Biodiesel Production: Technologies, Challenges, and Future Prospects

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Biodiesel
Production
Technologies, Challenges, and Future Prospects
Biodiesel Production: Technologies, Challenges,

and Future Prospects Task Committee
EDITED BY
R. D. Tyagi, Rao Y. Surampalli,

Tian C. Zhang, Song Yan,
and Xiaolei Zhang
Biodiesel Production
Technologies, Challenges, and Future
Prospects
Sponsored by
Biodiesel Production: Technologies, Challenges, and Future Prospects Task
Committee of the Technical Committee on Hazardous, Toxic, and Radioactive
Waste Engineering of the Environmental Council of the Environmental and
Water Resources Institute of the American Society of Civil Engineers
Edited by
R. D. Tyagi
Rao Y. Surampalli
Tian C. Zhang
Song Yan
Xiaolei Zhang
Published by the American Society of Civil Engineers

Library of Congress Cataloging-in-Publication Data
Names: Tyagi, R. D., 1952– editor.
Title: Biodiesel production : technologies, challenges, and future prospects / sponsored by
Biodiesel production: Technologies, Challenges, and Future Prospects Task Committee
of the Technical Committee on Hazardous, Toxic, and Radioactive Waste Engineering

of the Environmental Council of the Environmental and Water Resources Institute
of the American Society of Civil Engineers; edited by R. D. Tyagi, Rao Y. Surampalli,
Tian C. Zhang, Song Yan, Xiaolei Zhang.
Description: Reston, Virginia : American Society of Civil Engineers, [2019] | Includes
bibliographical references.
Identifiers: LCCN 2019017462 | ISBN 9780784415344 (print : alk. paper) | ISBN
9780784482285 (pdf)
Subjects: LCSH: Biodiesel fuels.
Classification: LCC TP359.B46 B5637 2019 | DDC 665/.37–dc23
LC record available at https://lccn.loc.gov/2019017462
Published by American Society of Civil Engineers
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Copyright © 2019 by the American Society of Civil Engineers.
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ISBN 978-0-7844-1534-4 (print)
ISBN 978-0-7844-8228-5 (PDF)
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Cover image courtesy of Arslan Enginery
Contents
Preface................................................................................................................................................ ix
Contributing Authors .................................................................................................................. xi
About the Editors........................................................................................................................xiii
Chapter 1 Biodiesel Basics ....................................................................................................1

1.1 Biodiesel History ............................................................................................................. 1
1.2 Properties of Biodiesel................................................................................................. 2
1.3 Biodiesel Standards.......................................................................................................5
1.4 Biodiesel versus Other Biorefinery Products ....................................................5
1.5 Biodiesel Blending, Storage, and Transport ..................................................... 9
1.6 Performance of Biodiesel as a Fuel and Demonstration of
its Usage .......................................................................................................................... 12
References............................................................................................................................... 13
Chapter 2 Biodiesel: Variations, Properties, and Comparison with

Diesel........................................................................................................................15
2.1 Introduction ................................................................................................................... 15
2.2 Variations in Biodiesel............................................................................................... 16
2.3 Physicochemical Aspects......................................................................................... 17
2.4 Contaminants in Biodiesel...................................................................................... 22
2.5 Particulate Emissions ................................................................................................. 23
2.6 Conclusions .................................................................................................................... 24
References............................................................................................................................... 24
Chapter 3 Biodiesel Production by Transesterification ...................................27

3.1 Introduction ................................................................................................................... 27
3.2 Transesterification of Oil/Fat to Biodiesel....................................................... 28
3.3 Transesterification of Oil-Bearing Substances to Biodiesel.................... 39
3.4 Biodiesel Production.................................................................................................. 41
3.5 Summary.......................................................................................................................... 44
3.6 Acknowledgments ...................................................................................................... 47
References............................................................................................................................... 47
Chapter 4 Enzyme-Catalyzed Transesterification for Biodiesel

Production ............................................................................................................53
4.1 Introduction ................................................................................................................... 53
4.2 Feedstocks and General Methods for Biodiesel Production................. 54
4.3 Enzyme-Catalyzed Transesterification for Biodiesel Production.......... 58
iii
iv CONTENTS
4.4 Cost Analysis of Immobilized Lipase-Based Transesterification .......... 80

4.5 Summary.......................................................................................................................... 81
References............................................................................................................................... 81
Chapter 5 Plant Oil to Biodiesel.....................................................................................89

5.1 Introduction ................................................................................................................... 89
5.2 Feedstocks for Biodiesel Production ................................................................. 90
5.3 Physical and Chemical Properties of Plant Oils........................................... 92
5.4 Transesterification Processes of Plant Oils ..................................................... 94
5.5 Challenges in Biodiesel Production from Plant Oils ...............................113
5.6 Future Work and Prospects .................................................................................114
5.7 Summary........................................................................................................................114
References.............................................................................................................................116
Chapter 6 Animal Fat Biodiesel ................................................................................... 123

6.1 Introduction .................................................................................................................123
6.2 Sources of Animal Fats for Biodiesel Production .....................................124
6.3 Comparative Studies of Free Fatty Acids .....................................................133
6.4 Effects of Metals on Tallow, Lard, Poultry, and Fish Fat.......................137
6.5 Conversion of Animal Fats into Biodiesel Using Charcoal
and CO2 .........................................................................................................................139
6.6 Measuring the Economic Impact of Animal Fat Biodiesel...................140
6.7 Pros and Cons of Animal Fat Biodiesel .........................................................142
6.8 Summary........................................................................................................................143
6.9 Acknowledgments ....................................................................................................144
References.............................................................................................................................144
Chapter 7 Biodiesel from Waste Cooking Oil ..................................................... 151

7.1 Introduction .................................................................................................................151
7.2 Sources of Waste Cooking Oil............................................................................152
7.3 Biodiesel from Waste Cooking Oil....................................................................152
7.4 Comparison between Waste Cooking Oil and Virgin Oil .....................166
7.5 Cost Analysis of Biodiesel from Waste Cooking Oil................................169
7.6 Summary........................................................................................................................171
References.............................................................................................................................171
Chapter 8 Microalgae Oil Biodiesel ........................................................................... 175

8.1 Introduction .................................................................................................................175
8.2 Microalgae for Biodiesel Production ...............................................................176
8.3 Impact Factors of Microalgae Production and Oil Accumulation ....180
8.4 Microalgae Cultivation Systems.........................................................................188
8.5 Microalgae Biomass Harvest................................................................................191
8.6 Possibility of Microalgae Biodiesel ...................................................................193
8.7 Summary........................................................................................................................193
References.............................................................................................................................193
CONTENTS v
Chapter 9 Single Cell Oil Biodiesel............................................................................ 201

9.1 Introduction .................................................................................................................201
9.2 Characteristics of Single Cell Oil .......................................................................202
9.3 Oleaginous Microorganisms for Single Cell Oil Production ................204
9.4 Substrates Utilized for Single Cell Oil Production....................................204

9.5 Factors Affecting Production of Single Cell Oil.........................................208
9.6 Degradation of Lipids in Carbon Limitation ...............................................210
9.7 Biochemistry of Single Cell Oil Production in Yeast...............................212
9.8 Genetic Engineering for Single Cell Oil Production................................217
9.9 Cost and Economic Consideration in Single Cell Oil Production.....218
9.10 Challenges and Prospects ..................................................................................220
9.11 Summary.....................................................................................................................221
References.............................................................................................................................222
Chapter 10 Biodiesel Production from Oleaginous Microorganisms

with Organic Wastes as Raw Materials ........................................ 229
10.1 Introduction...............................................................................................................229
10.2 Organic Wastes for Oleaginous Microorganism Cultivation.............230
10.3 Parameters Affecting Lipid Accumulation .................................................243
10.4 Case Studies ..............................................................................................................251
10.5 Challenges and Future Perspectives.............................................................252
10.6 Summary.....................................................................................................................253
10.7 Acknowledgments..................................................................................................253
References.............................................................................................................................253
Chapter 11 Oil Extraction Using Wastewater Sludge .................................... 263

11.1 Introduction...............................................................................................................263
11.2 Methods of Sludge Disposal.............................................................................264
11.3 Sludge Characterization ......................................................................................266
11.4 Valuable Products from Wastewater Sludge ............................................266
11.5 Recent Studies on Oil Extraction Using Wastewater Sludge ...........266
11.6 Advantages and Disadvantages of Different Oil Extraction
Processes.....................................................................................................................277
11.7 Comparison of Sludge-Based Oil with Other Oils .................................277
11.8 Techno-Economic Evaluation............................................................................279
11.9 Recent Advancements and Future Perspectives ....................................283
11.10 Conclusions .............................................................................................................284
References.............................................................................................................................285
Chapter 12 Biodiesel Production Using Fermented Wastewater

Sludge–Derived Lipids............................................................................. 289
12.1 Introduction...............................................................................................................289
12.2 Characteristics of Wastewater Sludge..........................................................290
12.3 Biodiesel from Wastewater Sludge–Derived Lipids ..............................291
12.4 Factors Affecting Lipid Production Using Wastewater Sludge .......293
vi CONTENTS
12.5 Lipid Accumulation in Oleaginous Microorganisms Fed with

Sludge ..........................................................................................................................296
12.6 Challenges and Future Perspectives.............................................................298
12.7 Summary.....................................................................................................................300
References.............................................................................................................................300
Chapter 13 Conversion of Crude Glycerol to Lipid and Biodiesel ........ 305

13.1 Introduction...............................................................................................................305
13.2 Characteristics and Composition of Crude Glycerol.............................307
13.3 Rationale for Use of Crude Glycerol in Lipid Production ..................308
13.4 Metabolism for Glycerol Uptake .....................................................................309
13.5 Recent Studies on Lipid Production from Crude Glycerol................313
13.6 Analysis of Studies Reported in Literature................................................325
13.7 Major Findings from Previous Studies and Future Perspectives ...333
13.8 Summary.....................................................................................................................336
References.............................................................................................................................336
Chapter 14 Lignocellulosic Biomass: The Future Renewable

Low-Cost Carbon Source for Microbial Lipid
Production....................................................................................................... 341
14.1 Introduction...............................................................................................................341
14.2 Lipid Production from Lignocellulosic Biomass ......................................342
14.3 Fermentation.............................................................................................................345
14.4 Types of Fermentation for Lipid Production............................................348
14.5 Pretreatment Inhibitors and Their Effect on Microbial
Growth and Lipid Accumulation ....................................................................352
14.6 Summary.....................................................................................................................355
References.............................................................................................................................355
Chapter 15 Lipid Extraction Technologies............................................................ 359

15.1 Introduction...............................................................................................................359
15.2 Cell Disruption .........................................................................................................360
15.3 Physical Technologies of Lipid Separation................................................363
15.4 Chemical Technologies of Lipid Separation .............................................365
15.5 Summary.....................................................................................................................377
References.............................................................................................................................377
Chapter 16 Milking of Lipids from Oleaginous Microorganisms ........... 383

16.1 Introduction...............................................................................................................383
16.2 Microbial Potential for Lipid Production ....................................................385
16.3 Milking Methods to Extract Lipids.................................................................387
16.4 Summary and Future Prospects......................................................................392
References.............................................................................................................................393
CONTENTS vii
Chapter 17 Application of Nanotechnology in Biodiesel

Production....................................................................................................... 397
17.1 Introduction...............................................................................................................397
17.2 Lipid Production Using Nanotechnology ...................................................398
17.3 Lipid Extraction Using Nanotechnology .....................................................399

17.4 Nanotechnology in Selective Purification of Hydrocarbons
from Oil .......................................................................................................................400
17.5 Nanotechnology in the Transesterification Reaction ...........................402
17.6 Conclusions................................................................................................................416
References.............................................................................................................................417
Chapter 18 Genetic/Metabolic Engineering and Synthetic Biology

Applications to Improve Single Cell Oil Accumulation...... 421
18.1 Introduction...............................................................................................................421
18.2 Basic Gene Transfer Mechanisms...................................................................422
18.3 Advances in Genetic Engineering Tools.....................................................425
18.4 Multiple Knowledge Needed for Planning the Genetic
Engineering................................................................................................................426
18.5 Genetic Engineering Strategies for Lipid Production ..........................430
18.6 Genetic Engineering in Algae ..........................................................................437
18.7 Improvement in Biodiesel Production by Genetic Engineering.....440
18.8 Importance of Gene Constructs and Synergy of Operons................442
18.9 Challenges in Designing Metabolic Pathways.........................................443
18.10 Summary and Future Prospects ...................................................................445
References.............................................................................................................................446
Chapter 19 Recovery and Purification Technologies of Biodiesel......... 453

19.1 Introduction...............................................................................................................453
19.2 Biodiesel Separation Techniques....................................................................455
19.3 Biodiesel Purification Techniques...................................................................458
19.4 Quality Testing of Biodiesel ..............................................................................473
19.5 Storage, Stability, and Transportation..........................................................477
19.6 Future Work and Prospects...............................................................................478
19.7 Summary.....................................................................................................................479
References.............................................................................................................................480
Chapter 20 Purification of Biodiesel Using Resins and Adsorbents .... 485

20.1 Introduction...............................................................................................................485
20.2 Recent Case Studies..............................................................................................487
20.3 Comparative Studies among Adsorbents, Ion-Exchange
Resins, and Wet-Washing Methods...............................................................494
20.4 Research on Improvement of Ion-Exchangers and Absorbents
for Biodiesel Purification.....................................................................................498
20.5 Conclusions................................................................................................................501
References.............................................................................................................................501
viii CONTENTS
Chapter 21 Management of Coproducts from Biodiesel Production ..... 503

21.1 Introduction...............................................................................................................503
21.2 Bioresidue Generated after Harvest and Extraction of Oil
from Agricultural Biomass..................................................................................504
21.3 Coproduct Management.....................................................................................508

21.4 Biorefinery Concept...............................................................................................517
21.5 Conclusions................................................................................................................518
References.............................................................................................................................519
Chapter 22 Economic Evaluation of Biodiesel Production

Processes.......................................................................................................... 527
22.1 Historical Account of Biodiesel Industry Development ......................527
22.2 Necessity of Techno-Economic Evaluations ..............................................529
22.3 Evaluations with Vegetable Oil and Waste Cooking Oil ....................531
22.4 Techno-Economic Studies with Algal Oil ...................................................535
22.5 Advent of Single Cell Oil and Techno-Economic Shortcomings....538
22.6 Cost Considerations in Downstream Processing....................................544
22.7 Cost Consideration in the Transesterification Process ........................547
22.8 Post-Transesterification Purification Cost Consideration ....................550
22.9 Evaluation of Economic Feasibility ................................................................565
22.10 Global Biodiesel Economy and Roadmap Ahead................................574
22.11 Conclusions .............................................................................................................577
References.............................................................................................................................578
Chapter 23 Biodiesel Impact on Environment and Health ........................ 583

23.1 Introduction...............................................................................................................583
23.2 Effect on Air Quality..............................................................................................584
23.3 Effect on Water Resources.................................................................................601
23.4 Effect on Soil.............................................................................................................612
23.5 Effect on Biodiversity............................................................................................618
23.6 Greenhouse Gas Emissions................................................................................624
23.7 Public Safety and Health ....................................................................................628
23.8 Challenges ..................................................................................................................639
23.9 Summary.....................................................................................................................639
23.10 Acknowledgments ...............................................................................................640
References.............................................................................................................................641
Chapter 24 Biodiesel: Socioeconomic and Political Aspects ..................... 653

24.1 Introduction...............................................................................................................653
24.2 Policy Impetus to Biodiesel Production in Various Countries .........653
24.3 The Oil Sector: Its Political Power and Sensitivity .................................656
24.4 Social and Environmental Aftermath of Biodiesel Production........659
24.5 Conclusions................................................................................................................661
References.............................................................................................................................662
Index.............................................................................................................................................. 665
Preface
As industrialization and population are increasing, the need for energy is also
increasing. Petroleum, coal, natural gas, nuclear, and hydro are the basic available
sources for this energy. Petroleum diesel is being used as a major fuel worldwide.
However, using petroleum-based fuels possesses many disadvantages, including that
fossil fuel reserves are decreasing because of increased energy consumption, and
atmospheric pollution is also increasing because the combustion of petroleum diesel
leads to emissions of greenhouse gases (GHGs) and air contaminants including SOx,
NOx, CO, volatile organic compounds (VOCs), and particulate matters. These
pollutants have significant negative impacts to the global environment and human
health. Therefore, it is imperative to develop alternate fuels and renewable sources of
energy that are environmentally sustainable, with biodegradability, low toxicity,
renewability, and less reliance on petroleum products. One of these energy sources is
biodiesel. Over the last few years, biodiesel has obtained the political and scientific
acknowledgment owing to its major benefits over petroleum diesel, including (1)
significant reduction in GHG emissions, (2) nonsulfur emissions, (3) nonparticulate
matter pollutants, (4) low toxicity, (5) biodegradable, and (6) obtainable from
renewable sources like vegetable oils, animal fat, and others. Therefore, biodiesel can
complement petroleum-based fuels.
This book presents approaches, technologies, and source materials for biodiesel
production, as well as socioeconomic and political impacts of biodiesel applica-
tions. Chapter 1 introduces biodiesel basics, the history, its composition, physi-
cochemical properties, and standards for biodiesel. Chapter 2 compares the
biodiesel with petroleum diesel. Chapters 3 and 4 focus on the general methods
for biodiesel production. The major part of this book includes several chapters
addressing applications of various raw materials in biodiesel production, including
plant oils (Chapter 5), animal fat (Chapter 6), waste cooking oil (Chapter 7), algal
oil (Chapter 8), single cell oil (Chapter 9), organic waste (Chapter 10), wastewater
sludge (Chapters 11 and 12), crude glycerol (Chapter 13), and lignocellulosic
biomass (Chapter 14). Chapter 15 discusses various technologies involved in the
extraction of lipids from the oil-bearing materials. Chapter 16 addresses the
oleaginous microbes involved in lipid milking. Chapters 17 and 18 focus on
applications of nanotechnology and genetic engineering in biodiesel production,
respectively. Chapter 19 discusses various conventional methods for purification
of biodiesel, whereas Chapter 20 focuses on the use of resins and adsorbents for its
purification. The last part of the book consists of four chapters (Chapters 21 to 24).
Chapter 21 addresses the management of coproducts formed during the process of
biodiesel production. Chapter 22 presents the economic evaluation of biodiesel
production processes. Chapter 23 discusses the impact of biodiesel on the
ix
x PREFACE
environment and human well-being, and Chapter 24 presents socioeconomic and

political aspects of biodiesel. This book will help the readers readily find the
information they are looking for.
The editors acknowledge the hard work and patience of all authors who have
contributed to this book.

RDT, RYS, TCZ, SY, XLZ
Editors
Contributing Authors
G. S. Anisha, Government College, Chittur, Palakkad, India

Bharti Bhadana, INRS, Université du Québec, Québec, QC, Canada
I. Bhattacharya, Summerfresh Co. Ltd., Woodbridge, ON, Canada
V. Cheernam, Sardar Patel College of Engineering, Mumbai, India
J. Chen, INRS, Université du Québec, Québec, QC, Canada
W. Dong, Harbin Institute of Technology (Shenzhen), Shenzhen, China
P. Drogui, INRS, Université du Québec, Québec, QC, Canada
J. P. Hettiaratchi, University of Calgary, Calgary, AB, Canada
R. P. John, INRS, Université du Québec, Québec, QC, Canada
R. Kaur, INRS, Université du Québec, Québec, QC, Canada
L. Kumar, INRS, Université du Québec, Québec, QC, Canada
L. R. Kumar, INRS, Université du Québec, Québec, QC, Canada
M. Kuttiraja, Mara Renewables Corporation, Dartmouth, NS, Canada
J. Li, Harbin Institute of Technology (Shenzhen), Shenzhen, China
T. T. More, University of Calgary, Calgary, AB, Canada
H. Panidepu, Indian Institute of Technology, New Delhi, India
S. Pilli, National Institute of Technology, Warangal, India
S. K. Ram, INRS, Université du Québec, Québec, QC, Canada
B. Sellamuthu, INRS-Institut Armand-Frappier, Laval, QC, Canada
R. Y. Surampalli, University of Nebraska–Lincoln, Lincoln, NE, USA
B. Tiwari, INRS, Université du Québec, Québec, QC, Canada
R. D. Tyagi, INRS, Université du Québec, Québec, QC, Canada
Z. Wu, Harbin Institute of Technology (Shenzhen), Shenzhen, China
J. S. S. Yadav, Research Scientist, BioVectra, Charlottetown, PEI, Canada
S. Yan, INRS, Université du Québec, Québec, QC, Canada
S. K. Yellapu, INRS, Université du Québec, Québec, QC, Canada
Tian C. Zhang, University of Nebraska–Lincoln, Lincoln, NE, USA
X. L. Zhang, Harbin Institute of Technology (Shenzhen), Shenzhen, China
xi
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About the Editors
R. D. Tyagi, Ph.D., is an internationally recognized professor at Institut National

de la Recherche Scientifique—Eau, Terre, et Environnement (INRS-ETE), Uni-
versité du Québec, Canada. He holds Canada Research Chair (senior) on
“Bioconversion of wastewater and wastewater sludge to value added products.”
Professor Tyagi is the member of Hall of Excellence of University of Quebec
(Canada) and is also a member of European Academy of Sciences and Arts;
Academician. He conducts research on hazardous/solids waste management,
water/wastewater treatment, sludge treatment/disposal, and bioconversion of
wastewater and wastewater sludge into value-added products. He has developed
the novel technologies of simultaneous sewage sludge digestion and metal
leaching, bioconversion of wastewater sludge (biosolids) into Bacillus thuringien-
sis–based biopesticides, bioplastics, biofertilizers, and biocontrol agents. He is
recipient of the Outstanding Scientist Award of The International Bioprocessing
Association (an International Forum on Industrial Bioprocesses) for significant
contributions in the area of environmental biotechnology. He is also a recipient of
the ASCE State-of-the-Art of Civil Engineering Award, ASCE Rudolph Hering
Medal, ASCE Wesley W. Horner Medal, and ASCE Best Practice-Oriented Paper
Award. He received the Superior Award, Excellence in Environmental Engineer-
ing Award of American Academy of Environmental Engineers in 2015. He also
received 2010 Global Honor Award (applied research) of International Water
Association (IWA), 2010 Grand Prize (university research) of American Academy
of Environmental Engineering, Life-Time Achievement Award (Specialist medal,
2007) for outstanding research contributions in wastewater sludge management
by the IWA and Excellence Award of Natural Sciences and Engineering Research
Council of Canada for industry-university collaborative research. Dr. Tyagi has
either published or presented more than 600 papers in refereed journals and
conference proceedings and is the author of 13 books, 80 book chapters, 10
research reports, and nine patents.
Dr. Rao. Y. Surampalli, Ph.D., P.E., Dist.M.ASCE, received his M.S. and Ph.D.
degrees in Environmental Engineering from Oklahoma State University and Iowa
State University, respectively. He is a Registered Professional Engineer in the
branches of Civil and Environmental Engineering, and also a Board Certified
Environmental Engineer (BCEE) of the American Academy of Environmental
Engineers and Scientists (AAEES) and a Diplomate of the American Academy of
Water Resources Engineers (D.WRE). He is an Adjunct Professor in five univer-
sities and distinguished/honorary visiting professor in five universities. Currently,
he serves, or has served, on more than 70 national and international committees,
review panels, or advisory boards including the ASCE National Committee on
xiii
xiv ABOUT THE EDITORS
Energy, Environment and Water Policy. He is a Distinguished Engineering

Alumnus of both the Oklahoma State and Iowa State Universities, and is an
elected Fellow of the American Association for the Advancement of Science, an
elected Member of the European Academy of Sciences and Arts, an elected
Member of the Russian Academy of Engineering, an elected Fellow of the Water

Environment Federation and International Water Association, and a Distin-
guished Member of the American Society of Civil Engineers. He also is Edi-
tor-in-Chief of the ASCE Journal of Hazardous, Toxic, and Radioactive Waste, and
past Vice-Chair of Editorial Board of Water Environment Research journal. He has
authored more than 600 technical publications in journals and conference
proceedings, including more than 300 refereed journal articles, 15 patents, 20
books, and 136 book chapters.
Dr. Tian C. Zhang, Ph.D., P.E., is a professor in the Department of Civil
Engineering at the University of Nebraska–Lincoln (UNL). He received his Ph.D.
in environmental engineering from the University of Cincinnati in 1994 and
joined the UNL faculty in August 1994. Professor Zhang teaches courses related to
water/wastewater treatment, remediation of hazardous wastes, and non-point
pollution control. Professor Zhang’s research involves fundamentals and applica-
tions of nanotechnology and conventional technology for water, wastewater, and
stormwater treatment and management, remediation of contaminated environ-
ments, and detection/control of emerging contaminants in the environment.
Professor Zhang has published more than 120 peer-reviewed journal papers,
72 books chapters, and 14 books since 1994. Professor Zhang is a Diplomate of
Water Resources Engineer (D.WRE) of the American Academy of Water
Resources Engineers, a Board Certified Environmental Engineer (BCEE) of the
American Academy of Environmental Engineers, an elected Fellow of American
Society of Civil Engineers (F.ASCE), an elected Fellow of American Association
for the Advancement of Science (F.AAAS), and an elected member of European
Academy of Sciences and Arts (EASA). Professor Zhang is an Associate Editor of
Journal of Environmental Engineering (since 2007), Journal of Hazardous, Toxic,
and Radioactive Waste (since 2006), and Water Environment Research (since
2008). He has been a registered professional engineer in Nebraska since 2000.
Dr. Song Yan. Ph.D., is currently working as a research associate at Institut
National de la Recherche Scientifique—Eau, Terre, et Environnement (INRS-
ETE), Université du Québec, Canada. She received her Ph.D. in Water Science
from INRS, University of Quebec. Dr. Yan’s research focuses on the production of
bioplastics, biodiesel, value-added products using wastewater and sludge (bioso-
lids), greenhouse gas emissions from wastewater and sludge treatment processes;
and wastewater and sludge treatment process control. Dr. Yan has published 76
papers in refereed journals and presented 60 papers in national and international
conferences. She is author of two books, 40 book chapters, and many scientific
reports. Dr. Yan was part of the team that received the Superior Achievement
Award for Excellence in Environmental Engineering and Science in 2010 and 2015
from American Academy of Environmental Engineers and Scientists (AAEES) for
University Research work. She was selected as an Outstanding Reviewer of ASCE’s
ABOUT THE EDITORS xv
Journal of Environmental Engineering in 2012 and holds membership in Canadian

Association for Water Quality (CAWQ) and International Water Association
(IWA).
Dr. Xiaolei Zhang, Ph.D., received her doctorate degree in Water Science from
INRS University of Québec in 2013. She is currently working at the Harbin

Institute of Technology (Shenzhen) as assistant professor. Her research work
focuses on bioconversion of municipal waste and industrial organic waste to
bioenergy; green technology for bioenergy generation; and techno-economic
assessment of bioenergy production. Dr. Zhang was part of the team that received
the Superior Achievement Award for Excellence in Environmental Engineering
and Science (the highest award of the 2015 competition) from American Academy
of Environmental Engineers and Scientists (AAEES) for University Research work
on “Bioconversion of Wastewater Sludge and Glycerol to Biodiesel.” Dr. Zhang
has published more than 20 peer-reviewed journal papers, nine book chapters, and
many conference presentations.

CHAPTER 1
Biodiesel Basics
R. Kaur
S. K. Yellapu
Bharti Bhadana
L. R. Kumar
R. D. Tyagi
Rao Y. Surampalli
Tian C. Zhang
1.1 BIODIESEL HISTORY
In the 1890s, Rudolf Diesel invented the diesel engine in Germany. The engine can
run by using different vegetable oils and fuels. At the Paris Exposition in 1900,
very few people were aware of the peanut oil–based new diesel engine. However,
because of very low-cost fossil fuels, the demand for vegetable oil as a fuel
decreased. During World War II, several countries were so curious to use
vegetable oil–based fuel in internal combustion engines because of the very high
viscosity of vegetable oil. In the 1930s, the process was developed to break down
fatty acids from glycerol to reduce viscosity and to produce thinner product with
properties similar to petroleum diesel. In 1937, the first patent was filed on
biodiesel (Anastasov 2009) and a Belgian patent was granted for the work on
conversion of palm to ethyl esters, which is known as biodiesel. The term biodiesel
means the monoalkyl esters of long-chain fatty acids from different feedstocks
such as plant oil, animal fats, and others. The first biodiesel automobile was routed
between Brussels and Louvain (Knothe 2005).
The word biodiesel was first used about 1984 (Van Gerpen et al. 2004), and
the first manufacturing plant was started at Austria Agricultural College in 1985.
Later, biodiesel production was well established in Europe where Germany is the
largest producer. In the United States, biodiesel was first commercially produced
in Kansas City in 1991. The University of Idaho in 1995 supplied biodiesel to
trucks in Yellowstone National Park, and they traveled several miles without
damage to engines (Pahl 2008). Because biofuels such as biodiesel help reduce
1
2 BIODIESEL PRODUCTION
environmental pollution, net carbon emissions, and dependence on fossil fuels,

nowadays governments are encouraging the use of biofuels and offering tax credits
for their use. Production of biodiesel has been increasing rapidly since 2004. There
are 200 biodiesel companies listed on national biodiesel boards. Worldwide, a
similar phenomenon of a rapid increase in biodiesel production was observed

from 1 billion liters in 2001 to 6 billion liters in 2006 (Pahl 2008).
The Energy Policy Act of 2005 was intended to reduce the need for imported
fossil fuels, and the US government turned up to purchase alternative fuels for
automobiles. When the US Department of Energy finalized biodiesel as an
altentaive fuel for automobiles without changing engine configuration, biodiesel
became an attractive option for some operators. Today, several feed stocks
(e.g., vegetable oil, nonedible plant oil, waste cooking oil, animals fats, and others)
are being utilized worldwide to produce biodiesel based on availabilty and cost.
Depending on the environment protection agency (EPA420-B-07-019) 2% to 20%
blended biodiesel is approved for use in automobiles.
1.2 PROPERTIES OF BIODIESEL
1.2.1 Chemical Composition

The constituents of biodiesel—in other words, carbon, hydrogen, oxygen, and C/H
ratio—are highly influenced by the feedstock and vary from one feedstock to
another. Biodiesel is mainly composed of fatty acid methyl and ethyl esters, and
their numbers depend on the feedstock from which biodiesel is produced. These
esters are made up of carbon, hydrogen, and oxygen atoms, which can form a linear
chain with single bonds called saturated fatty acids or a linear chain with double
bonds called unsaturated fatty acids. They are usually represented in the form of
Cnc:nd. Taking C18:2 as an example, nc (i.e., 18) is the number of carbon atoms,
and nd (i.e., 2) is the number of double bonds present in the fatty acid. C18:1, C18:2,
and C18:3 are present in the highest number followed by C18:0 (Barabás 2011).
Other properties of biodiesel are presented in the following sections.
1.2.2 Cetane Number

Cetane number (in the range of 15 to 100) is a dimensionless parameter or
indicator, which characterizes the diesel fuel quality. It is determined according to
ASTM D613 and is related to the ignition delay time a fuel experiences after
injection into the combustion chamber of a diesel engine. The shorter the delay
time of ignition, the higher the cetane number of the fuel. Reference hydrocarbon,
hexadecane, which is a linear chain molecule with the molecular formula being
C16H34, has short ignition delay and therefore has an assigned cetane number of
100. However, another hydrocarbon called 2,2,4,4,6,8,8-heptamethylnonane
(strongly branched chain molecule) with the same molecular formula (C16H34)
has long ignition delay and therefore has an assigned cetane number of 15 (Knothe
and Ryan 1997).
BIODIESEL BASICS 3
Diesel fuel with a very low cetane number makes it difficult to start the engine
at low temperatures, causes the engine to function improperly with incomplete
combustion, and can increase the pollution of the engine with hydrocarbon
emissions. Whereas diesel fuel with a very high cetane number can cause the
ignition to start even before completely mixing with the air, may also result in
incomplete combustion, and can further increase the exhaust smoke (Knothe
2002). Therefore, an optimal cetane number is required for the diesel fuel to
maintain the engine for a long time, and it should be between 41 and 56 but should
never be higher than 65.
Chain length, saturation or unsaturation of the molecule, as well as branching
affects the cetane number of the fuel. Higher cetane number has been observed for
the long-chain compounds (e.g., lauric and stearic acids) as well as for highly
unsaturated molecules like oleic, linoleic, and linolenic acids (Ali et al. 2013).
Cetane number determination for the monocylindrical engine is very expensive
via a lengthy procedure. Therefore, another alternative method, which is a cheaper
and faster procedure, is to derive the cetane number through ignition delay using a
constant column combustion chamber.
1.2.3 Oxidative Stability

Oxidative stability is important for any fuel because it prevents degradation
during storage while waiting for distribution as well as within the vehicle engine
(Van Gerpen et al. 2004). Oxidative stability determines the ability of fuel to resist
the chemical changes during long-term storage. Biodiesel stability is strongly
affected by oxidation during storage (owing to contact with air) and by hydrolytic
degradation (owing to contact with the water). Oxidative stability is highly
influenced by biodiesel composition because polyunsaturated fatty acids undergo
oxidation very fast and form hydroperoxides after oxidation, which can further
polymerize to form the larger molecules and may increase the viscosity of the fuel.
Oxidation has many disadvantages such as increasing the acid value, causing color
changes from yellow to brown, increasing the solid deposits in the engine, and
reducing the lubricity and heating value of the biodiesel. An increase in the acid
value because of hydrolysis of esters to long-chain fatty acids after contact with the
water can further cause the reverse transesterification reaction. Oxidation stability
is measured with the Rancimat method, which measures the stability index of the
oils (Barabás and Todoruţ 2011).
1.2.4 Heat of Combustion

The heat of combustion (heating value) at a constant volume of a fuel containing
only the elements carbon, hydrogen, oxygen, nitrogen, and sulfur is the quantity of
heat liberated when a unit quantity of the fuel is burned in oxygen in an enclosure
of constant volume, with the products of combustion being gaseous carbon
dioxide, nitrogen, sulfur dioxide, and water, and the initial temperature of the
fuel and the oxygen and the final temperature of the products at 25 °C. The unit
quantity can be mole, kilogram, or normal square meter. Thus, the units of
measurement of the heating value are kJ/kmol or kJ/kg. The volumetric heat of
combustion, that is, the heat of combustion per unit volume of fuel, can be
calculated by multiplying the mass heat of combustion by the density of the fuel
(mass per unit volume). The volumetric heat of combustion, rather than the mass
heat of combustion, is important to volume-dosed fueling systems, such as diesel
engines.
1.2.5 Density of Biodiesel

Fuel density is the mass per unit volume, measured in a vacuum. Because density is
strongly influenced by temperature, the quality standards state the determination
of density at 15 °C. Fuel density directly affects fuel performance, because some of
the engine properties, such as cetane number, heating value, and viscosity are
strongly connected to density. The density of the fuel also affects the quality of
atomization and combustion. Because diesel engine fuel systems (the pump and
the injectors) meter the fuel by volume, modification of the density affects the fuel
mass that reaches the combustion chamber, and thus the energy content of the fuel
dose, altering the fuel/air ratio and the engine’s power. Knowing the density is also
necessary in the manufacturing, storage, transportation, and distribution process
of biodiesel because it is an important parameter to be considered in the design of
these processes.
1.2.6 Viscosity of Biodiesel

The viscosity of liquid fuels is their property to resist the relative movement
tendency of their composing layers due to intermolecular attraction forces
(viscosity is the reverse of fluidity). Viscosity is one of the most important
properties of biodiesel. Viscosity influences the ease of starting the engine, the
spray quality, the size of the particles (drops), the penetration of the injected jet,
and the quality of the fuel-air mixture combustion (Alptekin and Canakci 2009).
Fuel viscosity has both an upper and a lower limit. The fuel with a viscosity that is
too low provides a very fine spray with the drops having a very low mass and
speed. This leads to insufficient penetration and the formation of black smoke
specific to combustion in the absence of oxygen (near the injector) (Băţaga et al.
2003) A biodiesel that is too viscous leads to the formation of drops too large,
which will penetrate to the wall opposite the injector. The cylinder surface being
cold, it will interrupt the combustion reaction and blue smoke, which is the
intermediate combustion product consisting of aldehydes and acids with pungent
odor, will form (Băţaga et al. 2003).
1.2.7 Cold Flow Properties

In general, all fuels for a compression ignition engine (CIE) may cause starting
problems at low temperatures, because of worsening of the fuel’s flow properties
at those temperatures. The cause of these problems is the formation of small
crystals suspended in the liquid phase, which can clog fuel filters partially or
totally. Because of the sedimentation of these crystals on the inner walls of the
fuel system’s pipes, the flow section through the pipes is reduced, causing poor
engine fueling. In extreme situations, when low temperatures persist longer
BIODIESEL BASICS 5
(e.g., overnight), the fuel system can be completely blocked by the solidified fuel.
The cloud flow performances of the fuels can be characterized by cloud point (CP),
the pour point, the cold filter plugging point (CFPP), and viscosity. An alternative
to CFPP is the low-temperature flow test (LTFT). Recently, the United States
introduced a new method for assessing the cold flow properties of biodiesel called
cold soak filtration test (CSFT).
1.2.8 Iodine Value

The iodine value (IV) or iodine number was introduced in biodiesel quality
standards for evaluating their stability to oxidation. The IV is a measurement of
total unsaturation of fatty acids measured in grams of iodine/100 g of biodiesel
sample, when formally adding iodine to the double bonds. Biodiesel with high IV is
easily oxidized in contact with air. The IV highly depends on the nature and ester
composition of the feedstocks used in biodiesel production. Therefore, the IV is
limited in various regions of the world depending on the specific conditions: 120 in
Europe and Japan; 130 in Europe for biodiesel as heating oil; and 140 in South
Africa. In Brazil, the IV is not limited. In the United States, Australia, and India, the
IV is not included in the quality standard (instead, it would exclude feedstocks like
sunflower and soybean oil). Biodiesel with high IV tends to polymerize and form
deposits on injector nozzles, piston rings, and piston ring grooves. The tendency of
polymerization increases with the degree of unsaturation of the fatty acids.
1.3 BIODIESEL STANDARDS
The produced biodiesel is tested to determine fuel characteristics based on ASTM

and European Union (EN) Biodiesel Fuel Quality Assurance Standard Tests
(Table 1-1). Biodiesel produced from various methods should be in its limits.
1.4 BIODIESEL VERSUS OTHER BIOREFINERY PRODUCTS
Biodiesel and other biorefinery products, such as bioethanol, have similar

characteristics to petroleum diesel (Table 1-2). The performance and emission
features of CIEs depend on the inner nozzle flow and spray performance. Inner
nozzle flow and spray performance in an engine controls the air-fuel mixing,
which is necessary for the process of combustion. Because of differences in the
physical properties of biodiesel and petro-diesel, the inner nozzle flow and spray
structure are expected to be significantly altered and, consequently, the perfor-
mance and emission features of the diesel engine. Som et al. (2010) reported that
because of lower vapor pressure of biodiesel, it was observed to cavitate less than
petro-diesel. A reduction in injection velocity and loss of flow efficiency were also
observed because biodiesel viscosity is higher.
Table 1-1. Biodiesel Standard Specifications per EN 14214 and ASTM D6751.
EN 14214 ASTM D6751
Limits Limits
Characteristics Minimum Maximum Test method Minimum Maximum Test method
Flash point (°C) >101 — ISO CD 3679e 130 — D 93

Acid value (mg KOH/g oil) — 0.50 Pr EN 14104 — 0.50 D 664
Density at 25 (°C) (g/cm3) 0.860 0.900 EN ISO 3675/ — — —
BIODIESEL PRODUCTION
EN ISO 12185
Kinematic viscosity at 40 (°C) 3.50 5.00 EN ISO 3104 1.9 6.0 D 445
(mm2 s−1)
Cetane number 51 — EN ISO 5165 47 ≥ 51 D 613
Soap content (soap/oil) (g) — — — — — —
FAME content (%) 96.5 — Pr EN 14103d — — —
Linolenic acid methyl ester content — 12.0 Pr EN 14103d — — —
(%)
Water content (mg/kg or % v/v) — 500 EN ISO 12937 0.050 D2709
Methanol content (% by weight) — 0.20 Pr EN141101 — — —
Free glycerol content (% by weight) — 0.02 Pr EN 14105m/ — 0.02 D 6584
Pr EN 14106
Total glycerin content (% by weight) — 0.25 Pr EN 14105m — 0.240 D 6584
Triglyceride content (% by weight) — 0.20 Pr EN 14105m — 0.20 —
Diglyceride content (% by weight) — 0.20 Pr EN 14105m — — —
Monoglyceride content (% by — 0.80 Pr EN 14105m — — —

weight)
(Na+ K) content (mg/kg) — 5.0 Pr EN 14108/ pr EN — 5.0 —
14109
(Ca + Mg) content (mg/kg) — 5.0 EN 14538 — — —
P content (mg/kg) — 10.0 Pr EN 14107p 0.001 D 4951
S content (mg/kg) — 10.0 — — 0.050 D 5453
Sulphated ash (% mol/mol or — 0.02 — 0.020 D 874
% w/w)
Total contamination (mg/kg) — 24 EN 12662 — — —
Iodine value (g/g) — 120 Pr EN 14111 — — —
Distillation temperature for 10% — 0.3 EN ISO 10370 — 360 (°C) D 1160
remnant (% m/m)
Copper strip corrosion [3 h, 50 (°C)] Class 1 Class 1 EN ISO 2160 — 0.020 D 130
(rating)
Oxidation stability at 110 (°C) (h) 6 — Pr EN 14112 k — — —
BIODIESEL BASICS
7
Table 1-2. Comparison of Properties between Biodiesel, Low Sulfur Diesel, and Ethanol.
Properties Biodiesel Low sulfur diesel Ethanol
Fuel material Fats and oils from sources such as Crude oil Corn, grains, or agricultural waste
(feedstocks) soy beans, waste cooking oil, (cellulose)
animal fats, and rapeseed
Chemical structure Methyl esters of C12 to C22 fatty C8 to C25 CH3CH2OH
acids
Gasoline gallon B100 has 103% of the energy in 1 gal. of diesel has 113% 1 gal. of E85 has 73% to 83% of the
equivalent 1 gal. of gasoline or 93% of the of the energy of 1 gal. energy of 1 gal. of gasoline
energy of 1 gal. of diesel; B20 has of gasoline. (variation due to ethanol content
109% of the energy of one gal. of in E85); 1 gal. of E10 has 96.7% if
gasoline or 99% of the energy of the energy of 1 gal. of gasoline.
1 gal. of diesel.
Energy content (lower 119,550 Btu/gal for B100 128,488 Btu/gal. 76,330 Btu/gal. for E100
heating value)
Physical state Liquid Liquid Liquid
Cetane number 48–65 40–55 0–54
Flash point 212 oF–338 oF 165 oF 55 oF
Autoignition 300 oF 600 oF 793 oF
temperature
Energy security impacts Biodiesel is domestically produced, Manufactured using oil, Ethanol is produced domestically.
renewable, and reduces of which nearly 1/2 is E85 reduces life cycle petroleum
petroleum use 95% throughout imported use by 70% and E10 reduces
its life cycle. petroleum use by 6.3%.
BIODIESEL BASICS 9
Fallahipanah et al. (2011) analyzed biodiesel fuel and its compounds using
laws of thermodynamics and finite-time thermodynamics to investigate the
performance of biodiesel in an engine that completes a specific cycle; they showed
that when biodiesel is applied as a fuel in the engine, similar results were obtained;
in some cases, even better results were obtained over petro-diesel fuel. Ilkılıç et al.
(2011) experimented on a diesel engine using biodiesel fuel produced from
sunflower oil with petro-diesel. They found that, in comparison to using petro-
diesel, using biodiesel increased the brake-specific consumption but decreased the
generation of pollutants such as particulate matter and carbon monoxide. The
poor lubricity of petro-diesel fuel has led to the failure of engine parts, such as fuel
injectors and pumps, because these parts are lubricated by the fuel itself. It was also
reported that neat biodiesel possesses inherently greater lubricity than petro-
diesel, especially low-sulfur petro-diesel, and that adding biodiesel at low blend
levels (1% to 2%) to low-sulfur petro-diesel restores lubricity to the latter
(Goodrum and Geller 2005).
One of the major technical advantages of biodiesel over petro-diesel is
lubricity (Knothe 2005). Knothe and Steidley (2005) reported that biodiesel has a
better lubricity than petro-diesel hydrocarbons because of the polarity that is
introduced with the presence of oxygen atoms that is lacking in petro-diesel. It
was reported that lubricity improves with the chain length and the presence of
double bonds.
Brake thermal efficiency (BTE) is defined as the ratio between power output
and energy introduced through fuel injection (Enweremadu et al. 2011). Reddy et al.
(2010) compared the BTE of diesel fuel and cottonseed methyl ester (biodiesel)
blends and found that the BTE of biodiesel blends was always lower than that of
petro-diesel. Enweremadu et al. (2011) also showed that blending biodiesel with
petro-diesel decreased the BTE but, conversely, increased the brake-specific fuel
consumption (BSFC), which is defined as the rate of fuel consumption divided by
the power produced. They also found that the BSFC of biodiesel and biodiesel
blends was higher compared with that of petro-diesel, which could be attributable to
the lower viscosity, density, and higher heating value of the petro-diesel.
1.5 BIODIESEL BLENDING, STORAGE, AND TRANSPORT
1.5.1 Biodiesel Blending

Biodiesel fuel composition varies depending on the feedstock used in fuel
production. Often, these compositions make fuel unsuitable for specific applica-
tions. Fuels of different origins can be blended together with petroleum-derived
diesel fuels to meet specific requirements. EN 590, EN 166 and ASTM D975 and
D 167 fuel specifications for petroleum-based diesel fuels permit blending with
biofuel content of up to 5% and 20% (v/v), respectively. Biodiesel fuel is usually
blended with petroleum diesel to form a B20 blend, although other blends can be
used up to B100. B20 fuel blend can be used in existing engines and fuel injection
equipment with little impact on operating performance. The viscosity and flash
point for B100 were 6.0 mm2/s and 93 °C, respectively. Blending with petroleum-
based diesel B20 results in lower viscosity (4.1 mm2/s), lower flash point (52 °C),
and lower cloud point (93 °C), making its application feasible in cold countries
also (Anitescu and Bruno 2011).

Ethanol contains only 67% the energy of gasoline, but it has a higher
antiknock index. Hence it can be used in blending with biodiesel to achieve a
high antiknock index. Pure biodiesel (B100) has been reported to have blends with
5%, 10%, and 15% (v/v) ethanol (Anitescu and Bruno 2011). The results showed
that ethanol in biodiesel influences the most important fuel properties of the
blended fuel such as fuel stability, density, viscosity, cold filter plugging point,
pour point, flash point, filter plugging tendency (FPT), corrosiveness, and so on. In
one of the studies, biodiesel, diesel, and ethanol were blended in different ratios to
result in reduced emissions (Kwanchareon et al. 2007). Fuel properties of the
blended mix along with emission performance in a diesel engine were examined
and compared with base diesel. It was found that a blend ratio of 80% diesel,
15% biodiesel, and 5% ethanol was most suitable for diesohol production because
of acceptable fuel properties and reduction in emissions. Although the blend
resulted in lower flash points, the high cetane value of biodiesel compensated for
the decrease in the cetane number of blends caused by the presence of ethanol.
In another study, Jatropha and Palm biodiesel were examined to achieve
low-temperature properties with improved oxidation stability (Sarin et al. 2007).
Jatropha-based biodiesel has poor oxidation stability but good temperature
properties, whereas palm biodiesel has good oxidative stability but low tempera-
ture properties. The blend of Jatropha and palm biodiesel results in a synergistic
effect of improved temperature profile along with oxidative stability. The stability
of biodiesel is very critical. Biodiesel requires antioxidant to meet storage
requirements and to ensure fuel quality at every point along the distribution
chain. For biodiesel, 200 ppm of antioxidant is required, which is higher than
petroleum-based diesel. To minimize the dose of antioxidant, appropriate blends
of Jatropha (60%–80%) and palm biodiesel (20%–40%) are made.
The biodiesel fuels have different chemical characteristics compared with
petroleum-derived hydrocarbons. Biodiesel molecules (esters) are all polar and
associating. Property science is currently focused on pure fluids and simple
mixtures. Accurate property data for mixtures with hundreds of chemically
dissimilar components is required for implementation of blending of biofuel
with petroleum-based diesel or ethanol. Meeting this challenge requires new
approaches to modeling and measurements. Measurements must be performed
over wide ranges of thermodynamic conditions that are required for a successful
model development.
1.5.2 Storage and Transport

Once biodiesel is produced at a manufacturing plant/blending site, it needs to be
transported to storage facilities through pipeline transport. Because biodiesel is
produced from biofeedstocks, they are more prone to oxidation than other fuels.
BIODIESEL BASICS 11
The storage stability of fuels based on fatty acid methyl esters (FAMEs) is a critical
issue. Standardized chemical analysis indicates that the shelf life of pure biodiesel
fuels obtained from different sources ranges widely from 4 weeks to 4 months
(Anitescu and Bruno 2011).
1.5.2.1 Thermal and Oxidative Stability of Biofuels

The thermal/oxidative stability of finished biofuels depends on the feedstocks used
for production. The stability is much greater for biodiesel fuels with a high
saturated fatty acid content and decreases with the degree of fatty acid unsatura-
tion. FAMEs containing fatty acids have stability in the following sequence:
palmitic (0) > oleic (1) > linoleic (2) > linolenic (3). Analytical tests measuring
properties like peroxide values (ASTM D3307), induction period Rancimat
(EN 14112), level of insolubles and gum formation (ASTM D2274), acid value
(ASTM D664), and viscosity (ASTM D445) can help to determine the oxidative
stability of biodiesel (Abou-Nemeh 2008). Currently, EN 14214 prescribes the use
of EN 14112 with 6 h minimum for biodiesel stability. On the other hand,
the ASTM D6751 requires a minimum 3 h. The IV is another chemical property of
the biodiesel fuel for predicting the oxidative stability. This property is a
quantification of the number of double bonds of the fatty acids in FAME.
EN 14111 specifies an IV of 120 for canola or rapeseed-based biodiesel fuel, but
for soybean-based biodiesel fuel, the IV is about 130. There are no specifications
for IV in ASTM D6751.
Several approaches can be applied to improve the oxidative stability of
biodiesel fuels. One of them is to avoid contact of the biofuel with oxygen
(Al-Zuhair et al. 2007). Another is to prevent their contact with materials and
substances that promote or catalyze the reaction of oxidation such as prooxidants,
trace metals, higher temperature, light, and so on. These are mostly preventative
measures, but they may not be fully applicable to the real transport/storage
environment. Synthetic anti-oxidants are used in the biodiesel industry because
they possess robustness, cost-effectiveness, and superior chemical and thermal
stability.
1.5.2.2 Bacterial Degradation of Biofuels

Biodiesel fuel is susceptible to both aerobic and anaerobic biodegradation by wild-
type bacteria commonly present in natural environments (Lutterbach and Galvão
2010). Degradation of biodiesel during fuel storage is detrimental to fuel quality.
The biodiesel is subjected to hydrolysis, resulting in free fatty acids (FFA); thus,
more caution is required when integrating this fuel with the existing infrastruc-
ture. Biodiesel fuel must have a low moisture content (<0.05% v/v) to achieve less
biological growth and fuel-filter plugging. As the biodiesel industry continues to
develop, the need for better testing equipment will steadily increase. For this
reason, the photochemiluminesence method appears to be a promising way
forward, but more research is required.
1.6 PERFORMANCE OF BIODIESEL AS A FUEL AND

