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Engineering Principles in Biotechnology
Engineering Principles in Biotechnology

Wei-Shou Hu

Department of Chemical Engineering and Materials Science

University of Minnesota, USA
This edition first published 2018
© 2018 by John Wiley & Sons Ltd

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Library of Congress Cataloging-in-Publication Data

Names: Hu, Wei-Shou, 1951- author.

Title: Engineering Principles in Biotechnology / by Wei-Shou Hu.
Description: First edition. | Hoboken, NJ, USA : John Wiley & Sons, Inc.,
2018. | Includes bibliographical references and index. |
Identifiers: LCCN 2017016663 (print) | LCCN 2017018764 (ebook) | ISBN
9781119159032 (pdf ) | ISBN 9781119159049 (epub) | ISBN 9781119159025
(cloth)
Subjects: | MESH: Bioengineering | Biotechnology
Classification: LCC R855.3 (ebook) | LCC R855.3 (print) | NLM QT 36 | DDC
610.285–dc23
LC record available at https://lccn.loc.gov/2017016663

Cover design by Wiley

Cover images: (Background) © STILLFX/Gettyimages; (Illustrations) Courtesy of Wei-Shou Hu

Set in 10/12pt WarnockPro by SPi Global, Chennai, India

10 9 8 7 6 5 4 3 2 1
This book is dedicated to Jenny, Kenny, and Sheau-Ping.
 vii

Contents

Preface xvii
About the Companion Website xix

1 An Overview of Bioprocess Technology and Biochemical

Engineering 1
1.1 A Brief History of Biotechnology and Biochemical Engineering 1
1.1.1 Classical Biotechnology 1
1.1.2 Recombinant DNA 4
1.1.3 A Typical Bioprocess 6
1.1.4 Biochemical Engineering and Bioprocess Technology 8
1.2 Industrial Organisms 10
1.2.1 Prokaryotes 12
1.2.1.1 Eubacteria and Archaea 12
1.2.2 Eukaryotic Microorganisms 12
1.2.2.1 Fungi 13
1.2.2.2 Algae 13
1.2.3 Multicellular Organisms and Their Cells 13
1.2.3.1 Insect Cells 13
1.2.3.2 Plant Cells, Tissues, and Organs 13
1.2.3.3 Animal Cells, Tissues, and Organs 14
1.2.4 Transgenic Plants and Animals 14
1.3 Biotechnological Products 15
1.3.1 Metabolic Process 15
1.3.2 Metabolites 18
1.3.3 Cells, Tissues, and Their Components 19
1.3.3.1 Viruses 20
1.3.4 Secreted Enzymes and Other Biopolymers 20
1.3.5 Recombinant DNA Products 20
1.3.5.1 Heterologous rDNA Proteins 20
1.3.6 Metabolic Engineering and Synthetic Pathways 22
1.4 Technology Life Cycle, and Genomics- and Stem Cell-Based New
Biotechnology 23
1.4.1 The Story of Penicillin and the Life Cycle of Technology 23
1.4.2 Genomics, Stem Cells, and Transformative Technologies 25
Further Reading 26
Problems 26
viii Contents

2 An Introduction to Industrial Microbiology and Cell

Biotechnology 29
2.1 Universal Features of Cells 29
2.2 Cell Membranes, Barriers, and Transporters 30
2.3 Energy Sources for Cells 31
2.3.1 Classification of Microorganisms According to Their Energy Source 32
2.4 Material and Informational Foundation of Living Systems 34
2.4.1 All Cells Use the Same Molecular Building Blocks 34
2.4.2 Genes 34
2.4.3 Genetic Information Processing 36
2.5 Cells of Industrial Importance 36
2.5.1 Prokaryotes 38
2.5.2 Eubacteria 38
2.5.2.1 Cell Wall and Cell Membrane 38
2.5.2.2 Membrane and Energy Transformation 40
2.5.2.3 Differentiation 41
2.5.3 Archaea 42
2.5.4 Eukaryotes 43
2.5.4.1 The Nucleus 44
2.5.4.2 Mitochondrion 45
2.5.4.3 Endoplasmic Reticulum and Golgi Apparatus 46
2.5.4.4 Other Organelles 47
2.5.4.5 Cytosol 48
2.6 Cells Derived from Multicellular Organisms 49
2.7 Concluding Remarks 50
Further Reading 50
Problems 50

3 Stoichiometry of Biochemical Reactions and Cell Growth 53

3.1 Stoichiometry of Biochemical Reactions 53
3.1.1 Metabolic Flux at Steady State 58
3.1.1.1 NAD/NADH Balance in Glycolysis 59
3.1.1.2 Oxidative Metabolism and NADH 60
3.1.2 Maximum Conversion of a Metabolic Product 63
3.2 Stoichiometry for Cell Growth 66
3.2.1 Cell Composition and Material Flow to Make Cell Mass 66
3.2.1.1 Composition and Chemical Formula of Cells 66
3.2.1.2 Material Flow for Biomass Formation 69
3.2.2 Stoichiometric Equation for Cell Growth 70
3.2.2.1 Yield Coefficient 71
3.3 Hypothetical Partition of a Substrate for Biomass and Product Formation 73
3.4 Metabolic Flux Analysis 74
3.4.1 Analysis of a Chemical Reaction System 74
3.4.1.1 Setting Up Material Balance Equations 74
3.4.1.2 Quasi–Steady State 76
3.4.1.3 Stoichiometric Matrix, Flux Vectors, and Solution 76
3.4.2 Analysis of Fluxes in a Bioreaction Network 77
 Contents ix

3.4.3 Metabolic Flux Analysis on a Cellular System 81

3.4.3.1 Selecting Reactions for Analysis 81
3.4.3.2 Compartmentalization 83
3.4.3.3 Biomass 83
3.4.3.4 Limitations on Accounting of Materials 84
3.4.3.5 Solution and Analysis 84
3.5 Concluding Remarks 85
Further Reading 85
Nomenclature 86
Problems 86

