of renewable resources
will be one of the strong drivers for sustainable products. Reduced energy con-
sumption, waste generation, and emission of greenhouse gases will take on
greater emphasis. Polylactic acid is the first commodity plastic to incorporate
these principles and Cargill Dow LLC was awarded the 2002 Presidential Green
Chemistry Award for their process to produce NatureWorks PLA. The published
literature on PLA is extensive and has been reviewed in detail in several recent
publications [10].
Fig. 14.4 Polymerization routes to polylactic acid.

Fig. 14.5 Schematic of PLA production via prepolymer and lactide.
Fig. 14.6 Non-solvent process to prepare polylactic acid.
14.3.1
Polymerization of Lactide
Many catalyst systems have been evaluated for polymerization of lactide, includ-
ing complexes of aluminum, zinc, tin, and lanthanides. Even strong bases such
as metal alkoxides have been used with some success. Depending on the cata-
lyst system and reaction conditions, almost all conceivable mechanisms (cation-
ic [11], anionic [12], coordination [13], etc.) have been proposed to explain the ki-
netics, side reactions, and nature of the end groups observed in lactide polymer-
ization. Tin compounds, especially tin(II) bis-2-ethylhexanoic acid (tin octoate),
are preferred for bulk polymerization of lactide, because of their solubility in
molten lactide, high catalytic activity, and low rate of racemization of the poly-
mer. Conversions > 90% with less than 1% racemization can be achieved while
providing polymer with high molecular weight.
The polymerization of lactide using tin octoate is thought to occur via a coor-
dination–insertion mechanism [13] with ring opening of the lactide to add two
lactyl units (a single lactide unit) to the growing end of the polymer chain, as
shown schematically in Fig. 14.7. The tin catalyst facilitates the polymerization,
but hydroxyl or other nucleophilic species are the actual initiators. There are
usually several hundred ppm of hydroxyl impurities in the lactide from water,
lactic acid, and linear dimers and trimers.
High molecular weight polymer, good reaction rate, and low levels of racemi-
zation are obtained with tin octoate catalyzed polymerization of lactide. Typical
conditions for polymerization are 180–210 8C, tin octoate concentrations of 100–
1000 ppm, and 2–5 h to reach 95% conversion. The polymerization is first-order
in both catalyst and lactide. Hydroxyl-containing initiators such as 1-octanol are
frequently used to both control molecular weight and accelerate the reaction.
In addition to the work performed on catalysts and comonomers, a significant
amount of work has been devoted to designing the optimum polymerization
process from a cost and versatility perspective. Most of this work has used lac-
tide dimer as the starting point. Batch polymerization and continuous processes
can be used. Continuous processes (Fig. 14.6) have a cost and productivity ad-
vantage and thus are the focus of most work. Stirred tank or pipe reactors have
been evaluated alone and in combination. Because of the low energy of the
ring-opening polymerization and the potential to obtain high rates of polymer-
ization, melt extruders have been extensively evaluated as reactors to produce
PLA. Several groups have reported or patented specific aspects of the use of ex-
truders or combinations of several reactor concepts to produce PLA [14–27].
Dupont [15] recognized the importance of high rates of lactide polymerization
on the economic viability of using extruders. They investigated the issues sur-
rounding hydroxyl initiators, catalysts, and acidic impurities. Dupont’s patent
Fig. 14.7 Coordination-insertion chain growth mechanism of

lactide to PLA; R = growing polymer chain.
[15] (US 5,310,599) provides examples showing the dramatic (negative) effect of
acidic impurities, such as the linear dimer (DP2), etc., on the rate of lactide po-
lymerization. The acid impurities coordinate with the tin catalyst and render it
ineffective as a ring-opening site of polymerization. As an example, 389,000
MW PLA can be made with a 5-min residence time (6000 : 1 monomer:Sn octo-
ate) at 180 8C in the absence of acidic impurities and 98% conversion is ob-
tained. The conversion in 5 min drops to 50% when the impurty:catalyst ratio is
2:1 and to 12% in 5 min if the ratio is 6:1.
The rate of polymerization of lactide can be accelerated by use of hydroxyl ini-
tiators such as butanol or by higher catalyst loading. Increased initiator levels
will, however, reduce the molecular weight while more catalyst will reduce the
thermal stability and increase the color of the PLA.
14.4
Control of Crystalline Melting Point
PLA is a hard, brittle plastic which can exist in either the amorphous or semi-
crystalline state, depending on the stereochemistry and thermal history. Com-
mon physical properties such as density, heat capacity, and mechanical and rhe-
ological properties are very dependent on its transition temperatures. For amor-
phous PLA, the glass transition (Tg) determines the upper use temperature for
most commercial applications. For semi-crystalline PLA, both the Tg (*58 8C)
and melting point (Tm), 130–230 8C, (depending on structure) are important for
determining the usage temperatures for different applications. Both of these
transitions, Tg and Tm, are highly affected by overall optical composition, pri-
mary structure, thermal history, and molecular weight. Several excellent reviews
have been written which include details of the properties and characteristics of
PLA [28–32].
Having two optical arrangements for lactic acid (l-lactic acid, d-lactic acid),
and three optical arrangements for lactide (l-lactide, d-lactide, meso-lactide), the
variety of primary structures available for PLA is substantial. In addition, many
other monomers have been copolymerized with lactic acid and/or lactide, a top-
ic which will not be covered in this chapter. Techniques for measuring PLA
stereo-sequences by NMR have been published by Thakur et al. [33].
The most common commercial polymers of PLA are optical copolymers of
predominantly l-lactide, with small amounts of d- and meso-lactides, made by
bulk polymerization with tin octoate catalyst by ring-opening polymerization
(ROP). Although these copolymers are usually described as random, there is
evidence of some stereo selectivity. The selectivity of tin octoate is discussed by
Thakur et al. [34]. Condensation polymerization of, mostly, l-lactic acid with
small amounts of d-lactic acid, polymerized in solution, has also been used.
The optical comonomers introduce “kinks” in PLA’s natural helical conforma-
tion and “defects” in the crystal arrangement, which results in depression of the
melting point, reduction in the level of attainable crystallinity, and reduction in
14.4 Control of Crystalline Melting Point 395
the rate of crystallization. Optical purity (OP) is common nomenclature for de-
scribing polymers of this variety. As OP decreases, crystallization eventually be-
comes impossible and the polymer is amorphous (occurring about when OP
<0.78). For the purposes of this chapter, the designation a-PLA will be used for
PLA made with insufficient optical purity to crystallize, and c-PLA will be used
to denote PLA that are crystallizable. Stereo-selective catalysis has been used to
develop optical copolymers with a highly tapered concentration of optical purity
(OP) along the chain [35].
Random copolymers made from meso-lactide result in an atactic primary
structure referred to as poly(meso-lactide) and are amorphous. Random optical
copolymers made from equimolar amounts of d-lactide and l-lactide are com-
monly referred to as PDLLA or poly(rac-lactide). PDLLA is also essentially atac-
tic, but the primary structure is segregated into optical doublets of the lactyl
group, and it is also amorphous.
For l-rich polymers with small amounts of d- and meso-lactides, there is an
observed decrease in the equilibrium melting point with decreasing OP. At least
two possible explanations for this have been considered. In one extreme case it
is assumed that the optically impure units are incorporated into the crystal
structure as defects. This results in a decreasing melting point (Tm), because of
the decrease in the enthalpy of fusion. In the other extreme case it is assumed
that the optically impure units are rejected from the crystal structure, causing a
reduction in Tm because of entropy effects. Runt [36] studied the degree of crys-
tallinity, spherulitic growth rate, lamellar thickness, and melting temperature of
a series of optical copolymers of this variety, comparing the effects of d-lactide
and meso-lactide optical impurities. Small-angle X-ray scattering (SAXS) results
showed that lamellar thickness decreases with decreasing OP at a given degree
of super cooling. It was proposed that a critical sequence length that is free of
stereo defects is necessary for crystallization, and that the stereo defects are ex-
cluded from the crystalline regions. In a study by Munson et al. [37] it was
shown that, at least for long crystallization times, a certain fraction of optical
impurity (in this case, a small percentage of labeled l-lactide copolymerized into
a polymer of predominantly d-lactide) is incorporated into the crystal lamellae.
Munson went on to develop a model for determining the fraction of crystalliz-
able PLA based upon a critical chain length necessary for crystallization that en-
ables stereo defects to be included in the crystalline region if they are sur-
rounded by a sufficient isotactic run length. The spherulite structure for l-lac-
tide-rich PLA was studied by Pyda et al. [38] using AFM and polarized light mi-
croscopy.
The observed melting point by DSC after isothermal treatment at the crystalli-
zation temperature Tc has been studied as a function of crystallization tempera-
ture for a series of poly(l-lactide-co-d-lactide)s [36]. Experimental equilibrium
melting points for a series of l-lactide-rich polymers are shown in Fig. 14.8.
During normal processing and crystallization procedures, polymers do not
reach their equilibrium melting temperature. A practical equation describing
melting point for PLA of predominantly l-lactide with meso-lactide is simply
Fig. 14.8 Equilibrium melting point for PLA L-lactide rich

homopolymers as a function of increasing %D.
Tm ( 8C) & 175 8C 300wmeso, where wmeso is less than 0.18 [28]. The melting
point is depressed by approximately 3 8C for every 1% initial meso-lactide.
An enthalpy of fusion of 93.1 J g–1 is used for 100% crystalline PLLA or
PDLA homopolymers (i.e. DHm8 = 93.1 J g–1) [39] having infinite crystal thick-
ness. However, because Tm8 decreases with decreasing OP, it is expected that
using a constant value for DHm8 across all optical compositions will introduce
error. It is well known that for PLA of essentially random optical copolymers of
predominantly l-lactide, with small amounts of d- and meso-lactides, the attain-
able percentage crystallinity (xc) decreases with decreasing optical purity (OP),
and crystallization is essentially non-existent about when OP < 0.78.
The crystallization kinetics of PLA are highly dependent on the optical copoly-
mer composition and molecular weight. The degree of crystallinity, nucleation rate,
and spherulite growth rate decrease substantially with decreasing optical purity
(OP). Kolstad showed that under quiescent crystallization conditions the bulk crys-
tallization half-life increases by roughly 40% for every 1% w/w increase in meso-lac-
tide [40]. PLA has the highest rate of crystallization between 100 and 130 8C.
14.5
Rheology Control by Molecular Weight and Branching
Rheological properties are useful in evaluating thermoplastics for their perfor-

mance during processing operations. The literature reports a wide variety of
processes and products using PLA, spanning from high-orientation processes
such as fiber spinning and oriented films to lower orientation processes such as
14.5 Rheology Control by Molecular Weight and Branching 397
thermoforming and foams. The time scales of these processes are very different.
Considerations for material flow characteristics, orientation and crystallization
must be made to gauge and predict the resulting properties of the products
made from these processes. Intrinsic factors affecting the flow characteristics of
PLA are molecular weight distribution, degree and type of branching, optical
composition, optical block length distributions, and melt stability.
14.5.1
Melt Rheology of Linear PLA
PLA, like many thermoplastics, is a pseudoplastic, non-Newtonian fluid. Above

the melting point, PLA behaves as a classic flexible chain polymer across all op-
tical compositions [41]. PLA’s zero shear viscosity and shear thinning behavior
have been measured by a variety of groups. Complicated by the array of optical
copolymers studied, the difficulty associated with measuring the absolute molec-
ular weight of PLA, and the issues of melt stability, however, interpretation of
PLA’s rheology has not always been consistent.
The most comprehensive studies of linear PLA in the melt were performed
by Janzen and Dorgan [42–44] who showed that zero shear viscosity (go) scales
very close to the usual 3.4 power with Mw, and that PLA obeys the Cox–Merz
rule well into the shear thinning region when in the linear visco-elastic range.
The plateau modulus for PLLA is approximately 1 MPa, which corresponds to a
critical molecular weight of about 9000 g mol–1. In previous work Cooper-White
and MacKay [45] measured the rheological properties of PLLA with molecular
weights ranging from 2000 to 360,000 and reported a dependence of zero shear
viscosity (go) on chain length to the power 4.0 (M4.0
w ); only a very small number
of samples were used to build the 4.0 power relationship, however. Dorgan also
observed strain-hardening of PLA during extension in the melt, a quality neces-
sary for high-speed fiber spinning.
14.5.2
Melt Rheology of Branched PLA
By introduction of branching, the rheological properties of PLA can be signifi-

cantly modified. Because the linear polymer has very little melt strength, for
some applications it is desirable to increase the melt strength by introducing
long-chain branching. Figure 14.9 shows the effect of branching on the complex
viscosity of PLA. Several routes have been used to achieve long-chain branching.
These routes can be achieved either in the polymerization process itself, or in
post polymerization steps in an extruder.
Fig. 14.9 Complex viscosity of

branched PLA made by peroxide
treatment.
14.5.3
Branching Technology
14.5.3.1 Multi-functional Polymerization Initiators

These have been used to make star and comb-shaped molecules. Several studies
have examined the reaction of lactides with branched polyols since first pro-
posed by Schindler et al. [46] Dorgan [47] published rheological characterization
of linear and star PLA. Zhao [48] made starburst structures by polymerizing lac-
tide on to dendrimer macroinitiators. From 16 to 21 polylactide arms were at-
tached to the surface of the initiator. Molecular weight is controlled by the ratio
of monomer to initiator.
14.5.3.2 Hydroxy Cyclic Ester and/or Carbonate Polymerization Initiators

These comprise an initiation group (e.g. hydroxyl) and a ring capable of copo-
lymerizing with a growing PLA chain. Drumright et al. [49] reported polymeriz-
ing PLA with hydroxy carbonate initiators resulting in long chain branching
and enhanced melt elasticity. Tasaka [50] made branched PLA with mevalonic
lactone initiator.
14.5.3.3 Multi-cyclic Ester, Multi-cyclic Carbonate

and/or Multi-cyclic Epoxy Comonomers
These comprise multiple rings (most commonly two) capable of copolymerizing
with a growing PLA chain. Drumright [51] also reported on the polymerization
of PLA with bicyclic diesters and/or dicarbonates to control rheological proper-
ties. The bicyclic diesters and dicarbonates copolymerize easily with lactide,
forming copolymers that can have tailored levels of branching. The copolymers
had excellent rheological properties, including increased melt tensions and im-
proved shear thinning, compared with the analogous linear polymers. A typical
polymer was manufactured by polymerization of l-lactide at 130–185 8C with
14.6 Melt Stability 399
0.1% a,a-2,5-dioxabicyclo[2.2.2]octane-3,6-dione. Because multi-cyclic lactones do

not contain an initiator, the chemistry can theoretically be used to cross-link
PLA to infinite molecular weight. A patent by Gruber et al. [52] describes rheo-
logical modification of PLA by copolymerization with a multi-functional epoxi-
dized oil.
14.5.3.4 Free Radical Cross-linking

This has been accomplished in the melt by treating PLA with peroxides [53].
Small amounts of peroxide result in long chain branches. The polymer remains
melt-processable and is stable. Large improvements in melt elasticity have been
measured using die swell, melt tension, and parallel plate rheometry. Blends of
linear and branched PLA had good compatibility and melt rheology predictable
by use of the mixing rule [54].
14.6
Melt Stability
Issues related to PLA melt stability during processing and rheological testing
have been cited by Witzke [28], Dorgan [42], Ramkumar [55], and Feng [56]. Re-
actions leading to poor melt stability include chain scission, because of hydroly-
sis, chain scission because of thermal instability, and lactide formation by the
well documented “back biting” mechanism. Hydrolysis can be reduced or essen-
tially eliminated with conventional drying operations in which water is removed
before melting. Recommended drying conditions are available for commercial
PLA. When PLA is exposed to a humid environment after drying, moisture is
quickly absorbed into the polymer up to its equilibrium level of swelling. Ki-
netics for melt hydrolysis depend on water concentration, acid or base catalysis,
and polymerization catalyst concentration.
The proton in the CH group of the main chain of PLA is labile. It has been
suggested that the proximity of this labile proton to the ester group affects the
thermal sensitivity of the polymer [55]. The proton can be abstracted, resulting
in the breakdown of the molecular chain. In practical extrusion processes, ther-
mal stability becomes important at temperatures greater than 250 8C.
Using PLA polymerized with tin octoate catalyst, Cicero, et al. [57] showed
that the melt stability of PLA can be improved by addition of small amounts of
tris(nonylphenyl) phosphite (TNPP), probably as a result of balancing chain ex-
tension with degradation reactions. Addition of TNPP during rheological testing
greatly stabilized the polymer, and increased the time available for testing.
The thermodynamic equilibrium between polylactide and lactide monomer
varies with temperature, and is about 4.3% lactide at 210 8C [28]. Because lactide
imparts undesirable characteristics, for example fuming, during processing, it is
desirable to depress the lactide-formation kinetics and stabilize the polymer.
Gruber et al. [58] showed that lactide formation could be substantially reduced
under normal processing conditions by complexing the catalyst with additives,

resulting in a melt-stable PLA polymer.
14.7
Applications and Performance
PLA resins can be tailor-made for different fabrication processes, including in-
jection molding, sheet extrusion, blow molding, thermoforming, film forming,
or fiber spinning. The key is controlling molecular properties in the process, for
example branching, d isomer content, and molecular-weight distribution. The
ability to selectively incorporate l-, d- or meso-lactide stereoisomers into the poly-
mer backbone enables PLA to be tailored for specific applications. The ease of
incorporation of various defects into PLA enables control of both crystallization
rate and ultimate crystallinity.
Typical properties of NatureWorks PLA from Cargill Dow LLC for injection
molding and extrusion applications are shown in Table 14.4. The various grades
of NatureWorks PLA differ in stereochemical purity, molecular weight and addi-
tive packages. Each grade is optimized for both processing and end-use perfor-
mance in its intended application.
Table 14.4 Properties of various grades of NatureWorks PLA.
PLA polymer ASTM PLA polymer ASTM

2000D a) method 3010D b) method
Physical properties
Specific gravity (g cc–1) 1.25 D792 1.21 D792
Melt index, g/10 min (190 8C/ 4–8 D1238 10–30 D1238
2.16 kg)
Clarity Transparent Transparent
Mechanical properties
Tensile strength at break, psig 7700 (53) D882 7000 (48) D638
(MPa)
Tensile yield strength, psig (MPa) 8700 (60) D882
Tensile modulus, kpsig (GPa) 500 (3.5) D882
Tensile elongation (%) 6.0 D882 2.5 D638
Notched izod impact, ft-lb in–1 0.24 (0.33) D256 0.3 (0.16) D256
(J m–1)
Flexural strength, psig (MPa) 12,000 (83) D790
Flexural modulus, kpsig (GPa) 555 (3.8) D790
a) 2000D is a product of Cargill Dow LLC designed as an extru-

sion/thermoforming grade; properties typical of extruded
sheet
b) 3010D is a product of Cargill Dow LLC designed as an injec-
tion molding grade; properties typical of injection molded
tensile bars
14.8 PLA Stereocomplex 401
Extrusion-thermoforming is optimized at a d isomer-content that does not al-

low crystallization to occur during the melt processing steps, with 4–8% d con-
tent being the effective range. Branching can be introduced by a variety of
methods [50–53], thus enhancing melt strength during fabrication and opening
up new application opportunities in areas such as foams and extrusion coating.
In short, the rheological characteristics and physical properties of PLA can be
tailored for use in a variety of processes and applications [59].
14.8
PLA Stereocomplex
Blends of PLA optical copolymers also have a variety of new properties. The
most notable is that of the stereocomplex PLA, which is made by solvent or
melt blending PLLA and PDLA and in which the polymers undergo stereocom-
plexation or racemic crystallization. This phenomenon has been an area of in-
tense study since first described by Ikada [60]. PLA stereocomplex is expected to
open markets in consumer electronics, automobile manufacturing, and other
durable goods, because of excellent heat stability combined with relatively low
cost and other benefits such as PLA’s inherent flame retardency and biodegrad-
ability.
PLA stereocomplex occurs when PLLA is mixed with PDLA and the homo-
polymer segments co-crystallize into a new arrangement with a markedly higher
melting point. The interesting features of the stereocomplex crystal will be dis-
cussed later. Tsuji and Ikada [61] reported that the Tg of stereocomplex PLA is
5 8 higher than that of the non-blended components. The equilibrium melting
temperature of PLA stereocomplex has been reported by Tsuji to be 279 oC [62],
a very marked improvement over homocrystals, with the minimum tacticity se-
quencing needed to produce stereocomplex for the l-rich and d-rich PLA to be
15 lactyl (or 7.5 lactide) units [63]. The heat of fusion for pure stereocomplex
has been determined to be 142 J g–1 by Loomis [64]. This was attributed to the
strong interaction between the l-lactyl and d-lactyl unit sequences in the amor-
phous region of the blends, resulting in a more dense chain packing.
Melting points for stereocomplex are dictated by the optical purity (OP) of
both the l-lactide-rich and d-lactide-rich polymers (in terms of optically pure
run lengths) and molecular weight and blend ratio. Maximum regularity is at-
tained with a blend of PLLA and PDLA. The greater the deviation in OP of the
l-rich and/or d-rich chains, the greater the decrease in stereocomplex melting
peak. This follows the equivalent behavior of PLA homocrystals, but is shifted
to higher temperature. A practical upper attainable melting temperature that
would be achievable for commercial resins approaches 230 oC.
Melting point (Tm) and enthalpy of melting (DHm) of the stereo complex de-
pends on the ratio of l-lactide-rich polymer and d-lactide-rich polymer. For a
constant optical purity of racemic polymers, the Tm varies only by a few degrees
across the possible blend ratios, whereas the stereocomplex DHm varies directly
with the blend ratio with maximum at 1:1. Tsuji et al. have published an excel-
lent series of papers on the crystallization, mechanical behavior and hydrolysis
properties of PLA stereocomplexes [65–76].
14.9
Fossil Resource Use and Green House Gases
PLA made today in Blair, Nebraska, uses corn as the feedstock, natural gas to
heat the manufacturing process, and coal fired electricity. Vink et. al., have pub-
lished a life-cycle inventory (LCI). This LCI indicates that PLA production gener-
ated 56% less greenhouse gas emissions than production of polyester. Yet, even
though PLA uses fixed carbon dioxide from the atmosphere as the carbon
source, it still contributes 1.2 kg CO2 kg–1 PLA to greenhouse gases because of
the use petrochemical energy sources to heat and operate the manufacturing
plant.
Integrated into a lignocellulosic biorefinery PLA production is a carbon diox-
ide sink at –0.3 kg CO2 kg–1 PLA while using traditional sources of electricity.
The advantage of 1.5 kg CO2 kg–1 PLA is because of the displacement of natural
gas by lignin as the fuel source to heat the manufacturing process. With im-
provements in “green” sources of electricity, PLA would even be a greater sink
at –1.7 kg CO2 kg–1 PLA.
14.10
Summary
Because all of the carbon of PLA originates from carbon in the atmosphere, it
is regarded a renewable resource-based material. This, combined with useful
material properties and processability, makes it attractive.
PLA takes advantage of a biological system to perform chemistry that tradi-
tional production techniques cannot perform efficiently. Fermentation of sugar
to lactic acid produces chiral lactic acid in high yield. Not only does bioproces-
sing and fermentation provide chiral lactic acid, it does it inexpensively while
conforming to the principles of Green Chemistry. Control of chirality enables
producers to change the polymer performance by changing the optical activity
of the lactic acid units in the polymer backbone. The result is a family of prod-
ucts, all made from lactic acid, with properties that can reach the wide range of
applications previously discussed. It is not possible, with all the technology
known today, to economically produce commercially useful PLA from petro-
chemical feedstocks. Production of PLA depends on bio-based sources of lactic
acid because the stereo-isomer content must be controlled.
PLA is currently produced using corn as a feedstock. The corn-based sugar
raw material can account for approximately 25–40% of the total cost of PLA. To-
day PLA made from corn is competitive with traditional petrochemical-based
Abbreviations 403
polymers given its combination of performance in applications, cost, and the

environmental benefits.
Lignocellulosic biorefineries are important to PLA because of the improve-
ments in both the cost of raw material and the overall environmental footprint.
In a lignocellulosic biorefinery, in which multiple value-added co-products are
produced and the lignin is used for energy, the cost of PLA would be less, pri-
marily because of the lower cost of sugars. In this case the cost of sugar raw
materials could be as low as 15–25% of the total cost of PLA production. Impor-
tantly, the environmental footprint would improve because of the displacement
of petrochemical-based energy by lignin-based renewable energy to drive the
manufacturing process. PLA made from a lignocellulosic biorefinery in the fu-
ture will be competitively advantaged.
Biorefineries utilizing lignocellulosic feedstocks hold the promise of lower net
feedstock costs combined with an improved environmental footprint. PLA al-
ready has an advantage over some traditional petrochemical based polymers
such as PET. Improvements in cost will make it competitive with polypropylene
and polystyrene also. Improvements in the environmental footprint will take
PLA to a whole new level – that of a truly sustainable polymer system.
Abbreviations
PLA Polylactide, poly(lactic acid)

c-PLA Crystallizable PLA
a-PLA Amorphous and non-crystallizable and PLA
o-PLA Oriented PLA
PDLA Poly(d-lactide)
PDLLA Poly(d-lactide-co-l-lactide) at 1:1 molar ratio of l- and d-lac-
tides, Poly(rac-lactide)
PDxLyLA Polylactide composed of d-lactate mole fraction x and l-lactate
mole fraction c
PLLA Poly(l-lactide)
AFM Atomic force microscopy
bo-PLA Biaxially oriented PLA
DMA Dynamic mechanical analysis
DSC Differential scanning calorimetry
E' Storage modulus
E'' Loss modulus
G Crystal growth rate
GPC Gel-permeation chromatography
lc Crystalline lamellar thickness
LCA Life cycle analysis
Mn Number average molecular weight
Mn,0 Number average molecular weight at hydrolysis time zero
Mn,t Number average molecular weight at hydrolysis time t
Mw Weight average molecular weight

ROP Ring opening polymerization
tan d E''/E'
tc Crystallization time
Tc Crystallization temperature
Tg Glass transition temperature
Tm Peak melting temperature
Tm8 Equilibrium melting temperature

xc Fraction of crystallinity
[g] Intrinsic viscosity
Nature Works registered trademark of Cargill Dow
References
1 Lipinsky, E. S. and Sinclair, R. G., Chemi- M. L., Benson, R. D., Borchardt, R. L,

cal Engineering Progress, August 1986. U.S. Patent 5,247,058, 1992. Gruber,
2 Carothers, H., Dorough, G. L., and Van P. R., Hall, E. S., Kolstad, J. J., Iwen,
Natta, F. J., J. Am. Chem. Soc., 54, 761, M. L., Benson, R. D., Borchardt, R. L,
1932. U.S. Patent 5,247,059, 1993. Gruber,
3 Nef, J. U., Liebigs. Ann. Chem., 403, 204, P. R., Hall, E. S., Kolstad, J. J., Iwen,
1914. Pelouze, P. M. J., Ann. Chim. Phys., M. L., Benson, R. D., Borchardt, R. L,
13, 257, 1845. U.S. Patent 5,258,488, 1993. Gruber,
4 Plastics Technology, January, pp. 13–15, P. R., Hall, E. S., Kolstad, J. J., Iwen,
1998. M. L., Benson, R. D., Borchardt, R. L,
5 Carlson, T. L. and Peters, E. M. Low pH U.S. Patent 5,274,073, 1993. Gruber,
lactic acid fermentation, U.S. Patent P. R., Hall, E. S., Kolstad, J. J., Iwen,
6,475,759, 2002 M. L., Benson, R. D., Borchardt, R. L,
6 Baniel, A. M., Eyal, A. M., Mizrahi, J., U.S. Patent 5,357,035, 1994 Gruber,
Hazan, B., Fisher, R. R., Kolstad, J. J., P. R., Hall, E. S., Kolstad, J. J., Iwen,
and Stewart, B. F. Lactic acid production, M. L., Benson, R. D., Borchardt, R. L,
separation and/or recovery process, U.S. U.S. Patent 5,484,881, 1996.
Patent 5,510,526, 1996 10 Hartmann, M. H., Biopolymers from Re-
7 Datta, R. and Tsai, S. P. Esterification of newable Resources, ed. D. L. Kaplan, Chap-
fermentation-derived acids via pervapora- ter 13, Springer-Verlag, Berlin Heidel-
tion, U.S. Patent 5,723,639; 1998 berg New York, 1998. Dubois, P., Je-
8 Enomoto, K., Ajioka, M., Yamaguchi, A., rome, R., Lofgren, A., and Albertsson,
U.S. Patent 5,310,865, 1995. Kashima, T., A. C., J. M. S. Rev. Macromol. Chem.
Kameoka, T., Ajioka, M., Yamaguchi, A. Phys., C35(3), 379, 1995. Tsuji, H., Poly-
U.S. Patent 5,428,126, 1995. Ichikawa, lactides in Biopolymers, Chapter 5, Wiley-
F., Kobayashi, M., Ohta, M., Yoshida, Y. VCH, 2002. Drumright, R., Gruber, P.
Obuchi, S., Itoh, H., U.S. Patent and Henton, D. E., Advanced Materials,
5,440,008, 1995. Ohta, M., Yoshida, Y., 12(23),1841, 2000.
Obuchi, S., Yoshida, Y., U.S. Patent 11 Kricheldorf, H. R. and Krieser, I., Makro-
5,440,143, 1995. mol. Chem., 187, 1861, 1986.
9 Gruber, P. R., Hall, E. S., Kolstad, J. J., 12 Kurcok, P., Penczek, J., Franek, J. and
Iwen, M. L., Benson, R. D., Borchardt, Kedlinski, Z., Macromolecules, 25(9),
R. L., U.S. Patent 5,142,023, 1992. Gru- 2285, 1992.
ber, P. R., Hall, E. S., Kolstad, J. J., Iwen,
References 405
13 Nijenhuis, A. J., Du, Y. J., Van Aert, 32 Hartmann, M. H., High molecular
H. A. M. and Bastiaansen, C., Macromole- weight polylactic acid polymers, in: Bio-
cules, 28, 2124, 1995. H. R. Kricheldorf, I. polymers from Renewable Resources,
Kreiser-Saunders, and C. Boettcher, Poly- Springer-Verlag, Berlin, 367-411.
mer, 36(6), 1253, 1995. 33 Thakur, K. A. M., Kean, R. T., Hall, E. S.,
14 Feijen, J., J. Applied Polymer Science, 62, Doscotch, M. A., Munson, E. J., A quanti-
1295, 1996. tative method for determination of lac-
15 US Patent 5,310,599, 1994. tide composition in poly(lactide) using
16 Jacobsen, S., Fritz, H. G., Degee Ph., Du- 1H NMR, Analytical Chemistry, 69, 4303,
bois, Ph. and Jerome, R., Polymer, 41, 1997.
3395, 2000. 34 Thakur, K. M., Kean, R. T., Hall, E. S.,
17 Schmack, G., Jacobsen, S., Fritz, H. G., Kolstad, J.J., Munson, E. J., Stereochemi-
Vogel, R., Tandler, B. and Beyreuther, R., cal aspects of lactide stereo-copolymeri-
Journal of Biotechnology, 86, 15, 2001. zation investigated by 1H NMR: A case
18 Jacobsen, S., Fritz, H. G., Degee Ph., Du- of changing stereospecificity, Macromole-
bois, Ph., and Jerome, R., Journal of Poly- cules, 31, 1487, 1998.
mer Science, 37, 2413, 1999. 35 Spassky, N., Wisniewski, M., Pluta, C.,
19 Jacobsen, S., Fritz, H. G., Degee Ph., Du- Le Borgne, A., Highly stereoselective po-
bois, Ph., and Jerome, R., Industrial lymerization of rac-(d,l)-lactide with a
Crops and Products, 11, 265 2000. chiral Schiff’s base/aluminum alkoxide
20 Jacobsen, S., Fritz, H. G., Degee Ph., Du- initiator, Macromolecular Chemistry and
bois, Ph., and Jerome, R., Macromol. Physics, 197, 2627, 1996.
Symp., 144, 289, 1999. 36 Baratian, S., Hall, E. S., Lin, J. S., Xu, R.,
21 Jacobsen, S., Fritz, H. G., Degee Ph., Du- Runt, J., Crystallization and solid-state
bois, Ph., and Jerome, R., Macromol. structure of random polylactide copoly-
Symp., 153, 261, 2000. mers: poly(L-lactide-co-D-lactide)s, Macro-
22 T. Fukushima, Intern. Polymer Processing, molecules, 34, 4857, 2001.
XV, 4,2000. 37 Zell, M. T., Padden, B. E., Paterick, A. J.,
23 R. Miyoshi, Intern. Polymer Processing, Hillmyer, M. A., Kean, R. T., Thakur,
XI, 4, 1996. K. A. M., Munson, E. J., Direct observa-
24 Miyoshi, R., et al., U.S. Patent 5,574,129, tion of stereodefect sites in semicrystal-
1996. line poly(lactide) using 13C solid-state
25 Narayan, R., et al., U.S. Patent 5, NMR, Journal of the American Chemical
906,783, 1999. Society, 120, 12672, 1998.
26 Reichert, D., et al., U.S. Patent 38 Pyda, M., Buzin, A., Wunderlich, B.,
5,378,801, 1995. Bopp, R. C., Morphology of poly(lactic
27 Jansson, K., Koskinen, J., and Selin, J.-F., acid) by AFM and calorimetry, Proceed-
U.S. Patent 6,214,967, 2001. ings of the NATAS Annual Conference on
28 Witzke, D.R. Introduction to Properties, Thermal Analysis and Applications, 30th,
Engineering, and Prospects of Polylactide 463, 2002.
Polymer, Ph.D. thesis, Chemical Engi- 39 Fischer, E. W., Sterzel, H. J., Wegner, G.,
neering, Michigan State University, Investigation of the structure of solution
1997. grown crystals of lactide copolymers by
29 Garlotta, D., A literature review of poly means of chemicals reactions., Kolloid
(lactic acid), J. of Polym. and the Environ- Zeitschrift & Zeitschrift fuer Polymere, 251,
ment, 9, 63, 2002. 980, 1973.
30 Tsuji, H. and Ikada, Y., Physical proper- 40 Kolstad, J. J., Crystallization kinetics of
ties of polylactides,. Current Trends in poly(L-lactide-co-meso-lactide, Journal of
Polymer Science, 4, 27, 1999. Applied Polymer Science, 62, 1079, 1996.
31 Tsuji, H. Polylactides, in: Biopolymers 4 41 Janzen, J., Dorgan, J. R., Knauss, D. M.,
Polyesters III Applications and Commercial Hait, S. B., Limoges, B. R., Fundamental
Products., Wiley-VCH, Weinheim, Ger- solution and single-chain properties of
many, 2002, chap. 5.
polylactides, Submission to Macromole- 51 Drumright, R.E.; Hartmann, M., Wolf,