DEMONSTRATION OF ITS USAGE
The environmental impact of biodiesel depends on several factors, such as the raw
materials from which the biodiesel was produced, different production processes,
and the final use that can determine the environmental balance of biodiesel
introduction (Nanaki and Koroneos 2012). Replacing petro-diesel fuel with
biodiesel may have environmental advantages as well as disadvantages. The
prominent advantage is that biodiesel has the potential of reducing most exhaust
emissions that have regulations, excluding nitrogen oxides (NOx).
A heavy-duty 2003 six-cylinder 14 L diesel engine with exhaust gas
recirculation was used to analyze neat methyl laurate, neat methyl palmitate,
and technical grade methyl oleate, and for exhaust emissions (Knothe 2005). The
three fatty acid methyl esters were compared with pure dodecane and hexade-
cane, and pure biodiesel sample as well as petro-diesel with low sulfur content.
All fuels were analyzed and tested for exhaust emissions. Emissions of particulate
matter were found to decrease to about 77% and 73% for biodiesel and methyl
oleate, respectively. Similarly, the reduction in particulate matter emissions
for methyl laurate and methyl palmitate was even greater—83% and 82%,
respectively—in comparison to petro-diesel. An increase of about 12% NOx
emissions was observed for biodiesel, whereas an increase of about 6% was
observed for methyl oleate, but methyl palmitate and methyl laurate particulate
matter emission was reduced by about 4%–5% relative to those of the base fuel
(Knothe et al. 2006). Overall, biodiesel fuel and the fatty compounds consider-
ably reduced particulate matter emissions by 75%–83% when compared to the
petro-diesel base fuel, whereas the two hydrocarbons components found in
petro-diesel—dodecane and hexadecane—reduced particulate matter emission
by only 45%–50%. Similarly, Rubianto et al. (2015) found that biodiesel has a
contribution to reduce particulate emission by 29.7% from combustion on a
boiler burner. This percentage of reduction is significant to decrease air pollu-
tion, creating a better and healthier environment. Blending biodiesel with petro-
diesel at various ratios (from B0 to B10) would lead to a decrease in the amount
of particulate matter being emitted from 0.6276 to 0.5061 g, but not for B15 and
B20 (Rubianto et al. 2015).
Ali (2011) reported that in an assessment of the environmental hazards
caused by using fossil fuels, biodiesel is the best fuel for diesel engines, because
burning biodiesel and its blends produces the lowest GHG (Greenhouse gas)
emissions on a life cycle basis. The emission of carbon monoxide gas is reduced by
using biodiesel as a fuel. The use of biodiesel in blends or its pure form reduces
emissions, such as particulate matter, visible smoke, odor, and polyaromatic
hydrocarbon emissions. Particulate soot emissions, which have an adverse health
effect in terms of respiratory impairment and related illness, are significantly
reduced with the use of biodiesel or its blends. Furthermore, biodiesel does not
contain an undesirable element like sulfur compared with petro-diesel. Therefore,
BIODIESEL BASICS 13
biodiesel is a fuel that is clean and environmentally friendly, which can supple-
ment or replace petro-diesel as a fuel in the future (Ali 2011).
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diesel engine fueled with methyl ester of shea butter.” World Acad. Sci. Eng. Technol.
5 (7): 142–146.
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of the performance of Biodiesel, Diesel, and their compound in Diesel air standard
irreversible cycles.” In Vol. 15 of Proc., Int. Conf. on Environmental and Agriculture
Engineering, Chengdu, China, 7–13.
Goodrum, J. W., and D. P. Geller. 2005. “Influence of fatty acid methyl esters from
hydroxylated vegetable oils on diesel fuel lubricity.” Bioresour. Technol. 96 (7): 851–855.
Ilkılıç, C., S. Aydın, R. Behcet, and H. Aydin. 2011. “Biodiesel from safflower oil and its
application in a diesel engine.” Fuel Process. Technol. 92 (3): 356–362.
Knothe, G. 2002. “Current perspectives on biodiesel.” INFORM - International News on
Fats, Oils and Related Materials, 13 (12): 900–903.
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esters.” Fuel Process. Technol. 86 (10): 1059–1070.
Knothe, G., and T. W. Ryan. 1997. Cetane numbers of fatty compounds: Influence of
compound structure and of various potential cetane improvers: SAE technical paper.
Warrendale, PA: Society of Automotive Engineers.
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neat methyl esters, and alkanes in a new technology engine.” Energy Fuels 20 (1):
403–408.
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biodiesel–ethanol blend, its fuel properties, and its emission characteristics from diesel
engine.” Fuel 86 (7): 1053–1061.
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In Applied microbiology and molecular biology in oilfield systems, 229–235. New York:
Springer.
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and gasoline for transportation.” J. Cleaner Prod. 20 (1): 14–19.
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Green Publishing.
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brake thermal efficiency and brake specific fuel consumption of diesel engine fuelled with
bio-diesel.” Int. J. Eng. Technol. 2 (5): 305–309.
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waste frying oil to biodiesel conversion.” Indonesian Green Technol. J. 4 (1): 11–17.
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blends: An optimum mix for Asia.” Fuel 86 (10–11): 1365–1371.
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and spray characteristics of biodiesel with petrodiesel.” Fuel 89 (12): 4014–4024.
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analytical methods, 37–47. Golden, CO: National Renewable Energy Laboratory.
CHAPTER 2
Biodiesel: Variations,
Properties, and Comparison

with Diesel
H. Panidepu
S. K. Ram
V. Cheernam
R. D. Tyagi
2.1 INTRODUCTION
In this chapter, consideration will be given to the various technical aspects of

biodiesel that qualify it to be an alternative fuel, the details regarding the
physicochemical properties of biodiesel, the cause and sometimes the need for
variation in the biofuel and its impact on engine performance, the effect of such a
change in the fuel characteristics on various quantifiable outputs, and a conclusive
examination of the overall performance and potential of biofuels in comparison to
conventional mineral oil.
The necessity to compare biodiesel and conventional petroleum diesel is
foundationally premised on the fact that the former could substitute for the
latter. Elaborately, biofuels have several physical and chemical properties such
as their liquid state, specific energy, density, viscosity, and combustion
characteristics more similar to gasoline or diesel than for other alternatives.
They are combustible in existing internal combustion engines with minor
modifications. As a result, adapting to biofuel-based infrastructure (at least at
low levels of blending, perhaps 10% or 20%) can be achieved more cost
effectively than adapting to hydrogen, battery, or natural gas–based automo-
biles (Fulton 2004).
15
2.2 VARIATIONS IN BIODIESEL
The technical definition of biodiesel is a fuel suitable for use in compression

ignition (diesel) engines that is made of fatty acid mono-alkyl esters derived from
biologically produced oils or fats including vegetable oils, animal fats and micro-
algal oils (Benea 2013). Fatty acid methyl esters (FAME) closely resemble
biodiesel. This definition of biodiesel enlarges the domain of biofuels. These
varied sources of oil naturally result in variation of biofuel properties thus formed.
This however is not a bottleneck, because a closer look at the existing fossil fuels in
the market show some variability in physicochemical properties, and thus there is
a possibility for innovation in this space with new combinations to improve upon
existing fuels either in terms of efficiency or environmentally benevolent in the
21st century. For instance, there are categories in diesel just as there are for
gasoline. On the basis of efficiency, diesel is classified by its cetane number just as
gasoline is classified on the basis of its octane number. A higher cetane number
indicates higher combustion efficiency and more volatility. Standard diesel fuel is
available as Diesel No.1 (or 1-D) and Diesel No.2 (or 2-D) or Grade 1 and Grade 2
diesel oil, respectively. Grade 2 diesel oil is lower in cetane number compared with
Grade 1 oil. However, Grade 2 diesel fuel is typically used to power vehicles for
reasons of better fuel economy. Just like any other fuel, the diesel thickens at low
temperatures, and sometimes it gels and refuses to flow. Thus, depending on the
climatic conditions of a region, Grade 1 and Grade 2 diesel oils are blended in an
appropriate proportion to elicit an efficient performance of the vehicles in the
region.
Similarly, in the case of biodiesel, one type of variation is formed when it is
blended with diesel in requisite proportions. In the United States and Canada,
B-5 is primarily used, which is 5% biodiesel blended with 95% diesel fuel.
Although the engines might still work when the blending percentages increase
to 30%, that would violate the engine’s manufacturer’s warranty. In addition, there
is much variation with respect to source oil. Palm, olive, peanut, rapeseed,
soybean, sunflower, grape, high oleic sunflower, almond, and corn oil are some
of the oils that could be extracted from their respective feedstock. These oils differ
in fatty acid composition and thereby affect the quality of biodiesel produced, thus,
they can be broadly classified as polyunsaturated fatty acids and monounsaturated
fatty acids.
Eventually, depending on the difference in their physicochemical properties,
biodiesel has a number of variations and is therefore denoted by different names.
Some standards exist for biodiesel quality based on unique specifications. These
specifications for biodiesel are based on ester content, density at 15 °C, viscosity at
40 °C, flash point, sulfur content, cetane number, sulfated ash content, water
content, total contamination, copper band corrosion, oxidation stability, acid and
iodine values, linolenic acid methyl ester, polyunsaturated methyl ester, methanol
content, monoglyceride content, diglyceride content, triglyceride content, free
glycerin, total glycerin, phosphorous content, and Group I and Group II metals.
BIODIESEL: VARIATIONS, PROPERTIES, AND COMPARISON WITH DIESE 17
Each of these particular parameters is codified in a unique terminology. For

instance, the European standard EN 14214, ASTM International D6751, and the
Canadian standard CAN/CGSB 3.524 are all comparable, although they are known
by different nomenclatures in different countries. A fuel certified by any of these
standards qualifies to be used as a fuel in unmodified diesel engines. Similarly, each

country has its own preferred standard way for biodiesel production. For instance,
the European Union primarily relies more on rapeseeds for production of biodiesel
rather than soybean and palm oil, because the latter (according to EU standards)
will not yield pure biodiesel (Mukonza and Nhamo 2016).
2.3 PHYSICOCHEMICAL ASPECTS
Cloud point, the temperature at which biodiesel starts to appear cloudy on

reduction of temperature, is an important aspect of the biodiesel product.
The cloud point of the FAME is estimated by the percentage weight of the
C-16 content in the fatty acid. Technically with the reduction of temperature,
the fuel starts to become amorphous crystals, and when the size of the crystal
becomes one-quarter the wavelength of the light, the biodiesel starts to appear
cloudy (Park et al. 2008).
Cold filter plugging point (CFPP), the temperature at which the biodiesel is
unable to flow through a 45-μm mesh on reduction of temperature, is another
important characteristic. There are different standards for this mesh size in
different countries. If the standard for mesh size is smaller, it would require the
cloud point for biodiesel to be at a much lower end of the temperature scale,
although there are additives available to reduce the CFPP of biodiesel. Similarly,
blending biofuels with Grade-2 mineral-diesel or low-sulfur diesel is also another
alternative to lower CFPP. Palm oil and beef tallow1 have higher CFPP of 16 °C
compared with canola oil (−10 °C), which makes canola oil a better biodiesel in
winter than beef tallow and palm oil.
The fatty acid content of biodiesel is a key parameter that influences its
physicochemical properties. A study conducted on three biodiesel oils—palm,
rapeseed, and soybean—is a case in point. Palm oil has higher oxidation stability
than rapeseed, which has higher oxidation stability than soybean, whereas the
CFPP of these oils follows the reverse trend. The blending experiment of these oils
in varied proportions is premised on the logic of changing the fatty acid
composition, has improved the oxidation stability2 and decreased the CFPP of
the resulting mixture, thereby suggesting that the decrease in unsaturated fatty
1
A hard-fatty substance made from rendered animal fat, used (especially formerly) in making candles
and soap.
2
Oxidation stability is a chemical reaction that occurs with a combination of the lubricating oil and
oxygen. The rate of oxidation is accelerated by high temperatures, water, acids, and catalysts such as
copper.
acid content like linolenic acid in oils is associated with increase in the oxidation
stability of the mixture while the CFPP increases. Blending of oils alters the
properties of biodiesel thus formed and provides a rough estimate of properties of
biodiesel from new sources, like waste oils, after knowing its fatty acid composi-
tion; it also enables the performance of new prospective biofuels to be judged

(Park et al. 2008). A blending study focused on karanja blended oil to evaluate the
torque, power, specific fuel consumption.3 Emissions (such as CO, smoke density,
and nitrous oxide) of the engine showed an increase in torque, brake power, brake
thermal efficiency, and reduction in brake-specific fuel consumption4 on blending
in a B20 and B40 manner (Raheman and Phadatare 2004).
Software tools are available that predict the performance of the FAME
produced after the user inputs the fatty acid composition of the FAME, which
is the output of a gas chromatography (GC), directly into the web interface.
Biodiesel Diesel Analyzer 1.1 is one such software that takes in the fatty acid
composition as the input and provides the estimated properties under the
following tabs: unsaturation level, including the amount of the saturated and the
unsaturated fatty acids5 and the degree of unsaturation (DU); the cetane number;
the cold flow properties, including the cloud point (CP) and the CFPP; the
oxidation stability, including the allylic (APE) and the bis-allylic position equiva-
lents (BAPE); the higher heating value (HHV)6; the kinematic viscosity7; and the
density (Talebi et al. 2014).
Kinematic viscosity is another important aspect of performance of biodiesel,
in regards to the physicochemical properties of biodiesel with change in tempera-
ture. The kinematic viscosity is dependent on the percentage of each specific fatty
acid present in the oil and the number of double bonds present in it. If the
biodiesel becomes significantly viscous with decrease in temperature, then higher
viscosity necessitates excessive fuel injection pressures during engine warm-up.
The engine could also be starved for fuel at low temperatures as the fuel moves
slowly through the fuel filter and fuel lines (Tat and Van Gerpen 1999). Typically,
the kinematic viscosity of Grade No. 2-D8 diesel fuels is around 2 to 6 centistokes
when the temperature is about 37.8 °C. Tables 2-1, 2-2, and 2-3 provide the
physical properties of biodiesel and their specific characteristics.
3
Specific fuel consumption is one of the determining criteria of an engine that quantifies the thrust
produced per unit fuel consumption.
4
The rate of fuel consumption divided by the power produced. It is typically used for comparing the
efficiency of internal combustion engines with a shaft output.
5
An unsaturated fat is a fat or fatty acid in which there is at least one double bond within the fatty acid
chain.
6
The higher heating value (also known as gross calorific value/gross energy) of a fuel is defined as the
amount of heat released by a specified quantity (initially at 25 °C) once it is combusted and the products
have returned to a temperature of 25 °C, which considers the latent heat of vaporization of water in the
combustion products.
Dynamic viscosity of a fluid per unit density.
7
8
includes the class of distillate oils of lower volatility
Table 2-1. Physical and Chemical Properties of Biodiesel.

Kinematic Lower
Vegetable oil viscosity Cetane heating value Cloud point Flash point Density Sulfur (percent
methyl ester (mm2/s) number (MJ/L) (°C) (°C) (g/L) by weight)
Peanut 4.9 (37.8 °C) 54 33.6 5 176 0.883 —

Soybean 4.5 (37.8 °C) 45 33.5 1 178 0.885 —
Soybean 4.0 (40 °C) 45.7–56 32.7 — — 0.880 (15 °C) —
Babassu 3.6 (37.8 °C) 63 31.8 4 127 0.879 —
Palm 5.7 (37.8 °C) 62 33.5 13 164 0.880 —
Palm 4.3–4.5 (40 °C) 64.3–70 32.4 — — 0.872–0.877 —
(15 °C)
Sunflower 4.6 (37.8 °C) 49 33.5 1 183 0.860 —
Tallow — — — 12 96 — —
Rapeseed 4.2 (40 °C) 51–59.7 32.8 — — 0.882 (15 °C) —
Used rapeseed 9.48 (30 °C) 53 36.7 — 192 0.895 0.002
Used corn oil 6.23 (30 °C) 63.9 42.3 — 166 0.884 0.0013
Diesel fuel 12–3.5 (40 °C) 51 35.5 — — 0.830–0.840 —
(15 °C)
JIS-2D (gas oil) 2.8 (30 °C) 58 42.7 — 59 0.833 0.05
BIODIESEL: VARIATIONS, PROPERTIES, AND COMPARISON WITH DIESE
19
20
Table 2-2. General Parameters of Biodiesel Quality.

Czech Republic France Germany

Parameters Austria (ON) (CSN) (journal official) (DIN) Italy (UNI) USA (ASTM)
Density at 158 °C g/cm3 0.85–0.89 0.87–0.89 0.87–0.89 0.875–0.89 0.86–0.90 —

Viscosity at 40 mm2/s 3.5–5.0 3.5–5.0 3.5–5.0 3.5–5.0 3.5–5.0 1.9–6.0
Flash point 100 110 100 110 100 130
CFPP 0/K5 K5 — 0–10/K20 — —
Pour point (°C) — — K10 — 0/–5 —
Cetane Number R49 R48 R49 ≥ 49 — R47
Table 2-3. Specific Parameters of Vegetable Oils for Biodiesel Quality.

Austria Czech Republic France Germany
Parameters (ON) (CSN) (journal official) (DIN) Italy (UNI) USA (ASTM)
Methanol/ethanol (% mass) ≤0.2 — 0.1 0.3 ≤0.2 —

Ester content (% mass) — — ≥96.5 — ≥98 —
Monoglyceride (% mass) — — 0.8 0.8 ≤0.8 —
Diglyceride (% mass) — — 0.2 0.4 ≤0.2 —
Triglyceride (% mass) — — 0.2 0.4 ≤0.1 —
Free glycerol (% mass) 0.02 0.02 0.02 0.02 0.05 0.02
Total glycerol (% mass) 0.24 0.24 0.25 ≤0.25 — 0.24
Iodine number ≤120 — ≤115 ≤115 — —
BIODIESEL: VARIATIONS, PROPERTIES, AND COMPARISON WITH DIESE
21
The cetane number is a function of saponification value9 and iodine value.10

Both saponification and iodine values are empirically related to the number of
double bonds and single bonds present in the fatty acid, the molecular weight of
the fatty ester, and the percentage of fatty ester present in the oil sample. Current
1-D11 and 2-D diesel fuels have a cetane rating between 40 and 45. The long chain
saturation factor and the CFPP are estimated based on percentage of C-18, C-24,
C-16, among others. The degree of unsaturation is calculated on the basis of
monounsaturated fatty acid12 (MUFA) and polyunsaturated fatty acid content
(PUFA) present in the oil. APE13 and BAPE14 are dependent on the position of the
allylic and bis-allylic groups in the specific fatty acid (Talebi et al. 2014).
2.4 CONTAMINANTS IN BIODIESEL
Contaminants cause property changes in biodiesel. The contaminants typically

present include water, free glycerin, bound glycerin, alcohol, free fatty acids, soaps,
catalysts, unsaponifiable15 matter, and the products of oxidation. Among all, the
contaminants having significant impact are water, bound glycerin, and oxidation
products of biofuels. Bound glycerin increases the viscosity of biodiesel. Oxidation
of biodiesel produces certain chemical components that increase the cetane
number of the biodiesel, thereby having a higher calorific value; however, this
may cause increased acidity of biodiesel.
Water is a significant contaminant in biodiesel, which can absorb 40% more
water than mineral diesel due to the hygroscopic16 nature. Also, because of
incomplete transesterification17 reaction, monoglycerides and diglycerides that are
left after the production process act as an emulsifier and allow the mixing of
biodiesel and water. In addition, water might creep into the storage stock as a
result of condensation in the storage tank. Water significantly alters the combus-
tion efficiency of biodiesel. It causes delayed start of engines and loss of power. The
9
Saponification value is expressed by potassium hydroxide in mg required to saponify one (1) gram of
fat. It depends on the kind of fatty acid contained in the fat.
10
The iodine value in chemistry is the mass of iodine in grams that is consumed by 100 grams of a
chemical substance. Iodine numbers are often used to determine the amount of unsaturation in fatty
acids.
11
1-D comprises the class of volatile fuel oils from kerosene to the intermediate distillates.
12
Mono-unsaturated fatty acids have only one double carbon bond in the fatty acid.
13
Allylic position equivalent
14
bis-allylic position equivalent
15
Unsaponifiables are components of an oily (oil, fat, wax) mixture that fail to form soaps when blended
with sodium hydroxide (lye) or potassium hydroxide.
16
Tending to absorb moisture from the air.
17
Transesterification is the process of exchanging the organic group R″ of an ester with the organic
group R′ of an alcohol.
presence of water causes pitting in the pistons, which is the consequence of

rusting. The microbes present in water cause the paper element in the filter to rot.
Smoke emissions also increase owing to water contamination. During winter or
when cold conditions prevail, water freezes to form ice crystals, which act as
nucleation sites and accelerate the gelling of biodiesel (Bockey 2004). Previously,
water content present in biodiesel was difficult to measure because water and oil
are immiscible. Now, however, water–oil sensors make it possible to estimate the
water content in a sample (Van Gerpen et al. 1997).
Oxidation of biofuels is a major glitch for advancing biofuels as an alternative
because the products of oxidation, such as aldehydes and ketones, thicken the
biodiesel, technically increasing the kinematic viscosity and thereby clogging the
pumps and filters of the engine and eventually corroding it. Oxidation of biodiesel
occurs because of the presence of trace metals, oxygen, unsaturated fatty acids, and
high temperatures (Dantas et al. 2011).
2.5 PARTICULATE EMISSIONS
Biodiesel is reported to cause less particulate pollution compared with fossil diesel,
cause less pollution to water bodies, contain less sulfur, and to be more biode-
gradable than fossil diesel. Biodiesel may also show reduced dermal toxicity and
reduced immunogenicity.18 These results were obtained on experiments per-
formed in a single-cylinder engine with both fuels (Kalligeros et al. 2003). In
addition, reduction in hydrocarbons and carbon monoxide emissions were shown
(Demirbas 2008). However, study also reported an increase in nitrous oxide
emissions when used in unmodified diesel engines. Study also reveal imperceptible
power loss and the increase in fuel consumption, among others (Xue et al. 2011).
For these reasons, blending and other modifications of biofuel are being consid-
ered by scientists hitherto. Among the numerous studies conducted on particulate
matter emissions from a diesel combustion engine, The study (Agarwal et al. 2011)
focused on the particle-size distribution, metals, benzene soluble organic fraction,
elemental and organic carbon fractions, particle number, and morphology. Dark
and stickier exhaust is primarily present in the output of mineral diesel combus-
tion, whereas benzene soluble organic fractions dominate the exhaust on biodiesel
combustion. The metal output of biodiesel exhaust was predominantly calcium,
magnesium, iron, and zinc for almost all operating conditions, whereas the output
of mineral diesel exhaust particulate was primarily copper, lead, chromium, and
nickel. The metal output was found to be more in the case of mineral diesel
because of engine wear, accentuated because of the non-lubricating properties of
mineral diesel (Agarwal et al. 2011).
18
Immunogenicity is the ability of a particular substance, such as an antigen or epitope, to provoke an
immune response in the body of a human or animal.
Toxicological studies on biodiesel emissions also point toward some evidence

that exposure to biodiesel emissions elicits more vascular, inflammatory, and
mutagenic responses from the body, although some studies also signify an
opposite trend. Therefore in vitro tests, studies on animal or human exposure
in a controlled fashion, and the associated impacts should be performed (Madden

2016). There is a scope for improvement, and there has always been room for up-
gradation of biofuel production technologies. Table 2-1 highlights physical and
chemical properties of biodiesel, whereas Tables 2-2 and 2-3 provide general and
specific parameters for the quality of biodiesel.
2.6 CONCLUSIONS
Rudolf Diesel is credited as the inventor of the first diesel engine, and it was
originally designed to run on fuel derived from peanut oil (Jääskeläinen 2019). He
reportedly said, “The diesel engine would help considerably in the development of
agriculture of the countries which use it” (Vrabie 2016). That being said, the effect
of biodiesel on engine power, economy, durability, regulated and nonregulated
emissions, as well as the corresponding effect should be carefully examined in
great detail. Further research should focus on optimization and modification of
engines, on low temperature performance of engines, and development of new
instrumentation and methodology for measurements before petroleum diesel is
substituted completely by biodiesel.
References
Agarwal, A. K., T. Gupta, and A. Kothari. 2011. “Particulate emissions from biodiesel
vs diesel fuelled compression ignition engine.” Renew. Sustain. Energy Rev. 15 (6):
3278–3300.
Benea, B. C. 2013. “Study regarding the effect of biodiesel on diesel engine emission.” Acta
Technica Corviniensis – Bulletin of Engineering tome vi – Fascicule 3 [July–September]
ISSN 2067-3809, 127–129.
Bockey, D. 2004. Biodiesel flowerpower: Facts, arguments, tips, 2nd ed., 10. Berlin: Union for
the Promotion of Oil and Protein Plants.
Dantas, M., A. Albuquerque, A. Barros, M. Rodrigues Filho, N. Antoniosi Filho,
F. Sinfrônio, et al. 2011. “Evaluation of the oxidative stability of corn biodiesel.” Fuel
90 (2): 773–778.
Demirbas, A. 2008. “The importance of bioethanol and biodiesel from biomass.” Energy
Sources Part B 3 (2): 177–185.
Fulton, L. 2004. “Driving ahead-biofuels for transport around the world.” Renew. Energy
World 7 (4): 180–189.
Jääskeläinen, H. 2019. “Early history of the diesel engine.” Accessed May 20, 2019. https://
www.dieselnet.com/tech/diesel_history.php.
Kalligeros, S., F. Zannikos, S. Stournas, E. Lois, G. Anastopoulos, C. Teas, et al. 2003. “An
investigation of using biodiesel/marine diesel blends on the performance of a stationary
diesel engine.” Biomass Bioenergy 24 (2): 141–149.
Madden, M. C. 2016. “A paler shade of green? The toxicology of biodiesel emissions: Recent
findings from studies with this alternative fuel.” Biochim. Biophys. Acta (BBA)-General
Subjects 1860 (12): 2856–2862.
Mukonza, C., and G. Nhamo. 2016. “Institutional and regulatory framework for biodiesel
production: International perspectives and lessons for South Africa.” Afr. J. Sci. Technol.
Innovation Dev. 8 (1): 1–11.
Park, J.-Y., D.-K. Kim, J.-P. Lee, S.-C. Park, Y.-J. Kim, and J.-S. Lee. 2008. “Blending effects
of biodiesels on oxidation stability and low temperature flow properties.” Bioresour.
Technol. 99 (5): 1196–1203.
Raheman, H., and A. Phadatare. 2004. “Diesel engine emissions and performance from
blends of karanja methyl ester and diesel.” Biomass Bioenergy 27 (4): 393–397.
Talebi, A. F., M. Tabatabaei, and Y. Chisti. 2014. “BiodieselAnalyzer: A user-friendly
software for predicting the properties of prospective biodiesel.” Biofuel Res. J. 1 (2):
55–57.
Tat, M. E., and J. H. Van Gerpen. 1999. “The kinematic viscosity of biodiesel and its blends
with diesel fuel.” J. Am. Oil Chem. Soc. 76 (12): 1511–1513.
Van Gerpen, J. H., E. G. Hammond, L. Yu, and A. Monyem. 1997. Determining the influence
of contaminants on biodiesel properties: SAE technical paper. Warrendale, PA: Society of
Automotive Engineers.
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emissions.” Renew. Sustain. Energy Rev. 15 (2): 1098–1116.

CHAPTER 3
Biodiesel Production by
Transesterification
X. Zhang
S. Yan
R. D. Tyagi
R. Y. Surampalli
3.1 INTRODUCTION
Several technologies have been developed to produce biodiesel including pyrolysis,

microemulsions, and transesterification (Doll et al. 2008, Macala et al. 2008,
Suarez et al. 2009). Microemulsion is the oldest method to produce biodiesel from
oils or fats by blending with cosolvents, mostly short-chain alcohols such as
methanol and ethanol, and amphiphiles (ionic or nonionic). Adding a cosolvent
reduces the viscosity of the oil or fat, and hence the product (considered as
biodiesel) can be used directly to power diesel engines. In the early 1980s, the
ethanol microemulsion in soybean oil was investigated in a short block engine, and
the blended biodiesel fuel showed excellent adaption with the engine (Goering
et al. 1982b). Both soybean oil and triolein blending with amphiphile, 2-octanol,
using methanol as immiscible liquid, gave good performance in diesel engine
(Ma and Hanna 1999, Schwab et al. 1988). Recently, microemulsion is frequently
used in blending diesel with ethanol (Bilgin et al. 2002).
Pyrolysis, also called cracking, is a thermal decomposition process that cracks
long alkyl chains into small molecules at high temperature under oxygen-free
conditions. Biodiesel production through pyrolysis can be simplified as follows
with Equations (3-1) and (3-2):
300−500 °C
Feedstocks ðoils=fatsÞ !gas þ mixture ðliquidÞ (3-1)
catalyst
Separation ðdistillationÞ
Mixture ðliquidÞ!biodiesel (3-2)
27
Various feedstocks, such as plant oils (Doll et al. 2008, Fortes and Baugh 1999,
Lima et al. 2004, Vitolo et al. 2001), fats (Adebanjo et al. 2005), and waste oils
(Nerín et al. 2000) were reported in biodiesel production through pyrolysis.
However, the large amount of energy consumption resulting from the high
temperature requirement in the reaction becomes the concern (Nasikin et al.

2009).
Transesterification is the most common and vital process in which oils are used
to react with short-chain alcohol (methanol or ethanol) to form biodiesel (Boz et al.
2009, Dizge et al. 2009, Singh and Singh 2010). Both catalytic and noncatalytic
transesterification exist, depending on the presence or absence of a catalyst.
Noncatalytic transesterification takes advantage of high pressure or temperature
to achieve a high conversion rate; hence, it is considered a high energy consumption
approach. In general, catalytic transesterification promotes the biodiesel conversion
rate with a short time (several hours), but downstream treatment is more complex
than that of using noncatalytic. The selection of a catalyst in transesterification is
determined by the raw oil or fat properties. Free fatty acid (FFA) content in the oil or
fat is the major factor because it causes soap formation in an alkaline catalyst
reaction, which consumes the catalyst and reduces biodiesel yield. Normally,
alkaline catalyst reaction is not preferred when FFA content is higher than 2%
of total oil/fat. Otherwise, a two-step transesterification can be used, with the first
step to convert FFA to biodiesel in the acidic catalyst condition and the second
alkaline catalyst step (Chen et al. 2012, Sánchez et al. 2011). In catalytic transesteri-
fication, technologies such as ultrasonication and microwave have been applied to
accelerate the conversion rate (Deng et al. 2010, Veljković et al. 2012). The addition
of ultrasonication or microwave generates pressure and heat, and thus enhances
mass transfer and increases the rate.
Apart from biodiesel synthesis through transesterification of oil extracted
from biomass, transesterification of oil-rich biomass to biodiesel, also called in situ
transesterification has been reported (Ehimen et al. 2012, Ehimen et al. 2010, Qian
et al. 2013) and is gaining more and more attention because of the elimination of
oil extraction, which is energy and cost consuming.
In this chapter, biodiesel production by transesterification will be addressed.
Parameters affecting the transesterification have been discussed. The new tech-
nologies and their applications in transesterification have been reviewed.
3.2 TRANSESTERIFICATION OF OIL/FAT TO BIODIESEL
Transesterification is known as the most popular approach for biodiesel

manufacture. It uses oil/fat (triglyceride) to produce fatty acid methyl esters
(FAMEs) and glycerol by reacting with alcohols. Among all alcohols, methanol is
preferable because of the cost consideration. Catalyst type, alcohol type, oil type,
alcohol-to-oil ratio, and assistance technology addition are significant parameters
of transesterification, and Equation 3-3 describes this reaction as follows:
BIODIESEL PRODUCTION BY TRANSESTERIFICATION 29
CH2 COOR1 − CHCOOR2 − CH2 COOR3 ðtriglycerideÞ

ðcatalystÞ
þ 3EtOHðmethonal=ethanolÞ!CH3 COOR1 þ CH3 COOR2
þ CH3 COOR3 þ CH2 OH − CHOH − CH2 OHðglycerolÞ
(3-3)
where R1, R2, and R3 are fatty acid chains. The products,CH3 COOR1 ,
CH3 COOR2 , and CH3 COOR3 represent alkyl (methyl, propyl, or ethyl) esters.
3.2.1 Catalyst Impact

According to the absence or presence of catalyst in the process (Equation 3-3),
biodiesel production can be divided into noncatalytic and catalytic biodiesel
production.
Noncatalytic biodiesel synthesis, as the name suggests, is to produce biodiesel
without catalyst addition. In the late 1990s, it was reported that the conversion rate
could be up to 85% after 10 h reaction when temperature was set at 235 °C (in a
catalytic system, the required temperature is around 50 °C) in biodiesel synthe-
sizing with soybean oil and methanol in a catalyst-free system (Diasakou et al.
1998). When excess and supercritical methanol was used, the conversion rate was
also increased up to 95% (Saka and Kusdiana 2001). In the study, the supercritical
methanol was achieved by treating methanol for a period of 3 min under a
pressure of 45 to 65 MPa at temperature of 350° to 400 °C. It was observed that
excess alcohol and critical conditions, such as high temperature (Diasakou et al.
1998), irradiation (Melo-Junior et al. 2009), and supercritical treatment (Saka and
Kusdiana 2001), were needed in the noncatalytic biodiesel production to achieve a
high conversion rate. However, it would also lead to high synthesis cost (owing to
the addition of large amounts of alcohol) and high energy input.
Catalytic biodiesel synthesis uses a catalyst in the reaction to urge the
conversion to complete. To some extent it is considered superior to the non-
catalytic method because the reaction can occur in mild conditions. The catalysts
that have been applied for biodiesel production are described next.
Homogeneous alkali-catalyzed transesterification is the most common bio-
diesel production process because of the low cost compared with enzyme and
heterogeneous catalysts, and high efficiency compared with acids (Grepen 2005).
In general, small amounts of water and FFA exist in oils and fats. As previously
mentioned, when alkalis are used, soap can be formed through the reaction
occurring between alkalis and free fatty acids (Equation 3-4). Therefore, the actual
required quantity of base to be added will be more than theoretically required,
according to the following:
RCOOH þ KOH=NaOH → RCOOK=NaðsoapÞ þ H2 O (3-4)

where R represents fatty acid chains.
Homogeneous acid catalytic biodiesel synthesis requires strong acid (con-
centrated H2SO4), a relative high temperature (around 65 °C), and a long reaction
time (>24 h) compared to the base catalytic system (Canakci and Gerpen 2003,
Vicente et al. 2009). The use of aggressive reagents (strong acid) demands high
attention to the operation and high corrosive resistance material in the reactor.
Enzymatic catalysts have attracted a growing attention in biodiesel produc-
tion because the process is more effective, selective, and environmentally friendly
(fewer by-products) than acidic or alkali catalyst (Chen et al. 2011, Du et al. 2004,
Park et al. 2008, Shaw et al. 1991). Enzyme (lipase) used in the biodiesel synthesis
process can be generated by all living organisms, such as microorganisms, animals,
and plants (Akoh et al. 2007). Among all the lipase sources, microorganisms have
shown a great advantage because of the high lipase yield. So far, many micro-
organisms such as Candida antarctica (Watanabe et al. 2007), Thermomyces
lanuginose (Xie and Ma 2009), Chromobacterium viscosum (Shah et al. 2004),
Penicillium sp., Pseudomonas sp., and Rhizopus sp. (Sellappan and Akoh 2005)
have been investigated to produce lipase. The mechanism of lipase catalytic
transesterification process is illustrated in Figure 3-1. Lipase (a polar substance),
which can be activated in water, catalyzes the reaction by entering the substrates
from the interface formed between lipid (insoluble in water) and alcohol (soluble
in water). It reflects that enzymatic synthesis allows the presence of water in the
reaction, which is an inhibition in the acid or base catalytic synthesis system.
Lipase catalytic biodiesel synthesis has been widely studied, but the high lipase cost
hampers its industrial application.
To reduce the production cost, two solutions have been reported: microbe
whole-cell utilization and immobilized lipase utilization (Li et al. 2008, Salis et al.
2008, Xie and Ma 2009). Microbe whole-cell utilization is a method to use the
whole microbial cells that contain a large amount of lipase in the cells instead of
using pure lipase as catalyst. Therefore, the production cost would be reduced
because of the avoidance of lipase separation and purification that are the major
causes of high lipase cost. Li et al. (2008) found that greater than 90% oil
conversion is accomplished by using Rhizopus oryzae whole cells as catalyst,
indicating that the lipase efficiency is comparable to free lipase.
The immobilized lipase catalytic process offers a cost-effective way for
biodiesel production by developing lipase reuse capacity. Several carriers, such
Interface
Alcohol
Lipid
Lipase
Water
Figure 3-1. Mechanism of enzymatic biodiesel synthesis.

as fiber cloth, acrylic resin, silica gel, hydrotalcite, nanoparticles, and macroporous
and microporous materials, have been reported for lipase immobilization (Bai et al.
2006, Dizge et al. 2009, Gao et al. 2006, Noureddini et al. 2005). Some studies
showed that the reused lipase could perform as stable and active as the initial one
(Jegannathan et al. 2008, Noureddini et al. 2005, Salis et al. 2008). Biodiesel synthesis
with free lipase, whole-cell lipase, and immobilized lipase is compared in Table 3-1.
The immobilized lipase shows a comparable performance in biodiesel yield.
Apart from whole-cell catalytic or immobilized catalytic biodiesel synthesis,
the combination technology, which is to immobilize the whole-cell catalyst onto
carriers, has also been investigated (Fukuda et al. 2008, He et al. 2008, Zeng et al.
2006). This method could be more cost-effective in biodiesel production when
compared to the production using whole cells or free lipase immobilized onto
carriers alone as catalyst.
Heterogeneous catalytic transesterification is another efficient method of
biodiesel production that requires no neutralization at the end of the process
and could keep the catalyst remaining in the reaction system by filtration.
Heterogeneous catalysts are usually alkaline and are also called solid base catalysts;
they include, for example, calcined Li-CaO (Watkins et al. 2004), Mg-Al hydro-
talcites (Xie et al. 2006), magnesia-rich magnesium aluminate spinel (Wang et al.
2008), Mg/Zr (Sree et al. 2009), and so on. CaO and MgO are the most often
investigated catalyst in transesterification (Table 3-2). Based on the studies,
nanoparticle-sized catalysts have shown an enormous advantage because of their
high efficiency. In fact, 99% biodiesel conversion was obtained by adding 0.6% by
weight (based on the oil weight) of nanocrystalline calcium oxides (Reddy et al.
2006). Boz et al. (2009) reported that 99.84% biodiesel yield was achieved by
adding 3% by weight (based on the oil weight) of nano-γ-Al2O3 catalyst particles
(<50 nm). It is evident that the use of nanoparticle catalysts has dramatically
reduced the catalyst addition quantity resulting in even higher biodiesel yield
(10% by weight addition is needed for normal size catalysts) (Veljković et al. 2009).
Overall, base catalytic synthesis is the major source of biodiesel in the market
because of the low price and developed technology. However, the drawbacks, such
as soap formation, large base consumption, and complicated separation and
purification of biodiesel, require the development of alternative approaches.
Immobilized lipase enzymatic synthesis and heterogeneous catalyst synthesis
have great potential in industrial production of biodiesel when nanotechnology
is used (Table 3-1 and 3-2).
3.2.2 Effect of Alcohol Type

Short-chain alcohols such as methanol and ethanol are used in the transester-
ification because the increase in chain length of alcohol lowers the biodiesel
formation rate (Hanh et al. 2009). The biodiesels produced by transesterification
with methanol and ethanol are called methyl esters and ethyl esters, respectively.
Methanol is the most applied alcohol in current biodiesel production because it is
cheaper, has smaller polarity, and provides a high conversion rate (Kulkarni et al.
32
Table 3-1. Summary of Biodiesel Synthesis Catalyzed by Enzyme.

Catalyst
addition
Catalyst C/O ratio Temperature Time Yield
type Lipase source/name Feedstock (% by weight) (ºC) (h) (%) Reference
Free lipase Candida antarctica Rapeseed oil 5 40 24 76.1 Jeong and Park
(2008)
Candida rugosa Rapeseed oil 40 45 24 97 Linko et al. (1998)
Candida cylindracea Waste-activated 10 37 3 <100 Kojima et al.

bleaching (2004)
earth
Chromobacterium Jatropha oil 10 40 8 62 Shah et al. (2004)
viscosum
Cryptococcus spp. Rich bran oil 10 30 96 80 Kamini and Iefuji
(2001)
Pseudomonas cepacia Palm kemel oil 10 40 8 72 Abigor et al.
(2000)
Rhizomucor Soybean oil NA 30 12 95 Guan et al. (2010)
miehei + Penicillium
cyclopium
Rhizopus oryzae Soybean oil 4−30 35 72 80−90 Kaieda et al.
(1999)
Rhizopus oryzae Palm oil 13 37 72 55 Lara Pizarro and
Park (2003)
Whole-cell Fusarium heterosporum Palm oil 3.1 30 96 98 Adachi et al.

lipase (2011)
Rhizopus oryzae Soybean 3.6 35 72 90 Ban et al. (2001)
Rhizopus oryzae Rapeseed oils 10 35 48 90 Li et al. (2008)
Rhizopus oryzae Waste vegetable 4.5 30 72 80 Jin et al. (2009)
oil
Virgin canola oil 75
Brown grease 55
Immobilized Canadida antarctica Soybean oil 4 30 24 93.8 Watanabe et al.
lipase (2002)
Canadida antarctica Cottonseed oil 1.7 50 24 97 Royon et al. (2007)
Candida antarctica Jatropha oil 10 50 12 91.3 Modi et al. (2007)
Karanj oil 90
Sunflower oil 92.7
Candida antarctica Acid oil 1 30 24 98 Watanabe et al.
(2007)
Candida antarctica Soybean oil 2 50 12 80 Ha et al. (2007)
Candida Soybean oil 20 45 3 99.13 Lee et al. (2011)
rugosa + Rhizopus
oryzae
Candida rugosa Palm oil 1 35 2 85 Moreno-Pirajàn
and Giraldo
BIODIESEL PRODUCTION BY TRANSESTERIFICATION
(2011)
Candida sp Rapeseed oil 5 40 36 98 Deng et al. (2003)
(Continued)
33
34
Table 3-1. Summary of Biodiesel Synthesis Catalyzed by Enzyme. (Continued)

Catalyst
addition
Catalyst C/O ratio Temperature Time Yield
type Lipase source/name Feedstock (% by weight) (ºC) (h) (%) Reference
Candida sp. Salad oil 20 40 6 96 Nie et al. (2006)

Lipozyme Soybean oil — 30 — 95 Du et al. (2003)
Lipozyme Soybean oil, 10 40 36 90 Du et al. (2005)
Pseudomonas cepacia Soybean oil 4.75 35 1 67 Noureddini et al.

(2005)
Pseudomonas cepacia Jatropha oil 10 50 8 93 Shah and Gupta
(2007)
Pseudomonas cepacia Tallow tree oil 2.7 41 24 97 Li and Yan (2010)
Pseudomonas Safflower oil, 0.3 50 25 99 Iso et al. (2001)
fluorescens
Rhizopus miehei Soybean oil 25 36.5 6.3 92.2 Shieh et al. (2003)
Thermomyces Canola oil 1 50 24 97 Dizge et al. (2009)
lanuginosus
Thermomyces Soybean oil 60 50 30 90 Xie and Ma (2009)
lanuginosus
Thermomyces Pomace oil 5 25 24 93 Yücel (2011)
lanuginosus
Note: NA = not available.
Table 3-2. Summary of Biodiesel Synthesis Catalyzed by Heterogeneous Catalyst.

Catalyst
addition C/O
ratio (% by Temperature Time Yield
Catalyst type Lipase source/name Feedstock weight) (ºC) (h) (%) Reference
Other catalyst Al-MCM-41 Palmitic acid 0.6 130 2 79 Carmo et al. (2009)
Ba(OH) Canola oil NA 90 8 90 Dalai et al. (2006)
CaO (nanosized) Poultry fat 0.6 25 12 99 Reddy et al. (2006)
CaO Rapeseed oil 0.8 60 3 90 Kawashima et al.
(2009)
CaO Sunflower oil 1 60 2 98 Veljković et al.
(2009)
CaO Palm oil 7 60 1 94 Yoosuk et al. (2010)
CaO (nanosized) Soybean oil 16 60 6 93.5 Luz MartiÌnez et al.
(2011)
CaO/MgO Rapeseed oil 2 64.5 8 92 Yan et al. (2007)
KI/mesoporous silica Soybean oil 15 70 8 90.09 Samart et al. (2009)
KF/Al2O3 Canola oil 6.5 60 8 87 Xie and Chen (2006)
KF/Al2O3 (nano sized) Vegetable oil 3 NA 8 99.84 Boz et al. (2009)
KF/CaO–Fe3O4 Stillingia oil 4 65 3 95 Hu et al. (2011)
(nanosized)
KF/Zn(Al)O Vegetable oil 3 65 3 95 Xu et al. (2010)
Li-CaO (nanosized) Karanja oil 5 65 1 100 Kaur and Ali (2011)

Jatropha oil 2 100
35
(Continued)
36
Table 3-2. Summary of Biodiesel Synthesis Catalyzed by Heterogeneous Catalyst. (Continued)

Catalyst
addition C/O
ratio (% by Temperature Time Yield
Catalyst type Lipase source/name Feedstock weight) (ºC) (h) (%) Reference
MgO Soybean oil 5 180 2 72 Di Serio et al. (2006)

MgO (nano sized) Soybean oil 1.5 70 6 90 Verziu et al. (2008)
Na2MoO4 Soybean oil 5 65 3 95 Nakagaki et al.
(2008)
SrO Soybean oil 3 70 0.5 95 Liu et al. (2007)
WO3/ZrO2 Soybean oil 20 75 3 93 Park et al. (2010)
Note: C/O ratio presents the catalyst-to-oil ratio; NA = not available.
2007). Ethanol is receiving increased attention because it is nonpoisonous and

results in higher biodiesel lubricity compared with methanol (Peterson et al. 1992).
To compromise the advantage and disadvantage of using solo methanol and
solo ethanol, a mixture (1:1 mol/mol) was used in the transesterification (Kim et al.
2010, Kulkarni et al. 2007). Studies showed that no difference was observed in the
conversion rate when using the mixture of methanol and ethanol, single methanol,
or single ethanol. However, the lubricity of the produced biodiesel with the mixture
of methanol and ethanol was improved compared with that of single methanol.
3.2.3 Effect of Oil Type

Vegetable oils and animal fats are the current biodiesel production feedstocks.
The increased price of vegetable oil and animal fat shifts the focus of biodiesel
production from vegetable oil and animal fat to waste cooking oil and microbial
oil. The suitability of waste cooking oil and microbial oil must be evaluated in
terms of the property of the feedstock (oil or fat) which includes fatty acid
composition, FFA content, water sulfur, and phosphorus content as they deter-
mine the transesterification catalyst selection and the product properties.
Fatty acid composition plays an important role in biodiesel qualities related to
viscosity, oxidation stability, cetane number (CN) (an indicator of ignition
quality), cold flow property, flash point, calorific value (also called heat content
or energy density), and density of biodiesel. Viscosity indicates the fuel features of
spray, mixture formation, and combustion process. High viscosity can cause early
injection and increase combustion chamber temperature. Normally, viscosity
increases with the increase in the chain length and in fatty acid saturation level,
whereas better oxidation stability requires a high level of fatty acid saturation
(Goering et al. 1982a, Içingür and Altiparmak 2003). Cetane number shows the
similar trend as viscosity, which implies that CN increases as an increase in chain
length and saturation of fatty acid (Içingür and Altiparmak 2003, Knothe 2005).
Cold flow properties depend on the saturation level of the feedstock oil. The higher
the saturation level, the poorer the cold flow properties (Chapagain and Wiesman
2009, Ramos et al. 2009). The flash point will be low when the chain length is short
(Karmakar et al. 2010). It is predicted that greater saturation will give higher
calorific value (Karmakar et al. 2010). The level of polyunsaturation seems to be in
proportion to the density according to (Karmakar et al. 2010).
As discussed before, alkaline catalytic transesterification is not suitable for
feedstock with high FFA content (more than 2%), such as animal fat or used
cooking oil, because of the concern of soap formation. In general, biodiesel
production from oil or fat with a high FFA content requires a two-step conversion
with the first step of esterification (FFA to biodiesel with acidic catalysis) and the
second step to finally complete the transesterification with alkali catalysis.
Water content in feedstock oil has a great impact on the biodiesel synthesis
because water may cause the hydrolyzation of triglyceride to FFA (Anderson et al.
2003, Sanford et al. 2009). The FFA formation would lead to soap generation when
base is used as catalyst. In addition, emulsions could occur with the presence of
water in the feedstock oil. Therefore, a water removal step is required to be
performed prior to the transesterification when the water content in feedstock is

more than 0.05% (wt/wt) (Sanford et al. 2009).
Phosphorus can induce the damage on catalytic converters used in emissions
control systems of the vehicles (del Río 2007). Normally, phosphorus content in
biodiesel (from feedstock) needs to be controlled to conserve the functionality of

the exhaust gas treatment systems during the operational life of the vehicle, and
thereby reduce the level of emission pollutants in the environment.
The presence of sulfur in biodiesel greatly increases the risk of choking up the
catalytic converter and harming the emission control system of the vehicle. In
general, sulfur content in biodiesel produced from animal fat and plant seed oil is
nearly zero, which is why that biodiesel is used to blend with petro-diesel for
reducing the sulfur content in diesel (Sanford et al. 2009).
3.2.4 Alcohol-to-Oil Ratio

Equation (3-3) shows that 1 mol triglyceride requires 3 mol alcohol in transes-
terification. To drive the reaction to proceed to the right, excess alcohol is
required, which is the reason why normally a 6-to-1-molar ratio of alcohol to
oil is used in industrial biodiesel production from transesterification (Boz et al.
2009, Dizge et al. 2009). However, it is not true that a higher molar ratio gives a
better conversion rate. A high alcohol-to-oil molar ratio could increase the
solubility of biodiesel and result in difficulty separating glycerol and biodiesel.
When glycerol remains in the system it would lead the reaction to go toward the
left (dissociation of biodiesel). Researchers have investigated the effect of the molar
ratios of methanol to oil from 3:1 to 12:1 on transesterification of Jatropha oil to
biodiesel and observed that 9:1 was the best ratio with a conversion rate greater
than 93% (Vyas et al. 2011). The highest conversion rate (93.5%) was obtained in
the methanol-to-oil molar ratio of 6:1 among 3:1, 6:1, and 8:1 in the transester-
ification of duck oil to biodiesel (Liu and Wang 2013). The investigation showed
that the methanol to waste cooking oil molar ratio at around 6:1 provided the
better conversion rate than at 3:1, 9:1, and 12:1 (Kawentar and Budiman 2013).
3.2.5 Assisted Transesterification

Ultrasonic cavitation or hydrodynamic cavitation has been reported for enhancing
transesterification (Gogate 2008, Ji et al. 2006, Stavarache et al. 2005). In these
studies, high conversion biodiesel yields (98% to 99%) were obtained within a
short time (10 to 30 min), and only a half quantity of catalyst (0.5% wt/wt) was
required compared with the conversional base catalyzed process (several hours
reaction time). Cavitation caused by ultrasound or flow microscopically generates
high temperatures (227° to 14,727 °C) and pressures [(100 to 5,000 atm)
(1.013 × 107 − 5.065 × 108 pa)] locally, while the overall system keeps atmospheric
conditions (T: 25 °C and P: 1 atm). The locally high temperature and pressure
enhance the transesterification biodiesel synthesis (Suslick 1989). Therefore,
ultrasonic or hydrodynamic cavitation can be used in the biodiesel production
industry to reduce the reaction time.
Microwave-assisted transesterification has also been reported. To achieve a

similar conversion rate, microwave assistance could significantly reduce the
reaction time from several hours to several minutes (Azcan and Danisman
2008, Azcan and Yilmaz 2013). In fact, it is the process to utilize microwave
irradiation to rapidly heat up the system and accomplish the conversion. However,
the effect of the high temperature on feedstock oil should be studied.
3.3 TRANSESTERIFICATION OF OIL-BEARING SUBSTANCES

TO BIODIESEL
The general transesterification is to convert oil or fat to biodiesel. Recently,

directly transferring an oil-bearing substance to biodiesel without the step of oil
separation/extraction from the substance has been studied. This process is also
called in situ transesterification. The technology becomes promising by the
avoidance of oil extraction, which is a high-energy and cost-requiring process.
The difference between in situ and normal transesterification is to perform
the transesterification by using oil-bearing substances instead of oil. In normal
transesterification, oil directly contacts the methanol and catalyst; hence the
reaction is easier than in situ transesterification. In in situ transesterification,
either long reaction time or addition of a large amount of methanol has to be
provided or cell disruption technologies have to be performed simultaneously
to achieve the transesterification. Using soy flakes to produce biodiesel through
alkali transesterification at 60 °C (8 h) required three times longer to achieve
the similar conversion rate as using normal transesterification oil to biodiesel
(less than 2 h) under the same conditions (Haas et al. 2004). Another study
reported that the time would be 16 h (Haas and Scott 2007). In addition, review
of in situ transesterification showed that a very large methanol-to-oil molar
ratio (300:1 to 900:1) was required to obtain a high conversion rate (Samuel and
Dairo 2012).
The high reaction time and large amount of alcohol addition requires
improvement of the current in situ transesterification. Treatment for cell disrup-
tion, which enhances the contact between oil and reactant (alcohol), would assist
the process. There are many methods (homogenization, ultrasonication, micro-
wave, among others) for cell disruption. Those suitable for in situ transesterifica-
tion are adding solvent, bead milling, ultrasonication, and microwaving. In fact,
methanol is a reactant as well as a solvent in in situ transesterification. Methanol is
polar (Methanol makes the cell wall weak) and oil is nonpolar, thus methanol
cannot pull oil from the cells. Solvent addition can be used to enhance oil transfer
from cell to outer environment, and thus achieve high biodiesel yield in in situ
transesterification (Mondala et al. 2009). Hexane and toluene can be used. It was
reported that toluene addition highly improved the biodiesel yield to 86% from
27% and reduced the reaction time to 1 h from 4 h compared with that without
solvent addition (Xu and Mi 2011).
Using ultrasonication during in situ transesterification is to create vigorous

mixing and enhance mass transfer. The formation and collapse of micro-bubble
causes rapid variation in pressure and temperature at microscopic local, which
thus enhances the mass transfer. One study found that 93% conversion rate was
achieved in 15 min at 60°C with a methanol:oil molar ratio of 315:1 under

ultrasonication in in situ transesterification of microalgae biomass to biodiesel
(24 kHz, 200 W) and only 32% conversion was obtained in normal in situ
transesterification (Ehimen et al. 2012). The combination of ultrasonication and
cosolvent addition could further increase the conversion rate to 99% with much
less methanol addition (methanol-to-oil molar ratio of 79:1) (Ehimen et al. 2012).
It was observed that the biodiesel from in situ transesterification had a similar
profile as the biodiesel obtained from traditional transesterification of oil (Haas
et al. 2004, Samuel and Dairo 2012). The remarkable advantage of the technology
is the simplification of the process (Figure 3-2), and the disadvantage is the large
amount of excess alcohol demand (79:1 for in situ and 6:1 for normal transester-
ification). Great effort is required to reduce the addition of alcohol because it is
associated with the energy and cost consumption of the process.
Oil bearing substances
Oil extraction In situ transesterification
Oil separation from

Biodiesel separation from
residue
mixture
Oil Biodiesel
Transesterification
Biodiesel separation from

mixture
Biodiesel
Figure 3-2. Normal transesterification and in situ transesterification for biodiesel
production from oil bearing substances.
3.4 BIODIESEL PRODUCTION
As discussed, biodiesel can be synthesized through transesterification or in

situ transesterification. The major flow charts of different transesterification
processes for synthesis of biodiesel are shown in Figure 3-3. In general, the
process highly depends on the feedstock characteristics. With vegetable oil
and animal fat as feedstock, biodiesel could be synthesized with base as the
catalyst because of the low FFA content in these oils or fats [Figure 3-3(a)].
After transesterification, two phases—an upper phase as crude biodiesel and a
lower phase as crude glycerol—are present. As aforementioned, methanol is
normally greatly in excess in the transesterification process to enhance the
conversion efficiency of the oils or fats to biodiesel. Therefore, after the
reaction, methanol recovery is normally performed. However, in real practice,
the recovery is usually avoided because of its low cost and high energy
demand in the recovery process. The upper phase and lower phase will be
sent for purification to finally obtain high quality biodiesel (99%) and glycerol
(90%) after methanol recovery.
Because of the increasing price of vegetable oils and animal fats, an attempt
has been made to use waste cooking oils to produce biodiesel. Acids are normally
used to catalyze the reaction of transferring waste cooking oil for biodiesel, which
is attributed to the high content of free fatty acids (leading to the formation of soap
with base as catalyst). During the reaction, the free fatty acids will be converted to
biodiesel and water, and the triglyceride will be turned to biodiesel and glycerol
[Figure 3-3(b)]. After purification in both phases, pure biodiesel and glycerol
would be obtained, respectively.
Microalgae have been widely reported to be a potential oil source for
biodiesel production. The main steps of biodiesel production from microalgae
are microalgae cultivation, microalgae biomass harvesting, lipid extraction
from biomass, and the remaining steps were similar to the process of biodiesel
production from vegetable oils and animal fats [Figure 3-3(c)]. There is also a
simple step in which the lipid extraction and transesterification are combined,
and in situ transesterification. In the biodiesel production from microalgae
with in situ transesterification, the biomass obtained after microalgae harvest-
ing with or without drying is transferred to biodiesel [Figure 3-3(d)]. In
general, hexane extraction has to be conducted to extract the biodiesel from
the mixture, and the pure biodiesel will be obtained after removal of the
solvent (hexane).
Apart from the homogeneous catalyst, heterogeneous catalyst can also be
used in transesterification because it is easy to separate from the product; however,
no related process has been applied in practice owing to the cost concern and
operation difficulties. With the similar reaction condition, heterogeneous catalyst–
assisted transesterification required a higher cost compared with the one assisted
by a homogeneous catalyst (Table 3-3). The current focus of biodiesel production
is in the utilization of waste cooking oils by alkali transesterification, and studies
showed that the cost was also more favorable.
(a) Biodiesel production from vegetable oils and fats

KOH
Heating
Oil/fat Transesterification Mixing
Methanol
Phase separation
Methanol
recovery
Lower phase Upper phase
Purification Washing
Saponified Refined Waste

products Biodiesel
glycerol water-alkaline
(b) Biodiesel production from waste cooking oils
Waste Purification (removal of

cooking particles)
oils H3PO4
Esterification/ Mixing
transesterification
Methanol
Phase separation
Methanol
recovery

Biodiesel
products glycerol water-acidic
Figure 3-3. Major processes of biodiesel production from different sources of
feedstock.
(c) Biodiesel production from microalgae lipid by

transesterification
Microalgae cultivation
Microalgae harvesting
Wet microalgae biomass
Microalgae drying
Lipid extraction
KOH
Heating
Transesterification Mixing
Methanol
Phase separation
Methanol
recovery

products Biodiesel
glycerol water-alkaline
Figure 3-3. (Continued)

(d) Biodiesel production from microalgae lipid by in situ

transesterification
Microalgae cultivation
Microalgae harvesting
Wet microalgae biomass
Microalgae drying Heating KOH
In situ transesterification Mixing
Methanol
Extraction
Lower phase Solvent phase
Purification Evaporation
Residual Refined Extraction

biomass Biodiesel
glycerol solvent
3.5 SUMMARY
Transesterification is the most applied biodiesel synthesis route. It is the process

that 1 molar triglyceride reacts with 3 molar alcohol to form 3 molar biodiesel
(FAMEs) and 1 molar glycerol. In the presence of catalyst (homogeneous acid or
base, enzyme, and heterogeneous catalyst), the reaction is faster and the
Table 3-3. Different Biodiesel Production Comparison.