4 Kinetics of Biochemical Reactions 95

4.1 Enzymes and Biochemical Reactions 95
4.2 Mechanics of Enzyme Reactions 96
4.3 Michaelis–Menten Kinetics 98
4.4 Determining the Value of Kinetic Parameters 101
4.5 Other Kinetic Expressions 104
4.6 Inhibition of Enzymatic Reactions 106
4.7 Biochemical Pathways 108
4.7.1 Kinetic Representation of a Reaction Pathway 108
4.7.2 Linearity of Fluxes in Biochemical Pathways 110
4.8 Reaction Network 114
4.9 Regulation of Reaction Rates 114
4.9.1 Flux Modulation by Km 114
4.9.2 Allosteric Regulation of Enzyme Activities 115
4.9.3 Regulation at Transcriptional and Posttranslational Levels 117
4.9.4 Modulation of Resource Distribution through Reversible Reactions 118
4.10 Transport across Membrane and Transporters 120
4.10.1 Transport across the Cell Membrane 120
4.10.2 Transport of Electrolytes 121
4.10.3 Transport of Charged Molecules across Membrane 122
4.10.4 Types of Transporters 123
4.10.5 Kinetics of a Facilitated Transporter 124
4.11 Kinetics of Binding Reactions 126
4.11.1 Binding Reactions in Biological Systems 126
4.11.2 Dissociation Constant 127
4.11.3 Saturation Kinetics 128
4.11.4 Operator Binding and Transcriptional Regulation 129
4.11.5 Kinetics of Transcription and Translation 131
4.12 Concluding Remarks 135
Further Reading 136
Nomenclature 136
Problems 138

5 Kinetics of Cell Growth Processes 145

5.1 Cell Growth and Growth Kinetics 145
5.2 Population Distribution 148
x Contents

5.3 Description of Growth Rate 149

5.4 Growth Stage in a Culture 150
5.5 Quantitative Description of Growth Kinetics 151
5.5.1 Kinetic Description of Substrate Utilization 153
5.5.2 Using the Monod Model to Describe Growth in Culture 155
5.6 Optimal Growth 156
5.7 Product Formation 158
5.8 Anchorage-Dependent Vertebrate Cell Growth 159
5.9 Other Types of Growth Kinetics 161
5.10 Kinetic Characterization of Biochemical Processes 162
5.11 Applications of a Growth Model 163
5.12 The Physiological State of Cells 164
5.12.1 Multiscale Model Linking Biotic and Abiotic Phases 166
5.13 Kinetics of Cell Death 168
5.14 Cell Death and the Sterilization of Medium 169
5.15 Concluding Remarks 171
Further Reading 172
Nomenclature 172
Problems 173

6 Kinetics of Continuous Culture 183

6.1 Introduction 183
6.2 Kinetic Description of a Continuous Culture 185
6.2.1 Balance Equations for Continuous Culture 185
6.2.2 Steady-State Behavior of a Continuous Culture 187
6.2.2.1 Monod Kinetics 187
6.2.2.2 Steady-State Concentration Profiles 187
6.2.2.3 Washout 189
6.2.3 Productivity in Continuous Culture 190
6.3 Continuous Culture with Cell Recycling 193
6.3.1 Increased Productivity with Cell Recycling 193
6.3.2 Applications of Continuous Culture with Cell Recycling 196
6.3.2.1 Low Substrate Levels in the Feed 196
6.3.2.2 Low Residual Substrate Concentration 197
6.3.2.3 Labile Product 197
6.3.2.4 Selective Enrichment of Cell Subpopulation 197
6.3.2.5 High-Intensity Mammalian Cell Culture 197
6.4 Specialty Continuous Cultures 199
6.4.1 Multiple-Stage Continuous Culture 199
6.4.2 Immobilized Cell Culture System 200
6.4.3 Continuous Culture with Mixed Populations 201
6.5 Transient Response of a Continuous Culture 202
6.5.1 Pulse Increase at the Substrate Level 203
6.5.2 Step Change in Feed Concentration 204
6.6 Concluding Remarks 205
 Contents xi

Further Reading 205

Nomenclature 205
Problems 206

7 Bioreactor Kinetics 217

7.1 Bioreactors 217
7.2 Basic Types of Bioreactors 218
7.2.1 Flow Characteristics in Idealized Stirred-Tank (Well-Mixed) and Tubular
(Plug Flow) Reactors 219
7.2.2 Reaction in an Idealized CSTR 220
7.2.3 Reaction in an Idealized PFR 222
7.2.4 Heterogeneous and Multiphasic Bioreactors – Segregation of Holding
Time 225
7.3 Comparison of CSTR and PFR 225
7.3.1 CSTR versus PFR in Conversion Yield and Reaction Rate 225
7.3.2 CSTR versus PFR in Terms of Nutrient Depletion and Scale-Up 226
7.3.3 CSTR versus PFR – A Perspective from Residence Time Distribution 227
7.4 Operating Mode of Bioreactors 229
7.4.1 Batch Cultures 229
7.4.2 Fed-Batch Cultures 229
7.4.2.1 Intermittent Harvest 229
7.4.2.2 Fed-Batch 230
7.5 Configuration of Bioreactors 231
7.5.1 Simple Stirred-Tank Bioreactor 231
7.5.2 Airlift Bioreactor 233
7.5.3 Hollow-Fiber Bioreactor 233
7.6 Other Bioreactor Applications 233
7.7 Cellular Processes through the Prism of Bioreactor Analysis 235
7.8 Concluding Remarks 236
Further Reading 236
Nomenclature 237
Problems 237

8 Oxygen Transfer in Bioreactors 241

8.1 Introduction 241
8.2 Oxygen Supply to Biological Systems 242
8.3 Oxygen and Carbon Dioxide Concentration in Medium – Henry’s Law 243
8.4 Oxygen Transfer through the Gas–Liquid Interface 244
8.4.1 A Film Model for Transfer across the Interface 244
8.4.2 Concentration Driving Force for Interfacial Transfer 245
8.4.3 Mass Transfer Coefficient and Interfacial Area 246
8.5 Oxygen Transfer in Bioreactors 248
8.5.1 Material Balance on Oxygen in a Bioreactor 249
8.5.2 Oxygen Transfer in a Stirred Tank 251
8.6 Experimental Measurement of KL a and OUR 253
xii Contents