cules, Feb. 11, 2003. R., Copolymers of monocyclic esters and
42 Dorgan, J. R., Lehermeier, H., Mang, M., carbonates and methods for making
Thermal and rheological properties of same, PCT Int. Appl., 2002
commercial-grade poly(lactic acid)s, Jour- 52 Gruber, P.R., Kolstad, J. J., Witzke, D. R.,
nal of Polymers and the Environment, 8, 1, Hartmann, M. H., Brosch, A. L., Viscosi-
2000. ty-modified lactide polymer composition
43 Janzen, J. and Dorgan, J. R., A novel and process for their manufacture, U.S.
approach to modeling viscoelastic prop- Patent 5,594,095, 1997.
erties of thermoplastic polymer melts, 53 Gruber, P. R., Kolstad, J. J., Ryan, C. M.,
with applications to polylactides, Annual Hall, E. S., Eichen-Conn, R. S., Paper
Technical Conference – Society of Plastics having a melt-stable lactide polymer
Engineers, 60, Vol. 1, 929, 2002. coating and process for manufacture,
44 Palade, L., Lehermeier, H. J., Dorgan, U.S. Patent 5,338,822, 1995.
J. R., Melt rheology of high L-content 54 Lehermeier, H. J. and Dorgan, J. R., Melt
poly(lactic acid), Macromolecules, 34, rheology of poly(lactic acid): conse-
1384, 2001. quences of blending chain architectures,
45 Cooper-White, J. J. and MacKay, Michael, Polymer Engineering and Science, 41,
E., Rheological properties of poly(lactides). 2172, 2001.
Effect of molecular weight and tempera- 55 Ramkumar, D. H. S. and Bhattacharya,
ture on the viscoelasticity of Poly(L-lactic M., Steady shear and dynamic properties
acid), Journal of Polymer Science: Part B: of biodegradable polyesters, Polymer En-
Polymer Physics, 37, 1803, 1999. gineering and Science, 38, 1426, 1998.
46 Schindler, A., Hibionada, Y. M., Pitt, 56 Feng, Q. and Hanna, M. A., Rheological
C. G., Aliphatic polyesters. III. Molecular properties of amorphous and semicrys-
weight and molecular weight distribu- talline polylactic acid polymers, Industrial
tion in alcohol-initiated polymerizations Crops and Products, 10, 47, 1999.
of e-caprolactone, Journal of Polymer 57 Cicero, J. A., Dorgan, J. R., Dec, S. F.,
Science Polymer Chemistry, 20, 319, 1982. Knauss, D. M., Phosphite stabilization ef-
47 Dorgan, J.R., Williams, J. S., Lewis, fects on two-step melt-spun fibers of
D. N., Melt rheology of poly(lactic acid): polylactide, Polymer Degradation and Sta-
Entanglement and chain architecture ef- bility, 78, 95, 2002.
fects, Journal of Rheology, 43, 1141, 1999. 58 Gruber, P. R., Kolstad, J. J., Hall, E. S., Ei-
48 Zhao, Y., Cai, Q., Jiang, J., Shuai, X., chen-Conn, R. S., Ryan, C. M., Melt-
Bei, J., Chen, C., Xi, Fu., Synthesis and stable lactide polymer compositions and
thermal properties of novel star-shaped their manufacture, PCT Int. Appl., WO
poly(L-lactide)s with starburst PAMAM- 9407949, 1994.
OH dendrimer macroinitiator, Polymer, 59 P. Gruber, D. Henton, J. Lunt, J. Randal,
43, 5819, 2002. Polylactic Acid Technology in Biopoly-
49 Drumright, R., Barker, H, Beaudoin, D., mers and Their Biocomposites, CRC
Berler, N, Broomall, C., Leibig, C., Hart- Press, 2005.
mann, M, Henton, D., McCarthy, K., Ol- 60 Ikada, Y., Jamshidi, K., Tsuji, H., Hyon,
son, M., Randall, J., Syverud, K., Rheolo- S.H., Stereocomplex formation between
gy modification of poly(lactic acid), ACS enantiomeric poly(lactides), Macromole-
regional conference, Grand Rapids, MI, cules 20, 904, 1987
2001. 61 Tsuji, H. and Ikada, Y., Stereocomplex
50 Tasaka, F., Ohya, Y., Ouchi, T., One-pot formation between enantiomeric poly-
synthesis of novel branched polylactide (lactic acid)s. XI. Mechanical properties
through the copolymerization of lactide and morphology, Polymer 40, 6699, 1999.
with mevalonolactone, Macromolecular 62 Tsuji, H. and Ikada, Y., Crystallization
Rapid Communications, 22, 820, 2002. from the melt of poly(lactide)s with dif-
ferent optical purities and their blends,
References 407
Macromolecular Chemistry and Physics, 70 Tsuji, H., Hyon, S. H., Ikada, Y., Stereo-
197, 3483, 1996. complex formation between enantio-
63 Tsuji, H. and Ikada, Y., Stereocomplex meric poly(lactic acids). 5. Calorimetric
formation between enantiomeric poly- and morphological studies on the stereo-
(lactic acid)s. XI. Mechanical properties complex formed in acetonitrile solution,
and morphology of solution-cast films, Macromolecules, 25, 2940, 1992.
Polymer, 40, 6699, 1999. 71 Tsuji, H., Horii, F., Nakagawa, M., Ika-
64 Loomis, G. L. and Murdoch, J. R., Poly- da, Y., Odani, H., Kitamaru, R., Stereo-
lactide stereocomplexes, Polymer Prepr. complex formation between enantio-
31, 55, 1990. meric poly(lactic acid)s. 7. Phase struc-
65 Okihara, T., Kawaguchi, A., Tsuji, H., ture of the stereocomplex crystallized
Hyon, S. H., Ikada, Y., Katayama, K., Lat- from a dilute acetonitrile solution as
tice disorders in the stereocomplex of studied by high-resolution solid-state car-
poly(L-lactide) and poly(D-lactide), Bulle- bon-13 NMR spectroscopy, Macromole-
tin of the Institute for Chemical Research, cules, 25, 4114, 1992.
Kyoto University, 66, 271, 1988. 72 Tsuji, H. and Ikada, Y., Stereocomplex
66 Tsuji, H., Horii, F., Hyon, S. H., Ikada, formation between enantiomeric poly-
Y., Stereocomplex formation between en- (lactic acid)s. 6. Binary blends from co-
antiomeric poly(lactic acid)s. 2. Stereo- polymers, Macromolecules, 25, 5719,
complex formation in concentrated solu- 1992.
tions, Macromolecules, 24, 2719, 1991. 73 Tsuji, H. and Ikada, Y., Stereocomplex
67 Okihara, T., Tsuji, M., Kawaguchi, A., formation between enantiomeric poly-
Katayama, K., Tsuji, H., Hyon, S. H., Ika- (lactic acids). 9. Stereocomplexation from
da, Y., Crystal structure of stereocomplex the melt, Macromolecules, 26, 6918, 1993.
of poly(L-lactide) and poly(D-lactide), 74 Tsuji, H., Ikada, Y., Hyon, S. H., Kimura,
Journal of Macromolecular Science, Phys- Y., Kitao, T., Stereocomplex formation
ics, 30, 119, 1991. between enantiomeric poly(lactic acid).
68 Tsuji, H., Hyon, S. H., Ikada, Y., Stereo- VIII. Complex fibers spun from mixed
complex formation between enantio- solution of poly (D-lactic acid) and poly
meric poly(lactic acid)s. 3. Calorimetric (L-lactic acid), Journal of Applied Polymer
studies on blend films cast from dilute Science, 51, 337, 1994.
solution, Macromolecules, 24, 5651, 1991. 75 Tsuji, H., In vitro hydrolysis of blends
69 Tsuji, H., Hyon, S. H., Ikada, Y., Stereo- from enantiomeric poly(lactide)s part 1.
complex formation between enantio- Well-stereocomplexed blend and non-
meric poly(lactic acid)s. 4. Differential blended films, Polymer, 41, 3621, 2000.
scanning calorimetric studies on precipi- 76 Tsuji, H. and Suzuki, M. In vitro hydro-
tates from mixed solutions of poly(D-lac- lysis of blends from enantiomeric poly-
tic acid) and poly(L-lactic acid), Macro- (lactide)s. 2. Well-stereocomplexes fiber
molecules, 24, 5657, 1991. and film, Sen’i Gakkaishi, 57, 198, 2001.
409
15
Biobased Consumer Products for Cosmetics
Thomas C. Kripp
15.1
Introduction and Historical Outline
15.1.1
Cosmetics Past and Present
The concept of cosmetics as agents for cleansing, grooming, and embellishing

the human body is as old as mankind. Prehistoric remains found in Alicante
and Lescaux provide evidence that women applied red dye to their faces in those
times. In Ancient Egypt, cosmetic products were widely used and highly devel-
oped. Henna and indigo were used for coloring the hair, malachite was applied
to the eyelids, and stibium to the eyebrows, and the lips and cheeks were also
colored. The raw materials for these early cosmetics were derived from nature.
Chemical transformation of natural substances to obtain more effective prod-
ucts was also known in antiquity. The earliest written evidence of soap is on Su-
merian clay tablets (2500 BC): Vegetable oil was boiled with potash to produce
soap, a detergent still in use today.
The manufacture of soap was doubtless the beginning of the industrialization
of cosmetics. Early as during the reign of Charlemagne there were fairly large
soap-boiling factories. Around the Mediterranean Sea, Savona, Venice and Mar-
seille were important centers of a flourishing soap industry. The advancement
of science in the Industrial Age enabled the manufacture of cosmetic products
in large quantities and at affordable prices. Because of progress in chemical
know-how, the results of perming and coloring the hair became better and bet-
ter; new detergents in shampoos ensured increasingly gentler cleansing of the
hair, and special ingredients even added a conditioning effect. Resins based on
elaborate synthetic polymers, sprayed on the hair with micro-fine nozzles, give
hold to today’s hair styles [1].
ISBN: 3-527-31027-4
410 15 Biobased Consumer Products for Cosmetics
15.1.2
Bionics: Learning from Nature
A relatively new branch of science called bionics is concerned with understanding

amazing mechanisms in Nature and using them to develop engineering solutions
for humans. Bionics studies, for example, the hydrodynamics of fast-swimming
fish to develop ships with optimum dimensions. The Velcro principle of fastening
is a direct “offshoot” of Nature’s sticky burr. Actually, only part of the numerous
inventions in world history are products of the human mind alone. The wheel,
the plough, and the ball-point pen are prominent examples. Many inventions
are based on models found in nature which man has used – consciously or uncon-
sciously – and adapted, where necessary, to suit his needs. Gliders, suction hooks,
and the microphone are nothing but modified applications of primeval principles
such as the use of thermal currents by birds, the suckers of tree frogs’ feet, or the
eardrum of most vertebrates. The transmission of information by means of light
impulses was employed by glow-worms millions of years before Morse developed
his telegraph, and electricity was present on earth, for example as lightning, nerve
impulses, and weapons of some animals such as the electric eel, long before hu-
mans were able to put it to practical use. SONAR (an ultrasonic positioning sys-
tem for ships) is, in fact, a principle used by bats since their arrival on earth for
orientating themselves in the dark. Jellyfish and squid were using recoil long be-
fore there were engineers thinking of jet propulsion. When man started to cover
water-sensitive objects with protective layers of wax or lacquer and varnish, fruits,
leaves, and flowers had long been used to protecting themselves against drying
out, decay, and other attacks from outside with water-repellent wax coverings [2].
Wella is a company that is very much concerned with utilizing nature’s crea-
tiveness and capacity with to obtain new materials for developing new products.
Although many natural substances, both animal and vegetable, are known to be
highly toxic – for example snake venom, spotted hemlock, autumn crocus,
deadly nightshade, and some fungi – a wealth of life-giving, protecting, and car-
ing substances have also evolved. Some of these have been used in cosmetics
for a long time (soaps on the basis of natural fats, glycerol, vegetable extracts,
lanolin, essential oils, henna . . . ), and others – examples are given below –
found their way into cosmetics only a few years ago.
15.2
Betaine, The Conditioner Made from Sugar Beet [3]
15.2.1
Occurrence
Betaine was first discovered as a component of sugar beet (Beta vulgaris) more
than a hundred years ago. Betaine (trimethylglycine) has an important role in
life-sustaining systems in Nature. It is present throughout the ecosystem, in mi-
croorganisms, plants, all animals, and in humans. It is an important intermedi-

ary stage in the complex biochemical reactions within cells. It is, for instance,
instrumental in controlling breathing and in osmoregulation (control of the
water content of cells or the organism) and for oxygen fixation by a variety of
useful bacteria. The work of microorganisms that fix nitrogen in the soil is
stimulated by betaine [4, 5].
Betaine is present not only in sugar beet but also in a variety of other plants,
for example spinach, wheat, barley, beans and oranges. Biologists have discov-
ered that these plants synthesize betaine as a protection against changes in their
environment. Cereals, for example, use betaine to withstand increased salt con-
tent of the soil without impairment of growth [6]. Betaine also seems to protect
the cell membranes of plants against the cold [7] and to lessen the damaging ef-
fect of sulfur dioxide on the leaves of trees. It accumulates, especially, in chloro-
plasts which are responsible for the green color of plants and which provide
their cells with energy.
In animal bodies betaine has a function similar to that of vitamins closely re-
lated to the effect of folic acid and vitamin B12. It is found in the liver, stomach,
heart, and other organs of a large number of species, including humans. Be-
taine is a so-called lipotropic factor that reduces the risk of fatty degeneration of
the liver [8, 9]. In the kidneys it seems to play a key role in protecting the cells
against urine [5].
15.2.2
Chemical Properties
Betaine is a stable quaternary ammonium compound. It has the character of a

zwitterion with interesting chemical and physical properties. It is water-soluble
and highly hygroscopic. It has no toxic effect whatsoever, it is even approved as
a food additive. Betaine is a close relative of the amino acid glycine from which
it is formed through biosynthesis (Fig. 15.1). As a by-product of sugar beet pro-
duction, which amounts to approximately 270 million tons a year, betaine is
widely available and it is also biodegradable.
15.2.3
Production
On an industrial scale betaine is produced by extraction from sugar beet

(Fig. 15.2). The highest concentration is in the upper part of the beet; the total
content varies between 1 and 1.5% to the dry weight.
Fig. 15.1 Chemical structure of be-

taine (left) and glycine (right).
Fig. 15.2 Production of betaine.
In the first stage of sugar production, betaine and sugar (saccharose) are ex-
tracted from the crushed beet by dissolution in hot water. Betaine is a white
substance and after physical purification of the sugar up to 5% betaine is pres-
ent in the remaining molasses. Further clean-up, e.g. by chromatographic sepa-
ration on industrial columns, is necessary for separation of the betaine. It is fi-
nally recrystallized repeatedly from pure water.
15.2.4
Use and Fields of Application
Betaine is used in foods, for instance as an additive in energy drinks and food-
stuffs, and as a flavor enhancer in seafood. Many vitamin mixtures for humans
and for animals contain betaine. It is approved as a dietary supplement in a
variety of countries.
In acid solution betaine has a positive charge; in other words it has a cationic
character. In hair care, cationic compounds are used to improve the combability
of damaged hair. The mode of action is as follows. Hair suffers oxidative dam-
age as a result of a variety of effects, for example sun and sea water, e.g. during
a seaside holiday, but also as a result of different kinds of treatment. The result
is accumulation of negatively charged groups on the hair surface. This negative
charge enables deposition of positively charged – i.e. cationic – compounds,
which improve the properties of the hair surface (Fig. 15.3).
Examples of such cationic compounds are long-chain alkyltrimethyl ammo-
nium salts. These so-called cationic surfactants may be replaced by betaine in
special formulations of hair rinses and deep-conditioning products. Because
conventional cationic surfactants also act as emulsifiers, a formulation contain-
ing betaine must also include – in addition to an acid – an emulsifier system to
regulate the viscosity, which fortunately also enhances the conditioning effect of
Fig. 15.3 Depositing cationic

compounds on the hair surface.
the product. Wella has obtained patents [10] for this kind of formulation and
they have been successfully used in hair-care products for many years.
An interesting discovery was made when the skin tolerance of betaine prod-
ucts was investigated. Not only were betaine-containing skin-cleansing lotions
Fig. 15.4 Elbow washing test. Left:

product without betaine; right: prod-
uct with betaine.
well tolerated by the skin, they even proved to have a smoothing effect on the
skin compared with formulations without betaine.
Proof of the good skin tolerance is provided by the elbow washing test
(Fig. 15.4) according to P. Frosch. The products (one with betaine and one with-
out betaine) were each tested on a person with sensitive skin. The products
were applied one to the right and one to the left arm (crook of the elbow) of
one person twice daily on five consecutive days under standardized conditions.
The results were impressive – see the photos of the test person.
15.2.5
Innovation Through Combination: Betaine Esters
The cationic surfactants mentioned in the previous paragraph are usually also
quaternary ammonium compounds which are known to be biologically “hard”,
that is they do not biodegrade easily. By using betaine derivatives, however, it is
possible to manufacture hair conditioners based on biologically “soft” surfac-
tants [11]. The chemical structure of betaine contains a cationic group and an
anionic acid group, depending on the pH value. The group with a positive
charge, responsible for the excellent substantivity to hair, makes the molecule
readily soluble in water. A surfactant molecule, however, also has a hydrophobic
part to make it “feel at home” in both worlds – those of water and oil – and to
make it able to mediate between them. By applying appropriate methods a so-
called “hydrophobic chain” of any length can be attached to the acid group by
linking the betaine to a fatty alcohol. Chemically speaking, this is an ester bond
that forms when an acid combines with an alcohol with elimination of water.
The structural formulas (Fig. 15.5) of betaine esters and “normal” cationic sur-
factants are very much alike.
There is, however, a difference that has far-reaching consequences. It is pre-
cisely the reaction by which the betaine ester was formed that can – under cer-
tain conditions – take a reverse course to form the original materials. A wel-
come property of this class of substance is the fairly good stability of the ester
bonds in acid media, for example in a hair conditioner, whereas at neutral or,
especially, alkaline pH the bonds are quickly cleaved. Thus it is possible to for-
Fig. 15.5 Structures of a normal cationic surfactant (a) and of a betaine ester (b).
mulate hair conditioners with excellent conditioning properties and sufficient

stability, whose surfactant ingredients undergo degradation in the neutral to al-
kaline medium of sewage treatment plants. The resulting fragments are betaine
and a fatty alcohol, completely biodegradable natural substances [10–14] that are
borrowed from Nature for a short period and returned in a reusable form to
complete the natural cycle.
15.2.6
Summary and Prospects
Betaine is a very interesting compound for formulating hair conditioners and

rinses. It also has skin-soothing properties and is used as an ingredient of a
wide variety of cosmetic products.
Additional advantages are its global availability as a by-product of sugar beet
processing and its biodegradability. A natural substance present not only in su-
gar beet but also in microorganisms, plants, and mammals, it is also found in
our food and is even approved as a dietary supplement. When used in body-care
products betaine is withdrawn from its natural cycle for a short time only.
Betaine may be used in hair-care products in its natural form or it may be
transformed into customized derivatives by esterification.
15.3
Chitosan, Hair-setting Agent from the Ocean [15]
15.3.1
Chitin, a Precursor of Chitosan
Carbohydrates are the most abundant class of organic substance on earth. The
most common carbohydrate is cellulose, a polymer of glucose, and it forms the
skeletal structure of most plants. Among the nitrogenous polysaccharides, chi-
tin, a polymer of acetylglucosamine, is most important (Fig. 15.6). Like cellu-
lose, its role in nature is that of a skeletal and membrane substance. It does,
however, not occur in higher plants but rather in lower plant life and inverte-
brates, the arthropods, in different amounts. The term “chitin” is derived from
the Greek word “chiton” which means “armor” [16].
The properties of chitosan and its production from chitin are described below;
chitosan also occurs in several lower fungi, e.g. Mucor rouxii.
15.3.2
Occurrence of Chitin (Fig. 15.6)
A chitin content of up to 45% is characteristic of many lower and higher fungi.

Chitin is found, for example, in classes such as algal fungi (phytomycetes), sac
fungi (ascomycetes) – which include yeasts and mold – and club fungi (basidio-
Fig. 15.6 Chitin-producing living organisms.
mycetes) – including the well-known mushrooms, toadstools, and honey mush-

room. In these fungi the biopolymer is used for the cell walls and the mem-
branes of the mycelium [17]. Chitin is also present in some forms of algae.
In the animal kingdom, huge quantities of chitin are found in arthropods, a
phylum of diverse, highly developed animals of more than 900,000 species. The
group includes crustaceans such as crabs, shrimps, lobsters, and krill, and also
insects. The shells of crustaceans have a chitin content of up to 85% [17, 18]. In
insects, chitin is the structural material of the hard wings of beetles, e.g. the po-
tato beetle or the cockchafer, or the wing sheaths of dragonflies. In addition to
its supporting function and its protection of the soft parts, the chitin armor also
prevents water loss – without this armor scorpions, for example, could not sur-
vive in the desert [19].
It is estimated that the global quantity of chitin formed by Nature is more
than one billion tons per year [20]. Today, most chitin and chitosan production
facilities are located in the United States and in Japan. The raw material is
obtained from the shellfish industry from which enormous amounts of crab
and shrimp shell are available. Chitin production is, therefore, closely connected
with ocean fishing. In the past few years the annual quantity of chitin has
grown steadily. In the USA and Japan dumping of waste from shellfish proces-
sing, the chitin, at sea or in landfills, is not allowed and so the search for new
uses of chitin and its derivative chitosan provides the opportunity to gain a new
raw material from seemingly useless by-products. We see that “waste” can easily
become a valuable secondary raw material.
15.3.3
Production
15.3.3.1 Purification of Chitin (Fig. 15.7)

In nature, chitin does not occur in an isolated form. Proteins are often found
with chitin. The chitin tissue of a crab shell, for example, also contains major
quantities of calcium carbonate, which accounts for up to 75% of the weight of
the shell. For purification, first the proteins are removed from the pre-dried,
ground crab shells in a sodium hydroxide bath. The biopolymer is then treated
with hydrochloric acid to eliminate mineral salts such as calcium carbonate and
calcium phosphate. After washing it is dried as flakes [21].
Fig. 15.7 Purification of chitin.
Chitin and cellulose have similar structures, the difference being that in chitin
the hydroxy group on the C-2 atom is replaced by an acetylamino group. The acet-
ylglucosamine units are connected through b-glycoside linkages (Fig. 15.9).
Chitin is insoluble in water, dilute acid, dilute alkalis, alcohol, and other or-
ganic solvents. It dissolves in concentrated hydrochloric acid or methanesulfonic
acid, although with partial cleavage of the biopolymer.
15.3.3.2 Production of Chitosan

When chitin is treated with concentrated sodium hydroxide solution the N-ace-
tyl bond is cleaved and the deacetylated product is called chitosan (Fig. 15.8).
With its free amino groups, chitosan is insoluble in water. Water-soluble sub-
stances can, however, be obtained from the salts formed on reaction with acids
such as hydrochloric, acetic, or lactic acid. In acid medium each of chitosan’s
numerous amino groups is positively charged, i.e. it is a polyelectrolyte or poly-
cation (Fig. 15.8). Because each glucosamine unit has a charge, the biopolymer
has a very high charge density. Many materials, for example water-soluble pro-
Fig. 15.8 Deacetylation of chitin to chitosan and its protonation by salt formation.
Fig. 15.9 Chemical structure (section) of cellulose (top),

chitin (center), and chitosan (bottom).
teins or the hair keratin, are negatively charged, which enables interaction with
the positively charged chitosan.
15.3.4
Chitosan in cosmetic products
The positive charge of chitosan (in an acid solution) is the cause of its substan-
tivity (i.e. its ability to become attached to the hair, especially damaged hair),
which improves the combability, luster, and feel of the hair (Fig. 15.10). Wella
has obtained patents [22] in many countries for use of chitosonium salts in hair
conditioners and special-formulation shampoos.
As early as in 1976, Wella researchers discovered that chitosonium salts were well
suited as film-forming agents in hair-setting products, and chitosan has been suc-
cessfully used in Wella products ever since. The biopolymer has several advantages
over synthetic polymers. It contains no harmful monomers or by-products from
polymerization, the polymer films do not tend to become tacky, even at high relative
humidity, and the concentrations required to achieve a firm hold are relatively low.
These properties are particularly useful in setting mousses. The quality of a
setting product is expressed in terms of its ability to retain curl in hair strands.
An uncomplicated method (hair relaxation or curl retention test, Fig. 15.11) is
used to show that curl retention in hair swatches treated with a chitosan setting
product is significantly better than in swatches treated with other products, and
the more so for untreated hair.
In this test the relaxation of swatches of curled hair, that is the change in
length of the swatches, is measured over a period of time and the results are re-
laxation curves characteristic of the product applied to the wound hair. The
comparison criteria are “no product applied”, “chitosan setting product applied”,
Fig. 15.10 Substantivity of cationic polymers to hair.