Feedstock Catalyst Reaction condition Cost (US$/L) Reference
Canola oil NaOH Oil to methanol molar ratio: 1:9; 1.000 Lee et al. (2011)
Temperature: 70 °C;
Reaction time: 3 h
Jatropha curcas oil Alkali CaO Oil to methanol molar ratio: 1:9; 0.665 Labib et al. (2013)
Reaction time: 3 h
Palm oil H2SO4 Oil to methanol molar ratio: 1:3; 0.813 Simasatitkul and
Temperature: -; Arpornwichanop (2017)
Reaction time: -
Waste cooking oil H2SO4 Oil to methanol molar ratio: 1:30; 0.600 Zhang et al. (2003)
Reaction time: 69 h
Waste cooking oil NaOH Oil to methanol molar ratio: 1:30; 0.762 Lee et al. (2011)
Reaction time: 69 h
Waste cooking oil Tin(II) oxide Oil to methanol molar ratio: 1:6; 0.935 Abubakar et al. (2015)
Temperature: 25º and 70 °C;
Reaction time: -
(Continued)
45
46
Table 3-3. Different Biodiesel Production Comparison. (Continued)

Feedstock Catalyst Reaction condition Cost (US$/L) Reference
Waste cooking oils NaOH Oil to methanol molar ratio: 1:6; 0.747 Hussain et al. (2016)
Temperature: -;
Reaction time: -
Waste cooking oil NaOH Oil to methanol molar ratio: 1:6; 0.716 Glisic et al. (2016)
Reaction time: 1.8 h
Microalgae NaOH Oil to methanol molar ratio: 1:30; 0.970 Nagarajan et al. (2013)
Reaction time: 69 h
conversion rate is higher at mild conditions (50°–60 °C, 0.101 MPa) compared to a
noncatalyst reaction, which requires high temperature (around 200 °C) and
pressure (50 MPa).
Parameters including catalyst type, feedstock oil property, and methanol-to-
oil molar ratio have great impact on the transesterification. Alkali catalytic
transesterification is widely applied in industrial biodiesel production because of
the short reaction time required (less than 2 h). However, the method is not
suitable for transesterification of oil with an FFA content greater than 2%; thus,
acidic catalyst transesterification should be used in biodiesel synthesis. Enzyme,
free or immobilized, has also been used in the reaction, which has high efficiency
but the high cost hampers its application. Heterogeneous catalysts are the solid
acid or base, and its disadvantage is of mass transfer. Nanosized heterogeneous
catalyst is the solution to the problem and would be a promising catalyst because it
is easily recovered and could contact the reactant very well. A methanol-to-oil
molar ratio of 6:1 or 9:1 is normally sufficient to achieve high efficiency
transesterification, although it could alter as the reaction condition (temperature
and pressure) changes.
To reduce the reaction time, assisting technologies such as microwave
irradiation and ultrasonication have been applied in the transesterification. They
provide a high conversion rate with short time (several minutes).
In situ transesterification draws growing interest owing to the avoidance of oil
extraction. The technology directly converts oil located in oil-bearing substances
to biodiesel without affecting the biodiesel profile (fatty acid ester composition).
The problem of the process is the long reaction time and high alcohol-to-oil ratio.
To solve the problem, solvent, ultrasonication, or microwave irradiation can be
used, which would be widely applied in biodiesel production in the future.
3.6 ACKNOWLEDGMENTS
We thank the Natural Sciences and Engineering Research Council of Canada

(Grant A 4984, Canada Research Chair) for their financial support. The views and
opinions expressed in this chapter are those of the authors.
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CHAPTER 4
Enzyme-Catalyzed
Transesterification for
R. Kaur
S. K. Yellapu
R. D. Tyagi
4.1 INTRODUCTION
Owing to certain limitations associated with chemical catalysts, researchers

have gained interest in development of more promising technology for biodiesel
production using biocatalysts (Fjerbaek et al. 2009). Use of lipases as biocatalysts is
more attractive due to production of high-quality biodiesel, requirement of
mild reaction conditions, and no soap formation, less consumption of energy,
easy product recovery, and reusability compared with chemical catalysts
(Brun et al. 2011). The mechanism of enzyme transesterification is similar to
general transesterification, but acid or base catalyst is replaced by lipase enzyme
(Figure 4-1). Despite several advantages, the enzyme biodiesel production is very
costly, which is always a barrier for commercialization. Researchers are devoted to
improving the technology to make this process economical. The use of immo-
bilized lipase, improvements in the immobilization techniques, screening,
and production of more stable enzyme and developments of efficient bioreactors
can improve the process and can make it a commercial possibility (Peralta-Yahya
et al. 2012).
In this context, this chapter critically reviews the enzyme-catalyzed biodiesel
production, focusing on lipase as the biocatalyst, factors affecting lipase-catalyzed
transesterification, advancements in immobilization techniques, and development
of bioreactors for competent transesterification with economic considerations for
developed technology.
53
Figure 4-1. Enzyme transesterification reaction.
4.2 FEEDSTOCKS AND GENERAL METHODS FOR BIODIESEL

PRODUCTION
4.2.1 Potential Feedstocks

The feedstocks, which contain fats or oils, are represented as raw materials for
biodiesel production. The choice of feedstock depends on the abundance of raw
materials at a particular region, such as soybean oil in the United States, rapeseed
and sunflower oil in Europe (Knothe 2013), canola oil in western Canada (Ahmad
et al. 2011), palm oil in Indonesia and Malaysia, Jatropha oil in India and Africa
(Khan et al. 2009), or coconut oil in Philippines (Ahmad et al. 2011). However,
because the major concern with biodiesel production is to cut down the produc-
tion cost, it is important to choose efficient and economical feedstocks for
biodiesel production because the feedstocks can add up to 75% of the total
production cost for biodiesel (Tan et al. 2013).
The feedstocks are divided into three categories: first, second, and third
generation. First-generation feedstocks include edible oils like soybean, rapeseed,
sunflower, coconut, palm, sesame, groundnut, peanut, barley, sesame, and safflower
(Ahmad et al. 2011, Lin et al. 2011, Shahid and Jamal 2011, Kumar and Sharma
2014, Verma and Barrow 2015). Use of edible oils as potential feedstock has been
criticized because they are an essential part of human food, and a food-versus-fuel
crisis may arise due to the enormous requirement of land, water, and fertilizers for
the growth of the plants required for biodiesel production (Gui et al. 2008, Ahmad
et al. 2011). Moreover, the large requirement of land for cultivation of biofuel crops
can extend to deforestation and may lead to a negative impact on the environment
(Atabani et al. 2012, Poppe et al. 2015). Therefore, to deal with major environmental
concerns, it was necessary to find economical feedstocks, which led to the use of
second-generation low-cost feedstocks for biodiesel production.
The second-generation feedstocks include nonedible oils (Jatropha, tobacco,
mahua, jajoba, tall, salmon, castor) (Rattanaphra and Srinophakun 2010), animal
fats (poultry fat, fish oil, beef tallow, pork lard), waste cooking oil, and restaurant
grease (Ahmad et al. 2011). Because of the release of toxic substances, nonedible
oils are not consumed by humans and can be cultivated on wasteland. Therefore,
their use as feedstock can eliminate food-versus-fuel crisis (Ahmad et al. 2011,
Verma and Barrow 2015). However, there are several disadvantages of second-
generation feedstocks, such as the presence of more saturated fats in animal fats,
which makes them inefficient to be used at cold temperature. Moreover, animal
ENZYME-CATALYZED TRANSESTERIFICATION FOR BIODIESEL PRODUCTION 55
fats may be contaminated and cause major concerns to health. To grow, many
nonedible plants also require land with more humid soil (Janaun and Ellis 2010).
Waste cooking oils are good feedstock because they do not require land to grow, and
their use can also address the waste disposal issue (Verma and Barrow 2015).
Millions of tons of waste cooking oil (e.g., ~10 million tons/year in the United States,
0.12 million tons/year in Canada, 4.5 million tons/year in China) is available each
year; therefore, availability of feedstocks for biodiesel production can easily compete
with diesel fuel. However, both nonedible oils and waste cooking oils contain
enormous amounts of free fatty acids and a high water content that may require
further chemical treatment steps and lead to high production cost (Upham et al.
2009, Leung et al. 2010). Restaurant grease can be considered as efficient feedstock
because collection of grease is around 2.6 billion poundsyear (1.17 billion kg/year).
Moreover, restaurant grease [(yellow grease: USD 0.09–0.20/lb (USD0.20-0.44/kg);
brown grease: USD 0.01–0.03/lb.(02–0.06/kg)] is an inexpensive source compared
with vegetable oils (Canakci 2007).
Unsustainability and requirement of land for growth of crops leads to
exploration of third-generation feedstocks, which includes oleaginous microor-
ganisms like bacteria (Rhodococcus, Streptococcus, Nocardia, Acinetobacter,
Arthrobacter, Bacillus alcalophilus, Mycobacterium, Gordonia sp.); microalgae
(Chlorella protothecoides, Chlorella vulgaris, Botryococcus braunii, Schizochytrium
sp., Scenedesmus sp., Nannochloropsis sp., Isochrysis galbana, Pithophora, Spiro-
gyra, Microcystis); yeasts (Candida curveta, Lipomyces starkeyi, Cryptococcus
albidus, Rhodosporidium toruloides, Rhodotorula glutinis, Yarrowia lipolytica);
and fungus (Cunninghamella echinulata, Aspergillus terreus, Aspergillus oryzae,
Claviceps purpurea, Tolyposporium, Mortierella alpina, Humicola lanuginose,
Mortierella isabellina, Mucorales, Mortierella vinacae) (Kumar and Sharma
2014, Singh et al. 2014, Verma and Barrow 2015, Xu et al. 2017). Microorganisms
that accumulate lipids more than 20% are considered as oleaginous microorgan-
isms (Christopher et al. 2014). Some microorganisms can accumulate lipids up to
80% under nitrogen-deficient conditions, which are sufficient to efficiently
compete with primary and secondary feedstocks (Certik et al. 1999). In addition,
oleaginous microorganisms have fast growth and productivity compared with
crops required for biodiesel production (Minowa et al. 1995). Furthermore,
valuable by-products produced during growth like proteins, carbohydrates, and
biomass can also be used as fertilizers (Ahmad et al. 2011). Despite many
advantages, the fatty acid methyl esters (FAMEs) produced by using oil from
oleaginous microorganisms are not of high quality as those derived from plant
oils; therefore, further research is needed to improve the quality of biodiesel.
4.2.2 Overview of Transesterification Methods

High viscosity and low volatility of feedstocks make them inefficient to be directly
used in diesel engines. This may lead to other problems like oil ring sticking,
deposits of high carbon, gum formation, high cloud and pour points, trumpet
formation on the injectors, gelling of lubricating oil, and engine abrasion (Meher
et al. 2006, Ghaly et al. 2010, Christopher et al. 2014). To avoid these problems
associated with feedstocks, the triglycerides and free fatty acids (FFA) present in
the oil are converted into fatty acids alkyl esters (FAAE), which have properties
similar to conventional diesel (Fjerbaek et al. 2009). Four methods are available for
conversion of oil into biodiesel: blending of diesel fuel with oil, microemulsion,
pyrolysis, and transesterification.

Direct use by diluting vegetable oil with diesel fuel to improve the viscosity of
oil has been investigated (Ahmad et al. 2011). Different ratios (oil:diesel fuel of
1:10 and 2:10) are found to be effective for short-term use, but long-term use
caused high wear problems as well as gum formation (Fukuda et al. 2001, Leung
et al. 2010, Koh and Ghazi 2011). Another method, microemulsion involves using
solvents like methanol, ethanol, butanol, hexane, and octanol to reduce the
viscosity of oils by forming microemulsions (Fernando and Hanna 2005).
However, disadvantages of this method include heavy carbon deposits and
incomplete combustion (Fukuda et al. 2001). The third method, pyrolysis or
thermal cracking, involves extreme heating to convert triglycerides into FAAEs in
the absence of air. However, the cost of equipment, production of low value
materials, and gasoline remain the major barriers to implement this technology at
large scale. Among all the methods, transesterification has been found to be the
most cost effective, high yielding, and ecofriendly method of converting trigly-
cerides into FAAEs by using alcohol in the presence of catalyst (Ghaly et al. 2010,
Ahmad et al. 2011, Christopher et al. 2014).
Transesterification is defined as the catalytic or noncatalytic process of
exchanging the alkoxy group of an ester with alcohol, which leads to the
conversion of triglycerides to FAAEs along with the release of glycerol as the by-
product (Christopher et al. 2014). It is basically a sequence of three reversible
steps, including conversion of triglycerides to diglycerides, monoglycerides, and
then finally glycerol (Meher et al. 2006, Ghaly et al. 2010). The most commonly
used alcohols for transesterification reaction are methanol and ethanol due to their
cheap price and properties like short chain lengths (Fernando and Hanna 2005).
The catalyst increases the rate of reaction by increasing the surface contact and
further increases the biodiesel yield (Ahmad et al. 2011). Catalysts are mainly
divided into two categories: chemical (acid and alkali) and enzymatic.
4.2.2.1 Alkali-Based Transesterification

At high FFA concentration, base catalysts react and form soap, which leads to low
yield of FAMEs and inhibition of glycerol separation from biodiesel (Meher et al.
2006). In addition, high water content causes saponification that leads to
consumption of catalyst for formation of soap and enhances viscosity of the
reaction. High viscosity ultimately causes gel formation and makes the separation
of by-products difficult (Meher et al. 2006, Helwani et al. 2009, Ahmad et al. 2011).
4.2.2.2 Acid-Based Transesterification

Major drawbacks of base catalysts like sensitivity to high FFA content and soap
formation are overcome by acid catalysts (Balat and Balat 2010). The most
commonly used acids for transesterification are sulfuric acid, hydrochloric acid,
and sulfonic acid. The main advantages of using acid catalysts are that low-cost
feedstocks can also be used, and esterification and transesterification can take
place simultaneously by using acidified alcohol (Ahmad et al. 2011). Moreover,
using an acid catalyst gives a high yield of esters and eliminates the two-step
process (Helwani et al. 2009; Ahmad et al. 2011). Nevertheless, acid catalysts are
susceptible to even a low concentration (0.1 percent by weight) of water, and also
the reaction rate is very slow. Acid catalysts require a high molar ratio of alcohol to
oil (30:1) to achieve reaction in a short time with a high yield (Lam et al. 2010).
Other limitations include corrosion of equipment, high temperature, formation of
salts during neutralization of unreacted catalyst, weak catalytic activity, longer
reaction time, and difficulty to recycle (Bacovsky et al. 2007, Hoang et al. 2013).
4.2.2.3 Enzymatic Transesterification

The enzymatic transesterification process has gained considerable attention by
researchers in recent years compared with other chemical methods because of the
lower energy requirement, reduced limitation of feedstocks, insensitivity to the
presence of free fatty acids, production of high purity products, easy separation
from other by-products, biodegradability, and less generation of wastewater
(Oliveira and Mantovani 2009)
As a biocatalyst, lipase is a carboxylesterase (i.e., a family of enzymes that
hydrolyzes carboxylic esters) that can enhance the chemical reaction. Lipolytic
enzymes are currently more attractive for industrial applications (Jegannathan
et al. 2008). The lipase enzyme was initially determined from Rhizomucor miehei
and in human pancreases in 1990. The molecular weight ranges from 19 to
60 kDa, and all lipases have a characteristic folding pattern known as the three-
dimensional (3D) structure of the bacterial lipase (Figure 4-2). The central lipase is
composed of β strands that are connected to α helices. The catalytic site consists of
amino acids such as histidine, serine, and aspartate. The serine (nucleophilic)
residue is placed near the C-terminal end of strand β5, which is a very highly
conserved region and forms a β-turn-α motif named the nucleophilic elbow.
Substrate (triglycerides) hydrolysis is initiated with a nucleophilic attack by the
catalytic site (Yuce-Dursun et al. 2016). Lipases are considered as biocatalysts
because of their ability to hydrolyze the ester bond in the triacylglycerides (TAGs)
and to convert them into fatty acids, glycerol, and di- and monoglycerides.
Lipases are used in enzymatic transesterification owing to (1) their ability
to work in different reaction systems, including hydrophobic or hydrophilic
solvents, (2) bulk production of enzyme and versatility, (3) their ability to perform
transesterification with long- and branched-chain alcohol, (4) high thermostabili-
ty and specificity, (5) easy separation of products as well as by-products, and
(6) ability to perform transesterification with a wide range of FFAs (0.5% to 80%)
(Fjerbaek et al. 2009, Ghaly et al. 2010, Canet et al. 2017).
Despite several advantages, the enzymatic transesterification process has still
not been commercialized due to certain limitations, primarily its high production
cost (Zhao et al. 2014). Other limitations include requirement of high catalyst
concentration, slow reaction rate, alcohol inhibition, and inability to use enzyme
Figure 4-2. Three-dimensional lipase enzyme (Pseudomonas aeruginosa) struc-

ture. α-Helices are coils with blue or yellow; the yellow helix represents cap over
the active site; and β-sheets are represented as dark red arrows. The active site
residues D229, Ser82, and His251 are labeled; and the green ball is a Ca2+ ion.
catalyst repeatedly. For the last few years, continuous efforts have been made by
researchers to deal with the demerits and to increase the efficiency of the reaction
(Cesarini et al. 2013, Wang et al. 2013).
4.3 ENZYME-CATALYZED TRANSESTERIFICATION FOR BIODIESEL

PRODUCTION
4.3.1 Sources of Lipases

In general, lipase enzymes are of plant, animal, and microbial origin. Of those,
microbial lipases are more commonly used in biodiesel production due to their
high stability, bulk production, and availability at low cost (Ghaly et al. 2010).
Lipase- producing microorganisms have been screened from a variety of sources
like soil, marine waste, wastewater, and industrial waste. Microbial lipases have
been well characterized in terms of their properties and structure. Techniques to
isolate these microorganisms to increase the production of lipases have been well
established. Using fermenters for growth and selection of microbes in continuous

enrichment cultures has overcome the shortcomings of using time-consuming
batch cultures on agar substrates for the same purposes (Abramić et al. 1999).
Lipases are usually isolated from bacterial and fungal species like Bacillus
subtilis, Bacillus licheniformis, Streptomyces aureus, Clostridium, Corynebacteri-

um violaceum, Geobacillus, Streptococcus vibrio, Pseudomonas fluorescens,
Pseudomonas cepacia, Aspergillus niger, Penicillium roqueforti, Rhizomucor
miehei, Rhizopus oryzae, Rhizopus arrhizus, Thermomyces lanuginosus, Fusari-
um heterosporum, and Candida antarctica (Gupta et al. 2004, Akoh et al. 2007).
An example of extremophilic lipases includes Lip A and Lip B from hyperther-
mophillic anaerobe Thermosyntropha lipolytica, from psychrophiles Psychrobac-
ter okhotskensis, and from halophilic archaeon Haloarcula marismortui. Among
the yeasts, lipases from Candida rugosa are considered as commercially important.
4.3.2 Lipase Enzyme Production

Microbial lipase production takes place in different types of fermentation strate-
gies, such as batch, repeated-batch, fed-batch, and continuous mode. In most
cases, the mode of operation is, to a large extent, dictated by the characteristics of
the product of interest.
Carbon sources serve as important substrates for energy production in
microorganisms. In general, oil-rich carbon sources serve as inducers and are
essential for obtaining a high lipase yield. Pandey et al. (1999) reported that
C. rugosa lipase production increased with an increase in concentration of olive
oil, and the maximum lipase production was achieved at the 10% (v/v) oil
concentration. Production of a thermostable lipase from thermophilic Bacillus
sp. strain Wai 28A 45, in the presence of tripalmitin at 70 °C, was described by
Sharma et al. (2001). Media with tripalmitin, tristearin, and trimyristin carbon
sources were tested, and tripalmitin was found to be the best inducer of lipase
activity. Enzyme activity was not detected when glucose was the sole carbon
source, confirming that the presence of an inducer is necessary for Penicillium
aurantiogriseum to produce lipases (Lima et al. 2003). However, the requirement
for sugar as a carbon source in addition to lipids varies with the microorganism. In
general, media supplemented with glucose along with triglycerides stimulate the
lipase production in Rhizopus nigricans as reported by Ghosh et al. (1996). Both
olive oil and Tween-80 stimulated the production of extracellular lipase in
Penicillium citrinum at 0.1 and 0.7% (v/v), respectively. Paiva et al. (2000) showed
that Tween-80 stimulates both lipase biosynthesis and its secretion in C. rugosa.
The yeast Pseudozyma hubeiensis HB85A and Schizochytrium sp. S31 lipase was
strongly stimulated by 150.8% in the presence of Tween-80 compared to non-
Tween-80-supplemented media (Fernandez-Lafuente 2010). Carbon sources are
one of the major factors for the production cost of the enzyme. Therefore, to
decrease production cost, a cheap source such as oil-rich wastewater from the food
industry was an alternative carbon source (Sun and Hu 2017).
Municipal wastewater can be another alternative carbon source due to its oil
content. In activated sludge-based biological wastewater treatment processes,
specific TAGs accumulating organisms excrete extracellular lipases, catalyzing the

lipid hydrolysis present in the surrounding wastewater before its assimilation.
Microthrix parvicella possesses eight lipases. Because of the complexity of lipid
mixtures in wastewater and extensive inter-organismal competition, these
enzymes must have broad substrate specificity and high enzymatic efficiency.
These enzyme characteristics should prove very useful for transesterification
reactions involved in biodiesel production using a range of different lipid
feedstocks.
Lipase coproduction with other products such as microbial lipid and citric
acid using Yarrowia lipolytica in hydrophobic carbon substrates (vegetable fat,
animal fat) is an economical approach for industrial biodiesel production.
Aguieiras et al. (2017) investigated the potential of the utilization of raw agro-
industrial fat for production of lipase and citric acid by the yeast Yarrowia
lipolytica; after 6 days fermentation, the maximum lipase activity was found to be
2,760 units/mL.
4.3.3 Reaction Mechanism of Lipase-Assisted Transesterification

Transesterification reaction caused by lipase enzyme for each ester bond of
triglyceride involves two steps: (1) hydrolysis of the ester bond and release of
alcohol moiety; and (2) esterification with the other substrate.
The kinetics of the enzyme-catalyzed reaction is commonly described in
terms of the ping-pong model with competitive substrate inhibition. The mostly
accepted model for lipase transesterification is ping-pong Bi-Bi mechanism
(Figure 4-3), in which binding of one substrate changes the shape of the enzyme
and enables it to bind with another substrate (Al-Zuhair et al. 2007). Product is
released after binding of one substrate. For validation and fitting of experimental
results, Michaelis–Menten kinetics is used. Several kinetic studies have been
explained by different researchers(Stroberg et al., 2016).
The kinetics of triglyceride hydrolysis by lipase is dependent on various
factors, such as the type of lipase, type of organic solvent present, concentration of
reactants, mass transfer limitations, as well as the effect of temperature on
deactivation and formation and conversion of intermediates (Paiva et al. 2000,
Dossat et al. 2002).
Figure 4-3. Schematic representation of the ping-pong Bi-Bi mechanism.

4.3.4 Extracellular Lipases

Lipases are classified as extracellular lipases or intracellular lipases; both are used
in different industries. Extracellular lipases are those extracted from microorgan-
isms and purified for further use. After completion of fermentation, enzymes are
purified by using various techniques depending on the source and structure of

enzyme (Jegannathan et al. 2008). The most commonly used immobilized lipases
are extracellular, which involve Lipozyme TL IM produced by Thermomyces
lanuginosus, Lipozyme RM IM produced by Rhizopus mucor, and Novozyme 435
produced by Candida antarctica (Robles-Medina et al. 2009).
4.3.5 Intracellular Lipases and Whole-Cell Immobilization

High separation and purification costs of extracellular lipases have led to use of
intracellular lipases, which remain either inside the producing microorganism or
in the cell walls. Using microbial whole cells containing the intracellular lipases as
biocatalysts can produce ester with a high yield at a relatively low cost by
eliminating costly separation and purification steps (Jegannathan et al. 2008).
The fungal cell immobilization by using biomass support particles (BSPs) is an
effective technique. BSPs (polyurethane and polypropylene foam) are used
because they do not require chemical additives and particles are reusable and
resistant to mechanical shearing. Immobilization of whole cells does not require
extended immobilization techniques as in the case of extracellular lipases.
Rhizopus oryzae, Aspergillus, and Candida antarctica are commonly used as
whole-cell biocatalysts. Using intracellular enzymes increases the conversion
efficiency because they are relatively stable and with limited mass transfer
resistance (Ghaly et al. 2010).
4.3.6 Parameters Affecting Lipase-Catalyzed Transesterifications

To obtain high biodiesel yield, the transesterification process and rate of reaction
can be affected by different parameters, such as (1) chain length of alcohol,
(2) alcohol to substrate molar ratio, (3) use of solvents, (4) reaction temperature,
(5) water content, and (6) catalyst concentration.
4.3.6.1 Chain Length of Alcohol

The carbon chain length of the alcohol affects the reactivity of the transesterifica-
tion process. The most commonly used alcohols for the transesterification reaction
are short-chain alcohols, ethanol and methanol, because of their commercial
availability. Du et al. (2004) reported the use of short-chain alcohol acetate for
enzymatic transesterification because it does not have inhibitory effects on
enzymatic activity. Chulalaksananukul et al. (1992) investigated the reactivity of
alcohol (esters) such as ethyl acetate, methyl acetate, propyl acetate, butyl acetate,
isobutyl acetate, and amyl acetate of various chain lengths ranging from C3 to C10
and observed that the Michaelis constant and maximal velocities (Vmax/KM)
increased with an increase in chain length up to C5 (propyl carbonate), and
thereafter the reaction rate became constant (Table 4-1).
Table 4-1. Effect of Chain Length on Vmax/KM.
Alcohol Structure Vmax μmol h-1g-1) Vmax /KM
Ethyl acetate 389 432

Methyl acetate 375 293
Propyl acetate 448 772
Butyl acetate 438 584
Isobutyl acetate 439 399
Amyl acetate 322 388
Other researchers explained that activity of the lipase enzyme depends on the
chain length of the alcohol (Hama and Kondo 2013). Irrespective of the chain
length, branching of the carbon chain directly affects the rate of reaction, probably
due to the steric hindrance (collision) of the molecule. However, methanol and
ethanol are short-chain alcohols used as acyl acceptors in the transesterification
reaction.
Recently, the use of ethyl acetate and methanol for enzymatic transesterifica-
tion of rubber seed oil using Rhizopus oryzae lipase was reported; it was found that
ethyl acetate gave a better biodiesel yield even at high molar ratios compared with
methanol (Vipin et al. 2016). It was observed that when using molar ratios of 1: 4
for both alcohols (methanol and ethanol), ethyl acetate attained 27.9% conversion
and methanol attained 22.05% conversion. Furthermore, even stepwise addition of
methanol did not give better results compared with methanol, which may be
because of the short-chain alcohol (methanol and ethanol) inhibition action of
enzyme.
4.3.6.2 Alcohol-to-Substrate Molar Ratio

The molar ratio of alcohol to substrate (FFA, lipids, and so forth) is a very crucial
factor for complete conversion of oil to methyl esters. Because the transesterifica-
tion reaction is reversible, the higher concentration of alcohol shifts the rate of
reaction toward the product formation. After transesterification, residual alcohol
forms the layer on the active sites of the lipase enzyme, which can cause
deactivation of the enzyme. However, when hydrolysis of triglycerides occurs due

to enzyme binding, methanol solubility is a major factor, which affects the enzyme
activity (Hanefeld et al. 2009). Because of this limitation, Tan et al. (2010)
investigated the approach of stepwise alcohol addition for optimizing molar ratio
using solvent free systems. The conventional transesterification explains that

increase in alcohol concentration can further enhance the rate of reaction
(Tan et al. 2010).
Several researchers reported the effect of alcohol and oil ratio on FAMEs yield
by immobilizing lipase for transesterification of plant oil such as canola oil (Janaun
and Ellis 2010), microalgae oil (Ghaly et al. 2010), and cotton seed oil (Royon et al.
2007). The FAMEs yield obtained by several researchers was greater than 90%
using immobilized lipase as a catalyst with a methanol and oil molar ratio of
approximately 6:1 using different vegetable oils (Christopher et al. 2014). How-
ever, the scale of the experiment was less than 0.1 L as shown in Table 4-2.
Therefore, large transesterification experiments need to be conducted to meet the
technical challenges that could arise during the scale-up.
Methanol is a commonly used alcohol for the transesterification process due
to the nature of its reaction, less cost, and volatile nature. It is one of the by-
products from a nonrenewable source (fossil fuels). Lipase enzymes are inactivated
in the presence of organic solvent, which affects their catalytic activity (Chen and
Chen 2016). The increase in carbon chain and branched chains of alcohols inhibit
activity of lipase enzyme because solubility of alcohol is lower than the stoichio-
metric ratio (Antczak et al. 2009).
The lipase-catalyzed transesterification of Ocimum basilicum using Novo-
zyme 435 was also studied. It was reported that the methanol-to-oil ratio has a
significant effect on the rate of transesterification, and the optimum ratio varies
according to the process (Sankaran et al. 2016). The methyl ester conversion was
increased with an increase in the methanol to oil molar ratio from 3:1 to 12:1,
whereas conversion efficiency started to decrease when the ratio was increased
greater than 12:1, which was due to inactivation of the enzyme resulting from
excess methanol.
4.3.6.3 Organic Solvent

The influence of hydrophobicity of organic solvents on lipase-based catalysis is to
overcome the inhibiting effects of lower chained alcohols and to increase the rate
of the transesterification reaction. The solubility of nonpolar TAGs was higher in
nonpolar solvents such as propanol and butanol compared with polar solvents
such as methanol and ethanol (Soumanou and Bornscheuer 2003). The possible
reason behind the increase in transesterification rate using organic solvents is the
protection of enzymes from denaturation by a high concentration of methanol and
ethanol. The specific selection of the organic solvent impacts the effectivity of the
enzyme catalytic property and also affects the several physical and chemical
properties such as polarity, boiling point, molar mass, viscosity, and acidity. The
other parameters include relative permittivity, Hildebrand solubility parameter,
the dielectric constant, the partition coefficient, and the dipole moment.
Table 4-2. Recent Literature on Different Lipases and Feedstocks Used for Enzymatic Biodiesel Production.
64
M/O Reaction Time Yield

Oil Lipase System ratio Alcohol volume (L) (h) (%) References
Ocimum bacilicum Novozyme 435 Solvent free 10:01 Methanol 0.1 68 94.58 Jegannathan et al.
seed oil) (2008)
Canola oil Candida rugosa Solvent free 5:01 Methanol Nr* 1 94.3 Shao et al. (2008)
Soap nut tree oil Candida sp. Solvent free Nr* 0.1 8 83 Azócar et al. (2010)
Microbial oil Rhizopus oryzae t-butanol 4:01 Methanol 0.0001 24 90.1 Tan et al. (2010)
Microbial oil Novozyme 435 t-butanol 4:01 Methanol 0.0001 48 94.7 Tan et al. (2010)
Rubber seed oil Candida antarctica t-butanol 6:01 Methanol 0.1 24 84.69 Meunier et al. (2015)
lipase B
Microbial oil Novozyme 435 Solvent free Nr* Methanol 0.1 5 95 Vipin et al. (2016)
Waste cooking oil Thermomyces tert-Butyl 1:07 Ethanol 0.015 12 92.1 Vescovi et al. (2016)
lanuginosus
Soyabean oil Candida rugosa hexane 4:01 Methanol 0.005 30 87 Xie and Ma (2010)
Soyabean oil Thermomyces Solvent free Nr* Methanol 0.0096 12 92.3 Xie and Ma (2010)
lanuginosus
olive oil Thermomyces n-hexane cetyl 0.006 2 87 Sankaran et al. (2016)
lanuginosus
Lard oil Candida antarctica Solvent free 1:03 tert-amyl Nr* — 90 Zhao et al. (2015)
Waste cooking oil Geotrichum sp. Solvent free Nr* Nr* — 85 Yan et al. (2011)
Waste cooking oil Candida sp. Solvent free Nr* Methanol 0.15 0.2 Thangaraj et al. (2016)
Microbial oil Rhizomucor miehei n-hexane 1:03 Methanol 0.003 24 90 Poppe et al. (2015)
Soyabean oil Aspergillus niger Solvent free Nr* Methanol 0.1 — — Tan et al. (2010)
Soap stock oil Candida antarctica Solvent free 5:01 Methanol 0.005 6 95.2 Tan et al. (2010)
Soyabean oil Candida antarctica Solvent free 3:01 Ethanol 0.1 14 90.4 Cervero et al. (2014)
*Nr. = No ratio
The hydrophobic solvents such as tert-butanol (logP = 0.83), cyclohexane

(logP = 3.1), and n-hexane (logP = 3.5) are mostly suitable for biodiesel produc-
tion (Soumanou and Bornscheuer 2003). Polar (or) hydrophilic solvents interact
and are miscible with water and enzyme molecule; therefore, hydrophilic solvents
are much less useful in an enzyme-catalyzed reaction (Royon et al. 2007). Sharma
et al. (2001) reported that yield of fatty acid esters was very low (5% to 20%) when
logP was −0.24 (acetone). However, with an increase of logP > 3.0 using a
hydrophobic solvent, yield of fatty acid esters increased to 80%. Chen et al.
(2014) investigated continuous biodiesel production using soybean oil in a butanol
solvent system catalyzed by Novozyme 435 with methanol, and obtained a
conversion yield of 83%, with no specific decrease in lipase activity during
continuous operation for 30 days.
The ionic liquids are very expensive, and erudite solvents are also used in
biodiesel production (Royon et al. 2007). Two ionic liquid solvent systems,
1-butyl-3-methylimidazolium hexafluorophosphate [BMIm] [PF6] and tert-
butanol was investigated using microalgae oil catalyzed by different lipases
obtained from Penicillium expansum and Candida antarctica. Ionic liquid
[BMIm] [PF6] showed a more efficient biodiesel yield of 90.7% compared with
48.6% obtained by using commonly used solvent tert-butanol.
The effect of organic solvent t-butanol on transesterification of crude canola
oil using immobilized Candida antarctica lipase B (CALB), Thermomyces lanu-
ginosus lipase (TLL), and Rhizomucor miehei lipase (RML) was reported (Babaki
et al. 2016). Organic solvents increase the solubility of alcohol and decrease the
viscosity of the reaction mixture. t-Butanol increased the biodiesel yield and gave
95% FAMEs in 72 h. In a solvent-free system, only 24% FAMEs yield was obtained
within the same reaction time, which was due to elimination of the inhibitory
effects of alcohol on the enzyme.
4.3.6.4 Reaction Temperature

Stability of an enzyme is an important property for its industrial application.
Advantages of high-temperature-stable enzymes involve high reaction rates at
temperature ranges from 25° to 80 °C. In general, an increase in reaction
temperature can cause denaturation and inactivation of enzyme in the system.
However, enzymes isolated from drastic environment can maintain stability at
higher temperature (Lukovic et al. 2011). Research during the last few years was
used to identify and rectify the physical problems (effect of pH and temperature)
to lipase catalytic property for biodiesel production.
A large spectrum of temperature operational strategies was studied from
25° to 55 °C for lipase from C. antarctica and from 20° to 70° C for B. cepacia
lipase, but optimum temperature for maximum catalytic activity was 40oC for
C. antarctica lipase and 50 °C for B. cepacia lipase, respectively. The optimum
temperature for a mixture of lipase enzymes (R. oryzae and C. rugosa) was 45 °C.
Most researchers identified the optimum temperature for catalytic activity of
enzyme was 40 °C (Chen et al. 2014).
The immobilization technique can give solid support to decrease the effect of
temperature deactivation, which leads to improved thermal ability (Fjerbaek et al.
2009). The rate of reaction increases with temperature, but the conversion rate
from oil to FAMEs decreases because of the denaturation of enzyme at the
temperature greater than optimal. Sun and Hu (2017) performed the transester-
ification of ethyl acetate (Burrett et al. 2009) using Novozyme 435 lipase
immobilized on polyacrylic resin and showed that immobilization improved the
thermal stability of enzyme as the EC (Enzyme conversion) conversion rate was
increased with an increase in temperature from 50° to 70°C, and 98% conversion
was achieved at 70 °C after 6 h of transesterification reaction. As the temperature
was further increased from 70 °C to 90 °C, EC conversion was maintained at a
higher level but decreased to 5.2% with a further increase in temperature, which
was due to complete deactivation of the enzyme at higher temperature.
4.3.6.5 Water Content

The water content present in the waste cooking oil and animal fat is higher
compared with the other oils. Numerous studies were conducted on enzymatic-
based transesterification with different water content in the reaction system
(Jegannathan et al. 2008). The presence of water content can increase the catalytic
activity of enzyme and hence FAMEs yield.
In addition, water maintains the 3D structure of the enzyme (Oliveira and
Mantovani 2009) and can also affect the activity. The amount of water bound to
enzyme is defined as water activity (Antczak et al. 2009). The hydrolysis reaction
of transesterification is catalyzed by increasing and maintaining the optimum
enzyme concentration. However, no reaction will occur in the absence of water in
the lipase oil complex. At optimum water content, maximum hydrolysis and
maximum transesterification will occur (Jegannathan et al. 2008).
Optimization of the water content has been done by several authors. At
optimum water content, the presence of catalyst increases the hydrolysis of ester
linkage and ensures the maximum yield of biodiesel. The water content is
optimized along with the lipase enzyme concentration and reaction composition
of the medium. Sivaramakrishnan and Incharoensakdi (2017) used lipase to
catalyze direct transesterification of algae oil and observed that addition of water
influenced the yield of methyl esters. However, the presence of excess of organic
solvent strips the water molecule from the lipase enzyme, which may deactivate
the catalytic property of an enzyme. Paula et al. (2011) evaluated lipases from
Rhizopus oryzae and Candida rugosa immobilized on polypropylene EP-100. The
Rhizopus lipase expressed the highest activity and maximum conversion rate at the
lowest water activity (aw = 0.06), whereas lipase obtained from Candida achieved
high activity at aw = 0.53. Thus, the optimum concentration of water activity varies
with the source of enzyme.
4.3.6.6 Lipase Concentration

In general, the optimum concentration of enzyme is very crucial to achieve
product yield. For efficient transesterification reaction, 2% to 10% w/w lipase
loading is necessary. Increased enzyme loading may produce high biodiesel yield,
but it will add up to the overall production cost. For the efficient enzymatic
transesterification at industrial level, a minimum concentration of enzyme, which
is able to produce high biodiesel yield is required. Sankaran et al. (2016) optimized
the enzyme concentration for transesterification of Ocimum basilicum using

Candida antarctica lipase immobilized in acrylic resins and found that enzyme
loading of 6% w/w increased the FAMEs conversion by 1%–to 6%.
Vipin et al. (2016) also optimized the Rhizopus oryzae lipase concentration for
transesterification of nonedible rubber seed oil. As expected, the increase in
enzyme loading from 5% to 10% increased the conversion rate drastically;
thereafter, there was a small increase in the conversion rate when enzyme loading
was increased more than 10%. Because enzyme loading was different for every
system, optimization of enzyme concentration is very essential for enzyme catalyst
biodiesel production.
4.3.7 Immobilization of Lipases for Transesterification

Enzyme immobilization is a promising technology because enzyme is confined to
certain space because of its solid attachment and retains its catalytic activity
(Jegannathan et al. 2008). This technology offers certain advantages over free
lipase such as enzyme reusability, easy glycerol recovery, improved tolerance to
pH, temperature and organic solvents, enhanced lipase stability, and ability for
continuous operations (Cipolatti et al. 2016; Sankaran et al. 2016). This method is
especially useful for large-scale industrial operations and reduces solubility
problems. However, there are certain limitations involved in this technology
such as expensive carriers for immobilization, decrease in enzymatic activity due
to discharge (or deactivation) of enzyme from carriers, less durability in oil–
aqueous systems, and requisite of novel reactors for efficient conversion of oil to
biodiesel.
Lipase cost is up to 90% of the total production cost for enzyme-catalyzed
transesterification processes, the main portion of which includes expense of the
carrier chosen for immobilization (Ghaly et al. 2010). Therefore, it is crucial to
choose an applicable support or carrier that is less expensive, allows the high
catalyst activity, adequate mass transfer, and does not offer limitations for
substrate and product diffusion (Robles-Medina et al. 2009). Other properties
of the carrier must include durability, mechanical strength, stability, adequate
porosity, microbial resistance, hydrophilic or hydrophobic properties, and func-
tionality (Jegannathan et al. 2008). Various materials explored for immobilization
of lipases include microspheres, carbon nanotubes, and magnetic particles.
Polymers such as silica, ceramics, celite, and resins have also been used for
immobilization of lipase enzyme (Tan et al. 2013).
4.3.7.1 Immobilization Techniques

Several techniques have been explored for immobilization of enzyme with the
carrier or support. The technique to be used depends on the required enzyme
activity, prerequisite of final properties of immobilized enzyme, and limitation of
Figure 4-4. Various techniques for enzyme immobilization.
cost (Tan et al. 2010). Immobilization techniques are divided into two groups
(Figure 4-4), that is, reversible and irreversible techniques. Physical adsorption,
affinity, and chelation bonding are included in the reversible method of immobi-
lization, in which enzyme is attached to support by noncovalent bonding and can
be detached (Zhang et al. 2012). In contrast, entrapment, encapsulation, covalent
bonding, and cross-linking are listed as irreversible immobilization techniques,
which are defined as methods in which enzyme is attached to support by covalent
bonds and cannot be detached without destroying enzyme activity or the carrier
(Zhao et al. 2015). Each technique has its own advantages and disadvantages
(Table 4-3), and the immobilization of different lipases using different techniques
is shown in Table 4-4.
4.3.7.2 Immobilization by Physical Adsorption Technique

Physical adsorption is simple and the most commonly used technique for
immobilization of lipase. It is defined as the attachment of lipase to a carrier
by using noncovalent interactions such as Van der Waals forces, electrostatic
forces, affinity adsorption, hydrophilic and hydrophobic interactions, and bios-
pecific adsorption (Al-Zuhair et al. 2007; Zhao et al. 2015). The excess enzyme,
which is not attached to a carrier, is washed away after some time of immobiliza-
tion (Yücel et al. 2012). The most commonly used carrier materials are polypro-
pylene, acrylic, celite, sepharose, polyacrylonitrile, sephadex, polyacrylate,
polymethacrylate, toyonite, textile membranes, polystyrene, spherosil, and sili-
conized glass (Jegannathan et al. 2008, Ghaly et al. 2010). Of these materials,
Table 4-3. Advantages and Disadvantages of Different Immobilization Techniques.

Immobilization technique Advantages Disadvantages References
Physical adsorption Simple and cheap method; Weak bonding between enzyme Ghaly et al. (2010)
requirement of mild conditions; and carrier; leaching of enzyme
chemical additives not required; from carrier; difficult to reuse
high catalytic activity; easy enzyme
recycling or reuse of carrier;
high mass transfer rate of
substrate
Covalent bonding Strong interactions between Use of toxic coupling agents for Jegannathan et al. (2008)
enzyme and carrier; thermal and pre-operation conditions; loss of
operational stability; stability enzyme activity during
toward high pH immobilization; high cost
Cross-linking No requirement of carrier or Reactions performed under harsh Jegannathan et al. (2008)
support; direct immobilization conditions; change of lipase
from fermentation broth; high confirmation and hence low
stability in aqueous solutions; activity; low yield
effective toward high pH and
temperature; low production
cost
Entrapment High stability; simple operation Low mass transfer; conversion Sivaramakrishnan and
method;easy recovery; required efficiency lower than adsorption Incharoensakdi (2017)
mild conditions and cross-linking; limitation of
ENZYME-CATALYZED TRANSESTERIFICATION FOR BIODIESEL PRODUCTION
free diffusion of reactants and

products
Encapsulation Easy separation of enzyme; Low converison efficiency; Sivaramakrishnan and
improved mass transfer; no membrane may be clogged and Incharoensakdi (2017)
69
leaking of enzyme hence low enzyme activity

70
Table 4-4. Immobilization of Different Lipases Using Different Techniques.

Conversion efficiency
Carrier Lipase Technique (% w/w) References
Macroporous acrylic resin Novozyme 435 Covalent bonding 72.2 Tan et al. (2010)
Acrylic resin Thermomyces lanuginosus Covalent bonding 69.2 Vescovi et al. (2016)
Magnetic chitosan Candida rugosa Covalent bonding — Xie and Ma (2010)
Magnetic Fe3O4 Thermomyces lanuginosus Covalent bonding 90 Xie and Ma (2010)
Acrylic resin Candida antarctica Covalent bonding 50.5 Zhao et al. (2014)
Sol–gel Rhizomucor miehei Entrapment — Macario et al. (2008)
Silica gel Candida antarctica Cross-linking 87.2 Bonazza et al. (2018)
Macroporous resin Candida antarctica Covalent bonding — Cervero et al. (2014)
polymers (e.g., polyacrylate, polymethacrylate, polystyrene, polyacrylonitrile, and

polypropylene) are easily available and economically beneficial. The immobiliza-
tion efficiency is affected by carrier properties such as porosity, polarity, surface
area, hydrophilic to hydrophobic group’s molar ratio, and particle size (Culver
et al. 2016). Highly porous support is more advantageous because enzyme can
bind both on the surface and within the support materials, resulting in high
immobilization efficiency. According to Leung et al. (2010), activity of immobi-
lized enzyme was 1.6−3.4 times enhanced after incubation of immobilized (by
adsorption) lipases with polar organic solvents prior to lyophilization. Therefore,
using polar compounds for immobilization increases the immobilization efficien-
cy. Along with the polarity, hydrophobicity of the carrier also shows a high impact
on immobilization, and the enzyme shows higher activity when attached to a
hydrophobic carrier compared with a hydrophilic carrier (Poppe et al. 2015).
Immobilization conditions, including temperature, pH, contact time, ionic
strength, enzyme loading, and carrier-to-enzyme ratio, have significant influence
on the immobilization efficiency. The increase in temperature helps in expansion
of the pores and hence more efficient attachment of the enzyme to the support
(Al-Zuhair et al. 2007). The pH of the buffer used for adsorption of enzyme is
crucial because the more efficient interaction takes place at pH that is close to the
isoelectric point. According to Wang et al. (2013), the contact time between
enzyme and carrier affects the immobilization in an effective way. The hydrolytic
activity of Rhizopus oryzae lipase increased when R. oryzae lipase was immobilized
on anion resin Amberlite IRA-93 for 10 to 20 min, but decreased gradually when
the duration of immobilization was more than 20 min.
The ionic strength influences the immobilization of lipase on several supports
(Ding et al. 2015). According to Subhedar and Gogate (2016), the immobilization
yield (ratio of protein removed from the solution during the immobilization
process to the total amount of protein used for immobilization) was not affected
by ionic strength between 5 and 200 mM when lipase from Thermomyces
lanuginosus was immobilized on mesoporous resin, but a slight decrease in
enzyme activity was observed at the ionic strength of more than 200 Mm. This
was due to the change in isoelectric point as a result of interaction between ions
and ionic groups of enzymes that changed the 3D structure of the enzyme.
Enzyme loading also affects the immobilization of enzyme in a significant manner.
According to Yang et al. (2016), 0.6 mL/g of enzyme loading was sufficient to
achieve high specific activity for immobilization of Rhizomucor miehei lipase on
macroporous resin because of reduction of pore diameters of the support, which
led to the increase of diffusion limitation.
Novozyme 435 is the most commercially utilized enzyme that is obtained by
immobilization of Candida antarctica lipase enzyme on the acrylic resin carrier. It
has been successfully used for transesterification of various feedstocks such as
vegetable oils, waste cooking oils, algae oils, fats, and yeast lipids, and 90%
transesterification efficiency has been achieved (Zhao et al. 2015).
Despite several advantages associated with the physical adsorption technique
(e.g., easy operation, nonrequirement of chemical additives, reusability of carrier,
and high catalytic activity), this technique has various limitations (e.g., leaching of
enzyme from carrier molecule due to weak interactions and loss of enzyme),
making it susceptible to further improvement (Yücel et al. 2012, Sankaran et al.
2016).
4.3.7.3 Immobilization by Covalent Bonding Technique

Immobilization by covalent bonding is an irreversible technique that is defined as
the formation of covalent bonds between active amino acid residues present on the
surface of the enzyme and the aldehyde groups present on the surface of the carrier
(Yücel et al. 2012, Costa-Silva et al. 2017). The amino and thiol groups present on
the surface of the enzyme usually take part in covalent bonding with the carrier.
The binding stability between enzyme and carrier is affected by the direction of the
enzyme involved in binding. It has been reported that immobilized enzyme has
higher activity and stability when active center amino acids are not involved in the
covalent bonding (Mohamad et al. 2015). Several functional groups can be added
to the support to make it more reactive toward enzyme. Glutaraldehyde is the
most commonly used activating agent, which helps to establish the covalent bond
between enzyme and carrier (Barbosa et al. 2015). Carriers that are most
commonly explored for covalent bonding are silica gel, ceramic, agarose, chitosan,
nylon fibers, polypeptides, chitin, porous glass, sand, and metallic oxides
(Ognjanovic et al. 2009, Zhao et al. 2015). According to a recent study by
(Yuce-Dursun et al. 2016), a new carrier or support, ultraviolet-curable hybrid
epoxy-silica polymer film was successfully prepared and used for immobilization
of lipase from Candida rugosa. Immobilized enzyme was able to retain 82%
hydrolytic activity compared with free enzyme.
There are several advantages associated with covalent bonding over physical
adsorption technique of immobilization such as strong interactions between
enzyme and carrier resulting in high operational, pH, and thermal stability
(Ozturk and Kilinc 2010). However, loss of enzyme activity due to alteration in
structure of enzyme is a major disadvantage of this method (Zhang et al. 2012). In
the recent study by (Babaki et al. 2016), physical adsorption and covalent bonding
were combined to gain benefits of both methods. Two coupling linkers such as
Octyltriethoxysilane (OTES) and (3-Glycidoxypropyl)trimethoxysilane (GPTMS)
were used for hydrophobic interaction and covalent linkage, respectively. Immobi-
lization of Rhizomucor miehei lipase on octyl-functionalized supports showed 1.5 to
2 times higher specific activity than free enzyme. Therefore, this newly developed
method was able to overcome the limitations of both physical adsorption and
covalent linkage.
4.3.7.4 Immobilization by Cross-Linking Technique

Immobilization of enzyme by cross-linking is an irreversible technique, in which
formation of intermolecular and intramolecular cross-linkage takes place by the
addition of bifunctional or multifunctional cross-linking agents (Chen and
Chen 2016). The cross-linking technique does not require any support materials
or carriers, and hence pure enzymes are obtained. The most commonly used
cross-linking agent is glutaraldehyde (GLA), but hexamethylene diisocyanate and

bisdiazobenxidine are also used (Ghaly et al. 2010). In this technique, lipase
molecules are cross-linked by precipitation using organic solvents such as ethanol,
ammonium sulfate, and acetone to form cross-linked enzyme aggregates (CLEAs),
which are further cross-linked by using bifunctional or multifunctional cross-

linking agents (Hanefeld et al. 2009, Mohamad et al. 2015). The formed aggregates
are stable at high temperature and pH in aqueous solutions. Cross-linking of
Geotrichum sp. lipase increased pH, thermal stability, and gave 85% biodiesel yield
compared with free enzyme that was agglomerated and showed lower catalytic
efficiency (Yan et al. 2011). It was due to the formation of water when esterified with
alcohol because high free fatty acid content was present in the waste cooking oil.
It was observed that when CLEAs are further cross-linked by using glutaral-
dehyde, it may decrease activity of CLEAs due to binding of excess GLA with
amino acids present in the active site of enzyme (Rehman et al. 2016). To
overcome this limitation of glutaraldehyde, a milder cross-linking agent, ethylene
glycolbis (succinimidylsuccinate) (EG-NHS) was used in the study by Rehman
et al. (2016). It was found that EG-NHS cross-linked lipase showed superior
hydrolytic activity (52.08 ± 2.52%) and esterification (64.42%) compared with
GLA cross-linked lipase enzyme.
Despite several advantages, the cross-linking technique has several limitations
such as loss of enzyme activity due to harsh reaction conditions required during
cross-linking reactions and low immobilization yield (Yang et al. 2010). To
overcome these limitations, cross-linking is further followed by the adsorption
process using polymer membrane and macroporous resin (Zhao et al. 2015).
4.3.7.5 Immobilization by Entrapment/Encapsulation Technique

Immobilization of lipase by entrapment is a fast and cheap method, in which
enzyme is captured within a polymeric matrix that allows the substrate and
products to pass through but traps the enzyme (Zhang et al. 2012). When enzyme
is trapped within gel polymers (beads or capsules), this technique is called
encapsulation (Ghaly et al. 2010). The most commonly used carriers for immo-
bilization are acrylic polymers, resins, celite, and gels such as calcium alginate,
kappa carrageenan and methylenebisacrylamide (Yücel et al. 2012). This tech-
nique provides more stability to the enzyme and minimizes enzyme leaching;
therefore, this method is better than physical adsorption.
Lipase enzyme was encapsulated in mesoporous matrix by the sol–gel
method, and catalytic activity of encapsulated enzyme was checked for transes-
terification reaction. The FAME yield of 80% was obtained after 70 h, compared
with 50% for free lipase, which was attributable to the increased stability of the
enzyme and decreased leaching (Macario et al. 2008). There are some disadvan-
tages of sol–gel immobilization, which involves denaturing of the enzyme from
shrinkage and drying of the gel as well as slow diffusion of the substrate toward the
encapsulated enzyme (Huang et al. 2008). According to a recent study (Bonazza
et al. 2018), epoxy functionalized silica support was prepared by modification of
silica gel that strengthened the interaction between enzyme and carrier by covalent
bonding, which prevented the denaturation of the enzyme. Candida antarctica

lipase B enzyme was immobilized with increased catalytic activity and high
operational stability being observed. The major drawback of this technique is
limitation of mass transfer due to clogging of the membranes. It occurs because of
adhesion of the produced glycerol on the surface of the support, which further
limits the diffusion of the reactants and products (Zhao et al. 2015).
4.3.7.6 Immobilization Using Nanotechnology

Enzyme immobilization has revolutionized with the advent of nanotechnology
(Sankaran et al. 2016). The use of nanobiocatalysts in transesterification for
biodiesel production is a novel research approach. Researchers have explored
nanomaterials such as nanoparticles, nanotubes, nanofibers, nanopores
nanosheets, and nanocomposites for immobilization of enzymes (Verma and
Barrow 2015). Of the different immobilization methods discussed earlier, nano-
materials and enzyme interactions by covalent bonding provide more stability and
high operational efficiency. Magnetic nanoparticles have been used as a popular
carrier for immobilization of enzyme by physical adsorption or covalent bonds
(Thangaraj et al. 2016). There are several advantages associated with the use of
magnetic nanomaterials, including a large surface-to-volume ratio for high
enzyme loading, hence high catalytic activity; low mass transfer resistance; easy
separation of product and enzyme; easier preparation and handling; flexibility in
reactor design; high mechanical strength; high thermal and chemical stability; and
low toxicity (Verma and Barrow 2015, Thangaraj et al. 2016, Zhu et al. 2016)
It is reported that naked magnetic nanoparticles are not able to effectively
attach with the enzyme, so surface modifications are required (Cipolatti et al.
2016). In the literature, some reported modifications include using 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDAC), coating with polymers, use of
glutaraldehyde, coupling with agarose and chitosan, using silica compounds, and
using (3-aminopropyl)triethoxysilane (APTES) (Chen et al. 2014, Xie and Ma
2010, Zang et al. 2014).
The use of polyamidoamine (PAMAM) dendrimer for the modifications of
magnetic nanoparticles was reported (Zhu et al. 2016). Candida rugosa lipase was
immobilized onto modified nanoparticles. The immobilized lipase showed high
pH and thermal stability, and 90% activity was retained even after 10 recycles. The
high stability was due to the 3D structure of the dendrimer.
In another study (Ziegler-Borowska et al. 2017), Lipase NS81006 was
immobilized on Fe3O4 magnetic nanoparticles modified with compounds APTES
and (3-mercaptopropyl)trimethoxysilane (MPTMS). Glutaraldehyde was used as
the cross-linking agent for covalent binding of lipase on modified nanoparticles.
Transesterification reaction was performed by using immobilized lipase, and 89%
and 81% biodiesel yield was obtained by using lipase immobilized on APTES-
Fe3O4 and MPTMS-Fe3O4 magnetic nanoparticles, respectively, after 12 h of
reaction time. The lipase was easily recovered by external magnetic field after use.
The novel approach of using squaric acid (SqA) as a cross-linking reagent was
studied (Ziegler-Borowska et al. 2017) and compared with glutaraldehyde
(Ochsenreither et al. 2016) as the cross-linking reagent. Six types of magnetic

nanoparticles (MNPs) were prepared, which were coated with chitosan (CS)
(Belafi-Bako et al. 2002), collagen (Coll), and by CS/Coll together. Further
polymeric stabilization was done by using the cross-linking agent squaric acid
(SqA) and glutaraldehyde (Glu). Candida rugosa lipase was immobilized on

prepared nanoparticles. The twofold higher specific activity was obtained for
enzyme immobilized on MNPs using SqA for surface cross-linking compared with
immobilization of enzyme using Glu.
The use of magnetic nanoparticles for immobilization of Thermomyces
lanuginosus enzyme was studied and further used for transesterification reaction
(Raita et al. 2015). Different covalent modification methods were used for
attaching the enzyme to the magnetic matrix. Of the many modification methods,
immobilized lipase prepared on Fe3O4-AP-EN-LIP showed the highest catalysis
for hydrolysis of p-nitrophenyl palmitate, and 97.2% (w/w) FAMEs yield was
obtained by transesterification of refined palm oil.
There are several advantages of using nanomaterials for lipase immobilization
and further transesterification, but several challenges, such as high production
cost, nonuniformity, and lack of knowledge about nanoparticles, still need to be
addressed (Shuai et al. 2017).
4.3.8 Reactors for Immobilized Lipase-Based Transesterification

For commercialization of enzyme-based biodiesel production, the enzymatic
transesterification reaction should be performed at the reactor level (Zhao et al.
2015). The design of the reactor requires knowledge of mechanisms of mass
transfer, kinetics of reactions, and hydrodynamics of the system. Until now,
various advancements have been accomplished in the design of reactors. The novel
reactors include expanded-bed reactor, packed-bed tubular, rotating packed-bed
reactor, stimulated moving reactor, basket-type stirred tank reactor, and tubular
reactors with static mixers (Xu et al. 2017). The most commonly used reactors
for enzyme-based biodiesel production are stirred tank reactor (STR), packed-
bed reactor (PBR), and fluidized-bed reactor (FBR) as shown in Figure 4-5
(Ochsenreither et al. 2016).
4.3.8.1 Stirred Tank Reactor

STR is the most commonly used reactor because of its simple construction, easy
operation and maintenance, efficient substrate dispersion, and suitability for
multiphasic systems (Poppe et al. 2015). STR [Figure 4-5(a)] consists of the
reactor vessel with a propeller (the stirring system) and temperature and pH
control. The reactor is operated in both batch and continuous mode. In this
reactor, agitation is used to disperse the free or immobilized enzyme. At the end of
the process, centrifugation separates the solid and liquid phases and recovers the
immobilized enzyme (Christopher et al. 2014).
The transesterification of sunflower oil using Novozyme 435 lipase was
performed, and reaction conditions for transesterification process were optimized
(Ognjanovic et al. 2009). FAME yield of more than 99% was obtained within 50 h
Figure 4-5. Most commonly used reactors for enzyme-based biodiesel production:
(a) batch stirred tank reactor (STR); (b) packed-bed reactor (PBR); and (c) fluidized-
bed reactor (FBR) (Ochsenreither et al. 2016).
reaction time, but poor stability was observed. Therefore, methyl acetate as an acyl
receptor was used to improve stability, and further verification for industrial scale
was done by using batch STR equipped with six-bladed turbine impellers. High
biodiesel yield of 99.7% was obtained at the methyl acetate-to-oil ratio of 12:1
within 24 h of reaction time with only 3% of immobilized lipase. The high yield
was because the reactor with six-bladed turbine impellers improved the dispersion
of the biocatalyst, increased the contact between substrate and catalyst, and
lowered mass transfer resistance. However, enzyme activity of the catalyst
decreased with reuse, which may be attributed to high shear stress and mechanical
damage to the enzyme (Sankaran et al. 2016).
To overcome the limitation of enzyme damage associated with mechanical
shearing, it was proposed that the STR contain a central basket in the reactor
vessel, and transesterification of milk fat with soybean oil was performed using
Rhizopus oryzae lipase immobilized on polysiloxane-poly(vinyl alcohol) (SiO2-
PVA) (Paula et al. 2011). A flat blade stirrer and an inclined blade stirrer were
tested for their mixing efficiency since insertion of the basket could affect the
homogenization of the reaction mixture. The consistent biocatalyst stability was
observed for 10 cycles, each lasting for 6 h. Both evaluated stirrer designs were
potentially successful for industrial use. The high operational stability of immo-
bilized lipase was also attributed to the lipase immobilization on carrier SiO2-PVA
using the sol–gel method (Paula et al. 2011).
However, the laborious steps of separation of products after each batch,
cleaning, reloading and low productivity, long reaction times, and requirement of
high volume tanks make the STR vulnerable at the industrial scale and make the
use of continuously stirred tank reactor (CSTR) more appreciable (Poppe et al.
2015).
Continuous systems have certain advantages compared with batch reactors
such as high efficiency to control the reaction parameters and the system, high
productivity, high energy efficiency, few scale-up steps and unit operations, low
cost for optimization of reaction conditions, complete automation, as well as being
faster and more economical (Fjerbaek et al. 2009, Gog et al. 2012)
In the CSTR, enzyme is retained in the reactor and membrane technologies
are used as a filter at the outlet of the reactor. By using the CSTR, more efficient
use of enzyme preparations can be done because tanks can be operated using
enzymes having different ages and activities. For simultaneous removal of glycerol
formed during the transesterification reaction, different units can be installed
between the reactors (Freire et al. 2011).
The process for improvement of enzymatic production of biodiesel was
developed and a multienzyme system was used to overcome the enzyme deacti-
vation as in the case of batch STR (Basheer et al. 2011). Two consecutive STRs
were assimilated together with a bottom sintered glass filter and a settling tank
between the two reactors. The first reactor contained single or multiple lipases.
Excess water and glycerol formed during the transesterification reaction were
removed continuously using a settling tank, which increased the conversion
efficiency to 98% in the second reactor within 4 h reaction time, and the
immobilized enzyme was reused for 100 consecutive cycles.
4.3.8.2 Packed-Bed Reactor

PBR is a fast and economical reactor for industrial-scale biodiesel production and
mostly used for continuous processes (Wang et al. 2013). In PBR [Figure 4-5(b)],
immobilized enzyme is usually fixed inside the cylindrical column, and reactant
components are passed through the column to react with the catalyst (Xu et al.
2017). The specific flow rate of the reactants determines the reaction time with
respect to the column volume (Fernandez-Lafuente 2010). Flow rate is one of the
most significant operational factors, and its range must be selected wisely to
provide conciliation between reasonable pressure drop, minimization of the
diffusion layer, and efficient conversion yield (Poppe et al. 2015). There are
several advantages associated with the PBR, including large reacting surface area,
high productivity, low substrate-to-enzyme ratio, high reaction performance,
lower shear stress to immobilized enzyme, and high stability (Hama and Kondo
2013, Zhao et al. 2015).
An important limitation of this system is the reduced biodiesel yield during
long-term operation. Long-term operation is further associated with another
major problem of glycerol accumulation that increases the mass transfer resistance
between substrate and enzyme and hence decreases the transesterification rate
(Tan et al. 2010). The glycerol produced after transesterification reaction is highly
viscous, which accumulates on the surface of the enzyme. The hydrophilic layer
on the surface makes the immobilized enzyme inaccessible to the hydro-
phobic substrates, thereby decreasing the biodiesel yield (Antczak et al. 2009,
Zhao et al. 2015). Furthermore, accumulated glycerol causes clogging of the

column and increases pressure drop inside the reactor. In addition, unreacted
alcohol from the reaction mixture mixes with the glycerol layer and accumulates
on the surface of the immobilized enzyme and hence causes deactivation of
enzyme owing to excess alcohol (Tran et al. 2016).