8.6.1 Determination of KL a in a Stirred-Tank Bioreactor 253

8.6.2 Measurement of OUR and qO2 254
8.7 Oxygen Transfer in Cell Immobilization Reactors 256
8.8 Concluding Remarks 256
Further Reading 256
Nomenclature 256
Problems 258

9 Scale-Up of Bioreactors and Bioprocesses 265

9.1 Introduction 265
9.2 General Considerations in Scale Translation 266
9.2.1 Process and Equipment Parameters Affected by Scale-Up 266
9.2.2 Scale Translation for Product Development and Process
Troubleshooting 266
9.2.3 How Scale-Up Affects Process Variables, Equipment, and Cellular
Physiology 267
9.2.4 Scale-Up of Equipment and Geometrical Similarity 267
9.3 Mechanical Agitation 268
9.4 Power Consumption and Mixing Characteristics 269
9.4.1 Power Consumption of Agitated Bioreactors 269
9.4.2 Other Dimensionless Numbers 272
9.4.3 Correlation of Oxygen Transfer Coefficient 273
9.5 Effect of Scale on Physical Behavior of Bioreactors 273
9.6 Mixing Time 276
9.6.1 Nutrient Enrichment Zone: Mixing Time versus Starvation Time 276
9.6.2 Mixing Time 277
9.6.3 Mixing Time Distribution 278
9.7 Scaling Up and Oxygen Transfer 279
9.7.1 Material Balance on Oxygen in Bioreactor 279
9.7.1.1 Aeration Rate and the Oxygen Transfer Driving Force 280
9.8 Other Process Parameters and Cell Physiology 281
9.9 Concluding Remarks 282
Further Reading 283
Nomenclature 283
Problems 284

10 Cell Culture Bioprocesses and Biomanufacturing 289

10.1 Cells in Culture 289
10.2 Cell Culture Products 290
10.2.1 Vaccines 290
10.2.2 Therapeutic Proteins 291
10.2.3 Biosimilars 292
10.3 Cellular Properties Critical to Biologics Production 294
10.3.1 Protein Secretion 294
10.3.1.1 Folding in the Endoplasmic Reticulum 294
10.3.1.2 Membrane Vesicle Translocation and Golgi Apparatus 295
10.3.2 Glycosylation 296
 Contents xiii

10.3.3 Protein Secretion and Glycan Heterogeneity 296

10.4 Nutritional Requirements 299
10.4.1 Chemical Environment In Vivo and in Culture 299
10.4.2 Types of Media 300
10.4.2.1 Basal Medium and Supplements 300
10.4.2.2 Complex Medium, Defined Medium 301
10.5 Cell Line Development 301
10.5.1 Host Cells and Transfection 301
10.5.2 Amplification 302
10.6 Bioreactors 304
10.6.1 Roller Bottles 304
10.6.2 Stirred-Tank Bioreactors for Suspension Cells 305
10.6.3 Stirred-Tank Bioreactor with Microcarrier Cell Support 306
10.6.4 Disposable Systems 307
10.7 Cell Retention and Continuous Processes 307
10.7.1 Continuous Culture and Steady State 307
10.8 Cell Culture Manufacturing – Productivity and Product Quality 308
10.8.1 Process and Product Quality 308
10.8.2 Product Life Cycle 309
10.8.3 Product Manufacturing 311
10.8.3.1 Platform Process 311
10.8.3.2 Manufacturing 311
10.9 Concluding Remarks 312
Further Reading 312
Problems 313

11 Introduction to Stem Cell Bioprocesses 319

11.1 Introduction to Stem Cells 319
11.2 Types of Stem Cells 320
11.2.1 Adult Stem Cells 320
11.2.1.1 Hematopoietic Stem Cells 321
11.2.1.2 Mesenchymal Stem Cells 323
11.2.1.3 Neuronal Stem Cells 323
11.2.2 Embryonic Stem Cells 324
11.2.3 Induced Pluripotent Stem Cells and Reprogramming 324
11.3 Differentiation of Stem Cells 326
11.4 Kinetic Description of Stem Cell Differentiation 328
11.5 Stem Cell Technology 331
11.6 Engineering in Cultivation of Stem Cells 332
11.7 Concluding Remarks 335
Further Reading 335
Nomenclature 336
Problems 336

12 Synthetic Biotechnology: From Metabolic Engineering to Synthetic

Microbes 339
12.1 Introduction 339
xiv Contents

12.2 Generalized Pathways for Biochemical Production 340

12.3 General Strategy for Engineering an Industrial, Biochemical-Producing
Microorganism 342
12.3.1 Genomics, Metabolomics, Deducing Pathway, and Unveiling
Regulation 342
12.3.2 Introducing Genetic Alterations 343
12.3.3 Isolating Superior Producers 345
12.3.3.1 Screening of Mutants with the Desired Phenotype 345
12.3.3.2 Selection of Mutants with the Target Trait 345
12.3.4 Mechanisms of Enhancing the Biosynthetic Machinery 347
12.3.4.1 Relaxing the Constriction Points in the Pathway 347
12.3.4.2 Channeling Precursor Supply 348
12.3.4.3 Eliminating Product Diversion 350
12.3.4.4 Enhancing Product Transport 350
12.3.4.5 Rerouting Pathways 350
12.3.5 Engineering Host Cells – Beyond the Pathway 352
12.3.5.1 Altering Substrate Utilization 352
12.3.5.2 Manipulating the Time Dynamics of Production 352
12.3.5.3 Increasing Product Tolerance 354
12.4 Pathway Synthesis 356
12.4.1 Host Cells: Native Hosts versus Archetypical Hosts 356
12.4.2 Expressing Heterologous Enzymes to Produce a Nonnative Product 357
12.4.3 Activating a Silent Pathway in a Native Host 359
12.5 Stoichiometric and Kinetic Considerations in Pathway Engineering 359
12.6 Synthetic Biology 367
12.6.1 Synthetic (Cell-Free) Biochemical Reaction System 367
12.6.2 Synthetic Circuits 369
12.6.2.1 Artificial Genetic Circuits 369
12.6.2.2 Synthetic Signaling Pathway 369
12.6.3 Synthetic Organisms 371
12.6.3.1 Minimum Genome and Reduced Genome 371
12.6.3.2 Chemical Synthesis of a Genome 372
12.6.3.3 Surrogate Cells from a Synthetic Genome 374
12.7 Concluding Remarks 374
Further Reading 374
Problems 375