Fig. 15.11 Curl-retention test: chitosan reduces the sensitivity

of hair setting products to humidity.
and “non-chitosan setting product applied”. Although after four hours at a rela-
tive humidity of 30% there is not much difference in relaxation, the results vary
widely at 70% relative humidity. Curl retention by swatches treated with a chito-
san setting product is far better than by the other samples.
The water-retentive properties of films based on chitosonium salts depend on
the acid used for neutralization. The so-called water vapor sorption is deter-
mined from the difference in weight of the films at 30% and at 65% relative hu-
midity. Comparison with the results obtained with hyaluronic acid, a moistur-
izer frequently used for skin-care products indicates that some chitosonium
salts have similar effects (Fig. 15.12).
Many other uses of chitosan in cosmetics have been suggested in the relevant
patent literature. The potential uses proposed for chitosan include coating of
cosmetic pigments, and as a skin-protecting agent and moisturizer in cosmetic
products [23–26]. Chitosonium salts have also been proposed for oral hygiene
purposes to prevent dental caries and halitosis [27].
It is also possible to use chitosan as a basis of ingredients of other products.
The free NH2 and OH groups shown in the graphic formula can be derivatized
under suitable conditions, which enables its properties to be tailored to the in-
tended use. Qualities such as solubility, substantivity, conditioning, and film-
forming properties, etc., can be adapted to suit the most diverse needs
(Fig. 15.13). Although such derivatives are no longer pure natural substances (a
definition is given in Section 15.4.1), the fact that most of the molecule has
been “borrowed” from Nature enables their classification as “renewable raw ma-
terials” with all their well-known advantages.
Fig. 15.12 Water vapor sorption of chitosonium salts.
Fig. 15.13 Different derivatives of chitosan for various types of product.
15.3.5
Summary and Prospect
The natural substances chitin and chitosan have been known for a long time.
Professor Joseph Nagyvary from Texas goes so far as to argue that Stradivari
was the first to discover the biopolymer – he is said to have used chitosan ob-
tained from crushed dragonfly wings as the varnish for his violins. It is, how-
ever, in the past 25 years that, because of its structure and its properties, chito-
san has proved a versatile raw material.
The polymer is an excellent basis for synthesis of customized materials for
the most diverse applications. Depending on the type and position of substi-
tuents the products may be incorporated into hair and skin-care products. The
growing volume of the patent literature indicates that the number of cosmetic
and non-cosmetic uses of chitosan and its derivatives will continue to increase
in years to come.
15.4
From Energy Reserve to Shampoo Bottle: Biopol [28]
15.4.1
Biodegradable Packages
As described in the introductory section, renewable materials have always been

widely used as ingredients for cosmetics. In this section we will deal with at-
tempts to produce packaging materials for cosmetics from renewable materials.
Packages made of renewable materials are nothing special – paper, cardboard,
Cellophane, etc., are examples everybody knows. But also composite materials
such as Tetra Pak cartons fulfill some of the criteria. In the past two decades
many materials have been developed on the basis of substances derived from
natural materials and intended for new types of packaging. In addition to their
undisputed advantage of being obtained from renewable resources, they are re-
quired to be biodegradable. This is doubtless true for natural substances that
have not undergone any chemical change or for derivatives that are readily de-
composed into biocompatible fragments by hydrolysis or oxidation under nor-
mal environmental conditions. Such derivatives, e.g. Mater-Bi, based on starch,
are difficult to distinguish from their petroleum-based counterparts by the lay-
man. Unlike the latter, however, in compost works they are degraded to com-
post, water, and carbon dioxide within a couple of weeks. A major drawback of
these materials in comparison with conventional plastics is that they cannot
hold liquids. Even completely non-aggressive liquids like pure water cause the
starch-based materials to swell very quickly.
There is, by nature, a reciprocal relationship between the biodegradability of a
material and its stability during use. On the following pages we give an account
of a “man-made” material that combines the allegedly opposing qualities of re-
sistance to liquids and biodegradability. It is a genuine natural substance and a
renewable material. These two terms are not necessarily synonyms. The follow-
ing definition explains our concept of the two expressions.
· Natural substance. A substance which – irrespective of its origin – also occurs
in that particular form in animated nature. Because the properties of a sub-
stance such as melting point, solubility, degradability, etc., are independent of
its origin, this definition only concerns the question of whether the substance
flows through biological metabolism. As an example, salicylic acid is basically
a natural substance, even if a sample of it is produced by synthesis instead of
by extraction from wicker bark. The food industry has coined the term “na-
ture-identical” for synthetically produced natural substances.
· Renewable material. A substance which is obtained fully or at least chiefly
from biomass, including after chemical modification, when the resulting
product is not found in animated nature. Examples include nitrocellulose,
made from cellulose, and fatty acid methyl ester (“biodiesel”), made from veg-
etable oil. The resulting products are not known to be present in animated
nature, but they are based mainly on renewable materials.
15.4.2
What is “Biopol”?
When food is abundant, each living organism uses specific substances as energy
reserves. Whereas humans and animals deposit surplus nutritional energy mostly
in the form of body fat, plants also use carbohydrates, in particular starch. The seed
pods of plants, in particular, are rich in carbohydrates and fats, the energy supply
used by the germinating seedling. Although these two classes of substance are cer-
tainly the most important for energy storage, others are used for the purpose, espe-
cially in microorganisms. Many bacteria form polyhydroxy-b-alkanoate, PHA
(Fig. 15.14), as reserve or food-storage material which provides carbon and energy
when needed. Its monomer constituents include b-hydroxybutyric acid, hydroxyva-
leric acid, and lactic acid. The linear polymer poly-b-hydroxy butyric acid (PHB) is
accumulated within the cell in the form of grains or granules and it plays a pro-
minent part among the polyhydroxyalkanoic acids. It is a chloroform-soluble poly-
ester with an average of 60 (up to 25,000) units of hydroxybutyric acid.
Fig. 15.14 Formula of 3-hydroxybutyric acid (a), 3-hydroxy-

valeric acid (b), and (section) poly-3-hydroxybutyric/valeric
acid (Biopol) (c).
Fig. 15.15 Accumulation of poly-b-hy-

droxybutyric acid in the cells of Alcali-
genes eutrophus [29].
Poly-b-hydroxybutyric acid was discovered as an intracellular reserve material

in Bacillus megaterium by Lemoigne in 1926. The polymer was then forgotten
until in the early 1960s when it was found in aerobic bacilli, anaerobic photo-
trophic bacteria (e.g. Chromatium okenii), cyanobacteria, and soil bacteria (Alcali-
genes eutrophus) by Schlegel and co-workers in Göttingen, Germany. Polyhy-
droxyalkanates are exclusively formed in bacteria. Under certain conditions of
growth PHB accounts for up to 80% of the dry weight of Alcaligenes eutrophus
(Fig. 15.15). Such conditions are fulfilled by a sufficient supply of high-carbon
nutrients (sugar, starch, etc.) available to the bacteria and a deficiency of an im-
portant biogenic element, e.g. nitrogen or phosphorus to prevent utilization of
the nutritional source for increasing the biomass (multiplication). Instead the
excess nutrient is mostly used to build the reserve polymer.
PHB can be extracted from dried cells by use of organic solvents such as
chloroform, dichloromethane, 1,2-dichloroethane, or ethylene carbonate, and
subsequently precipitated with methanol, ethanol, ether, or hexane, etc. [30].
15.4.3
Biodegradability of Biopol
It is an empirical fact that each substance generated by Nature is normally de-

graded by Nature. If this were not so the undegradable material would have ac-
cumulated in immense amounts over the millions of years since living organ-
isms appeared on the earth. One example is petroleum which is not degraded,
at least under anaerobic conditions, and thus accumulates in huge reservoirs be-
neath the earth, unaffected by atmospheric oxygen.
Fig. 15.16 Degradation of a Biopol

shampoo bottle [31].
Table 15.1 Physical and mechanical properties of Biopol [32].
Molar mass 10 000 to 3.0 ´ 106

Melting temperature (Tm) 173–180 8C
Glass transition temperature (TG) 10 8C
Decomposition temperature ³ 205 8C
Heat of fusion 100 J g–1
Specific gravity 1.25 g cm–3
Crystallinity 55 to 65%
Oxygen permeability 45 cm3 m–2 at–1 day–1 for 25-lm films
Tensile strength (TB) 1500–2500 N cm–2
Elastic modulus 4.6 Gpa
Izod impact strength 0.3 J m–1 (notch radius 1 mm)
Extensibility to break 5%
Modulus (M) 800–1000 N cm–2
Piezoelectric coefficient 1.67 pC N–1
Being a polyester, Biopol is, of course, hydrolysable; the resulting hydroxy

acids are integrated into the usual degradation processes of fatty acids. In con-
trast with hydrocarbons, Biopol is degraded not only under aerobic conditions
with elimination of carbon dioxide and water, but also in an anaerobic environ-
ment, with methane as a by-product (Fig. 15.16). Extensive research into its de-
gradation behavior in soil and in and under water have shown that Biopol is
completely degradable under both aerobic and anaerobic conditions. Its physical
and mechanical properties are listed in Table 15.1.
15.4.4
The Long Way to the Shampoo Bottle
Many problems had to be solved on the way from the first discovery of the bio-
polymer to its use as a packaging material. The first PHB samples obtained by
mechanical processing were dirty brown in color, mainly because of impurities
in the biomass. It was, in particular, the vacuole membranes of the bacteria that
impaired the appearance and mechanical properties of the new raw material.
Initially it was not possible to re-melt the biopolymer, although it is a thermo-
plastic, which meant that leftovers from the production of shampoo bottles were
useless waste. The material was very brittle, difficult to print on using commer-
cial processes, was of limited availability, and above all was very expensive.
All these problems, except the high price in comparison with petroleum-
based polymers, were solved within approximately six years. Optimization of the
fermentation conditions and of the isolation and purification methods made the
product increasingly cleaner. The technological features improved with progress
in lightening the color. Specific fermentation conditions were chosen to produce
not pure polyhydroxybutyric acid, but a copolymer of PHB and polyhydroxyva-
leric acid (PHV). This further improved the properties of the material.
The natural brittleness of the PHB-PHV copolymer was reduced by addition
of suitable plasticizers, which were, of course, also required to be readily biode-
gradable. Small amounts of triacetin (glycerol triacetate) enhanced the elasticity
of the material. Printing of the shampoo bottles was facilitated by the use of
special UV-hardening varnishes. Finally the plastic properties of the new materi-
al were largely comparable with those of polyethylene.
15.4.4.1 Product Development

The high motivation to develop a biodegradable bottle was reason enough for
Wella to modify the standard procedure of product development. Usually a new
cosmetic product is developed with the best possible performance in mind. The
package is designed to optimally suit the product with regard to material, shape,
and presentation. Because the composition of Biopol is completely different
from that of polyethylene, however, despite their physical resemblance, several
particularities had to be taken into consideration.
It is an essential prerequisite that cosmetic products are safe to use for three
years from manufacture. This applies both to the contents and to the packaging.
Because of the ester structure of Biopol, such a long life cannot be guaranteed
in the presence of hydrolytic agents. Thus the material is not suitable as packag-
ing for alkaline products (for example perms or semi-permanent colors) or
strongly acid products (perm neutralizers, rinses). Alcoholic solutions (setting
lotions, hair sprays) are also out of the question, because the alcohol would at-
tack the material over time. The only products that seemed well-suited were
shampoos with their slightly acid pH between 5.0 and 6.0.
There was still one problem to be solved – the plasticizer used for bottle man-
ufacture. Triacetin is also an ester, so decomposition of small amounts of the
material over the course of time cannot be prevented. Although the amounts
are so small that the plasticizing function will not be impaired, the human nose
is extremely sensitive to the smell of acetic acid which is released in the pro-
cess. Consequently every effort was made to find a perfume combination that
optimally masks traces of acetic acid. This was certainly the only example of a
perfume being chosen for its compatibility with the package.
15.4.4.2 Market Launch

In a pilot project in cooperation with Biopol-manufacturer ICI, Wella introduced
the world’s first biodegradable shampoo bottle in 1990. In the consumer market,
the Sanara range was launched in a Biopol bottle, and Linie N was the correspond-
ing professional brand. One of the objectives was to demonstrate a possibility of
curbing the flood of packaging. Another aim was to arouse the interest of manu-
facturers from other industries with a view to encouraging large-scale production
of the material to make it globally available at a favorable price.
This latter goal was achieved. The acceptance level among consumers, traders,
and hairdressers was exceptionally high. Other companies became interested in
the product, which led to further market launches and to completion of a larger
Biopol-manufacturing plant to ensure sufficient supplies of the polymer. In the
meantime the price of Biopol has dropped dramatically, although it has not
reached the level of the fossil-based plastics [28].
In the first stage Wella bore the extra cost of the material of the Biopol bottle
without charging the consumer. The response was overwhelming, and Biopol
production quantities soon fell short of the demand. When all Biopol bottles of
the initial phase were used up, many inquiries for other Biopol products fol-
lowed. The concept of a biodegradable bottle had quite obviously hit the bull’s
eye. In the early 1990s, protection of the environment and economical use of
fossil fuels were a major concern among the population. The fall of Berlin Wall
offered prospects of enormous economic growth, and the charms of a package
that neither increased rubbish heaps nor depleted scarce raw materials seemed
to be the ultimate solution for unrestricted consumption.
Encouraged by the success of its bio-package and backed up by various mar-
ket research reports of positive acceptance tests, Wella started another project
involving Biopol bottles. While efforts were being made to achieve an acceptable
price, meet consumer needs, and ensure technical feasibility, the Biopol project
was conclusively stopped for the time being, at least at Wella.
What had happened? In Germany, the so-called “Duales System” for material re-
cycling (with the green dot identifying recyclable materials) had been started in
1991, and consumers were no longer troubled ecologically, because they collected
conventional plastics in a separate disposal system (“Yellow Bag Program”). This
had a bad effect on the advantages of bio-based plastics, because there is still no
full coverage with appropriate disposal systems. Although the intended amend-
ment to the German bio-waste management ordinance included plans for a nation-
wide collection system for bio-waste [33], there was a good reason for not assigning
Biopol packages to the bio-waste disposal route, despite its high compostability – it
was precisely the result of many years of in-depth research, i.e. making the bacte-
rial energy reserve material look and perform like a “conventional” plastic material,
that made it difficult, not to say impossible, for the average consumer to differenti-
ate between Biopol and other polymers. Mistakes seemed bound to occur and, be-
cause neither the “Duales System” nor biowaste collection provided a practical and
practicable disposal method, the promising approach ended up a blind alley.
15.4.5
Quo vadis, Biopol?
Because of disposal problems, activity centered around production of biodegrad-

able materials was booming in the 1990s. A yoghurt pot marketed by Danone
was another attempt to promote the use of biodegradable plastics for packaging
in Germany. The yoghurt pot was made of polylactic acid, a polymer which,
though based on renewable materials, was synthesizable and thus significantly
cheaper [33]. Its resistance to hydrolysis was far inferior to that of Biopol, but
there is no requirement to keep yoghurt for three years. Despite the favorable
overall conditions, this packaging material did not find a firm footing in the
marketplace and disappeared after a year or so [34].
In addition to the merely mechanical and ecological problems to be solved
when launching biodegradable packages, the issue must be viewed in terms of
disposal. The frequently advertised disposal on the user’s compost cannot be re-
garded as a serious proposition. Apart from the fact most users do not make
their own compost, the same objections as to municipal composting can be
raised – users cannot be expected to know biodegradable polymers from conven-
tional plastics, even if the labeling were clear and prominent.
As matters stand, the “Duales System Deutschland” would have to assume re-
sponsibility for separating and composting the biodegradable polymers. Ther-
mal technology, e.g. the production of electricity and heat in waste-fuelled power
stations, are applicable to biopolymers as they are to mass-produced plastics,
although in this disposal method the former do not have major advantages over
the usual (at least the halogen-free) plastics. Even its neutral behavior with re-
gard to carbon dioxide balance is less of an asset in the light of the considerable
additional transport and processing expenditure.
The market maturity of Biopol was, perhaps, too late, coming at a time when
the Duales System Deutschland was already in the pipeline and when, as a con-
sequence, there was not much interest in organizing a sensible disposal system.
Or perhaps the time is not yet ripe for that kind of material. It may well happen
that some time in the future the current point of view will be changed and it
will no longer be considered expedient to spend so much on the organization,
technology, and energy for collecting, separating, and processing plastics waste
merely to produce minor-quality raw materials and products. Added to this is
Fig. 15.17 Cartoon [35].
the costly petroleum fuel needed in residual waste combustion plants, because
the waste does not contain enough combustible materials. More than 60 years
elapsed between the discovery of polyhydroxybutyric acid and the first techno-
logical application. Maybe the continuing increase in oil prices and the increas-
ing reluctance to subsidize waste processing will eventually be reason enough
for a breakthrough of biodegradable polymers . . .
15.5
Natural Apple-peel Wax: Protection for Hair and Skin [36]
15.5.1
Raw Material Source
The apple, the epitome of fruit since time immemorial, is still a symbol of
youthfulness, freshness, health, beauty, lushness, naturalness, and vitality. In
the garden of Eden it was the coveted fruit from the tree of knowledge, the
cause of the Fall. In Greek mythology, Paris, prince of Troy, judged Aphrodite,
the goddess of love, to be “the fairest”, preferring her to Hera and Athene, and
awarded her the “apple of discord”. William Tell had to shoot an apple off his
son’s head to save his and his son’s lives.
The apple appears in proverbs and sayings (“the apple never falls far from the
tree”, “the apple of someone’s eye”), it is a metaphor of love or immortality. Apples
are healthy and rich in vitamins (“an apple a day keeps the doctor away”), they can
be eaten raw, cooked, as juice, or in a fermented form as cider or applejack, and
they are sort of model for other kinds of fruit – pineapple, “love apple” (for toma-
to), “pomegranate” (from the French word “pomme” for apple) and so on.
15.5.2
Apple-peel Wax
Apples are coated by nature with a very thin wax layer (Fig. 15.18) which accounts
for the shine that appears when an apple is wiped with a soft cloth. It is widely
believed that a man-made wax coating is applied to apples before they are mar-
keted, but it is usually the natural wax we see and feel. In many countries, includ-
ing Germany, apple skin waxing is not allowed by law. Pre-sale waxing would, any-
way, be applied only to fruit intended to look “good”. The apple-peel wax we are
dealing with is obtained from juice-making residues that contain apple skin,
and the appearance of apples used for this purpose does not matter at all.
15.5.3
Observations
The valuable ingredients of apples are a rich nourishment not only for humans
but also for microorganisms, especially for mold and yeast (it is the special quality
of apple juice to ferment easily which is exploited in making cider and applejack).
The inside of the apple is highly sensitive to atmospheric oxygen, light, and
drying out but although apples consist of perishable and highly sensitive com-
ponents, if handled carefully they stay in good condition for a surprisingly long
Fig. 15.18 An apple in a water jet.

Fig. 15.19 Schematic structure of the

plant cuticle. A, surface wax; B, cutin–
wax composite; C, cutin–wax–cellu-
lose–lignin composite; D, pectin;
E, epidermal cell wall [37].
time – quite unlike peaches, apricots, or strawberries, etc. They owe their ro-
bustness partly to the extremely clever composition of their skin, a composite of
mechanically resistant polymers (cutin, cellulose) and hydrophobic waxes pro-
viding effective protection against all types of attack from outside (Fig. 15.19).
That apples, on the one hand, quickly take on an unsightly brownish color,
dry out, and rot when the skin is lacerated but, on the other hand, can be stored
for a long time when their skin is intact, has encouraged investigation of the
natural surface wax of apple peel. It turned out that it is not the skin but the
natural wax coating that accounts for the long-lasting protection of apples
against drying out and microbial attack: In a laboratory-scale test the wax coat-
ing was removed from one half of an apple with an alcohol-soaked cotton pad
without abrading its skin. Within a very short time of storage in a normal office
environment the de-waxed half looked brownish and shriveled, as shows
Fig. 15.20. It seems that without the wax coating the apple skin is porous and
deprived of its protective function.
Fig. 15.20 Left: removal of the wax from half of an apple.

Right: the same apple after ten days.
15.5.4
Production of Apple-peel Wax
It would, of course, be far too labor-intensive and expensive to isolate the wax from
intact apples. Dried apple pomace, a by-product of juice and pectin production,
has proved to be a favorably priced source with guaranteed availability throughout
the year. Depectinized pomace (Fig. 15.21) is a sufficiently enriched raw material
with the additional advantage that the apples being intended for juice-making and
are thus usually smaller than dessert apples (and consequently have a relatively
larger wax-coated surface). They also contain significantly less pesticide residues
(because their appearance does not matter – see above).
Wella has developed a process for isolating the wax from the dried pomace
and for removing chlorophyll and pesticide residues to obtain a rich, yellow, un-
scented raw material. For purification, as for extraction, purely physical pro-
cesses are used (adsorption, filtration, distillation), which means no chemical
change is involved.
Pomace is usually dried and used as a high-nutrition animal feed. Because
ecological aspects are considered throughout the manufacturing process, in par-
ticular with regard to the solvent used, the residual pomace after extraction of
the wax (which has hardly any nutritional value for the animals anyway) can
still be fed to animals. We could say that the pomace is only “borrowed” for wax
extraction. The process is illustrated in Fig. 15.21.
The apple-peel wax is extracted and purified using supercritical carbon diox-
ide, a solvent which is completely harmless and may be employed for food treat-
Fig. 15.21 Production of apple-peel wax [38].

ment. The yield of apple-peel wax suitable for incorporation in cosmetic formu-
lations is approximately 2%.
15.5.5
Chemical Composition
Apple-peel wax is not a chemical compound proper, but – as is typical of natural

extracts – a mixture of different classes of substance with a wide distribution of
homologs. The chief components are wax esters, hydrocarbons, and fatty acids
(Fig. 15.22).
As is commonly observed for natural lipids, the fatty acids are composed of
even numbers of carbon atoms whereas the hydrocarbons, formed biosyntheti-
cally by decarboxylation of fatty acids, mostly have odd carbon-chain lengths.
The fatty alcohols are different – these occur in roughly equal numbers of even
and odd homologs (Fig. 15.23).
The term “undefined substances” includes triglycerides, xanthophylls, and ter-
penes. The analytical data presented differ from those of references [39–41] in
several respects. Apple-peel wax is, for instance, said not to contain triglycerides,
but triterpenes, especially ursolic acid in addition to a little oleanolic acid. There
are two reasons for these variations. First, in contrast with the references cited,
the raw material source is not the apple skin (see above), but the pomace,
which means that parts of the apple inside, in particular the seeds and thus the
seed lipids are inevitably included in the extract. Second, that only minute
quantities of ursolic acid and similar compounds are present is because of the
extraction method – such terpenes are not soluble in supercritical CO2 and only
traces are extracted. If polar solvents (e.g. ethanol) were used, an amount of ur-
solic acid and related compounds approximately equal to the 2% in apple-peel
wax would be found in the extract. Because the triterpene fraction largely resists
incorporation, however, and also has other inconvenient properties in apple-peel
wax for use in cosmetics, it is not, anyway, a desirable constituent.
15.5.6
Mode of Action and Uses
Many measurements were taken to discover whether the natural action of ap-
ple-peel wax could be transferred to cosmetic applications.
Fig. 15.22 Analysis of apple-peel wax

by classes of substance.
The homologous distribution of fatty
acids (free and those of wax esters
and triglycerides), fatty alcohols, and
hydrocarbons is shown in Fig. 15.23.
Fig. 15.23 Left: fatty acid spectrum of apple-peel wax. Center:

fatty alcohols in apple-peel wax. Right: aliphatic hydrocarbons
in apple-peel wax.
15.5.6.1 Skin Cosmetics

The protective effect on the skin has been proved by determination of the trans-
epidermal water loss (TEWL) in occlusion tests (occlusion meaning “covering”
or “sealing”) and by examining skin protection in an ammonia irritation test. In
the occlusion test the rate of evaporation of the skin’s moisture is measured
after application of a variety of cosmetic materials. The occlusive action of ap-
ple-peel wax is approximately like that of the mineral substance Vaseline and
superior to that of most vegetable and synthetic waxes and oils (Fig. 15.24).
15.5.6.2 Hair Care

Surface analyses of hair strands treated with different products showed that
even small amounts of apple-peel wax added to shampoo and conditioner for-
Fig. 15.24 Occlusion testing of a variety of cosmetic lipids.

mulations, the typical rinse-off products, result in remarkable protection. A mi-

crofine coating of apple-peel wax prevents aggressive environmental factors
which result the presence of water (drying out, effect of chlorinated and sea
water, oxidative stress, . . .) from affecting the light-sensitive and easily oxidized
components of the hair.
Evidence of the hydrophobic coating was furnished by X-ray photoelectron spec-
troscopy (XPS or ESCA), a method for analyzing material surfaces to determine
the atomic percentage composition and the binding energy of the elements in
the outer molecular layer (penetration depth approx. 10 nm). The method detects
all chemical elements with an atomic number ³ 3. The accuracy of measurement
is approximately 0.5 atom percent. All XPS measurements were performed at the
Deutsches Wollforschungsinstitut in Aachen, Germany [42].
Procedure Standardized hair swatches were washed twice with 0.5-mL volumes
of lauryl ether sulfate solution (14%), while being moved to and fro, for one
minute each time. They were then rinsed under running water at 40 8C while
being combed through. The hair conditioner to be tested (0.5 mL) was then
applied to the pre-treated hair swatches – application time 30 s – and left to act
for 5 min. The hair was then rinsed under running water at 40 8C while being
combed. The swatches were then dried with a blow-drier. The reference was a
swatch pre-treated in the same way but without after-treatment.
Interpretation of the Results Interpretation of the results obtained was based on

the fact that hair mostly consists of the chemical elements carbon (C), hydrogen
(H), oxygen (O), nitrogen (N), and sulfur (S), whereas the conditioning agents
tested by XPS are composed of C, H and O only. As already mentioned, hydro-
gen cannot be detected for technical reasons. As the surface area coated with
conditioner expands, the typical hair elements C, H, O, N, and S are increas-
ingly masked by the elements of the conditioner. This results in an increase in
the carbon content and a reduction of the amount of oxygen (owing to the lower
O/C ratio of the raw materials) and – particularly important – a reduction of the
amounts of (protein-specific) nitrogen and sulfur. Consequently a measure of
the protective action of a material is the rate of reduction of the nitrogen and
sulfur content of the surface layer of the hair in comparison with that of hair
not treated with the substance. It does not matter which of the two elements is
determined, they provide the same information. The largely parallel reduction
rate of the nitrogen and sulfur content is, however, instrumental in corroborat-
ing the reliability of the results.
Because the measuring beam (about 1 mm in diameter) is large in relation to
the sample (hair diameter between 50 and 90 lm) and furnishes identical re-
sults at different sites of the sample, it is safe to assume that the lipophilic con-
ditioning agents do not become attached to the hair surface in an irregular pat-
tern but form a more or less uniform coating.
Fig. 15.25 Protective action (= percentage reduction of detect-

able nitrogen and sulfur) of different conditioning agents on
hair keratin.
Reference [36] contains a detailed description of the measurement technique

and the evaluation of the results. The protective effect of the conditioning
agents tested is represented in Fig. 15.25.
The attachment of hydrophobic substances to the hair as a protein-covering
film after the product is rinsed off can be interpreted as protective function. On
the basis of the results described above the graph shows the relative decline of
the keratin exposed (determined as protein-specific nitrogen and sulfur) after
application of the respective conditioning agent as a relative protective factor
(Nrel and Srel). Among the materials tested, the apple-peel wax leaves the best
coating (about 80%) after rinsing.
15.5.7
Market Launch
The first use of apple-peel wax as a natural protection and conditioning princi-
ple was in shampoos, rinses, and hair tip repair fluids of Wella’s hair care
ranges “Sanara”, “Wella Balsam”, and “Wellapon”. According to EU regulations,
the botanical name of the raw material source, “Pyrus malus”, is used for INCI
labeling on the product package.
15.5.8
Apple-peel wax is a natural protective and conditioning agent. Being the chemi-
cally unaltered component of a common staple food, it has no toxic potential
whatsoever. It is a renewable material extracted from waste available in unlim-
ited quantities, throughout the year, from juice and pectin production. Wax es-
ters of long-chain components and hydrocarbons impart high hydrophobicity to

this raw material.
The unique protective properties of the wax on fruit, for example:
· selective, breathable protection against moisture loss,
· sealing against water from outside (protection against chemical attacks),
· protection against microbial attack, and
· protection against light,
can be translated to the hair and skin [43]. Potential applications are skin-care
and hair-conditioning products and shampoos. Products containing apple-peel
wax have been marketed nearly everywhere in the world.
15.6
Ilex Resin: From Shiny Leaves to Shiny Hair [44]
15.6.1
Holly
Holly (Ilex species) is found in numerous varieties almost throughout the

world. In moderate climates it is an evergreen hardy ornamental shrub (Ilex
aquifolium) with beautifully shaped, usually sharp-pointed leaves whose color
ranges from dark green, through light green, to bright yellow (e.g. the “Golden
Queen” variety – Fig. 15.26). In autumn the female plants are full of golden to
fire-red berries.
In Anglo-Saxon countries holly branches are traditionally used for Christmas
decoration – their unusually shaped, glossy leathery leaves make a noble and
festive ornament.
Other evergreen plants are also used for interior decoration, but at surviving
– apparently unaffected – indoors without water at room temperature and low
relative humidity neither fir nor pine or box compare with holly. Holly leaves
Fig. 15.26 Ilex aquifolium, Var. “Gold-

en Queen”.
seem to have a highly effective coating that not only imparts the deep green col-
or and brilliant shine, but also protects them against drying out even under ex-
tremely untoward conditions, and against the action of atmospheric oxygen. A
holly leaf torn from the stem can be kept for years before it looks withered, the
(very sensitive) chlorophyll fades, and the leaf’s glossy surface shrivels.
15.6.2
Extraction of a Resin Fraction
The observations described – like those for apple peel [45] – led to closer investi-
gation of ilex leaves. Relatively simple extraction and purification methods were
sufficient for isolation of a hydrophobic, tacky, highly refractive, viscous sub-
stance (Fig. 15.27). Owing to its specific consistency the material is called “re-
sin”.
Because the leaves of the Central European holly (Ilex aquifolium) are difficult
to obtain on an industrial scale (the first holly leaves used for investigation were
from the author’s garden), better sources of the raw material were sought. It
was found that the leaves of the closely related Paraguay tea shrub (Ilex para-
guariensis) furnishes a cosmetic raw material with similar properties. The Para-
guay tea shrub or maté is not a hardy shrub in Central Europe, but the leaves
are commercially available throughout the year. Chemically, ilex resin – the fol-
lowing statements apply to the material derived from maté – is a complex mix-
ture of numerous hydrophobic substances, with a fraction containing hydrocar-
bons, terpenes (for example squalene and amyrin), and terpene esters being the
largest. Figure 15.28 shows the relative amounts of the main ingredients.
The large proportion of high-molecular-weight hydrophobic substances is re-
sponsible for the plant’s enormous water-retaining capacity which accounts for
the extreme protection against drying out.
A new, little-known raw material is always a special (i.e. inverse) challenge –
it may be the answer to a question that has not yet been asked. Ilex resin has
Fig. 15.27 Sample of native (left) and dechlorophyllized (right) ilex resin.
Fig. 15.28 Main components of ilex

resin.
proved to have many uses and, depending on the type of product, has a variety
of sometimes quite unexpected effects [46].
15.6.3
Effects in Cosmetics
15.6.3.1 Skin Care

Human skin is exposed to several unwholesome conditions. Environmental fac-
tors such as dry and cold air have a damaging effect, as do occupational factors,
for example for housewives, doctors, hairdressers, and people of many other
groups who are required to clean their hands frequently. Repeated washing with
wetting or extracting materials and contact with chemical substances are harm-
ful to the skin. Pure water alone, and more so aggressive chemicals such as
chlorinated water, salt-water, acid rain, oxidative stress, and heat also cause skin
damage.
To lessen the effect of such exposure, substances forming a protective film
may be applied to the skin. They must be hydrophobic, because most of the
damaging action occurs in combination with water. Ilex resin has been added to
a variety of skin-care creams (both o/w and w/o types), making the seem
“richer”. When added to oil-in-water emulsions the new substance particularly
improves the basic formulation. Depending on the concentration of the resin,
the improvement of the conditioning effect is such that it justifies the descrip-
tion “protective function”. Ilex resin can be used for skin-care products, both
creams and (very rich) lotions.
15.6.3.2 Hair Care