Several approaches have been used to overcome these limitations, including
stepwise addition of methanol to decrease the inhibition of enzyme, continuous
separation of accumulated glycerol using the so-called dialysis membrane sepa-
ration method (Belafi-Bako et al. 2002), use of silica gel in the reactor system to
adsorb glycerol, use of solvents such as n-hexane and tert-butanol to decrease the
viscosity of glycerol and make it soluble (Royon et al. 2007). However,
these techniques require additional investment and make the process expensive
(Tran et al. 2016).
An economical process was developed by integrating the PBR with the
glycerol separation system for methanolysis of plant oils catalyzed by Novozyme
435 without using solvents (Hama and Kondo 2013). Methyl ester content
increased while glycerol accumulation decreased on the immobilized enzyme.
The PBR can be operated for a longer time with efficient biodiesel yield. In spite of
certain advantages associated with the modified PBR, addition of excess methanol
decreased the activity of lipase.
The methanolysis of sunflower oil in a series of three PBRs with glycerol
removal devices was conducted (Tran et al. 2016). Immobilized enzyme denoted
as celite-alkyl-lipase was used for transesterification of sunflower oil. Biodiesel
conversion was enhanced to 85% by using serial three PBRs compared with 67%
using a single PBR. Low conversion in the single PBR was because of glycerol
accumulation and high mass transfer resistance, which was rectified by using serial
PBRs with continuous removal of glycerol. Moreover, celite-alkyl-lipase was
developed for more stability and for working under ambient conditions, and
thus gave high biodiesel yield.
In another study (Canet et al. 2017), FAMEs and monoacylglycerols were
simultaneously synthesized from olive oil using a 1(3)-positional specific recom-
binant Rhizopus oryzae lipase (rROL). This immobilized enzyme synthesizes
monoacylglycerols as a by-product instead of glycerol; thus, inactivation of enzyme
due to accumulation of glycerol was prevented. A comparison between STR and
PBR was made. PBR was better as it produced a higher yield of 49.1% compared
with 33.6% by STR owing to desorption of enzyme by shear stress in the STR.
The biodiesel production catalyzed by Burkholderia cepacia lipase immobi-
lized on SiO2-PVA using macaw palm oil was reported (Ramos et al. 2016). Two-
stage PBRs with a glycerol extraction column was developed, and glycerol was
continuously removed by adsorption with commercial resin Lewatit GF 202. The
output of the first PBR was connected to a glycerol removal column, and the
glycerol-free feed was added into the second PBR, which reduced the enzyme
inactivation and produced biodiesel with a high yield of 96.3% (w/w).
The applicability of a rotating packed-bed reactor (RPBR) for production of
biodiesel from soybean oil using lipase from the Candida sp. 99-125 was studied
(Xu et al. 2017). The high hydrolysis yield of 97% (w/w) after 24 h and
esterification yield of 96% were achieved after another 6 h. The RPBR provided
a good dispersion and homogenization for the reaction system and efficiently
intensified the mass transfer.
4.3.8.3 Fluidized-Bed Reactor

The FBR is a variation of PBR in that PBR is operated in down-flow mode whereas
FBR is operated in up-flow mode and the biocatalyst particles are not in proximity
with each other (Sheldon 2007). In FBR [Figure 4-5(c)], reactants are usually fed
from the bottom and are uplifted by the sufficient flow rate. The individual
catalysts are always in motion due to continuous feed of the substrate. The
pressure drop of the fluid flow maintains or supports the weight of the bed in a
significant way (Kosseva et al. 2009). The fluidization is usually mediated by liquid
or gas. When the immobilized enzymes require oxygen, air is used for fluidization
(Poppe et al. 2015).
The FBR has certain advantages, such as efficient solid–fluid mixing and mass
transfer, lower pressure drop, and less formation of superior channels (Fidalgo
et al. 2016). However, there are certain disadvantages associated with FBR,
including low residence time and hence lower yield, requirement of a lower
amount of enzyme and decrease of reactor efficiency, as well as unexpected
changes in conversion rate due to changes in flow patterns (Poppe et al. 2015). The
major disadvantage of FBRs is the difficulty to scale it up, that is, it can be scaled
up only 10 to 100 times compared with PBRs, which can be scaled up to more than
50,000 times (Kosseva et al. 2009).
Ricca et al. (2009) reported the transesterification of olive husk oil by
Rhizomucor miehei lipase immobilized on ion exchange macroporous resin using
FBR. The reactor generated high productivity compared with PBR because of
effective mixing of catalyst with oil and improvement in heat and mass transfer. The
FBR reactor was also operated in recycle mode to check out its performance. It was
observed that increasing and decreasing of the recycle ratios, that is, recycle flow rate
over feed flow rate (R/F), influenced the productivity and conversion rate in an
effective way. Thus, optimization of the R/F ratio should always be considered.
Zhou et al. (2014) also proposed the biodiesel production from soybean oil
using immobilized Rhizopus oryzae lipase in a magnetically stabilized FBR
(MSFBR). The maximum FAMEs content of 91.3% w/v was achieved at a flow
rate of 25 mL/min. In addition, MSFBR showed excellent stability and reusability
even after six batches. Certain advantages of MSFBR over conventional FBR
include avoiding destruction of enzyme structure, elimination of requirement of
solid mixing, and decrease in pressure drop. Fidalgo et al. (2016) reported the
production of biodiesel by ethanolysis of babassu oil using immobilized lipase
Novozyme 435 in a FBR. It was assimilated with a glycerol removal column with
cation exchange resin. The high biodiesel yield of 98.1% was attained using 12%
biocatalytic loading within 8 h reaction time, which was higher than those
reported in literature. The system was run for 15 days continuously without any
loss of catalytic activity.
4.4 COST ANALYSIS OF IMMOBILIZED LIPASE-BASED

TRANSESTERIFICATION
Economical evaluation of any process technology is a key driving force for

successful implementation at the commercial level. Although there are a number

of studies available for economical evaluation of alkali- and acid-catalyzed
biodiesel production, only a few are reported for economic evaluation of
enzyme-catalyzed biodiesel production. The major cost factor for industrial
biodiesel production is cost of feedstock, which adds up to 70% to 95% of the
total production cost. The price of enzyme and alcohol also affects the economic
assessment of the enzymatic biodiesel production in a significant manner.
Fjerbaek et al. (2009) calculated the catalyst cost, which was USD 0.14/kg of
biodiesel for lipozyme IM and USD 0.006/kg for alkali catalyst. In addition to
enzyme and alcohol cost, various other parameters such as oil to biodiesel
conversion, retention time, lipase lifetime, and biodiesel recovery yield also affect
the economic feasibility of enzyme-catalyzed biodiesel production (Zhao et al.
2014). Enzymes are more expensive and are slow reacting catalysts compared with
alkali and acid catalysts, but produced biodiesel is much easier to purify.
The comparison of economic assessment for alkali, soluble enzyme, and
immobilized enzyme-based biodiesel production for a 1,000 ton plant capacity was
conducted (Jegannathan et al. 2008). The procedure for economic assessment
involves development of process flow sheets, process time charts, and equipment
list. Thereafter, estimation of equipment cost, plant cost, and manufacturing cost
is done. The production cost per ton of biodiesel was estimated. It was USD
2,414.63 for the immobilized lipase-catalyzed process; USD 7,821.37 for the
soluble lipase-catalyzed process; and USD 1,166.67 for alkali-catalyzed process.
Reusability of the enzyme plays a major role in the production cost for the
immobilized lipase-catalyzed process. The stability and reusability of enzyme
could decrease the production cost in an efficient manner and can compete with
the alkali-catalyzed process.
The economic assessment for enzymatic biodiesel production using rapeseed
oil was also conducted and concluded that a solvent-free process is appreciable for
biodiesel production at the industrial level (Sotoft et al. 2010). The estimated
biodiesel production cost was USD 0.73 to 1.49/kg biodiesel with current enzyme
price of USD 762.71/kg using cosolvent, whereas USD 0.05 to 0.75/kg with future
enzyme price of USD 7.627/kg using a solvent-free system. The high energy
consumption for cosolvent production makes this process too expensive. It
requires extra distillation facilities, which adds more cost than extra reactors and
decanters required for the solvent-free system.
More research on the solvent-free process and improvements in the immo-
bilized enzyme techniques should be focused on high retention time, enzyme
stability, and activity as well as the increase in enzyme life time and yield to make
this process economically acceptable for large-scale enzyme-catalyzed biodiesel
production.
4.5 SUMMARY
Diminishing fossil fuel reserves and concerns of environmental pollution caused

by burning fossil fuels led to interest of finding alternative renewable fuels, and
biodiesel seems to be a proficient substitute for petroleum-based fuels. Biodiesel is

a renewable energy resource with nontoxic, easily biodegradable, and environment
friendly properties. Conventionally, biodiesel was produced by using alkali and
acid catalysts, which enhances the reaction rate of fatty acids with alcohol called
transesterification reaction. The use of biocatalysts, especially lipase, for biodiesel
production overcomes the limitations of alkali and acid catalysts such as soap
formation and low reaction rate. The trend of using lipase for biodiesel production
is increasing owing to high-quality biodiesel production and easy recovery and
purification processes.
Biodiesel production using lipase as catalyst has been established since the last
decade. Considerable research is being performed to increase stability and activity
of lipase as well as to optimize the reaction conditions for efficient biodiesel
production. Despite many advantages, the enzyme-catalyzed biodiesel production
depends on very expensive enzymes; thus, it has limited industrial-scale applica-
tions. Researchers are continuously developing new techniques to make this
process economical. Immobilization of lipase using different methods for enhanc-
ing the lipase stability, reusability, and use of acyl receptors to decrease the
inhibitory effects seem to be attractive options for augmenting biodiesel produc-
tion. Further improvements can be done by designing and developing novel
reactors to enhance biodiesel yield and to enable this technology to be
commercialized.
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CHAPTER 5
Plant Oil to Biodiesel
B. Bhadana
B. Tiwari
R. D. Tyagi
P. Drogui
5.1 INTRODUCTION
Vegetable oils have proven to be a best substitute for fossil fuels because they
provide good heating power, emit no sulfur content and aromatic polycyclic
compounds, and their burning makes complete recycling of CO2 (Singh and Singh
2010). They can be obtained from various sources. Currently, various vegetable
edible oils are being used as the major feedstock for biodiesel production, for
example, soybean oil in the United States, rapeseed oil in Canada, palm oil in
Southeast Asia, sunflower oil in Europe, and coconut oil in Philippines, among
others. However, the edible oils pose various limitations for its continuous use in
biodiesel or fatty acid methyl ester (FAME) production because of (1) higher cost
of edible oils influencing the global imbalance to the food supply and market
demand, and (2) reduction in food sources and the growing commercial plant
capacities. Thus, it creates the crisis of food versus fuel. Hence, researchers in the
various countries are looking for cheap nonedible oils (e.g., jatropha, castor,
linseed, karanja, coffee, neem, tobacco, jojoba, Moringa oleifera, and others) to
make biodiesel production more economical (Kafuku and Mbarawa 2010, Ahmad
et al. 2011, Balat 2011, Kibazohi and Sangwan 2011, Kumar and Sharma 2011,
Bora and Baruah 2012, Borugadda and Goud 2012, Atabani et al. 2013). There are
various advantages of using nonedible vegetable oils for biodiesel production,
including (1) low cost of plantation compared with edible oils, (2) not for human
consumption, thereby, overcoming the food versus fuel problem, (3) easy culti-
vation in wastelands, (4) more efficient, (5) reduce deforestation rate, and (6) more
eco-friendly and very economical compared with edible oils (Silitonga et al. 2011,
Atabani et al. 2012, Banković-Ilić et al. 2012).
Biodiesel production from vegetable oils can be achieved by any of four
methods: blending, pyrolysis, microemulsions, and transesterification.
89
Transesterification is the most common way to produce biodiesel from plant oils. In
this reaction, oils or triacylglycerides (TAGs) are reacted with alcohol (methanol or
ethanol), resulting in the formation of ester and glycerol. Transesterification of
vegetable oils can be done with or without catalysts. Catalytic alcoholysis includes
chemical alcoholysis (in the presence of homogeneous or heterogeneous acid or

base) and enzymatic alcoholysis (catalyzed by lipases). Noncatalytic alcoholysis is
performed at high temperatures and pressures (i.e., under supercritical conditions).
Vegetable oils also pose the disadvantage of higher viscosity, low volatility, and
reactivity of unsaturated carbon chains that limit their direct use in diesel engines.
The problem of high viscosity can be overcome by chemical treatment of vegetable
oils (i.e., transesterification) (Demirbas 2009, Singh and Singh 2010).
The choice of feedstock plays a key role in biodiesel production, and it
depends on the process chemistry and economy. Oil content in the feedstock is the
most important consideration for biodiesel production due to its fundamental
significance (Karmakar et al. 2010). Various nonedible oils have been found to be
promising feedstocks to replace edible oils. These include jatropha, neem, mahua,
karanja, castor, linseed, and others, but their applicability for biodiesel is limited
due to high economic value and concern regarding the food versus fuel problem.
Because initial evaluation of physicochemical properties of feedstocks is necessary
to assess their viability for future biodiesel production, this chapter accounts for
physical and chemical properties of edible and nonedible oils, their transester-
ification, and the properties of their methyl esters.
5.2 FEEDSTOCKS FOR BIODIESEL PRODUCTION
Depending on the climate and soil conditions, biodiesel is being produced from
different kinds of vegetable oils. Edible oils like rapeseed, sunflower, soybean,
peanut, palm, and others are major feedstocks for biodiesel production through-
out the world. Various nonedible oils have been found to be promising feedstocks
to replace edible oils. These include jatropha, neem, mahua, karanja, castor,
linseed, and others (Kafuku and Mbarawa 2010, Ahmad et al. 2011, Balat 2011,
Kibazohi and Sangwan 2011, Kumar and Sharma 2011, Bora and Baruah 2012,
Borugadda and Goud 2012, Atabani et al. 2013).
5.2.1 Commonly Used Edible Oils

Rapeseed oil or canola oil (Brassica napus L.) is the potential feedstock for
biodiesel production used by industry in the European Union and Canada.
Rapeseeds are small in size, and their oil content can be limited by hot dry
conditions. About 1.1 L of canola oil gives 1 L of FAME production. Canola seeds
contain 38% to 46% (percent by weight) oil content. Presently, nitrogen-containing
fertilizers are used when rapeseed is grown. This results in N2O generation (a potent
greenhouse gas); thus, it has become a great concern to use rapeseed oil for biodiesel
production (Karmakar et al. 2010). Palm oil (Elaeis guineensis) is rich in myristic
PLANT OIL TO BIODIESEL 91
and lauric fatty acids. Both its flesh and seed contribute to oil derivation.
It has a sharp melting point with excellent oxidative stability. Malaysia is the
largest country worldwide for palm plantation, which covers two-thirds of the
agricultural land. It is an efficient biodiesel feedstock. About 6,000 L of palm oil/ha
produces 4,800 L of biodiesel (Karmakar et al. 2010). Rice bran oil (Oryza sativum)
is an inexpensive, nonconventional, and low-grade vegetable oil for FAME
production. This oil is rich in protein. It is derived from the inner husk and
germ of rice. Rice bran consists of 15% to 23% oil content that gives an oil yield of
828 L/ha/year (Atabani et al. 2012). Coconut oil (Cocos nucifera) is widely
harvested in the tropical coastal areas. It is extracted from copra (dried flesh of
the nut). Coconut oil burns very neatly in diesel engines. It contains 63%−65% oil
content that gives an oil yield of 2,689 L/ha/year (Atabani et al. 2012). Peanut oil
(Arachis hypogaea) is native to Mexico, South America, and Central America.
Approximately, 1,170 L/ha biodiesel is produced from peanut oil. Because peanut
oil is more valuable for human consumption in the world market, it is not an
economical feedstock for biodiesel production (Karmakar et al. 2010). Similarly,
soybean oil (Glycine max), sunflower oil (Helianthus annuus), sesame oil (Sesa-
mum indicum L.), corn oil (Zea mays), passion fruit seed oil (Passiflora edulis),
and others are being used at the commercial level for biodiesel production.
However due to their high cost and increased concern under the food versus fuel
problem, these edible oils are not economically viable for biodiesel production.
5.2.2 Commonly Used Nonedible Oils

Oils extracted from various nonedible crops have proven to be potential feedstocks
for biodiesel production, such as jatropha, karanja, neem, mahua, tobacco, rubber,
castor, and others. Jatropha (Jatropha curcas) oil is the most promising potential
feedstock for FAME production in India, Africa, Central and South America, and
Southeast Asia. It can grow under different climatic conditions, on waste, saline,
and sandy soils and under low or high rainfall. After first plantation, jatropha
survives for 30 to 50 years, which eliminates its yearly replantation. Its oil content
range is 46% to 58% in kernels and 40% to 60% in seeds (Kumar and Sharma
2011). Because no modification is required in the engine with jatropha oil, it has
great potential to be used as an alternative fuel (Jain and Sharma 2010). Castor
(Ricinus communis) plant is native to Brazil, China, India, and Thailand. India
contributes about 0.73 Mt/year of castor oil to the world market (Kumar and
Sharma 2011). Mahua (Madhuca indica) and neem (Azadirachta indica) trees are
mostly found in India and Burma. Neem and mahua trees contribute 100,000 and
180,000 t, respectively, of oil per year (Jain and Sharma 2010). Karanja (Pongamia
pinnata) is the nitrogen-fixing tree. Significant oil content is produced from its
seeds. Karanja is grown in the United States, Indonesia, India, Malaysia, Philippines,
and Australia. Its annual production is 55,000 t (Jain and Sharma 2010). Soil quality
can also be improved by its cultivation on agricultural land (Gui et al. 2008).
Several other nonedible oils have also been extracted from various plants such
as Argemone mexicana, Thlaspi arvense, M. oleifera, Thevetia peruviana, Pistacia
chinensis bge, Datura stramonium, Euphorbia lathyris, Sapium sebiferum.
5.3 PHYSICAL AND CHEMICAL PROPERTIES OF PLANT OILS
Any potential biodiesel feedstock must be evaluated for its physical and chemical
properties to assess its viability for future biodiesel production. Oil content (%) and
yield per hectare are the most important parameters for a feedstock to be a biodiesel
source (Kumar and Sharma 2011, No 2011, Usta et al. 2011, Wang et al. 2011, Atabani
et al. 2012, Wang et al. 2012). The oil content and yields of different edible and
nonedible oil feedstocks is shown in Table 5-1. Biodiesel contains many fatty acids
with different levels of unsaturation. This fatty acid composition of any feedstock
defines the fuel properties of the biodiesel (Karmakar et al. 2010). Fatty acid profiles
of different edible and nonedible oil feedstocks are presented in Table 5-2. The
Table 5-1. Oil Content and Yields of Different Plant Oils.
Oil content Oil yield

Feedstock (percent by weight) (L/ha/year)
Coconut 63−65 2,689

Jatropha curcas Seed = 20−60 1,892
Kernel = 40−60
Madhuca indica (mahua) Seed = 35−50 —
Kernel = 50
Datura stramonium 10.3−23.2 —
Moringa oleifera Seed = 33−41 —
Kernel = 2.9
Palm oil 30−60 5,950
Soybean 15−20 446
Rice bran 15−23 828
Pongamia pinnata (karanja) Seed = 25−50 —
Kernel = 30−50
Nicotiana tabaccum (tobacco) Seed = 36−41 —
Kernel = 17
Hevea brasiliensis (rubber tree) Seed = 40−60 —
Kernel = 40−50
Rapeseed oil 38−46 1,190
Olive oil 45−70 1,212
Azadirachta indica (neem) Seed = 20−30 —
Kernel = 25−45
Sapium sebiferum 12−29 —
Simmondsia chinensis (jojoba) 45−55 1,818
Ricinus communis (castor) 45−50 1,413
Sunflower 25−35 952
Peanut oil 45−55 1,059
Corn 48 172
Table 5-2. Fatty Acid Composition of Plant Oils.

Myristic Palmitic Stearic Oleic Linolenic Linoleic Arachidic
Oil (C14) (C16) (C18) (C18:1) (C18:3) (C18:2) (C20)
Sunflower <1 3−6 1−3 14−35 <1.5 44−75 0.6−4.0

Soybean <0.5 7−11 2−6 19−34 5−11 43−56 <1
Rapeseed — 4.9 1.6 33 7.4 20.4 —
Palm oil 0.5−2.0 32−45 2−7 38−52 — 5−11 —
Peanut oil — 6−9 3−6 52−60 — 13−27 2−4
Olive oil 0.1−1.2 7−16 1−3 65−80 — 4−10 0.1−1.3
Coconut 13−19 8−11 1−3 5−8 — 0−1 0−0.5
Rice bran 0.4−1.0 12−18 1−3 40−50 0.5−1 29−42 <2.5
Sesame oil — 7−9 4−5 40−50 — 35−45 0.4−1.0
Corn 0.2−1.0 8−12 2−5 19−49 <2.0 34−62 —
Castor — 2 1 7 — 5 —
Linseed — 4−7 2−5 12−34 35−60 17−24 0.3−1.0
Coffee — 34 7 9 — 44 —
Karanja — 3.7−7.9 2.4−8.9 44.5−71.3 — 10.8−18.3 2.2−4.7
Mahua — 20−25 20−25 41−51 — 10−14 0−3.3
Jatropha 0.5−1.4 12−17 5−9.5 37−63 — 19−41 0.3
Neem 0.2−2.60 13.6−16.2 14.4−24 49.62 — 2.3−15.8 0.8−3.4
Tobacco — 9.6 6.3 21.7 — 55.6 —
Moringa oleifera — 6.5 6 72.2 — 1 4
PLANT OIL TO BIODIESEL
Jojoba — — — 0.55−0.77 — — 28−31

93
amount of free fatty acid (FFA) present in the feedstock influence the transester-
ification process selection. During transesterification, FFAs react with alkali and
cause saponification that decreases biodiesel yield and increases the production cost
because extra energy will be used for ester purification (Karmakar et al. 2010). Heat
content or calorific content is another property of vegetable oil. It is the energy

content of feedstock oil that is directly related to heat content of FAME. More
unsaturated fuels show lower energy content, whereas more saturated fuels show
higher energy content (Karmakar et al. 2010). Another property, moisture, impuri-
ties, and unsaponifiables (MIUs), indicates the amount of water, filterable solids, and
other nontriglycerides (not converted to monoalkyl fatty esters by esterification or
transesterification) in the vegetable oil. Hence, MIUs should be removed during ester
purification by extra processing steps such as filtration, centrifugation, and heating
(Karmakar et al. 2010). Average amounts of MIUs in different edible and nonedible
oils are presented in Table 5-3. Titer is another important factor affecting the
selection of biodiesel feedstock. Titer is the temperature at which the crude oil
changes from solid to liquid. Because transesterification is a liquid process, the oils
with high titer require heating. This leads to an increase in energy requirements and
thereby production cost for biodiesel plant (Karmakar et al. 2010). Various physico-
chemical properties of edible and nonedible vegetable oils are provided in Table 5-3.
5.4 TRANSESTERIFICATION PROCESSES OF PLANT OILS
There are four ways to produce biodiesel from plant oils: blending, pyrolysis,
microemulsions, and transesterification. The most common method of conversion
of TAGs from vegetable oils to biodiesel is the reversible transesterification
reaction. This method also overcomes the problem of high viscosity oil. There
are many factors that affect transesterification reaction and ester yield, such as type
of alcohol, molar ratio of alcohol:oil, type and amount of catalyst, reaction time,
temperature and pressure, moisture content, and FFA content in oils. The
transesterification reaction can take place in the presence or absence of a catalyst
(acid, base, or enzyme). Transesterification reaction can be categorized into
homogeneously catalyzed or heterogeneously catalyzed based on the catalyst
solubility in a reaction mixture. Depending on FFA content in the oil, these
reactions can be performed in a one-step (acid or base) or two-step (acid/base)
process. When more than 1% FFA content is present in a feedstock, the two-step
process is recommended. Noncatalytic alcoholysis is accomplished at high
temperature and pressure (supercritical transesterification) (Ilham and Saka
2010). Figure 5-1 shows the flow diagram for the common transesterification
processes.
5.4.1 Homogeneously Catalyzed Transesterification Process

Homogeneously catalyzed transesterification of edible and nonedible plant oils by
one-step and two-step processes is frequently applied at the industrial level for
Table 5-3. Physical and Chemical Properties of Plant Oils.

High
Kinematic heating
Density viscosity Cetane value Flash Saponifiable Iodine Titer MIU (percent
Oil (Kg/m3) (mm2/s) number (°C) (MJ/Kg) point (°C) value value (°C) by weight)
Soybean 913.8 28.87 37.9 39.6 254 195.30 128−143 22−27 0.77
Sunflower 916.1 35.84 37.1 39.6 274 193.14 125−140 16−20 0.65
Palm 918 44.79 42 — 267 208.63 48−58 42−45 0.03
Peanut 902.6 39.60 41.8 39.8 271 191.50 84−100 26−32 —
Corn 909.5 30.75 37.6 39.5 277 183.06 103−128 14−21 1.67
Rice bran 918.5 36.68 — — — 201.27 90−108 24−28 2.73
Sesame 913.3 36 41.8 39.4 260 196.50 103−116 21−24 —
Coconut 918 27.26 — — — 267.56 7.5−10.6 20−24 2.74
Jatropha 940 33.9 — 38.65 225 200.8 82−98 31 0.16
Neem 918.5 50.3 — — — 209.66 65−80 35−36 2.16
Karanja 936.5 43.61 — — — 188.5 81−90 30−31 0.72
Mahua 960 24.50 — 36 232 190.5 58−70 23−31 —
Linseed 923.6 25.75 34.6 39.3 241 187.63 — 19−21 0.64
Castor 955 251.20 42.3 37.4 — 191.08 83−86 3 0.41
Tobacco 917.5 27.70 — — — 191.50 125−154 16−81 —
95
Catalyst/alcohol
Transesterification Glycerol
Vegetable oil reactor
Biodiesel
Figure 5-1. Transesterification process.
biodiesel production. Various feedstock characteristics including FFA content and

fatty acid composition decide the process type to be applied for biodiesel
production.
5.4.1.1 One-Step Process

The FFA content or acid value available in oil feedstock influences the selection of
the type of catalyst (acid or base) used in the process. Source of feedstock,
cultivation type, and oil storage mechanism influence the amount of FFA content
in the oil feedstock, for example, acid value found in jatropha oil varies from 0.92
to 28 mg KOH/g (Achten et al. 2008, Chitra et al. 2005). During the transester-
ification reaction, FFAs react with base catalyst and cause saponification that leads
to reduction in biodiesel yield, loss of catalyst, and problems in phase separation
(Juan et al. 2011; Parawira 2010). Therefore, base catalyst is preferred for the
feedstocks with less than 1% FFA content (Demirbas 2009). However, when more
than 1% FFA content is available in the feedstock, acid catalyst is recommended
due to its low susceptibility to FFA because it can catalyze both FFA esterification
and transesterification reactions simultaneously (Abbaszaadeh et al. 2012, Vyas
et al. 2009). However, the reaction rate with acid catalyst is slow and requires a
long reaction time to reach high biodiesel yield (Koh and Ghazi 2011). Therefore,
acid catalysts are rarely applied in the one-step transesterification reaction.
Table 5-4 presents the optimum reaction conditions and the catalyst type
applied in various studies for one-step homogeneously catalyzed transesterifica-
tion of different vegetable oils. Primarily, alkali hydroxides (KOH and NaOH) and
sulfuric acid are used in the transesterification process. Methanol or rarely ethanol
is applied in alcoholysis reactions, but the temperature is set below the boiling
point of alcohol (Berchmans and Hirata 2008, Cavalcante et al. 2010, Kumar et al.
2011, Singh et al. 2011, Berchmans et al. 2013, Fadhil and Ali 2013). Base catalysts
have various advantages over acid catalysts: they are less expensive, possess high
catalytic activity, and give high quality biodiesel production in a short reaction
time (Helwani et al. 2009, Ranganathan et al. 2008).
Most of the studies have reported more than 90% biodiesel yield in this one-
step process (Table 5-4). According to de Lima da Silva et al. (2009), ethanolysis of
Table 5-4. Homogeneously One-Step Catalyzed Transesterification Process of Plant Oils.

Operating conditions
Catalyst loading
Alcohol/alcohol to (percent by Temperature Time Yield
Feedstock oil molar ratio weight) (°C) (min) (%) References
Jatropha Methanol/6:1 1% KOH 45 180 95 Kumar et al. (2011)

Methanol/6:1 1% KOH 65 180 91 Sahu et al. (2011)
Methanol/6:1 1% KOH 50 120 97.1 Berchmans and Hirata (2008)
Methanol/6:1 1% NaOH 60 60 47.2 Deng et al. (2010)
Methanol/0.4:1 (v/v) 1% H2SO4 60 240 92.8
Methanol/0.7:1 3.3% NaOH 65 120 55 Berchmans and Hirata 2008)
Neem Methanol/6:1 2% KOH 60 60 83.4 Ragit et al. (2011)
Methanol/10:1 0.7% NaOH 60–75 6.5–8 h 88.94 Nabi et al. (2008)
Cottonseed Ethanol/7:1 1−2% NaOH 80 60 86 Keera et al. (2011)
Methanol/6:1 1% NaOH 60 60 98.5
Methanol/7:1 2% NaOH 60 20 95 Georgogianni et al. (2008)
Mahua Methanol/6:1 1% KOH 45 180 99 Kumar et al. (2011)
Ethanol/9:1 6% H2SO4 80−85 300 92 Saravanan et al. (2010)
Butanol/9:1 6% H2SO4 118−120 300 95.4
Tobacco Methanol/10:1 1% KOH 50 5 98 Parlak et al. (2009)
Methanol/10:1 0.5% NaOH 50 5 98
Castor Ethanol/16:1 1% C2H5ONa 30 30 99 de Lima da Silva et al. (2009)

Sea mango Methanol/6:1 1.5% KOH 60 30 91.4 Azcan and Danisman (2007)
(Continued)
97
98
Table 5-4. Homogeneously One-Step Catalyzed Transesterification Process of Plant Oils. (Continued)
Catalyst loading
Feedstock oil molar ratio weight) (°C) (min) (%) References
Fodder radish Ethanol/6:1 1.3% 30 70 97.9 Hasheminejad et al. (2011)

NaOCH2CH3
Moringa oleifera Methanol/3:1 1% KOH 60 60 82 Kafuku and Mbarawa (2010)
Karanja Methanol/6:1 1% KOH 65 180 89 Sahu et al. (2011)

Sesame Methanol/6:1 0.5% NaOH 60 — 74 Saydut et al. (2008)
Rice bran Methanol/9:1 0.75% NaOH 55 60 90.2 Moser et al. (2009)
Soybean Methanol/20:1 2M 120 5h 98.4 Miao et al. (2009)
trifluoroacetic
acid
Methanol/6:1 1% NaOH 65 60 90 Keera et al. (2011)
castor oil in the presence of C2H5ONa as a catalyst and at the 16:1 molar ratio of
alcohol:oil gave 99% ester yield. Similarly, methanolysis of jatropha and tobacco
oils (at a molar ratio 6:1 and 10:1, respectively) using KOH catalyst led to 99% and
98% biodiesel yield at 45 °C and 50 °C reaction temperature, respectively (Kumar
et al. 2011; Parlak et al. 2009). Conversely, about 90% ester yield has been reported
with acid catalysts as well. Saravanan et al. (2010) reported that ethanolysis of
mahua using 6% H2SO4 at the 9:1 molar ratio of ethanol:oil gives 92% ester yield.
Liao and Chung (2011) also studied transesterification of mahua oil in the
presence of butanol (at the 9:1 molar ratio) using 6% H2SO4 and reported
95.4% biodiesel yield. Figure 5-2 represents the flow diagram for a one-step base
homogeneously catalyzed transesterification process for biodiesel production.
Methanol recycle
Vegetable oil
Catalyst (NaOH/KOH) Glycerol

Transesterification
/ Alcohol /alcohol Vacuum distillation
(methanol/ethanol) @ phase
60 C, 1.4-4.0 bar
Ester phase
H3PO4 Catalyst neutralization
Separator Aqueous Phase
Filtration and water washing
Vacuum distillation Glycerol
Biodiesel
Figure 5-2. One-step base homogeneously catalyzed transesterification process for

The use of homogeneous catalysts also poses many drawbacks, such as

hazardous effect of alkali hydroxide and the need for a large amount of water
for purification steps to meet specific product quality that converts it into
wastewater. This wastewater requires appropriate treatment that increases the
overall process cost. Thus, there is a need to develop alternative methods (Helwani
et al. 2009).
5.4.1.2 Two-Step Process

When the FFA content in the oil feedstock is greater than 1%, the two-step (acid/
base) transesterification process is recommended for biodiesel production. This
process consists of two steps: (1) acid-catalyzed FFA esterification, that is, a
pretreatment step to reduce FFA content to less than 1%, and (2) base-catalyzed
transesterification to achieve high ester yield in short reaction time under mild
reaction conditions compared with the one-step process. During esterification
reaction in the presence of acid catalyst (e.g., H2SO4), the problem of soap
formation is eliminated. However, in comparison to the one-step process, the two-
step process contributes to higher production cost. Various studies on two-step
homogeneously catalyzed transesterification processes of different plant oils are
presented in Table 5-5. The type of oil feedstock, type of alcohol, molar ratio of
alcohol to oil, catalyst loading, reaction time, and temperature are the important
variables that affect the acid value in the first step and biodiesel yield in the second
step of the process. Crude deodorized jatropha oil contains 15% FFA content,
which does not fall in the acceptable limit for alkali-catalyzed transesterification.
Thus, a two-step process is utilized for this oil.
A comparison between the one-step and two-step process for ester yield
shows that a higher yield (90.1%) (Jain and Sharma 2010) is achieved by the two-
step catalyzed esterification/transesterification method than the one-step alkali-
catalyzed process (55%) (Berchmans and Hirata 2008). Similarly, Deng et al.
(2010) reported that jatropha oil with 10.45% FFA content gave reduced ester yield
of 47.2% in one-step base-catalyzed reaction, whereas a much higher FAME yield
(96.4%) was achieved in the two-step process.
The major alcohol used in both steps of the process is methanol (Deng et al.
2010, Liao and Chung 2011, Patil and Deng 2009, Patil et al. 2009). Transester-
ification of castor oil is an exception, in which ethanol was used (at 40:1 ethanol to
oil in the first step and 20:1 in the second step) to achieve 95.3% ester yield
(Hincapié et al. 2011). In other reported studies, methanol was used in the range of
6:1 to 18:1 for both steps. In the first step, increase in the molar ratio of methanol:oil
led to sharp reduction in acid value (Liao and Chung 2011; Wang et al. 2011, 2012)
with a continuous increase in ester yield (Patil and Deng 2009). For the second step,
a lower ethanol:oil molar ratio is required for further acid value reduction because
major FFA content of feedstock was esterified in the first step only.
In a number of studies, H2SO4 (0.4%−5%) has been used as an acid catalyst in
the first step, and KOH or NaOH (0.5%−2%) are used as base catalysts in the
second step. The optimum concentration of catalysts (0.5% H2SO4 and 2% NaOH
or KOH) should be applied to get the maximum ester yield (Patil and Deng 2009,
Table 5-5. Homogeneously Two-Step Catalyzed Transesterification Process of Plant Oils.

Catalyst loading
Feedstock Step oil molar ratio weight) (°C) (min) (%) References
Jatropha I Methanol/0.32:1 3% H2SO4 60 — 91.4 Liao and Chung (2011)

II (v/v) 1.2% NaOH 60 10 99.38
Methanol/8:1
I Methanol/8:1 0.4%H2SO4 60 30 92 Wang et al. (2011)
II Methanol/6:1 1% KOH 60 30 86.2
I Methanol/0.4:1 4% H2SO4 60 60 88.5 Deng et al. (2010)
II (v/v) 1.4% NaOH 60 30 96.4
Methanol/0.24:1
(v/v)
I Methanol/3:7 (v/v) 1% H2SO4 65 180 95 Jain and Sharma (2010)
II Methanol/3:7 (v/v) 1% NaOH 50 180 90.1
I Methanol/6:1 0.5% H2SO4 45 120 93 Patil and Deng (2009,
II Methanol/9:1 2% KOH 60 120 95 Patil et al. (2009)
(Continued)
101
Table 5-5. Homogeneously Two-Step Catalyzed Transesterification Process of Plant Oils. (Continued)
102
Catalyst loading
Feedstock Step oil molar ratio weight) (°C) (min) (%) References
Karanja I Methanol/6:1 1% H2SO4 60 — 95 Lakshmi et al. (2011)

II Methanol/6:1 1% KOH 60 60 97
I Methanol/6:1 0.5% H2SO4 65 — 91 Naik et al. (2008)
II Methanol/6:1 1% KOH 65 — 97
I Methanol/6:1 1% H2SO4 50 45 94 Patil and Deng (2009)
II Methanol/9:1 0.5% KOH 50 30 80
Kusum I Methanol/10:1 1% H2SO4 (v/v) 50 60 96 Sharma and Singh (2010)
II Methanol/8:1 0.7% KOH 50 60 95
Castor I Ethanol/40:1 1% H2SO4 60 60 — Hincapié et al. (2011),
II Ethanol/20:1 1% KOH 60 60 95.3 Wang et al. (2012)
Datura I Methanol/8:1 0.6% H2SO4 60 30 89 Wang et al. (2012)
stramonium II Methanol/6:1 1% KOH 60 30 87
Sapium I Methanol/8:1 0.4% H2SO4 60 30 — Wang et al. (2011)
sebiferum II Methanol/6:1 1% KOH 60 30 88.3
Liao and Chung 2011, Wang et al. 2011). Otherwise, the desired acid value
reduction cannot be obtained if a lower concentration of acid catalyst is used,
whereas the higher concentration makes the product darker (Sharma et al. 2010;
Wang et al. 2011, 2012). On the other hand, base catalyst with a concentration
higher than the optimum value increases the saponification reaction and reduces
ester yield (Sharma et al. 2010, Wang et al. 2011). Figure 5-3 shows the block
diagram of a two-step catalyzed transesterification process.
5.4.2 Heterogeneously Catalyzed Transesterification Process
5.4.2.1 One-Step Process

Heterogeneous (solid) catalysts have proven to be eco-friendly for biodiesel
synthesis due to their various advantages: (1) product separation and purification
Methanol recycle
Vegetable oil with >1%
Base
Esterification
Acid catalyst/ alcohol Acid neutralization and
Reactor separation
60-70°C
Transesterification
Alkali catalyst/ alcohol reactor @
50-65°C
Methanol recycle
Filtration and water washing

Separator
tank
Vacuum distillation
Biodiesel Glycerol
Figure 5-3. Two-step catalyzed transesterification process.

is simple with reduction in wastewater generation; (2) easy regeneration and reuse,
thus making FAME production more economical; and (3) high catalytic activity
(Boey et al. 2011, Jagadale and Jugulkar 2012, Viriya-Empikul et al. 2010). These
catalysts can be prepared by washing, drying, crushing, and calcinating under high
temperature conditions. Table 5-6 presents catalyst types and optimum reaction
conditions for one-step catalyzed (heterogeneous) transesterification processes of
different plant oils.
The nature of the heterogeneous catalyst, its specific surface area, pore size
volume, and active site concentration, influence the catalytic activity of the
catalyst. If the carrier support is provided to the catalyst, then its performance
can be improved because the carrier provides the higher specific surface area
(Zabeti et al. 2009). Alternatively, catalyst could also be improved by its pretreat-
ment to increase its basicity and acidity (Corro et al. 2010, Supamathanon et al.
2011). According to Kaur and Ali (2011), Li/CaO obtained after impregnation of
Li2CO3 shows the highest basic strength and highest surface area, and therefore
results in the highest activity, giving more than 99% FAME yield in 0.75 h at 65 °C
reaction temperature. Applied reaction conditions have a great influence on
FAME yield by heterogeneously catalyzed transesterification. This process
requires a higher initial methanol:oil ratio, catalyst loading, and reaction temper-
ature with much longer reaction time to obtain ester yields comparable to a
homogeneously catalyzed reaction. In general, Mg/Zr-mixed oxides with a 2:1
weight ratio of Mg/Zr (Sree et al. 2009), Li/CaO (Kaur and Ali 2011), and mixture
of acid (Fe2(SO4)3) and base (CaO and Li-CaO) catalysts (Endalew et al. 2011)
show the highest catalytic activity under mild reaction conditions with 100%, 99%,
and 100% ester yield, respectively (Table 5-6).
Easy regeneration and reuse of heterogeneous catalysts enables the
development of continuous process. Aluminum oxide-modified Mg-Zn
(Mg0.7Zn1.3Al2/3O3) catalyst can be recycled five times, but shows a drop of
3% in FAME yield during regeneration and 6% during reusability (Olutoye and
Hameed 2011). Mg/Zr-mixed oxide has proven to be an effective and stable
catalyst that can be recycled four times (Sree et al. 2009). CaMgO can be reused
three times with constant conversion of more than 80% (Taufiq-Yap et al. 2011).
However, some heterogeneous catalysts show significant decrease in ester yield
after recycling three times (Bokade and Yadav 2009, Vyas et al. 2009, Endalew
et al. 2011). This can be due to three major reasons: (1) adsorption of product
and byproduct on the catalytic surface; (2) leaching of active sites into the
solution; and (3) collapsed catalytic structure (Deng et al. 2011). To avoid the
blockage of catalytic active sites, various regeneration methods have been
applied: (1) washing with methanol and drying (Kumar et al. 2011, Olutoye
and Hameed 2011, Sree et al. 2009); (2) washing with methanol and hexane,
followed by drying and calcination at optimum temperature (Taufiq-Yap et al.
2011); (3) drying and calcinations (Vyas et al. 2009); and (4) recycling of catalyst
with glycerol (Endalew et al. 2011).
Table 5-6. Heterogeneously One-Step Catalyzed Transesterification Process of Plant Oils.

Alcohol/alcohol Catalyst loading Temperature Time Yield
Feedstock to oil molar ratio (percent by weight) (°C) (h) (%) References
Jatropha Methanol/12:1 4.8% Montmorillonite KSF 160 6 68 Zanette et al. (2011)

Methanol/16:1 12% K on NaY 65 3 73 Supamathanon et al.
(2011)
Methanol/15:1 4% CaMgO 65 6 83 Taufiq-Yap et al.
(2011)
Methanol/12:1 6% KNO3/Al2O3 70 6 84 Vyas et al. (2009)
Methanol/55:1 Mg-Zr mixed oxide with 65 0.75 100 Sree et al. (2009)
Mg/Zr weight ratio of 2:1
Methanol/11:1 8.68% Mg0.7Zn1.3Al2/3O3 182 6 94 Olutoye and Hameed
(2011)
Methanol/6:1 CaO + Fe2(SO4)3; Li- 60 3 100 Endalew et al. (2011)
Cao + Fe2(SO4)3
Methanol/15:1 20% DTPA (Dodecatungesto- 170 8 93 Bokade and Yadav
phosphoric acid) supported (2009)
on K-10 clay
Karanja Methanol/12:1 5% Li/CaO with 0.75 percent 65 1–2 99 Kaur and Ali (2011)
by weight of Li ion
Cerbera odollam Methanol/8:1 6% SO42−/ZrO2 180 3 84 Kansedo et al. (2009)

Castor Methanol/29:1 5% Zn5(OH)8(NO3)2. 2H2O 60 3 20 Zieba et al. (2009)
Methanol/6:1 10% Al2O3/ 50% KOH 60 0.08 85 Perin et al. (2008)
Methanol/18:1 7.5% Na2CO3 65 2 90 Yuan et al. (2011)
105
Moringa oleifera Methanol/19.5:1 3% SO42−/SnO2-SiO2 150 2.5 84 Kafuku et al. (2010)

5.4.2.2 Two-Step Process

This method of two-step heterogeneously catalyzed transesterification has not yet
been developed. However, many studies have been reported with this process
(Table 5-7). To make biodiesel synthesis process more economical, considerable
research has been performed to search for a cheaper catalyst. For example,
calcinations of waste chicken eggshells have been done to prepare CaO that has
been used to catalyze alcoholysis of mahua (Singh et al. 2011) and karanja (Sharma
et al. 2010) in the two-step process to achieve 95% ester yield at 65 °C in the
presence of 1.5% (v/v) H2SO4 in the first step and 2.5% (percent by weight) CaO in
the second step.
Similar to the one-step process, catalyst reusability is the most attractive point
in the two-step process to develop the continuous method. CaO (Singh et al. 2011)
can be recycled 10 times after washing and calcination without any significant loss
in ester yield, whereas hydrotalcite (Deng et al. 2011) can be reused for eight runs.
SiO2•HF (Corro et al. 2010) shows the best recycling performance of 30 runs
without requiring regeneration and with no change in ester yield.
5.4.3 Enzyme-Catalyzed Transesterification Process

It is well proved that lipases have the highest catalytic activity in nonaqueous
media. The lipase-catalyzed transesterification process involves the reaction
between TAG and alcohol in the presence of lipase enzyme that results in the
formation of biodiesel and glycerol. Enzyme-catalyzed processes show various
advantages: (1) simultaneous TAG alcoholysis and FFA esterification; (2) use of
high FFA content feedstock; (3) easy recovery of glycerol; and (4) less wastewater
generation (Ranganathan et al. 2008, Slomkowski et al. 2011). High cost of the
enzyme is the only drawback with this method (Leung et al. 2010). To recycle
enzyme catalyst, and hence to make the process more economical, lipases are
immobilized on the carriers, such as macroporous acrylic resin, celite, silica,
macroporous anion exchange resin, and reticulated polyurethane foam. Enzy-
matic transesterification of plant oils is performed with lipases from different
origins, including Chromobacterium viscosum (Kumar et al. 2011), Candida
antarctica (Kumar et al. 2011, Su et al. 2011, Tamalampudi et al. 2008, Wang
and Zhang 2010), Mucor miehei (Shah and Gupta 2007), pancreatic (Chatto-
padhyay et al. 2011), Pseudomonas cepacia (Kumari et al. 2007, Shah and Gupta
2007), and Rhizopus oryzae (Li et al. 2011, Tamalampudi et al. 2008).
In a study by Modi et al. (2007), transesterification of jatropha oil was
performed by lipase Candida antarctica immobilized on macroporous acrylic
resin in the presence of ethyl acetate as an acyl acceptor at 50 °C reaction
temperature. In this study, 91.3% ester yield was achieved in 12 h. A similar study
was done with karanja oil, and 90% ester yield was obtained (Modi et al. 2007).
Li et al. (2012) reported 94% biodiesel yield by methanolysis of P. chinensis bge with
lipase R. oryzae (F-AP15) immobilized on macroporous resin and anion exchange
resin at 37 °C in 60 h reaction time. Immobilized lipase (Lipozyme RMIM) has
been reported to catalyze alcoholysis of soybean oil under optimum conditions of
Table 5-7. Heterogeneously Two-Step Catalyzed Transesterification Process of Plant Oils.

Catalyst loading
Alcohol/alcohol to oil (percent by Temperature Time Yield
Feedstock Step molar ratio weight) (°C) (h) (%) References
Jatropha I Methanol/12 percent 1% H2SO4 70 1 91 Lu et al. (2009)

by weight
II Methanol/6:1 1.3% KOH 64 0.33 98
Jatropha I Methanol/12:1 10% SiO2.HF 60 2 96 Corro et al. (2010)
II Methanol/6:1 1% NaOH 60 2 99.6
Jatropha I Methanol/40 percent 4% H2SO4 (v/v) 60 1 88 Deng et al. (2011)
by weight
II Methanol/4:1 1% Hydrotalcite 45 1.5 95
Karanja I Methanol/6:1 1.5% H2SO4 65 1 91 Sharma et al. (2010)
II Methanol/8:1 2.5% CaO 65 2.5 95
Mahua I Methanol/6:1 1.5% H2SO4 (v/v) 55 1 91 Singh et al. (2011)
II Methanol/8:1 2.5% CaO 65 2.5 95
107
the 3.0 molar ratio of ethanol:oil, 50 °C reaction temperature, and 7% (w/w)

enzyme concentration (Bernardes et al. 2007). They achieved 60% ester yield after
1 h reaction time. Lipase-catalyzed transesterification of different plant oils is
presented in Table 5-8.
Reusability of lipases can make the alcoholysis process more economical.

When propan-2-ol and ethyl acetate are used as acyl acceptors, the immobilized
lipase can be reused for 12 runs without any significant loss in lipase activity. This
is due to better miscibility of these solvents in TAG; also, they are less polar than
linear-chain alcohols (Modi et al. 2007).
5.4.4 Supercritical Transesterification Process

The supercritical transesterification process (i.e., alcoholysis under high tempera-
ture and pressure) is a promising process for ester yield from plant oil feedstocks
using alcohols (methanol, propanol, ethanol, and butanol) without any catalyst
(Marulanda 2012, Shin et al. 2012, Glisic et al. 2016). In this method, alcohol is
used at a temperature and pressure above its critical point. At this state, its
dielectric constant decreases and creates single-phase formation instead of two
phases of oil-alcohol mixture as reported in the aforementioned transesterification
processes. This single phase favors the accelerated reaction due to an absence of
mass transfer limitation under such conditions. This process is advantageous due
to the following reasons: (1) it can overcome the initial reaction lag stage that is
caused by low alcohol solubility in the oil phase; (2) no soap formation; (3) no
waste generation; (4) higher reaction rate; (5) shorter reaction time; and (6) easy
glycerol recovery due to absence of catalyst. There are certain disadvantages of
supercritical reactions: (1) high cost of apparatus; (2) need for a large amount of
alcohol; and (3) high temperature and pressure that may cause degradation of
produced esters (Juan et al. 2011).
Table 5-9 presents the supercritical one- and two-step processes applied to
various plant oils. For oils containing high FFA content, there is a need for the
higher alcohol:oil molar ratio to push supercritical transesterification in the
forward direction (Campanelli et al. 2010). As the alcohol:oil molar ratio increases,
ester yield also increases, presumably because of increased contact area between
TAG and alcohol. Tang et al. (2007) obtained 90.5% ester yield by using a
24:1 molar ratio of methanol to jatropha oil at 250 °C reaction temperature and
7 MPa pressure. On the other hand, when the methanol:jatropha oil molar ratio
was increased to 43:1, 100% ester yield was achieved at 320 °C and 8.4 MPa
(Hawash et al. 2009).
Reaction temperature and pressure have great influence on supercritical
transesterification reaction. With an increase in reaction temperature, ester yield
also increases (Rathore and Madras 2007; Tang et al. 2007; Varma and Madras
2007; Demirbas 2008, 2009; Hawash et al. 2009; Valle et al. 2010; Micic et al. 2014).
For example, supercritical ethanolysis of linseed oil gave 86% ester yield at 250 °C,
whereas at 350 °C, 100% ester yield was achieved (Varma and Madras 2007,
Demirbas 2009). Increased pressure also influences the ester yield, such as the
supercritical methanolysis of jatropha oil at 7 MPa, which contributes to 90.5%
Table 5-8. Lipase-Catalyzed Transesterification Process of Different Plant Oils.

Lipase source/ Lipase

Alcohol/alcohol loading (percent by Temperature Time Yield
Oil to oil molar ratio weight) Carrier (°C) Solvent (h) (%) Reference
Jatropha Methanol/1:1 Rhizopus oryzae/6 Reticulated 30 — 60 80 Tamalampudi

polyurethane et al. (2008)
foam
Methanol/1:1 Candida antarctica Macroporous 30 — 90 76
(Novozyme 435)/2 acrylic resin
Methanol/4:1 Chromobacterium Silica activated — — 0.5 84.5 Kumar et al. (2011)
viscosum/5 with
ethanolamine
Methanol/4:1 Enterobacter aerogenes/ Silica activated 55 t-butanol 60 94 Kumari et al. (2009)
50 units with
ethanolamine
Diethyl carbonate/ Candida antarctica — 45 — 13.3 96.2 Su et al. (2011)
3.75:1 (Novozyme 435)/13.7
Ethyl acetate/11:1 Candida antarctica Macroporous 50 — 12 91.3 Modi et al. (2007)
Karanja Ethyl acetate/11:1 Candida antarctica Macroporous 50 — 12 90
(Continued)
109
Table 5-8. Lipase-Catalyzed Transesterification Process of Different Plant Oils. (Continued)

110
Lipase source/ Lipase

Alcohol/alcohol loading (percent by Temperature Time Yield
Oil to oil molar ratio weight) Carrier (°C) Solvent (h) (%) Reference
Cottonseed Methanol/4:1 Candida antarctica — 40 — — 95 Wang and Zhang

(Novozyme 435)/3.5 (2010)
Methanol/15:1 Pancreatic/0.5 — 37 t-butanol 4 75−80 Chattopadhyay
et al. (2011)
Rice bran Methanol/2:1 Candida sp. 99-125/20 — 40 n-hexane 12 87.4 Zheng et al.
(2010)
Pistacia chinensis Methanol/5:1 Rhizopus oryzae (F-AP15)/ Macroporous 37 — 60 94 Li et al. (2012)
7 IU/g acrylic resin and
anion exchange
resin
Soybean oil Ethanol/3.0 Immobilized lipase — 50 — 1 60 Bernardes
(Lipozyme RMIM)/7 et al. (2007)
Rapeseed Methanol/4:1 Lipozyme TLIM/3 — 35 t-butanol 12 95 Li et al. (2006)
Table 5-9. Supercritical Transesterification Process of Different Plant Oils.