13 Process Engineering of Bioproduct Recovery 381

13.1 Introduction 381
13.2 Characteristics of Biochemical Products 382
13.3 General Strategy of Bioproduct Recovery 385
13.3.1 Properties Used in Bioseparation 385
13.3.2 Stages in Bioseparation 387
13.3.2.1 Cell and Solid Removal 387
13.3.2.2 Product Isolation (Capture) and Volume Reduction 387
13.3.2.3 Product Purification 388
13.3.2.4 Product Polishing 388
 Contents xv

13.4 Unit Operations in Bioseparation 389

13.4.1 Filtration 389
13.4.2 Centrifugation 390
13.4.3 Liquid–Liquid Extraction 393
13.4.4 Liquid Chromatography 395
13.4.5 Membrane Filtration 396
13.4.6 Precipitation and Crystallization 397
13.5 Examples of Industrial Bioseparation Processes 398
13.5.1 Recombinant Antibody IgG 398
13.5.2 Penicillin 401
13.5.3 Monosodium Glutamate 404
13.5.4 Cohn Fractionation 404
13.6 Concluding Remarks 404
Further Reading 406
Nomenclature 407
Problems 408

14 Chromatographic Operations in Bioseparation 413

14.1 Introduction 413
14.2 Adsorbent 415
14.2.1 Types of Adsorbent 415
14.2.2 Ligand and Mechanism of Separation 418
14.2.3 Types of Liquid Chromatography 419
14.3 Adsorption Isotherm 420
14.3.1 Adsorption Equilibrium: Langmuir Isotherm 420
14.3.2 Isotherm Dynamics in Adsorption and Desorption 421
14.4 Adsorption Chromatography 425
14.4.1 Discrete-Stage Analysis 425
14.4.2 Breakthrough Curve 427
14.4.3 An Empirical Two-Parameter Description of a Breakthrough Curve 429
14.4.4 One-Porosity Model for an Adsorption Process 431
14.4.5 Elution of Solutes from an Adsorption Column 433
14.5 Elution Chromatography 435
14.5.1 Discrete-Stage Analysis 435
14.5.2 Determination of Stage Number 441
14.5.3 Effect of Stage Number and Number of Theoretical Plates 442
14.5.4 Two-Porosity Model, Mass Transfer Limitation 444
14.6 Scale-Up and Continuous Operation 447
14.6.1 Mass Transfer Limitation and the van Deemter Equation 447
14.6.2 Scale-Up of Chromatography 448
14.6.3 Continuous Adsorption and Continuous Elution Chromatography 450
14.7 Concluding Remarks 454
Further Reading 454
Nomenclature 454
Problems 456

Index 471
 xvii

Preface

Bioprocesses use microbial, plant, or animal cells and the materials derived from them,
such as enzymes or DNA, to produce industrial biochemicals and pharmaceuticals. In
the past two decades, the economic output from bioprocesses has increased drasti-
cally. This economic growth was the result of the translation of numerous discoveries to
innovative technologies and manufactured products. The success has brought together
numerous scientists and engineers of different disciplines to work together to break
new ground. The task of taking biotechnological discoveries to a successful product
or process requires a multidisciplinary team consisting of engineers and chemical and
biological scientists to work synergistically. The success of a project, a team, or even
a company in biotechnology often hinges on the ability of scientists and engineers of
different specialties to work effectively together. This book has been written with this
important characteristic of the bioprocess industry in mind. A major goal of the book
is to give students the necessary vocabulary and critical engineering knowledge to excel
in bioprocess technology.
This textbook is based on a biochemical engineering course that has been offered
at the University of Minnesota for a number of years. The contents are intended for a
semester course of about 14 weeks of three lecture-hours a week. Although the majority
of the students taking this course are senior undergraduate and graduate students from
chemical engineering and bioengineering, nearly one-third are graduate students from
a life science background. An emphasis of the content and writing of the book is thus
the fundamental engineering principles, the quantitative practice, and the accessibil-
ity of analysis for students of different backgrounds. The target audience of the book is
not only students taking the biochemical engineering or bioprocess engineering courses
given in chemical engineering or bioengineering programs but also students in biotech-
nology programs that are outside of the chemical engineering disciplines, especially in
countries outside North America.
In writing this book, I assumed that the students have had at least one biology course,
and have fundamental knowledge of carbohydrates, DNA, RNA, proteins, and other
biomolecules, as is the case for most engineering students nowadays. Nevertheless,
students from both engineering and life science backgrounds will encounter new
vocabulary and new concepts that will help them in cross-disciplinary communication
once they join the biotechnology workforce.
Chapters 1 and 2 give an overview of organisms, cells and their components, how they
become the product, and what the bioprocesses that produce them look like. Chapters
3 and 4 use basic biochemical reactions, especially the energy metabolism pathways, to
xviii Preface

familiarize engineering students with analysis of biochemical systems and to introduce