In hair care formulations such as shampoos, but particularly in conditioners
and rinses, ilex resin improves the feel and the combability with the wet hair.
When incorporated in specific conditioners it also deepens hair color. The hair
is easier to style and has more elasticity and bounce. It was also found that a
thin but measurable film on the hair surface provided some protection.
15.6.3.3 Styling
Styling products are usually “leave-on” products and thus the favorable effect of
the ilex resin in styling formulations is especially conspicuous. Use of hair sprays,
styling gels, and, particularly, shine gels containing the resin resulted in improved
hair luster and color richness which was occasionally noticeable, otherwise often
measurable (Fig. 15.29). It was amazing that in some hair sprays, setting mousses,
and styling gels it also enhanced the elasticity of the hair and the hold of the hair
style. In principle, ilex resin is suitable for all kinds of styling product, except per-
haps hair sprays with a high water content (>10%). It can be used for setting
mousses, hair sprays, gels, and waxes and for hair tip repair fluids, i.e. formula-
tions preventing split ends, and for leave-on conditioners.
15.6.4
Ilex resin is a new natural material with many useful cosmetic properties. As
an active ingredient it enhances the performance of numerous product groups
in hair and skin care and, because it is derived from a very decorative ornamen-
tal and useful plant and has an obvious active principle working in its natural
habitat, its efficacy is readily substantiated.
Ilex resin is extracted from a plant cultivated in many countries as an agricul-
tural crop. There are no supply problems, and being renewable the substance is
available in virtually unlimited amounts. The resin is obtained by a purely phys-
ical solvent-free process with and without undergoing chemical change, simply
by extraction of maté leaves with supercritical carbon dioxide. Because it is non-
toxic, this process has recently largely replaced conventional solvent extraction
for foodstuffs (e.g. decaffeination), spices, and drugs. Biodegradability, climate
Fig. 15.29 Increase of hair luster by application of a product containing an ilex resin.
References 441
safety, and sparing of fossil resources are additional benefits of all chemically
unaltered natural substances.
Ample toxicological testing has shown that ilex resin is not an irritant to the
skin and the eye mucosa, that it is not mutagenic, and has no sensitization po-
tential. The first hair conditioners and shampoos containing ilex resin were
launched under Wella’s “Lifetex” brand in 1998 and hair-care products contain-
ing this natural material are now marketed nearly everywhere in the world. The
trade name is “Ilex Gloss”, and – according to EU legislation – the INCI term is
the botanical name of the raw material source, that is “Ilex paraguariensis”.
References
1 Lang, G., Clausen, T., Chitosan and Be- 16 Römpp Chemie Lexikon, Thieme Verlag
taine, Wella AG Darmstadt, company Stuttgart, Online DE Version (2002–
brochure, revised edition (2004). 2004).
2 Kripp, T. C., Von der Naturstoffisolierung 17 Zechmeister, L.; Toth, G.: Fortschritte
bis zur Trendformulierung. SOeFW-Jour- Chem. Org. Naturstoffe II (1939) p 212.
nal 123, 20–27 (1997). 18 Muzzarelli, R.A.A.: Chitin. Pergamom
3 Lang, G., Clausen, T., Chitosan and Be- Press, New York (1977), p 5 ff.
taine, Wella AG Darmstadt, company 19 “Panzerknacker”, High Tech 7 (1988),
brochure, revised edition (2004). p 29 ff.
4 Imhoff, J. F.; Rodriguez-Valera, F.: Jour- 20 Zikakis, J. P. (Ed.): Chitin, Chitosan and
nal of Bacteriology (1984), pp 478–479. Related Enzymes. Academic Press Inc.
5 Bagnasco, S.; Balaban, R.; Fales, H. M.; (1984), S. XVIII.
Yang, Y.-M.; Burg, M.: The Journal of 21 Sandford, P. A.; Hutchings, G. P.: Prog.
Biological Chemistry, Vol. 261, No. 13 Biotechnol. 3 (1987) p 363.
(1986) pp 5872–5877. 22 Groß, P.; Konrad, E.; Mager, H.: DE PS
6 Grattan, S. R.; Grieve, C. M.: Plant and 26 27 419 (1976).
Soil 85, pp 3–9 (1985). 23 Bernadet, M.: FR 1552076 (1969).
7 Coughlan, S. J.; Heber, U.: Planta (1982), 24 Gleckler, G. C.; Goebel, J. C.: US
156: pp 62– 69. 4035267 (1977).
8 Semmler, F.: Ther. d. Gegenw. 116 25 Yanagida, T.: JP 61/210014 A2 [86/
(1977), pp 2113–2124. 210014] (1986).
9 Babucke, G.; Sarre, H.: Med. Klin. 68 26 Konrad, E.; Gross, P.; Mager, H.: EP PS
(1973), pp 1109–1113. 0 002 506 (1987).
10 Schroeder F., Konrad, E., Lang, G.: Wella 27 Waki, M.; Tsuruta, E.: Jpn. Kokai Tokkyo
AG DE 3527974 (1987). Koho, JP 62/223108 A2 [87/223108]
11 Bimczok, R., Racky, E. D., Lang, G.: (1986).
Wella AG EP 747468 A1 (1996). 28 Kripp, T.: Industrielle Erfahrungen mit
12 Bimczok, R., Lang, G.: Wella AG EP Verpackungen aus Nachwachsenden
0750904 A1 (1997). Rohstoffen. Vortrag und Tagungsband
13 Racky, E.-D.: Wella AG DE19640086 C2 des Zentrums für Europäische
(1998). Wirtschaftsförderung und der IHK
14 Lang, G., Bimczok, R., Czigler, T., Kripp, Karlsruhe, (1994) pp 44–53.
T.: Wella AG EP 826661 A2 (1999). 30 Schlegel, H. G.: Allgemeine Mikrobiolo-
15 Lang, G., Clausen, T., Chitosan and Be- gie, Thieme Stuttgart, New York, 5. Au-
taine, Wella AG Darmstadt, company flage (1981), p 65.
brochure, revised edition (2004). 29 Römpp Chemie Lexikon, Thieme Verlag
Stuttgart, Online DE Version (2002–
2004).
31 Universität Stuttgart, Institut für Sied- tion and fractionation of cuticular waxes,
lungswasserbau, Wassergüte und Abfall- Ann. Appl. Biol., 53, 43–58 (1964).
wirtschaft. 42 Kaufmann, R., Höcker, H., XPS, eine
32 Nachr. Chem. Techn. Lab. 39 (1991) Methode zur Charakterisierung von Fas-
Nr. 10 p 1112. erveränderungen in der Oberfläche.,
33 www.umweltfibel.de/lexikon/pq/lex_p_ Fachz. Lab. 4, 348–352 GIT-Verlag,
polymilchsaeure.htm. Darmstadt (1994).
34 Danone, personal communication 5 July 43 Lang, G., Hoch, D., Konrad, E., Geibel,
2004. W., Wendel, H., Kripp, T.. EP 0 583 ;438 B1
35 Financial Times 15 February 1994. “Verfahren zur Gewinnung von Apfel-
36 Kripp, T. C., Von der Naturstoffisolierung wachs, durch diese Verfahren erhältliches
bis zur Trendformulierung. SOeFW-Jour- Apfelwachs sowie apfelwachshaltige kos-
nal 123/1 (1997) pp 20–27. metische Mittel” (1993).
37 Kolattukudy, P.E., Cutin, suberin and 44 Kripp, T. C., Ilex Resin, a Novel Renew-
waxes. Comprehensive biochemistry of able Raw Material for Cosmetics.
plants, IV, 571–645, Academic Press, SOeFW-Journal 126, 18-21 (2000).
New York (1980 b). 45 Kripp, T. C., Von der Naturstoffisolierung
38 Herbstreith & Fox, Neuenbürg, Firmen- bis zur Trendformulierung. SOeFW-Jour-
prospekt “Von der Apfelernte zum Pek- nal 123, 20-27 (1997).
tin”. 46 Kripp, T. C., Bormuth, H., Franzke, M.,
39 Sando, C. E., Constituents of the wax-like Baecker, S., Schröder, F. and Kischka,
coating on the surface of the apple, J. K. H., Cosmetic Agent with an Ilex resin
Biol. Chem., 56, 457–468 (1923). content, process for extraction of Ilex re-
40 Chibnall, A. C., Piper, S. H., Pollard, A.., sin and Ilex resin obtained thereby. WO
Smith, J. A. B., Williams, E. F., The wax 97/30680. Wella AG, Berliner Allee 65,
constituents of the apple cuticle, Bio- D-64274 Darmstadt (DE).
chem. J. 25, 2095–2110 (1931).
41 Fernandes, A.., Baker, E., Martin, J.,
Studies on plant cuticle, VI. The isola-
Part III
Biobased Industry:
ISBN: 3-527-31027-4
445
16
Industrial Biotech –
Setting Conditions to Capitalize on the Economic Potential
16.1
Introduction
Today’s pharmaceutical industry would be almost unthinkable without biotech-

nology. Practically every new drug has relied on biotech tools for discovery and
development and an increasing number of drugs themselves are biological in
nature. This is a growing trend. Biotechnology has also increased in importance
in agriculture. In the United States, for example, approximately 80% of soy and
corn plants grown today are genetically modified. The use of biotechnology in
food production is not without controversy, however, and these products face
consumer skepticism especially in Europe.
After these inroads made by “red” and “green” biotechnology, a third wave is
beginning to spread – “white” or industrial biotechnology. It enables companies
to manufacture new, innovative products or existing products more effectively
and efficiently than with conventional processes. Biotechnology mostly uses re-
newable materials and copies tried-and-tested natural processes to produce in-
dustrial goods. This often saves resources, reducing the burden on the environ-
ment. Biotechnology lays a foundation for sustainable development in which so-
cial, ecological, and economic concerns can all be reconciled.
The potential for industrial biotech is broadly recognized, and chemical and
biotech companies are starting to move into this space and increase their pres-
ence. The success of biotechnological processes for vitamin production, the
growth of the enzyme industry, and the introduction of cost-competitive biopoly-
mers, for example, all hint at the possibilities. Major challenges still lie ahead
in transforming this potential into economic value, however, and here we set
out some of these challenges and discuss ways to tackle them.
ISBN: 3-527-31027-4
446 16 Industrial Biotech – Setting Conditions to Capitalize on the Economic Potential
16.2
Time to Exploit the Potential
For those who remain unconvinced that biotech is more than just a passing
fad, approximately 5% of the estimated US$ 1.2 trillion total chemical sales al-
ready depend on biotech. (Here, “dependency” is defined as at least one produc-
tion step using biotechnology; 100% of the sales of a chemical product were ac-
counted for in a multi-step production process.) The global market for bio-based
ethanol is worth US$ 15 billion alone; other basic organic molecules, for exam-
ple citric acid (US$ 2 billion) and lactic acid, are produced by fermentation, and
so are all but three amino acids (approx. US$ 4 billion). Various basic, advanced,
and active pharmaceutical ingredients produced by the fine chemical industry
are worth US$ 7.5 billion; the attractive enzyme market has reached US$ 2 bil-
lion in sales and is growing by more than 5% per year, and specialty chemicals
for flavors, fragrances, and other applications add several more billion dollars.
Biotech is changing industrial production in three specific ways. First, sugars,
vegetable oils, and ultimately waste biomass are replacing fossil fuel feedstock
(oil and natural gas). Second, bioprocesses such as fermentation, biocatalysis,
and, in the future, plant- and animal-based production may replay chemical syn-
theses. Last, new bioproducts are emerging including bio-based polymers, en-
zymes for use in textiles or feed, and innovative nutritional ingredients.
16.2.1
How Far Can it Go?
McKinsey & Company has analyzed the market potential by looking at the tech-
nological and market trends in the chemical industry, taking an inventory of ex-
isting commercial and research and development biotech activities, estimating
the likely penetration in different chemical market segments, and finally by con-
ducting interviews and discussions with more than 100 industry executives
(Fig. 16.1).
The results suggest that biotech could have an impact on 10% of chemical
sales by 2010, double what it is today. Initial analyses at the beginning of the
decade indicated that 20% might have been achievable in the same timescale.
Today this looks highly unlikely, and even the 10% figure will not be reached
without effort.
One of the most important factors affecting this development is the price dif-
ference between fossil fuel feedstocks and biological carbohydrate feedstocks,
especially for those products with a high relative feedstock cost. Consumer ac-
ceptance is also vital – as we have seen in agricultural biotech with the wide-
spread rejection in Europe of genetically modified foods, despite their accep-
tance in the US. Thus far there is no significant consumer movement against
industrial biotech – indeed, as we show later, it actually has strong environmen-
tal benefits. Consumer and environmental organizations have not actually pub-
licly endorsed industrial biotech, however.
Fig. 16.1 Biotech adoption could more than double by 2010.
The regulatory framework is another important factor. Supportive regulatory

procedures, active government support (e.g. grants, and renewable purchasing
policies) and the extent to which less sustainable production methods need to
compensate for their environmental impact will all be a fundamental prop if
the industry is to grow. This sets the context for the final success factor – invest-
ment by the chemical companies. Without this industrial biotech will remain
no more than a dream.
The promise of industrial biotech has been around for decades, and failures
far outnumber success stories. So it is not surprising that chemical companies
are hesitant to move forward. The prospects look brighter than ever, however,
because of three key drivers – advances in technology, environmental and eco-
nomic benefits, and the need for innovation in the chemical industry.
16.2.2
Better Technology, Faster Results
A broad spectrum of enzymes and fermentation systems are already available to

the market, and the number is increasing all the time. The pace of biotech de-
velopment has picked up to the extent that it can now take a matter of weeks
rather than years to develop new, highly specific, and efficient enzymes. Until
recently the slow pace of development has hindered the use of enzymes in phar-
maceutical production, where drugs need to be developed quickly. Now DSM’s
Pharmaceutical Product Unit, for example, is exploiting them systematically as
a competitive advantage. Enzymes are also becoming more resistant to harsh

environments such as heat and acidity, and are cheaper to produce, opening in-
roads into other industrial production processes, for example pulp and paper,
oil exploration, and textile processing.
16.2.3
Environmentally and Balance-sheet Friendly
The increased pressure for sustainable production is also helping to spur the in-
dustry’s prospects. Two reports – one authored by the OECD (OECD Report
“The Application of Biotechnology to Industrial Sustainability” (2001), which in-
cludes 21 case studies on the impact of biotechnology on the environment), the
other by a consortium of companies, industry associations, the Öko-Institut,
and McKinsey – clearly demonstrated that industrial biotech can help create
jobs, boost profits, and benefit the environment (Fig. 16.2).
Fig. 16.2 Case studies demonstrate sustainability.
Green Economics
Several studies of the environmental impact of replacing chemical synthesis

with biotech routes have demonstrated the benefits of industrial biotech. The
German chemical company BASF was able to adopt biotech processes to
transform production of Vitamin B2. Traditionally, this requires a compli-
cated eight-step chemical process, but biotech reduces that to just one step.
Soy oil is fed to a mould and Vitamin B2 is recovered as yellow crystals di-
rectly from this fermentation process. This has cut production costs by 40%
and reduced CO2 emissions by 30% and waste by 95%.
The antibiotic Cephalexin has been produced on an industrial scale by the

Dutch chemicals firm DSM for several years. Metabolic pathway engineering
helped establish a bio-route that reduced substantially the number of steps
needed in the process. The biotech process uses 65% less energy, 65% less
input chemicals, is water-based, and generates less waste. In total, it has
halved the variable costs of the process.
Novozymes, a Danish biotech company, produces enzymes for the scouring
process in the textiles industry. Scouring, which removes the brown, non-cel-
lulose parts of cotton, traditionally requires a harsh alkaline chemical solu-
tion. Use of enzymes not only reduces discharges into the water by 60%, but
reduces energy costs by a quarter. Environmental and economic benefits go
hand in hand: the new process is also 20% cheaper than the chemical treat-
ment.
Cargill Dow’s bio-polymer PLA made from corn requires 25 to 55% fewer
fossil resources than the conventional polymers against which it competes.
With the help of biomass and potentially other forms of renewable energy
for processing, the joint venture between Cargill and Dow Chemical believes
PLA could even become a net carbon sink.
In the near future DuPont’s Sorona® polymer will be based on propane-
diol (PDO) produced by fermentation, in collaboration with Tate and Lyle.
This is estimated to reduce greenhouse gas emissions by approximately 40%.
When oil and gas are used as production feedstock, carbon is removed from the
earth and bound into the chemicals that constitute plastics and other materials.
Eventually, the plastic is discarded and burned and CO2 is released into the at-
mosphere – a one-way cycle that produces significant pollution. When biomass
is used, the CO2 released helps provide the raw material for further production,
because it can be absorbed by plants, which can in turn be used as new feed-
stock. This closed cycle theoretically results in a neutral carbon balance, thereby
reducing greenhouse gas emissions.
The increased energy efficiency that comes from replacing fossil fuels has led
McKinsey to estimate that 7% of the Kyoto target on emissions could be
achieved (based on the full range of case studies in the reports mentioned
above, and the figure of 10% of chemical product sales relying on biotech).
When we reach the stage where 20% of chemical products are produced using
biotechnology, the contribution will increase from 7 to 20%.
The economic benefits alone are also driving the adoption of biotechnology.
The case studies for Vitamin B2 and Cephalexin (see box), and dozens of phar-
maceutical intermediates suggest that cost savings of 50% and more are not un-
likely. The savings can come directly from lower variable costs, but also from re-
duced capital expenditure for simpler production assets, or from reduced scale
and therefore lower risk, transportation costs, and/or overcapacity.
16.2.4
Rekindling Chemicals Innovation
Finally, interest in biotech has increased recently because of its role in product
innovation. At a time of increasing competition from Asia in established prod-
ucts and the subsequent commoditization and strong price decline, chemical
companies are once again looking at innovation as a key source of differentia-
tion. At the 2004 World Economic Forum in Davos, the leaders of the world’s
largest chemical companies generally shared the view that biotechnology will be
the predominant driver of innovation for their organizations, and placed it
among the key drivers of change for the decade ahead.
The importance of stimulating innovation can be seen by looking at the intro-
duction of new polymers. Over the course of the 20th century the development
of fossil-fuel-based polymers increased steadily through to the post-war period,
stimulated by the abundance and low cost of basic petrochemicals. It has, how-
ever, declined dramatically since 1960. Innovation in the traditional polymer in-
dustry today is mainly related to the application and blending of these poly-
mers, rather than the invention of new ones (Fig. 16.3).
Just as low-cost petrochemical building blocks such as ethylene, propylene,
and butadiene became available with the introduction of crackers in the 1930s,
we are seeing the emergence of new bio-based building blocks today. These in-
clude lactic acid, which can be polymerized to the biopolymer PLA. PLA has
started to replace polyester because of its competitive cost and new applications.
Lactic acid can also be processed into chiral drugs, acrylic acid, propylene glycol,
food additives, and more (Fig. 16.4).
Fig. 16.3 The innovation potential for fossil-based building

blocks appears to be exhausted.
Fig. 16.4 Bioethanol: an early beneficiary of low-cost biomass.
Other examples of innovation abound – Cargill is exploring the potential of 3-

hydroxyproprionic acid as a new building block; BASF is looking into new
chemistry around the simple organic molecule succinic acid; and DuPont will
use cheap PDO as a monomer for its Sorona® polymer. Overall, we are seeing
the emergence of a green chemistry that complements the traditional product
trees and that gives the industry more innovation headroom.
16.2.5
Increasing Corporate Action in all Segments
All these economic, regulatory, and environmental factors are encouraging com-
panies in all major chemical market segments to make more definite moves In
fine chemicals, half of all life science chemicals at DSM are based on biotech –
a wolume which is worth US$ 1.5 billion. BASF has committed more than
1 500 million to explore the potential of plant-based biotechnology. DuPont has
invested more than US$ 500 million in the development of Sorona®, and Car-
gill Dow’s investments for PLA are of a similar scale. Ciba Specially Chemicals
has introduced an enzymatic process for acrylic acid, BP is exploring industrial
biotech for oil exploration and basic chemicals, Degussa has recently opened a
second project house dedicated to biotechnology, Givaudan is using biotech ex-
tensively for new flavors and aromas, and Novozymes has an annual research
and development budget of more than 1 100 million for new and enhanced en-
zymes. Finally, dedicated industrial biotechnology companies such as Codexis,
Diversa, and Genencor have established themselves in the market and have en-
couraged a new generation of start-ups to emerge.
16.3
The Importance of Residual Biomass
Early introductions of bio-based products have shown that the willingness to pay a
high “green” premium is limited to a small portion of the customer base. For broad-
based adoption, new products must be competitive with existing offerings. In this
context, the use of alternative low-cost feedstock could give industrial biotech an-
other boost (as, of course, will the recent increases in the price of natural oil and gas).
16.3.1
Why Waste Biomass Works
The most promising alternative feedstock is waste biomass. It comes primarily

from agricultural sources such as straw and corn stover. This material is abun-
dant, cheap, and largely serves no other purpose. It is also obviously renewable
and contains three useful raw materials:
· cellulose and hemicellulose, which can be turned into sugars;
· proteins that can be used in animal feed and for industrial products such as
hydrolysates; and
· lignin that may be used as a combustible fuel; indeed it can power the very
biorefineries that process biomass, making them virtually self-sufficient.
Bioethanol is one of the first and the largest markets to profit from cheap bio-
mass feedstock. Ethanol is usually produced from dextrose, which in the US
tends to be derived from corn. The first ethanol biorefinery based on waste bio-
mass is already online. It is a Canadian venture operated by Iogen and receiving
investment from Shell, Petro Canada, and the Canadian government. With an
annual capacity of 700,000 liters it is semi-commercial in scale and too ineffi-
cient to compete on cost with conventional ethanol refineries. The technology is
expected to improve quickly, however.
The process sounds relatively simple. Straw is delivered to the refinery where it is
chemically and/or physically pretreated. Enzymes then decouple the cellulose and
hemicellulose chains, breaking them down into individual sugars. Most of the sugar
yielded will be used as feedstock for fermentation. As bacteria or yeast cells break
down the sugar it is fermented into ethanol. The organisms can also be modified to
produce vitamins, organic acids, and other substances. The primary products that
emerge from the biorefinery are often subject to further downstream processing.
16.3.2
Economic Benefits and Regulation
Biomass-based biorefineries have the potential to reduce sugar costs from about
8 to 9 cents per pound in the US today to approximately 4 cents per pound in
three to four years, and lower still as the biorefineries become more integrated.
It is even possible to see a case where the net cost of producing sugar in an in-
16.3 The Importance of Residual Biomass 453
Fig. 16.5 Bio-based building blocks emerge as a source of new products.
tegrated biorefinery is zero because by-products such as lignin and proteins gen-
erate the value. It is important to realize that the price of conventionally pro-
duced sugar will also fall as genetic modification makes a variety of productivity
improvements possible.
The technology for the cost-competitive, large-scale application of ethanol
could be ready in the next few years, and the value-creation potential for shift-
ing from corn to biomass for ethanol is estimated to be US$ 10 to 16 billion by
2010 (Fig. 16.5). (This figure is derived from industry estimates for the increas-
ing use of biomass as feedstock. After calculating marginal and overall cost sav-
ings, we multiplied the result by typical profit multiples for the industry. We as-
sumed that the price of ethanol remains stable. The resulting change in market
capitalization is the value creation.)
Legislation is already in place to support production of fuel ethanol in coun-
tries such as Canada and Brazil, and the EU has committed to ensuring that
5.75% of all transportation fuel in the EU will be bio-based – a target that can-
not be achieved by biodiesel alone. Several European companies, for example
Abengoa and Südzucker, have therefore announced aggressive plans for expan-
sion in ethanol in the coming years. Estimates for the US market suggest that
15 billion gallons (57 billion liters) of bioethanol will be produced from a com-
bination of biomass and corn-based feedstock by 2020; and the cost of ethanol
could come down from approximately $ 1.20 per gallon (32 cents per liter) today
to as low as 40 cents per gallon (11 cents per liter) by 2010.
16.3.3
Still a Long Way to Go
For this to happen, all steps of the process need to undergo major efficiency im-
provements. The cost of enzymes must fall by 90% from its 2003 level. This
might sound unrealistic, but it is not unusual to increase the effectiveness of
enzyme production by factors of 10, 100 or even 1000. By modifying the amino
acid sequences of the cellulase and hemicellulase enzymes, biochemists from
Genencor and Novozymes have been able to make them dramatically more ef-
fective, and recent advances suggest the cost target for the enzymes will be ex-
ceeded in due course.
The US Department of Energy predicts that 2 billion gallons (7.5 billion li-
ters) of ethanol will be derived from biomass by 2010. This figure, depends,
however, on whether companies are willing to invest. Despite major govern-
ment support, especially in the US, no major companies have yet aggressively
pursued this. Cargill, DuPont, Deere, Shell, and others are putting in moderate
investments of US$ 10 to 15 million often related to matching government
grants, thereby reserving an option to play in the future. The competitive threat
is still perceived as low, and companies are afraid to risk being the first mover.
Only the technology companies, in particular Novozymes and Genencor, and
the Spanish pioneer Abengoa have publicly announced aggressive growth plans
and commitments to biomass-based ethanol.
It is not just the chemical or the biotech companies that need to make the in-
vestments; it goes all the way down the value chain. Farmers need the right sort
of equipment, storage facilities for corn stover must be built, pretreatment
needs investment, etc. No one owns the full value chain, so collaborating with
partners is essential. In fact, the value chain does not yet exist, and collabora-
tion between different parties is required if it is to be built.
16.3.4
Collaboration Will Push Biomass Conversion Forward
There are three collaboration models. The first, which prevails today, is where
multiple players are involved in diverse activities with very little explicit collabo-
ration and without enough investment to launch a new value chain. In the sec-
ond, entrepreneurial pioneers invest substantially to bundle research and devel-
opment efforts and build the new value chain. This is a high-risk approach, but
has potentially high rewards. It also enables the company to assume a clear lea-
dership role. The final model is a structured network in which multiple players
from different parts of the value chain cooperate closely. The risks – and there-
fore the returns – are shared out and each company can focus on its core com-
petency. Such networks have yet to emerge. When they do, they will have much
to accomplish, including collecting, warehousing, and treating biomass, and
constructing biorefineries. One possible drawback of this model is the introduc-
tion of governance and interface inefficiency.
16.4 Overcoming the Challenges Ahead 455
It will be interesting to see how governments and the various players along
the value chain will act in the coming years, and whether a real breakthrough
in the broad commercial use of waste biomass will happen. Next to the advan-
tages for the environment, political stability, and jobs in rural areas, it could
give a welcome boost to the broader adoption of industrial biotech.
16.4
Overcoming the Challenges Ahead
The path from awareness to profit for companies engaged in biotech is long
and arduous (Fig. 16.6). Most companies have already taken the first step and
are aware of the opportunities and the threats of biotech. Some have even made
the commitment to invest; but from there onward is a long staircase with many
internal and external challenges to manage.
16.4.1
Internal Obstacles
The first challenge for chemical companies in turning the biotech promise into
reality is to create the awareness of its economic potential and benefits for sus-
tainability. We have already come a long way in achieving this. The next chal-
lenge is to convert the awareness into a commitment to act. Investors and se-
nior managers have recently demonstrated their willingness to act if they are
Fig. 16.6 Staircase of challenges from awareness to profit.

presented with good opportunities. This has held true particularly for incremen-
tal investments. Those high-risk/high-return areas that require major invest-
ments, for example biomass conversion, still face great skepticism. Further-
more, very few companies have progressed beyond an opportunistic use of bio-
tech and have formulated a true biotech strategy, with a clear focus on where
and how to compete. To execute a strategy, a company needs the right assets,
capabilities, and networks, and no one has all the requirements in-house. Iden-
tifying where the gaps lie and understanding how best to fill them is not easy,
and feedback on partnerships in this field has been mixed.
The next step is to identify and select the right opportunities. There is usually
no shortage of ideas, as illustrated, for example, by the impressive theoretical
product trees rooted in the new bio-based building blocks. The difficulty is se-
lecting the right ones; as mentioned earlier, the list of failed biotech invest-
ments is much longer than the list of successes.
Failure cannot be avoided. The trick – as in the world of venture capital – is
to manage the uncertainty and to build a portfolio of good prospects. And, as
with venture capital, the starting point is a solid business case for the opportu-
nities deemed right for investment. The most common mistakes are overesti-
mating the addressable market size and market uptake (mostly by defining it
too broadly), and underestimating the investments, in particular for ongoing
market development and application development after the initial research and
development investment.
With a portfolio of projects in hand, there remains the challenge of separating
the biotech wheat from the biotech chaff. Companies often hang on to second-
tier projects far too long, rather than focusing their scarce resources on the few
most promising. Investors are also no longer willing to tie their money up in
projects that might take a decade to break even. Research and development
work in biopolymers, for example, began in the 1980s, so we need to find ways
to accelerate the whole process.
The launch and market development might be the last steps, but they are also
the ones that can make all the difference. The art is to focus on the right mar-
ket segments and customers with the right value proposition, to align the value
chain to adopt your innovation, and to use partners appropriately. Indeed, man-
aging partnerships plays a role in several steps of this staircase of success.
Surrounding this set of internal challenges are external pressures that also
need close attention, but which can be met.
16.4.2
External Challenges
There are four distinct external challenges.