Operational conditions
Type of acyl acceptor/acyl Temperature Pressure Time Yield
Feedstock acceptor:oil molar ratio (°C) (MPa) (min) (%) References
Jatropha Methanol/24:1 with 0.8% micro 250 7 28 90.5 Tang et al. (2007)
NaOH
Methyl acetate/42:1 345 20 50 100 Campanelli et al. (2010)
Methanol/43:1 320 8.4 4 100 Hawash et al. (2009)
Methanol/50:1 400 20 30 95 Rathore and Madras (2007)
Ethanol/50:1 400 20 20 94
I step- Water/217:1 270 27 25 — Ilham and Saka (2010)
II step- Dimethyl carbonate/14:1 300 9 15 97
I step- Water/- 270 11 60 92.1 Chen et al. (2010)
II step- Methanol/3:1 (v/v) 270 11 15 99
I step- Water/10:1 (v/v) 290 11 60 94.8 Chen et al. (2010)
II step- Methanol/3:1 (v/v) 290 11 9 98.3
Karanja Methanol/50:1 400 20 20 95 Rathore and Madras (2007)
Ethanol/50:1 400 20 30 94
Castor Methanol/40:1 350 20 30 100 Varma and Madras (2007)
Linseed Ethanol/40:1 350 20 40 100
Methanol/41:1 250 — 8 98 Demirbas (2009)
Ethanol/41:1 250 — 8 86
(Continued)
111
Table 5-9. Supercritical Transesterification Process of Different Plant Oils. (Continued)

112
Operational conditions
Type of acyl acceptor/acyl Temperature Pressure Time Yield
Feedstock acceptor:oil molar ratio (°C) (MPa) (min) (%) References
Fodder radish Methanol/39:1 317 18.3 27 97 Valle et al. (2010)

(Raphanus Ethanol/39:1 319 12.5 22 97.5
sattivus)
Cottonseed Methanol/41:1 250 — 8 98 Demirbas (2008)
Ethanol/41:1 250 — 8 86
Rapeseed oil Methanol/42.0 350 43 4 98.5 Kusdiana and Saka (2004)
Methanol/42:1 350 12 15 93 Micic et al. (2014)
Ethanol/42:1 350 12 20 91.9
1-propanol/42:1 350 12 25 91.1
Sunflower seed oil Methanol/41:1 252 24 20 95 Demirbas (2007)
Methanol with 0.3% CaO/41:1 252 24 17 95
Methanol with 5% CaO/41:1 252 24 13 100
Coconut oil Methanol/42:1 350 19 7 95 Bunyakiat et al. (2006)
Palm kernel oil Methanol/42:1 350 19 7 96
ester yield that was increased to 100% at 8.4 MPa (Tang et al. 2007, Hawash et al.
2009). This may be due to the increased density of oil with pressure that may
increase the solvent power of the supercritical fluid (Campanelli et al. 2010). The
reported optimum conditions for supercritical transesterification of rapeseed oil
are 350 °C reaction temperature, 43 MPa pressure, and 240 s reaction time with a
42.0 molar ratio of supercritical methanol:oil (Kusdiana and Saka 2004). This
study led to 98.5% FAME yield.
Recently, many studies have been done for the two-step supercritical process
as an alternative to the one-step process. In the first step, TAGs are hydrolyzed in
supercritical water at 270 °C; in the second step, the separated fatty acids are
supercritically esterified in supercritical methanol (Chen et al. 2010) or dimethyl
carbonate (Ilham and Saka 2010). Ilham and Saka (2010) reported 97% ester yield
by the two-step process. During first step, a 217:1 molar ratio of supercritical
water:jatropha oil was used to hydrolyze TAGs at 270 °C and 27 MPa pressure for
25 min. Then, the subsequent esterification was done in dimethyl carbonate at
300 °C and 9 Mpa for 15 min. Similarly, methanolysis of jatropha oil by the
two-step supercritical reaction led to 99% FAME yield (Chen et al. 2010). Thus,
FAME yield can be increased by a supercritical process in comparison to the
conventional alkali-catalyzed method.
5.5 CHALLENGES IN BIODIESEL PRODUCTION FROM PLANT OILS
Biodiesel is an alternative to fossil fuel that meets the growing demand of energy
supply and provides energy security. The reduced emission of greenhouse gases,
biodegradable, and nontoxic characteristics of biodiesel makes it an even more
promising substituent for conventional fuel. In the current scenario, the share of
biodiesel as the global transportation fuel is 1%, and increasing its share has
several challenges which need to be considered. The major obstacle in biodiesel
production from plant oil are limited quantity of feedstocks, land crisis for
afforestation, impact on environment owing to deforestation and the formation
of byproducts during transesterification processes.
The major contributor to cost in biodiesel production is feedstock, and in the
case of plant oil, the price increases with demand. The increasing demand of
feedstock can be met by increasing production, which in turn requires the large
suitable area for crop production. To meet growing demand for feedstock,
deforestation of rainforest for the harvesting of feedstock crops (palm oil, maize,
soybean) has been reported in many countries, for instance, in Malaysia (Anuar
and Abdullah 2016). Although, biodiesel stabilizes the carbon cycle in the
environment by reducing greenhouse emissions, the deforestation may destabilize
it and affect the biodiversity of the environment. Moreover, the supply of
feedstock (plant oils) is not continuous and varies with the change in climate,
and the storage of plant oil deteriorates its quality due to the biodegradation or
gum formation. The storage of biodiesel also raises concern regarding the
formation of acidic compounds due to oxidation, which has a negative impact on

engine performance. The use of edible plant oils for biodiesel production raises a
concern regarding the food-versus-energy problem. The high demand for edible
plant oil from maize, soybean, sunflower, and canola is raised with the prolifera-
tion of biodiesel producing industries. This proliferation causes food inflation and
fights between hunger and energy. The pretreatment processes during oil extrac-
tion and removal of the excess chemical by washing generate a large amount
wastewater, which may cause environmental pollution.
5.6 FUTURE WORK AND PROSPECTS
The limitation of biodiesel, for example, low energy content, high viscosity, high
price, degradation in fuel quality while in storage, are the major challenges that
require optimization and simulation of the biodiesel production technique. The
transesterification process is time-consuming and requires many toxic chemicals.
Moreover, washing and drying processes for the removal of chemicals generate a
large amount of wastewater. Research efforts toward catalyst regeneration,
enzyme-based catalysis, and supercritical transesterification processes may lead
to the development of and highly efficient technology for biodiesel production.
The 90% production of biodiesel attributed to the edible oil may create a food
crisis and also have negative environmental impact. The nonsophisticated edible
oils such as algal oil, jojoba oil, tall oil, and castor oil have the potential to become
feedstock for the biodiesel; however, the specific technique for different feedstock
is required. Biodiesel quality can be enhanced by assessing and optimizing the
factors affecting combustion quality, corrosion, and engine durability. Thus,
shifting toward the second-generation feedstock with the optimized technique
for biodiesel production can fulfill the energy demand and ensure energy security.
5.7 SUMMARY
Biodiesel has proven to be a promising alternative fuel that can be used in diesel
engines because it is a nontoxic, renewable, biodegradable, and eco-friendly fuel,
and also shows the superior flash point and high combustion efficiency. Biodiesel
is reported to decrease the greenhouse gases emission by 58%. First-generation
feedstocks are responsible for 90% production of biodiesel. Various nonedible oils
have been found to be promising feedstocks to replace edible oils. These include
jatropha, neem, mahua, karanja, castor, linseed, and others, but their applicability
for biodiesel is limited due to high economical value and concern regarding the
food versus fuel problem. The choice of feedstock plays a key role in biodiesel
production, and it depends on the process chemistry and economy. The determi-
nation of physical and chemical properties of feedstock such as oil content, fatty
acid composition, cetane number, flash point, titer, and iodine value is required at
the initial stage to assess their viability for future biodiesel production.
The commonly applied approach for conversion of plant oil into biodiesel
is the reversible transesterification reaction. Types of alcohol, the amount of
catalyst, reaction time, temperature and pressure, moisture content, and FFA
content in oils are the factors influencing the transesterification reaction, which
is divided into two classes—namely, homogeneously catalyzed or heterogeneous-
ly catalyzed on the basis of solubility of the catalyst. FFA content in the feedstock
(oil) determines the reactions step that is to be taken, and it has to be performed
in a one-step (acid or base) or two-step (acid-base) process. Source of feedstock,
cultivation type, and oil storage mechanism influence the amount of FFA
content, which in turn determines the selection of catalyst (acid or base) in
the reaction. In the one-step homogeneously catalyzed reaction, acid catalyst is
used when the FFA content is greater than 1%. The commonly used catalyst for
the one-step process is alkali hydroxide (KOH and NaOH) and sulfuric acid.
The homogeneously catalyzed one-step process has biodiesel yield greater than
90%. The two-step process is performed when the FFA content in oil is low.
Although acid catalyst in the one-step process overcomes the problem of
saponification, which is prevalent with the use of the base catalyst, the transes-
terification process is slow and requires a long reaction time compared with base
catalyst. In a two-step process, the acid-catalyzed pretreatment step is used to
reduce FFA content to less than 1%, and then the second step involves base-
catalyzed transesterification to achieve high ester yield in a short reaction time
under mild reaction conditions. However, the two-step process contributes to
higher production cost.
The homogeneous catalysts have many drawbacks, such as the hazardous
effect of alkali hydroxide and additional purification steps to meet specific product
quality. The high catalytic activity, ease in separation and purification step, and
catalyst generation make the heterogeneous (solid) catalysts eco-friendly for the
production of biodiesel. Regeneration and reuse of heterogeneous catalysts enable
the development of the continuous process; however, the process may suffer a
drop of 3% in FAME yield during regeneration and 6% during reusability due to
adsorption of product and byproduct on the catalytic surface, leaching of active
sites into the solution, and collapsed catalytic structure.
Immobilized enzyme-catalyzed transesterification can reduce harmful con-
sequence associated with the use of the chemical catalyst. The supercritical
transesterification process is performed with alcohol above its critical temperature
and pressure point for biodiesel production without any catalyst. In spite of many
advantages like higher reaction rate and shorter reaction time, the supercritical
transesterification process has high production cost because of the high cost of
apparatus, requirement of large amounts of alcohol, and maintenance of high
temperature and pressure. Challenges in biodiesel production is majorly associ-
ated with land crisis, deforestation, and price spike of feedstocks. Undoubtedly,
biodiesel is an eco-friendly fuel that provides energy security; however, research
toward the optimization of biodiesel quantity and quality is required.
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CHAPTER 6
Animal Fat Biodiesel
I. Bhattacharya
S. Yan
R. D. Tyagi
R. Y. Surampalli
Tian C. Zhang
6.1 INTRODUCTION
Biodiesel refers to a vegetable oil– or animal fat–based diesel fuel consisting of

long-chain alkyl (methyl, ethyl, or propyl) esters. Biodiesel is most commonly used
as a blend with petroleum diesel. Conventional diesel fuel blended with biodiesel
at a concentration of up to 5% by volume [Blend 5 (B5)] can be used in any
application. Blend 20 (B20; 20% by volume) is the most commonly used biodiesel
blend in the United States because it provides a good balance between material
compatibility, cold weather operability, performance, emission benefits, and costs.
Blend 20 is the minimum blend level allowed for compliance with the US Energy
Policy Act of 1992 (EPACT) (Prasad and Dhanya 2011).
The use of animal fat to produce biodiesel is an age-old technology. Fats are
classified as greases and oils, including the yellow grease, white grease, edible or
inedible tallow, lard, trap grease, poultry fat, vegetable oil, and fish oil (Groschen
2002). When vegetable oil and animal-derived waste fats are being compared, the
application of waste frying oil has a limited usage and is highly restricted to small-
scale projects. On the other hand, animal fats are being produced in large
quantities at various slaughterhouses in meat processing industries (Dias et al.
2012). Every country has its own set of rules to deal with these animal fats. For
example, in Brazilian meat chains, mostly fats from animals are generated in
rendering plants, and products from the rendering industry have a lower market
value. Materials of sanitary or aesthetic manifestations that are not suitable for
human food are integrated for feedstock for rendering purposes (Feddern et al.
2011). In Korea, each year nearly 400,000 tons of animal fat are further
transformed to 315,000 kL of biodiesel, which is equivalent to 25% of total
tax-free consumption of fuel (= ∼1,264,000 kL) that are made from protected
123
crops by production industry. In Korea, fats from cows and swine are the major
resources for animal fat biodiesel production (Kim et al. 2012); in Brazil, biodiesel
from fats are being produced from chicken fats, tallow, and lard.
Recently, utilization of animal fat as animal feed has been decreased because
animal fat possibly transmits various animal diseases. Compared with biodiesel
from vegetable oil, biodiesel from animal fat has advantages because of its higher
calorific value and cetane number (CN). However, animal fat biodiesel is less
stable to oxidation, because of the absence of natural antioxidants and has a major
cold filter plugging point because of a diverse amount of saturated fatty acids.
When additions of other raw materials are obtained, the fuel characteristics may
be improved (Dias et al. 2012). Without any transformation, animal fat has been
used to improve the rheological properties of fuel oil. The utilization of waste
animal fat to produce biodiesel opens a door to recycle the waste that otherwise
would have been washed down the drain or as a feedstock for the soap industry.
Many animal fats are low-quality feedstock compared with vegetable oil because of
the huge quantity of free fatty acids (FFAs) in animal fats. Using animal fat to
produce biodiesel can be considered as a reasonable way to minimize the cost of
biodiesel production. Other factors determining the feasibility of using animal fats
to produce biodiesel are the engine performance and harmful components derived
after using biodiesel produced from animal fats. Another factor is safe utilization
of animal fat biodiesel because animal fats are usually contaminated with prions
(Canoira et al. 2008).
Until now, it has been estimated that the total US biodiesel production is
more than 3.79 billion L) in 2012, of which, production of biodiesel from animal
fat accounts for nearly half. This chapter is designed to cover all possible aspects of
animal fat biodiesel. The chapter provides the sources of animal fats and the
factors affecting rendering techniques and other chemical parameters. It also
introduces comparative studies of the FFAs from various animal fat sources to get
an estimation of maximum saturated and unsaturated fatty acid production. The
presence of various metals in animal fat and the techniques for using natural
resources to convert biodiesel are described. Finally, the economic impacts as well
as the pros and cons of using animal fats for biodiesel production are discussed.
6.2 SOURCES OF ANIMAL FATS FOR BIODIESEL PRODUCTION
The use of animal wastes as raw materials for biodiesel production has three major
advantages: (1) does not compete with the food market; (2) recycles waste; and
(3) reduces production costs. Concerns exist for the utilization of low-cost waste
sources for biodiesel production, which otherwise cannot be used as human food,
such as waste animal fats, which are either from meat, chicken, or fish processing
industries. Biodiesel production from animal fats are usually environmentally
friendly and are a lower-cost alternative. Waste animal fats of tallow, lard, and
poultry fat are usually collected from slaughterhouses and meat processing
ANIMAL FAT BIODIESEL 125
industries. At the laboratory scale, these are melted and filtered for removal of gums,
residues of protein, and other suspended particles (Mata et al. 2010). Other resources
are the aquatic resources (e.g., fish oil) for producing biodiesel. The conversion
process of biodiesel from fish oil is simple. After the fish oil has been produced from
the leftover waste of the fishing industry, the oil is cleaned with the addition of
caustic soda and then purified with methanol for production of biodiesel. An
estimate shows 1 kg of fish waste can produce up to 1.3 L of biodiesel (Piccolo 2009).
In general, all biodiesels produced from animal fats have lower iodine values
when compared with the value fixed by European standard EN 14214. Animal fats
are usually incorporated with vegetable oils to achieve a product with an iodine
value according to the specifications (Encinar et al. 2011). Animal fats are usually
classified as tallow, lard, chicken fats, and various mixed animal fats. Tallow,
which is extracted from residues of bovine slaughter, can be filtered and contains
90% of total fatty acids with unsaponifiable impurities up to 1.5%. It does not
contain any FFA. Lard is extracted from swine slaughterhouses, and the specifi-
cation of lard is the same as that of tallow. Chicken fats are extracted from broiler
slaughterhouses, again containing 90% of total fatty acids and nearly 3% of
unsaponifiable impurities without any FFA. Animal fat mixtures are extracted
from slaughter residues of mammals and birds with 2% unsaponifiable impurities
(Feddern et al. 2011).
6.2.1 Edible and Inedible Tallow Products

Tallow is defined as the rendered form of beef that is processed from the hard fatty
tissue. At room temperature, it is solid and can be stored for a long time. It basically
consists of triglycerides, primarily steric and oleic acids. Edible tallow products
include margarine, cooking oil, and baking products, some of which are listed under
American Fats and Oils Association Ltd (Swisher 2006). The inedible tallow
products include candles, soap, and other lubricants like biodiesel. Tallow is derived
from rendering cattle fat. Production of tallow is directly proportional to the cattle
slaughtered, that is, countries producing the largest numbers of cattle are the largest
tallow producers (Swisher 2006). Edible tallows are usually consumed, whereas
inedible tallows are used to produce biodiesel. A report by Tyson Fresh Meats
(2013) suggests that 9 Code of Federal Regulations (CFR) 310 inspects each carcass
carefully and individually, where they are suitable enough for human consumption.
Inedible beef tallow and fats are prohibited for human consumption, per 21 CFR
Part 589.2000, and are used to produce lubricants. Tallows (beef or sheep tallow) are
considered to produce biodiesel and other oleochemicals because of the changing
habits of people and, apparently, because the soap industry cannot utilize all the
excess animal tallow. A study has shown that sheep tallow is a very suitable low-cost
feedstock to produce biodiesel (Bhatti et al. 2008).
6.2.1.1 Rendering Types

The rendering process is the separating process of the tallow by evaporating of
the water content, and tallow is then extracted mainly by centrifugation. There
are several popular methods, including the pressure-cooking and fat-melting
methods. In the pressure-cooking method, the hazardous material is inactivated,

prions are destroyed, and more qualitative tallow is achieved. Prion proteins are
not inactivated under the conventional rendering process, but their infectivity
reduces after pressure cooking. Prions are completely inactivated when the
rendered materials are cooked at 132 °C around 3 bar (45 psi) for 4.5 h (Taylor
2000). In the European Union (EU), pressure cooking is one of the efficient
methods. However, in North America, it is not considered as an effective method.
In the fat melting method, only discrete adipose tissues are used, and it produces a
high-end product, which is too fine to justify its use for biodiesel production
because very clean fats are never considered for biodiesel production (Baribeau
et al. 2007). Both biodiesel and direct firing of the tallow has been proposed as low-
risk end uses for tallow from livestock (Nelson 2006). In a rendering method
developed by Ejikeme et al. (2013), the thick piece of lard or tallow was obtained
by cutting the lard or tallow into small pieces and then melting it at 40 °C. NaOH
and methanol were added to it and heated at 40 °C again for 90 min. It was then
immersed in cold water, and then its lower layer was washed consecutively four
times to remove the unreacted catalyst. Furthermore, properties like flash point,
heating temperature, and pH values were determined.
Rendering products leave the slaughtering process and are considered as a
waste stream. These rendered products are then processed in the rendering units
to yield meat, bone meal and tallow. Furthermore, this tallow is then transported
to the biodiesel plant for transesterification prior to production of biodiesel
(Niederl and Narodoslawsky 2004). Certain rendering types include dry and wet
rendering, but for most tallow-based products, dry rendering is the suitably
preferred approach. The difference lies in the rendering methods in which the
system uses either wet or dry rendering principles. The system, where low
temperature is maintained for rendering, is called wet rendering, and this process
is usually continuous, whereas the system for which high temperature is main-
tained for rendering is called dry rendering and these processes are often
continuous or in batch. The rendering products are beef and sheep tallow, meat,
and bone meal because they are meant for biodiesel production. People involved
with rendering must be aware that the final usage affects the required product
quality and specific requirements to produce the end product. Significant para-
meters for the usage of tallow in biodiesel are plastics, moisture, impurities, and
unsaponifiables (Meat and Livestock Australia 2009).
6.2.1.2 Viscosity and Density

The density of biodiesel has been determined as 860 to 890 kg/m3 according to the
standards set in EN 14214. Inedible animal tallow methyl ester has a slightly lower
density among other methyl esters, and henceforth the densities of others are close
to each other. Density of biodiesel fuels has not changed much, because the
densities of methanol and oil are close to the density of the produced biodiesel.
The animal fat–based methyl esters have a density of around 870 kg/m3. Densities
of biodiesel fuels will vary with the fatty acid composition and their purity.
The specific gravity decreases slightly with increased saturation in the ester
composition. The viscosity and density of biodiesel fuels are important parameters
because they influence the key properties in any diesel engine. Different blends of
biodiesel fuels were prepared to check the fuel characteristics. It was observed that
viscosities of the animal fat–derived fatty acid methyl esters are higher than that of
the esters based on soy (Altun et al. 2010).

Oner and Altun (2009) prepared different blends of biodiesel directly from
Blend 100 to Blend 50, and Blend 20 and Blend 5, and injected the different grades
of biodiesel into the combustion chamber of the engine. The higher density of the
biodiesel along with its viscosity leads to the utilization of a larger volume of
biodiesel and poor atomization of the fuel (Xue et al. 2011). Biodiesel prepared
from animal tallow or fat, although an inexpensive and cheap source, leads to
highly viscous biodiesel and a high content of saturated fatty acids. When density
and viscosity of the biodiesel and diesel fuel were compared, it was observed that
diesel fuel has 3.6663 mm2/s of viscosity at 40 °C, whereas biodiesel has viscosity of
5.072 mm2/s, and density of diesel fuel is 843.51 kg/m3. Nearly 1.8 billion lbs
(3.53 billion kg) of animal fats are produced annually in the United States.
Experimental investigations have proved that beef tallow can be easily esterified to
components with properties similar to that of the esters from vegetable oil. Beef
tallow’s transesterification leads to the formation of a gel-like material with a high
concentration of saturated fatty acids components in beef tallow or cow tallow
because of poor agitation and low conversion of triglycerides, which leads to
incomplete reactions (Muniyappa et al. 1996, Ghazavi et al. 2012).
6.2.1.3 Cetane Number

The higher the CNs, the more efficient the fuel is. CNs rate the ignition properties
of diesel fuels, just as octane numbers determine the quality and value of petrol.
CNs are the ability of the fuel to ignite when it is in compressed state. Biodiesel has
a higher CN than petrodiesel because of its oxygen content. The ignition quality
affects engine performance, cold starting, warm up, and engine combustion
roughness (Department of Agriculture and Food 2006). The CN of biodiesel
depends on the distribution of fatty acids in the original oil or fat from which it is
produced (Bamgboye and Hansen 2008).
Biodiesel made from tallow and fats has a higher CN compared with soy oil.
In addition, the CN of animal tallow methyl ester is much higher than those
of waste cooking oil biodiesel and diesel fuel. The CN per ASTM D6751 and
EN 14214 biodiesel standards has been determined to be 47 and 51 min,
respectively. These CNs agree with ASTM D6751 and EN 14214. Furthermore,
higher viscosity of inedible animal tallow methyl ester causes poor injection.
However, inedible animal tallow is more effective than waste cooking oil (Altun
2011). Biodiesel production from inedible animal tallow and its usability were
investigated and further compared with pure biodiesel and the petro-diesel fuel
blends diesel engine. Tallow methyl esters as biodiesel fuel were prepared by base-
catalyzed transesterification of the fat with methanol in the presence of NaOH as
the catalyst. The cetane index is proportional to density value. It was observed that
the cetane index of the diesel alcohol mixture decreases with increasing amounts
of ethanol because ethanol has a very low CN (approximately 5 to 8). The CNs of
biodiesel from animal tallow and diesel are 56 to 58.8 for biodiesel, whereas CN is
47 for diesel (Altun et al. 2011, Darunde and Deshmukh 2012).
6.2.2 Lard Products

Pig fat, either in rendered or unrendered form, is called lard. The qualities of lard
vary somewhat, depending on the part of the pig from which the fat is taken and
how the lard is processed. Lard can be obtained from any part of the pig if there is a
high concentration of fatty tissue. Lard may be rendered by either of two
processes: wet or dry. In wet rendering, pig fat is boiled in water or steamed
at a high temperature, and the lard, which is insoluble in water, is skimmed off the
surface of the mixture or it is separated in an industrial centrifuge. In dry
rendering, the fat is exposed to high heat in a pan or oven without the presence
of water (a process similar to frying bacon). The two processes yield somewhat
differing products. Wet rendered lard has a more neutral flavor, a lighter color,
and a high smoke point. Dry rendered lard is equivalent to brown in color and
flavor and has a lower smoke point. The highest grade of lard, known as leaf lard,
is obtained from the fat deposit surrounding the kidneys. Leaf lard has little pork
flavor, making it ideal for use in baked goods, where it is valued for its ability to
produce flaky, moist pie crusts. The next highest grade of lard is obtained from
fatback, the hard fat between the back skin and muscle of the pig. The lowest grade
(for purposes of rendering into lard) is obtained from the soft fat surrounding
digestive organs, making it an efficient resource for biodiesel production. Pig lard
has similar composition as that of tallow; however, they have different fatty acid
content and iodine value (Gatlin et al. 2002).
The waste frying oils are known to be scarce, but waste animal fats such as pig
lard are more abundant in the environment. These pig lards had a common
application as human feeds, but over the years, certain practices of using pig lard
simultaneously decreased as the possibility of severe animal disease and the
consequent obligation to effectively discard or recycle the waste fats in the
environment increased. Also, animal fats have a significant content of saturated
fatty acids, and the fuel produced has a higher cold filter plugging point compared
with biodiesel from vegetable oils. Therefore, it might not be appropriate to use it
purely (100%) in vehicles during cold weather. It has been observed that biodiesel
production from waste lard by acid catalysis might have higher yields than basic
catalysis; however, greater amounts of sulfuric acid (25% to 50% of fat weight), a
higher molar ratio of methanol to fat (30:1), and much longer reaction time (24 h)
were used compared with basic transesterification. Pork wastes (mainly consisting
of fat and residual skin and meat) were collected at a local butchery, and the fat
extraction was made by heating until the fat was melted and separated from the
solid remaining residue. This product was finally filtered at reduced pressure.
The extracted fat was kept in a freezer at 4 °C during the experimental period. The
waste lard had an acid value of 14.57 mg KOH/g. Basic transesterification of lard at
65 °C, 1.0% [weight by weight (w/w)] NaOH, and a 6:1 molar ratio of methanol
resulted in immediate soap production (Singh et al. 2006).
A mixture of the waste lard with soybean oil was also studied as possible raw
material for biodiesel production. Biodiesel production from acid lard was
effectively enabled by a two-step synthesis. The influence of the pre-esterification
conditions in biodiesel quality was studied, and two predictive models were
further obtained. The catalyst amount and temperature mostly affected biodiesel
quality. The selected conditions, which led to a product viscosity of 4.81 mm2/s
and a purity of 99.6% by weight were 65 °C, 2.0% by weight H2SO4, and 5 h.
A mixture of waste lard and soybean oil was used to produce biodiesel using a one-
step synthesis, and the good results obtained indicated that blending might be an
interesting alternative to recycle wastes and even improve some product proper-
ties. Due to the existence of C17:0, in some animal fats, a correction should be
performed to the method proposed by EN 14214 for biodiesel purity determina-
tion (Dias et al. 2009).
Lee et al. (2002) fractionated lard with acetone to reduce the saturated fatty
acid content in the recovered fractions that were further used for cold tempera-
ture–resistant biodiesel production. However, by dry-fractionation, the unsatu-
rated fatty acid content increased only 1.9% to 4.6% from that of neat lard. Alkyl
ester as biodiesel needs stable performance at cold temperatures. Thus, solvent
fractionation was conducted to reduce the content of saturated fatty acid from lard
owing to their relatively high melting point.
In a pretreatment of pork lard as the raw material, the pork lard was first
heated at 100 ºC to eliminate residual water and then cooled to near the reaction
temperature (60 ºC). Synthesis of biodiesel was made by transesterification. The
mixtures of waste frying oil and lard were prepared considering the increase in the
fat fraction of the mixture (Buzetzki et al. 2010), varying from 0−1 (w/w), in
0.2 intervals. The fat was weighed and added to the reactor, which already
contained the necessary amount of oil. A defined amount of methanol (6:1 molar
ratio to oil) premixed with NaOH (0.8% by weight) was added to the reactor,
which already had 100 g of the raw material mixture, preheated at the reaction
temperature. At the point the reaction started, the reactor consisted of a 1 L flat
bottom flask immersed in a temperature controlling bath, equipped with a water-
cooled condenser and a magnetic stirrer. The biodiesel quality was obtained when
the minimum purity (96.5% by weight) was closely obtained only when waste
frying oil was used alone and when 0.2% of lard was incorporated in the raw
material (96.3% by weight); however, it ranged from 93.9 to 96.3 (% by weight)
being always close to the limit. Concerning the influence of raw material
composition in biodiesel quality, it was postulated that the parameter of the
biodiesel obtained from the mixture corresponded to the weighted average of the
parameter of biodiesel resulting from each component. The equation was reported
in the literature (Dias et al. 2008a).
6.2.2.1 Iodine Value

Iodine value numbers are often used to determine the amount of unsaturation in
fatty acids. This unsaturation is in the form of double bonds, which react with
iodine compounds. The higher the iodine value, the more C=C bonds are present
in the fat. High iodine value indicates a higher potential for biodiesel degradation,
either through thermal oxidation or free radical attack. The traditional method for
the determination of iodine value uses the methodology described by Johnston
and Li (2011) and adopted by American Oil Chemist Society (AOCS). The
polyunsaturated fatty acids (PUFAs) to saturated fatty acids (SFAs) ratio is

commonly referred to as the iodine value (IV), which is based on the laboratory
procedure using iodine to measure the number of double bonds (degree of
unsaturation) in fats and oils (Jordahl 2012). Raw material (pork lard) was
previously heated to melt. The reaction was carried out under constant stirring
for 40 min. The reaction mixture was heated up to 80 °C for 15 min until complete
ethanol evaporation. Hexane was added to extract the biodiesel phase. Then, the
hexane was evaporated, and the biodiesel recovered was dried in anhydrous
sodium sulfate and characterized.
The IV of the biodiesel samples was calculated by means of the equation in
which area of one hydrogen and the molecular weight is needed. It has been
suggested that 1H nuclear magnetic resonance (NMR) method is a reliable
alternative for the determination of the iodine value for ethylic biodiesel samples.
Based on the formula, pig lard was calculated as 70.6 using 1H NMR method and
71.0 using the AOCS method, which is far lesser than the IV of canola, corn, or
soybean samples (Reda et al. 2007). Although at certain places, the IV of pork lard
was calculated as 67 g I2/100 g (Sathiyagnanam et al. 2012), whereas the IV of
waste frying oil (waste cooking oil) was 117 g I2/100 g (Dias et al. 2008a).
6.2.2.2 Acid Value

The acid number is used to quantify the amount of acid present, for example, in a
sample of biodiesel. It is the quantity of base, expressed in milligrams of KOH, which
is required to neutralize the acidic constituents in 1 g of sample (Vermani and Narula
2005). Acid value of pork lard was found to be 0.71 mg KOH/g in comparison to
waste frying oil, which is 0.82 mg KOH/g. Pork lard represents a low acid value,
probably resulting from pretreatment processes; the commonly referred value for
commercial lard is slightly higher at 1.3 mg KOH/g fat (Dias et al. 2008b).
The acid value (AV) of fat or pork lard was determined by titration with
potassium hydroxide. FFA conversion (%) was calculated by the formula in which
AV at reaction time must be subtracted from the initial AV. These crude fats, with
an AV equivalent to 7.3 (mg of KOH/g of fats), were used for biodiesel production
by a two-step process. To ensure an efficient conversion of high AV crude fat into
biodiesel, an acid-catalyzed esterification (1% H2SO4 catalyzed) was initially used
to convert the FFAs present in the crude fat into biodiesel and to lower the AV.
Then, an alkaline-catalyzed transesterification comprising 0.8% NaOH catalyzed
were applied to produce biodiesel from animal fat (Li et al. 2011).
6.2.3 Poultry Fat

The use of vegetable oil leads to shortage of food, although use of animal fat for
human consumption is a health hazard. Rendered animal fats and restaurant
waste oils are appealing feedstock to produce biodiesel, and they are sold
commercially as animal feed. Investigations have shown that chicken fat is a

promising feedstock for biodiesel production. Biodiesel was produced from
chicken fat as waste that even included chicken feathers, blood, offal, and trims
after the rendering process with high FFA. Feather meal contains a significant
amount of chicken fat. The fat content of the feather meal varies from 2% to 12%,
depending on the kind of feathers used (Alptekin and Canakci 2011). Chicken
feather contains nearly 11% of fat, whereas duck and turkey feathers contain 6.7%
of fat (Lopes 2011).
Optimization of the transesterification reaction was investigated with different
methanol:oil ratios, reaction temperature, reaction time, and the catalyst amount.
The obtained ester was characterized by determining its fuel properties according to
standard test methods. The solid chicken fat was melted at around 65 °C, filtered,
centrifuged, and decanted to remove other suspended particles. The processed fat
was homogeneous in nature and was stored in airtight opaque plastic jars to prevent
oxidation. The percentages of yield of methyl esters are presented as graphs for
different conditions (Panneerselvam et al. 2011, Fadhil et al. 2012). High ester yields
up to 99% were obtained from chicken fat after 24 h in the presence of sulfuric acid
(Alptekin et al. 2011). The percentage of yield of methyl esters is calculated by the
equation in which methyl esters are divided by the melted chicken fat. Henceforth,
the biodiesel yield is calculated similarly, that is, mass of biodiesel divided by the
mass of the raw material (Phalakornkule et al. 2009).
The study on the biodiesel production process and optimization of chicken fat
showed that the quantity of catalyst, amount of methanol, reaction temperature,
and reaction time are the main factors affecting the production of methyl esters.
The optimal reaction conditions for production of methyl esters from chicken and
mutton fat were established as follows: the reaction time of 90 min at 60 °C; the
6:1 molar ratio of methanol to oil; and 0.38 g of KOH/g of oil for chicken fat for
50 mL of chicken fat. The use of chicken fat is very suitable as low-cost feedstocks
for biodiesel production (Panneerselvam et al. 2011). Chicken fat density variation
was experimentally measured in the temperature range from 25 °C to 100 °C; it is
said that chicken fat is thermally stable up to ∼350 °C. Given the negligible
volatility of chicken fat, a linear variation was obtained and then extrapolated to
account for densities at reaction temperatures.
It has also been reported that a preheating step of chicken fat feedstock of up to
350 °C can be used without a significant thermal decomposition (Marulanda et al.
2010), and the associated chicken fat has an acid number of 26.89 (Alptekin et al.
2011). The yield of fat extraction from poultry wastes was 40 % by weight. The
characterization of the fat showed an AV of 0.92 mg KOH/g and an iodine number
of 80 g of I2/100 g. The AV of the raw material has a good alkaline transesterification
due to reduced risk of catalyst consumption by FFAs (Moreira et al. 2010).
6.2.4 Fish Waste

To produce biodiesel, fish fat as an economic resource are considered eventually
(Clifford et al. 2008). Using fish waste to produce biodiesel is a highly viable
alternative. The use of animal waste and oil to produce biodiesel is not a new
technology, but the adaptability of this technology to aquatic resources has only
recently attracted public interest. The process is relatively simple. The production
of biodiesel starts with crushing the fish waste. This allows the oil to be extracted,
which is mixed with methanol (roughly 9%) and caustic soda (to separate the
glycerin from the biodiesel). Aquafinca, which specializes in processing of tilapia

fish, is estimated to be producing more than 15,000 L of biodiesel from tilapia fish
oil a day. The biodiesel is then used to produce electricity at its production facility
as well as running its vehicles. Aquafinca operates a tilapia farming facility in the
El Cajon dam as well as at Yojoa Lake. Andersen and Weinbach (2010) reported
that 623 kt by-products from fish are the theoretical maximum available amounts
that could be utilized for biodiesel production. However, 166 kt of the byproduct is
being dumped, of which 48 kt are dumped on shore. Biodiesel from fish residue/
waste could also promote more efficient use of aquatic living resources and
generate additional income for fishing and fish farming communities.
Fish oil biodiesel indicates significant sensitivity to oxygen exposure, leading
to the formation of varnish on the surfaces of the fuel handling system (Witmer
2010). According to Taku Renewable Resources (2010), salmon oil has a rapid
oxidative rate and a high free fatty acid content, which necessitates the addition of
antioxidants (such as citric acid or phosphoric acid) to the oil prior to transester-
ification and in relatively high quantities if the oil is going to be stored even for just
a few days prior to biodiesel production (Taku Renewable Resources 2010). In a
study by El-Mashad et al. (2006), salmon oil extracted from acidified waste and
salmon oil extracted from rendered waste were used as feedstock. The researchers
found virtually no difference between the two feedstocks. Both salmon oils
required a sulfuric acid pretreatment to bring down the high AV of the oil, and
both oils yielded approximately the same amount of biodiesel for the same cost.
Based on the weight of the salmon oils prior to transesterification, a maximum
99% biodiesel yield was achieved. However, the researchers calculated that up to
15% of the esters were lost during the final washing and drying steps due to the
formation of emulsion in the biofuel. Finally, a preliminary economic analysis
revealed that the cost of salmon oil biodiesel production was almost twice the cost
of soybean oil biodiesel production (El-Mashad et al. 2006). Salmon oil and
salmon oil biodiesel have similar characteristics of corn biodiesel from corn oil.
Unisea, based in Dutch Harbor, has been processing and burning fish oil as
waste in its boilers for decades, along with other Pacific Rim seafood processors.
According to Steigers (2002), since 2001, Unisea has also used burned fish oil as
petrol-diesel blends in their electrical generators. In addition, as part of the Alaska
Biodiesel Demonstration Project, Unisea shipped more than 18,000 gal. (68,137 L)
of walleye pollock oil to Hawaii in 2005, and Pacific Biodiesel processed this fish
oil into biodiesel before it was shipped back to Alaska for testing (Witmer and
Schmid 2008). This project established fish oil–based biodiesel as a viable engine
product for engine operability in Alaska, but also identified critical storage and
handling problems with the fish oil–based biodiesel. Aquafinca is a tilapia farm,
rendering plant, and biodiesel production facility located approximately 125 miles
(201.0 kms) from San Pedro Sula, the administrative capital of Honduras.
The farm currently harvests roughly 200,000 lbs (90,718 kg) of tilapia a day, of
which approximately 54% [(108,000 lbs) (48,987 kg)] is waste including viscera,
heads, frames, and skins. The skins are immediately separated from the rest of the
byproducts, dried, and sold to China to produce gelatin. Viscera, heads, and
frames are ground and rendered at high heat to approximately 16,000 lbs
(7,257 kg) of fish oil (8% yield) and 22,000 lbs (9,979 kg) of fishmeal
(11% yield). Of the 16,000 lbs (7,257 kg) of fish oil, approximately 10,000 to
12,000 lbs (4,536 to 5,443 kg) or 1,200 to 1,500 gal. (4,542 to 5,678 L) are converted
to biodiesel daily. It has been demonstrated that 3,000 gal. (11,356 L) of biodiesel
was produced in a day from the catfish byproducts, which are nearly 110,000 lbs
(49,895 kg) per day and salmon byproducts produced each day are 50,000 lbs
(22,679 kg). The oil content of the biodiesel feedstock is high, ranging from
8% (farmed tilapia) to 30% (farmed Atlantic salmon) (Taku Renewable Resources
2010).
Some fish oils contain essential fatty acids like omega-3, which is a highly
valued commodity especially in the pharmaceutical industry. Therefore, care must
be taken on which types of fish are used when producing the fish oil. According to
Piccolo (2009), one of the lowest in the omega-3 content but high in oil is catfish,
with an omega-3 content of 0.18 g/100 g of fish. Another note of care is the acid
content of the oil extracted. For example, salmon oil is high in acid, and this acid
needs to be removed. Therefore, an additional step in removing this acid is
required. Sulfuric acid is added to reduce the AV of the oil. Once this has been
done, the process of transesterification can begin. In 2007, the National Techno-
logical Centre for the Canning of Fish Products in Spain was looking for ways to
produce biodiesel with fish fat available in wastewater generated by the canning
industry (Piccolo 2009). Any marine fish oil methyl ester must have density of
0.860 g/cm3 and viscosity of 7.20 at 40 °C with a CN of 50.9, which are much less
than the CN of waste oil methyl ester (Fok et al. 2012).
6.3 COMPARATIVE STUDIES OF FREE FATTY ACIDS
Different feedstock oils are used to produce biodiesel. The effect of various
operating and processing parameters on transesterification depends on quality
and source of the feedstock oil. According to FFA content, different technological
approaches can be used. One approach is the alkali-catalyzed process because the
FFA content in collected frying oil was less than 1%; , but however, when FFA
content is a higher value, like on bovine tallow, it is a significant step to do a two-
step process, which are esterification and transesterification (Ribeiro et al. 2011).
To produce biodiesel, low-cost animal wastes with relatively high FFA are used,
after reduction of FFA by 1% in the pretreatment reaction. Chicken fat is a low-
cost feedstock for biodiesel production compared to high-grade vegetable oils. Fat
is usually extracted from feather meal, which is prepared from chicken wastes,
including chicken feathers, blood, offal, and trims after the rendering process.
Feather meal contains a significant amount of chicken fat. The fat content of the
feather meal varies from 2% to 12%, depending on the kind of feathers used.
However, they often contain significant amounts of FFA. The fats with high FFA
cannot be converted to biodiesel using alkaline catalysts. Mostly, sulfuric acid is
considered as the best catalyst for reducing the FFA level. Another method is the
removal by KOH in the form of soap (Alptekin et al. 2011). The FFA reacts with
alcohol to form ester of biodiesel through
Hþ
..
.
R1 -COOH þ ROH → R-O-CO-R1 þ H2 O
FFA Alcohol Fatty acid ester water (6-1)
The water content of the fats is relatively low; however, conversion can be
affected. For the alkaline-catalyzed method, the conversion was slightly reduced
when more water was added. However, in the acid-catalyzed method, only as little
as 0.1% of added water would lead to some reduction of the yield of methyl esters.
Most properties of biodiesel depend on the feedstock used. It has been reported by
Knothe (2006) that biodiesel viscosity and density can be predicted based on
feedstock fatty acid profile. High viscosity is expected because the main fatty acids
have a long chain of carbon and a low degree of unsaturation, and thus, no high
density is expected for the same reasons. Density increases with a decrease in chain
length and an increase in the number of double bonds (Encinar et al. 2011). The
production of poultry meat has increased significantly in recent years, and the
recycling of fatty wastes resulting from its slaughter has been the subject of study
since their incorporation in animal diets was restricted. Consequently, poultry fat
is currently considered a potentially good low-cost raw material for biodiesel
production. Biodiesel composition, which relates to the composition of the waste
poultry fat used, was determined by gas chromatography. The methyl esters
contained in the biodiesel were identified by comparing retention times with those
of chromatographic standards. Water content was always higher than the maxi-
mum set by EN 14214, independent of the transesterification conditions used.
This fact showed that the drying method selected should be improved or replaced
by others of greater effectiveness, such as evaporation under reduced pressure
(Moreira et al. 2010).
The fatty acid methyl ester content, or ester content, is an indicator of the
purity of biodiesel, and EN 14214 indicates a minimum of 96.5% by weight to
ensure quality. This parameter is very important to prevent an illegal mixture of
other substances. Biodiesel production from recycled wastes represents lower
yields than when virgin oils are used. Accordingly, when using poultry fat as raw
material, the yields were not very high, that is, from 73 to 86% by weight. However,
such disadvantages can be compensated by the value added to the wastes through
their recycling, the cost reduction on waste treatment, and the reduction of
greenhouse gases emissions (Chhetri et al. 2008, Moreira et al. 2010). Fish oil is
composed mainly of unsaturated fatty acids (USFAs) and is particularly rich in
PUFAs; thus, it can be used as raw oil for biodiesel production to improve fuel flow
fluidity at low operating temperatures. In general, biodiesel fuels produced from

raw oil with longer chain fatty acids have a higher CN and thus superior
compression ignition characteristics. The deterioration rate of a lipid or oil is
significantly influenced by the amount and composition of unsaturated fatty acids
and by the number and location of the double bonds in the chemical compounds.
The oxidation instability starts with the methyl group (-CH2-) carbons attacking
the olefinic carbons as part of the peroxidation chain mechanism. The USFA
content may be converted to form primary oxidation products such as hydro-
peroxides and conjugated dienes, which may be either isolated or accumulated at
an accelerated rate. The marine fish oil biodiesel contained 37.06% by weight of
saturated fatty acids and 37.3% by weight of long carbon chain (in the range of
C20−C22) fatty acids and is likely to have a greater kinematic viscosity than
biodiesel made from general vegetable oils (Lin and Lee 2010).
Clifford et al. (2008) compared the chemistry of diesel fuel with that of biodiesel
and found that biodiesel typically has different fats and oils; biodiesel contains
common types of fatty acids, ranging from 12 to 22 carbon atoms, with 16 to
18 carbons being the most common. Some of the chains are saturated; others are
monosaturated; the rest are polyunsaturated. Within the limits of specifications, the
differing levels of saturation can affect biodiesel fuel properties. Beef tallow can
undergo oxidation reactions in the presence of oxygen, high temperatures, and light
because of the presence of USFAs. This decreases the biodiesel stability, because the
ester derivative of oleic acid is monounsaturated and may suffer reactions from
photo-oxidation and autoxidation. The boiling point is a significant aspect of the
biodiesel, and it relates to the quality of the biodiesel. In beef tallow, decomposition
occurs at higher temperatures, because of high predominance of the fatty acids with
higher molecular weight (usually they are the ones with C18:0). Among beef tallow,
low thermal stability has been observed, as 70.60% of saturated esters are present.
Eventually, cetane numbers, heat of combustion, melting point, and viscosity of
neat fatty acids increase with increasing chain length and decrease with increasing
unsaturation (Araujo et al. 2010).
Table 6-1 lists FFAs available in various animal and fish fat wastes. Crude
lipids have impurities such as phospholipids, FFAs, aldehydes, ketones, and
pigments. For improving stability and quality, numerous degrees of purifications
were implemented. Common methods are (1) bleaching to remove pigments,
minerals, FFAs, aldehydes, and ketones; (2) degumming to remove phospholipids;
(3) deodorizing to remove FFAs; and (4) neutralization to remove water. For the
palatability of the lipids, winterizing is a common technique to improve trans-
parency, which later impacts the brightness. Triglycerides consist of a glycerol
backbone esterified to three fatty acids, which are either essential or nonessential.
The energy liberated depends on the carbon chain length and the degree of
unsaturation of the fatty acids. Longer chains yield more energy than shorter
Table 6-1. Composition of FFAs from Different Sources of Animal, Poultry, and Fish Wastes.
136
FAME
composition C10:0 C12:0 C14:0 C16:0 C16:1 C17:0 C18:0 C18:1 C18:2 C18:3 C19:0 C20:1 C20:4 C20:5 C22:6 References
Inedible/edible — 13.147 18.48 23.57 — — 28.175 27.471 27.108 — — 30.05 — — — Trushenski and
tallow Lochmann (2009),
Araujo et al. (2010),
Bayraktar and Bayir
(2012)
Lard — — 1.3 20.66 1.98 0.48 10.91 39.13 19.55 1.21 — 0.91 — 0.12 0.2 Robles et al. (2003),
Rohman et al.
(2012), Tang et al.
(2008)
Poultry fat — — 1.5 27.6 4.5 — 12.1 42.8 10.5 0.7 — — 0.37 — — Marulanda et al.
(2010), Alptekin
and Canakci
(2011), Alptekin
et al. (2011),
Encinar et al.
(2011)
Fish fat — — — 19.61 5.16 — 5.24 20.94 — — — — — 3.7 15.91 Lin and Lee (2010)
Other fat waste 3.1 35.6 7.6 14.8 3.8 — 3.6 23.6 5.8 — 1.4 — — — — Li et al. (2011)
Biodiesel — — 0.6 2.4 5.8 — 5.5 41 21 1.4 — 0.4 — — — Moreira et al. (2010),
Panneerselvam
et al. (2011)
EN 14103 — — — — — — — — — Maximum — — — — — Lin and Lee (2010)
12.0
Figure 6-1. % of unsaturated fatty acids and saturated fatty acids in different
animal fats, poultry fats and fish wastes: (a) USFAs; and (b) SFAs.
chains, and saturated chains (with no double bonds) yield more energy than
unsaturated chains (Trushenski and Lochmann 2009).
The presence of more SFAs crystallizes easily for the biodiesel fuel, which
further causes fuel filter plugging and other performance hazards (Gerpen 2005).
FFAs have double bonds and are unsaturated FFAs. These double bonds are
unstable and can eventually break in the presence of water and heat; therefore,
USFAs tend to spoil faster than SFAs (Popescu and Ionel 2011). Often, biodiesel
produced with such oils tends to break down quickly, and biodiesel made from
such USFAs tends to degrade overnight. Figure 6-1 shows the results of a
comparative study on various percentages of USFAs and SFAs. Tallow has the
lowest number of USFAs, whereas lard, poultry fat, and fish fat have the highest
(Cunha et al. 2012).
6.4 EFFECT OF METALS ON TALLOW, LARD, POULTRY,

AND FISH FAT
Sulfur content in biodiesel is affected by the sulfur content of different feedstock.

Utilization of ultralow sulfur diesel (ULSD) has become mandatory since 2006
(EPA 2006). Biodiesel must meet the similar ULSD standards, in which the total
sulfur content is set at 15 ppm maximum. When biodiesel is compared with fossil
diesel, biodiesel contains a lesser amount of sulfur. Question has been raised about
the sulfur content of the feedstock from which biodiesel is prepared. Sulfur content
in animal fats and oils are higher according to ASTM D5453 and ASTM D7039
(Sanford et al. 2009). Sulfur content varies from one animal waste to another. The
highest ones are the mustard and rapeseeds ranging from 9,000 to 15,000 ppm,
respectively; whereas animal fats have much higher sulfur levels, usually more
than 15 ppm. Feedstocks, which are usually higher at FFA, are treated with sulfuric
acid for the reduction of FFA prior to transesterification. In that case, phase
separation is a must for the removal of sulfur from the fuel layer (He et al. 2009).
The sulfur from the animal fat usually originates from sulfur-containing amino
acids, which are associated with proteins and carried off from the rendering
process. The sulfur contents in tallow, fish, and poultry are the maximum; whereas
sulfur content in lard is a minimum by 1 ppm as shown in Figure 6-2 (Choi et al.
2012, Janchiv et al. 2012).
Phosphorus does damage to catalytic converters that are being used in
emission control systems. Therefore, to produce biodiesel, low phosphorus must
be maintained. At a study done by Ostrovsky et al. (2006), the European Union
leads the way to environmental protection and implementation of the production
of biodiesel. At a tolerance level of 10 ppm, the Inductively coupled plasma mass
spectrometry (ICP) system is ensured to reliably meet the anticipated lower levels
per regulations now. A low amount of phosphorus is present in lard, compared
with the rest of the animal wastes. In biodiesel (according to ASTM D6751),
phosphorus limitation is just 10 ppm (Moser et al. 2013), whereas the combined
amount of magnesium and calcium must be less than 5 ppm according to the test
method by EN 14538. Accordingly, one must even measure calcium, magnesium,
sodium, and potassium in the biodiesel to avoid any issues about ionization
interferences among the alkali metals. Tallow and poultry fat have the highest
phosphorus and even higher calcium (Atabani et al. 2012), whereas calcium in lard
is the lowest (0.1 ppm), and phosphorus in fish fat is 49 ppm (Sugiura 2009).
Figure 6-2. Concentrations of sulfur, phosphorus, calcium, and magnesium in

tallow, lard, poultry fat, and fish fat. For biodiesel, the accepted concentration
must to be 15 ppm for sulfur, 10 ppm for phosphorus, and 5 ppm combined for
magnesium and calcium.
The ICP technology usually uses manganese or cobalt as internal standards.