the concepts of material balance and reaction kinetics to students with a life science
background. For all students, these chapters introduce them to kinetic analysis of bind-
ing reactions, gene expression, and cellular membrane transport.
Chapters 5 and 6 cover the quantitative description of cell growth and the steady-state
behavior in a continuous bioreactor. This paves the way for dealing with different types
of bioreactors. Chapters 7, 8, and 9 are the core of bioreactor engineering, dealing with
subjects important to process development. These chapters draw upon extensive prac-
tical interactions with industry to make them more relevant to bioprocess technology.
The next three chapters – 10, 11, and 12 – discuss three segments of bioprocesses.
Cell culture processes, the subject of Chapter 10, currently produce goods valued over
US$100 billion per annum. After introducing cell culture processes, the evolution of
biomanufacturing and its life cycle is discussed. Chapters 11 and 12 look to the future
on cell-based therapy and on the technologies arising from synthetic biology. In dealing
with stem cells, the kinetic description of cellular differentiation is also introduced, and
in discussing synthetic pathways the importance of using a stoichiometric relationship
to determine the maximum conversion yield is reiterated. The last two chapters, 13 and
14, highlight the bioseparation processes. The overall strategy and the key concepts of
various unit operations in bioseparation are covered briefly in Chapter 13. Chapter 14
focuses on the basic quantitative understanding of chromatography.
Writing this book has been a long undertaking. Many of my former and current
graduate students have helped in formulating the problem sets and the examples. In
preparing the book, I also took ideas from many textbooks on biochemical engineering,
especially Bioprocess Engineering: Basic Concepts by Shuler and Kargi; Biochemical
Engineering Fundamentals by Bailey and Ollis; Fermentation and Enzyme Technology
by Wang, Cooney, Demain, Dunnill, Humphrey, and Lilly; and Biochemical Engineering
by Aiba, Humphrey, and Millis. I extend my gratitude to my colleagues at the University
of Minnesota, especially Arnold G. Fredrickson, Friedrich Srienc, Edward Cussler, Ben
Hackel, Kechun Zhang, Samira Azarin, Efie Kokkoli, Yiannis Kaznessis, and Prodromos
Daoutidis, for their stimulating discussion that helped shape the book. Finally, I thank
Kimberly Durand for her editorial devotion to this book.
 Overview of Bioprocess Technology & Biochemical Engineering 11

Industrial microorganisms can be classified according to the class of products they

produce or by their taxonomy (Panels 1.2 and 1.3). Some genera have a certain propen-
sity for producing certain classes of compounds. For example, members of Streptomyces
are known for being producers of antibiotics, while species in Corynebacterium are most
amenable for producing amino acids.

Panel 1.2 Industrial Organisms

Prokaryote
Eubacteria
Unicellular
Mycelial – Actinomycetes
(Streptomyces, Nocardia)
Archaea (Extremophiles)
Eukaryotic Cells
Fungi
Yeast
Mold
Algae
Protozoa
Insect cells
Plant cells, tissues, and organs
Animal cells, tissues, and organs
Transgenic Animals and Plants

Panel 1.3 Biotechnological Products

Metabolic Process
Food
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Mississippi river. We can now look across a gorge from the coaches
of the Pennsylvania Railroad, beyond Altoona, and see the grade of
the old Portage Railway.

Fig. 28. Broad Street Station, Philadelphia: Pennsylvania

Railroad
The canal almost put out of business the Conestoga wagons on
the dusty pike which had seen so much travel by way of Carlisle and
Bedford. But the people did not stop with a canal. Like the men of
New York, they wanted something even better than that. They
wished to have a railroad all the way, and in 1846 the Pennsylvania
Railroad Company was incorporated. By this time it was very well
known that railroads were successful both in America and in
England, and that steam was better than horses.
 Over the Allegheny Front a route was found where the grades
were not too steep for locomotives. The grade, of course, had been
the one great hindrance to the whole project, and when this difficulty
was overcome there was no reason why passengers should not be
carried from Philadelphia to Pittsburg, or a load of iron from Pittsburg
to Philadelphia, without changing cars. In the year 1854 the
Pennsylvania people triumphed, for they had conquered the
mountains and could run trains from the banks of the Delaware to
the Ohio river.
If we leave Philadelphia by the great Broad street station of the
Pennsylvania Railroad, we shall pass out among the pleasant homes
of West Philadelphia and through the fine farms of the Pennsylvania
lowlands, until we come, in about an hour and a half, to the staid old
city of Lancaster. We have been here before, to learn of turnpikes
and Conestoga freighters.
The next stop, if we are on an express train, will be at
Harrisburg, a little more than a hundred miles from Philadelphia. We
have now come from the Delaware to the Susquehanna, and are
close to the mountains. Before we go in among them let us see
Harrisburg. It is a city of fifty thousand people, and lies along the
east bank of the Susquehanna, which here is a great river a mile
wide, having gathered its tribute of waters from hundreds of
branching streams in Pennsylvania and New York.

Fig. 29. Bridge, Pennsylvania Railroad, above Harrisburg

 Not far to the east a small stream runs parallel to the main river,
and the larger part of Harrisburg is on higher ground between the
two. On the highest part of this ridge is the state capitol, a great
building but recently finished. Harrisburg is at the right point for the
state government. It is not in the center of the state, to be sure, but it
is at the rear of the lowlands which reach in from the sea, and is just
outside the great gateway where roads from all the northern,
western, and central uplands come out on the plain. It is a
convenient center for coal and iron, and hence one sees along the
river below the city many blast furnaces, rolling mills, and factories.
To the northeast rich, open lands stretch along the base of Blue
mountain, and railroads join Harrisburg to Reading, Allentown,
Bethlehem, and Easton. To the southwest bridges cross the
Susquehanna, and roads run to Carlisle, Hagerstown, and other
cities of the Great Valley (Chapter XI).
Thus the Pennsylvania Railroad, running northwest from
Philadelphia, crosses at Harrisburg other roads that run to the
southwest. As hamlets often gather about “four corners” in the
country, so cities grow up where the great roads of the world cross
each other.

Fig. 30. Pennsylvania

Railroad Shops, Altoona
 Leaving Harrisburg behind, we pass the splendid new bridge of
the Pennsylvania Railroad, across the Susquehanna (Fig. 29), and
go through the gap in Blue mountain. Soon we turn away from the
main river and enter the winding valley of the Juniata. The grades
are easy, the roadbed is smooth, and by deep cuts through the rocks
the curves have been made less abrupt. It is only when one looks
out of the car window that the land is found to be rugged and
mountainous.
All the greater valleys and ridges of the mountain belt of
Pennsylvania run northeast and southwest. The last of these to be
crossed on our journey is Bald Eagle valley, from which the
Allegheny Front rises to the northwest.
In this valley, near the place where the Portage Railway began to
scale the heights, and a little more than a hundred miles from
Pittsburg, the Pennsylvania Railroad Company in 1850 founded a
town and called it Altoona. Here they started shops, which have now
grown to notable importance. The town became a city eighteen
years after it was begun, and has to-day about forty thousand
inhabitants. In the railway shops alone may be found nine thousand
men repairing and building locomotives, passenger coaches, and
freight cars. The Pennsylvania Railroad Company is now founding a
great school in Altoona, where young men may be taught to become
skillful and efficient in railway service.