· Consumer acceptance is not yet a big issue, but is one to monitor. Supermar-
ket chains in the UK have refused a biopolymer because it was derived from
genetically modified plants, even though it is an eco-friendly material. There
is even a discussion as to whether vitamins produced by fermentation should
be labeled GM. The environmental NGOs and pressure groups have been rel-
atively quiet on industrial biotech so far, but it would only take one of the
more influential groups putting it on the agenda for lengthy delays to be pos-
sible. The industry therefore needs to invest resources in educating people on
the benefits.
· The cost differential between hydrocarbon (oil, natural gas) and carbohydrate
(sugar, biomass) feedstock is clearly a moving target. In a climate of rising oil
prices, carbohydrates look even more appealing but such a situation cannot
be relied upon permanently. Companies need to consider potential price
changes, and analyze the sensitivity of biotech investment cases against var-
ious assumptions on future feedstock costs.
· The regulatory situation is certainly liable to change but is also subject to the
influence of various interest groups. The proponents of industrial biotechnol-
ogy, represented, for example, by the BIO and EuropaBio industry associa-
tions, need to maintain or even increase the level of activity on this front.
This, in turn, requires commitment from their members.
· The success of company strategies and investment decisions ultimately de-
pends on the moves of competitors. Products must be distinctive; intellectual
property must be in place to introduce a new biotech process; and an eye
must be kept out for opportunities to align interests and join forces. It is
therefore essential to watch and anticipate competitor moves as well as possi-
ble.
16.5
Overcoming Challenges
A growing number of case studies demonstrate that all these challenges can be
overcome. Usually, the trick is to tailor well-established good management prac-
tices and problem-solving approaches from comparable situations to the specific
needs of industrial biotech. These approaches do not have to come from the
chemical industry itself. For example, chemical companies have looked at risk
management tools in financial services to learn how to deal with uncertainty.
Here, we set out a number of cases with which McKinsey has been involved,
and in which companies have managed to capture value from industrial bio-
tech.
16.5.1
Case 1: Building a Biotech Strategy
After years of internal discussions, and smaller investments with promising re-
sults, the board of a chemical company decided to take a major step in indus-
trial biotech. It looked at a number of different growth fields and biotech
emerged as the one with the highest innovation potential and a good fit with
the company’s broader capabilities and position in the value chain. There were
Fig. 16.7 Building the biotech strategy for a chemical company.
many potential entry points and business opportunities, some of which had in-
ternal champions and others that did not seem worth pursuing, but none had a
clear rationale. The company approached the challenge from two sides. It deter-
mined its distinctive skills and assets through a benchmarking process. At the
same time, it assessed the relevant opportunities and threats – both competitors’
recent or announced moves and the potential for competitors if the company
chose not to enter a specific area.
This produced a list of strategic options – a combination of areas that seemed
most attractive for the client to enter and different business models and value
chain positions that would match these areas. The next step was to assess these
different strategic options along a set of criteria that included economic value,
feasibility, risk, investments, fit with overall strategy, and portfolio of initiatives
(Fig. 16.7). The final decision was made after extensive discussions and inter-
views both within and outside the company. It is too early to determine the fi-
nancial success, but the company has already achieved alignment between the
overall strategy, the level of investment and the organizational setup.
16.5.2
Case 2: Identifying the Right Opportunities
Another chemical company already had a clear biotech strategy in place and
had built the capabilities, assets, and networks required to implement it. Execu-
tion was already successfully underway in several business units and new bio-
based products and processes had started to generate healthy profits. The com-
pany was, however, wondering how best to apply biotechnology to a recently ac-
quired business. In particular, it was seeking ways to change the old chemical
production processes to new, more competitive synthesis routes.
The project scope expanded to include a complete review of the company’s
core product strategies, which included a detailed assessment of competitor
moves, market trends, etc. This was important because a new biotech process
can easily take five years or more to develop, so it is critical to understand
whether it will result in a distinctive cost position after that time. Regulations
and customer sensitivities also change – would there be a “bio-based” premium
or a “genetically modified” discount for a product produced by fermentation?
The scope was also extended on the technology side. While one team investi-
gated the potential for new biotech routes, a competing team tried to optimize
the existing process, including analyzing different locations, and a third team
searched for the best alternative chemical routes. In the end, biotech was just
one of the solutions. Each potential solution was assessed, and the final one
was chosen based on the basis of the best risk/reward ratio. This led to an im-
plementation roadmap and a decision tree, as further technical feasibility stud-
ies were required to prove the concept and assist in making the final choice.
Of the fifteen products under investigation, biotech was the best solution for
five of them; in four cases a new chemical process was found; incremental im-
provements to the existing chemical process were made in three further cases;
two products were moved to China, one was stopped completely and bought in-
stead from a low-cost producer. So even though the impetus for the project was
to see how biotech might have an impact on the company’s processes, the out-
come was an improvement of every process in a variety of ways. By 2010, it is
predicted that costs will have fallen by 60% on average for these fifteen pro-
cesses.
16.5.3
Case 3: Managing Uncertainties
This company was building a portfolio of industrial biotech projects but it was
difficult to predict the future cost and revenues of each one and to determine
which ran a relatively high risk of failure. The company wanted to make the
risks more transparent and to improve its decision making against a back-
ground of high uncertainty.
The solution was inspired by riskmanagement practices used in the venture
capital and pharmaceutical industries. The basis of many of these tools is a
Monte Carlo simulation, which randomly combines potential parameters to as-
sess the probability of certain resulting values. It was used to estimate the pro-
cess economics of a new biotech process. Instead of using a best guess for yield,
cycle times and other key process characteristics, a best-, base-, and worst-case
scenario were each assigned a certain probability of occurrence. The Monte Car-
lo simulation combined these randomly and generated a graph that showed the
potential total production costs against its probability of occurrence. For exam-
ple, it turned out that there was a 60% chance that a new process was going to
be at least 30% cheaper than the old one. Based on the investment required,
this was still considered to be too risky and the project was terminated. In an-
other case, the analysis revealed a spread of potential outcomes that did not en-
able any conclusive recommendation. A more detailed assessment showed that
the range of outcomes was extremely broad for one process characteristic, and
the company decided to invest in another six months of laboratory work to un-
derstand this better and then repeat the assessment with a better defined range
of potential outcomes. This is just one example demonstrating that uncertainty
and risk are not reasons to refrain from rational decision making.
16.5.4
Case 4: Preparing the Launch and Market Development
This company was on the very last step of the staircase towards profitability in
industrial biotech. It had a strategy, a highly capable organization, a great prod-
uct, a new production facility, and some funding. Its problem was that the po-
tential market applications for its new bio-product were so numerous that it
was impossible to pursue them all simultaneously with its limited resources. It
was also becoming clear that the product was not equally suited to all applica-
tions and that each market required a different product positioning. The com-
pany was looking for a go-to-market strategy for its new blockbuster.
The full list of potential applications and addressable market segments were
assessed against:
1. the relative strength of the new product’s value proposition to the customer
in terms of price and performance compared with existing offerings;
2. the size and attractiveness of the addressable market; and
3. the ease of capturing the value, i.e. the time and effort it would take to devel-
op the product applications and markets and the hurdles for adoption of the
product along the value chain.
The last point required much attention because consumers had already ex-
pressed considerable interest in some of the applications and the economics
looked attractive. Interviews with companies along the value chain and support-
ing analyses showed, however, that the investment of intermediaries in the val-
ue chain made adoption very unlikely. In other market segments, retailers were
concerned about the brand risk and were not willing to proceed without further
demonstration of fitness-for-use and safety. These segments were therefore put
on the back burner, but will be reignited when results from other segments
support this market case.
In the end, a handful of segments were chosen as top-priority targets for im-
mediate focus and specific targets, marketing strategies, and implementation
plans were put in place. In some other market segments and geographies part-
nering was the preferred strategy, mostly because partners had better customer
access or application technologies than the company. The remaining segments
were put on hold. It is too early to measure the success in financial terms, but
16.6 More Needs to be Done 461
the company now feels it has a clear focus and funding has recently been re-
newed by investors, so the new go-to-market strategy can be implemented.
16.5.5
Case 5: Building a Favorable External Environment
The previous case referred to overcoming internal hurdles, but the final case
study illustrates how companies and industry associations can work together to
tackle external challenges: to educate opinion leaders and decision makers and
avoid a negative atmosphere that could prevent the economic and environmen-
tal potential from being realized.
In 2003, EuropaBio decided to launch a project together with BIO (its Ameri-
can big sister) and some of its most prominent industrial members. BASF, Car-
gill Dow, DSM, DuPont, Genencor, and Novozymes all committed funds, data,
and resources to put together case-studies on recent industrial biotech innova-
tions. The German Öko-Institut, which has a good reputation and credibility
with environmental interest groups, was asked to ensure that the environmental
impact assessment was sound, and McKinsey was brought in to manage the
process and perform the economic analyses. The resulting report included case
studies and high-level recommendations for policy makers. It was presented at
several prestigious conferences, and is still cited regularly in the trade and gen-
eral press. Moreover, it has opened the door for EuropaBio to enter a dialogue
with the key policy makers in the European Commission. One of the outcomes
was to establish round-table discussions with the key stakeholders, including in-
dustry, academia, governments, and consumers. Several consumer interest
groups are meanwhile supporting industrial biotech, and, more importantly,
none is campaigning against it!
16.6
More Needs to be Done
The potential of industrial biotech to benefit the triple P – “profits, planet, and
people” – has been broadly recognized and companies are starting to turn it
into reality. There are significant challenges in making this happen, but the
cases shown here encourage us to think that they can and will be overcome.
Nevertheless, the effort required from chemical companies in terms of making
and managing investments is significant. The speed and extent to which the po-
tential of industrial biotech is realized will largely depend on how determined
and successful chemical companies are in forging ahead. Another part of the
equation is government support. Various US institutions, especially the Depart-
ment of Energy, have supported industrial biotech significantly in the past. It is
important that this level of investment is sustained or even increased. There are
rumors that part of this funding will be redirected towards other technologies,
but this would send a negative signal that could have repercussions far beyond
the direct loss of investment.
In contrast to the situation in the US, the EU has yet to implement any ma-
jor initiatives to support industrial biotechnology. Such support would be more
than desirable and should concentrate on three specific areas. First, the EU
should formulate a vision and a long-term strategy for developing the biotech-
nology industry and all the relevant groups should be included in drafting such
a strategy. Second, legislators should be challenged to create a favorable environ-
ment, which can include temporary support for lower prices for carbohydrate
feedstock that Europe is not producing competitively. Third, the EU should pro-
mote research to intensify technological development in Europe.
It feels as if we are teetering on the edge of an economic breakthrough in in-
dustrial biotech. If governments help create a conducive environment and com-
panies understand how to move forward without taking unnecessary risks, this
could be the beginning of a genuine revolution in the application of science. It
is an exciting time to be involved in this industry, but there are still challenges
ahead that we must all rise to meet if industrial biotech is to fulfill its sizeable
potential.
463
Subject Index
Numbers in front of the page numbers refer to Volume 1 and 2: e.g., 2: 282 refers
to page 282 in volume 2
a
A. niger CBX-209, levoglucosan fermenta- acyclic sugar derivatives 2: 48
tion 1: 229 acyl glutamate, synthesis 2: 305
A. rhizogenes 2: 275 acylated proteins 2: 304
absorption, acoustic 2: 242 addition
acetaldehyde, glucose product family 1: 21 – cationic 2: 264–265
acetate, biorefinery concept 2: 210 – copper-initiated 2: 262–263
acetic acid, glucose product family 1: 21 – perfluoroalkyl iodides 2: 263–264
Acetobacterium woodii 1: 235 additive chemicals 1: 91
acetogenic bacteria, metabolic pathways additive replacement, ethanol 1: 357
1: 234 adhesions prevention, post-surgical 2: 238
acetogens 1: 233 adhesive films 1: 282
acetoin, biomass building blocks 1: 22 adhesive tack 2: 186
acetone, glucose product family 1: 21 adhesives 2: 86
acetyl-CoA 1: 233, 236 advanced materials, protein-based polymeric
acetylated starches 2: 80 materials 2: 220
acid addition, lactic acid 2: 386 aerobic storage, potato juice 1: 300, 309
acid-catalyzed dehydration 1: 91 agarose gel gene ladder 2: 229
acid-catalyzed hydrolysis, cellulose 1: 133 age of sustainability, modeling tools 1: 57–
acid-catalyzed stages, biofine process 60
1: 144 agribusiness, integrated production 1: 8
acid conversion, cellulose 1: 129 agricultural applications, lignin 2: 192
acid cooking, straw 1: 193 agricultural crop residues 1: 117
acid-forming anaerobes 1: 235 agricultural ecosystem modeling 1: 57–60
acid hydrolysis, carbohydrate polysaccha- agricultural land, net requirement 1: 54–55
rides 1: 144 agricultural varieties, oil qualities 2: 276
acid hydrolysis of polysaccharides 1: 140 agricultural waste 1: 68
acid hydrolysis process 1: 130–133, 199 agriculture residue collection 1: 317–344
acid-insoluble ligneous components, feed- agrification 1: 96
stock 1: 146 Agrobacterium tumefaciens 2: 275
acid prehydrolysis 1: 200 agroindustry, sugar 1: 209–211
acidification, lactic acid 2: 386 agrosector, dutch 1: 96
acidogenic anaerobes 1: 235 air-blown gasification 1: 232
acids air classification 1: 176
– dilute 1: 362 – oat grain 1: 183
– sugar-derived 2: 5 Alcaligenes eutrophus, PHB accumulation
aconitic acid, glucose product family 1: 21 2: 424
acoustic absorption, elastic protein-based alcell demonstration plant 2: 180
polymer 2: 242 alcell process 2: 179
acrylic acid, glucose product family 1: 21 alcohol commodities, sugar-based 2: 36–37
ISBN: 3-527-31027-4
464 Subject Index
alcohols amylose
– microbial conversion 2: 32–34 – physical structure 1: 140
– microbial fermentation 2: 33 – starch synthesis 2: 71
– production 1: 235–236 anaerobe bacteria, lactic acid fermenta-
– sugar-based 2: 42 tion 1: 298
– sugar-derived 2: 5 anaerobes, acidogenic 1: 235
alfalfa 1: 254 anaerobic fermentations, succinic acid
– chlorophyll extraction 2: 329 2: 35
– cultivation 1: 260 anaerobic production, succinic acid 2: 369
Alfalfa New Products Initiative see ANPI anaerobic storage, potato juice 1: 300, 310
alfaprox procedure 1: 257 analytical assays, antioxidant activities
algal fungi, chitin occurrence 2: 415 1: 186
aliphatic diols, high molecular weight analytical methods, lactic acid fermenta-
2: 298 tion 1: 299
alkali pretreatments 1: 200 1,6-anhydro-b-d-glucose, chemical struc-
alkaloids 1: 268 ture 1: 245
alkanes 1: 268 d-anhydroglucopyranose units 1: 140
– thermal addition 2: 264 anhydrosugar 1: 229, 243, 245
alkyl polyglycoside carboxylate 2: 307 animal bedding, stover 1: 325
alkyl polyglycosides 2: 272, 305 animal feed, nutrient-enriched 1: 170
– emulsifiers 2: 308 animal feed supplements
– manufacturing processes 2: 306 – antioxidants 2: 188
– synthesis 2: 306 – lignin 2: 196
– see also APG animal health 2: 195–196
allylic C–H Bonds, oxidation 2: 269–270 anions of fatty acids, oxidative coupling
alternative life, GJ Drinks 1: 275–277 2: 266
American Society for the Testing of Materi- anodic coupling, fatty acids 2: 267–269
als see ASTM ANPI 1: 260
american straw, chemical composition anthracenes 1: 118
2: 107 antibiotic-resistant bacterial strains 2: 195
amino acid-based product family trees antibiotics 2: 15
2: 201–216 antidiarrheic effects, dietary lignin 2: 195
amino acid composition, lucerne 1: 275 antifeedants 1: 277
amino acid production, microbial antifreeze protein 1: 269
2: 201–216 antimutagenic effects, chlorophyll 2: 336
amino acid residues, hydrophobicity antioxidant activity, oat bran-rich frac-
scale 2: 234–235 tions 1: 186
amino acid units, proteins 1: 122 antioxidants 1: 186
amino acids 1: 267, 2: 304 – animal feed supplements 2: 188
– analysis 1: 299 – ferulic acid 1: 179
– brown juice content 1: 305 – lignin 2: 187–189
– fermentation 2: 203 – lubricants industry 2: 188
– markets 2: 207 – rubber industry 2: 188
d-aminolevulinic acid see DALA – synthetic 2: 188
ammonia fiber explosion, pretreatment antisense RNA approach 2: 275
1: 362 APG 2: 11–12, 272
ammonium lactate 2: 387 – synthesis 2: 13
amphiphilic drugs, controlled release apolar groups, exothermic hydration
devices 2: 240 2: 218
amylases 1: 197 apolar–polar repulsive free energy of hydra-
amylopectin tion 2: 218
– physical structure 1: 140 apple-peel wax 2: 430–432
– starch synthesis 2: 71 – components 2: 434
Subject Index 465
– market launch 2: 436–437 bagasse 1: 91

– natural 2: 429–437 – brazil production 1: 210
– production 2: 432–433 – energy source 1: 222
– skin protection 2: 434 – world production 1: 51
aquatic biomass 1: 91 bagasse storage, case study 1: 321
aqueous media, proteins 2: 218 bale storage 1: 322
aqueous phase hydrogenation 2: 375 – corn stover 1: 320
arabinanes 2: 109 bale transport, Iowa Corn Stover Collection
arabinose, reaction 1: 199 Project 1: 319
arabinoxylans 1: 178 baling 1: 333
Arachis hypogaea 2: 277 – dry material 1: 332
aromatic chemicals, sugar-based 2: 29 BAS 1: 268
aromatic compounds 1: 118 basic chemicals
– renewable raw materials 2: 259 – cellulose 1: 17
– transition metal-catalyzed syntheses – glucose 1: 17
2: 259 – starch 1: 17
aromatic functionality, char 1: 156 basic principles, biotechnology 2: 349–351
Arthrobacter 1: 398 basic substances, biorefinery 1: 18
arthropods, chitin occurrence 2: 416 batch fermentation, brown juice 1: 299–300
arylglycerol units 2: 156 BBI 2: 322
ascorbic acid, d-sorbitol 2: 9 14-BDO 2: 373, 375
ash components, feedstock 1: 146 beer streams 1: 134
asparagine residues, biodegradable thermo- benzene 1: 87
plastics 2: 241 benzene derivatives 1: 118
aspartic acid (ASP) 2: 34 – cyclotrimerization 2: 259
– basic biobased chemicals 1: 22 benzene–toluene–xylene see BTX
Aspergilli 1: 181 Bergius, F. 1: 5
Aspergillus 1: 202 Berzelius, J. J. 1: 5
Aspergillus itaconicus, IA 2: 36 Beta vulgaris 2: 410
Aspergillus oryzae 2: 20 betaine 2: 410–415
– cellulase development 1: 366 – chemical properties 2: 411
– thermostabilization 1: 370 – chemical structure 2: 411
Aspergillus succinoproducens 2: 35 – usage 2: 412–414
Aspergillus terrous, IA 2: 36 betaine esters 2: 414–415
asphalt emulsifiers, lignin-based BG 1: 203, 364
2: 192–193 BG Supplement 1: 366–367
ASTM, tests 2: 232 binder, starch 2: 84
austria-wide concept, biomass usage 1: 284 bio-alcohols, sugar conversion 2: 32
austrian-concept, biorefinery 1: 273 bio-based building blocks 2: 453
autoadhesive tack 2: 186 – emergence 2: 450
autotrophic acetogens, syngas fermenta- biobased consumer products, cosmetics
tion 1: 233 2: 409–442
autotrophic bacterium biobased economy 2: 138
AVGVP, biocompatibility 2: 232 – 3-pillar model 1: 3
axioms, phenomenological 2: 232–234 – existing 1: 43
– growth 1: 44–45
b – historical outline 1: 42–45
B-starch 2: 68 Biobased Industrial Products, initiative
Bacillus megaterium 2: 424 group 1: 16
bacteria, biodegradation 1: 363 bio-based industry, transition 1: 93–96
bacteria cellulosome 1: 365 bio-based materials 2: 354
bacteria destruction, lignin 2: 196 biobased oleochemicals, industrial develop-
bacteriochlorophylls 2: 326 ment 2: 291–314
466 Subject Index
biobased poly(lactic acid) 1: 296 biofuel cells 1: 379

biobased production, integrated 1: 8–12 biofuels 1: 182
biobased products – directive 1: 15
– market opportunity 2: 353 – promotion 1: 94
– markets 2: 348 biogas 1: 30, 377
biobased technology, current 2: 375–377 biogenic amorphous silica see BAS
bio-cascade, biorefinery concepts 2: 355 biological inhibitors, fast pyrolysis 1: 249
biocatalysts 1: 68 biological raw materials, product classes
– development 1: 108 1: 13
– genetically engineered 2: 37 biomass
– improvement, 3-HPA 2: 35 – availability 1: 99–101
biocatalytic routes – commercialization 1: 317–344
– chemicals production 1: 385–406 – components 1: 22
– ethanol production 1: 390–393 – composition 1: 16, 119, 359, 2: 108
biochemical refinery, secondary 1: 104–106 – compositional variety 1: 45
biochemicals 1: 13 – conversion 2: 151–163, 350, 455–456
biocompatibility 2: 230–232 – definition 1: 12–14
biocomposites 2: 362 – depolymerization 1: 123
bioconversion – diversification 1: 54–55
– biomass processing 1: 98 – hydrolysis 1: 129–138
– fermentation 1: 104 – hydrolyzate 1: 78
– starch 2: 89–91 – industrial 1: 13
bio-counterpart, petroleum-derived poly- – industrial chemicals 2: 347–365
mers 2: 41 – key sugars 2: 3–59
biocrude 1: 98, 2: 351 – lignocellulose 2: 97
biodegradability – local 1: 56–57
– general aspects 1: 213 – multi-quality 1: 92
– intrinsic 2: 348 – policy targets 1: 85
biodegradable films, gluten 1: 181 – polysaccharide-containing 1: 105
biodegradable lubricants, european potential – pretreatment 1: 107, 361–363
market 2: 301 – recycling 1: 117
biodegradable packages 2: 422–423 – refining 1: 41–66, 107, 227–252
biodegradable plastics 1: 182 – sustainability 1: 93–97, 106
– sugar cane 1: 212–216 – technology 1: 14–16, 93–97
biodegradable polymer 1: 91 – thermochemical processing 1: 249
biodegradable thermoplastics, programma- biomass-based industrial products 1: 87
ble 2: 241 biomass-based products, estimated EU po-
biodegradation tential 1: 89
– definition 1: 213 biomass carbon resources 1: 116
– white-rot fungi 2: 160 biomass chemistry, comparison with petro-
biodiesel 1: 116, 152 leum 1: 118–122
– production 1: 126 biomass content, classes 2: 4
bio-ethanol 2: 451–452 biomass feedstocks 1: 45
bio-fertilizer, grasses 1: 283 – costs 1: 48–50
biofine char 1: 155–158 – required properties 1: 50
– insoluble components 1: 146 biomass flux, The Netherlands 1: 99
biofine plants biomass fuels 1: 103
– byproducts 1: 145 biomass gasifiers, types 1: 231
– costs 1: 159 biomass industry, chemical production
biofine process 1: 139–164 numbers 1: 284
– advantages 1: 146 biomass-nylon-process 1: 26
– economics 1: 158–161 Biomass Research and Development Techni-
– yields 1: 145–146 cal Advisory Committee 1: 135
Subject Index 467
biomass streams 1: 100 – primary research areas 1: 101–103

biomass substitution volume 1: 85 – principles 1: 16
biomass suppliers 1: 118 – raw material 1: 45–47, 253
Biomass Technical Advisory Committee see – sugar-based 1: 209
BTAC – supply 1: 45–52
biomass value 1: 324–328 – technological development 1: 53–56
biomaterials 1: 13 – wet mill-based 1: 28
bionics 2: 410 – wheat based 1: 167–183
bio-oil – whole-crop 1: 24, 26–29
– characteristics 1: 243 biorefinery complex, cost estimates 1: 118–
– fermentation 1: 229, 244 122
– yield 1: 241 biorefinery concepts 1: 98–99, 2: 355–356
Biopol 2: 44, 422–424 – definition 1: 166
– biodegradability 2: 424 – elements 1: 81
– future 2: 428–429 biorefinery context 2: 315–324
biopolyesters, synthetic 2: 41 biorefinery evolution 1: 69
biopolymers 2: 40–47 biorefinery I, sucrose-based 1: 68
– cellulose 2: 104 biorefinery II, starch-based 1: 69
bioprocessing, consolidated 1: 56 biorefinery III 1: 69
bioproduct opportunities, industrial 1: 379 biorefinery lignin, substitution 2: 182
bioproduction biorefinery model 1: 68
– highlights 2: 223 biorefinery process, integrated 1: 102
– mechanistic foundations 2: 217–251 biorefinery products 1: 11
– protein-based polymers 2: 223–227 biorefinery research, current 1: 11
bioproducts, classification 2: 356–357 biorefinery supply, transport options
bioreactor engineering 1: 108 1: 338
biorefineries biorefinery systems 1: 3–40, 23
– basic principles 1: 17 – history 1: 4–16
– bio-oil based 1: 229 – sustainability 1: 56–65, 60–65
– Brazil 1: 71 – whole crop 1: 165–191
– building-block concept 2: 202–204 biorefinery technology developments,
– cellulosic 1: 55–56 milling industries 1: 345–353
– chlorophyll disregard 2: 338 biorefinery two platforms concept 1: 24
– conceptual schematic diagram 1: 239 biorefinery wastes 1: 56
– definition 1: 19–22, 116, 227, 358 biosyngas 1: 98
– development 1: 67–83 biosynthesis, poly(3-hydroxybutyric acid)
– disadvantage 1: 46 1: 224
– fuel-oriented 1: 193 biosynthesis genes, Escherichia coli 2: 44
– future integration 1: 380 bio-synthetics, car production 1: 9
– generations 1: 19–20 biotech, industrial 2: 445–462
– green see green biorefinery biotech adoption 2: 447
– integration 2: 201–216 biotech development, pace 2: 447
– lignin 2: 177 biotech strategy 2: 457
– lignocelluloses 2: 110–115 biotechnological processes, typical prob-
– lignocellulosic 1: 115–128 lems 1: 388–389
– lignocellulosic feedstock 1: 24–26, biotechnology, predictions 2: 32
129–138 biphenyl units, lignin 2: 158
– MAAP 2: 209 1,5-biphosphatecarboxylase/oxygenase
– near future production 1: 317 1: 255
– oats based 1: 183–187 bisphenol A see BPA
– phase III 1: 19 black liquors
– plant juice 1: 295–314 – Kraft pulping 2: 170
– possible products 1: 45–47 – soda pulping 2: 171
468 Subject Index
blood plasma substitutes, starch 2: 89 1,2,4-butanetriol 2: 37

blue starch 2: 62 n-butanol, glucose product family 1: 21
Boehringer, A. 1: 7 Butyribacterium methylotrophicum 1: 228
bonding patterns 2: 156–159 – representatives 1: 235
Boudouard reaction, syngas production c-butyrolactone see GBL
1: 230 by-products
Bowman–Birk inhibitor see BBI – animal feed 1: 100
BPA 1: 148 – biorefinery 1: 23
Brabender viskograph 2: 75 – brown juice 1: 311
Braconnot, H. 1: 5
bran, wheat milling 1: 170 c
Branched PLA, melt rheology 2: 397 C1 compounds 1: 233
branching, rheology control 2: 396 – syngas fermentation 1: 228
branching technology 2: 398 C5-carbon sugars, product categories
Brassica napus 2: 277 2: 358–360
Brazil, agroindustry 1: 209–211 C6-carbon sugars, product categories 2: 358
breeding material, fatty acid variants 2: 279 C–C coupling, radical 2: 266–269
Brevibacterium 2: 35 C–C double bonds, oxidative cleavage 2: 258
British gums 2: 62 C. glutamicum 2: 205, 210
bromination, LA 1: 150 – phosphorus supply 2: 211
Brookfield viscometer 2: 75 C–H bonds, functionalization 2: 269–270
brown juice (BJ) 1: 271, 274, 300–308 C. milleri 2: 212
– average composition 1: 301–302 C-nucleosides 2: 25
– batch fermentation 1: 299–300 (C-x)-chemicals 1: 21
– carbohydrate addition 1: 311 C2 anions, oxidative coupling 2: 266
– composition of nutrients 1: 305 C2 building-block chemical, ethanol 2: 132
– fermentation medium 1: 298 C3–C5 carboxylic acids, microbial fermenta-
– lactic acid fermentation 1: 305–306 tion 2: 33
– lactic acid source 1: 295 C3 plants
– quality variations 1: 302–305 – protein yield 1: 253
– storage alternatives 1: 298 – yield 1: 258
BTAC 1: 14 C4 plants
BTX 2: 29 – protein yield 1: 253
building-block concept 2: 204 – yield 1: 258
– biorefinery 2: 202 C7 plant acids, potential generation 2: 32
– metabolic engineering 2: 204, 206 CAFI 1: 136
building blocks 1: 22–23, 98 calcium lactate 1: 106
– biobased 2: 450, 453 cancer chemopreventive agents 2: 322
– heterocyclic 2: 26 cancer therapies, DALA 1: 150
– protolignins 2: 152 Candida antarctica, lipase B 2: 256
– succinic acid 2: 367–379 Candida bombolica 2: 274
– sugar derived chemicals 2: 34 Candida tropicalis 2: 273
building chemistry 2: 87 Candida tropicalis DSM 3152 2: 274
bulk chemicals 1: 386 Candida tropicalis M 25 2: 274
– production routes 1: 385–406 capital costs, biorefinery 1: 240
Burkholderia spp, poly(3-hydroxybutyric acid) carbohydrate-based product lines 2: 3–59
biosynthesis 1: 224 carbohydrate-based surfactants 2: 305
business structure 1: 117–118 carbohydrate composition, lignocellulosic
butadiene feedstock 2: 109
– 1,4-BDO 2: 373 carbohydrate content, changes 1: 266
– glucose product family 1: 21 carbohydrate esters, lipase-catalyzed syn-
1,4-butanediol 1: 149 theses 2: 272
2,3-butanediol, glucose product family 1: 21 carbohydrate homopolysaccharides 1: 139
Subject Index 469
carbohydrate polymers, cellulose 1: 55 carton production 2: 84

carbohydrate polysaccharides, acid hydroly- case studies, sustainable production
sis 1: 144 2: 448
carbohydrate refining 1: 351 catabolism, chlorophyll 2: 330–331
carbohydrate source, addition to brown catalysis technology 2: 349
juice 1: 311–312 catalysts
carbohydrate stream, corn refinery 1: 349 – bio-oil production 1: 244
carbohydrates 1: 89–90, 2: 108 – carbohydrates conversion 1: 403
– annually renewable 2: 6 – metal-based 1: 228, 233
– biorefinery 1: 18 catalytic decarbonylation, furan 2: 9
– catalytic oxidation 1: 403–404 catalytic hydrogenation
– chemical catalytic conversion 1: 402 – LA 1: 151
– contained in biomass 2: 3 – sorbitol 2: 130
– heating 2: 24 catalytic pulping, wood 2: 118
carbon catalytic routes, chemicals production
– recycling 1: 116 1: 385–406
– renewable 1: 43 catalytic transformations 2: 270–272
carbon-14-labeled Escherichia coli, purifica- – succinic acid 2: 372–375
tion 2: 228–229 catechol 2: 30
carbon-based plant material, yearly cationic addition, Lewis acid-induced
amount 1: 43 2: 264–265
carbon dioxide cationic polymers, hair 2: 419
– glucose product family 1: 21 cationic surfactants 2: 412
– recycling 1: 117 – structure 2: 414
carbon dioxide sink, PLA 2: 402 CBH 1: 203, 364
carbon fibers CBH-EG-BG System, optimization
– annual demand 2: 197 1: 366–371
– porous 1: 283 CBH I 1: 76
– vehicle production 2: 196–197 CBH I (Cel7A) variants, thermal activity
carbon-oxygen reaction, syngas produc- 1: 368
tion 1: 230 CBM, cellulase families 1: 364
carbon-processing industries 1: 42–44 CC 1: 61
carbon sequestration 1: 62 cell contents 1: 265–269
carbon sources 2: 209 cell-immobilization 1: 392
– fermentable 1: 78 cell removal 2: 388
– industrial 1: 67 cell wall, structural constituents 1: 260–265
– reduced cost 2: 204 cellobiohydrolase see CBH
carbon sugars 2: 358 cellobiohydrolase I see CBH I
carbon-water reaction, syngas produc- cellulase development 1: 366–375
tion 1: 230 cellulase enzyme performance 1: 74
carbonate polymerization 2: 398 – improved 1: 76–77
carboxylic acids 2: 34–36 cellulase enzyme production 1: 194
– addition 2: 262 cellulase enzymes 1: 74, 2: 177–178
– chemical conversion 2: 37–40 – costs 1: 72–73
– microbial conversion 2: 32–34 – production 1: 201–202
– sugar-based 2: 43 – superior 1: 205
carboxymethylation 2: 77 – thermal stability 1: 76
cardboard, from press cake fibers 1: 282 cellulase expression inducers, disaccharide
care additives, multifunctional 2: 309 sophorose 1: 76
carotene 1: 257 cellulase mix, lignocellulosic conversion
– industrial production 1: 9 1: 374
carotenoids 2: 320 cellulase production economics, im-
Carothers, W. H. 1: 8 proved 1: 74–77
470 Subject Index
cellulase production strain, enhance- cereal fractionation plants, categories 1: 167

ment 1: 374 cereal fractionation processes see CFP
cellulase saccharification, plant develop- cereal grains, baling 1: 333
ment 1: 134 cereal waste, LCF biorefinery 2: 111
cellulases cereals 1: 26, 165–191
– biodegradation 1: 364 – starch sources 2: 63
– commercial status 1: 202 cetiol CC 2: 311
– expression 1: 374–375 CFP 1: 166
– improvements 1: 367 chain length, cellulose 1: 195
– novel 1: 367–370 char 1: 145
cellulolyteomics 1: 374 – biofine process residual 1: 155
cellulolytic fungi chemical composition, apple-peel wax
– protein gels 1: 372 2: 433
– secretome 1: 371–373 chemical conversion, sugars 2: 37–40
cellulose 1: 6, 71–73, 90, 121 chemical degradation, chlorophyll 2: 333
– accessibility 1: 55 chemical digestion, intracellular poly(hy-
– acetate 1: 243 droxyalkanoates) 1: 218
– acid conversion 1: 129 chemical fractions, lignocellulose 1: 24
– biosynthesis 1: 90 chemical industry 1: 97
– chemical composition 1: 359 – biorefineries 1: 85–111
– chemical conversion to LA 1: 144 – renewable raw materials 2: 253–289
– chemical structure 2: 418 chemical modification, naturally produced
– conversion rates 1: 130 structures 2: 349
– digestibility 1: 261 chemical pulping 2: 166
– enzymatic hydrolysis 2: 115 – environmentally friendly 2: 179
– fermentation 2: 177–179 – LCF 2: 114
– glucan source 1: 139 chemical sources, grasses 1: 282–283
– high-vacuum pyrolysis 2: 23 chemical transformation steps, petrochem-
– history 2: 100 ical industry 1: 88
– hydrolysis 1: 26, 194, 199, 202–205 chemicals 1: 22–23
– isolation 2: 127 – basic 2: 5
– plant content 1: 261 – biobased 1: 22
cellulose-based biorefinery III 1: 69 – biomass compounds 1: 119
cellulose-based product family tree, indus- – fossil sources 1: 120
trial 2: 129 – from biomass 1: 108
cellulose-based product lines 2: 127 – from renewable resources 2: 367–379
cellulose-binding modules see CBM – glucomannan derived 2: 120
cellulose derivatives 2: 357 – lignocellulose-based 2: 97–150
– principal 1: 90 – low-molecular-weight 2: 160
cellulose fiber, pretreatment 1: 72 – organic 1: 124
cellulose-hydrolyzing enzymes 1: 17 – product family tree 2: 124–126, 132
cellulose saccharification 2: 99 – production 1: 386
cellulosic biomass 1: 68, 197 – production routes 1: 385–406
– conversion to fuel 1: 52 – special 1: 378
– ethanol production 1: 193 chemo-enzymatic epoxidation 2: 254
– recalcitrance 1: 56 chemo-enzymatic self epoxidation, reaction
cellulosic biomass conversion 1: 71 principle 2: 256
cellulosic biorefineries, process develop- chemoattractant, peptides 2: 238
ment 1: 55–56 chemopreventive agents, cancer 2: 322
cellulosic feedstocks, hydrolysis 1: 140 chemurgy 1: 9
cement 2: 87 chiral purity, lactic acid 2: 383
CENTURY model 1: 60–65 chitin 1: 182
cereal fractionation, advanced 1: 173 – chemical structure 2: 418
Subject Index 471
– chitosan precursor 2: 415 competitive prices, biobased products 2: 49