When testing feedstock for metals using the ICP technology, the entire feedstock
must be pretreated if the concentration of phosphorus, calcium, or magnesium is
higher than the accepted amount (= 10 ppm) (Sanford et al. 2009). This measure
must be taken because sodium, calcium, magnesium, and potassium are oriented
toward forming deposits in the equipment, and hence catalyze undesired reactions
and simultaneously poison the control equipment. Therefore, to avoid that,
combinations of metals are made in a category in which Na + K are considered
and Ca + Mg are considered and quantified (US Department of Energy 2009).
If poultry fat methyl ester has a high CN, its disadvantages include high freezing
point and viscosity even if the poultry fat methyl ester is noncorrosive, clean, and
renewable. These disadvantages limit the usage of biodiesel fuels in diesel engines.
To improve the biodiesel, magnesium-based additives of a 16 mmol/L concen-
tration were added to the poultry fat methyl ester to lower the freezing point and
viscosity. These additions support the idea of the catalytic cracking effect of the
additives, which are with smaller chains of hydrocarbons (Guru et al. 2010).
Sodium and potassium may be present in biodiesel as abrasive solids or
soluble metallic soaps. Sodium and potassium are usually added as catalysts in the
form of sodium and potassium hydroxide for transesterification as they have
excellent stability and activity (Ali et al. 2012). However, the drawbacks of such
catalysts are that they are insoluble and corrode the apparatus. As a solid catalyst,
it can decrease the cost of biodiesel and even the steps of purification (Jiang et al.
2010). However, sodium and potassium do not directly come from the animal
waste. Another supported catalyst used is potassium carbonate. After transester-
ification is complete, potassium carbonate can be extracted easily from the leftover
biomass with the help of inexpensive classical extraction technology (Baroi et al.
2009). Singh et al. (2006) showed that the potassium-based catalyst gave better
yields than the sodium-based catalyst. Usage of sodium and potassium hydroxide
flakes are common because they are inexpensive. The residual catalyst of sodium
and potassium hydroxide should be removed from the biodiesel through succes-
sive washes with water, after which quantification of metals must be done to
ensure the quality of the biodiesel product (Jesus et al. 2008). Increasing usage
of biodiesel worldwide implies the necessity for controlling the quality of
the biodiesel to avoid any undesirable mechanical and environmental effects
(Demirbas 2009, Caland et al. 2012).
6.5 CONVERSION OF ANIMAL FATS INTO BIODIESEL USING

CHARCOAL AND CO2
6.5.1 Using CO2

Biodiesel conversion reaches to 98% after 30 min in the presence of CO2. CO2 is
used in the reaction medium for the transesterification reaction. However, the role
of CO2 is still under investigation (Kwon et al. 2012). The pyrolysis process is
enhanced in the presence of CO2 as the reaction medium and bond disassociation,
which is accelerated at temperatures greater than 550 °C in the presence of CO2.
6.5.2 Using Charcoal

The conventional pyrolysis has a high charcoal content if biomass is associated

with the product of interest (Demirbas 2008). Ecomotion Sternberg plant
(Germany), which has an oil mill and glycerin production facility, is actively
using charcoal as activated charcoal filter for glycerin production (Andreas 2006).
Many times, charcoal comes as impurities in animal waste for biodiesel and must
be filtered out (Banga and Varshney 2010). In a real continuous flow system to
produce biodiesel, charcoal was packed into the tubular reactor to reach the
desired temperature. Apart from that, methanol along with tallow and lard are fed
into the tubular reactor; CO2 was used in the reaction medium, and hence
noncatalytic transesterification was carried out. Hence, it is an alternative for the
biodiesel conversion (Kwon et al. 2012). Hussain et al. (2011) reported that
the major advantages of such a pyrolysis process to produce hydrocarbons are the
simplicity and high charcoal burnout leading to high decomposition efficiencies.
However, further work must be done when charcoal is considered for noncatalytic
biodiesel conversion processes (Kwon et al. 2012).
6.6 MEASURING THE ECONOMIC IMPACT OF ANIMAL FAT

BIODIESEL
Production of biodiesel from animal wastes or fats is an economical approach and

environmentally friendly, because the disposal of these may create problems in the
environment and even contaminate the water resources (Albalawi et al. 2011).
Since 2006, nearly 20% of the biodiesel manufactured in the United States was
produced from animal fats. Purchase of animal fat to produce biodiesel increased
simultaneously. In 2006, animal fats were priced at USD 16.13/lb (USD 35.6 /kg);
by 2008, that value had more than doubled to USD 33.22/lb (USD 73.3/kg)
(Schettler 2009). According to USDA market news service, costs of animal fats in
December 2012 were 34.29 /lb (in Minneapolis) and 30.42/lb (in Central United
States) USD 75.7/kg and USD 67.15/kg respectively; but the prices of animal fats
are still lower than the prices quoted for cottonseed, canola, and sunflower seed
(Johnson 2013). Animal fats have a great potential as feedstock for biodiesel
production because they have low market values (Feddern et al. 2011).
The major feedstocks for biodiesel production in 2012 were (1) corn oil with
a production rate of 59 million lbs (26.77 million kg); (2) yellow grease with a
production rate of 57 million lbs (25.85 million kg); and (3) inedible/edible tallow
with a production rate of 44 million lbs (20.0 million kg) (Rayer 2012). The
biodiesel production rate in the United States was 75 million gal. (284 million L).
until October 2012. As shown in Table 6-2, in the United States alone, contribu-
tions for the year of 2010, 2011, and 2012 were 100, 240, and 212 million lbs
(45.35, 109, and 96.16 million kg) by poultry fat and 170, 431, and 371 million lbs
(77.11, 195.4, and 168.283 million kg) by tallow, respectively (US Energy Informa-
tion Administration 2012). For a long time, the European Union was the world’s
largest biodiesel producer, representing nearly 70% of the entire biofuel market in
the transport industry. For 2010, 2011, and 2012, the total animal fat contribution to
produce biodiesel was 390, 420, and 335 million tons (Table 6-2) for the grand total
of biodiesel production of 9.4, 9.4, and 9.65 million tons, respectively (Peters et al.
2011, Flach et al. 2012). For 2013, the animal fat contribution was estimated to be
340 million tons to produce 10.1 million tons biodiesel (Flach et al. 2012). To
understand the economic impact of the usage mandatory of various blends of
biodiesel produced in Canada to date, Stiefelmeyer et al. (2006) did a study by
focusing on the oilseed sector and the rendered animal fats industry, which were
supposed to be the major feedstocks for the biodiesel production. Certain para-
meters were estimated with respect to the nature of the markets of the feedstocks.
Other estimations were the economic effects of 2% and 5% biodiesel blend with
fossil fuel diesel. As shown in Table 6-2, for 2010, 2011, and 2012, total biodiesel
production by Canada only was 139, 158, and 284 million L, of which one of the
feedstock animal fat contributed at 95, 105, and 84 million tons, respectively. For
2013, of the estimated 538 million L, 135 million tons were contributed by animal fat
for biodiesel production (Brent Evans and Dessureault 2012).
Despite the wide variety of feedstocks that are potentially useful to produce
biodiesel, soybeans have nearly 77% of total biodiesel feedstock, followed by animal
fat, which accounts for 16% (Barros 2012). In 2012 and 2013, total biodiesel
production in Brazil was 2.7 and 2.760 (forecasted) billion L. Brazilian National
Agency for Petroleum, Natural Gas and Biofuels (ANP) has even estimated that 1 kg
of animal fat is equivalent to 1.064 L of biodiesel. Australia has biodiesel industries
also, and the main source is from animal fat and waste cooking oil. For 2010, the
total biodiesel produced was approximately 180 million tons. After 2010, many
Australia-based biodiesel manufacturers were shut down because the economic
viability of the operations in the industries decreased (Department of Resources,
Energy and Tourism Australia 2011). However, earlier in 2004 and 2005, total
production of biodiesel was approximately 1 and 4 million L (Paech 2006). Biodiesel
Table 6-2. Contribution of Animal Fats in the Worldwide Production of Biodiesel.
Animal fat contribution for biodiesel production

(million tons)
Country distribution 2010 2011 2012 2013 (forecast)
United States 170 431 371 ND

European Union 390 420 335 340
Canada 95 105 84 135
Australia 180 ND ND ND
Note: ND = not determined.
units are set in even smaller countries like Turkey, which has many small biodiesel
manufacturing facilities to meet the needs of the local population. Very large
biodiesel facilities in order to meet the needs of industries are very few, but it is a
promising business for the European Union, who promote biodiesel facilities in the
nation and in the worldwide arena. Along with favoring economics and increasing
demand, smaller nations are coming up for the production of biodiesel (Kleindorfer
and Öktem 2007).
6.7 PROS AND CONS OF ANIMAL FAT BIODIESEL
Like any biofuel counterpart, biodiesel has its own pros and cons, depending on
the type of feedstock used for the preparation of biodiesel. The universal benefit
would be the reduction of the reliance of fossil fuels and henceforth, simultaneous
reduction of the amount of emissions of greenhouse gases. One concern may be
the interference of biodiesel and its feedstock with the human food chain or factors
like deforestation. The significant advantage is the reduction of CO2 in the
environment. However, a gradual increase in the production of nitrogen oxide
(NOx) has been reported (Colomar et al. 2000, Chapman et al. 2003). Other
factors that affect the pros and cons of biodiesel include the following:
• Physical and chemical characteristics of animal fats: The properties of various
animal fats (tallow, lard, poultry fat, and fish fat) affect the resulting biodiesel
product properties. Certain standards were set for biodiesel that are desig-
nated under ASTM D6751; biodiesel from animal fats must meet those
specifications (Groschen 2002). Temperature is an importance characteristic
of the biodiesel property and the commodity from which biodiesel was
prepared. Biodiesel has the tendency to solidify in cold weather. For instance,
if it is made from animal fats, the biodiesel will primarily be in the solid phase
at room temperature, and the final product has the potential to further solidify
at relatively cold temperature. For instance, tallow (rendered form of beef)
processed from suet is always solid at room temperature. In fact, tallow can be
kept for an extended period without refrigeration to avoid decomposition, but
it is supposed to be kept in an airtight container for oxidation prevention
(Meat and Livestock Australia Limited 2011). This factor is dependent on the
presence of SFAs (Fontana 2009).
• Price rate of animal fats in various market places: Considering that animal fats
can be used in biodiesel production, the availability of animal fats as tallow,
lard, poultry fat, or fish fat will have an impact on the price of the raw
materials. Depending on the region, the prices may vary compared with the
other feedstock such as sunflower, canola, and soybean. According to
Minnesota’s biodiesel law, diesel fuel sold in the United States must contain
approximately 2% of biodiesel (Groschen 2002).
• Tax regulations: The United States and the European Union have benefited from
biodiesel consumption subsidies. The subsidies vary from United States and
European Union. In the European Union, the tax exemption is at the retail fuel
pump; however, in the United States, the tax credit is at price/3,785 L of biodiesel
fuel, which has since been discontinued. Any policy being released has impacts
on the market. Because the EU tax exemption is much higher than the US tax
credits, the worldwide biodiesel market prices are set on the EU parameters. The
prices must tally with the world oil prices (de Gorter et al. 2010).
6.8 SUMMARY
The production of biodiesel is an accepted contributor to the fuel industry, and

obviously it minimizes the fossil fuel utilization. Biodiesel has fewer emissions
compared with that of diesel. Various engines and motors can use up to 20%
blends of biodiesel along with diesel. Each country has a set of rules for the
marketing of biodiesel as a product. Although animal fats may have the risk of
certain contamination (like prions or other contaminants) and are unsuitable for
human consumption, they can be effectively used for biodiesel production. In a
few years, animal fats (as tallow, lard, and poultry fat and fish waste) will be an
influential industrial material for biodiesel production.
Production of biodiesel depends on certain parameters, such as rendering
types, viscosity, and density of the animal fat, cetane number, iodine value, as well
as acid value. Further properties for consideration are flash point, pH, and the
heating temperature. The interference might be impurities present in the animal
fats. Removal of impurities must be performed by various pretreatment methods
before the final treatment. Another factor influencing the biodiesel production is
the presence of FFAs as unsaturated fatty acids or saturated fatty acids. Animal
fats, as an inexpensive source, are considered for biodiesel production after the
FFAs are reduced to 1% in the final product.
Under the law EN 14214, the purity of biodiesel has been stated as 96.5%
exclusive of all impurities. More saturated fatty acids in the biodiesel fuel leads to
crystal formation, which further causes plugging of the fuel filter. However, the
presence of unsaturated fatty acids can destabilize down the line as the double
bonds tend to break in the presence of heat and various treatments. Tallow is the
maximum saturated fatty acid producer, and fish fat is the highest unsaturated
fatty acid producer. Certain metal might be associated with the fats, such as sulfur,
phosphorus calcium, magnesium, and potassium. However, animal fats may not
have sulfur content, depending entirely on where they are being bred. Phosphorus
in animal fats damages the catalytic convertors; hence, a lower concentration of
phosphorus must be maintained. These factors entitle themselves as the cons of
biodiesel production from animal fats. The physical and chemical characteristics
determine whether an animal fat can be used for biodiesel production. Price rates
of animal fats vary from country to country; however, animal fat is still an
inexpensive endeavour when compared with corn oil, soymeal, and sunflower oil
for biodiesel production.
6.9 ACKNOWLEDGMENTS
The authors sincerely thank the Natural Sciences and Engineering Research
Council of Canada (Grant A4984, RDCPJ 379601, Canada Research Chair) for
financial support. The views and opinions expressed in this chapter are strictly
those of the authors.
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CHAPTER 7
Biodiesel from Waste
Cooking Oil
B. Bhadana
R. D. Tyagi
7.1 INTRODUCTION
Over the last few years, biodiesel has received political acknowledgment, owing to
its major benefits over petroleum diesel, including (1) significant reduction in the
emissions of greenhouse gases (GHGs), (2) nonsulfur emissions, (3) nonparti-
culate matter pollutants, (4) low toxicity, (5) biodegradable, and (6) obtainable
from renewable sources like vegetable oils and animal fat, among others (Banerjee
et al. 2014, Thirumarimurugan et al. 2012). Therefore, many researchers have tried
to develop the vegetable oil–based derivatives to complement petroleum-based
diesel fuel. It has been well reported that biodiesel obtained from canola and
soybean oil acts very well as a diesel fuel substitute (Campanelli et al. 2010, Dizge
et al. 2009). Different mechanisms can be used to produce biodiesel, including
direct use or blending of the diesel fuel, microemulsions, thermal cracking of
vegetable oils, and transesterification. The most common mechanism to produce
biodiesel is transesterification, that is, a reaction between vegetable oil and alcohol
in the presence of a catalyst that results in the formation of fatty acid alkyl ester
and glycerol (Raqeeb and Bhargavi 2015). The most important crops identified for
biodiesel production include sunflower, palm, canola, corn, olive, soybean, peanut,
and rapeseed oils (Demirbas 2009). However, the key issue in commercialization
of biodiesel production using vegetable oil is its high manufacturing cost, which is
because of the higher cost of virgin vegetable oil (VVO) that contributes to 70% to
95% of total production cost. The cost of vegetable oil has a crucial role in the
economics of biodiesel (Tan et al. 2011).
Waste cooking oil (WCO) is a promising alternative to vegetable oil for
biodiesel production because it is much less expensive than VVO. Recent studies
on the production cost of biodiesel using WCO as feedstock shows that the overall
production costs of biodiesel can be reduced by more than half compared with
151
VVO because using WCO can effectively reduce the feedstock cost to nearly
60% to 70% (Escobar et al. 2009). Hence the high cost of feedstock can be
overcome if WCO is used for biodiesel production, that is, WCO significantly
enhances the economic viability of biodiesel production. Cost of catalyst also
affects the overall biodiesel production cost. Several studies have been done so that
waste materials can be used to prepare low-cost catalyst to develop suitable
biodiesel production processes (Stacy et al. 2014).
This chapter critically discusses the sources of WCO, factors affecting WCO,
methods for preparation of biodiesel from WCO, fuel characterization, compari-
son of WCO and VVO for biodiesel production, and economic assessment for
biodiesel production from WCO.
7.2 SOURCES OF WASTE COOKING OIL
WCO is a byproduct from fast food restaurants, hotels, shops selling fritters, and
an operating vegetable oil refinery. The quantity of the WCO generated per year
by any country is huge. In many countries, domestic WCO is generated in huge
quantity; for example, 0.7 to 10 million tons/year domestic WCO is generated in
Europe. Similarly, this quantity reaches 10.0, 0.12, 0.153, 4.5, 0.5, 0.45–0.57, and
0.07 million tons/year in the United States, Canada, Ireland, China, Malaysia,
Japan, and Taiwan, respectively (Kalam et al. 2011).
The disposal of WCO is problematic, because its disposal methods may
contaminate the environment. Many countries have set the policies that penalize
the disposal of WCO through the water drainage system (Chen et al. 2009). The
production of biodiesel from WCO is one of the better ways to utilize it efficiently
and economically. In fact, using WCO reduces the dependence on biodiesel-
producing crops and competition for food. Biodiesel production from WCO is the
same as with VVO, except that WCO contains certain debris that requires it to be
filtered first, followed by a pretreatment step (esterification) before transester-
ification because the WCO undergoes heating several times that leads to formation
of free fatty acid (Fadhil et al. 2012).
7.3 BIODIESEL FROM WASTE COOKING OIL
7.3.1 Factors Affecting Waste Cooking Oil

Healthy food production is very important for human consumption and health.
Heat plays a crucial role for food preparation. During cooking, boiling, and frying,
different degrees of heat are applied. Based on the degree of heating, food
constituents undergo many physical and chemical changes (Adaileh and AlQdah
2012). Because frying improves the taste of food, it has become a common method
in food preparation. While frying, oil is heated at a temperature of 160° to 190 °C
under atmospheric conditions for a long duration (Adaileh and AlQdah 2012).
BIODIESEL FROM WASTE COOKING OIL 153
The same cooking oil is also used several times over for economic reasons.
However, continuous use of the same cooking oil for frying may lead to
various physical and chemical changes, for example, (1) an increase in viscosity,
(2) a change in surface tension, (3) an increase in specific heat, and (4) a change
in color (Wen et al. 2010). Table 7-1 shows typical chemical and physical
characteristics of WCO in comparison to VVO.
7.3.1.1 Thermolytic Reaction

This reaction occurs at very high temperatures and in the absence of oxygen. If the
triglycerides containing saturated fatty acids are heated at very high temperature
(180 °C) in the absence of oxygen, then a series of normal alkanes, alkenes,
symmetric ketones, lower fatty acids, oxopropyl esters, CO, and CO2 is produced.
Unsaturated fatty acids form dimeric compounds, like dehydrodimers, polycyclic
compounds, and saturated dimers. Unsaturated fatty acids also react with the
other unsaturated fatty acids through the Diels–Alder reaction and form dimers
and trimers. In the case of glycerides, this reaction happens between acyl groups
within the same molecule (Omar and Amin 2011a, 2011b).
7.3.1.2 Oxidative Reaction

Unsaturated fatty acids can react with molecular oxygen (O2) through a free-
radical mechanism (Figure 7-1). During the reaction, hydroperoxide is formed as a
primary product that may further lead to formation of many other compounds,
such as isomeric hydroperoxides containing conjugated diene groups. Hydro-
peroxides are also involved in the production of many chemicals with a significant
variation in flavor threshold, molecular weight, and biological significance. The
alkoxy radical is formed by scission of the O–O bond of the hydroperoxides. This
alkoxy radical can lose or gain protons (H+) to form the keto or hydroxy
derivatives, respectively. Various chemicals such as hydrocarbons, aldehydes,
acids, and semialdehydes are formed by decomposition of the alkoxy radicals.
In the presence of excess oxygen, alkoxy and peroxy radicals can be transformed
into dimeric and oligomeric compounds (Tan et al. 2011).
7.3.1.3 Hydrolytic Reaction

The steam production during the preparation of food leads to the hydrolysis of
triglycerides, and results in the formation of free fatty acids (FFAs), glycerol,
monoglycerides, and diglycerides (Adaileh and AlQdah 2012). The change in
oil composition by the hydrolytic reaction can be quantified by measuring the
monoglyceride and diglyceride content and not the FFA content of oil, because
some of the FFAs are lost during frying (Tan et al. 2011). The amount of heat
and water during frying triggers the hydrolytic reaction of triglycerides that
causes a growth in FFAs in WCO (Adaileh and AlQdah 2012, Tan et al. 2011).
During oxidation and polymerization reactions, the viscosity and saponification
number of WCO increase compared with VVO. Furthermore, during frying,
the transport of matter and heat occurs between the vegetable oil and the
frying food, which increases the water content in WCO. At the time of
Table 7-1. Comparison of Main Properties of WCO and VVO.
Property Unit WCO VVO
Density g/cm3 0.91–0.924 0.919

Kinematic viscosity mm2/s 39.5 35.6

Water content % 0.42 0.1–0.2
Acid value mg KOH/g 2.66 0.25
Iodine value g I2/100 g 89 93
Peroxide value meq./kg 16.61 10–20
Total polar material % 24.5–30 11
Heating content kJ/kg 39,741 36,000
Flash point K 485 374–429
Pour point K 284 275
Cetane number — 49 37–42
Sulfur content % 0.09 0.0096
Carbon residue % 0.46 <0.1
Unsaponifiable material % 1.70 1.5
Saponifiable index mgKOH/g 188.2–207 184–196
Ash % 0.006 0.015
Refractive index — 1.4700 1.4677–1.4705
Fatty Acid Composition
Percent by Percent by
Fatty Acid Formula weight weight
Myristic C14:0 0.9 0.1
Palmitic C16:0 20.4 5.5
Palmitoleic C16:1 4.6 1.1
Stearic C18:0 4.8 2.2
Oleic C18:1 52.9 55
Linoleic C18:2 13.5 24
Linolenic C18:3 0.8 8.8
Arachidic C20:0 0.12 0.7
Eicosenic C20:1 0.84 1.4
Behenic C22:0 0.03 0.5
Erucic C22:1 0.07 0.4
Tetracosanic C24:0 0.04 0.3
Mean molecular weight g/mol 856 882
transesterification, presence of water in WCO leads to hydrolysis, whereas

higher content of FFAs and higher saponification number cause the saponifi-
cation reaction. Both of these reactions (hydrolysis and saponification) lead to
low biodiesel yield and the high catalyst consumption (Omar and Amin 2011a,
Tan et al. 2011).
Figure 7-1. Simplified mechanism for oil oxidation reactions during frying.
If the oil is used repeatedly, then these three reactions result in the formation
of many harmful compounds. If WCO is to be made a feedstock for the biodiesel
production, then the amount of polar compounds in the WCO, especially FFA,
must be taken into consideration because they will greatly affect the transester-
ification reaction. Refined oil contains less than 0.5% by weight FFA, whereas
for WCO, FFA contents range between 0.5% and 15% by weight (Raqeeb and
Bhargavi 2015). To remove the undesirable compounds in WCO, the pretreat-
ment (esterification) of the same is required before the transesterification reaction.
Esterification is used to convert FFAs to fatty acid methyl esters (FAMEs), or
biodiesel, and thus reduces the amount of FFAs in WCO (Raqeeb and Bhargavi
2015). FFA reacts with alcohol (mainly methanol) in the presence of acid catalyst
to yield FAMEs
RCOOH þ CH3 O ↔ RCOOCH3 þ H2 O

Fatty acid Methanol FAME Water
ðacid catalystÞ
This process actually minimizes the acid value of WCO. Usually it is an acid-
catalyzed process in which acids, such as hydrochloric acid, sulfuric acid, sulfonic
acid, and butyl-methyl imidazolium hydrogen sulfate (BMIMHSO4), are used as
acid catalyst (Javidialesaadi and Raeissi 2013). Titration is used to determine the
acid value of WCO by titrating the mixture of the oil with ethanol and diethyl
ether (1:1) against KOH with phenolphthalein as an indicator. The following
formula is used to calculate acid value:
Acid value = 56.1 × CV∕m (7-1)
where C = concentration of KOH (M); V = volume of KOH (mL); and

m = weight of oil sample (g). Based on the acid value, catalyst is selected for
esterification. According to Javidialesaadi and Raeissi (2013), 2% v/v H2SO4 gives

the maximum conversion. Because this reaction is reversible, the equilibrium
causes the inhibition in the completion of the esterification reaction. Preheating
WCO in an oven reduces the water content and thereby minimizes the FFA
(Javidialesaadi and Raeissi 2013). Excess of methanol (5.6 to 7.8 M) can be applied
to shift the equilibrium in the forward direction during pretreatment (Anitha and
Dawn 2010). The major parameters that affect the esterification reaction include
catalyst, the methanol-to-catalyst ratio, the amount of catalyst used, and the
reaction temperature (Adaileh and AlQdah 2012, Javidialesaadi and Raeissi 2013).
The next section describes different transesterification methods for the efficient
and economical production of biodiesel from WCO.
7.3.2 Transesterification
Transesterification refers to the reaction of the triglyceride component of oil with
alcohol (methanol or ethanol) in the presence of NaOH or any other catalyst
resulting in the formation of ester and glycerol (Figure 7-2). In general, there are
two systems of transesterification—chemical and enzymatic systems (Mumtaz
et al. 2012). WCO is reacted with alcohol. Primarily, methanol is used because of
its better efficiency (Adaileh and AlQdah 2012, Banerjee et al. 2014, Farooq et al.
2015, Raqeeb and Bhargavi 2015, Stacy et al. 2014, Thirumarimurugan et al. 2012).
It has been reported that this process depends on several parameters, including
reaction temperature and pressure, rate of agitation, reaction time, type and
concentration of catalyst used, type of alcohol used, the molar ratio of alcohol to
oil as well as the concentration of moisture, and FFA in the feed oil. The optimal
values of all these parameters depend on the physical and chemical properties of
the feedstock oil for attaining higher conversion (Adaileh and AlQdah 2012).
Figure 7-2. Schematic of the transesterification reaction.

7.3.2.1 Alkali-Based Transesterification

Currently, alkali-catalyzed transesterification is used for biodiesel production
from vegetable oils (Adaileh and AlQdah 2012, Banerjee et al. 2014, Javidialesaadi
and Raeissi 2013d, Thirumarimurugan et al. 2012). Many researchers have used
NaOH and KOH for biodiesel production due to their various advantages, such as
(1) being able to catalyze the reaction at lower reaction temperature and
atmospheric pressure, (2) giving higher conversion in less time, and (3) being
readily available and thus, economical (Atapour and Kariminia 2011, Demirbas
2011). However, this process also has some limitations, including high energy
consumption, which ultimately increases capital equipment costs and safety
issues. This method is also highly sensitive to FFA and water content in the
feedstock. High moisture content can lead to saponification of the reaction that
reduces ester yield, causes difficulty in glycerol separation from FAME, increases
the viscosity, and leads to the formation of emulsion (Omar and Amin 2011a,
Tan et al. 2011). Therefore, different pretreatment methods are used to remove
water and reduce FFA content in WCO, including steam injection, column
chromatography, neutralization, film vacuum evaporation, vacuum filtration, and
esterification (Talebian-Kiakalaieh et al. 2013). Because esterification is more
economic compared with other methods, it is preferred for pretreatment of WCO.
Leung et al. (2010) used the esterification method to reduce FFA content of WCO
by reacting FFA of WCO with methanol in the presence of sulfuric acid as catalyst.
Acid catalysts are actually insensitive to FFAs, and they also give better results in
converting FFAs of WCO into FAME.
As shown in Figure 7-3, the usual procedure for esterification is as follows:
Methanol and H2SO4 (acid catalysts) are properly mixed in the mixer before
transfer to the esterification reactor. The temperature of the mixing tank is usually
maintained at 60 °C (Morais et al. 2010). The catalyst mixture and preheated
feedstock are then loaded into the esterification reactor. Temperature of the
esterification reactor is usually maintained between 80° and 90 °C and the pressure
at 1 atm (101325 Pa). Products from the esterification reactor are cooled to 45 °C,
and the catalysts are removed or neutralized before being put into the settling tank
to remove the methanol and water mixture. The methanol–water mixture is
removed from the top of the settling tank, and then taken to the distillation
column for separation of methanol from the methanol–water mixture, and the
distilled methanol is reused. The bottom product (biodiesel) of the separating
vessel is taken for the transesterification reaction.
Alkali catalyst and methanol are properly mixed in the mixer, and products
obtained from the pretreatment process are transferred into the transesterification
reaction column along with alkali catalyst and methanol mixture. The temperature
of the reactor is usually maintained at 65 °C, 1 atm (101325 Pa) pressure, and a
6:1 molar ratio of methanol to esterified oil. The products of the transesterification
reactor are then fed into a separator. In the separation tank, the biodiesel and
methanol mixture is distilled to separate the methanol and biodiesel. From the
distillation column, methanol is recycled and reused. Then biodiesel is washed
Figure 7-3. Esterification reaction.
with hot water and sent to the separator to separate water and biodiesel. Biodiesel
is then sent to a storage tank from the third separator tank. Bottom products
(methanol and glycerol) of the separator are moved to the methanol and glycerol
distillation column. Methanol is recycled from the top of the distillation column.
Bottom product (glycerol) of the distillation column is taken as a byproduct.
Figure 7-4 shows the schematic representation of this process.
Many studies have been done using this process as shown in Table 7-2. Fan
et al. (2009) produced biodiesel from WCO (canola oil) by the two-step acid and
alkali-catalyzed transesterification method using H2SO4 and KOH as catalysts. In
the first step, acid-catalyzed esterification was performed at optimum conditions
of the 40:1 molar ratio of methanol to oil, 5% (w/w) H2SO4, 55 °C reaction
temperature, and 1.5 h reaction time. At this step, FFA content was reduced to
0.41%. After esterification, alkali-catalyzed transesterification was performed at a
6:1 molar ratio of methanol to oil using 1% (w/w) KOH at 65 °C for 1 h. This study
led to 96.3% yield of biodiesel. Omar et al. (2009) also achieved an 81.3% biodiesel
yield using ferric sulfate and KOH catalysts to catalyze the reactions. In another
study, Wang et al. (2006) produced biodiesel from WCO under optimum
conditions of 95 °C reaction temperature, the 10:1 molar ratio of methanol to
oil using 2% (w/w) ferric acid for 2 h to catalyze esterification and 65 °C reaction
Figure 7-4. Transesterification reaction.
temperature, and the 6:1 methanol-to-oil ratio with 1% (w/w) KOH for 1 h to
perform transesterification. Hence, they reached 97% FAME production.
According to Patil et al. (2010), a 96% biodiesel yield was obtained when
WCO was esterified at 100 °C with a 9:1 molar ratio of methanol to oil at 2% (w/w)
ferric sulfate, for 2 h, followed by transesterification with 0.5% (w/w) KOH using a
75:1 molar ratio of methanol to oil at 100 °C for 1 h. Charoenchaitrakool and
Thienmethangkoon (2011) used this method to produce biodiesel from WCO
under optimum conditions of the 6.1:1 molar ratio of methanol:oil, 0.68% (w/w)
H2SO4, 51 °C reaction temperature and 1 h reaction time during esterification, and
the 9.1:1 methanol:oil ratio, 1% (w/w) KOH, 55 °C reaction temperature, and 1 h
reaction time for transesterification. This led to a 96.3% FAME yield.
This procedure (i.e., acid esterification of FFA as pretreatment followed by
transesterification reaction to generate FAME) has been found very efficient for
the transesterification of WCO with high FFA content. Thus, the utilization of the
acid and alkali catalysts in the first and second stages, respectively, has overcome
the problem of a slow rate of reaction with the acid catalyst and formation of soap
with an alkaline catalyst and hence increases the ester yield. However, this method
has a drawback in removing the catalyst in both stages (esterification and
160
Table 7-2. Alkali-Based Transesterification under Different Reaction Conditions.

Reaction Conditions
Temperature Alcohol used and molar Catalyst loading Reaction Yield
Catalyst (°C) ratio of alcohol to oil (percent by weight) time (h) (%) References
Ferric sulfate Acid: 95 Acid:Methanol (10:1) Acid: 2 Acid: 2 97 Wang et al. (2006)
followed by KOH Base: 65 Base:Methanol (6:1) Base: 1 Base: 1
Ferric sulfate Acid: 100 Acid:Methanol (9:1) Acid: 2 Acid: 2 96 Patil et al. (2010)
followed by KOH Base: 100 Base:Methanol (7.5:1) Base: 0.5 Base: 1
Ferric sulfate Acid: 60 Acid:Methanol (7:1) Acid: 0.4 Acid: 3 81.3 Omar et al. (2009)
followed by CaO Base: 60 Base:Methanol (7:1) Base: not specified Base: 3
Sulfuric acid Acid: 55 Acid:Methanol (40:1) Acid: 5 Acid: 1.5 96.3 Fan et al. (2009)
followed by KOH Base: 65 Base:Methanol (6:1) Base: 1 Base: 1
Sulfuric acid Acid: 51 Acid:Methanol (6.1:1) Acid: 0.68 Acid: 1 90.56 Charoenchaitrakool and
followed by KOH Base: 55 Base:Methanol (9.1:1) Base: 1 Base: 1 Thienmethangkoon
(2011)
transesterification). In the first stage, this problem can be solved by neutralizing

the acidic catalyst, with the addition of extra alkaline catalyst in the second stage.
However, the use of extra catalyst will add to the cost of the biodiesel.
7.3.2.2 Enzymatic Alcoholysis

Chemical-catalyzed transesterification of WCO has problems, such as pretreat-
ment of the feedstock, recovery of glycerol, removal of catalyst, and the energy-
intensive nature of the process, that is, high stirring speed and temperature
required for good conversions. Enzyme (such as lipase)-catalyzed reactions have
many advantages over the traditional chemical-catalyzed reactions: (1) the
generation of no byproducts; (2) easy product recovery; (3) mild reaction
conditions; and (4) catalyst recycling. Also, enzymatic reactions are insensitive
to FFA and water content in WCO. Thus, the reactions catalyzed by enzymes are
considered as green reactions. However, this method has not yet been used at the
industrial scale due to several reasons: (1) high cost of the lipases, (2) enzyme
inhibition by methanol, (3) adsorption of glycerol on lipase, and (4) long reaction
time (Ranganathan et al. 2008, Talukder et al. 2011).
Lipase enzyme is extracted from various sources, including microorganisms
(fungi and bacteria), plants, and animals. Microorganisms are the most common
source for lipase extraction due to their low production cost and the simple
modifications (Gog et al. 2012). This enzyme is used in the immobilized form so
that it can be reused in many batches. Several studies have been done in which
lipases have been immobilized onto several supports, including hydrophobic
sol–gel support (entrapment), macroporous support (adsorption), chitin (chemi-
cal binding), and silica aerogel (encapsulation) (Dizge et al. 2009). Many lipases
have been used in biodiesel production from WCO, such as Thermomyces
lanuginosus, Mucor miehei, Candida antarctica (Novozyme 435), Geotrichum
sp., Aspergillus oryzae, Penicillium expansum, Bacillus subtilis, Rhizopus oryzae,
Pseudomonas cepacia, Candida rugosa, and Pseudomonas fluorescens (Gog et al.
2012, Tongboriboon et al. 2010). However, it has been proven through several
investigations that Novozyme 435 is most efficient in the biodiesel production
due to its two most important properties: (1) high conversion percentage, and
(2) reusability. In its immobilized form, Novozyme 435 can be reused for more
than 50 reaction batches (Hama et al. 2011, Talukder et al. 2011).
The reaction catalyzed by lipase can be classified into two stages—hydrolysis
and synthesis reactions (Figure 7-5). Lipase catalyzes hydrolysis in an aqueous
system. It hydrolyzes the ester bonds in triglycerides to produce fatty acids and
glycerol. Synthesis reaction is subdivided into two reactions—esterification and
transesterification. Esterification is the reverse of the hydrolysis reaction. It is
catalyzed in the microaqueous system (i.e., where water content is very low). It
converts fatty acids back into ester. Transesterification is further categorized into
acidolysis and alcoholysis. Acidolysis is the reaction between an acid and an ester,
whereas alcoholysis occurs between an alcohol and an ester, resulting in FAME
production (Yaakob et al. 2013).
Figure 7-5. Reaction classifications of lipase activity.
Table 7-3 presents the enzymatic production of biodiesel from WCO under
various reaction conditions with various lipases. Ranganathan et al. (2008)
investigated the biodiesel production from WCO using immobilized lipase
(extracted from P. expansum) under optimum conditions of the 1:1 methanol-
to-oil molar ratio, 35 °C reaction temperature, and 7 h reaction time. The FAME
yield of 92.8% was obtained in this study. Al-Zuhair et al. (2009) used WCO to
produce biodiesel using lipase-catalyzed transesterification and got greater than a
67% yield at 45 °C with 10% (w/w) catalyst loading, the 3:1 ethanol-to-oil molar
ratio for 8 h. In another study done by Chakraborty et al. (2010), lipase extracted
from C. antarctica was used to catalyze the reaction at a 3:1 molar ratio of
propanol to oil at 45 °C for 0.833 h, and a 95% biodiesel yield was obtained.
Talukder et al. (2011) produced biodiesel from WCO using enzyme-catalyzed
transesterification (lipase extraction from R. oryzae) under optimum conditions of
a 4:1 methanol-to-oil molar ratio, 30% (w/w) catalyst loading, 40 °C reaction
temperature, and 30 h reaction time. This investigation led to a 90% biodiesel
yield. According to Gog et al. (2012), a 90% FAME yield was achieved from WCO
feedstock when 3% (w/w) catalyst loading (from B. subtilis) was used with a
1:1 molar ratio of methanol to oil at 40 °C for 72 h. However, this method has not
been used at the industrial scale for two major reasons: it is time consuming and
comes at high cost.
7.3.2.3 Factors Affecting Acid/Alkali-Catalyzed Transesterification

Major factors affect transesterification, including reaction temperature, reaction
time, the methanol-to-oil molar ratio, type and amount of catalyst, and FFA and
moisture content. These are briefly discussed in the following:
1. Reaction temperature: It is the most important factor that affects biodiesel
yield. At high reaction temperature, the reaction rate is increased while the
Table 7-3. Enzymatic Biodiesel Production from WCO under Various Reaction Conditions with Various Lipases.
Reaction Conditions
Reaction
Catalyst loading Reaction temperature Yield
Lipase origin Alcohol to oil ratio (percent by weight) time (h) (°C) (%) References
Penicillium expansum Methanol (1:1) Not specified 7 35 92.8 Ranganathan et al.

(2008)
Rhizopus oryzae Methanol (4:1) 30 30 40 90 Talukder et al.
(2011)
Bacillus subtilis Methanol (1:1) 3 72 40 90 Gog et al. (2012)
Candida antarctica Propanol (3:1) Not specified 0.833 45 95 Chakraborty et al.
(2010)
Candida rugosa/Penicillium Ethanol (3:1) 10 8 45 >67 Al-Zuhair et al.
fluorescens (mixed) (2009)
Candida antarctica Methanol (25:1) 25 4 50 89.1 Omar and Amin
(2011)
BIODIESEL FROM WASTE COOKING OIL
163
reaction time is shortened. This occurs due to reduction in viscosity of oil at

high reaction temperature. However, beyond the optimum level, it can also
decrease the biodiesel yield because saponification reaction accelerates at
higher reaction temperature (Mathiyazhagan and Ganapathi 2011) and also
causes vaporization of methanol that gives a decreased yield (Anitha and

Dawn 2010). As reported in various studies, the optimum reaction temper-
ature range varies from 50 °C to 60 °C, that is, below the boiling point of
alcohol so that alcohol evaporation can be prevented (Mathiyazhagan and
Ganapathi 2011).
2. Reaction time: It has been observed that when the reaction time is increased,
the fatty acid conversion to their corresponding esters is also increased.
At the beginning, the reaction rate is slow because of mixing and dispersion
of oil and alcohol. After this step, the rate of reaction becomes very fast,
but the maximum conversion is achieved in less than 90 min. Further
increase in time can lead to yield reduction because the transesterification
reaction is reversible and may result in soap formation and loss of esters
(Mathiyazhagan and Ganapathi 2011).
3. Methanol-to-oil molar ratio: It is the most important parameter that affects
biodiesel yield. Stoichiometrically, a 3:1 molar ratio of methanol to oil is used
to produce 3 moles of FAME and 1 mole of glycerol. It may vary from 5.6 to
7.8:1. FAME yield can be increased by applying excess methanol to shift
equilibrium toward the product side (Anitha and Dawn 2010).
4. Type and amount of catalyst: Catalyst concentration also affects FAME
yield. NaOH and KOH are the most commonly used catalysts (Mathiyaz-
hagan and Ganapathi 2011). The type and concentration of catalyst depend
on the quality of feedstock and the method of transesterification. For pure
feedstock, any catalyst can be used, but for a feedstock with high FFA and
moisture content, acid catalysts work better, because the alkali catalysts can
lead to saponification reaction (Omar and Amin 2011a, 2011b, Tan et al.
2011). FAME yield increases with increased catalyst concentration, but it is
not economical due to the high cost of the catalyst. Thus, similar to the
methanol-to-oil molar ratio, it is also necessary to optimize the amount of
catalyst for the transesterification process (Jagadale and Jugulkar 2012).
5. FFA and moisture content: The FFA and moisture content in the feedstock
play a vital role in transesterification. It has been reported that the acid value
of the feedstock must be less than 1; otherwise, a higher concentration of
catalyst will be required to neutralize FFAs. Feedstock must also be kept
anhydrous because moisture can lead to soap formation, which may
consume catalyst and thereby reduce the catalyst activity. This saponifica-
tion may increase the viscosity of the reaction mixture and sometimes may
cause gel formation that makes it difficult to separate glycerol from ester
(Mathiyazhagan and Ganapathi 2011). Thus, FFA and moisture content in
WCO must be kept to a minimum for transesterification because these
parameters can deactivate catalysts and may cause problems in the separa-
tion of pure products (Jagadale and Jugulkar 2012). If WCO feedstock has a
high moisture content, preheating of WCO in an oven to above 100 °C
would reduce the water content. Alternatively, vacuum distillation can also
be used (Javidialesaadi and Raeissi 2013).
7.3.3 Purification of Biodiesel

As the transesterification reaction is completed, biodiesel and glycerol are
separated by phase separation. Among the two phases, the glycerol phase is
much denser and thus, makes the biodiesel float. Thus, a settling vessel is used to
separate the two phases by gravity. Glycerol is collected at the bottom and the
crude biodiesel is drawn off from the top of the settling tank. This separation can
also be done by using a centrifuge (Garlapati et al. 2013). The crude biodiesel is
contaminated with free glycerol, water, unreacted alcohol, residual catalyst, and
soaps. To purify biodiesel, the following approaches have been adopted (for
detailed discussion, see Chapters 18 and 19):
1. Water washing: Water washing is most effective in the removal of glycerol
and alcohol, because both contaminants are highly soluble in water. Any
residual sodium salts and soaps can also be removed by this approach.
Distilled warm water or softened water is the primary requirement for this
method (Berrios et al. 2011, Hingu et al. 2010, Lam and Lee 2011). Warm
water prevents the precipitation in FAME and also retards emulsification by
gentle washing action. Softened water helps in the elimination of magnesium
and calcium contamination and also neutralizes the remaining base catalysts
(Banerjee and Chakraborty 2009). With repeated washing (4 to 5 times), the
water phase becomes completely clear, that is, free of contaminants. Then
the FAMEs can be separated from water by phase separation (or using
separating funnel in the laboratory) (Thirumarimurugan et al. 2012) or a
centrifuge (Berrios et al. 2011). Then the remaining water in the FAME can
be removed by passing it over the heated Na2SO4 overnight and then filtered
to obtain purified FAME (Berrios et al. 2011). However, there are many
drawbacks to this method, including (1) increase in cost and production
time, (2) pollution in the liquid effluent, (3) loss of product, and
(4) formation of emulsion in the case of WCO or acidic feedstock (Berrios
et al. 2011).
2. Dry washing: Researchers have used dry washing with a magnesium silicate
powder or an ion exchange resin to eliminate the impurities (Hingu et al.
2010). These two methods are effective in removing soaps and reducing the
free glycerol level. Both of these dry washing methods are advantageous over
the water washing method because they do not use water for FAME
purification. Although this approach is effective in methanol removal, it
does not meet the ASTM standard limits (Hingu et al. 2010, Predojević
2008).
3. Membrane extraction: Several studies have been done in which hollow fiber
membranes, such as polysulfone, have been used to extract contaminants,
such as soap and water. This membrane is filled with distilled water and
immersed into a reactor at 20 °C temperature. The crude biodiesel is then
pumped into the membrane at a flow rate of 0.5 mL/min at an operating

pressure of 0.1 MPa. After this step, FAME is passed over the heated Na2SO4
and then filtered to remove the remaining water (He et al. 2006). Thus, this
approach eliminates the emulsification and product loss during the refining
process. This approach leads to 90% purity of FAME and meets all ASTM
standards. Hence, it is a very effective method for purifying FAME.
4. Molecular distillation: This approach has also been used to purify FAME
from crude biodiesel so that the washing step can be replaced to reduce the
effluent of the process after transesterification (Azcan and Yilmaz 2013,
Wang et al. 2010). This method enriches the biodiesel in methyl ester
content by removing impurities such as monoglycerides, diglycerides, and
triglycerides at optimum evaporation temperature (160° to 220 °C) and feed
flow rate (Azcan and Yilmaz 2013, Wang et al. 2010). When WCO with a
high acid value is used as a reactant for biodiesel production, molecular
distillation is introduced for the final purification of FAME to remove all
impurities and unpleasant odor (Enweremadu and Mbarawa 2009). Wang
et al. (2006) has used distillation to purify FAME produced from WCO by
using ferric sulfate as a catalyst in transesterification. They performed
distillation in a 500 mL one-necked round bottom flask, including a
temperature controller, a receiver flask that is connected to a vacuum gauge,
and a condenser. The vacuum was provided by the pump connected to the
condenser. Then the crude biodiesel was transferred to the flask, and the
vacuum was set to 45 mm Hg. Using this method, 93% pure FAME was
obtained. Zhang et al. (2003a) has designed a process to distillate FAME with
different reflux ratios and theoretical stages. This system was operated under
vacuum so that the temperature can be kept low enough to prevent FAME
degradation (Figure 7-6). This approach is advantageous for several reasons:
(1) it needs low distillation temperature, which ensures no polymerization
and decomposition of FAME, and thereby, leads to high yield of final
product; and (2) no water washing is required, and thus it is more eco-
friendly. Conversely, this approach is expensive over the aforementioned
methods (Azcan and Yilmaz 2013).
7.4 COMPARISON BETWEEN WASTE COOKING OIL AND VIRGIN OIL
Biodiesel is the mixture of methyl esters with the long-chain fatty acids and made
up from vegetable oil, animal fats, or even WCO. It has been reported that large-
scale production of biodiesel from edible oil can lead to a global imbalance in the
food supply, thereby increasing the prices of edible oils. Because 95% or more of
Figure 7-6. Molecular distillation unit.
the current biodiesel is being made from the edible oils, many problems have
arisen accordingly. Thus, the accessible utilization of WCO will be a promising
way to decrease the use of edible oils (Stacy et al. 2014). WCO is relatively cheap
and it is considered to be a potential feedstock for the biodiesel production.
WCO contains high contents of FFAs and water, which leads to a more
complex procedure and worse fuel properties compared with biodiesel produced
from VVO. Alkali catalysts are preferred in conventional biodiesel industries
because their transesterification efficiency is very high. When the feedstock
with high FFA content is used for biodiesel production, the alkali catalyst
would be consumed by the FFAs that leads to saponification, and thereby, results
in the low biodiesel yield (Omar and Amin 2011a, 2011b). Hence, WCO is
converted to biodiesel by a two-step process, that is, esterification followed by
transesterification.
WCO and VVO have been compared on the basis of various parameters:
(1) their initial properties (Table 7-1), and (2) fuel properties of the biodiesel
produced (Tables 7-4 and 7-5). The feedstock properties about WCO and VVO
are given in Table 7-1. Acid value of WCO is 2.66 mg KOH/g which corresponds
to 1.33% by weight FFA content. Acid value of WCO is significantly higher than
that of VVO (0.25 mg KOH/g). It is usually caused during frying and cooking
processes at higher temperature, during which some triglycerides undergo
Table 7-4. FAME Compositions of WCO and VVO Biodiesels.
WCO biodiesel VVO biodiesel

FAME composition Formula (w/w%) (w/w%)
Methyl myristate C14:0 0.6 0.6

Methyl palmitate C16:0 27.3 26.2
Methyl stearate C18:0 4.4 4.2
Methyl oleate C18:1 37.1 35.9
Methyl linoleate C18:2 27.0 28.9
Methyl linolenate C18:3 2.9 3.6
Methyl arachidate C20:0 0.4 0.4
Methyl eicosenoate C20:1 0.2 0.2
Saturated FAMEs — 32.7 31.4
Unsaturated FAMEs — 67.2 68.6
Table 7-5. Fuel Properties of WCO and VVO Biodiesels and Compared with ASTM
D6751.
WCO VVO
Property Unit biodiesel biodiesel ASTM D6751
Acid value mg KOH/g 0.18 0.10 0.5 maximum

Cold filter plugging °C 8 8 —
point
Density kg/m 881.1 879.4 —
Ester content percent by weight 98.8 99.3 —
Iodine value g I2 100/g 90 93 —
Kinematic viscosity mm2/s 4.4 4.3 1.9–6.0
Oxidation stability, hours 0.2 2.6 3.0 minimum
110 °C
oxidation to generate FFAs, thereby increasing acid value. The kinematic viscosity
(KV) of WCO is 39.5 mm2/s, higher than VVO’s KV of 35.6 mm2/s. it has been
reported in literature that the increase in the KV occurs due to polymerization,
resulting in the formation of compounds with a higher molecular weight (Canakci
2007). In contrast, iodine value (IV) of WCO is 89 g I2/100 g, smaller than VVO’s
IV of 93 g I2/100 g. The IV is directly related to the number of double bonds in the
fatty acids (Encinar et al. 2007). Thus, with the oxidation of unsaturated fatty
acids, more FFAs are generated and hence, the IV of WCO is decreased.
Table 7-4 gives FAME composition of WCO and VVO biodiesel. Primary
FAMEs of the biodiesel are methyl oleate, methyl palmitate, and methyl linoleate.
WCO biodiesel shows 37% (w/w) methyl oleate, 27% (w/w) methyl palmitate, and
27% (w/w) methyl linoleate, with saturated and unsaturated FAMEs of 32.7% and
67% (w/w), respectively. Conversely, VVO biodiesel shows a higher content of
methyl oleate (35.9% by weight) and methyl linoleate (29% by weight) with
polyunsaturated C–C double bonds. The fatty acids with these bonds show poor
oxidation stability and hence, are easily decomposed during use (Gui et al. 2008).
Table 7-5 shows fuel properties of WCO and VVO biodiesel in comparison to
ASTM standard. Both WCO and VVO biodiesels have the same cold filter
plugging point (CFPP) value of 8 °C, which is in excess according to the standard.
This may be due to the higher content of saturated FAME. Therefore, it is
necessary to add antifreeze to improve low temperature properties of both
biodiesels. In addition, WCO and VVO biodiesels show moderate density,
KV, IV, and high ester content, which completely satisfy the biodiesel standard
(Gui et al. 2008).
7.5 COST ANALYSIS OF BIODIESEL FROM WASTE COOKING OIL
To date, several studies have been done for biodiesel production at the minimum
total production cost to compete with conventional diesel (Tan et al. 2011). In all
these studies, it has been observed that the feedstock cost contributes to more than
70% of the total cost. Hence, the researchers started searching for an alternative to
VVO and turned to WCO, which has proven to be a promising alternative
(Banerjee et al. 2014). Table 7-6 shows the average cost of some selected oils used
for biodiesel production (Banerjee et al. 2014).
Several feasibility studies on the biodiesel production using WCO as feedstock
have been carried out. According to Van Kasteren and Nisworo (2007), cost of
feedstock (71% to 80%) and capital cost (15% to 16%) are the major contributors
to the high biodiesel cost. However, glycerol production as a byproduct can reduce
the biodiesel cost by 22% to 36% of the total cost because it can be used in many
applications, such as cosmetics, pharmaceuticals, lubricants, and food industries
(Pagliaro et al. 2007). In another study by Zhang et al. (2003b), major economic
criteria in biodiesel production were the capital cost, the manufacturing cost, and
Table 7-6. Average Cost of Selected Oils Used for Biodiesel Production.
Source Oil Cost (US$/ton)
VVO Soybean oil 771

VVO Crude palm oil 703
VVO Yellow grease 412
VVO Rapeseed oil 824
Used oil WCO 224
the biodiesel break-even price. Table 7-7 summarizes the economic criteria for the
alkali-based transesterification process (Van Kasteren and Nisworo 2007).
With the current flow in crude oil price, the economics of biodiesel produc-
tion warrants a new look in the world market. According to the US Energy
Information Administration, the current market price of diesel is USD 2.569/gal.

in the United States, and USD 2.953/gal. in California. Conversely, the biodiesel
(Blend 20) price is USD 2.46/gal. as reported by the US Department of Energy in
its October 2016 alternative fuel price report. Thus, biodiesel may become a more
feasible alternate energy source.
Table 7-7 Itemized prices (in US$/) of Biodiesel Production from WCO Based on
Different Economic Criteria.
Plant Capacity (tones/year)

Criteria 8,000 80,000 125,000
Fixed capital cost 1,997,721 7,953,072 10,395,058

Working capital cost 313,729 1,513,014 1,661,348
Start-up cost 941,187 4,539,042 4,984,045
Total capital cost 3,252,638 14,005,128 17,040,452
Annual valuable cost
Raw material
WCO 1,709,129 16,784,050 26,068,993
Methanol 278,400 2,736,000 4,218,750
Total raw material cost 1,987,529 19,520,050 30,287,743
Utilities
Electricity 45,670 456,699 713,592
Cooling water 8,217 67,103 102,708
Biodiesel for reboiler 198,371 872,157 978,359
Total utilities cost 252,257 1,395,958 1,794,660
By-product credit
Glycerol 1,017,600 6,234,000 15,973,500
Fixed cost
Operating labor 1,020,000 1,020,000 1,020,000
Maintenance 371,143 371,143 485,103
Plant overhead 890,229 890,229 913,021
Taxes and insurance 39,954 159,061 207,901
Total fixed cost 2,321,326 2,440,433 2,626,024
Total operating cost 3,538,992 17,134,165 18,789,736
Capital charges 1,207,569 4,353,483 5,314,381
Required Selling Price (RSP) 4,983,890 22,562,030 25,309,323
RSP (US$/tones) 623 282 202
7.6 SUMMARY
As an effective alternative and renewable fuel to conventional diesel, biodiesel is

nontoxic, biodegradable, and does not contribute to the net carbon emission to the
atmosphere. Currently, biodiesel is being produced through the transesterification

reaction from VVO such as soybean, rapeseed, and palm oil. The high prices of
these oils in the global market have sharply increased the overall biodiesel
production cost. Thus, it is not economically viable compared with petrol-based
diesel. Furthermore, the oils are necessary commodities in the human food supply
chain and therefore its conversion to biodiesel over the long run may not be
sustainable. In contrast, WCO is a cheap and economical feedstock for biodiesel
production. Because WCO contains high FFA content, it leads to a decrease in the
overall yield. In this case, esterification is to be done before transesterification.
Apart from that, enzymatic transesterification is another possible way to produce
biodiesel from WCO because of its high stability toward FFA content in oils.
However, the enzyme should be synthesized in a cheaper way, and it should be
available for commercial use. Currently, WCO has become an economic booster
for petrol-based diesel and a supplement for the current dilemma of environ-
mental sustainability and dependence on the petroleum recourses. It is a major
cost-saving raw material.
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CHAPTER 8
Microalgae Oil Biodiesel
X. L. Zhang
J. Chen
Z. Wu
S. Yan
R. D. Tyagi
J. Li
W. Dong
R. Y. Surampalli
8.1 INTRODUCTION
Vegetable oils have been commercially used as the feedstock of biodiesel produc-
tion for years. The increasing price of the oils urges researchers to seek alter-
natives. Certain microalgae contain impressive oil content (up to around 70% dry
weight) (Schmidt et al. 2005, Han et al. 2016a, Morales-Sánchez et al. 2016).
Moreover, microalgae has a rapid growth rate compared to plants (Yu et al. 2015).
Most of microalgae could use CO2 as a carbon source to grow and synthesize
intracellular products such as proteins, lipids, and carbohydrates. Therefore,
microalgae are a potential feedstock for biodiesel production. Utilization of
microalgae for biodiesel production can produce renewable energy, and thus
mitigate the energy crisis that the society is currently facing. In addition, it would
also help with capturing greenhouse gases from the environment.
Utilization of microalgae oil for biodiesel production includes the following
steps: microalgae cultivation, harvesting, biomass drying, lipid extraction, and
lipid conversion to biodiesel. Each step is important in biodiesel production, and
added together, these steps decide the cost of the biodiesel final product.
Cultivation conditions mainly impact the microalgae biomass and lipid produc-
tivity and thus the cost (Kings et al. 2017). Harvesting is the process of recovering
microalgae from the cultivation medium. The method of harvesting directly
affects the recovery efficiency and the cost (Feng et al. 2016). Drying is used
sometimes to enhance lipid extraction efficiency because high water content in
175
biomass has a negative effect on the extraction (Song et al. 2016). Lipid extraction
right now is still used in biodiesel production from microalgae because in situ
transesterification (the process combining lipid extraction and transesterification)
is still at the research stage (Wang et al. 2016). Transesterification is the process
used to convert lipid to biodiesel by reaction with methanol in the presence of

catalysts.
This chapter reviews oleaginous microalgae that are suitable for biodiesel
production and the main lipid composition of these microalgae. The chapter
discusses the microalgae cultivation mode, factors impacting productivity of
microalgae biomass and lipid, harvesting methods, the drying process, lipid
extraction approaches, and transesterification. The chapter also addresses the
current problems and future aspects of microalgae for biodiesel production.
8.2 MICROALGAE FOR BIODIESEL PRODUCTION
There is a type of microalgae that can accumulate high lipid content and reserve it
in the cell body to provide energy when carbon is not available in the medium. For
biodiesel production, it is desired that the microalgae used have great lipid
production capacity and that the lipid accumulated in the microalgae have similar
composition as vegetable oil and animal fats that are currently used in commercial
8.2.1 Microalgae Diversity

Microalgae are widespread in marine and freshwater. They can be divided into
prokaryotic and eukaryotic according to their cell structure (Hannon et al. 2014).
Based on the pigmentation, microalgae are classified as green algae, red algae,
and diatoms (Brennan and Owende 2010). Microalgae can also be grouped as
autotrophic, heterotrophic, mixotrophic, and photoheterotrophic when consider-
ing their type of metabolism (Sharma and Singh 2017).
Utilization of autotrophic microalgae is in fact the most energy- and cost-
effective way for biodiesel production because they only depend on light and CO2
for growth, which is a photosynthesis process. This type of cultivation can be
achieved by open pond and photobioreactor systems. CO2 can be supplied
through injecting fume which is CO2-rich waste gas (Collet et al. 2014). Producing
biodiesel from autotrophic microalgae helps to reduce the pressure in the need of
feedstock oil for biodiesel production. Moreover, fume as a waste gas, otherwise
with a potential to pollute the environment, can be treated through microalgae
cultivation in the process.
Heterotrophic microalgae can grow without light support. They consume
organic compounds as energy source. The advantage of this type of microalgae for
biodiesel production is that they could accumulate high lipid content within a
short time. They are easy to control but expensive (Schmidt et al. 2005). The
cultivation normally takes place in fermenters, and thus conditions can be
MICROALGAE OIL BIODIESEL 177
manipulated easily for optimizing biomass and lipid accumulation. Because an

organic substrate such as glucose is essential for heterotrophic microalgae, the
cultivation would be costly in terms of carbon source utilization.
Mixotrophic microalgae are not like the autotrophic or the heterotrophic
which strictly consume carbon dioxide or organic carbon, respectively. They can
use both as a carbon source. Thus, it is more flexible in practice and should be a
good option of biodiesel production feedstock. When using mixotrophic micro-
algae as biodiesel production feedstock, CO2 can be provided to the system when
light is available, and then switch to supply organic carbon when light is absent.
This increases the independency of the system and would be more efficient
compared to other systems.
The photoheterotroph is a type of microalgae that depends on organic
compounds as a carbon source and requires light as an energy source. Compared
to other types of microalgae, the cultivation of photoheterotrophic microalgae
would be more costly and complex. Thus, their utilization for biodiesel production
is not favorable.
Currently, autotrophic and heterotrophic microalgae are the two most used
and applied microalgae in biodiesel production compared to others (Mata et al.
2010). In fact, mixotrophic microalgae have great potential in the field, but due to
the limited availability of mixotrophics containing lipid, their utilization has not
gained enough attention (Table 8-1).
8.2.2 Composition of Microalgae Oil

Vegetable oils and animal fats are widely utilized in biodiesel production because
the biodiesel produced from them are comparable to petro-diesel and compatible
with diesel engines. The major compositions for both vegetable oils and animal
fats are palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid
(C18:2), and linolenic acid (C18:3); they are dominant fatty acids in most of these
oils and fats (90% w/w oils or fats). To be acceptable as biodiesel production
feedstock, microalgae oil should have similar composition as vegetable oils or
animal fats. Thus, one needs to compare the contents of the fatty acids in
microalgae oil with vegetable oils and animal fats to evaluate if microalgae oil
is suitable for biodiesel production.
Many types of microalgae have been studied for producing lipid and then
converting to biodiesel. The most commonly studied microalgae are Chlorella sp.,
Cylindrotheca sp., Dunaliella sp., Nannochloropsis sp., Nitzschia sp., Schizochy-
trium sp. (Sharma and Singh 2017). Among all, Chlorella sp., Dunaliella sp., and
Nannochloropsis sp. are used most frequenctly in biodiesel production, mainly due
to their high lipid accumulation capacity and they are easy to manipulate (Park
et al. 2015, Wang et al. 2016, Sharma and Singh 2017). Thus, their oil composi-
tions are compared with vegetable oils and animal fats (Table 8-2). The fatty acid
composition and saturation degree of Chlorella sp. are similar to plant (corn,
sunflower, pumpkin, Jatrophacurcas) seed oils, whereas those of Dunaliella sp. and
Nannochloropsis sp. are similar to animal fats and palm seed oil. Therefore, it has
178
Table 8-1. Comparison of Microalgae for Biodiesel Production.

Type Requirements Advantages Disadvantages
Autotrophic Light and CO2 Cheap, sequence greenhouse Light depending, low lipid
gases, treating fume and biomass productivities
Heterotrophic Organic compounds Easy control, high lipid and Expensive
biomass productivities
Mixotrophic Light and CO2 or organic Flexible, with high potential in Limited information and
compounds feasibility research
Photoheterotrophic Light and organic High lipid and biomass Costly and complex
compounds productivity potential
Table 8-2. Microalgae Oil Compositions.