Fig. 31. Horseshoe Curve, Pennsylvania Railroad

Altoona looks new, and with its endless freight yards, its noisy
shops, and its sooty cover of smoke from burning soft coal, it is very
different from quiet Lancaster, which was old when forests covered
the site of Altoona.
On our way to Pittsburg we are soon pulling up the Allegheny
Front by a great loop, or bend, which enables the tracks to reach the
summit more than a thousand feet above Altoona. Nestling within the
great bend is a reservoir of water to supply the houses and shops of
the city lying below. Passing the highest point, we find ourselves
descending the valley of the Conemaugh river to Johnstown, and
surrounded by the high lands of the Allegheny plateau.
Johnstown is much older than Altoona, for it was settled in 1791,
but it has not grown so fast, and has only about as many inhabitants
as the city of railroad shops. Most people know of Johnstown
because of the flood which ruined the place in 1889. Several miles
above the town was a reservoir more than two miles long and in
several places one hundred feet deep. After the heavy rains of that
spring the dam broke on the last day of May, and the wild rush of
waters destroyed the town. Homes, stores, shops, and mills were
torn away and carried down the river. Clara Barton of the Red Cross,
who went to Johnstown as soon as she could get there, says that the
few houses that were not crushed and strewn along the valley were
turned upside down.
More than two thousand men, women, and children lost their
lives, and those that were left were in mourning and poverty. The
whole land sent in its gifts of money, clothing, and food, and the town
was built up again into a prosperous city. Near the city are found
coal, iron, limestone, and fire clay, and these things make it easy to
establish iron works. The Cambria Steel Company gives work to ten
thousand men in its shops, mines, and furnaces.
The main line of the Pennsylvania Railroad runs down the
rugged Conemaugh valley through Johnstown, and is its chief
means of traffic. As we go on to the west we near Pittsburg, but first
we pass through a number of stirring towns. At one place fire bricks
are made, and the clay for molding them and the coal for burning
them are found in the same hill. In another town there are coal mines
and glass works. Farther west the Pennsylvania road has more
repair shops, and Braddock is the great Carnegie town. We shall see
why many thriving young cities have grown up in this region when
we take up Pittsburg, about which they are all clustered.
At Pittsburg we pull into one of the finest railway stations in the
United States. We may stop in the city of coal and iron, or we may
go on to the west, over one of the main arms of the Pennsylvania
Railroad system. If we take the northern branch, it will carry us
across Ohio to Fort Wayne in Indiana and to Chicago. If we board a
train on the southern arm, we shall go through Columbus and
Indianapolis, and be set down on the farther side of the Mississippi
river at St. Louis.
North and south from the great east and west trunk lines run
many shorter roads, or “spurs.” On the east there is a network of
short roads in New Jersey, and one of the busiest parts of the whole
system is that which joins Washington to Baltimore, Philadelphia,
and New York.
 Fig. 32. Rock Cut, along the Line of the Pennsylvania
Railroad
West from Philadelphia for a long distance there are four tracks,
and on either side may be seen neat hedges, such as one finds
along the railways of England. In the mountains it is often hard to
make a roadbed wide enough for four tracks, and hence there may
be only three or even two in some places. No doubt four will in time
be built through to Pittsburg, for many millions of dollars are spent in
improving the road. Instead of having a long circuit around the hills,
tunnels and vast cuts in the bed rock are made so as to straighten
the line. Thus both passenger and freight trains are able to make
better time, and the road can carry the stores of iron and coal which
are found in the lands on either side.
 Some of the freight yards are always crowded with cars, and at
Harrisburg the company is building separate tracks around the city,
so that through freight trains need not be delayed.
At New York the Pennsylvania Railroad now has its station on
the New Jersey side of the Hudson river, but it is building a tunnel
under the river. The company has already bought several city blocks
and has torn away the buildings. Here it will build one of the greatest
passenger stations in the world. The tunnel will run on to the east,
under the streets and shops of Manhattan, and under the East river.
Thus under New York and its surrounding waters trains can go to the
east end of Long Island.
Pennsylvania has told us the same story that we learned from
New York. We read it again: first, how the Indian’s path was beaten
deeper and wider by the hoofs of the pack horse, bearing goods to
sell and barter in the wilderness; then how strips of forest were cut
down to make room for the Conestoga wagons and the gay stages
that swept through from Philadelphia to Pittsburg. These in their turn
became old-fashioned when the canal and Portage Railway were
done, and now we sit in a car that is like a palace, and think canals
and Conestogas very old stories indeed. In future generations swift
air ships may take the wonder away from the Empire State Express,
and make us listen unmoved when a man, standing in the station at
Philadelphia, calls the limited train for Pittsburg, Cincinnati, and St.
Louis.
 CHAPTER VIII
THE NATIONAL ROAD