– deacetylation 2: 417 competitors, external challenges 2: 457
– occurrence 2: 415–419 components, cereals 1: 166
– purification 2: 416–417 composite materials, carbon fiber 2: 196
chitosan 2: 415–422 compositional variety, biomass 1: 45
– chemical structure 2: 418 concentration, lactic acid 2: 390
– production 2: 417 concept, all biomass is local 1: 57
chitosan derivatives 2: 421 concrete, self-leveling 2: 88
chitosonium salts, water vapor sorption concrete admixtures 2: 189–190
2: 421 conditioner, sugar beet 2: 410–415
chlorophyll 2: 325–343 conditioning agent, natural 2: 436
– biological catabolism 2: 330–334 coniferyl alcohol, oxidation 2: 158
– breakdown 2: 330 consolidated bioprocessing 1: 56
– chemistry 2: 327 Consortium for Advanced Fundamentals
– commercial production 1: 257 and Innovation see CAFI
– degradation 2: 331, 333 consumer acceptance, external chal-
– derivatives 2: 335–339 lenges 2: 457
– fundamentals 2: 326 consumer products 2: 409–442
– historical outline 2: 325 continuous cultivation see CC
– industrial production 1: 9 continuous fermentation 1: 300
– isolation 2: 328 controlled-release devices, design 2: 240
– new materials 2: 338 conversion, chlorin 2: 333
– reactivity 2: 328 conversion efficiency 1: 196
– structure 2: 327 conversion steps
chlorophyllin 2: 335 – biorefinery 1: 23
cholesterol level, decrease 1: 180 – lignocellulosic biorefinery 1: 24
cholesterol mediation 1: 277 conversion technologies 1: 108
cholesterol reduction, b-glucan 1: 185 – primary 1: 270, 2: 350
chopping, pretreatment 1: 361 cooking liquors 2: 166
chrisgas-project 1: 103 coordination–insertion mechanism, lactide
Chromatium okenii 2: 424 polymerisation 2: 393
circuit board resins 2: 194–195 copolyesters, PHB 1: 215
citrates 1: 91 copolymer, PHV-PHB 2: 426
citric acid, glucose product family 1: 21 copper-initiated additions 2: 262–263
Clostridium ljungdahlii 1: 228, 235 corn 1: 26
Clostridium methoxybenzovorans SR3 1: 179 – phytochemicals 2: 317
Clostridium thermoaceticum 1: 235 – wet milling 1: 28
Clostridium thermocellum 1: 365 corn continuous cultivation 1: 61
clothing, synthetic fibers 2: 190 corn dry milling, biorefinery example 1: 70
CO2 flux 1: 328 corn dry milling industry 1: 345–353
CO2 production, MAAP 2: 212 corn grain, export reduction 1: 43
CO2 sequestration 1: 173 corn oil, corn refinery products 1: 348
co-polymerization, starch 1: 27 corn pricing 2: 368
co-products 2: 370 corn refinery, modern 1: 348
– sugar fermentation 2: 375 corn refining 1: 346–347
coconut oil 2: 292 corn–soybean rotation 1: 61
collection, baling dry material 1: 332 corn starch, pearl 1: 351
collection cost, forage harvester 1: 334 corn-steep liquor 1: 349
commercial consideration, MAAP corn stover, world production 1: 51
2: 205–209 corn stover bale storage 1: 320
comonomers, multi-cyclic 2: 398 corn stover pricing 1: 319
company closures, lignosulfonate produc- corn stover structure 1: 74
ers 2: 173 corn syrup, carbohydrate refining 1: 351
472 Subject Index
corn tillage practice 1: 330 crystalline cellulose 1: 195

corn wet milling industry 1: 345–353 crystalline melting point, control 2: 394
corn wet milling process 2: 367 crystallinity, starch 2: 72
corncobs 1: 91 CSL 1: 349
corporate action, increasing 2: 451 cultivation temperature 2: 213
corrugating industry, starch usage curl-retention test 2: 420
2: 83–84 curled hair, swatches relaxation 2: 419
Corynebacterium efficiens 1: 80 cycle times, PHB 1: 216
Corynebacterium 2: 35 cyclization, methyl 17-octadecanoate 2: 259
cosmetic emulsion, oil-phase compo- cyclodextrins 2: 90
nents 2: 310 cyclotrimerization, benzene derivatives
cosmetic lipids, occlusion testing 2: 434 2: 259
cosmetics Cyprus papyrus 2: 98
– chitosan 2: 419
– consumer products 2: 409–442 d
– history 2: 409–410 Dactylis glomerata 1: 261
– ilex resin 2: 439 – alkanes 1: 268
– starch usage 2: 88–89 – amino acid composition 1: 267
cost components, ethanol production 1: 73 – sugar 1: 265
cost disadvantage, cellulose-based etha- DALA 1: 149–150
nol 2: 203 DDGS 1: 71
cost efficiency 1: 381 debranning apparatus 1: 174
cost estimates, biorefinery complex 1: 118 decomposition methods, primary refin-
cost generators, waste 1: 96 ery 1: 271
cost savings, biotechnology 2: 450–451 decorative laminates 2: 185
costs deformation energy, recovery 2: 220
– antioxidants 2: 188 degradation, chlorophyll 2: 331, 333
– biomass vs. petroleum 1: 48–50 – definition 1: 213
– feedstock 1: 196 degradation resistance, cellulose fibrils
– MAAP 2: 202 1: 140
– processing systems 1: 53 degree of polymerization, cellulose 1: 195
cotton 1: 90 demonstration process, iogen’s 1: 193
coupling, oxidative 2: 266 density, bales 1: 335
cover crops 1: 331 department of energy (DOE) 1: 19, 74
crop-drying industry, grass usage 1: 298 depolymerization, biomass 1: 123
crop residues 1: 45 designer proteins 1: 122
– commercial 1: 318 development lines, sugar-based chemi-
– world production 1: 51 cals 2: 14
cropping system 1: 61 development trap, underdeveloped coun-
crops, starch sources 2: 63 tries 1: 52
cross-linking dextrins 2: 79
– free radical 2: 399 dextrose 2: 128
– starch modifications 2: 81 – production 1: 44
cross-reactions 1: 145 – starch hydrolysis 1: 5
crotonaldehyde, glucose product family dextrose syrup, carbohydrate refining
1: 21 1: 351
crude drugs, juice fraction 1: 274 DFA III, production 1: 397
crude fiber, plant content 1: 261 diacids, replacement 2: 38
crude oil 2: 292 1,4-diacids 2: 34
– high prices 1: 115 – basic biobased chemicals 1: 22
crude petroleum, separation 1: 119 dialkyl carbonates 2: 311
crude starch milk 2: 66 – synthesis 2: 311
crushing, pretreatment 1: 361 diamines, sugar-based 2: 42
Subject Index 473
diammonium succinate 2: 376 dry reactions, starch modifications 2: 77

diastereomeric forms, lignin 2: 157 dry storage, bagasse 1: 321
dibenzodioxocin structures, lignin 2: 158 DSM, transition process 1: 93
Diels-Alder reaction, methyl conjugen- Duales System, biodegradable bottle 2: 427
ate 2: 260 Dutch Energy Research Strategy 1: 109
diesel 1: 119 dye dispersants 2: 190–192
– low-smoke formulation 1: 153 dyes
dietary lignin, antidiarrheic effects 2: 195 – biorefinery context 2: 315–324
diethyl ether, glucose product family 1: 21 – juice fraction 1: 274
diffraction patterns, starch 2: 73 dyestuff 2: 191
difructose anhydride 1: 397–402
digestibility, cellulose 1: 261 e
diglycerides, lipase-catalyzed syntheses E. coli see Escherichia coli
2: 270–272 E10-Fuel 1: 9
dihydropyranones 2: 20 ECN 1: 109
– disaccharide-derived 2: 24 ecological aspects, green biorefinery
dilactide, glucose product family 1: 21 1: 283–285
dilute acid hydrolysis 1: 200 ecological balance, fermentative produc-
dilute acids tion 2: 207
– pretreatment 1: 362 ecological compatibility, biobased oleochem-
– starch treatment 2: 76 icals 2: 293
dilute sulfuric acid, biofine process 1: 142 economic aspects, green biorefinery
dilute-sulfuric-acid hydrolysis, cellulose 1: 283–285
1: 132 economic barriers, biotechnology 1: 381
dimer acid 2: 297–298 economic benefits 2: 452–454
dimerdiols, dimer acid based 2: 297–298 economic clusters, new synthesis 1: 95
dimerization, radical 2: 267 economic forces 1: 41
dimethyltetrahydrofuran see DMTHF economic potential
diphenolic acid 1: 148 – biotechnology 2: 446–451
direct distillation 2: 389 – industrial biotech 2: 445–462
disaccharide sophorose 1: 76 economics, biofine process 1: 158–161
disaccharides, availability 2: 4–7 economies of scale 1: 159
disposal problems, Biopol 2: 428 – biodiesel plant 1: 127
dissociation, of industries from petrochem- economy, biobased 1: 41–66
ical 1: 94 economy growth 1: 67
distillation of lactate ester 2: 389 economy of scale
distillers dried grains and solubles see – biorefineries 1: 350
DDGS – furfural 1: 125
DM 1: 261 ecosystem modeling 1: 57–60
DMTHF 1: 152 edible films, gluten 1: 181
DOE see department of energy efficiencies, biofine process 1: 145–146
door binders 2: 185–186 efficiency improvements, biotechnology
downdraft gasifiers 1: 231 2: 454
downstream processing efficient energy conversion, elasticity pro-
– grass fiber fraction 1: 281 vides 2: 219
– poly(3-hydroxybutyric acid) 1: 218–220 EG (endoglucanase) 1: 203, 364, 2: 133
drilling fluids, starch derivatives 2: 91 – structure–function relationship 1: 370–
drugs, sugar derived 2: 14 371
dry fractionation, wheat 1: 176–183 Ekman, C. D. 1: 6
dry matter 1: 261 EL (ethyl levulinate) 1: 152–153
dry mill refinery 1: 346–347 elastic consilient mechanism 2: 223
dry milling 1: 27, 70 – protein-based polymer engineering
– operations 1: 166 2: 217
474 Subject Index
elastic mechanisms, coupling to hydropho- environmental improvements 1: 64

bic mechanism 2: 237–238 environmentally friendly, biotech 2: 448–
elastic moduli, fibers 2: 241 450
elastic protein-based polymers, temporary enzymatic conversion 1: 130
functional scaffoldings 2: 239 enzymatic digestion, poly(3-hydroxybutyrate-
elasticity, protein-based polymers 2: 219– co-valerate) 1: 219–221
220 enzymatic hydrolysis 1: 79, 147
Elbow washing test, betaine 2: 413 – cellulose 2: 115
electricity 1: 46 – improvements 1: 205
– biomass share 1: 14, 16 – reactions 1: 203
electricity generation, biomass 1: 44 enzymatic hydrolysis process 1: 134
electrodialysis, lactic acid purification 1: 312 enzymatic methods, LCF 2: 115
electrophilic substitution, furan 2: 9 enzymatic oxidizing systems 1: 178
emollients 2: 310 enzymatic processes, starch degradation
emulsifiers 2: 79
– lecithin 2: 318 enzymatic reactions 2: 270–274
– polyglycerol esters 2: 308 enzymatic synthesis, MAAP 2: 202
– vegetable oil 2: 301 enzymatic transport, improvements 2: 204
emulsion(co)polymerization process, starch enzyme-based plant development 1: 134
derivatives 2: 90 enzyme broth 1: 201
endoglucanase see EG enzyme catalysis, economic barriers 1: 381
endotoxins, removal 2: 230 enzyme cost reduction, ethanol produc-
enediol, dehydration 1: 142 tion 1: 377
Energie Onderzoeks Strategie see EOS enzyme dosage 1: 366
energy and protein coproduction 1: 55 enzyme immobilization 1: 399–402
energy balance, simultaneous processing of enzyme performance, cellulase 1: 76–77
sugars 1: 221 enzyme production 1: 202
energy conversion, efficient 2: 219 – bran 1: 172
energy costs, impacts 1: 115 enzyme recovery 1: 74
energy crops, renewable carbon 1: 44 enzyme requirement, increase 2: 178
energy efficiency, fossil fuels replace- enzyme screening 1: 398
ment 2: 449 enzyme system, optimisation 1: 74
Energy Research Center of the Netherlands enzymes 1: 357–383, 2: 90
see ECN – biodegradation 1: 363
energy sources, biomass-based 1: 380 – biomass conversion 1: 68
engine efficiency loss, diesel 1: 153 – cellulase 1: 201–202
engineered organisms 1: 68 – cost reduction 1: 56
engineering – markets 2: 446
– mechanistic foundations 2: 217–251 – nonhydrolytic 1: 365
– protein-based polymers 2: 219–220, – oxidative 1: 213
232–238 – recycling 1: 205
engineering principles – superior 1: 205
– fundamental 2: 222 – synergism 1: 365–366
– protein-based polymers 2: 220 – thermally stable 1: 368
entire barrel of biomass 1: 54–55 EOS 1: 109
entropic elastic force, proteins 2: 223 epoxidation
environmental aspects, biodegradable plas- – chemo-enzymatic 2: 254
tics 1: 212 – new methods 2: 254–257
environmental benefits 1: 117 epoxides 2: 254
environmental consideration, MAAP – polyols 2: 298–299
2: 205–209 – PVC stabilizers 2: 256
environmental impact, production pro- equilibrium concentration, protonated glyco-
cess 1: 92 side 1: 141
Subject Index 475
erosion 1: 327 exothermic hydration, apolar groups

– cover crops 1: 331 2: 218
– prevention 1: 61 expansin, enzymatic hydrolysis 1: 365
ERRMA 1: 89 expressed protein-based polymers
erucic acid, high 2: 280 2: 242–245
erythro form, lignin 2: 157 expression vector, gene 2: 227
Escherichia coli 1: 206, 2: 30 external challenges 2: 456
– bioengineered 2: 35–37, 44 external environment, biotechnology
– carbon-14-labeled 2: 228–229 2: 461
– cost of production 2: 242 extraction, chlorophyll 2: 328
– fermentation 2: 230 extraction methods, chlorophyll 2: 337
– inulin production 1: 398 extraction processes, PHB 1: 218
– recombinant 1: 399 extrusion cooking, starch modifications
– transformation 2: 227–230 2: 77
esparto grass, xylitol source 1: 283 extrusion processes, PHB 1: 216
esterification, starch 2: 80
esters, lubricant applications 2: 300 f
ETBE 2: 7 fabric coloring 2: 191
ethanol 1: 89, 104, 146, 209, 2: 7–8, 132 FAME 1: 152
– additive replacement 1: 357 farmer value 1: 325–327
– fermentation 1: 11, 2: 120, 351 FAS 2: 303
– global market 2: 446 – synthesis 2: 303
– glucose product family 1: 21 fast pyrolysis 1: 229, 241
– lignocellulose transformation 2: 115 – biorefinery 1: 246–248
– predictions 2: 454 – products 1: 242
– vapor pressure 1: 151 – reaction pathways 1: 243
– wood hydrolyses 1: 5 fast-pyrolysis plant, schematic diagram
ethanol production 1: 125, 130, 193, 1: 244
209–210, 389 fat hardening 1: 7
– advantages 1: 197 fats
– cellulase 1: 201 – microbial conversion 2: 274
– costs 1: 72, 246 – new syntheses 2: 253–289
– enzyme cost reduction 1: 377 fatty acid esters, biodegradable 2: 299–301
– sucrose 1: 70 fatty acid methyl esters see FAME
ethanol production plant, process design fatty acid oil seeds variants, commercially
1: 393 available 2: 278
ethanol recovery 1: 206–207 fatty acids 1: 90–92
ether structures, lignin 2: 158 – anodic coupling 2: 267–269
etherification, starch 1: 27, 2: 80 – apple-peel wax components 2: 433
ethyl t-butyl ether 2: 7 – chain length 2: 292
ethyl ester, LA derivatives 2: 10 – epoxidation 2: 254–257
2-ethyl hexanol, glucose product family – juice fraction 1: 274
1: 21 – microbial oxidation 2: 273–274
ethyl lactate, glucose product family 1: 21 – nucleophilic addition 2: 265
ethyl levulinate (EL), properties 1: 151 – oxidative coupling 2: 266
ethylene, glucose product family 1: 21 – triglycerides 1: 122
EU directives 1: 94 – unsaturated 2: 272–273
Eubacterium limosum 1: 235 – vic-dihydroxy 2: 257–258
Europe, biomass conversion 2: 351 fatty alcohol sulfate 2: 303
European grassland, yield 1: 259 fatty alcohols 2: 294
European Renewable Resources and Materi- fatty compounds
als Association see ERRMA – reactions 2: 266–270
excess water, removal 1: 219 – unsaturated 2: 254–266
476 Subject Index
FDCA (furan-2,5-dicarboxylic acid) 2: 38, – inhibitors 1: 78

133 – performance 1: 78
– basic biobased chemicals 1: 22 – PLA 1: 296–297
– synthesis 2: 134 fermenters, ethanol production 1: 201
feedstocks 1: 100, 105 fertilizer
– alternative 2: 452 – ash 1: 161
– composition 1: 195 – nitrogen 1: 325
– conversion 1: 68–73 ferulic acid 1: 178–179
– fibrous 1: 239 Festuca arundinacea 1: 261
– insoluble components 1: 146 – alkaloid production 1: 277
– pretreatment 2: 167, 178 – fructans 1: 267
– pricing 1: 327 – sugars 1: 266
– production 1: 181 Festuca pratensis 1: 261
– quality 1: 196, 334 – amino acid composition 1: 267
– selection 1: 194–198 – sugar 1: 265
– sucrose-based 1: 197 Festuca spp, proteins 1: 277
– supply 1: 317, 2: 368–369 fiber fraction 1: 278–285
– thermogravimetric analyses 1: 156 – corn 2: 369
fermentable-carbon-cost 1: 68 – grass 1: 281
fermentable carbon source 1: 78 fibers
fermentable sugars 1: 67–73 – biodegradable 2: 10
fermentation 1: 74, 85, 123, 146, 2: 356 – corn refinery products 1: 348
– E. coli 1: 399 – high-performance 2: 41
– Escherichia coli transformation 2: 227 – improved 2: 241
– amino acids 2: 203 – Kraft lignin 2: 197–198
– biomass processing 1: 98 – paper 2: 84
– bio-oils 1: 229 – press-cake components 1: 280–282
– cellulose 2: 177–179 fibrous biomass, fast pyrolysis 1: 246
– commercial lactic acid production 2: 384 film-forming agents, chitosan 2: 419
– continuous 1: 300 films, water-retentive properties 2: 420
– economics 2: 369 filter aids, purified biogenic silica 1: 278
– ethanol 1: 390–391, 2: 351 fine chemicals 1: 386
– fungal 1: 181 – production routes 1: 385–406
– glucose 1: 31 finishing agents 2: 86
– guidelines 1: 21–22 Fischer glycosidation, APG synthesis 2: 12
– lactic acid 1: 298, 2: 383 Fischer–Tropsch process 1: 158
– microbial 1: 181 flocculants, starch derivatives 2: 91
– PHB production 1: 217 flow dynamics, agricultural ecosystems
– rhizopus-based 2: 388 1: 60
– succinic acid 2: 369 fluidized bed gasifiers 1: 231
– syngas 1: 233–239 foams, production 2: 9
fermentation broth, no processing 1: 75 follow-up chemicals, ethanol 2: 7
fermentation by-products 2: 388 follow-up products, biorefinery 1: 24
fermentation ethanol 2: 7 food 1: 13
fermentation industry, starch 2: 89 food preservative, ferulic acid 1: 179
fermentation inhibitors, bio-oil 1: 245 forage crops 1: 45
fermentation medium forestry ecosystem modeling 1: 57–60
– brown juice 1: 298 forestry waste, furfural hydrolysis 2: 8
– pearled grain flour 1: 176 formic acid 1: 153–154, 2: 39
– plant juice 1: 295–314 – production 1: 139–164
– potato juice 1: 309–310 formulation 1: 74
fermentation organisms 1: 77–81 forward extraction, lactic acid 2: 389
fermentation process 1: 104–106 fossil-based raw material substitution 1: 85
Subject Index 477
fossil carbon-processing industries fungus, wood-rotting 1: 201

1: 42–44 fungus Z proteins 1: 373
fossil fuel substitution 1: 85 furan 2: 19
fossil fuels replacement, energy effi- – hydrophilic 2: 20
ciency 2: 449 – polyesters 2: 44
fossil organic raw materials 2: 347 furan commodity chemicals 2: 8
fossil resources, dependence 1: 92 furan compounds 2: 16
foundry, starch derivatives 2: 91 furan derivatives 2: 98
foundry resins 2: 184–185 furan-2,5-dicarboxylic acid (FDCA) 2: 38,
Fownes, G. 1: 6 133
fraction-I protein 1: 274 – basic biobased chemicals 1: 22
– economic interest 1: 255 – synthesis 2: 134
fraction-II protein 1: 275 furan polyamides 2: 46
fractionation, green crops 1: 272 furan resins 1: 154
fractionation process, oats based furanoid sugar derivatives 2: 48
1: 183–187 furfural 1: 6, 78, 91, 124–125, 154, 199,
free energy of hydration, repulsive 2: 218 2: 8, 121–127
free radical cross-linking 2: 399 – biomass building blocks 1: 22
friction materials 2: 184 – chemical structure 1: 143
Friedel-Crafts acylation 2: 265 – formation 2: 123
fructans 1: 267 – history 2: 101
– enzymatic decomposition 1: 302 – lignocellulosic products 1: 25
fructose 2: 131 – mass yield 1: 145
d-fructose, synthesis 2: 132 – production 1: 142
frying oil, byproducts 1: 100 – yields 2: 102
fuel additives 1: 150 furfural production 1: 125, 133, 139–164
– ethanol 1: 26 furfuryl alcohol 1: 154
fuel alcohol, production 1: 193 furfurylamines, conversion 2: 28
fuel cells 1: 378 future biorefineries, lignocellulosic materials
fuel ethanol, legislative support 2: 453 processing 2: 166
fuel ethanol program, Brazil 1: 210 future development lines, sugars 2: 3–59
fuel gas 1: 102
fuel-oriented biorefineries 1: 193–208 g
fuel production 1: 53 galactanes 2: 108
– starch 1: 181 gas-phase chlorination, photochemical
fuel source, sustainable 1: 115–128 2: 269
fuels 1: 13 gas to liquids see GTL
– biobased products 1: 376 gasification 1: 31, 85, 101, 123, 227, 2: 350
– biofine char 1: 155 – air-blown 1: 232
fumaric acid 2: 35 – bioproducts 2: 361
functional foods 1: 180 – bran 1: 172
functional group transformations, side – coal 1: 157
chains 2: 333 – fundamentals 1: 230
functional groups 2: 156–159 – seperation of value components 1: 103
– addition to hydrocarbons 1: 119 gasification-based systems, hybrid process-
– LA 1: 147 ing 1: 230–241
functionalization, C–H bonds 2: 269–270 gasifier temperatures 1: 232
fungal cellulolytic system 1: 365 gasoline 1: 119
fungal fermentations 1: 181 – replacement 1: 49
fungal genes, schematic representation gasoline market, USA 1: 71
1: 373 GBL 1: 149, 2: 373, 375
fungi, cellulolytic 1: 371–373 GEGVP, repulsive free energy 2: 237
fungicides, lignin-based dispersants 2: 193 gelatinization, starch 2: 78
478 Subject Index
gelatinization temperature 2: 75 d-glucose residues, Lolium perenne 1: 264

gene constructions 2: 225–227 glucose yields 2: 177
– expressed protein-based polymers b-glucosidase see BG
2: 242–245 glucosides 2: 130–133
gene ladder 2: 229 5-(glucosyloxymethyl)furfural 2: 16
– GVGVP 2: 226 glutamate, markets 2: 207
gene technology, plant breeding 2: 275, glutamic acid (GLU) 2: 34, 304
281 – basic biobased chemicals 1: 22
Genencor International 1: 74, 77 gluten, corn refinery products 1: 348
generation-I biorefinery 1: 19 glycanes 2: 108
generation-II biorefinery 1: 19 glycerol 2: 271
generation-III biorefinery 1: 19–20 – basic biobased chemicals 1: 22
genetic engineering 1: 374, 398 – biocatalytic route 1: 393–397
– ethanol production 1: 391 glycine, chemical structure 2: 411
genetically engineered organisms, fermenta- Glycine max 2: 277
tion 2: 7 glycolipids, microbial conversion 2: 274
genetically modified crops 1: 96 glycoside hydrolase family classification sys-
geographical distribution, refineries 1: 57 tem see GH
geoporphyrins 2: 331 glycosidic bonds 1: 140
germ, corn refinery products 1: 348 GMF 2: 17, 25
GGAP, biocompatibility 2: 232 gold catalysts 1: 403
GH 1: 364 grain 1: 45
GH families, Trichoderma reesei 1: 373 grain wet-milling, biorefinery example
Gibbs free energy, change 2: 218, 222, 232 1: 70
– phase transition 2: 234 grass
GJ see green juice – composition 1: 262–263
glass fiber, resins 2: 185 – key components 1: 260–269
GLNC, juice fraction 1: 274 – production costs 1: 284
global warming 1: 94 grass fibers
GLU see glutamic acid (GLU) – basic properties 1: 281
glucan 1: 139, 2: 108 – downstream processing 1: 281
b-glucan 1: 175–176, 185 – products 1: 282
glucanases, biodegradation 1: 364 grass press cake, major components 1: 279
glucaric acid 2: 38 grass silage juice, physicochemical charac-
– basic biobased chemicals 1: 22 teristics 1: 276
d-glucitol 2: 9 grassland feedstocks, availability 1: 259–
gluconic acid 1: 402 260
– biomass building blocks 1: 22 gravity separator, acid hydrolysis 1: 145
Gluconobacter oxydans 1: 80 green biorefiner concept 1: 253–294
glucosamine 1: 182 green biorefineries 1: 19, 24, 29–31
d-glucosamine, pyrroles synthesis 2: 25 – concept 1: 269–273, 296–297
glucose 1: 17, 2: 128–128 – ecological aspects 1: 283–285
– electrolytic reduction 2: 130 – economic aspects 1: 283–285
– microbial conversion 2: 274 – products 1: 31
– nitric acid oxidation product 2: 38 – raw materials 1: 258–269
– wood saccharification 1: 5 green chemistry, chlorophyll 2: 325–343
d-glucose green crop-drying plant 1: 270
– derivatives 2: 22 green crops, industrial use 1: 9
– one-pot conversions 2: 20 green cycle, sugar cane industry 1: 210–211
– reductive amination 2: 12 green harvesting residue material 1: 258
glucose fermentation, plant develop- green house gases 2: 402
ment 1: 134 green juice (GJ) 1: 30, 269, 271, 273–277
glucose-product family tree 1: 21–22 green leaf nutrient concentrate 1: 274
Subject Index 479
green natural gas 1: 103 – chemical composition 1: 360

green pellets, production amount 1: 260 – feedstock content 1: 359
green plant material, composition 1: 258 – history 2: 101
green plant parts, fractionation 1: 256 – hydrolysis 1: 362
greenhouse gas reduction 1: 62 – isolation 2: 119
greenhouse gases, emission 1: 197 – plant content 1: 261
gross visualization, phase separated prod- – quantities 1: 264
uct 2: 229 – removal 1: 199
ground water pollution, brown juice 1: 298 hemicellulose removal, advantages 2: 177–
growth, biorefining industry’s 1: 317 179
growth phase, fermentation 1: 218 hemicellulose-based product lines 2: 119
GTL technology 1: 158 hemicellulose concentrations, forage
guerbet alcohols 2: 311 grasses 1: 264
– synthesis 2: 312 hemicellulose content, stem tissue 1: 265
guinea-pig, protein-based polymer injec- hemicellulose polysaccharides, hydroly-
tion 2: 231 sis 1: 141
gum arabic, substitute 2: 62 herbaceous species 1: 50
GVGIP 2: 240 herbicidal treatment, highly selective 1: 150
– patches 2: 241 herbicides 1: 149
GVGVP 2: 223 – lignin-based dispersants 2: 193–194
– adhesions prevention 2: 238 heterocoupling, fatty acids 2: 267–269
– biocompatibility 2: 232 heteropolymeric sugars, hemicellulose
GVL 1: 151 1: 360
gypsum 1: 106, 2: 87, 386 hexadecanedioic acid, yield 2: 274
hexose sugars 1: 78
h HFCS 1: 351
Haarmann, W. 1: 7 HFRR 1: 153
hair, protection 2: 429–437 high frequency reciprocating ring test see
hair care 2: 434–436 HFRR
– ilex resin 2: 439–440 high-performance fiber 2: 41
hair conditioners, betaine derivatives high-value-added products, sugar-de-
2: 414 rived 2: 14–15
hair-setting agent 2: 415–422 high value pharmaceuticals 2: 40
hair surface, cationic compound deposi- higher-value products, levulinic acid 2: 39
tion 2: 413 HMF (hydroxymethylfurfural) 1: 130, 133,
hair swatches, standardized 2: 435 142, 2: 16, 133
Hale, W. J. 1: 9 – levulinic acid process 2: 100
half esters, homocoupling 2: 268 – manufacture 2: 131
Halobacterium sp NRC-1 1: 80 HMF based family tree 2: 135
2-halocarboxylates, copper-initiated addi- Holly 2: 437–438
tions 2: 262–263 holocellulose change, wet storage 1: 336
hammer mill 1: 176 homocoupling, fatty acids 2: 267–269
hardwood, composition 2: 106 homofermentative strain, Lactobacillus sali-
hardwood lignins 2: 154, 157 varius 1: 307
harvest cost 1: 333 hot-wash, pretreatment 1: 362
harvest transport 1: 338–339 3-HPA 2: 34–35
3-HBL see 3-hydroxybutyrolactone HTU process, liquid biofuels 2: 351
heating value, biofine char 1: 155 Humicola grisea var thermoidea 1: 77
Helianthus annuus 2: 277 Humicola insolens 1: 375
heliogerme 1: 179 Humicola 1: 202
hemicellulases 1: 364 hybrid processing, biomass 1: 227
hemicellulose 1: 72, 121, 198, 2: 104 hybrid thermochemical-biological process-
– accessibility 1: 55 ing 1: 227–252
480 Subject Index
hydration – basic biobased chemicals 1: 22