Fatty acids fractions (% w/w) Saturation
degree
Source C16:0 C18:0 C18:1 C18:2 C18:3 (% w/w) References
Corn oil 10–15 2–5 40–50 30-40 — 10–20 Gülüm and Bilgin (2015), Mardhiah et al. (2017)
Sunflower seed oil 5–8 2–5 15–20 70–80 — 2–10 Sajjadi et al. (2016)
Soybean oil 8–15 1–9 15–34 39–60 2–11 5–20 Lee et al. (2015), Nam et al. (2016)
Pumpkin seed oil 10–12 5–7 16–50 30–60 — 2–20 Potočnik et al. (2016)
Jatropha curcas 10–15 4–8 30–50 30–47 — 5–15 Wassner et al. (2016)
seed oil
Palm oil 40–45 3–5 30–40 5–10 — 40–50 Mardhiah et al. (2017)
Chicken fat 15–20 5–10 30–37 30–35 2–4 20–30 Alptekin et al. (2014), Riedel et al. (2015)
Tallow 20–28 18–24 41–48 2–4 — 35–50 Ito et al. (2012), Riedel et al. (2015)
Fish oil 20–30 4–8 20–40 2–14 1–2 20–40 Behçet et al. (2015)
Lard 20–30 5–8 40–45 15–22 — 25–40 Riedel et al. (2015)
Chlorella sp. 5–32 2–4 22–75 15–35 0–4 9–36 Thawechai et al. (2016), Wang et al. (2016)
Dunaliella sp. 32–45 2–5 5–17 13–20 10–43 35–50 BenMoussa-Dahmen et al. (2016)
Nannochloropsis sp. 30–42 1–3 25–35 6–15 16–20 35–45 Taleb et al. (2016), Thawechai et al. (2016)
MICROALGAE OIL BIODIESEL
179
been suggested that microalgae oils can be a replacement of traditional feedstock

oils (vegetable oils and animal fats).
8.2.3 Microalgae Oil Content and Productivities

Microalgae are suggested and widely studied for biodiesel production mainly due
to their high oil content and productivities. In general, plants have oil content of
2% to 55% w/w; however, certain microalgae could accumulate more than 70% oil
in dry cells (Table 8-3). It shows great advantages in using microalgae as biodiesel
production feedstock compared to plant seeds owingto the high oil contents.
Oil productivity is the oil produced in per area per time period. Compared to
crops, microalgae usually do not require arable land for growth, and they have a
much more rapid growth rate. Microalgae can be harvested within a few days, but
crops usually take months to be ripe. Microalgae show greater oil productivities
than plants. In fact, land for cultivating microalgae can be completely different
from that of plants because the cultivation could occur in marine water, a
freshwater body, or even in wastewater (Caporgno et al. 2015, Drira et al.
2016). It indicates that microalgae are a promising alternative to plants for
8.3 IMPACT FACTORS OF MICROALGAE PRODUCTION AND OIL

ACCUMULATION
Microalgae have the potential to accumulate oil in cells. The accumulation amount
of oil can be enhanced by a change in the cultivation conditions (Hannon et al.
2014). In general, the oil content in cells of microalgae will increase when
environment stress (low temperature, low nitrogen, high carbon, and so forth)
is applied to the system. When considering microalgae used for biodiesel
production, maximum lipid production in a given space and time is desired. To
obtain high oil production, microalgae should have a high oil producing capacity
and short life cycles. It is important in selecting the strain and cultivation
conditions (pH, light, temperature, nutrients, substrate, and salinity) to enlarge
the oil production.
8.3.1 Microalgae Strain

As shown in Table 8-3, different types of microalgae have a great variation in oil
content. It was reported that Schizochytrium sp. could even accumulate up to 77%
(w/w dry basis) oil in dry cells (Chisti 2007). Schizochytrium sp. are marine
microalgae and considered as a promising oil producer because of the high cell
density and high oil content. Study has revealed that Schizochytrium sp. S065
under the optimal condition with glucose as a carbon source could reach a cell
density of 42.5 g/L with lipid content of 95% w/w (Chen et al. 2015). Schizochy-
trium sp. S065 could grow in seawater and would leave almost no residual biomass
Table 8-3. Lipid Content and Productivities of Microalgae.

Average
oil content Average
(% w/w dry yield Harvesting Oil productivitiesa
Source cells) (kg/ha) time (d) (kg/ha/d) References
Corn 2.8 9,000 120 2.1 Song et al. (2015)

Sunflower seed 38 5,000 120 15.8 Zeng et al. (2016)
Soybean 20 1,800 120 3 Pannacci and Tei (2014)
Pumpkin seed 55 1,275 138 5.1 Hernández-Santos et al. (2016), Yavuz et al.
(2015)
Jatropha curcas seed 39 1,000 150 2.6 Wassner et al. (2016)
Palm 50 14,000 365 19.2 Beskow et al. (2015), Hossain et al. (2016)
Chlorella sp. 30 — — 28.5 Palabhanvi et al. (2016), Sharma et al.
(2016), Zhang et al. (2016), Sharma and
Singh (2017)
Cylindrotheca sp. 28 — — 22.1 Liu et al. (2011), Pruvost et al. (2011)
Dunaliella sp. 37 — — 30.5 Tang et al. (2011), BenMoussa-Dahmen
et al. (2016), Cho et al. (2016a, b)
Nannochloropsis sp. 45 — — 60.4 Kim et al. (2015), McKennedy et al. (2016),
Upadhyay et al. (2016)
Nitzschia sp. 45 — — 47.2 Cheng et al. (2014), Jiang et al. (2014),
Abomohra et al. (2016)
MICROALGAE OIL BIODIESEL
Schizochytrium sp 60 — — 64.3 Hong et al. (2013), Chen et al. (2015),

Panchal et al. (2016)
a
Lipid productivity can be calculated based on the yield of plant seed/microalgae and maturation period. For instance, the average corn yield is around 9 ton/ha
and the maturation period is around 120 days. Thus, the corn lipid productivity can be calculated as follows: Corn lipid productivity = corn yield × lipid content/
181
maturation period = 9,000 × 0.028/120 = 2.1 kg/ha-day (where 0.028 is the lipid content in the corn).
(95% oil w/w dry cell). Thus, production of Schizochytrium sp. S065 will leave no
worry on finding arable land and dealing with the residue. If the results are
reliable, Schizochytrium sp. S065 would completely change the current biodiesel
production situation, which heavily depends on vegetable oils, and open a new
pathway of commercially generating biodiesel. Chlorella sp. has also shown strong
ability in oil accumulation, and the two are the most studied microalgae for oil
producing. The study investigating Chlorella sp. FC2 IITG as biodiesel production
feedstock showed that the biomass concentration could be up to 90 g/L with the
lipid content of 39% w/w with glucose as a carbon source (Palabhanvi et al. 2016).
The high biomass concentration and high lipid content were achieved in the
fermentation of heterotrophic Chlorella sp. and Schizochytrium sp.
There are also other studies on evaluating the potential of Chlorella sp. in oil
production (Chen et al. 2012, Feng et al. 2016, Sharma et al. 2016). Autotrophic
Chlorella sp. cultivated in photobioreactors showed very low biomass concentra-
tion (0.5 to 5 g/L) compared to the heterotrophic ones. Many reports on using
Nannochloropsis sp. for biodiesel production have been released (Ma et al. 2014,
Zhu et al. 2014, Kim et al. 2015, McKennedy et al. 2016, Upadhyay et al. 2016).
Zhu et al. (2014) evaluated the potential of Nannochloropsis sp. for oil production
in a large scale. The cultivation was conducted in an 8,000 L open raceway
pond with flue gas as the carbon source. The final biomass concentration reached
0.35 g/L with oil content of 28% w/w.
Based on the preceding discussion, the same group of microalgae (such as
Chlorella sp.) but different strains (Chlorella sp. FC2 IITG, Chlorella vulgaris,
Chlorella variabilis) has shown significantly different ability of accumulating oil in
cells. Results indicate that heterotrophic ones have great advantages as oil
producers for biodiesel production. However, the problem is that utilization of
organic compounds in the cultivation would elevate the production cost. It is
necessary to develop the strain that can assimilate organic waste instead of costly
organic substrate while keeps its production ability.
8.3.2 Nutrient Source

As mentioned in previous sections, there are four types of microalgae including
autotrophic, heterotrophic, mixotrophic, and photoheterotrophic. Among them,
autotrophs and heterotrophs are the two most investigated ones. Autotrophic
microalgae relies on carbon dioxide, but heterotrophic microalgae depends on an
organic carbon source; both require nitrogen as nutrient for growth.
In autotrophic cultivation systems, carbon can be supplied by injecting pure
carbon dioxide. However, in open pond systems, it would be a great waste because
of the low utilization efficiency of carbon dioxide by microalgae. In photobior-
eactors, the carbon dioxide gas aerated into the system would be either trapped or
circulated inside the system so that there is no waste on the gas, and microalgae
can efficiently consume it. However, utilization of pure carbon dioxide would be
costly. Many researchers developed the system that used flue gas, which generates
from power plants, as the carbon dioxide source for microalgae cultivation (Zhu
et al. 2014). The flue gas primarily contains CO2 (10%−12% v/v), SOx (220 ppm),
and NOx (400 ppm) (Kroumov et al. 2017). Utilization of flue gas as a carbon
dioxide source for microalgae growth would reduce the cultivation cost, treat flue
gas, and sequence carbon dioxide as well.
Heterotrophic microalgae require an organic substrate as a carbon source.
Glucose and starch are the most utilized carbons in heterotrophic microalgae
cultivation, but they are expensive and would result in a high final cost in biodiesel
production from microalgae. Thus, to seek an alternative cheap carbon is
significantly necessary to lower the cost of biodiesel production from microalgae
oil. Some food and industrial wastes have been tested as the carbon source for
Chlorella sp. to produce oil (Lu et al. 2015, Venkata Mohan et al. 2015, Kumaran
et al. 2016, Zhu et al. 2017). Results showed that the biomass and oil productivities
could reach similar levels as that obtained with glucose and starch as the carbon
source. Thus, glucose and starch can be replaced by organic wastes to lower the
biodiesel production cost.
Nitrogen and phosphorus are essential nutrients of microalgae growth
because they play significant roles in cell synthesis. Fertilizers such as urea and
KH2PO3 can be used as nitrogen and phosphorus sources. Similar to using glucose
and starch, utilization of fertilizers would be a costly choice. To reduce microalgae
oil cultivation cost, wastewater rich in nitrogen and phosphorus have been
involved in microalgae cultivation (Caporgno et al. 2015, Drira et al. 2016, Ling
et al. 2016). Studies showed that many microalgae can efficiently extract and utilize
nitrogen and phosphorus from wastewater. These studies provided a cost-effective
way for microalgae oil production.
8.3.3 Cultivation Conditions

Microalgae cultivation conditions mainly include light, temperature, salinity,
carbon source, and nutrient stress (Hui et al. 2016, Singh et al. 2016). The
conditions have great impacts on biomass and lipid accumulation. Detailed
information is described in the subsequent sections.
8.3.3.1 Light
The light impact is not applicable to heterotrophic microalgae because they get
energy from consuming organic carbons. For autotrophic microalgae, light is
significantly important because it determines the photosynthesis process. Major
factors—namely, light intensity and photoperiod, could affect the lipid accumu-
lation because they could alter the lipid biosynthesis pathway (Wahidin et al.
2013). Researchers studied the lipid accumulation variation of Nannochloropsis sp.
under light intensity of 50, 100, and 200 μmol/m2-s and photoperiod of 24, 18, and
12 h/day. It was found that the maximum cell density (6.5 × 107 cell/mL) was
observed at light intensity of 100 μmol/m2-s, which almost doubled the concen-
tration of that under 50 and 200 μmol/m2-s. It indicates that 100 μmol/m2-s was
suitable for cell division. To enhance lipid production, a high cell density is
required because the system provides more cell bodies accumulating lipid than
that with low cell density. The study also displayed that the highest lipid content
(31.3% w/w) occurred in the cultivation with a photoperiod cycle of 18 h light

and 6 h dark. The results suggested that light plays an important role in lipid
accumulation.
It is known that sunshine duration varies according to the region and season.
For practical production of microalgae, it is not possible to maintain a constant

light duration and intensity if the system only depends on sunlight. Therefore,
artificial light such as applying a light-emitting diode (LED) was used in micro-
algae production (Ra et al. 2016, Tan et al. 2016). LED can provide the required
photoperiod length, light intensity, and wavelength. It was reported that a high
biomass density of strain Nannochloropsis oculata was achieved at a blue-light
wavelength of 465 nm and red-light wavelength of 660 nm with light intensity of
70 μmol/m2-s with a light cycle of 12 h light and 12 h dark; however, the maximum
lipid accumulation (56% w/w) was attained at a green-light wavelength of 520 nm.
LED was also used as a backup light source in microalgae cultivation (Tan et al.
2016). In the lipid accumulation system, photobioreactors get light during the
daytime and LED was switched on when sunlight was not available. It would be
more energy- and cost-effective if sunlight was used in the system.
Using dyes and paints in the cultivation is another strategy to enhance lipid
accumulation in terms of alteration in light. Adding dyes and paints in the
cultivation medium would create different adsorption on light wavelength
and thus provoke lipid accumulation. The study using rhodamine 101 and 9,
10-diphenylanthracene dye in Chlorella vulgaris cultivation has doubled the lipid
productivity (Seo et al. 2015). The same research group has also evaluated the
impacts of using yellow, red, blue, and green florescent paints on lipid accumula-
tion of Chlorella vulgaris. The results showed that the highest lipid content
(30% w/w) was obtained in blue paint cultivation (Seo et al. 2014).
Light influence on photoautotrophic microalgae growth is expected, but its
impact on lipid accumulation could stem from the metabolism shifting due to the
light stress from one wavelength to the other. Each strain of microalgae has a
specific demand on light intensity, photoperiod, and light wavelength to obtain
maximum lipid accumulation capacity. It implies that the selection of the strain
would finally determine the cultivation light condition.
8.3.3.2 Temperature
Temperature effect on lipid accumulation of microalgae is mainly due to its
stimulation on lipid biosynthetic metabolism. Many researchers have investigated
the effect of temperature on lipid accumulation of microalgae (Sibi et al. 2016, Tan
et al. 2016). In general, the normal growth temperature for microalgae is in a wide
range (0° to 40 °C). However, each strain has its suitable temperature for growth.
When the cultivation environment temperature is much lower or higher than the
suitable temperature, stress is created in the system (Renaud et al. 1995).
Environmental stress could upset the microalgae and thus send the signal to the
cell body to modify its synthesis of lipid.
It was revealed that Nannochloropsis salina normally lived in the temperature
from 21 °C to 26 °C. When it was exposed to temperature of 17 °C, lipid
accumulated was largely improved as the lipid content was increased from 43%
(21 °C) and 56% (26 °C) to 70% w/w (17 °C). A similar trend was been reported by
Converti et al. (2009). Decreasing the cultivation temperature of Chlorella vulgaris
from 30 °C (the suitable temperature) to 25 °C would increase the lipid content
from 5.9% to 14.7% w/w (Converti et al. 2009). For Nannochloropsis oculata
(suitable temperature is 20 °C), increasing cultivation temperature from 20 °C to
25 °C would increase the lipid content by onefold (from 7.9% to 14.9%) (Converti
et al. 2009). The major trend is that unfavorable temperature for cell growth
normally tends to result in lipid accumulation, but temperature effect on lipid
accumulation is in fact completely strain specific.
8.3.3.3 Salinity
Most microalgae are isolated from oceans; thus, they could tolerant a certain range
of salinity. Microalgae have the ability to produce certain metabolites (xyloglucan
endotransglucosylase/hydrolase; expansion proteins) that alter cell wall structure
to adapt the osmotic effect of a change of surroundings (Le Gall et al. 2015). In
addition, the change leads to stress on cells and thus stimulates lipid accumulation
in microalgae. Some researchers mentioned that the salinity stress induces reactive
oxygen species generation, which could be a cause of lipid biosynthesis (Chokshi
et al. 2015). Many reports have proved that salinity stress had great effects on
biomass and lipid accumulation (Pancha et al. 2015, Pérez et al. 2016, Sibi et al.
2016).
It has been reported that the lipid content of Dunaliella tertiolecta increased
to 70% w/w from 60% w/w when salinity increased to 1.0 M from 0.5 M NaCl,
respectively (Takagi et al. 2006). The study has also shown that too much high
salinity would inhibit cell growth. It implies that the two-stage salinity cultivation
(first with low and then with high salinity) can be performed in microalgae
cultivation to enhance lipid accumulation. In the first stage, low salinity enhances
cell division to obtain maximum cells; then in the second stage, the salinity can be
increased to press the cells to accumulate high lipid content. However, a different
trend has been observed on lipid accumulation enhancement due to salinity stress.
The lipid content of Scenedesmus sp. CCNM 1077 was 45% w/w with 0 Mm NaCl,
but it decreased to 24% w/w with 400 mM NaCl. The big reduction was mainly
due to the strain type because Scenedesmus sp. CCNM 1077 was freshwater
microalgae. Therefore, the salinity influence on lipid accumulation of microalgae
is strain-dependent and should be evaluated before the scale-up of lipid produc-
tion from microalgae in practice.
8.3.3.4 Carbon Source

Carbon is significantly important in microalgae growth. There is about 50%
carbon w/w in microalgae dry cell weight, which suggests that microalgae growth
demands large amounts of carbon. Thus, carbon supply has a great impact on
microalgae production cost. It was predicted that the carbon cost takes up to 27%
of the total cost of microalgae production (Li et al. 2013). The carbon source
required is different for autotrophic and heterotrophic microalgae.
Carbon dioxide is essential in the metabolism of autotrophic microalgae. It is

taken up and converted to carbohydrates and lipids in microalgae cells. Carbon
dioxide can be provided by sparging pure CO2 or flue gas into the cultivation
system. The CO2 fixation efficiency is dependent on the purity of the CO2,
temperature of the system, microalgae age, and the cultivation mode (open or
closed). Normally, microalgae could assimilate pure CO2 at a higher rate than that
with CO2 in mixed gas. CO2 solubility decreases with increasing temperature (Guo
et al. 2014, Ou et al. 2015). Thus, the CO2 utilization efficiency by microalgae
could be low in high temperature. However, microalgae activity is different at
different temperatures. It is hard to determine the consumption efficiency of CO2
based on temperature. Microalgae in lag phase normally have low efficiency taking
in CO2 compared to exponential phase and stationary phase. In a closed
cultivation system, CO2 has a longer retention time in the system than that in
the open system, and hence its utilization efficiency could be enhanced. To
improve the gas fixation efficiency, Zhang et al. (2016) developed a spraying
absorption tower to supply CO2 to the open pond system and observed that the
fixation efficiency increased from 11.4% when using the sparging method to 50%
when using the new technology. The CO2 fixation efficiency greatly impacts the
microalgae production. Thus, special attention should be given to the design of the
process of microalgae production to lower the cost. However, using pure CO2 for
microalgae production is an expensive option and should be well evaluated before
application.
Organic carbons such as glucose, glycerol, and starch, among others, are
required in heterotrophic microalgae cultivation. In fact, these carbon sources are
unaffordable in microalgae cultivation in practice. Cheap and abundant organic
wastes currently have been introduced in microalgae cultivation (Yee 2015, Zheng
et al. 2016, Yun et al. 2016, Zhu et al. 2017). The results indicate that microalgae
can efficiently assimilate these carbons to grow (Yee 2015). The finding is
important because it will lower the cultivation cost of heterotrophic microalgae.
High carbon concentration has positive impacts on lipid accumulation as carbon
is demanded in both biomass and lipid production. In the beginning of the
exponential phase, carbon is used to synthesize compounds for new cells. It is used
to accumulate lipid in cells when the microalgae are in stationary phase. A high
carbon-to-nitrogen ratio is normally required to enhance lipid accumulation in
cultivation. However, a carbon concentration too high should be prevented
because it may inhibit cell growth (Bilanovic et al. 2016, Che et al. 2016).
8.3.3.5 Nutrients
Nitrogen, phosphorus, magnesium, iron, sulfur, and silicon are considered the
nutrients of microalgae. Nutrients play irreplaceable roles in microalgae growth
because they participate in the formation of cell compounds (such as proteins) and
cell life activities (such as photosynthesis, respiration, and intracellular transpor-
tation). The nutrients have also great impacts on lipid production. The most
studied nutrient affecting lipid accumulation is nitrogen. A medium with limited
nitrogen but sufficient carbon usually induces high lipid content in microalgae
cells (Converti et al. 2009, Abomohra et al. 2016, Chiranjeevi and Mohan 2016).
Nitrogen depletion would reduce or cease cell divisions and then trigger lipid or
starch biosynthesis (Morales-Sánchez et al. 2016, Sibi et al. 2016). It was reported
that the lipid content of Chlorella sp. was largely increased from 32.6% w/w to
66.1% w/w as the urea concentration decreased from 0.2 to 0.025 g/L, respectively
(Hsieh and Wu 2009). Many others revealed a similar trend that nitrogen
limitation leads to high lipid accumulation (Xu et al. 2001, Li et al. 2008). Nitrate,
ammonia salt, and urea are the most utilized nitrogen sources in microalgae
cultivation. The nitrogen has to be in amino acid form before being taken up by
microalgae; hence, whichever is close to the form would be the most efficient one.
It was also proven that microalgae utilize ammonia nitrogen more efficiently than
using others (Xu et al. 2001).
Phosphorus starvation seems to have no influence on lipid content, but has a
great impact on total lipid composition (Khozin-Goldberg and Cohen 2006).
Phosphorus is mainly used to synthesize phospholipids of microalgae. Hence, the
change of phosphorus concentration in the medium would mainly alter the fatty
acid composition of microalgae lipid. Studies showed that phosphorus limitation
caused significant reduction of phospholipids (8.3% w/w total lipid with 175 μM
K2HPO4 to 1.4% w/w total lipid with 175 μM K2HPO4) in Monodus subterraneus
(Khozin-Goldberg and Cohen 2006). Moreover, the fraction of triacylglycerols
(TAGs), the major compounds of plant seed oils, in total lipid was increased when
phosphorus concentration decreased.
Iron effect has also been widely investigated (Liu et al. 2008, Yeesang and
Cheirsilp 2011). Liu et al. (2008) performed experiments to evaluate FeCl3 effects
on lipid accumulation of Chlorella vulgaris. It was observed that high iron
concentration (1.2 × 10−5 mol/L FeCl3) had increased lipid content up to
sevenfold of that obtained from an iron-free medium (Liu et al. 2008). Similar
information has been released in other studies (Yeesang and Cheirsilp 2011).
Cell density increase was seen when iron concentration increased in the
medium (Liu et al. 2008). It was predicted that iron could prolong cell
exponential phase and thus increase cell density. Iron effect on lipid accumula-
tion of microalgae was mainly attributed to the alternation of metabolic pathway
of lipid biosynthesis.
High silicon concentration indeed increased the microalgae growth rate, but
the lipid content reduced (Jiang et al. 2014). However, the lipid content was not
impacted by silicate concentration variations (Lynn et al. 2000). Hence, the
influence of Si on lipid accumulation of microalgae could be strain sensitive,
and further study should be performed to achieve better understanding.
Nutrients effect the cell division, biomass growth, and cell compound
formation. Nitrogen limitation and the presence of iron in the medium seemed
to have more solid influence on lipid accumulation than other nutrients such as
phosphorus, iron, and silicon. The condition (nitrogen-deficient and iron-rich)
should be created when the target of microalgae cultivation is to produce
lipid.
8.3.3.6 Others
Lipid production enhancement is rather important in biodiesel production from
microalgae because it directly impacts the final cost. To achieve biodiesel
production from microalgae in practice, the current study examines how to
enhance lipid production from microalgae. Apart from light, salinity, temperature,
carbon source, and nutrients, others such as pH and ultrasonic have also been
tested to enhance lipid accumulation (Han et al. 2016b). It was shown that pH had
no significant effect on lipid accumulation (Pérez et al. 2016). To evaluate the
ultrasonication effect on lipid accumulation, microalgae Anabaena variabilis after
cultivation for 11 days was subjected to ultrasonication for 10 min at a power of
200, 350, and 500 W. The highest lipid content (37.8% w/w) was obtained after
200 W of treatment. Further evaluation on the time (5, 10, 20, and 40 min) effect
with power constant (200 W) showed that the best lipid enhancement occurred at
5 min of treatment. A study pointed out that phenol addition to the cultivation
system could enhance lipid accumulation (Cho et al. 2016a, b). The cells of
Dunaliella salina exposed to phenol (150 mg/L) for a short period time and then
recultivated in the nonphenol condition showed great lipid yield enhancement
compared with that obtained without phenol exposure (from 100 mg/L without
phenol exposure to 126 mg/L with phenol exposure).
As mentioned previously, environmental stress could induce lipid accumula-
tion on microalgae. In fact, it is attributed to the formation of oxidative stress in
the microalgae growth condition (Chokshi et al. 2015, Pancha et al. 2015). Studies
have stated that changing the cultivation conditions of microalgae would first lead
to the generation of reactive oxygen species that trigger the lipid accumulation.
Many factors influence lipid accumulation, and some of them are not
practical to apply in the real plant of microalgae cultivation for biodiesel
production. For example, it is neither cost-effective nor environmentally accept-
able to use dyes and paints on large-scale microalgae cultivation. However,
improving lipid content of microalgae by limiting nitrogen concentration in the
system is easy to manipulate; thus, this strategy is more feasible to apply in practice
in biodiesel production from microalgae.
8.4 MICROALGAE CULTIVATION SYSTEMS
Microalgae have shown great potential to accumulate high lipid (up to 70% w/w);
however, biodiesel production from microalgae is very rare in the world so far.
The major obstacle is the high cost which is mainly attributed to substrate
utilization. Autotrophic microalgae can grow and synthesize lipid in cells without
the need for organic carbons. Carbon dioxide usually is free, abundant, and largely
available in the planet. Hence, the cost of lipid production would be lowered. Thus,
autotrophic microalgae are the most promising ones for biodiesel production
compared with other types. Open ponds, photobioreactors, and hybrid cultivation
are the most practiced systems for autotrophic microalgae production (Table 8-4).
Table 8-4. Microalgae Cultivation Mode.
Cultivation mode Advantages Disadvantages

Open raceway Economical; easy to Low cell density yield;

ponds construct; easy to low biomass and lipid
operate; low energy yield; difficult to
demand during recover biomass,
cultivation; feasible to control temperature,
scale up evaporation, and
lights; susceptible to
weather; easily
contaminated
Closed High cell yield; high Expensive; difficult to
photobioreactor biomass yield; scale up; high energy
efficient; low risk of consumption
contamination; saves
space; easy to harvest
Hybrid system Easy to enhance lipid Difficult to recover
accumulation; biomass; requires a lot
efficient; low risk of of space; susceptible to
contamination; saves weather
energy; more
economical than closed
photobioreactor.
8.4.1 Open Pond Cultivation System

Open pond cultivation is a well-known simple and cost-effective system for
microalgae production. The system is usually of a raceway type. The aeration and
mixing are normally accomplished by paddle wheels. The inclined paddle wheels
of 15 degrees performed better than the traditional paddle wheel in terms of
mixing and aeration (Zeng et al. 2015). Sloping baffles and deflectors have also
been applied in raceway ponds to enhance mixing and aeration (Huang et al.
2015). Apart from the paddle wheel–driven system, the airlift-driven pond has
been developed to enhance aeration, mixing, and illumination (Richmond and
Qiang 1997, Ketheesan and Nirmalakhandan 2011). The airlift-driven system was
observed to save energy up to 80% (Ketheesan and Nirmalakhandan 2011).
Researchers have also designed airlift-driven sloping raceway ponds to enhance
mixing and light penetration and save energy (Huang et al. 2016). The study showed
that the biomass concentration (0.89 g/L in airlift-driven sloping raceway and
0.70 g/L in paddle wheel–driven raceway) as well as productivity (10.52 g/m2-day in
airlift-driven sloping raceway and 7.66 g/m2-day in paddle wheel–driven raceway)
were increased about 27% and 37%, respectively, compared to the control (paddle
wheel–driven raceway). Flue gas is the most used carbon source in the system
because it is free and contains a remarkable fraction of CO2. Sunlight is sequenced
by microalgae as energy to grow.
Open raceway ponds have been widely used in microalgae production

because they are easy to run and scale up. However, the system is facing several
problems, including low biomass productivity and CO2 utilization efficiency
(compared to the closed photobioreactor); contamination; high cost of harvesting
due to the low cell density in the pond; difficult to control temperature,
evaporation, and lights; as well as weather susceptibility.
8.4.2 Closed Photobioreactor Cultivation System

The closed photobioreactor cultivation system is grabbing significant attention
due to its high biomass productivity and low risk of contamination. It is easy to be
controlled. The simplest closed system is laboratory glass fermenter (Pruvost et al.
2011). This system is normally used in laboratory research because it is difficult to
scale up due to the limitation of vessel size. In this case, light can be provided
externally because the diameter of the fermenter is small and light can penetrate.
A tubular reactor is one of the most applied photobioreactors in practice
(Abomohra et al. 2016). It is normally made of glass or plastic, and light can easily
go through. The tube can be placed horizontally or vertically, indoor or outdoor,
and illuminated with artificial light when sunlight cannot meet the requirement.
Normally, there is a central control system to manipulate the pump, sensors,
addition of nutrient, and substrate. Tubular photobioreactors can provide high
biomass yield and short cultivation time, but their construction, operation, and
maintenance costs are high.
The plate photobioreactor is also popular for microalgae cultivation (He et al.
2016). Glass and plastic are used to construct thin layers to optimize the light
supply. This type of photobioreactor could provide similar biomass yield to the
tubular one, but the construction is normally cheaper than the tubular one because
it requires less-expensive material.
To enhance biomass yield, other types of photobioreactors such as horizontal
photobioreactors and Christmas tree photobioreactors have also been developed,
but the utilization is less compared to others because they are not easy to be
constructed.
Because glass and plastic are costly, the foil photobioreactor has been
designed. The bioreactor made of polyvinyl chloride and polyethylene were used
to cultivate microalgae. The price of the photobioreactor has been largely reduced
but replacement is required because the materials are not sustainable. The closed
photobioreactor has shown great potential in commercial microalgae production
as it provides several-fold higher biomass yield compared to open raceway ponds
(Abomohra et al. 2016). To produce the same amount of microalgae, closed
photobioreactors would demand less cultivation volume compared to open
raceway ponds. When the microalgae are used to produce high-value materials
such as docosahexaenoic acid (DHA), vitamins, protein, and minerals, the process
is feasible. However, to produce lipid, there is still a distance due to the cost
concern.
8.4.3 Hybrid Cultivation Systems

As discussed in the preceding sections, an open pond is economical and the most
promising microalgae production route, but it is easy to be contaminated and
with a low cell density. Closed photobioreactors can achieve high cell density and
prevent pollution, but require large investment. Therefore, the hybrid cultivation
system has been developed, which is the combination of open ponds and closed
photobioreactors. In general, the cultivation is in two stages. The first stage is
performed in closed photobioreactors to get a high cell density; then the second
stage will be completed in open ponds. The design is more favorable as lipid
production from microalgae can be enhanced by two strategies: increase cell
number and enhance lipid accumulation. The two-stage cultivation can accom-
plish the task. In a closed photobioreactor, the system can be controlled in an
optimal condition for steady and rapid cell division; hence, high cell density can
be fulfilled, and contamination can be prevented. The lipid accumulation can be
achieved in open raceway by providing external stress to the system such as
nitrogen limitation. Although the cultivation is exposed to the open environ-
ment in the second stage, the contamination risk is low because nitrogen can be
limited whereas cell division requires nitrogen. It was reported that the lipid
content of microalgae Chlorella vulgaris was greatly enhanced in the hybrid system
(50% w/w) compared to the single photobioreactor (30% w/w). The life cycle
assessment showed that hybrid cultivation can save energy to produce the same
amount of biodiesel from microalgae compared to solo closed photobioreactors
(Adesanya et al. 2014, Heidari et al. 2016, and Tan et al. 2014). Therefore, the
hybrid cultivation system could be the solution of microalgae production for
biodiesel.
8.5 MICROALGAE BIOMASS HARVEST
Harvesting following cultivation is an important step of microalgae for biodiesel

production. The aim of harvesting is to separate biomass from water and send for
downstream processing. It is, in fact, a concentrating process. Harvesting has been
considered the bottleneck of biodiesel production from microorganisms attributed
to the large demand on cost and energy. It was reported that the harvesting cost
took up to 30% of total microalgae production cost (Sharma and Singh 2017). The
major problem is the low concentration (5 to 20 g/L) of microalgae in the
cultivation broth and their small size (3 to 30 μm). There are a few harvesting
technologies available currently, including gravity sedimentation, centrifugation,
filtration, and flotation.
Gravity sedimentation is the simplest harvesting technology because it

requires no energy input. The separation depends on the density of microalgae,
which is normally greater than water (1 kg/L); however, it is usually difficult since
the microalgae trend to be dispersed/suspended in water due to the negative
charges (Collet et al. 2014). Therefore, the sedimentation would be a slow process.
Consequently, large-volume retention tanks/space must be used, which will
increase the cost. It was suggested that gravity sedimentation was, in fact, not
acceptable in practice.
Centrifugation is the most efficient microalgae harvesting strategy because it
could remove up to 85% water within 10 to 15 min; however, it is a costly method
for harvesting microalgae and is well known to be difficult to scale up. The
technology is widely used in laboratory-scale studies. It would be a promising
harvesting method when a centrifugation with low energy consumption is
developed. However, before that, it is difficult to apply in large-scale microalgae
harvesting.
Filtration is the most frequently applied technique for the separation of solid
from liquid. It has also been used in microalgae harvesting. Microalgae can be
separated from water by filtration with or without the addition of chemicals. As
mentioned, microalgae are small and with negative charges, and thus very difficult
to separate. To achieve high efficiency of filtration, flocculants such as polymers,
ferric chloride, and aluminum sulfate are added to allow the microalgae to
aggregate to large flocs (Pérez et al. 2016). The addition of chemical could pollute
the microalgae and lead to low quality of the product biodiesel. Using micro-
filtration or an ultrafiltration membrane for microalgae harvesting would avoid
the addition of chemicals because their pore size is small enough to catch the
microalgae (Mata et al. 2010). However, clogging and membrane fouling are the
major obstacles of the practice, and the cost is high.
Flotation uses dissolved air to attach with microalgae and bring the cells to the
top of the liquid. It is less expensive than other harvesting methods and can be
applied in large-scale plants (Greenwell et al. 2010). It is considered to be the most
potential technology for microalgae harvesting in practice, but efficiency needs to
be increased by developing proper equipment and designing proper cultivation
systems (the bottom is made up of gravitational force).
There are also other newly developed harvesting methods, including electro-
phoresis and ultrasonic harvesting (Bosma et al. 2003, Tang et al. 2011). The
former has emerged based on the fact that microalgae are negatively charged. The
microalgae cells would move toward the positive field when an electric field is
applied; hence, cells are concentrated. Ultrasonic application in harvesting is
possible because ultrasound waves induce the aggregation of cells. This technology
is normally applied with gravity sedimentation to harvest microalgae. Both
methods are clean because no chemical is added. Moreover, ultrasonic harvesting
has shown high efficiency up to 92%; however, it is difficult to scale up. New
harvesting technology is still highly demanded. More effort should be made to
reduce the harvesting cost, lower the energy input, and improve the scale-up
issues.
8.6 POSSIBILITY OF MICROALGAE BIODIESEL
Microalgae have high capacity of oil accumulation, rapid growth rate, and no
competition on arable land. It indicates that microalgae oil is superior to plant seed
oils and animal fats as biodiesel production feedstock in many aspects. However,
currently biodiesel production from microalgae is still at the research stage,
primarily because of the high cost. Studies have revealed that the cost is about
USD 10 to 30/gal. which is about 2.5 to 7 times of the price of vegetable oil
converted biodiesel (Greenwell et al. 2010, Francavilla et al. 2015). The high cost is
mainly due to the low biomass concentration, high maintenance requirement, and
the difficulty of extracting the oil. To achieve the commercial production of
microalgae, work has to be done to reduce the cost.
8.7 SUMMARY
Microalgae show significant advantages in replacing plant seed and animals as

feedstock of biodiesel production. Microalgae oils have similar composition to
vegetable oils and animal fats. The major factors impacting oil accumulation in
microalgae are strains, cultivation mode, and cultivation conditions (light, salinity,
temperature, pH, substrate, and nutrients). Harvesting as the following step of
microalgae cultivation plays an important role in biodiesel production from
microalgae because the harvesting method directly influences the biodiesel quality
and cost. Centrifugation is efficient but expensive; flotation is cheap but not well
developed; gravity sedimentation is simple but inefficient; filtration might be a
wise choice currently for microalgae harvesting in practice even though it could
cause pollution on microalgae biomass. Microalgae have shown the potential to be
a biodiesel production source, but the cost as the only determining factor of
microalgae for biodiesel production in practice must be reduced before it can be
applied.
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CHAPTER 9
Single Cell Oil Biodiesel
S. K. Ram
R. D. Tyagi
9.1 INTRODUCTION
With surging global population and plummeting resources there is an apparent

demand of radical unprecedented innovation to cater to the basic needs of lives. As
explained by Maslow and Lewis (1987), food and energy are the most basic
requirements of living. Presently, we happen to be moving rapidly toward
industrialization, which indeed signifies a rapid consumption and further demand
of new energy resources. Realizing the nonrenewable nature of our energy
resources, new unconventional renewable energy resources like biodiesel have
been worked out during the last few decades. In fact, application of biofuel is
highly recommended by regulatory authorities. The European Union directive
2003/30/EC has recommended an addition of 5.75% w/w of biofuel in conven-
tional fuel since 2010 (Papanikolaou and Aggelis 2009).
Biofuel serves to be the most promising and sustainable answer for the
competitive need of food and fuel demands in an ecologically benign way.
Conventionally, first-generation biofuels were produced using sugars, starch, and
vegetable oils. Generated biofuels included ethanol, biodiesel, biogas, and
bioethers, among others. The biofuels consumed agricultural resources like edible
oil crops and land, which gave rise to an intense moral debate on food versus fuel.
To abate the dire consequences of biofuel generation on the food sector, the
second-generation biofuels or advanced biofuels were developed to use various
types of biomass, usually lignocellulose, as their carbon source. It did solve the
problem to a certain degree but still did not completely answer the food-versus-
fuel competition for resources like arable land until 1985, when for the very first
time, oil was demonstrated to be produced from Mucor circinelloides (Ratledge
2004). Today, many other microorganisms are known to produce oil, also termed
as single cell oil (SCO) or simply microbial oil. SCO overcomes various problems
associated with conventional methods of oil production from plant and animal
sources like seasonal variation, high cost of production, and consumption of a
large share of agricultural resources. It is thus essential to develop and improve oil
201
production technologies using these cellular factories for a sustainable solution for
all oil requirements.
In this chapter, we discuss the major aspects of microbial oil production. The
chapter focuses on the basic characteristics of SCO and its unique features. The
chapter discusses in detail the basic elements of microbial oil production such as
oleaginous organisms, substrates usually available for economic production of
SCO, and basic physicochemical factors for the production of SCO. The chapter
includes a dedicated section to discuss the various mechanisms and underlying
biochemical pathways for SCO production and its consumption. In the discussion
over the cost and economics of SCO, the chapter elucidates the bottlenecks of SCO
production, process development, and future targets to be realized. Although
much progress has been achieved by the biodiesel industry recently, there is still a
long way to go to reach a sustainable solution to the present energy crisis situation.
9.2 CHARACTERISTICS OF SINGLE CELL OIL
Oil can be produced from various sources like plants, animal fats, and microbes.
Before the development of microbial oil production processes, oil was convention-
ally produced from various oil crops like mustard, soybean, rapeseed, sunflower, and
jatropha, among others. Oil derived from plant sources are of two types: one type is
for the storage purpose used in developmental stages of the seed, and another type is
essential oil that is used by plants for secondary purposes like defense. Plant oil in
general contains more unsaturated fatty acids and is often considered to be the only
source of nutritionally important unsaturated fatty acids like linolenic acids.
Essential oils from plants consist of terpenes and fatty acid derivatives.
Animal oils are different from plant-source oil because they are relatively
more saturated and contains major amounts of myristic acid (14:0), palmitic acid
(16:0), stearic acid (18:0), palmitoleic acid (16:1, ω-9), linolenic acid (18:3, ω-3),
and arachidonic acid (20:4, ω-6). Because of the high degree of saturation of
animal oils, they are usually solid at room temperature.
SCO is termed to recognize that the oil is produced from microbial sources
using basic fermentation techniques in a bioreactor. SCO is produced to achieve
oil of high nutritional values like polyunsaturated fatty acids (PUFAs), cocoa
butter substitute (CBS), and biodiesel feedstock. SCO is distinct in its commercial
value owing to its compositional superiority in terms of PUFA composition, which
is unlikely to be found in plant source oils and animal fats (Nagoya 2016). SCO
contains some particular fatty acids—gamma linoleic acid, arachidonic acid,
eicosapentaenoic acid, and docosahexaenoic acid—of which industrial processes
are not available from conventional sources like plant oil, animal fats, and fish.
Microbial oil provides an opportunity to develop a process for up-scaled produc-
tion of selective PUFAs, which are clinically proven to be of high value considering
the health benefits they deliver (Neuringer et al. 1988). Gamma linolenic acid
(GLA 18:3), Arachidonic acid (ARA 20:4), di-homo-gamma linolenic acid
SINGLE CELL OIL BIODIESEL 203
(DHGLA 20:3), eicosapentaenoic acid (EPA 20:5), and docosahexaenoic acid

(DHA 20:6) are considered to be most valuable PUFAs available in microbial oil.
Microbial oil varies in composition depending on the substrate and mode of
production. Microbes usually accumulate lipids in the form of triacylglycerols
with esterified chains of fatty acids containing unsaturation, preferably at sn-2

positions of glycerol backbone. PUFAs are synthesized from the saturated or
monounsaturated precursor fatty acids with elongation and desaturation as shown
in Figure 9-1. Animals, including humans, are devoid of the enzyme Δ12 and Δ15
desaturases, which transform oleic acid to linoleic acid by Δ12 desaturase and
further into alpha linolenic acid by Δ15 desaturase. The absence of these enzymes
makes these fatty acids essential nutrients. Comparing animal source oil for
clinically important fatty acids like DHA with microbial oils, fish get their DHA
supplement from phytoplankton feed, which contains inevitable amounts of EPA.
EPA is known to depress growth rates in infants (Carlson et al. 1992). Microalgae
synthesize DHA via de novo and thus do not contain EPA (Kyle and Ratledge
1992). Thereby microbial oil can be designed to produce specific PUFAs and can
contain fewer variants of unsaturated fatty acids leading to higher stability and
shelf life.
Besides PUFAs, production of CBS is also one of the niche areas in which
SCO research has focused for the last two decades. Cocoa butter is exclusive in
the composition of its fatty acid having equal amounts of palmitic, stearic, and
oleic acids, which are rarely found in plant oils. Many oleaginous organisms have
been discovered that can give lipids with composition similar to cocoa butter
(Hassan et al. 1994). Intensive research is going on to realize the production of
SCO at scales competitive to conventional vegetable oil for second-generation
Figure 9-1. Higher fatty acids and PUFA synthesis.

SCO production has obvious advantages over plant oils because growing
unicellular yeast is easier in terms of labor input, space requirement, and rapid
growth rates, which make them attractive alternatives for feedstock in biodiesel
production. In the subsequent section, SCO production for biodiesel feed purpose
is discussed.
9.3 OLEAGINOUS MICROORGANISMS FOR SINGLE CELL OIL

PRODUCTION
Almost all the microbes, including all the bacterial prokaryotes and eukaryotes like
fungi, yeast, molds and algae, are known to produce lipids for regular cellular
metabolism and structural purposes. However, recent research has identified
many microbes, mostly yeast and algae, which can accumulate a significant
amount of intracellular lipids in the form of lipid vesicles, which account for
more than 20% of dry biomass weight. These organisms are classified as oleagi-
nous microorganisms (Ratledge 1991). The origin of SCO dates back to 1985 when
the first SCO was produced from Mucor circinelloides; since then, many new
microbes have been discovered, including most prominently Cryptococus sp.,
Lipomyces sp., Rhodosporidium sp., Rhodotorula sp., Trichosporon sp., Yarrowia
sp., Aspergillus sp., Mortierrela sp., Thamnidium sp., Candida sp., Zygosacchar-
omyces sp., Zygorhynchus sp., Mucor sp., Torulopsis sp., and Pichia sp. (Donot
et al. 2014). Table 9-1 gives a nonexhaustive list of the oleaginous microorganism
used to produce SCO so far.
9.4 SUBSTRATES UTILIZED FOR SINGLE CELL OIL PRODUCTION
SCO is produced mainly by unicellular yeast. Yeast by virtue of de novo pathway

synthesizes various cellular fatty acids from various substrates to yield high lipid
accumulation. Except for less used cellulose and methanol, a wide array of
substrates has been discovered in recent decades. Because the major cost of oil
production (75%) is due to the cost of substrate utilized for the production,
intensive research is going on to utilize substrates that are readily available and
economical to lower the cost of production to make the process competitive with
conventional oil production methods (Huang et al. 2013). Different substrates
utilized for the production include carbon sources as simple as glucose, fructose,
and maltose to complex polymeric forms as in starch, cellulose, and lignocellulosic
hydrolysates.
Compared with glucose, lesser studies on fructose, galactose, and xylose have
been reported. Xylose is considered important because it is abundantly available
after chemical hydrolysis of various lignocellulosic materials. Fermentation done
on xylose produces very low levels of lipids compared to glucose (Hansson and
Table 9-1. Oleaginous Organisms.

Organism Lipid DCW (%) Biomass (g/L) Time (h) Substrate References
Aspergillus oryzae A4 18.6 4.3 144 Microcrystalline Hui et al. (2010)

cellulose
Aspergillus niger LFMB 57.40 5.4 96 Crude glycerol André et al. (2010)
Cryptococcus curvatus 75.0 168 192 Hydrogen production Chi et al. (2011)
ATCC20509 effluent + acetic acid
66.0 15.5 72 Sorghum hydrolysate, Liang et al. (2012)
bagasse
35.9 7.6 72 Oligocellulose, Gong et al. (2014)
oligoxylose
Cryptococcus sp. 63.5, 61.3 9.4, 10.8 96, 7 day Glucose, corncob Chang et al. (2013)
(fed batch) (fed batch) fed batch hydrolysate
Cunninghamella 41.17 17.6 120 Glucose Fakas et al. (2009a)
echinulata
ATHUM 4411
Lipomyces starkeyi 56.39 13.3 220 Glucose Angerbauer et al.
DSM 70295 (2008a)
72 9.3 220 Glucose (with waste
water)
Lipomyces starkeyi 47 17.2 8 days Corncob acid Huang et al. (2014)
hydrolysate
SINGLE CELL OIL BIODIESEL
27.9 50 103 Cellobiose and Xylose Gong et al. (2012)

Mortierella isabellina 75 13.2 237 Glucose Papanikolaou et al.
ATHUM 2935 (2007a)
205
(Continued)
Table 9-1. Oleaginous Organisms. (Continued)

206
Organism Lipid DCW (%) Biomass (g/L) Time (h) Substrate References
Microsphaeropsis sp. 32.5 8 164 Hemicellulose Peng and Chen

(2012)
Phaffia rhodozyma 39.74 7.8 144 Glucose El Bialy et al. (2011)
NRRL Y10921
Rhodotorula graminis 52.31 15.1 144 Glucose Galafassi et al. (2012)
34 Corn stover
hydrolysate
Rhodosporidium 63.63 22 192 Cassava starch Wang et al. (2012)
toruloides 21167 hydrolysate

Rhodosporidium 69.66 26.7 160 Crude glycerol Xu et al. (2012)
toruloides AS2 1389
Yarrowia lipolytica 58.77 11.4 96 Sugarcane bagasse Tsigie et al. (2011)
Po1g hydrolysate
Zygorhynchus moelleri 43.24 3.7 192 Raw glycerol Chatzifragkou et al.
MUCL 1430 (2011)
Hydrophobic Substrates
Rhodotorula glutinis 38.18 7.07 72 Palm oil mill effluent Saenge et al. (2011a)
TISTR 5159
Yarrowia lipolytica 50 15 120 (fed Animal fat Papanikolaou et al.
LGAMS (7)1 batch) (2002a)
Zygorhynchus moelleri 40.22 8.7 240 (cont.) Lipid with glycerol
MUCL 1430
Dostálek 1988). Sugars like lactose from whey, maltose from starch hydrolysis, and
sucrose from molasses have been used and are reportedly giving significant lipid
accumulations.
According to stoichiometric estimates, 100 g of glucose will yield 1.1 mole of
acetyl-CoA, whereas xylose will result 1.2 moles of acetyl-CoA (Cohen et al. 2010).
Assuming all the acetyl-CoA is channeled to SCO production, glucose will have a
lipid yield of 0.32 g/g, whereas xylose will have 0.34 g/g of lipid yield. Likewise,
glycerol has a lipid yield value of 0.3 g/g. Various studies conducted on glucose
found that the actual yield for glucose is > 0.24 g/g (Papanikolaou et al. 2010).
Other sugars, like sucrose and xylose, are reported to have a yield value of around
0.23 g/g, but glycerol has very low yield values of 0.1 g/g compared to other sugars
(Fakas et al. 2009b). Extensive research on glycerol has been conducted, and two
studies reported yield values greater than 0.2 g/g for glycerol. Aspergillus sp. and
Thamnidium elegans produced high lipid content in the range of 60% to 70% dry
cell weight (DCW) having yields in the range of 0.16 to 0.22 g/g (Chatzifragkou
et al. 2011). Highly reduced substrates like ethanol also have the potential to be
high yielding substrates. The theoretical yield for ethanol is 0.54, and laboratory-
scale studies on ethanol for lipid accumulation report an actual yield of 0.31 g/g
(Cohen et al. 2010).
Because of ubiquitous and economic availability of agricultural biomass,
special attention has been given to utilization of cellulosic substrates, hemicellu-
losic sugars, wheat straw, rice bran, corncob liquor, among others (Huang et al.
2013). Crude glycerol, a byproduct of biodiesel production units, is also being
intensively used. Papanikolaou et al. (2002b) reported high accumulation of lipids
(about 43% w/w DCW) using Yarrowia lipolytica, which clearly shows the
potential of glycerol for SCO production contradicting the earlier negative reports
about the potential of using raw glycerol as a carbon source (Sajbidor et al. 1988).
In spite of its low yield values, glycerol is considered to be one of the major carbon
sources for SCO production due to its high-volume availability. Many researchers
have tried to find oleaginous strains for better assimilation of glycerol.
Many Zygomycetes species including Candida boidinii, C. curvata, C. oleophila,
C. pulcherrima, C. echinulata, M. isabellina, M. ramanniana, Mucor sp., Pichia
membranifaciens, Rhodotorula sp., T. elegans, Y. lipolytica, Zygosaccharomyces
rouxii, and Zygorhynchus moelleri have been screened, of which T. elegans was
found to be producing high levels of lipids (Chatzifragkou et al. 2011).
Waste streams such as tomato waste hydrolysate, glucose-supplemented
sewage sludge, and wastewater is being utilized for the readily available nutrients
in these waste streams (Donot et al. 2014). Citric acid, acetic acid, and other
organic acids are also being investigated. Acetic acid is majorly present in effluent
streams of the Fischer–Tropsch reaction (Du Preez et al. 1997). Further, the zero
or the negative cost of these waste streams make them more attractive options to
bring down the cost of production.
Hydrophobic substrates, like waste oils and fats obtained from various
industries, have been studied to biotransform the compositions and content of
lipids. A very limited number of species are known to assimilate oil-like substrates
as their carbon source. Various yeasts capable of utilizing oil as substrate are
strains from Torulopsis, Candida, Trichosporon, and Geotrichum genera. Few
species like Pichia methanolica, Apiotrichum, and Rhodosporidium toruloides are
also known for their oil utilization abilities (Koritala et al. 1987). Apparently for an
organism to metabolize triacylglycerols (TAGs) and other forms of lipids, it needs

to have a dedicated lipase system. Cultures grown on free fatty acids (FFAs) do not
have such obligations. Various strains of Y. lipolytica have been identified to have
lipase systems and can accumulate a significant amount of intracellular lipids
(Papanikolaou and Aggelis 2002). Hydrophobic substrates utilized include vege-
table oils, fatty acid esters, pure FFAs, crude fish oils, and soap stocks. Lipid
accumulated by the ex-novo pathway in the presence of hydrophobic substrates
are principally lower than the de novo pathway (Koritala et al. 1987). Lipid
produced by the ex novo pathway is similar in FFA composition of raw substrate
utilized. Most of the lipid fraction is isolated in the form of FFAs indicating low
conversion into TAGs. In a study to analyze intracellular lipid obtained by
biomass grown on glucose-supplemented media containing FFAs, it was found
that 93% of accumulated lipid contained FFA only (Aoki et al. 2002). Thus, it
indicates that ex novo pathways can be used for biotransformation and are the
potential method to scavenge and improve oil from waste effluent streams in oil
industries.
It is thus apparent from Table 9-1 that cheap substrates can potentially
contribute significantly to reduction in production cost without any significant
productivity trade-off. However, the important problem remains in the realization
of these processes, that is, how to optimize the pretreatment processes like
hydrolysis and detoxification that these raw materials require before they can
give maximum productivity without any cost compromise at the industrial scale
(Mussatto and Roberto 2004).
9.5 FACTORS AFFECTING PRODUCTION OF SINGLE CELL OIL
Multiple factors influence the total lipid accumulation. The carbon to nitrogen
(C-to-N) molar ratio is the most profound factor. It has been well established that
the C-to-N ratio value greater than 20 is inducible for lipid accumulation or
lipogenesis. Multiple studies have been conducted to address this issue, and it
was found out that neither too high nor too low is suitable for lipid accumulation.
For example, the study conducted with Lipomyces starkeyi with a fixed initial
glucose concentration and decreasing nitrogen concentrations revealed that
lipid accumulation increases with an increase in C-to-N ratio up to as high as
150 mole/mole (Angerbauer et al. 2008), whereas in another study with
R. toruloides (Cohen et al. 2010), the lipid accumulation decreases with increasing
C-to-N ratio (> 70), thus indicating there exists some optimum. Nevertheless, it
also depends on the strain of choice as in M. isabellina (Papanikolaou et al. 2004a),
in which high lipid accumulation was observed with a very high initial C-to-N
ratio (>340). During cultivation at a very high C-to-N ratio, the activity of
nitrogen-dependent enzyme, isocitrate dehydrogenase (ICDH), in the tricarbox-
ylic acid (TCA) cycle decreases to very low levels (Wynn et al. 2001). After
completion of incubation, a large quantity of residual sugar remains in the media.
The carbon uptake in the microbe is regulated by malic enzyme activity, which is
often the rate determining step in the complete lipogenesis process (Ratledge and
Cohen 2008). Reportedly, microbes with high specific carbon uptake rates
accumulate higher amounts of lipids, whereas those with lower specific carbon
uptake rates favor biomass generation and lower lipid accumulation (Papaniko-
laou et al. 2007a). It has been reported that the presence of proteins in the growth
media may affect the specific substrate rate. Fakas et al. (2008a) found that the
presence of protein in the media will enhance the uptake rate of carbon substrate,
and the media with these proteins removed displayed relatively lower uptake rates
of the substrate.
Secondarily, nitrogen is the second most important nutrient factor in SCO
production process. Various nitrogen sources have been used in combination with
carbon sources like yeast extract, peptone, ammonium sulphate, urea, L-arginine,
corn gluten, corn steep whey concentrate, and tomato waste hydrolysate (Fakas
et al. 2008a). The nature of the nitrogen source used affects the lipid accumulation
process. A study with Cryptococcus albidus (Hansson and Dostálek 1986)
with various nitrogen forms found that use of inorganic nitrogen enhances the
lipid accumulation process, whereas organic nitrogen sources like urea help to
grow biomass and negatively affect the lipid accumulation process. Contradicting
this result as a general scale of choice, it was found that R. toruloides grown
in asparagine instead of ammonium chloride enhanced the lipid from 18% to
51% w/w DCW (Evans and Ratledge 1984).
Temperature evidently plays a significant role in determining the lipid
accumulation capacity, biomass proliferation, and fatty acid composition of the
cellular lipid. Studies on Y. lipolytica revealed that for better biomass growth the
microbe can be maintained at temperature ranging from 24 °C to 33 °C, although
it is only at 28 °C that the highest lipid accumulation was observed (Papanikolaou
et al. 2002a). Similarly, C. albidus has a temperature optimum of 20 °C, whereas
Adiantum curvatum has the best responses at 30 °C (Hansson and Dostálek 1986).
Temperature also affects the fatty acid composition which can be accounted by the
corresponding activities of enzymes involved in fatty acid synthesis. It is reported
that with an increase in temperature from 10 °C to 25 °C, a significant increase in
the Δ9,12C18: 2 (stearic acid) fraction in the accumulated lipid was observed while
culturing C. lipolytica with glucose (Ferrante et al. 1983). Contrary to this result,
while studying growth of Rhodotorula glutinis on glucose for lipid accumulation, it
was observed that with an increase in incubation temperature the saturation
degree of accumulated lipid increased significantly (Saxena et al. 1998).
The pH of the growing media is another important factor affecting the lipid
accumulation process. In general, almost all the studies reported slightly acidic
to neutral pH suitable for lipid accumulation ranging from 5 to 7. Studies
conducted on Y. lipolytica reveals that optimum pH for lipid accumulation is
6.0 (Papanikolaou et al. 2002a). Another study with A. curvatum on effluent whey
reported that there was no significant change in lipid accumulation when pH
varied from 3.5 to 5.5; thus, this apparently provides an interesting insight into
developing continuous processes without the need of sterilization of media,
thereby reducing the cost of production.