The sea reaches inland almost to the northeast corner of the

state of Maryland. This long, wide arm of the ocean receives many
rivers and is known as Chesapeake bay. Near its north end is
Baltimore, one of the four great cities of our Atlantic coast. It is one
hundred and fifty miles from the open sea. If, instead of sailing up
the bay, we should turn toward the west, we could go up the
Potomac river, which is deep and wide. On our way we should pass
Washington’s estates at Mount Vernon, the old city of Alexandria,
and the national capital, Washington. We could not sail much farther
because there are falls in the Potomac which ships cannot pass. The
Potomac runs so close to Chesapeake bay that it is only forty miles
from Washington across to Baltimore.
Chesapeake bay is much like Delaware bay and the tidal
Hudson river, only it is larger than either. Baltimore is at a greater
distance from the open sea than Philadelphia is, and Philadelphia is
farther inland than New York, but each of these cities tried to get as
much of the western trade as it could.
The natural way for the men of Baltimore and Alexandria to go
across to the west was up the Potomac river and through its passes
in the mountains. But before they tried this they had settled much of
the low, flat land along the Potomac and about the Chesapeake in
Virginia and Maryland. This was often called “tide-water country,”
because the beds of the rivers are below sea level, and the streams
are deep enough for boats of some size.
 Fig. 33. Tollhouse West of Brownsville, Pennsylvania
When the land was first settled and the colonists found that they
could go almost everywhere by boat, they paid small heed to making
roads. They could visit their neighbors on other plantations and they
could load their tobacco and take it to market by the rivers. Many
plantations were beside rivers of such great depth that sailing
vessels bound for London could come up to the farmer’s wharf and
get his crop of tobacco.
In early days the members of the legislature were not always
given so much per mile to pay the stage fares between their homes
and the capital, but they were allowed the cost of hiring boats
instead. Many ferries were needed, and laws about them were made
before rules were laid down for bridges and roads. Several
lawmakers at one time would have been fined for their absence from
the legislature of the colony had they not been excused because
there was no ferry to carry them over the river which they would
have had to cross.
Around Annapolis “rolling roads” were made. These were wide
paths made as smooth as possible, in order that large hogsheads of
tobacco might be rolled, each by two men, to the market in that old
town.
 After a time the lowlands of the coast region began to fill up and
the people were pushing westward, just as they did in Pennsylvania
and New York. No man had so great a part in this westward
movement as the young surveyor, George Washington. In 1748 he
was sixteen years old, a tall, strong lad, full of courage and energy.
Lord William Fairfax, a rich English gentleman who had settled in
Virginia, had bought great tracts of forest land up the Potomac
behind the Blue Ridge mountains, and he was eager to have them
surveyed. Knowing that Washington had studied surveying, Fairfax
asked him to undertake the task. The boy consented; he went
beyond the Blue Ridge into the country along the Shenandoah,
camped in the woods, swam the rivers, toughened his muscles,
learned the ways of the red men, and three years later came back, a
grown man, ready for great things.
While Washington was getting his practice as a surveyor the
Ohio Company was formed to take up lands along the Ohio river,
and to keep the French from settling there. Lawrence, Washington’s
elder brother, was one of the chief men of this company. In 1753
Washington himself went west to the Ohio river. Day by day the
French were taking a firmer hold of that country, and Dinwiddie, the
old Scottish governor of Virginia, looked about for some one to carry
a warning letter to the commander of one of their new forts. The
messenger was also to keep his eyes open and report what the
French were doing on the upper waters of the Ohio. He chose
Washington, saying, “Faith, you’re a brave lad, and, if you play your
cards well, you shall have no cause to repent your bargain.”
Washington did not wait, but left on the day he received his
commission, late in October, 1753.
Christopher Gist, a famous frontiersman, was secured as guide,
and we can have no doubt that he and Washington formed a team,
ready to meet Frenchmen, red men, and the dangers of river and
forest. They made up their little party where the city of Cumberland,
Maryland, now stands. It is far up the Potomac, in the heart of the
mountains,—a long way beyond the Blue Ridge and the lands where
Washington had been surveying.
 At this place a large stream called Wills creek cuts through one
of the mountain ridges by a deep gorge and enters the Potomac. On
a hill, where these streams come together, was Fort Cumberland,
the great outpost of Virginia and Maryland. A fine church now stands
on the ground of the old fort, in the heart of the busy city of
Cumberland. This was the starting point for Washington’s expedition
and for many later ones into the western wilderness.
Washington made his dangerous journey with success. He
brought back a letter from the French commander, but of much
greater value was the story of all that he had seen. The colonists
now knew just what they would have to do to keep possession of the
Ohio lands.
It was not long before Washington went again as commanding
officer of a small army, and in 1755 he served under General
Braddock in the famous battle which resulted in the defeat of the
English and the death of their general. Washington, as we know,
brought off the troops with honor to himself. In each of these
expeditions something was done toward cutting away the trees and
grading a road from Fort Cumberland to the head of the Ohio river at
Pittsburg.
 Fig. 34. Milestone on the
Line of Braddock’s
Road, near Frostburg,
Maryland

On the line of Braddock’s road, a dozen miles west of

Cumberland, is a milestone, set up about a hundred and fifty years
ago. A photograph of it is shown above. It is a rough brown stone,
standing in a pasture half a mile outside the city of Frostburg, in
western Maryland. The stone was once taken away and broken, but
it has since been set up again and cemented into a base of concrete.
The view shows how it has been split up and down. On one side are
directions, and on the other are the words, “Our Country’s Rights We
Will Defend.”
Braddock’s journey from Alexandria to Fort Duquesne was an
uncomfortable one, to say nothing of its disastrous end. He bought a
carriage to ride in, but the road was not suited to a coach, as were
the roads he knew in old England. Beyond Cumberland, especially,
in spite of all the work his men could do upon it, it was so bad that he
was forced to take Washington’s advice and change the baggage
from wagons to pack horses.
 Gradually, as time went on, these rough paths were beaten down
into smoother thoroughfares. The same causes that led to the
development of the North were working also at the South. Along the
Potomac, as in New York and in Pennsylvania, the stream of colonial
life flowed westward. First the pioneers settled the lowlands around
Chesapeake bay and along the deep rivers; then as their strength
and courage reached beyond the mountains they found the forests
and fertile soil behind the Blue Ridge. Farther within the rugged
highlands they built Fort Cumberland and sent out discoverers and
armies to the Ohio river. When the woods were cleared and towns
and states grew up on the Ohio, there was frequent occasion to
cross the mountains for trade, for travel, and to reach the seat of
government, which in 1801 was moved to Washington on the
Potomac.