– microbial 2: 272–273 hydroxypropylation 2: 77
– repulsive free energy 2: 237–238 3-hydroxyvalerate see PHV
hydrocarbons
– apple-peel wax components 2: 433 i
– fossil 2: 6 IA see itaconic acid
– linear 1: 118 ideal elasticity 2: 219
hydrochloric acid, carbon hydrolysis 1: 131 – mechanism 2: 220
hydrogen bonding, cellulose 1: 360 ift gene 1: 398
hydrogenation reaction, syngas produc- IgG 2: 230
tion 1: 230 Ilex aquifolium 2: 437–438
hydrolases 1: 373–375 Ilex paraguariensis 2: 438
hydrolysability, biopol 2: 425 Ilex resin 2: 437
hydrolysis Ilex species 2: 437
– biomass 1: 129–138 imidazoles 2: 27
– furfural 2: 8 – hydrophilic 2: 27
– hemicellulose 1: 362 immobilization 1: 389
– starch 1: 5 – enzyme 1: 399–402
hydrolysis reactors, novel 1: 205 – lipase B 2: 256
hydrolytic enzymes, costs 1: 105 immobilization technology 1: 392
hydrolytic liquefaction 1: 123 immunoblot technique, western
hydrolyzate, biomass 1: 78 2: 230–231
hydrophilic imidazoles, d-fructose-derived immunoglobulin G 2: 230
2: 27 income generator 1: 96
hydrophilic side-chains 2: 24 inducing sugar 1: 201
hydrophilic/hydrophobic balance, sorbitan industrial biobased products 2: 359
esters 2: 11 industrial biomass 1: 13
hydrophobic association, Gibbs free energy industrial bioproducts, opportunities 2: 357
2: 218, 232 industrial biotech 2: 445–462
– input energy 2: 219 industrial chemicals
– inverse temperature transition 2: 219 – biomass-derived 1: 124, 2: 347–365
hydrophobic coating 2: 434 – fossil sources 1: 120
hydrophobic consilient mechanism 2: 222 – sustainable 1: 115–128
hydrophobic effect, comprehensive 2: 237 industrial concepts
hydrophobic hydration 2: 218 – biobased materials 2: 354–362
hydrophobic mechanism, protein-based – biomass 2: 347–365
polymer engineering 2: 217 industrial feedstock, baling 1: 333
hydrophobicity scale industrial product family, development
– Gibbs free energy 2: 234–235 1: 18
– prosthetic groups 2: 235–236 industrial products, biomass-based 1: 87
hydrothermal conditioning, granular industrial resources, historical 1: 4–8
starch 2: 78 industrial starch platform 2: 61–95
hydrothermolysis 2: 351 industrial uses, sugars 2: 7–14
hydroxy cyclic ester 2: 398 industries, fossil carbon-processing
hydroxyalkylation, starch 2: 80 1: 42–44
3-hydroxybutyrolactone (3-HBL) 2: 38, 40 infection, natural 2: 275
– basic biobased chemicals 1: 22 infrastructure investment 1: 340
hydroxymethylfurfural (HMF) 1: 78, 2: 16, inhibitors, enzyme activity 2: 322–323
133 initiators 2: 398
– formation 1: 143 injection processes, PHB 1: 216
– hydration 1: 143 injections, histological sections 2: 231
– lignocellulosic products 1: 25 innovation potential, fossil-based building
3-hydroxypropionic acid (3-HPA) 2: 34–35 blocks 2: 450
Subject Index 481
insulation materials, resins 2: 185 k

integrated biorefinery applications 1: 125 Kevlar 2: 46
integrated biorefinery process, detailed key chemicals, cellulose-based 2: 128
view 1: 102 key intermediates
integrated biorefining systems, sustainabil- – chlorophyll chemistry 2: 338
ity 1: 56–65, 60–65 – HMF 2: 133
integrated process chain approach, biomass key sugars 2: 3–59
processing 1: 98 – exploitation 2: 14
integrated processing facility 1: 45 Kirchhoff, G. S. C. 1: 5
integrated production, sugar 1: 209 Kirchoff 2: 62
intergeneric hybridization, plant breed- Klebsiella pneumoniae 2: 36
ing 2: 276 Klebsiella 1: 206
intermediate chemicals, HMF derived kojic acid 2: 20
2: 16 – glucose product family 1: 21
intermediate products, biorefineries 1: 46 Kolbe electrolysis 2: 267–269
intermediates 1: 386 Kraft black liquor 2: 175
– biofine process 1: 145 Kraft lignin, producers 2: 175
intermolecular order, proteins 2: 221 Kraft lignin recovery 2: 175
internal obstacles 2: 456 Kraft pulping 2: 175
intracellular poly(3-hydroxybutyric acid) Kraft pulping industry, lignin 2: 169–170
1: 218–219 Kyoto objectives, dutch 1: 85
intracellular reserve material, PHB 2: 424
intramolecular cyclization, addition 2: 263 l
inulase II gene 1: 398 LA see lactic acid, levulinic acid
inulin 1: 267 lactate ester, distillation 2: 389
– biocatalytic route 1: 397–402 lactic acid (LA) 1: 7, 11, 91, 2: 10–11, 382
– fructose source 2: 131 – biomass building blocks 1: 22
inulinase II, immobilization 1: 399 – composition 1: 308
inverse temperature transition – fermentation 1: 11, 298, 303–305, 305–
– hydrophobic association 2: 219 306, 378
– protein-based polymers 2: 227 – glucose product family 1: 21
– purification 2: 228–229 – manufacturers 2: 383
investment costs, poly(3-hydroxybutyric – production 1: 306, 312, 2: 382
acid) 1: 222–223 – sources 1: 296
investments, biomass supplies 1: 318 – usage 2: 10
Iogen’s demonstration process 1: 193 lactide, polymerization 2: 392–396
– schematic 1: 194 Lactobacillus buchneri, 1,2-propanediol
Iowa corn stover collection project 1: 319– 2: 37
321 Lactobacillus delbrueckii 2: 210
isoamyl alcohol, extraction of PHB 1: 219 Lactobacillus paracasei subspecies paracasei
isoascorbinic acid, glucose product fami- 1: 302
ly 1: 21 Lactobacillus plantarum 1: 302, 2: 273
isolation, lignin 2: 116 Lactobacillus salivarius 1: 305
isomaltulose, industrial production 2: 17 Lactococcus lactus 2: 210
isosorbide dinitrate 2: 14 laundry starches 2: 89–91
itaconic acid (IA) 2: 34, 36 lauric oils 2: 292
– basic biobased chemicals 1: 22 Lb salivarius BC 1001, fermentation
– glucose product family 1: 21 1: 299–300
LCA, polylactic acid 1: 284
j LCF see lignocellulosic feedstock
jet milling 1: 176 LCF-mannan 2: 120
jetcutting 1: 401 LCI, PLA 2: 402
juice fraction 1: 273–285 leaf dyes, first production 1: 257–258
482 Subject Index
leaf nutrient concentrate 1: 274 lignin-based product lines 2: 116–118

leaf protein concentrate 1: 254 lignin chemistry, biomass conversion
learning from nature, bionics 2: 410 2: 151–163
leaves, Ilex resin 2: 437 lignin content 1: 265
Leblanc, N. 1: 7 lignin isolation 2: 116
lecithin 2: 318 lignin polymer 2: 155
levoglucosan 1: 229, 243, 247 – growth 2: 154
levoglucosan hydrolysis, alternative 1: 245 lignin precipitation system 2: 171
levoglucosenone 2: 21 lignin processing 1: 194, 205–206
levuglucosan, biomass building blocks lignin production
1: 22 – historical outline 2: 168–172
levulinate esters 1: 153 – industrial 2: 165–200
levulinic acid (LA) 1: 6–7, 143–144, lignin products 2: 152
2: 38–39, 134, 361 – existing 2: 172–177
– basic biobased chemicals 1: 22 lignin recovery process 2: 171
– bromination 1: 150 lignin removal, advantages 2: 177–179
– catalytic hydrogenation 1: 151 lignin unit, different types 2: 156–159
– history 2: 100 lignocellulose 1: 74
– maximum theoretical yield 1: 145 – biorefinery 2: 111
– oxidation 1: 149 – enzymatic sequence 2: 210
– production 1: 139–164, 2: 111 – history 2: 102
– reaction to diphenolic acid 1: 148 lignocellulose-based chemical products
levulinic acid-based family tree 2: 135 2: 97–150
Lewis acid-induced cationic addition lignocellulose chemistry, historical out-
2: 264–265 line 2: 98–99
life cycle analysis 1: 57 lignocellulose structure 1: 121
life cycle assessment see LCA lignocellulose utilization
life-cycle inventory 2: 402 – industrial 2: 102
lignin 1: 7, 72, 121, 195, 2: 104 – technical aspects 2: 98
– antioxidant 2: 187–189 lignocelluloses 1: 10
– approximate composition 2: 154 – carbohydrates 2: 108
– biodegradation 2: 160 lignocellulosic, raw material 2: 103
– cell wall constituents 2: 151 lignocellulosic biomass, pretreatment
– chemical composition 1: 360 1: 361
– chemical linkages 2: 182 lignocellulosic biorefineries, PLA 2: 403
– commercially available 2: 176 lignocellulosic biorefinery 1: 115–128
– emerging markets 2: 194–198 – chemistry 1: 122–125
– feedstock content 1: 359 lignocellulosic feedstock (LCF) 1: 24, 125,
– gasification 2: 118 139–164
– history 2: 101 – biorefinery 1: 24–26, 129–138, 2: 111–113
– hydrolysis 2: 118 – chemical composition 2: 106–108
– Kraft pulping industry 2: 169–170 – conversion methods 2: 113–115
– markets 2: 166, 175, 181, 198 – definition 2: 103
– organosolv biorefinery 2: 179–181 – major groups 2: 103
– plant content 1: 261 – sources 2: 105
– press cake component 1: 279 lignocellulosic fractionation 1: 139–144
– purified 2: 167 lignocellulosic materials 1: 45, 105
– pyrolytic 1: 241 lignocellulosic technology, conventional
– soda pulping industry 2: 170–172 1: 146–147
– structural units 2: 105 lignosulfonate, dye dispersants 2: 191
– structure 2: 152–159 lignosulfonate producers 2: 173–174
– utilization 2: 117 lignosulfonates 2: 168–169, 172–175
– water-soluble 2: 189–194 – markets 2: 174
Subject Index 483
lignosulfonic acid, vanillin production 1: 7 lyondell propylene oxide, 1,4-BDO 2: 373

linear hydrocarbons 1: 118 lysine
linear PLA, melt rheology 2: 397 – biomass building blocks 1: 22
linear polymer, idealized structure 2: 49 – markets 2: 207
b-1 linkage, phenylpropane units 2: 159 lysine fermentation 1: 11
b-b linkage, phenylpropane units 2: 157 lysine preparations, commercially avail-
b-O-4 linkage, arylglycerol units 2: 156 able 2: 208
linseed 2: 281 lysine yield, C. glutamicum 2: 210
Linum usitatissimum 2: 277
lipase-catalyzed syntheses 2: 270–272 m
– carbohydrate esters 2: 272 MAAP 2: 201–202
lipase-catalyzed transformations 2: 270– – ecological impact 2: 207
272 – environmental and commercial considera-
lipid based bioproducts 2: 361 tion 2: 205–209
lipid layer enhancing effect, evaluation – technical constraints 2: 209
2: 310 MAAP processes
lipids 1: 7 – cultivation temperature 2: 213
– chemical composition 1: 361 – major steps 2: 208
lipotropic factor, betaine 2: 411 – nitrogen source 2: 211
liquefaction 1: 123, 126 macrocycle 2: 334
liquid biofuels 1: 49 macrocyclic ring system, reactions 2: 333
liquid epoxy polyol esters 2: 298 Madison-Scholler process 1: 132
liquid fuel production, missing part 1: 55 Maillard reaction 2: 24
liquid transportation fuels 1: 46 maize starch production 2: 66, 67
LNC, composition 1: 274 MALDI-TOF, polymer size 2: 229
load-and-go wagon 1: 320 maleic acid, conversion 2: 375
local ownership, biorefinery 1: 56 malic acid 2: 35
Lolium hybridum, press cake fibers 1: 281 – glucose product family 1: 21
Lolium multiflorum malonic acid, biomass building blocks
– alkanes 1: 268 1: 22
– silica 1: 268 maltol, glucose product family 1: 21
Lolium perenne 1: 261, 264 managing uncertainties, biotechnology
– alkaloid production 1: 277 2: 459
– alkanes 1: 268 mannan 2: 108
– amino acid composition 1: 267 mannan/mannose product lines 2: 119
– antifreeze protein 1: 269 d-mannitol 1: 267
– fructans 1: 267 margarine 1: 7
– minerals 1: 268 Marggraf, A. S. 1: 5
– silica 1: 268 market development, biotechnology 2: 460
– sugar 1: 265 market launch
– water-soluble carbohydrate 1: 266 – apple-peel wax 2: 436–437
low-cost production 2: 242 – biodegradable bottle 2: 427–428
low nutrient conditions, succinate fermenta- market potential 2: 446
tion 2: 370 – LA 1: 147
LPC 1: 268 – succinic acid 1: 149
– first industrial process 1: 256 market price, furfural 1: 154
– first production 1: 254–257 markets, lignin 2: 198
– quality 1: 258 mass spectra, polymer size 2: 229–230
LPS process 2: 171 material sources
lubricants, fatty acid esters 2: 299–301 – biomass-based 1: 380
lubricants industry, antioxidants 2: 188 – renewable 2: 355
lucerne, protein fractions 1: 275 materials design 1: 108
lyocell 1: 90 matrices, natural fibers 2: 295
484 Subject Index
matrix assisted laser desorption time-of- methyltetrahydrofuran see MTHF

flight spectrometry 2: 229 Michael addition 2: 81
MBTE 1: 357 Michaelis-Menten constant 1: 77
MDI 2: 299 Michaelis-Menten kinetics, enzymatic hydro-
mechanical pulping 1: 280 lysis 1: 204
mechanical separation, cereals 1: 26 microbial activity, wet storage 1: 334
mechanistic foundations 2: 217–251 microbial amino acid production see MAAP
media cost 2: 370 microbial bioconversions, milling byprod-
Medicago sativa L 1: 255 ucts 1: 172
– press cake fibers 1: 281 microbial biomass 1: 106
medical grade purity 2: 230 microbial biosynthesis 1: 104
Mellier, M. A. C. 1: 6 microbial conversions
Melsens, G. F. 1: 5 – oils/fats and glucose 2: 274
melt, polylactic acid synthesis 2: 391 – six-carbon sugars 2: 32–34
melt rheology – sugar 2: 30
– branched PLA 2: 397 – sugar-based 2: 36–37
– linear PLA 2: 397 microbial fermentation, drying bales
melt stability, PLA 2: 399 1: 321
melting enthalpy, PLA 2: 401 microbial oxidation, fatty acids 2: 273–274
membrane electrodialysis 1: 106 microbial polyesters 2: 44–45
membrane process, lactic acid purifica- microbial transformations 2: 272–274
tion 1: 312 microfibril 1: 195
metabolic engineering 2: 204 microorganisms
metabolic flux distributions, C. glutami- – acetyl-coa forming 1: 233
cum 2: 205 – commercial lactic acid production
metabolic pathways 2: 384–385
– optimize 2: 212 – important 1: 80
– PHB synthesis 1: 238 – usage 1: 146
– syngas fermentation 1: 234 middlings 1: 170
metal-based catalysts 1: 228, 233 mill water 1: 348
metal catalysts, aqueous phase hydrogena- milled wood lignin 2: 155
tion 2: 375 milling
metal complexes, chlorophyllin 2: 335 – industries 1: 345–353
methanation, syngas production 1: 231 – pretreatment 1: 361
methane, glucose product family 1: 21 – process flow diagrams 1: 347
methanol, glucose product family 1: 21 milling byproducts, wheat flour
methanol synthesis, syngas 1: 158 1: 169–173
methyl 17-octadecanoate, cyclization 2: 259 milling efficiency, increase 1: 173
methyl 2-iodopetroselinate, radical cycliza- milling operations 1: 166
tion 2: 263 minerals 1: 268
methyl conjugate, Diels-Alder reaction – analysis 1: 299
2: 260–261 – brown juice content 1: 304
methyl elaidate, enantioselective oxida- Mitscherlich, A. 1: 8
tion 2: 258 mix-polymerization, starch 1: 27
methyl epiminooctadecanoate, synthesis mixed sugars 1: 98
2: 257 model building block, succinic acid
methyl oleate 2: 367–379
– co-metathesis 2: 260 modeling, ecosystem 1: 57–60
– oxidative cleavage 2: 258 modern corn refinery 1: 348–350
methyl tertiary butyl ether 1: 357 molasses 1: 222
1-methylamino-1-deoxy-d-glucitol 2: 11 molasses fermentation 1: 131
methylene di(phenylisocyanate) 2: 299 mold temperature, PHB 1: 216
methylglucoside, synthesis 2: 131 molecular weight, rheology control 2: 396
Subject Index 485
monitoring technologies, toxicity 2: 212 natural fibers 1: 90

mono-septic operation 2: 209 natural oils
monocyclic aromatic hydrocarbons, biorefin- – formaldehyde additions 2: 264
ery by-product 2: 29 – improvements 2: 275–281
monoglycerides, lipase-catalyzed synthe- – industrial processing 2: 295
ses 2: 270–272 – polymer building blocks 2: 296
monomer genes natural substance, definition 2: 422
– concatenation 2: 226 natural vector transformation systems
– preparation 2: 225 2: 275
– production 2: 226 NatureWorks 2: 10
monomers 1: 46 near ideal elasticity 2: 219
– biorefinery 2: 315 – mechanism 2: 220
– quasi-aromatic 2: 46 net corn cost 1: 50
monosaccharide production 1: 180 network polymer, lignin 1: 121
monosaccharides neutralizing agent, lactic acid 2: 385
– availability 2: 4–7 nitrocellulose, history 2: 99
– conversion 2: 28 nitrogen leaching 1: 63
MSW Management, coupling with fuel pro- nitrogen source 2: 211
duction 1: 126 NMGA 2: 11–12
MTBE 1: 71 NMP 2: 373, 375–376
MTHF 1: 150, 2: 135 – rhodium catalytical production 2: 377
– formation from LA 1: 152 NMR, purity 2: 229
mulch till 1: 329 Nocardia cholesteriolicum 2: 273
Mulder, G. J. 1: 6 non-carbohydrate natural products, synthe-
multi-cyclic carbonate comonomers 2: 398 sis 2: 20
multi-cyclic epoxy comonomers 2: 398 non-food products
multi-cyclic ester comonomers 2: 398 – manufacture 1: 165–191
multi-functional polymerization initiators, – renewable resources 1: 11
branching 2: 398 non-food uses, sugars 2: 3–59
multi-quality biomass 1: 92 non-recyclable organic solid waste materials
multifunctional care additives 2: 309 see NROSW
multifunctional compounds 2: 135 non-starch polysaccharides 1: 175
multimer genes 2: 226 non-wood fibers 1: 280
multiple feedstock capability 1: 68 nonactivated C–H bonds, oxidation 2: 269
municipal solid waste 1: 116–117 nonhydrolytic proteins 1: 374
– management 1: 125 nontraditional microorganisms 1: 80
mutagenesis 1: 75–77 Normann, W. 1: 7
mutated spores, fermentation 1: 75 novel fatty acids synthesis, starting materi-
MWL 2: 155 als 2: 255
novel plastics, 1,3-propanediol 1: 182
n novolacs 2: 181
N-heterocycles, sugar-derived 2: 24 novozym 435 2: 256
Naegeli 2: 62 novozymes 1: 77
naltrexone, controlled-release devices NREL 1: 19, 22, 72, 74, 150
2: 240 NROSW 1: 126
naphtha 1: 86 NSP 1: 175
naphthalene sulfonate, dye dispersants nuclear magnetic resonance 2: 229
2: 191 nucleophilic addition, unsaturated fatty
naphthenic compounds 1: 118 acids 2: 265
National Farm Chemurgic Council 1: 9 nucleus exchange method, lignin 2: 154
National Renewable Energy Laboratory pro- nutrient replacement 1: 324–325
cess see NREL nutrients 2: 388
natural lignin, recovery 2: 179 – lactic acid 2: 385
486 Subject Index
– oat 1: 183 – analysis 1: 299

– replenishment 1: 327 – brown juice content 1: 304
– wheat 1: 168 – commercially important 1: 79
nutritional value, re-growth 1: 261 – production 1: 234–235
nylon 6, markets 2: 113 organic chemicals
nylon-6,6 2: 45 – bioproduction 1: 182
nylon process, furfural-based 2: 123 – fossil sources 1: 120
– industrial 1: 115–128, 124
o – levulinic acid 2: 134
oat based biorefinery, schematic 1: 184 – renewable carbon 1: 44
oat bran-rich fractions, value-added byprod- organisms, engineered 1: 68
ucts 1: 185–187 organization infrastructure 1: 340
oat composition 1: 183 organosolv biorefinery, lignin 2: 179–181
oat gum 1: 185 organosolv lignin, products 2: 183
occurrence, betaine 2: 410–411 organosolv pretreatment, lignin 2: 178
OFP 1: 151 oseltamir phosphate, synthesis 2: 30
oil 1: 98 oxalic acid 2: 99
– microbial conversion 2: 274 oxidation 2: 254–258
– new syntheses 2: 253–289 – enantioselective 2: 258
– thermochemical conversion 2: 361 – fatty compounds 2: 257–258
oil and lipid-based bioproducts 2: 356 – selective 2: 38
oil-based surfactants 1: 90 – starch 2: 79
oil crisis 2: 348 b-oxidation, fatty acids 2: 273–274
oil fruits, FAS 2: 304 x-oxidation, fatty acids 2: 273–274
oil industry, sections 1: 86 oxidation technology, development 2: 38
oil-like proteins, repulsion 2: 218 oxidative cleavage 2: 258
oil production, permanent decline 1: 42 – transition metal-catalyzed 2: 258
oil qualities 2: 276–277 oxidative coupling 2: 266
oils and fats, world production 2: 292 oxidative enzymes, biodegradable plas-
oilseeds 1: 45 tics 1: 213
olefin, metathesis 2: 259–260 oxidative metabolism, phosphorus sup-
olefinic polymers, sugar-based 2: 47 ply 2: 211
olefins, monosaccharide-derived 2: 47 oxidative polymerization, lignin polymeriza-
oleic acid 2: 254 tion 2: 153
oleochemical base materials 2: 294 oxidative states, changes 2: 236
oleochemical-based dicarboxylic acids 4-oxopentanoic acid 1: 6
2: 296 oxygen supply 2: 212
oleochemical industry 1: 122 ozone, cleavage of fatty compounds 2: 258
oleochemicals ozone-forming potential, P-Series fuels see
– biobased 2: 291–314 OFP
– polymer applications 2: 295
one-pass collection 1: 333–335 p
one-pass harvest 1: 332 P-Series fuels 1: 151
one step biochemical modification, naturally Pachysolen tannophilus 1: 147
produced structures 2: 349 Pacific Northwest Laboratory see PNL
one-way cycle, oil and gas feedstock 2: 449 Pacific Northwest National Laboratory see
OP 2: 395 PNNL
– PLA 2: 401 palm kernel oil 2: 292
operating costs panel binders 2: 185
– biofine plants 1: 160 panelboard adhesives 2: 183–184
– biorefinery 1: 240 paper
optical purity 2: 395 – adhesion 2: 87
organic acids 1: 78 – from press cake fibers 1: 282
Subject Index 487
paper industries, starch usage 2: 83 – dependence 1: 115

paper mill waste 1: 134 – structural shift 1: 116
parasorbic acid, glucose product family petroleum-based pathways, polyamides
1: 21 2: 45
partial glycerides 2: 270 petroleum chemistry, comparison with bio-
particle size, feedstock materials 1: 144 mass 1: 118–122
paste reactions, starch modifications 2: 77 petroleum costs 1: 48–50
pasture lands 1: 52 petroleum dependence, reduction 1: 71
patents petroleum feedstocks 1: 45
– protein-based polymers 2: 245–249 petroleum refineries 1: 16
– reexamination request 2: 245–249 petroleum refining industry, develop-
Payen, A. 1: 6 ment 1: 41
PC 1: 30, 269, 271 petroleum reserves, prognoses 1: 387
– downstream processing 1: 281 petroporphyrin formation 2: 332
PCB, lignin containing 2: 194 petroporphyrins 2: 331–332
PCR technique 2: 225 PF 2: 181
PCS-hydrolyzing cellulases, improve- PF resins, markets 2: 183
ments 1: 367 pH adjustment 1: 79
PD 1: 393–397 PHA 1: 182, 214, 236, 239, 2: 44
PDLA 2: 395 – accumulation 1: 236
PDO 1: 11 pharmaceuticals 1: 13, 2: 14–15
peanut 2: 281 – intermediate 2: 40
pearl corn starch, carbohydrate refining – preparation 2: 26
1: 351 – purification target level 2: 230
pearling 1: 173–176 – starch usage 2: 88–89
– oat grain 1: 183 phase III-biorefineries 1: 19–20
pectin substances 1: 265 phase separated product, gross visualiza-
Penicillium 1: 202 tion 2: 229
pentaerythritol esters 2: 308 phase separation, purification 2: 228
2,3-pentane dione, glucose product phase transition, Gibbs free energy 2: 234
family 1: 21 PHB 1: 209, 238
“pentanes-plus” 1: 151 – chemical structure 1: 214
pentosan change, wet storage 1: 336 – copolyesters 1: 215
pentosans, conversion 2: 28 – intracellular reserve material 2: 423
pentose fermentation 1: 206 – lifetime of products 1: 214
pentose sugars 1: 78 – synthesis 1: 237–238
pentoses 1: 91 – yield determination 1: 238
– conversion 2: 28 PHB-PHV copolymer, brittleness 2: 426
peptide sequences, repeating 2: 217 phenol–formaldehyde resin 2: 181
Peptostreptococcus productus 1: 235 phenol–formaldehyde resin markets,
perfluoroalkyl iodides, addition 2: 263–264 lignin 2: 187
perfluoroalkylated products, synthesis phenolic acids 1: 178
2: 263 phenolic–carbohydrate complexes, Lolium
performic acid procedure 2: 254 perenne 1: 264
pericarp 1: 183 phenolic molding compound market
– wheat 1: 167 2: 184
pericyclic reactions 2: 260–261 phenolic resins 2: 16, 181, 185
pesticides, lignin-based dispersants 2: 193 – biorefinery lignin 2: 181–183
PET 2: 133 phenomenological axioms, engineering pro-
petrochemical industry 1: 86 tein-based polymers 2: 232–234
– transformation steps 1: 88 phenyl-propanoid units, crosslinked 2: 181
petrochemical technology 2: 373 phenylpropane units 2: 157, 159
petroleum – bonding 2: 153–156
488 Subject Index
phloroglucinol, biosynthesis 2: 30 plasticizer, biodegradable bottle 2: 427

phospholipids 1: 361 plastics
phosphorus source 2: 211 – biodegradable 1: 182, 212–216
phosphorylation, changes 2: 236 – novel 1: 182
photochemical gas-phase chlorination platform chemical 1: 147
2: 269 platform molecules 1: 182
photodynamic therapy, chlorophyll deriva- PLLA 2: 395
tives 2: 336 plug-flow reactor 1: 144
photosensitizer, chlorophyll 2: 334 PNL process 1: 151–152
photosynthesis 1: 12, 42 PNNL 1: 22
photosynthesis enzyme, plant content poly(3-hydroxybutyric acid) polymer
1: 255 1: 214–216
photosynthetic bacteria 1: 229 poly(hydroxyalkanoate) production, future
photosynthetic pigments 1: 257, 2: 326 milestone 1: 224
phthalo green 2: 338 poly(hydroxyalkanoates) 1: 238, 2: 44
PHV 1: 237, 2: 426 poly(3-hydroxybutyrate-co-valerate), enzy-
phylogenetic tree, gene sequences 1: 367 matic digestion 1: 219
phytic acid 1: 170 poly(hydroxybutyrate)
phytochemicals, biorefinery context – monomer 1: 237
2: 315–324 – processing 1: 215–216
phytoestrogens 2: 321–322 – switchgrass 1: 283
phytosterols 2: 317–318 poly(3-hydroxybutyric acid-co-3-hydroxyvale-
Pichia yeast 1: 206 ric acid) 1: 214–215
Picrophilus torridus 1: 80 poly(3-hydroxybutyric acid) 1: 212–213
pigments – biosynthesis 1: 224
– biorefinery context 2: 315–324 – downstream processing 1: 218–219
– carotenoids 2: 320 – production process 1: 217–223
– chlorophyll 2: 336 – sugar fermentation 1: 217
pilot plants, biomass fermentation 1: 135 poly-b-hydroxy butyric acid 2: 423
Pirie, N. W. 1: 9 poly(lactic acid) 1: 8, 296
PLA 2: 10–11, 41, 381 poly(tetramethylene ether glycol) see
– biobased 1: 296–299 PTMEG
– high polymer 2: 391 poly(vinyl chloride), cements see PVC
– melt rheology 2: 397 polyamides 1: 122, 2: 45–47
– production 2: 390–396 polyesters
– properties 2: 400 – fiber 2: 36
– resins 2: 400 – furan containing 2: 44
– semi-crystalline 2: 394 – microbial 2: 44–45
– stereocomplex 2: 401 – production 1: 236–239
plant breeding, oil improvement polyetherpolyols, biodegradable 2: 9
2: 275–281 polyglucaramides, stereoregular 2: 46
plant cuticle, schematic 2: 431 polyglycerol ester, emulsifier 2: 308
plant development, biomass hydrolysis polylactic acid 2: 10, 41
1: 129–138 – non-solvent process 2: 392
plant infection, fungal endophytes 1: 277 – polymerization routes 2: 391
plant material – production capacity 1: 284
– usage 1: 90 – renewable resources 2: 381–407
– yearly amount 1: 43 polymer building blocks, natural oils
plant resources 2: 353 2: 296
plant usage, historical 1: 254 polymer development, protein-based
plasma cholesterol, reduction 2: 317–318, 2: 221–222
321 polymer industry, starch 1: 28
plasticization, starch 1: 27 polymer size, mass spectra 2: 229–230
Subject Index 489
polymerase chain reaction 2: 225 – solvent-based 1: 200

polymeric materials, protein-based – straw 1: 193
2: 220–221 price changes, external challenges 2: 457
polymeric products, polymeric lignin price difference, fossil fuel feedstocks
2: 160 2: 446
polymerizable sugar derivatives 2: 40–47 price swings, oil 1: 48, 52
polymerization initiators, multi-func- prices, renewable carbon feedstock 1: 50
tional 2: 398 primary antioxidants 2: 187
polymers 1: 46 primary conversion technologies 1: 270,
– biobased 1: 11 2: 350
– oils and fats 2: 291 primary refinery 1: 269, 272
– oleochemicals 2: 295 – wet fractionation 1: 271–273
– PHB 1: 209 primary starch 2: 85
– protein-based 2: 217–251 primary streams, raw materials 1: 92
polyol esters 2: 307 prime starch 2: 68
polyols, epoxides based 2: 298–299 printed circuit boards 2: 194
polyoses, history 2: 101 process economics 1: 53
polypeptide, protein definition 2: 217 process optimization, lignocellulose-based
polysaccharides 1: 89, 121, 277 operation 2: 203
– acid hydrolysis 1: 141–142 process scheme, integrated 1: 127
– repeating units 2: 4 processing technologies, neccessary for bio-
polytrimethyleneterephthalate see PTT refineries 1: 46
polyurethane foams, production 2: 9 processor value, stover 1: 327
polyurethane stretch fibers 1: 149 product development, biodegradable bot-
polyurethanes, oleochemical building tle 2: 426–427
blocks 2: 298 product diversification, biomass 1: 54–55
polyvinylsaccharides 2: 47 product family tree 2: 97–150
pomace 2: 432 – amino acid-based 2: 201–216
porous carbon fibers 1: 283 – glucose 1: 21–22
potato 2: 69–70 – hemicellulose-based 2: 121
potato juice 1: 309–310 – HMF and levulinic acid-based 2: 136–138
– lactic acid source 1: 295 – lignin-based 2: 117–118
– quality 1: 300 – syngas 1: 33
potato starch crystals 2: 73 product flow-chart, biobased 1: 23
potato starch industry, lactic acid producer product innovation, biotechnology 2: 450
1: 310 product integrity, verification 2: 229–230
potato starch production 2: 69 product lines
potential future market, formic acid 1: 154 – biobased 1: 375
potential screening 1: 22–23 – carbohydrate-based 2: 3–59
power technologies, renewable 1: 139 – hemicellulose-based 2: 119
precursors, biomass targets 1: 17 product spectrum, chemical compounds
preprocessing, biomass 1: 46 1: 105–106
press cake 1: 257, 269 product yield 1: 53–54
press cake fibers, basic properties 1: 281 prognoses 1: 387
press-cake fraction 1: 278–285 propanediol, glucose product family 1: 21
press juice 1: 257 1,2-propanediol, racemic form 2: 37
pressure ulcers, prevention 2: 240 1,3-propanediol 2: 36
pretreatment 1: 135, 198–200 propionic acid, biomass building blocks
– biomass 1: 107 1: 22
– cornstover 1: 245 propylene, glucose product family 1: 21
– dilute acid 1: 246 prosthetic groups, hydrophobicity scale
– LCF 2: 113 2: 235
– lignocellulosic biomass 1: 361 protease inhibitors 2: 322
490 Subject Index
protective action, hair keratin 2: 436 – protein-based polymers 2: 227–230

protective film, skin care 2: 439 purity
protein-based polymers 2: 217–251 – evaluation 2: 229
– charged side chains 2: 240 – medical grade 2: 231
– development 2: 221–222 PVC 1: 149
– elasticity 2: 219–220 PVC stabilizers, vegetable oil epoxides
– engineering 2: 217, 232–238 2: 256
– expression 2: 227–230 PX (protein-xanthophylls), production num-
– gene constructions 2: 242–245 bers 1: 271
– materials 2: 220–221 pyran, building blocks 2: 21
– order 2: 219 pyranoid, building blocks 2: 23
– patents 2: 245–249 pyranoid sugar derivatives 2: 48
– purification 2: 227–230 pyrazoles 2: 26
protein content, comparison between plants 3-pyridinols 2: 28
and animals 1: 255 pyrogallol 2: 30
protein–fatty acid condensates 2: 304 pyrolysis 1: 123, 230
protein gels, cellulolytic fungi 1: 372 – bioproducts 2: 362
protein generation, ethanol 2: 133 pyrolysis oil 1: 98
protein-hydrolyzates, extraction 2: 202 – production 1: 244
protein line 2: 201–216 pyrolysis products, cornstover 1: 246
protein repulsion 2: 218 pyrolytic char 1: 247
protein–xanthophylls 1: 271 pyrolytic lignin 1: 241
proteins 1: 46, 122, 268, 277, 2: 217 pyrolytic liquid, yield 1: 241
– acylated 2: 304 pyrones 2: 20
– analysis 1: 299 pyrroles 2: 24
– aqueous media 2: 218 pyrrolidone solvents, manufacture 1: 149
– biomass conversion 1: 371–375
– brown juice content 1: 305 q
– chemical composition 1: 361 quasi-aromatic monomers, sugar-based
– crude starch milk 2: 66 2: 46
– juice fraction 1: 274 quinoxalines 2: 28
– potato starch production 2: 70 – sugar derivative 2: 24
– thermodynamics 2: 218
protolignin 2: 152 r
protonated glycoside, cellulose hydroly- racemic mixture, lactic acid 2: 382
sis 1: 141 radical additions 2: 261
Pseudomonas fluorescens 2: 30 – malonic acid 2: 261
Pseudomonas putida 1: 245, 2: 37 – perfluoroalkyl iodides 2: 263
PTMEG 1: 149 radical C–C coupling 2: 266–269
PTT production 1: 393–396 radical cyclization, methyl 2-iodopetroseli-
pulp 1: 6 nate 2: 263
pulping rail transport, feedstock 1: 339
– environmentally friendly 2: 179 Ralstonia eutropha 1: 217, 237
– mechanical 1: 280 ranitidine 2: 14
– semi-chemical 1: 280 rapeseed 2: 277
pure chemicals, xylan derived 2: 122 rapeseed oil, industrial use 2: 280
purification 1: 218–219, 2: 388 rapid pyrolysis 1: 227
– chitin 2: 416–417 – cellulose 1: 243
– inverse temperature transition 2: 228– rate of biodegradation 1: 213
229 raw material costs 1: 53
– lactic acid 1: 312 raw materials
– phase separation 2: 228 – appropriate 1: 165
– PHB 1: 218 – aromatic compounds 2: 259
Subject Index 491
– biomass 1: 12–14 resin fraction, holly 2: 438–439

– biorefineries 1: 45–47 resin industry 2: 182
– costs 2: 110 resins
– oleochemicals 2: 292–293 – PCB 2: 194
– renewable 2: 253–289 – thermoset 2: 184
– sterilization 2: 209 resols 2: 181
– world market prices 2: 356 retroaldolization, imidazoles 2: 27
RBAEF Project 1: 43, 52 retrogradation 2: 75
reaction system costs 1: 53 reversed-polarity unsaturated fatty acids,
reactive sites, triglycerides 2: 294 nucleophilic addition 2: 265
recombinant DNA technologies RFG 1: 151
– application 2: 204 rheological properties
– gene construction 2: 225–227 – b-glucan 1: 185
– protein-based polymers 2: 217 – NSP 1: 175
recovery 1: 218–219 rheology control 2: 396
recycling 1: 106 Rhizomucor miehei 2: 271
– plastics 1: 212 Rhizopus arrhizus 2: 35
refined biomass 1: 98 rhizopus-based fermentation 2: 388
refineries rhodium catalyst, NMP production 2: 377
– hybrid biomass processing 1: 227–252 Rhodopseudomonas gelatinosa 1: 237
– thermochemical 1: 101–103 Rhodospirillum rubrum 1: 237, 239
refinery economy 1: 350 Rhodospirillus rubrum 1: 229
refining, biomass 1: 41–66, 107 ribulose 1: 255
reformulated gasoline see RFG rice, starch production 2: 71
regioselective syntheses, ricinoleic acid rice straw, world production 1: 51
2: 254 ricinoleic acid 2: 254
re-growth, nutritional value 1: 261 ridge-till 1: 329
regulatory framework, biotechnology right opportunities, biotechnology 2: 458
2: 447 ring-opening
regulatory situation, external challenges – chlorophyll 2: 330
2: 457 – nucleophilic 2: 257
reinforcers, lignin 2: 186 ring-opening polymerization 2: 394
renewable carbon feedstock prices 1: 50 ring structures, saccharides 1: 121
renewable energy law 1: 15 rings, lignin polymer 2: 155
renewable material, definition 2: 422 Ritter, E. A. 1: 323
renewable-power technologies 1: 139 rocket fuels 2: 37
renewable raw materials 2: 355 Role of Biomass in America’s Energy Future
– oils and fats 2: 253–289 see RBAEF Project
– optimized by breeding 2: 277–281 ROP 2: 394
renewable resources 2: 347 Rothamsted process 1: 256
– industrial conversion 1: 5 Roulle, H. M. 1: 254
– integrated utilization 1: 10–11 rubber industry, antioxidants 2: 188
– non-food products 1: 11 rubber processing, lignin 2: 186
– polylactic acid 2: 381–407 rubisco 1: 274
– prognoses 1: 387 – plant content 1: 255
– sources 1: 385 Rubrivivax gelatinosus 1: 236–237
repulsive free energy 2: 222 rye grasses, digestibility 1: 261
– apolar–polar 2: 218 ryegrass, fiber properties 1: 281
– hydration 2: 237
residual biomass, importance 2: 452–457 s
residual sugars, separation 2: 388 saccharides 1: 121
residue utilization 1: 283–285 saccharification 1: 32, 2: 128, 177–179
resin binders 2: 183 – cellulose 2: 99
492 Subject Index
– wood 1: 5–6 signal peptide effect 1: 375

Saccharomyces 1: 194 silage additive, formic acid 1: 153
Saccharomyces cerevisiae 1: 7, 146, 2: 209 silage juice 1: 276–277
Saccharomyces yeast 1: 206 silage residues, reusage 1: 283
saccharose, fructose source 2: 131 silage wet-fractionation, primary refin-
Saccharum officinarum 1: 210 ery 1: 271
salt splitting technology, lactic acid 2: 387 silica 1: 268, 277
saponins 2: 321–322 silicon carbide, rye grass 1: 278
satake pearling system 1: 174 simultaneous saccharification and fermenta-
saturated fatty compounds, reactions tion 1: 134
2: 266–270 sitosterol 2: 317
SAXS, lamellar thickness 2: 395 six-carbon sugars, microbial conversion
scaffoldings, temporary functional 2: 239 2: 32–34
scenarios, intgeration of industries 1: 94 sixth framework program, EU 1: 103
Scholler process 1: 131 size-exclusion chromatography 2: 72
Scholten, W. A. 2: 62 sizing agents 2: 85
screening 1: 22–23, 77 skin, protection 2: 429–437
screening methods 1: 75–76 skin care, ilex resin 2: 439
scutellum, wheat 1: 169 skin cosmetics 2: 434
SDS–PAGE 2: 228 slaughterhouse wastes, byproducts 1: 100
– purification 2: 228 slurry process, starch modifications 2: 76–
SEC 2: 72 78
second-grade starch 2: 68 small-angle X-ray scattering 2: 395
secondary biochemical refinery 1: 104–106 small-scale extractions, chlorophyll 2: 329
secondary biorefining processes, thermo- smell, obnoxious 1: 310
chemical 1: 103 soap 2: 409
secondary starch soap production, history 1: 7
secondary streams, raw materials 1: 92 soda process, lignin 2: 176
secretome, cellulolytic fungi 1: 371–373 soda pulping industry, lignin 2: 170–172
sectoral integration, bio-based industry sodium dodecyl sulfate polyacrylamide gel
1: 93–96 electrophoresis 2: 228
seed, wheat 1: 167 sodium lactate, salt splitting 2: 387
selective oxidation, carboxylic acids 2: 38 soft tissue augmentation 2: 238
self-leveling concrete 2: 87 soft tissue reconstruction 2: 239
semi-chemical pulping, lignin extraction soft tissue restoration 2: 238
1: 280 softwood, composition 2: 106
separation of biomass, technically feasible softwood lignins 2: 157
1: 17 soil bioactivators, grass juices 1: 283
separation system costs 1: 53 soil carbon equilibrium 1: 325
sequence integrity, evaluation 2: 229 soil carbon loss 1: 328
sequence verification 2: 226 soil coverage, stover 1: 329
sequenced genomes, microorganisms 1: 80 soil erosion control 1: 329
sequestration, carbon 1: 62 soil organic material 1: 328–329
serine, biomass building blocks 1: 22 soil organic matter see SOM
shampoo bottle soil quality 1: 324
– Biopol 2: 422 – models 1: 324
– degradation 2: 425 solid state bioprocessing see SSB
– market launch 2: 427 solubilization, cellulose 1: 359
Shell, transition process 1: 93 solubles removal, wet storage 1: 336
shellfish industry, chitin source 2: 416 solvent, selective 2: 9
shikimic acid, metabolic engineering 2: 31 solvent extraction 1: 219–221, 2: 388
side-chain, oxidativ shortening 2: 25 SOM, loss 1: 324, 328
side-streams, fermentation 1: 106 sorbic acid, glucose product family 1: 21
Subject Index 493
sorbitan esters 2: 11, 272 starch modification, types 2: 81

sorbitol 2: 129–130 starch modification technology 2: 76
– basic biobased chemicals 1: 22 starch platform, industrial 2: 61–95
d-sorbitol 2: 9 starch saccharification 2: 178
– dehydration 2: 11 starch water, modification 2: 76–81
Sorghum dochna 1: 255 steam 1: 46
sorona 2: 41 steam-alkaline pulping, lignocelluloses
sovermol 2: 299 2: 114
soybean 2: 277 steam explosion 1: 280
– phytochemicals 2: 317 – pretreatment 1: 198
– processing 2: 316 steam gasification 1: 156
– saponins 2: 321 stearic acid, photochemical gas-phase chlori-
spandex 1: 149 nation 2: 269
special ingredients 2: 315–324 steep liquor 1: 349
special sugars, juice fraction 1: 274 steeping, corn 1: 348
specialties 1: 386 steepwater 1: 349
specialty chemicals, bio-based 1: 91 stereoregular polyglucaramides 2: 46
Spirulina, chlorophyll extraction 2: 329 stereoselective syntheses, ricinoleic acid
spores, mutated 1: 75 2: 254
SSB, fungal 1: 172 steric hindrance, enzymatic hydrolysis
SSF 1: 71, 146, 203 1: 147
– process 1: 79 sterigel 1: 178
starch 1: 27, 67, 70–71, 121, 181, 268 sterols, soybeans 2: 317
– acetylated 2: 80 storage 1: 334
– bioconversion 2: 89–91 – bagasse 1: 321
– chemical composition 1: 360 – baling dry material 1: 332
– chemical source 1: 50 – brown juice 1: 298
– commercial 2: 71–76 – potato juice 1:
– common sources 2: 62 storage area, square bales 1: 335
– composition 2: 74 storage investment cost 1: 337
– corn refinery products 1: 348 storage loss 1: 335
– degraded 2: 79 storage polymer, PHA 1: 239
– ethanol raw material 1: 197–198 stover, economic benefit 1: 325
– glucan source 1: 139 stover field value 1: 326
– history 2: 61 stover revenue, farmers income 1: 319
– industrial production 2: 65 strain development 2: 371
– modification 2: 61–95 straw
– nitric acid oxidation product 2: 38 – baling 1: 333
– production 2: 61–95 – LCF biorefinery 1: 26
– properties changes 2: 78 – world production 1: 51
– quality 2: 73 straw species 2: 107
– raw materials composition 2: 65 straw waste, wood saccharification 1: 10
– syrups 2: 89 Streptomyces setonii 1: 179, 245
– tailor-made 2: 92–93 strip till 1: 329
– total consumption 2: 82 strong acid addition, lactic acid 2: 386
– world market 2: 64 structural features, lignin 2: 155
– yield 2: 69 structure-based design, enzyme improve-
starch-based biorefinery II 1: 69 ment 1: 369
starch derivatives 2: 82–91 structure–function relationship, EG 1: 370
starch ethers, building chemistry 2: 87 styling, ilex resin 2: 440
starch–gluten slurry, corn refinery 1: 349 substance classes, apple-peel wax 2: 433
starch granules, reshaping 2: 75 substrate recalcitrance 1: 204
starch hydrolysis 1: 5 succinate fermentation 2: 369
494 Subject Index
succinate strain FZ 21 2: 371 – sulfite pulp process 1: 8

succinic acid 1: 149, 2: 35 sugar mill
– catalytic transformations 2: 372 – Brazil 1: 210
– conversion 2: 375 – poly(3-hydroxybutyric acid) production
– derivatives 2: 373 1: 221
– fermentation 1: 378, 2: 369–372 sugar platform 1: 32
– model building block 2: 367–379 – intermediate 1: 31
– wheat flour milling byproducts 1: 172 sugar production 1: 5
sucrose 1: 70 sugar residues, Lolium perenne 1: 264
– catalytic oxidation 2: 39 sugar syrup 1: 222
– conversion 2: 18 sugar transformations, prototype 2: 19
– ethanol raw material 1: 197–198 sugarcane bagasse 1: 74
sucrose-6,6'-dicarboxylic acid 2: 46 sulfite pulp process, historical improve-
sucrose-based biorefinery I 1: 68 ment 1: 8
sucrose fatty acid monoesters 2: 13 sulfite pulping industry 2: 189
sugar 1: 209 – lignosulfonates 2: 168–169
– analysis 1: 299 sulfite pulping process 2: 172
– bulk-quantity prices 2: 4 Sulfolobus sulfataricus P2 1: 80
– chemical conversion 2: 37–40 Sulfolobus tokodaii strain 7 1: 80
– contained in biomass 2: 3–59 sulfonated Kraft lignin, dye dispersants
– fermentable 1: 68 2: 191
– fermentation problems 1: 146 sulfur, lignin structure 2: 171
– increasing demand 1: 67 sulfur-bearing gases, catalyst poisoning
– juice fraction 1: 274 1: 234
– mixed 1: 98 sulfur-containing components, removal
– non-food industrial uses 2: 7–14 2: 350
– nonionic surfactants raw materials 2: 306 sulfur emissions, diesel 1: 153
– plant contents 1: 265 sulfuric acid, hydrolysis of cellulose
– simple 2: 5 1: 130
– thermochemical conversion 2: 360 sunflower 2: 280
– yield 1: 130 surface cover, fields 1: 324
sugar acids, chemical route 1: 402–405 surfactants
sugar and starch bioproducts 2: 356 – carbohydrate-based 2: 305
sugar-based biorefinery 1: 209 – cationic 2: 412
sugar-based chemicals 2: 14 – classification 2: 301
sugar-based olefinic polymers 2: 47 – nonionic 2: 272
sugar-based surfactants 2: 11–12 – oil-based 1: 90
sugar beet 2: 410 – production 2: 302
sugar biorefinery 1: 70 – sugar-based 2: 11–12
sugar cane industry, Brazil 1: 209–211 – vegetable oil 2: 301
sugar cane processing 1: 211 – worldwide market 2: 303
– steps 1: 222 sustainability 1: 92, 96
sugar composition, dependence on harvest- – biomass 1: 106
ing time 1: 265 – biorefining systems 1: 56–65
sugar content, brown juice 1: 303 – economic drivers 1: 381
sugar conversion, efficiency 1: 136 – integrated biorefining systems 1: 60–65
sugar crops 1: 45 sustainable development 2: 253
sugar derivatives 2: 48 sustainable production 2: 448
– polymerizable 2: 40–47 sweet potato, starch production 2: 71
sugar feedstock, carbohydrate sources sweeteners, alternative 2: 62
2: 385 switchgrass, polyhydroxybutyrate source
sugar fermentation 1: 194, 206–207 1: 283
– poly(3-hydroxybutyric acid) 1: 217 swollenin, enzymatic hydrolysis 1: 365
Subject Index 495
syngas 1: 26, 30–31, 46, 98, 126, 157, 2: 361 Thermatoga maritima 1: 80
– composition 1: 232 thermochemical biorefinery concept,
– fermentation 1: 228–229, 233–239, 239– ECN 1: 104
241 thermochemical conversion 1: 31
– platform 1: 31–32 – biomass processing 1: 98
– product family tree 1: 33 – catalytic 2: 356
– technology 1: 240–241 – oils 2: 361
syntheses 1: 123 – optimization 1: 108
– lactic acid 2: 382 – sugars 2: 360
– lipase-catalyzed 2: 270–272 thermochemical liquefaction 1: 123
– petroleum compounds 1: 119 thermochemical processing, biomass 1: 249
– with oils and fats 2: 253–289 thermochemical refinery 1: 101–103
synthesis gas 1: 182 thermogravimetric analyses, feedstock
synthesis of structure, petrochemistry 1: 156
1: 123 Thermoplasma acidophilum, microrgan-
synthetic biofuels 1: 30 isms 1: 80
synthetic biopolyesters 2: 41 thermoplastic polymer, PLA 2: 381
synthetic rubber 1: 131 thermoplastics 2: 225
– adhesive film 1: 282
t – thermoplastic 1: 215
tablet coatings 2: 88 thermoset resins 2: 184
tack 2: 186 THF 1: 149, 2: 373
tall oil fatty acid 2: 297 thickeners, textile-printing 2: 86
Tamiflu, synthesis 2: 30 threonine
tapioca 2: 70–71 – biomass building blocks 1: 22
tapioca starch production 2: 70 – markets 2: 207
tar-formation, acid hydrolysis 1: 144 Tiemann, F. 1: 7
target chemicals, biobased 1: 14, 16 Tilgham, B. C. 1: 6
target crops, feedstock production 1: 15 tillage effect, soil carbon loss 1: 328
tars, undesirable 1: 231 tillage practice 1: 330–331
technical constraints, MAAP 2: 209 tin-catalyzed lactide polymerization 2: 391
technical prerequisite, cellulosic biorefin- tin hydride radical chemistry 2: 261
eries 1: 55–56 tin octoate catalyzed polymerization, lac-
technoeconomic factor, dominant 1: 53–54 tide 2: 393
technological outline, biorefinery sys- TNPP, melt stability improvement 2: 399
tems 1: 4–8 tocopherols 2: 319–320
technological pathways, transformation pro- toxicity 2: 212
cess 1: 87 TPA, synthesis 2: 134
temperature adjustment 1: 79 traffic congestion 1: 338
temperature transition, inverse 2: 219 traffic problems, feedstock 1: 338–339
temporary functional scaffoldings 2: 239 tragacanth, substitute 2: 62
terpenes 1: 91 transepidermal water loss 2: 434
terrestrial biomass, content 2: 3 transesterification, sucrose fatty acid mono-
tetrahydrofuran see THF esters production 2: 13
tetrahydroxybutyl side-chain, furans 2: 19 transition metal-catalyzed syntheses, aro-
tetrapyrrole structures 2: 328 matic compounds 2: 259
TEWL 2: 434 transition metal metathesis, olefins 2: 259
textile-glass-fiber-industry, starch usage transport
2: 91 – baling dry material 1: 332
textile industry, starch usage 2: 85 – crops 1: 337
The Netherlands, bio-based industry transportation fuels, biomass share 1: 14, 16
1: 93–96 Treibs’s scheme, petroporphyrin forma-
thermal addition of alkanes 2: 264 tion 2: 332
496 Subject Index
triacylglycerides 1: 121–122 value-added components, bran-rich wheat

Trichoderma 1: 201–202 fractions 1: 175
Trichoderma cellulase 1: 75 value-added products 2: 360
– enzymes 1: 204 value chain approach, biomass process-
Trichoderma reesei 1: 77, 130, 134, 365, 375 ing 1: 97
– cellulase development 1: 366 vanillin 1: 7, 179, 2: 30
– enzyme improvement 1: 369 vegetable oil
– GH families 1: 373 – emulsifiers 2: 301
– protein secretion 1: 372 – nonionic surfactants raw materials 2: 306
Trichoderma viride 1: 134 vegetable oil epoxides, PVC stabilizers
trichomes 1: 183 2: 256
Trifolium pratense vegetable oils 1: 121, 2: 254, 291
– economic importance 1: 284 – chemo-enzymatic epoxidation 2: 256
– press cake fibers 1: 281 vehicle production, lignin 2: 196–197
triglyceride oils, hydroxyl-functional 2: 298 Vertec 2: 10
triglycerides 1: 121–122, 2: 294 vic-dihydroxy fatty acids 2: 257–258
tris(nonylphenyl) phosphite 2: 399 vigorous mixing, starch modifications 2: 77
truck transport, feedstock 1: 338 vinegar-like proteins, repulsion 2: 218
tunneling, dry-jet process 2: 88 vinyl acetate, glucose product family 1: 21
turpentine, crude 1: 91 vinylsaccharides 2: 47
two platforms concept 1: 31 viscosity, starch 2: 73
– biorefinery 1: 24 viskose process, history 2: 102
two-use ethic 1: 116 vital wheat gluten 1: 180
two-uses ethics, MSW 1: 126 vitamin A source, carotenoids 2: 320
vitamin E source 2: 319
u vitamins 2: 14–15
Udic–Rheinau process 1: 131 – analysis 1: 299
Umbellularia californica 2: 280 – juice fraction 1: 274
unicarbonotroph 1: 239 – wheat 1: 169
unicarbonotrophic acetogens, syngas fer- vitriol oil 2: 99
mentation 1: 233 von Walden, P. 1: 8
United States, biomass conversion 2: 352–
353 w
unsaturated fatty acids wagons, self loading and unloading 1: 319
– dimerization 2: 297 waste biomass 1: 259, 2: 452
– epoxidation 2: 254–257 – biorefinery products 1: 11
– microbial hydration 2: 272–273 waste-products, processing 1: 8
– nucleophilic addition 2: 265 waste streams, cost generator 1: 96
unsaturated fatty compounds, reactions waste treatment costs 1: 53
2: 254–266 waste water treatment 1: 347
unsaturated N-heterocycles, sugar-de- wastes, biorefinery 1: 56
rived 2: 24 water, enantioselective addition 2: 273
updraft gasifiers 1: 231 water-gas shift reaction, syngas produc-
US patent and trademark office 2: 245– tion 1: 230
249 water-retentive properties, chitosonium
USPTO 2: 245–249 salts 2: 420
Ustilago maydis DSM 4500 2: 274 water-solubility, chitosan 2: 417
water-splitting electrodialysis 2: 387
v watersoluble carbohydrates see WSC
c-valerolactone see GVL wax, isolating 2: 432
value-added byproducts wax coating, apple 2: 431
– bran-rich wheat fractions 1: 178 wax esters, apple-peel wax components
– oat bran-rich fractions 1: 185–187 2: 433
Subject Index 497
waxy maize, starch production 2: 66 window of processibility, PHB 1: 216

western immunoblot technique, purity winter cover crop 1: 61
2: 230–231 wood chemicals 2: 357
wet fractionation 1: 257, 271–273 wood chemistry, origin 1: 10
– green biomass 1: 29–30 wood hydrolyses 1: 5
wet mill-based biorefinery wood-hydrolysis pilot plant, US 1: 131
– products 1: 29–31 wood processing, LCF biorefinery 2: 112
– whole crop 1: 28 wood saccharification 1: 5–6
wet mill refinery 1: 346–347 woody biomass 1: 245
wet-milling 1: 48, 70 woody crops 1: 45
– corn 2: 367 WSC 1: 146
wet oxidation, pretreatment 1: 363 – press cake 1: 257
wet storage
– bagasse 1: 323 x
– silage 1: 334 xanthophylls 1: 257, 2: 321
wheat 2: 66 XPS 2: 435
– chemical composition 1: 169 X-ray photoelectron spectroscopy 2: 435
– composition 1: 167 xylan 1: 129, 2: 108
wheat-based biorefinery, schematic 1: 177 xylan/xylose product line 2: 120
wheat flour, secondary processing 1: 169– xylitol 2: 121
173 xylitol/arabinitol, basic biobased chemi-
wheat flour milling byproducts cals 1: 22
– annual amount 1: 173 xylitol source, esparto grass 1: 283
– biorefinery 1: 171 xyloglucan 1: 360
wheat germ 1: 179 xyloidin, history 2: 99
wheat germ oil, purified 1: 179 xylonic acid, biomass building blocks 1: 22
wheat kernel xylose
– exploitation 1: 180–183 – crystals 2: 121
– morphology 1: 168 – fermentation problems 1: 146
wheat milling efficiency, increase 1: 173 – monomeric 1: 198
wheat separation processes, advanced d-xylose, pyrazole synthesis 2: 26
1: 173–176
wheat starch production 2: 68 y
wheat straw yeast
– ethanol production 1: 193 – ascomycetous 2: 7
– world production 1: 51 – ethanol production 1: 194, 206
wheat tillage practice 1: 331 yeast extract, MAAP processes 2: 211
wheatfeed 1: 170
whey 1: 296 z
white biotechnology 2: 445 Z. mobilis see Zymomonas mobilis
white-rot fungi 2: 151 zeolites 1: 278
whole-crop biorefinery 1: 24, 26–29, 165– zwitterion, betaine 2: 411
191 Zymomonas 1: 206
– products 1: 27 Zymomonas mobilis 1: 133, 2: 7, 108, 210

Biorefineries Industrial Processes and Products Status Quo and Future Directions PDF

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Biorefineries Industrial Processes and Products Status Quo and Future Directions PDF

Uploaded by

Copyright:

Available Formats

Biorefineries –

Industrial Processes and

Olah, G. A., Goeppert, A., Prakash, G. K. S.

Beyond Oil and Gas: The Methanol Economy

Bailey’s Industrial Oil and Fat Products

Oil Refineries in the 21st Century

Status Quo and Future Directions

Dr. Patrick R. Gruber

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA,

All rights reserved (including those of translation

Typsetting K+V Fotosatz GmbH, Beerfelden

Printed in the Federal Republic of Germany

Editors’s Preface XXI

List of Contributors XXVII

Part I Background and Outline – Principles and Fundamentals

1 Biorefinery Systems – An Overview 3

1.3.1 Some Current Aspects of Biorefinery Research

2 Biomass Refining Global Impact –

2.5.2 Agricultural/Forestry Ecosystem Modeling: New Tools for an Age

4 Biorefineries for the Chemical Industry –

4.3 Biomass: Technology and Sustainability 93

Part II Biorefinery Systems

Lignocellulose Feedstock Biorefinery

5 The Lignocellulosic Biorefinery –

5.5.1 Production of Ethanol and Furfural from Lignocellulosic

6 Lignocellulosic Feedstock Biorefinery:

7 The Biofine Process – Production of Levulinic Acid, Furfural,

7.4 Conclusion 161

Whole Crop Biorefinery

8 A Whole Crop Biorefinery System:

9 Iogen’s Demonstration Process for Producing Ethanol

9.5 Cellulase Enzyme Production 201

10.4.4 Investment and Production Cost of Poly(3-Hydroxybutyric Acid)

Biorefineries Based on Thermochemical Processing

11 Biomass Refineries Based on Hybrid

12 The Green Biorefiner Concept – Fundamentals and Potential 253

12.3.3.1 Structural Cell Wall Constituents 260

13 Plant Juice in the Biorefinery –

13.4.2 Fed Batch Fermentation of Brown Juice with Lb. salivarius

Part III Biomass Production and Primary Biorefineries

14 Biomass Commercialization and Agriculture Residue Collection 317

14.5.2.3 Storage Loss 335

15 The Corn Wet Milling and Corn Dry Milling Industry –

Part IV Biomass Conversion: Processes and Technologies

16 Enzymes for Biorefineries 357

16.2.2.1 Dilute Acid Pretreatment 362

17.3.4 Biocatalytic Route from Inulin to Difructose Anhydride 397

Subjcet Index 407

Part I Biobased Product Family Trees

Carbohydrate-based Product Lines

1 The Key Sugars of Biomass: Availability, Present Non-Food Uses and

2 Industrial Starch Platform – Status quo of Production,

3 Lignocellulose-based Chemical Products

Lignin Line and Lignin-based Product Family Trees

4 Lignin Chemistry and its Role in Biomass Conversion 151

5 Industrial Lignin Production and Applications 165

Protein Line and Amino Acid-based Product Family Trees

6 Towards Integration of Biorefinery and Microbial Amino Acid

Biobased Fats (Lipids) and Oils

8 New Syntheses with Oils and Fats as Renewable

9 Industrial Development and Application of Biobased

Special Ingredients and Subsequent Products

10 Phytochemicals, Dyes, and Pigments in the Biorefinery Context 315

nCO2 nH2 O ! CH2 On nO2