Fig. 35. Old Road House, Brownsville, Pennsylvania

These glimpses of colonial journeys will help us to understand
why the National Road came to be built. About one hundred years
ago the government began to take a great interest in opening roads,
especially across the Appalachian mountains, to Ohio, Kentucky,
and other parts of the Mississippi valley. Washington, who died in
1799, had said much about this work, for he not only wanted western
trade to come to Virginia instead of going to New Orleans, but he
also felt that so long as the mountains kept the East and the West
apart we should never have one common country, held together by
friendly feelings.
The people of Baltimore, like those of New York and
Philadelphia, were eager to have the best road to the West, that their
business might be benefited. Not far from Baltimore is an old place
called Joppa, and several roads are still known as “Joppa roads.”
The town is older than Baltimore and was once the chief trading
town in the northern part of Maryland; but Baltimore was well
situated on an arm of the great bay, and by this time had gone far
ahead of its old rival.
A number of good roads had been built in Maryland, among
them a famous one leading out westward to Frederick. This was in
the direction of Hagerstown, and still farther west was Cumberland.
The United States government decided to build a great road to Ohio,
beginning at Cumberland. To get the benefit of this, the men of
Baltimore went to work to push the Frederick pike westward to the
beginning of the National Road.
So it came about in 1811 that the first contracts were let for
building parts of the National Road. We remember that the Erie
canal was not started until six years later. The act of Congress which
ordered the making of the road provided that a strip four rods wide
should be cleared of trees, that it should be built up in the middle
with broken stone, gravel, or other material good for roads, and that
all steep slopes should be avoided. The road was opened to the
public in 1818, one year after the Erie canal was begun. The original
plan was to make it seven hundred miles long, reaching from
Cumberland to the Mississippi river, but it was never carried out.
The Maryland roads, as we have seen, ran west from Baltimore
and Washington to Frederick, east of the Blue Ridge; to Hagerstown,
in the Great Valley; and to Cumberland, in the mountains.
Cumberland is a stirring town of about twenty thousand people, and
with its great business in coal, iron, and railroads it seems like a
larger city. Thence the National Road runs through the gap in Wills
mountain (Fig. 36) to Frostburg, a dozen miles west and fifteen
hundred feet higher. The road soon bears northward into
Pennsylvania and crosses the Monongahela river at Brownsville,
about forty miles south of Pittsburg. Coal is mined here, and boats
were running in those early days, as coal barges and steamboats
run to-day, down to the great iron city.
From Brownsville the pike leads over the hills and comes down
to the Ohio river at Wheeling, West Virginia. It then passes on
through Ohio, touching Columbus, the capital, on the way to Indiana
and the Mississippi.
We sometimes admire the cars marked with the sign of the
United States post office, which we see drawn by a swift locomotive
at a speed of sixty miles an hour; but when the government put its
mail coaches on the National Road from Washington to Wheeling, no
doubt they seemed quite as wonderful to the people of that time. And
it was only twenty-five years since the people of Utica had thought it
so remarkable that six letters had come to them in one mail! Soon
passenger coaches were rushing along at ten miles an hour, and
sometimes even faster. There were canvas-covered freight wagons,
each of which carried ten tons, had rear wheels ten feet high, and
was drawn by twelve horses. In those days life was full of stirring
interest on the National Road.
 Fig. 36. Cumberland and the Gap in Wills Mountain
There were rates of toll for all sorts of animals and wagons. The
toll was higher for hogs than for sheep, and more was charged for
cattle than for hogs. If the wagons had very wide tires, no toll was
demanded. Drivers sometimes lied about the number of people in
their stages, so as to pay less toll. The stages were not owned by
the drivers but by companies, which bid for travelers and freight, as
railways do now. There were penalties for injuring milestones or
defacing bridges, showing that some people then were like some
people now. The companies had interesting names. There were the
“Good Intent,” “Ohio National Stage Lines,” the “Pilot,” “Pioneer,”
“June Bug,” and “Defiance.” Not one of them cared for mud or dust,
for horses or men, if only it could be the first to reach its destination.
There must have been dust enough, for twenty coaches with their
many horses sometimes followed one another in a close line.
 Fig. 37. Bridge and Monument, National Road, near
Wheeling, West Virginia
Henry Clay was one of the chief advocates of this road, and a
monument built in his honor may be seen near the bridge, shown in
Fig. 37. It is a few miles east of Wheeling. At Brownsville a small
stream called Dunlap’s creek flows into the Monongahela from the
east. Over it is an iron bridge on the line of the National Road.
According to a story told in Brownsville, Henry Clay was once
overturned as he was riding through the creek before the bridge was
built. As he gathered himself up he was heard to say, “Clay and mud
shall not be mixed here again.” The story goes that he went on
immediately to Washington and got an order for the building of the
bridge.
Whether this be true or not, it is certain that he and many other
statesmen traveled over the National Road. They could not have
private cars, nor did they go in drawing-room coaches, as we can if
we choose. Anybody might chance to sit beside these men of
national fame, as day after day they rode through the valleys and
over the mountains, stopping at the wayside hotels for food and rest.
Some of the old hotels, tollhouses, and bridges, as they look to-
day, are shown in the illustrations in this chapter. The road itself was
long ago given up to the different states and counties through which
it runs, but it still tells to the traveler who goes over it many a story of
the life of a hundred years ago.
 CHAPTER IX
THE BALTIMORE AND OHIO RAILROAD

Even after the Erie canal was built and long lines of boats were
carrying the grain and other products of the West to New York, the
men of Virginia and Maryland did not give up the notion of still
making the trade of the western country come their way. They
planned the Chesapeake and Ohio canal, to reach the Ohio river,
and thought that other canals across the state of Ohio would let them
into lake Erie. By the Ohio river they would connect with New
Orleans and the upper Mississippi river, and through lake Erie they
could reach the towns and farms that border lake Huron, lake
Michigan, and lake Superior.
A canal along the Potomac valley had been talked of several
years before the Revolution, when Richard Henry Lee laid a plan for
it before the Assembly of Virginia. Doubtless others thought of it too,
as of the Erie canal, long before it was made. At the end of the War
of the Revolution Washington made a long journey into the wild
woods of New York. He went to the source of the Susquehanna at
Otsego lake, visited the portage between the Mohawk and Wood
creek, and saw for himself that New York had a great chance for
navigation and trade. But he had a natural love for his own Virginia,
and he did not intend to let New York go ahead of his native state.
His journeys across the mountains as a surveyor and as a soldier
gave him a knowledge of the Ohio country, and as he had himself
taken up much good land there, he wished to have an easy way, by
land or water, from the sea to the rich Ohio valley. So he thought
much about a canal to run by the side of the Potomac, and he joined
with others who felt as he did to form the Potomac Company. They