Intensified research goes on to replace conventional substrates with low-cost
waste stream effluents. In the light of this idea, major impedance is the presence of
toxic compounds in the waste stream that may inhibit cellular proliferation.
Pretreated lignocellulosic biomass contains various components like acetic acid,
syringaldehyde, vanillin, furfural, and others, and each presents a different level of
toxicity to biomass growth. A study with R. toruloides reported that components
like hydroxufurfural and acetic acid present slight inhibition at high concentra-
tion, whereas components like furfural and vanillin have very low critical
inhibition concentration resulting in ceased biomass growth (Hu et al. 2009).
Many studies have been devoted to optimize the mode of fermentation
processes, aiming to maximize lipid accumulation. Researchers have developed
batch, fed batch, and continuous modes as well. Looking at demand and scale of
operation a continuous operation is essentially desirable. Reportedly, dilution rates
have significant impacts on lipid accumulation by the organism. It has been
suggested that a dilution rate less than 0.06 h−1is normally required for optimum
conversion (Papanikolaou and Aggelis 2002). Few studies report that using fed
batch mode very high cell concentration (100 g/L of biomass) can be achieved
(Li et al. 2007). Strains of Y. lipolytica in a highly aerated chemostat system with
low dilution rates of 0.04/h is reported to accumulate ~25% (w/w DCW) lipids
(Papanikolaou and Aggelis 2002).
9.6 DEGRADATION OF LIPIDS IN CARBON LIMITATION
Lipid accumulated by the microbes by either the de novo or ex novo pathway is

subjected to degradation by the cell for its own survival and basal biochemical
needs in the event of carbon-limited conditions. During this phase a lipid-free
biomass is generated. A major fraction of accumulated lipid is composed of TAGs
and steryl esters (STEs) (Ratledge 1994). To catabolize these lipid fractions,
microbes need to have a dedicated enzymatic arsenal in the form of lipases and
STE hydrolases. These enzymes can be constitutively expressed in the cell system
or can be induced by various factors (Alvarez et al. 2000). Lipid particle containing
the stored lipids are surrounded by a monolayer of lipid molecules. This
monolayer contains various proteins called perilipin in animals and oleosins in
plants. Although polypeptides found on lipid particles in yeast did not have any
resemblance with oleasins and perilipins, they are assumed to act as docking sites
or activators for STE hydrolases and lipases (Mullner and Daum 2004). Lipid
degradation is independent of the mode of lipid accumulation. Fatty acids released
from lipase actions are broken down by virtue of beta oxidation (Figure 9-2).
Figure 9-2. Beta oxidation for catabolism of fatty acids.
Acetate in the form of acetyl-CoA obtained from beta oxidation is used in the
Krebs cycle. The Krebs cycle utilizes glyoxylate shunt (Figure 9-3) to convert
acetyl-CoA into malate ions, and then to form oxaloacetate in cytosol. The
cytosolic oxaloacetate ultimately takes part in gluconeogenesis (Papanikolaou
et al. 2002a). Enzymes for the glyoxalate shunt pathway are segregated in the
glyoxysome where acetyl-CoA condenses with oxaloacetate to give a citrate ion.
The citrate ion in turn converts into isocitrate as in the TCA cycle. The enzyme
isocitrate lyase (ICL) cleaves isocitrate to succinate and glyoxalate ion. Glyoxalate
ion moves ahead in the cycle to condense with acetyl-CoA to form malate by the
Figure 9-3. Glyoxylate pathway for acetyl-CoA to gluconeogenesis pathway.

enzyme malate synthase (MS) and continue in the cycle to form oxaloacetate. The
succinate ion thus formed is transported into mitochondria where it is converted
into malate by TCA enzyme mechanisms. Malate is then secreted out to cytosol to
contribute into gluconeogenesis.
Glyoxalate shunt enzymes, ICL, and MS get expressed in the presence of

simple two-carbon substrates like ethanol that lead to formation of acetyl-CoA
from substrates like TAGs and FFAs via beta oxidation (Holdsworth et al. 1988).
These pathways are not followed when glucose is present in the media. Thus,
essentially lipid degradation takes place by the glyoxalate pathway in the absence
of glucose like sugars and in the presence of accumulated lipids to use as carbon
source. It has been reported that the absence of multiple nutrients like iron and
magnesium suppresses the ICL and MS expression, thus inhibiting SCO degra-
dation (Papanikolaou et al. 2004b), which might indicate a method to avoid SCO
degradation in the production process.
Detailed studies conducted on lipid degradation by C. echinulata and
Y. lipolytica reveal that during the lipid degradation a simultaneous lipid free
biomass growth takes place (Fakas et al. 2007). The microbes follow glyoxalate
anaplerotic pathway for carbon and nitrogen is supplemented via adenosine
monophosphate (AMP) deaminase. During this phase, selective degradation of
lipid takes place. All the saturated lipids get metabolized and leave the cell with
highly unsaturated lipids, whereas in Y. lipolytica unsaturated fatty acids get
consumed first and residual lipid is highly saturated. The lipid-free biomass
yield is high, in the range of 1.9, 0.8, and 0.6 g/g for C. curvata (Aggelis
and Sourdis 1997), Y. lipolytica (Papanikolaou and Aggelis 2003), and Mucor
circinelloides (Holdsworth and Ratledge 1988), respectively, during these lipid
degradation phases.
9.7 BIOCHEMISTRY OF SINGLE CELL OIL PRODUCTION IN YEAST
Oleaginous organisms mostly include yeast, fungus, molds, and microalgae. Only
one or two exceptions of bacterial microorganisms are included in this category.
Biochemically, the oil production is nothing but esterification of fatty acid
produced by the organism in cytosol using the enzyme complex called fatty acid
synthase (FAS) and glycerol backbone to give neutral triacylglycerol, followed by
their modification in endoplasmic reticulum and packing all the lipid molecules
together as globular structures.
The main reason for the oleaginous nature of these organisms is the presence
of ATP-citrate lyase (ACL) enzyme active in the cytosol, which cleaves the citrate
ions to produce acetyl-CoA and oxaloacetate ions. The acetyl-CoA produced acts
as a carbon source for fatty acid synthesis. The second important factor that makes
them oleaginous is the presence of AMP-dependent ICDH enzyme in TCA, which
gives rise to citrate accumulation in the event of nitrogen depletion from the
media. Furthermore, oleaginous yeasts have mechanisms to regenerate sufficient
Nicotinamide adenine dinucleotide phosphate (NADPH) as reducing power for

producing the fatty acids using various mechanisms discussed in the subsequent
sections.
The complete biosynthesis of lipids can be studied with two pathways: (1) the
de novo pathway; and (2) the ex-novo pathway. De novo pathway is production of
lipids from hydrophilic carbon substrates like simple sugars. In this pathway lipid
accumulation is a secondary metabolic process that takes place after nitrogen
limitation. The complete pathway can be studied in gradual steps of fatty acid
synthesis, glycerol synthesis, modification of fatty acid, esterification of glycerol
with fatty acids to give triacylglycerol, and finally, aggregation of neutral lipids into
lipid globules. Ex novo pathway is a dedicated pathway to utilize exogenous
hydrophobic substrates and lipids. This pathway is often adopted by yeasts that are
specialized in import and degradation of long-chain fatty acids. Species like Pichia
jadinii, Candida rugosa, Y. lipolytica, and Torulopsis colliculosa are capable of
assimilating lipid or lipid-like substrates. Microbes tend to develop special
superficial features to capture and emulsify the oil droplets to facilitate the easy
import using the action of lipases. The imported fatty acid is transported by the
action of various fatty acid binding proteins, which proceed to get complexed with
coenzyme A. The fatty acid coenzyme complex is either directly or in modified
form incorporated into neutral lipid form. The subsequent section discusses the
biochemical events occurring in lipid synthesis.
9.7.1 Fatty Acid Synthesis

After the depletion of nitrogen from the media (e.g., at a high C-to-N molar ratio),
the microbe starts scavenging nitrogen from the available nitrogen sources it has
in the form of AMP for its cell maintenance and basal metabolic activities
according to the following:
AMP → IMP þ NH4 þ (9-1)
Equation (9-1) is catalyzed by the enzyme AMP deaminase, which converts

adenosine monophosphate (AMP) into inosine monophosphate to liberate one
molecule of ammonia ion which is to be used as nitrogen source. This reaction
leads to a surge in the ADP/AMP fraction. The ICDH enzyme of the TCA cycle is
completely dependent on AMP concentration in the cell. With the falling
concentration of AMP, the activity of ICDH ceases, leading to the accumulating
concentration of isocitrate, which is in equilibrium with citrate via aconitate. This
leads to a build-up of citrate concentration in the cell mitochondria. The citrate
molecules are transported out in the cytosol actively using anti-port facility
(citrate-malate transporter) available in the mitochondrial membrane in exchange
of the malate ion back into the mitochondrial compartment as a part of citrate
malate shunt to regenerate NADPH that is required in conversion of citrate into
oxaloacetate and one molecule of acetyl-CoA in the cytosol by the ACL enzyme.
Oxaloacetate ion is converted into malate ion using malate dehydrogenase (MDH)
O C
C N NH
O
CH CH
O O C
H2C CH
S (CH2)4 C NH (CH2)4 CH
Carboxybiotin lysine NH
residue
Figure 9-4. Carboxyl biotin–ACC enzyme link by amide bond.
(Papanikolaou and Aggelis 2011). The acetyl-CoA thus produced is converted into
malonyl-CoA using acetyl-CoA carboxylase (ACC) enzyme in the cytosol.
The enzyme, ACC has two functional sites for two different activities. The
first site is occupied by biotin as a prosthetic group which is connected to the
enzyme by an amide bond (Figure 9-4) between the carboxyl-terminal end of
biotin and the ε-amine group from the lysine residue, rendering a long flexible
arm for its functionality. The free end of biotin interacts with the carboxy ion
COO− using one ATP and brings it to the second functional site, where acetyl-
CoA is available to complete the carboxylation to convert it to malonyl-CoA as
shown in Figure 9-5.
The enzyme activity is regulated by sensing the presence of energy, a very high
AMP level deactivates the enzyme by phosphorylating the enzyme by AMP-
activated kinases, thereby inhibiting the formation of functional tetramer form of
the enzyme. Other than AMP, the enzyme deactivation can be signaled by
epinephrine triggering a cascade of protein. The deactivation is initiated by
activating cyclic AMP kinase to deactivate the ACC enzyme.
The malonyl-CoA thus produced is condensed with the growing chain of fatty
acid starting with the first condensation of acetyl-CoA with malonyl-CoA using
the complex enzymatic system called FAS in the cytosol. Every condensation step
consumes two NADPH for reduction activity, thus making the overall equation of
conversion to be the following:
8acetyl − CoA þ 14NADPH þ 7ATP

→ Palmitate þ 14NADPþ þ 8CoA þ 7ADP þ 7Pi (9-2)
In Equation (9-2), seven ATP are used for seven conversions of acetyl-CoA to
malonyl-CoA by ACC enzyme; 14 NADPH are used for seven condensation steps
Figure 9-5. ACC enzyme converting acetyl-CoA to malonyl-CoA.

done by FAS enzyme complex. The huge requirement for NADPH is replenished
via the reaction of malate to pyruvate conversion by malic enzyme, resulting in
one NADPH in the cytosol. The fatty acids are transported into the endoplasmic
reticulum where the subsequent desaturation, elongation, and esterification take
place to form the lipid globules that bud out from the endoplasmic reticulum as
separate vesicles in the form of lipid globules.
In fungi and yeast, the most common fatty acids are oleic (18:1), linoleic
(18:2), palmitic (16:0), and palmitoleic (16:1) acid. Longer fatty acid molecules are
generated by a series of elongation and desaturation done to palmitic acid
(16-carbon fatty acid) to yield higher fatty acid chains up to as long as 22-carbon-
long docosahexaenoic acid (DHA) as depicted in Figure 9-1.
9.7.2 Glycerol Backbone Synthesis

Source of glycerol for lipid synthesis can be either a free glycerol molecule available
from the media that is actively transported inside the cell (Kayingo et al. 2009), if
the culture is grown directly on glycerol, or from glycolysis where the intermediate
product fructose 1, 6 bisphosphate is split into two interconvertible 3-carbon
molecules, that is, dihydroxyacetone phosphate (DHAP) and glyceraldehyde
phosphate (GAP). DHAP can be reversibly reduced to glycerol-3-phosphate by
glycerol-3-phosphate dehydrogenase (G3PDH) as depicted in Figure 9-6. The
obtained glycerol-3-phosphate acts as a backbone for the esterification with fatty
acids to form neutral lipid (TAGs) molecules. These lipid molecules are stored as
neutral triacylglycerides (Beopoulos et al. 2009).
9.7.3 Triacylglycerol Synthesis

The fatty acids produced in the cytosol by the FAS enzyme complex are
transported to the endoplasmic reticulum where the glycerol phosphate backbone
is esterified by these fatty acids. Free glycerol is phosphorylated by glycerol kinases
to glycerol-3-phosphate, or DHAP can be reduced by G3PDH as mentioned in the
previous section to give glycerol-3-phosphate (G3P). The resulting G3P molecules
are acylated with the first fatty acid by a G3P acyl transferase to form lysopho-
sphatidic acid, which gets phosphorylated again by 1-acyl G-3-P acyltransferase to
form phosphatidic acid. Alternatively, lysophosphatidic acid for this reaction can
come from reduction of the 1-acyl DHAP molecule using 1-acyl-DHAP reductase.
Phosphatic acid is a key structure molecule involved in formation of other
phospholipids like phosphatidylethanolamine and phosphatidylcholine. The
action of phospholipase on such lipids can yield more phosphatic acids. Before
the third acylation on phosphatic acid, the phosphate group is removed from the
Figure 9-6. Biosynthesis of glycerol backbone during glycolysis.

Figure 9-7. TAG synthesis pathways.
glycerol backbone by phosphatidic acid phosphohydrolase enzyme, resulting in

diacyl glycerol (DAG). Finally, a third acyl group transfer is done on the DAG
molecule to form triacyl glycerol as depicted in Figure 9-7 (Donot et al. 2014).
In microbial lipids it is worth noting that fatty acids are not randomly acylated;
rather, the distribution is quite stereotypical in the sense that most of the saturated
fatty acids occupy sn-1 positions, whereas the unsaturated fatty acids take up the
central sn-2 positions on the glycerol backbone (DeBell and Jack 1975).
9.7.4 Single Cell Oil Production Pathway from Hydrophobic

Substrates
Ex novo lipid production pathway is a growth-associated process, and it is
completely independent of nitrogen limitation in the media. It is biotransforma-
tion of existing lipid and fat molecules, either intracellular or extracellular, by
oleaginous organisms to new lipids, effectively changing the fatty acid composi-
tions of the lipid profile (Donot et al. 2014).
Lipid molecules in the media are degraded by the lipase system of the
microorganism, and the FFAs are actively transported inside the cell. The FFAs
are transported by carnitine transporters into the mitochondria and thus get
degraded by β-oxidation process to form acetyl-CoA, as illustrated in Figure 9-2
(Papanikolaou and Aggelis 2011). The resulting acetyl-CoA is directed to various
metabolic activities providing necessary energy for cell growth and maintenance.
Secondarily, the excess of carbon is channeled toward synthesis of organics,
precursor molecules, and storage materials (Papanikolaou and Aggelis 2011).
The FFAs are broken down to acetyl-CoA and Acyl-CoA by the action of
acyl-CoA oxidase (AOX). In yeast Y. lipolytica, the LIP2 gene operon codes for its
extracellular lipase system. The expression products of this operon help the yeast
to assimilate fatty substrates (Fickers et al. 2005). LIP2 gene translates to a
premature protein with a Lys-Arg cleavage site called Lip2p protein (Pignède et al.
2000). This precursor protein gets modified by endoprotease to ultimately become
active. Other proteins that are cotranslated with Lip2p are Lip7p and Lip8p. These
lipase proteins have different specificity for different chain lengths of fatty acids.
After the action of lipase enzymes, FFAs are uptaken by the yeast at different rates
depending on the cell’s preference. After their uptake, the FFAs are acted on by
intracellular AOX enzymes. Y. lipolytica has a set of five AOX enzymes—namely,

Aox1p, Aox2p, : : : , Aox5p encoded by POX1, POX2, : : : , POX5 genes, respec-
tively (Wang et al. 1999). Aox3p is specific to short-chain acyl-CoA, whereas
Aox2p acts on the long-chain. Other oxidases do not show high specificity. During
beta oxidation, one mole of NADH and FADH2 each is released per mole of
acetyl-CoA generated. These cofactors (NADH and FADH2) serve as reducing
power for the cell for its basal metabolism and respiration via the electron
transport chain (Ratledge 1994).
Because of the inhibition caused by the presence of fatty substrates, the
activities of FAS and ACL enzymes are significantly low; thus, the de novo
pathway for lipid formation is not followed (Alvarez et al. 1997). All the excess
acyl-CoA is channeled into lipid accumulation. The effect of nitrogen concentra-
tion in media has been studied by Papanikolaou et al. (2007b), and it was found
that a significant amount of lipid accumulation takes place, invariably in the
presence of various concentrations of nitrogen. Lipid is accumulated as intracel-
lular lipid droplets containing TAGs and steryl esters enclosed by a monolayer of
lipid accompanied with proteins to carry out transport and metabolism of lipid
bodies in various cellular responses (Mlíčková et al. 2004). It has been reported
that at high substrate concentrations, Y. lipolytica accumulate lipid up to 53% of
DCW (Papanikolaou et al. 2002a).
The extracellular lipid composition is usually similar to accumulated lipid
composition, indicating the direct incorporation of fatty acids. Another study with
Y. lipolytica ACA-DC 50109 reported that growth of yeast in stearin only gave
saturated lipids, whereas when grown in mixed substrate (with glucose) the lipid
accumulated contained unsaturated fatty acids, indicating de novo formation of
lipids (Papanikolaou et al. 2006). Two different behaviors have been observed by
yeast while grown on fatty substrates. One type of yeast tends to catabolize short-
chain saturated fatty acids, leaving highly unsaturated fatty acid for lipid accumu-
lation (Kinoshita and Ota 2001); another set of yeast prefers to use unsaturated fatty
acids, resulting in highly saturated lipid accumulation (Aggelis et al. 1997). Thus, the
ex novo mode of lipid accumulation can be strategically utilized to modify the fatty
acid composition and the saturation degree of accumulated lipids, which can be
scavenged out of waste streams. The presence of hydrophilic substrates may help to
change the composition of the accumulated lipids in the microbial oil.
9.8 GENETIC ENGINEERING FOR SINGLE CELL OIL PRODUCTION
To improve the microbial oil production yield, various attempts have been made
to improve the lipid accumulation capacities of the microbe at the genetic level.
Many yeast strains have been studied at the molecular level for the key enzymes
involved in substrate uptake, fatty acid synthesis, elongation, glycerol esterifica-
tion, and lipid degradation. As a general theme, all the key enzymes directing the
carbon channeling to lipid synthesis have been tried to overexpress, and those
diverting the carbon substrate away from the lipid synthesis route are tried to be
suppressed or knocked out at genomic levels using homologous recombination
with replacement gene segments. Heterologous gene copies have been incorpo-
rated, and significant amplified lipid accumulation response has been noted.
Copies of ACC enzyme are incorporated into nonoleaginous organisms,
Hansenula polymorpha, to achieve a 40% increase in total fatty acid content
(Ruenwai et al. 2009). Similar overexpression conducted with diacylglyceride
acyltransferase (DGAT) gene gave fourfold increase in lipid production in
Y. lipolytica (Tai and Stephanopoulos 2013).
Gene deletion study can reveal the significance of genes in lipogenesis. This
information can help in identifying sites of incorporating or removing extra genes
among various forms of the same enzyme in the genetic map of the organism
(Zaremberg and McMaster 2002). Gene deletion of an isoform of G3PDH, the
enzyme for the reverse conversion of glycerol-3-phosphate back to DHAP, gave a
significant threefold increase in lipid accumulation (Beopoulos et al. 2008).
Mlíčková et al. (2004) demonstrated the various effects of deletion of isoforms
of oxidase enzymes involved in lipid degradation on the lipid accumulation and its
various aspects.
9.9 COST AND ECONOMIC CONSIDERATION IN SINGLE CELL OIL

PRODUCTION
Global fuel demands are skyrocketing due to the rapid modernization and
industrialization events in recent decades. Various technical reports from stake-
holding agencies carry out estimates of annual energy demands and production to
implement energy policies and develop strategies. According to the International
Energy Agency oil market report (IEA 2014), global oil demand has increased
from 10,797 million L/day in 2012 to 11,133 million L/day in 2015 at a current
augmentation rate of 3.11% per year.
Following the annual demands, various new alternatives for biofuel have been
implemented. Endless efforts by researchers have resulted in an exponential
increase in biofuel production (Figure 9-8) over the last decade, from
9,176,000 t of oil equivalent in 2000 to 65,348,000 t oil equivalent in 2013 (Statista
Report 2015a).
Countries have been actively participating in biodiesel production according to
their capacities because of the various economic and ecological benefits that the
biodiesel production industry renders. Biodiesel is the greenest fuel and not only
contributes to reduce the annual GHGs, but also provides employment opportu-
nities and boosting the economy. The United States, Germany, Brazil, Argentina,
Indonesia, and France are reckoned among the top producers of biodiesel with a
Figure 9-8. Global biofuel production (Statista Report 2015a).
Figure 9-9. Biodiesel producing countries in 2013 (Statista Report 2015b).
more than 2 billion L production capacity in the year 2013 (Figure 9-9). Other
significant countries in biodiesel production are Thailand, Singapore, Poland,
Colombia, Netherlands, Australias and Belgium, all having biodiesel production
capacity between 0.4 and 1.0 billion L in 2013 (Statista Report 2015b).
To estimate the economic feasibility of any new production process devel-
oped, it is required that its cost analysis is done using realistic estimations and
values. Haas et al. (2006) developed a model for biodiesel production cost
estimation. The model simulates a production process plant with a capacity of
10 million gal (37.85 million L) per year using soy-based feed. The cost analysis
estimated production cost of USD 0.53/L of biodiesel produced. Overall, 88% of
the raw material cost is accounted for feedstock cost. Conventional petrochemical
diesel costs around USD 0.2−0.25/L, which is significantly lower than biodiesel
production cost. Thus, it becomes essential to look for cheaper feedstock alter-
natives like waste effluents from various nutrient-rich industries. The model
suggests that, for every USD 0.022/kg increase in feedstock cost, an approximate
increase of USD 0.02/L in production cost is realized, expressing a strong 1:1 cost-
dependence correlation between feedstock and production. Glycerol is the
coproduct of every biodiesel production unit and can be sold at USD 0.33/kg,
which then reduces 6% of the production cost. Recently, owing to the high volume
of biodiesel production, glycerol availability has increased, thus decreasing the cost
of glycerol, which in turn affects the cost of biodiesel production. For every USD
0.01/L decrease in the glycerol market price, there is an increase of USD 0.008/L in
the cost of biodiesel production.
SCO has a substrate yield of 33% on glucose (Ratledge 1988). Thus, coupling
production of other metabolites within the SCO production scheme provides an
economic advantage to the process. Many SCO-producing microbes are also known
for their abilities to produce other metabolites like single cell protein (Taniguchi
et al. 1982), carotenoids (Saenge et al. 2011b), and lipases (Papanikolaou et al.
2007a). Many oleaginous yeast strains are known for their citric acid and beta
galactosidase production capabilities. These organisms can be advantageously used
to lower the cost of production and increase the profits of SCO production.
9.10 CHALLENGES AND PROSPECTS
The importance of SCO production as feedstock for the fuel sector cannot be
overemphasized. SCO production caused by a rising demand for fuel and food will
remain to be the point of high interest in the purview of research and industrial
production. Because of intrinsic advantages of oil production via microbial routes,
it remains an obligation for researchers to develop an up-scaled biorefinery model
for oil production. Several technical challenges that are still to be answered in the
arena of microbial fuel production include the requirement of aseptic conditions,
optimization and maximization of the accumulation process by developing robust
strains with higher accumulation potentials by biochemical adaptation, genetic
engineering, selection of readily available and economical substrates, development
of low-cost pretreatment methods for cheap substrates (e.g., lignocellulose and
sewage sludge), reducing the cost intensifying factors (e.g., time of cultivation),
maintenance of aseptic conditions, and nutrient supplementation.
Undeniably, biodiesel is expensive compared with petroleum diesel and the
biodiesel industry has to overcome many economic challenges. At the industrial
level, other challenges remain, such as understanding the trade-off between the
cost of production and the value of the product (biodiesel) by factoring in the
environmental benefits in economic analysis; optimizing production parameters
like process design and transportation logistics of raw materials; and finding an
efficient downstream extraction method at an economically feasible cost. Devel-

opment of food-grade oil production from cheap hydrophilic substrates and used-
up waste oil by ex novo biotransformation modes are still at nascent stages.
Intense research/development is required to study the lipid metabolism at the
molecular level and enzyme engineering. Genetic engineering and microbial

metabolism engineering can give their inputs to study the metabolic flux,
proteomics, and genomics of the oleaginous organisms to develop better insight
into the SCO production process (Fakas et al. 2009a).
9.11 SUMMARY
SCO or microbial oil is the most promising source of feedstock oil for the biodiesel
industry. The inherited advantages associated with SCO and its method of
cultivation make it a standout technology for food, feed, and fuel. Production
of SCO is achieved using microorganisms like yeast and fungi. Many successful
efforts have been made to develop an easy and economically viable process
technology for microbial oil production. Scientists have been able to find robust
strains of yeast with superior efficacy and wide spectrum of biochemical assimi-
lation abilities of substrates. The range of substrates include expensive sugars to
inexpensive industrial effluent waste streams like glycerol, whey, lignocellulose,
used oils, and even wastewater sludge.
Studies conducted on various physicochemical factors influencing the pro-
duction of SCO elucidate the importance of nutrient media, the C-to-N ratio,
temperature, pH, and mode of cultivation including batch, fed batch, and
continuous mode. In-depth studies about the mechanism of lipid turnover or
degradation in the event of carbon starvation by glyoxalate shunt helps to develop
a process with appropriate nutrient conditions and fermentation modes to avoid
loss of accumulated lipids after lipogenesis.
SCO accumulation is possible via de novo pathway using hydrophobic
substrates like sugars and polymers of carbohydrates using the basic oleaginous
characteristics of microorganisms, including the presence of ACL and MS
enzymes. Significant results for lipid accumulation up to 60% to 70% lipid of
DCW have been achieved by de novo pathway using strains of Y. lipolytica,
T. elegans, and Aspergillus sp. Lipid accumulation has also been achieved from
used oils, animal fats, and FFA in significant quantity via ex novo pathways. In the
ex-novo pathway, lipid of fatty acid in media gets directly incorporated in the form
of lipids. Physicochemical factors like temperature and pH changes can result in
inconsistent expression of cellular enzymatic machinery, giving rise to variation in
fatty acid composition and saturation degrees of the accumulated lipid.
With the advent of advance molecular and genetic techniques, recombinant
strains of oleaginous yeast have been developed by knocking out futile genes from
the genome of yeast in favor of driving the maximum carbon flux toward lipid
accumulation. Genetic engineering can be used to alter the genes for various
desaturase and elongates to achieve short and saturated fatty acid in the lipid.
Multiple copies of desaturase or elongase can be incorporated to elevate the levels
of unsaturated fatty acids for the nutritional purpose.
In spite of all the promising advantages that come along with production of
SCO in terms of GHG emission reduction and fuel feedstock, the complete process
is outweighed by the cost of production owing to various cost-intensive process
steps, like pretreatment for substrate, feedstock oil, necessity of aseptic conditions,
and biotechnical limitation on yields of production from cheap resources.
Currently, the cost of biodiesel production is almost double the cost of production
of petrochemical diesel.
Even the retail prices of petrochemical diesel are cheaper than bulk cost of
production of biodiesel. This situation presents a pressing need for finding
alternate and cheap substrates and production technologies that can lower the
cost of production for biodiesel, and a synchronized effort is required to translate
and upscale the best of technologies of production at industrial scales.
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CHAPTER 10
Biodiesel Production from
Oleaginous Microorganisms
with Organic Wastes as Raw
Materials
J. Chen
X. Zhang
S. Yan
R. D. Tyagi
R. Y. Surampalli
Tian C. Zhang
10.1 INTRODUCTION
The current problem of biodiesel production is the high cost of feedstock,

vegetable oil, and animal fat. In 2014, around 35% crops (w/w) (almost the
maximum amount that can be used to produce biodiesel) of Canada had been used
to produce biodiesel. The production of biodiesel in 2014 increased 500 times
within 10 years compared with that produced (1 million tons) in 2004. It was
predicated that the production will continuously increase for a long time (around
30 years). The gap will widen between the biodiesel production (which is limited
by the availability of raw material) and the demand on biodiesel. By 2024, around
900 million tons of biodiesel will be required in Canada to satisfy demand.
To mitigate the deep dependence on the raw material (vegetable oil or animal
fat) availability, it is necessary to find some alternative feedstock to produce
biodiesel. It has been widely reported that oleaginous microorganisms could be
used for producing biodiesel (Sitepu et al. 2014, Munch et al. 2015, Patel et al.
2015). Oleaginous microorganisms including yeast, fungi, bacteria, or microalgae,
are those containing more than 20% lipid of the total dry biomass weight (Liang
and Jiang 2013). Some of these oleaginous microorganisms could accumulate high
229
lipid content in the cell under their optimized conditions as summarized in

Table 10-1. Compared to plant seeds such as soybean, sunflower, canola, among
others, which have oil content around 25%to 35% w/w, oleaginous microorganism
(lipid extract up to 70% w/w) is a promising replacement to the traditional raw
materials. In addition, the fatty acid compositions of the oleaginous microorgan-

ism oil are similar to that of vegetable oil or animal fat (Table 10-2). Once the
lipids were extracted from the cells, they could be directly used to produce
biodiesel by the similar process using vegetable oils and animal fat for biodiesel
production. The high lipid content of oleaginous microorganisms and the
convenience of direct utilization of their lipid to produce biodiesel have made
biodiesel production from oleaginous microorganisms very attractive.
The biggest obstacle of biodiesel production from oleaginous microorganism
is the high cost (Benemann et al. 2011, Amanor-Boadu et al. 2014, Santander et al.
2014). It requires around minimum USD 10 to produce each gallon of biodiesel
from oleaginous microorganisms, but it is only USD 3 to 4/gal. diesel or biodiesel
produced from vegetable oil and animal fat (Davis et al. 2011, Delrue et al. 2012,
Ramos Tercero et al. 2014). Autotrophic microorganisms (microalgae) use
sunlight to drive the conversion carbon dioxide to lipid, which requires zero
cost in carbon utilization. However, the cultivation requires large land occupation
and the lipid accumulation is slow (Meng et al. 2009, Bellou et al. 2014).
Heterotrophic microorganisms are promising to produce lipid because of their
ability to accumulate high lipid content (Table 10-1) and rapid growth rate. The
process requires carbon source (sugars) and other nutrients as raw materials. High
grade/purity carbon source and nutrients are costly, which is the main cause of
high biodiesel production cost (Koutinas et al. 2014). To lower the cost, alternative
carbon and nutrient sources are desired.
Organic wastes from agriculture or other industries and residential areas are rich
in carbon and nutrients, often available for free or with a low cost; thus, these have
been used as raw materials for culturing oleaginous microorganisms. This chapter
reviews the sources and properties of these organic wastes, their use for growing
oleaginous microorganisms, their bioconversion to biodiesel with the related tech-
nologies and processes, as well as the parameters that affect lipid accumulation.
10.2 ORGANIC WASTES FOR OLEAGINOUS MICROORGANISM

CULTIVATION
Organic wastes can be divided into agriculture, industrial, and residential wastes.
They usually contain abundant carbon and/or nitrogen and phosphorous, which
are essential in microorganism growth.
10.2.1 Bioconversion of Agriculture Wastes to Biodiesel

Agriculture wastes are mainly wood and crop residues. The major compositions of
these wastes are fibers (60% to 90%) which include cellulose, hemicellulose, and
Table 10-1. Typical Oleaginous Microorganism and Their Lipid Contents.

Type Name Lipid content (%) References
Bacteria Arthrobacter sp. 40 Meng et al. (2009a)

Acinetobacter calcoaceticus 38 Meng et al. (2009a)
Rhodococcus opacus 25 Meng et al. (2009a)
Bacillus alcalophilus 24 Meng et al. (2009a)
Microalgae Chlorococcum sp 32.5 Tongprawhan et al. (2014)
Isochrysis galbana 42 Converti et al. (2009)
Monoraphidium dybowskii 43.47 He et al. (2015)
Ankistrodesmus falcatus 59.6 Singh et al. (2015)
Chlorella sp. 33.6 Han et al. (2015)
Yeast Cryptococcus sp 61.53 Tanimura et al. (2014)
Pichia guilliermondii Pcla22 60.6 Wang et al. (2012a)
Kodamaea ohmeri 53.28 Kitcha and Cheirsilp (2011)
Yarrowia lipolytica 61.7 Tai and Stephanopoulos (2013)
Candida sp 56.58 Duarte et al. (2013a)
Fungi Alternaria alternata 40.7 Bagy et al. (2014)
Epicoccum purpurascens 70 Koutb and Morsy (2011)
Phaeodactylum tricornutum 57.8 Xue et al. (2015a)
BIODIESEL PRODUCTION FROM OLEAGINOUS MICROORGANISM
Mortierella alpina 60.4 Eroshin et al. (2000)

Rhodotorula glutinis 47.2 Liu et al. (2015)
231
232
Table 10-2. Fatty Acid Compositions in Oleaginous Microorganisms and Vegetable Oils.
Fraction in total (% w/w)
Lipid Chemical Component Plant seeds Microorganisms References
Palmitic acid C16H32O2 (C16:0) 7–16 7–37 Ykema et al. (1988),

Palmitoleic acids C16H30O2 (C16:1) 0–1 1–57 Meng et al. (2009a),
Stearic acid C18H36O2 (C18:0) 3–5 1–12 Kim et al. (2010a), Gao
Oleic acid C18H34O2 (C18:1) 17–75 20–81 et al. (2013), Ryu et al.
Linoleic acids C18H32O2 (C18:2) 7–67 3–40 (2013a), Zhang et al.
Linolenic acid C18H30O2 (C18:3) 1–13 1–30 (2014b)
Arachidic acid C20H40O2 (C20:0) 0–1 0–1
BIODIESEL PRODUCTION FROM OLEAGINOUS MICROORGANISM 233
lignin. There are also crude proteins (3% to 12%) and a small amount of metals
(Ca, K, and Na) and phosphate. Agriculture wastes have been used as bioenergy
since ancient times and are still widely used in rural areas today. The major
utilization is for heating and generating power by burning (Welling and Shaw
2013). The problems in these applications are the low efficiency and particulate
matter air pollution.
Pyrolysis is a popular way to use the lignocellulosic materials for bioenergy
and bio-oil. Traditional pyrolysis is a well-established process mainly for
producing charcoal. However, fast pyrolysis technology has been given significant
attention because of its high bio-oil yield (up to 80% w/w dry matters)
with byproducts of char and fuel gas, and no waste stream is produced (Guda
et al. 2015, Yildiz et al. 2015). The bio-oil can be used in boilers, furnaces, engines,
and turbines instead of diesel, and bio-oil can be used for food flavorings,
specialities, resins, agrochemicals, fertilizers, and emissions control agents through
extraction (Bridgwater 2012, Lu et al. 2013). In the process, strict control in
operation is highly demanded to achieve the desired products. In addition,
the drying and grinding are expensive. Liquefaction and gasification can also
convert lignocelluloses to bio-oil or gas fuels and chemicals (Zhang et al. 2011,
Kruusement et al. 2014, Elliott et al. 2015). These technologies have intensive
energy demand.
The bioprocessing of agriculture wastes into bioenergy such as bioethanol and
biogas is a mild way to utilize agriculture wastes. Consolidated bioprocessing
(CBP) is the hottest topic on using lignocellulose for producing biofuel. CBP
consists of four steps including saccharolytic enzyme production, polysaccharide
hydrolysis, hexose sugar fermentation, and pentose sugar fermentation, in a single
reactor (Hasunuma and Kondo 2012). The engineered Saccharomyces cerevisiae
and Paecilomyces variotii are the most studied for bioethanol production by CBP
(Van Zyl et al. 2007, Khuong et al. 2014, Zerva et al. 2014). In biogas production,
pretreatment to hydrolyze the complex cellulose and hemicellulose is normally
performed prior to anaerobic digestion to achieve biogas production (Salehian
et al. 2013, Zheng et al. 2014). The optimal C/N ratio of biogas production is
between 20 and 30 (Mao et al. 2015, Wang et al. 2015a). In general, agriculture
wastes have a very high C/N ratio of ~500 to 600; thus, the codigestion of the
lignocelluloses with dairy manure (with a C/N ratio of 8:1) can be a great choice
(Charles and Charles 2006).
Researchers have been inspired by studies on bioethanol and biogas produc-
tion with agriculture wastes being utilized by microorganisms for cell growth and
lipid production. The main components of agriculture biomass (cellulose, hemi-
cellulose, and lignin) have rather low biodegradability owing to their long carbon
chain, which is not easily used by the microorganisms. Hydrolysis is often
performed before utilization to degrade long carbon chains to comparable short
carbon chains such as sugar, amino acids, and fatty acids, which are ideal carbon
sources for the microorganisms. There are three main hydrolysis methods:
thermal hydrolysis, chemical thermal hydrolysis, and enzyme hydrolysis (Liu
2010). The simplest is thermal hydrolysis.
There are two thermal hydrolysis methods: mild temperature thermal hydro-
lysis (70 °C for 2 to 6 h) and high temperature thermal hydrolysis (110 °C or higher
for 30 min to 2 h) (Abelleira-Pereira et al. 2015, Huang et al. 2015, Urrea et al. 2015,
Xue et al. 2015b). The end products of these two thermal hydrolysis methods
are a little bit different. The mechanism of thermal hydrolysis includes proton
adsorption onto the waste biomass, the biomass hydrolysis reactions, soluble
substances dissolution, and liquid extraction. Proton adsorption onto the biomass
is the essential step in the thermal hydrolysis process. To enhance the performance,
addition of protons into the solution of biomass, such as acidic thermal hydrolysis
was reported (Hu et al. 2015, Mokni Ghribi et al. 2015). In acidic thermal hydrolysis,
pH was usually adjusted to 2 and then subjected to high temperature (>110 °C) for
0.5 to 2 h (Hu et al. 2015). Compared to solo-thermal hydrolysis, the obvious
improvement is that the acidic thermal hydrolysis breaks down the lignin more
efficiently and hydrolyzes the hemicellulose into perspective monosaccharides.
However, byproducts such as furfural and hydroxymethylfurfural, which are
inhibitors of microorganisms, can be generated during strong acidic thermal
pretreatment. Thus, strong acidic thermal pretreatment is rarely performed
(Ariunbaatar et al. 2014). Although acidic thermal pretreatment is efficiency and
reliable, this process has not been widely used because of high operation cost, high
amounts of acid residues, and high amounts of alkali required to neutralize the acid.
To reduce the cost and the consumption of chemicals, biological pretreatment
(via either anaerobic or aerobic processes) with addition of specific enzymes
(Table 10-3) has been widely applied. Because of higher enzyme production in
aerobic treatment, the aerobic biological pretreatment is more commonly used
than the anaerobic one. The critical work of enzyme hydrolysis is to obtain an
optimal enzymatic mixture (generated or added during pretreatment) that is used
to degrade the agriculture biomass (Bussamra et al. 2015). Compared to thermal
hydrolysis and acid thermal hydrolysis, the enzyme hydrolysis needs additional
specific enzymes, which may increase the cost and requires a long fermentation
time (120 to 200 h) (Ghorbanpour Khamseh and Miccio 2012).
Once the agriculture biomass is hydrolyzed, it can be separated into solid part
(residual biomass) and liquid portion. The residual biomass contains lipid, which
could be extracted and used as feedstock for biodiesel production. On the other
hand, the liquid part of the hydrolysis contains almost all the necessary nutrients
for the growth of microorganisms (Heller et al. 2015). It indicates that no
additional chemical media would be needed for microorganism growth. After
being diluted to a proper concentration, the liquid portion can be directly used to
feed the microorganisms. Several studies have successfully produced lipids by
cultivating oleaginous microorganisms in the liquid portion obtained by hydro-
lysis of agriculture biomass (Table 10-4).
Oleaginous yeast and fungi are the most used microbes for lipid production
owing to their high lipid accumulation (up to 60% w/w) within a short cultivation
time (30 to 288 h) and high lipid productivity (0.33 g lipid/g waste). However,
utilization of agriculture wastes for lipid production is still not widely studied
because the requirement of pretreatment is very complicated.
Table 10-3. Parts of Enzyme Hydrolysis.

Type of microbe
Enzyme name Substrate microbe (aerobic or anaerobic) References
β-galactosidase Lactose Bacillus circulans Anaerobic Das et al. (2015)

β-1,4-xylanases Cellulose pulps Aspergillus terreus Aerobic Moreira et al. (2015)
Amyloglucosidase Wheat bran Aspergillus niger Anaerobic Gupta et al. (2015)
ß-xylosidases Lignocellulose Neurospora crassa Aerobic Meleiro et al. (2015)
Cellulolytic enzymes Sugarcane bagasse Trichoderma reesei Aerobic Gasparotto et al. (2015)
Cellic CTec2 Corn stover Trichoderma reesei Aerobic Lin et al. (2015)
235
Table 10-4. Agriculture Wastes for Lipid Production with Oleaginous Microorganisms.
236
Lipid Lipid
Time content productivity
Agriculture Wastes Pretreatment Microbes (h) (% w/w) (g/g wastes) References
Fruit pulp Thermal Rhodosporidium 144 53.2 0.008 Patel et al. (2015)
kratochvilovae
Sweet potato leaves, Enzymatic hydrolysis Trichosporon fermentans 168 35 0.27 Zhan et al. (2013)
stems and stalks
Sweet potato leaves, Acidic thermal hydrolysis Trichosporon fermentans 168 65 0.23 Zhan et al. (2013)
stems and stalks
+wheat straw
Sugarcane bagasse Acidic thermal hydrolysis Yarrowia lipolytica 120 60 0.33 Tsigie et al. (2011)
Corncobs Enzymatic hydrolysis Trichosporon dermatis 168 40 0.17 Huang et al.
(2012a)
Rice straw Acidic thermal hydrolysis Trichosporon fermentans 192 40 0.12 Huang et al. (2009)
Sugarcane bagasse Acidic thermal hydrolysis Trichosporon fermentans 192 40 0.14 Huang et al.
(2012b)
Wheat straw Acidic thermal hydrolysis Cryptococcus curvatus 144 33.5 0.14 Yu et al. (2011)
Sweet sorghum — Mortierella isabellina 96 11 0.09 Economou et al.
(2010)
Corn fiber Enzymatic hydrolysis Mortierella isabellina 144 46 0.18 Xing et al. (2012)
Corncobs Acidic thermal hydrolysis Trichosporon 192 37.8 0.17 Huang et al. (2013)
coremiiforme
Acid- and alkali- thermal Mortierella isabellina 84 30 0.14 Ruan (2014)
followed by enzymatic
hydrolysis
Sugar beet pulp Enzymatic hydrolysis Trichosporon fermentans 96 34.4 0.11 Wang et al. (2015b)
Sugar beet pulp Enzymatic hydrolysis Cryptococcus curvatus 96 43.6 0.14 Wang et al. (2015c)
Sugar beet pulp Enzymatic hydrolysis Trichosporon cutaneum 96 42.1 0.14 Wang et al. (2015c)
Papaya waste Acidic thermal hydrolysis Chlorella protothecoides 110 60 0.021 Heller et al. (2015)
Corncob hydrolysate Acidic thermal hydrolysis Rhodotorula glutinis 72 36.4 0.05 Liu et al. (2015)
Sweet sorghum crops Acidic thermal hydrolysis C. curvatus 96 52.5 0.11 Cui and Liang
(2015)
Sweet sorghum — Lipomyces starkeyi 120 47.3 0.131 Matsakas et al.
(2014)
Cassava starch Hydrolysis by the crude Rhodosporidium 144 63.2 — Wang et al. (2012c)
amylase preparation toruloides
Algal residue Hydrodynamic cavitation Cryptococcus curvatus 30 21 0.08 Seo et al. (2014)
under alkaline
Sweet sorghum enzymatic hydrolysis Cryptococcus curvatus 72 63.98 0.11 Liang et al. (2012)
bagasse
Hemp seed aqueous — R. kratochvilovae 216 55.56 — Patel et al. (2014)
extract HIMPA1
Waste sweet potato Washed, air-dried, and Trichosporon 288 35.6 0.128 Zhan et al. (2013)
vines milled
Corncob waste liquor Acidic thermal hydrolysis Aspergillus sp 96 23.33 — Venkata Subhash
and Venkata
Mohan (2011)
Lignocellulosic Acidic thermal hydrolysis Mortierella isabellina 144 53 0.17 Zeng et al. (2013)
biomass
(Continued)
237
Table 10-4. Agriculture Wastes for Lipid Production with Oleaginous Microorganisms. (Continued)
238
Lipid Lipid
Time content productivity
Agriculture Wastes Pretreatment Microbes (h) (% w/w) (g/g wastes) References
Sugarcane Grinded to powdered Citrobacter youngae 72 53.9 — Maymandi and

Rahimpour
(2015)
Bagasse hydrolysate Acidic thermal hydrolysis T. fermentans 288 39.9 0.117 Huang et al.
(2012b)
Lignocellulosic palm — TSIP9 288 34.2 0.0375 Kitcha and

Cheirsilp (2014)
Sugarcane bagasse Acidic thermal hydrolysis Y. lipolytica 168 58.5 — Tsigie et al. (2011)
Sugarcane bagasse thermal hydrolysis C. protothecoides 192 34 0.093 Mu et al. (2015)
Corncob residues Acidic thermal hydrolysis T. cutaneum 120 29.1 0.144 Gao et al. (2014)
10.2.2 Bioconversion of Industrial Wastes to Biodiesel

Industrial practices bring significant benefits to the society; however, they may
generate huge amounts and enormous varieties of industrial wastes. To satisfy the
increasing human demands, industrial activities will continue to increase. As a
result, more and more industrial wastes will probably be generated in the future,
which may cause serious environmental problems (Cheirsilp and Louhasakul 2013).
Industrial wastes contain industrial waste air, wastewater, and waste residue. The
best way of managing or reducing industrial waste air effluent is to utilize renewable
energies to replace the fossil fuels, which are currently used in industries. Because of
the heavy dependence on industry in our generation, the management of industrial
wastewater and waste residues becomes a great challenge.
To meet the current environmental regulations, manufacturers have to sacrifice
part of their profits to pretreat their industrial wastes. However, the management
approach is usually costly because the treatment plant is of small-scale and low
efficiency. In addition, most of the treatments are aimed to get rid of the waste
quickly and neglect the possibility to reuse some of them to produce value-added
products (Lee et al. 2014). Many of the wastes (water or residue) have great value as
raw materials for producing new material or products. A large proportion of the
industrial wastes are organic wastes. Some of them, such as effluent from food
companies and commercial farms, crude glycerol, and so on (Table 10-5) have high
biodegradability and can be used by microorganisms for growth. Studies have been
performed to utilize industrial organic wastes (wastewater and waste residues) to
grow oleaginous microorganisms to produce lipid (Table 10-5). The process reduces
the waste amount and produces feedstock of biodiesel production.
Most of the industrial organic wastes are already ground and thermal hydro-
lyzed during the production process; thus, no further pretreatment is needed to
enhance their biodegradability. However, for some special wastes, pretreatment is
required to make the waste more suitable for growing microorganisms. For instance,
alkaline thermal hydrolysis is normally used to pretreat piggery wastewater caused
by its high concentration of nitrogen (Marjakangas et al. 2015). After being
pretreated, mostly nitrogen (up to 70%) can be removed by evaporation. The
residual solution can be used for oleaginous microorganism growth to produce lipid.
Because of the increase in biodiesel production, crude glycerol as an un-
avoidable byproduct of biodiesel production through transesterification has
gained more attention. Roughly 50 million tons of crude glycerol were generated
in Canada in 2014. Crude glycerol is a promising carbon resource for the
oleaginous microorganisms; it could easily be taken up by the strain to accumulate
high amounts of lipid (Table 10-5). Using crude glycerol as substrate to feed the
oleaginous microorganisms is attractive because this process not only manages the
byproduct of biodiesel, but also efficiently produces more lipids for biodiesel
production. The only problem is that crude glycerol does not have all the
substrates that should be provided for the growth of microbes. Some chemical
nutrients or other wastes should be added to supply all the necessary nutrients.
Apart from oleaginous yeast and fungi that are widely used to produce lipids,
microalgae are another type of microbes used for lipid production by consuming
Table 10-5. Industrial Wastes for Lipid Production with Oleaginous Microorganisms.
240
Lipid Lipid productivity

Time content (g/g wastes or
Industrial Wastes Pretreatment Microbes (h) (% w/w) L wastewater) References
Stearin, hydrolyzed — Yarrowia lipolytica 64 44 0.38 Papanikolaou et al.

oleic rapeseed oil (2001)
Starch wastewater — Rhodotorula glutinis 60 35 3.9 Xue et al. (2010)
Monosodium — Rhodotorula glutinis 72 20 13.6 Xue et al. (2008)
glutamate
wastewater
Butanol fermentation — Trichosporon cutaneum 192 19 0.081 Chen et al. (2012)
wastewater
Bagasse hydrolyzate — Activated sludge 168 47.3 0.139 Mondala et al. (2015)
microorganisms
Biodiesel-derived — R. diobovatum 120 50 0.118 Munch et al. (2015)
glycerol
Biodiesel-derived — R. babjevae 120 24.2 0.04 Munch et al. (2015)
glycerol
Brewer fermentation Thermal and Chlorella 168 50.6 — Feng et al. (2014)
waste and crude filtration protothecoides
glycerol
Microbial lipid — R. toruloides Y4 120 60 0.19 Yang et al. (2015)
production wastes
Flour-rich waste — A. awamori 2B 200 40.4 0.06 Tsakona et al. (2014)
streams
Piggery wastewater Alkalic thermal C. vulgaris 384–480 34.7 — Marjakangas et al.
hydrolysis (2015)
Ketchup — Ochromonas danica 60 20 — Lin et al. (2014)

Brewery waste — Yarrowia lipolytica 24 30.1 — Poli et al. (2014)
Dairy wastewater R. opacus 100 52 — Kumar et al. (2015)
Pectin-derived Enzymatic Cryptococcus curvatus 48 47.9 0.11 Wang et al. (2015b)
carbohydrates hydrolysis
Potato processing — A. oryzae 72 40 0.11–0.22 Muniraj et al. (2013)
wastewater
Effluent from seafood — Mixed culture 168 61.1 0.146 Cheirsilp et al. (2011)
processing plant Rhodotorula glutinis
and molasses from and Chlorella
Sugarcane plant vulgaris
Crude glycerol — Chlorella vulgaris 144 50 0.007 Sarma et al. (2014)
Bioethanol wastewater — Rhodosporidium 120 34.9 — Zhou et al. (2013)
toruloides Y2
crude glycerol Methanol and soap Cryptococcus curvatus 288 49 0.18 Cui et al. (2012)
removal
Brewery waste Acidic thermal Cryptococcus curvatus 35 31.1 — Ryu et al. (2013b)
hydrolysis
Starch wastewater — Rhodotorula glutinis 60 30 — Xue et al. (2010)
Piggery wastewater — Chlorella pyrenoidosa 240 50 0.21 Wang et al. (2012b)
Brewery wastewater Thermal hydrolysis Chlorella vulgaris 240 42 0.04 Farooq et al. (2013)
Nisargruna biogas — Monoraphidium sp. 624 31 — Tale et al. (2014)
plant effluent
241
industrial organic wastes (mainly organic wastewater). Organic wastewater gen-

erated in the industrial process is in large quantity. It is very difficult and costly to
transfer it from one place to another. Hence, it is preferable to treat/convert
industrial organic wastewater to other products in situ. Moreover, a simple process
and facilities would be more attractive for industries. Compared to other types of
microorganisms, microalgae need fewer extra facilities, and the operation process
is simple. After a long cultivating time (more than 200 h), the microalgae could
accumulate acceptable lipid content (40% to 60% w/w), but the lipid productivity
is sometimes low.
10.2.3 Bioconversion of Residential Wastes to Biodiesel

Residential waste that can be used to cultivate microorganisms contains waste
cooking oils, municipal solid waste, municipal activated sludge, and municipal
wastewater. Waste cooking oils can be directly transferred to biodiesel using
chemical methods (transesterification) after necessary refining. Using waste
cooking oils to produce biodiesel is economically attractive because the production
cost is lower than the market price. In fact, waste cooking oil is also a carbon
source which can be used to feed oleaginous microorganisms for producing lipid,
which can be then converted to biodiesel. However, biodiesel production through
this process would be less attractive than that directly using waste cooking oil for
biodiesel production because it costs more as a result of the extra process for
growing oleaginous microorganism (Yahyaee et al. 2013, Karmee et al. 2015).
Municipal solid waste is normally a mixture of various organics from food
processing plants, domestic and commercial kitchens, and cafeterias and restau-
rants (Uçkun Kiran et al. 2014). Municipal solid waste contains around 40% food
waste which contains up to 30% lipid. Lipid from food waste can be separated by
solvent extraction. Even though this method is effective and efficient, the recovery
of solvent and the lipid separation are costly; in addition, the solvent used to
extract the lipid is harmful to the environment (Karmee et al. 2015). Today,
anaerobic digestion is the common method used to manage the municipal solid
waste. The anaerobic digestion process could produce methanol as a source of
energy, and the residues could be used for fertilization (Suwannarat and Ritchie
2015). During the anaerobic process, some organic materials were biodegraded to
volatile free fatty acids, which could be used by oleaginous microorganisms to
accumulate lipid in the cell. Recent research showed that a lipid productivity of
0.15 g lipid/g free fatty acid could be reached by using the effluent of anaerobic
digestion (Vajpeyi and Chandran 2015).
Municipal activated sludge is widely generated in the municipal wastewater
treatment plant. To reduce the chemical oxygen demand, biological oxygen
demand, total nitrogen, total phosphorous, and trace elements in the municipal
wastewater, aerobic-activated microorganisms are commonly used to treat mu-
nicipal wastewater. Most of the environmental toxic elements are captured and
used by microorganisms. After removing these microorganisms (sedimentation),
the environmental toxic elements can be reduced, and the effluent of the municipal
wastewater treatment plant can be discharged into the receiving water body.
However, during wastewater treatment, there is a large quantity of wastewater

sludge generated. Because of this high quantity, sludge must be treated correctly and
then disposed of. Municipal activated sludge contains a very high concentration of
organic waste, which can be up to 90% w/w, and hazardous metals. Directly
dumping sludge to landfill would cause high greenhouse gas emissions because there
is no methane capture, and then leaches lead to groundwater pollution once they
reach groundwater. The main current technology used to manage municipal
wastewater sludge is anaerobic digestion. After being concentrated, at temperature
of 30° to 60 °C and oxygen limited conditions, sludge can be bioconverted to biogas
in a hermitic tank. The biogas generated can be used as heating energy. This
management can reduce the quantity of sludge; however, it is not efficienct for three
reasons: (1) although the sludge contains high organic wastes that are mainly the
cells of aerobic microorganisms, the biodegradability of the sludge is low, so that
either some pretreatments should be introduced or longer digestion time should be
performed; (2) because of the long digestion time, the hermitic tank used for
anaerobic digestion usually has a large dimension; and (3) because anaerobic
digestion mainly reduces organic wastes, the hazardous metals are still in the
residues and are concentrated. The further management of the residues is more
difficult to prevent pollution of hazardous metals.
Studies have revealed that municipal wastewater sludge can be used as substrate
to culture oleaginous microorganisms (mainly yeast and fungi). To increase the
biodegradability of the sludge, pretreatments such as thermal hydrolysis, acid
thermal hydrolysis, enzymatic hydrolysis, and alkali thermal hydrolysis are intro-
duced (Olkiewicz et al. 2015, Tian et al. 2015). When the concentration of the sludge
used to feed the microorganism is high, the possibility of inhibition in microor-
ganism growth is high because the amounts of metals in the sludge medium are
high. Pretreatments such as washing and centrifugation are used to remove the
inhibitors and to enhance the lipid accumulation (Zhang et al. 2014a).
Both the treatment on municipal wastewater and the management of
municipal wastewater sludge are complicated, so some researchers proposed to
use municipal wastewater directly to cultivate oleaginous microorganisms for lipid
production. Previous research has tested microalgae to accumulate the lipid with
wastewater (Cabanelas et al. 2013). However, there are still many problems in the
process, such as large cultivation area, long fermentation time, and the lipid
content (less than 30%) owing to the low concentration of carbon and nutrients.
To solve the problems, utilization of concentrated municipal wastewater to feed
oleaginous yeast or fungi should be studied.
10.3 PARAMETERS AFFECTING LIPID ACCUMULATION
Among all the aforementioned substrates, municipal wastewater sludge, wastes

from sugar production companies, and crude glycerol are the raw materials that
are frequently used as feed stocks of oleaginous microorganisms, owing to their
promising carbon resource and high daily generation amounts. Oleaginous yeast
and fungi are mostly used in the current research for lipid accumulation. As
shown in Tables 10-4 to 10-6, even using the same oleaginous microbe and the
same substrate, different results have been reported of lipid content and lipid
productivity. Because of the current low lipid accumulation, the lipid production
by oleaginous microorganisms is still in the laboratory scale. There should be some
essential factors which can highly affect the lipid accumulation. These factors are
determined as affecting parameters. By controlling the parameters in an optional
range, the lipid accumulation could be enhanced. Therefore, understanding the
effects of the parameters on lipid accumulation may take the lipid production by
oleaginous microorganisms into the industrial scale.
The parameters mainly affect the lipid production in two ways: one is on cell
growth and the other is on lipid accumulation. The bright side is, as reported, lipid
is a mixed growth–associated product of oleaginous microbes (Amaretti et al.
2010). More cells generated in the growth phase could lead to more lipid
accumulation in the lipogenous phase. The practical method is to grow the cells
as much as possible in the growth phase and to let them accumulate lipid by
controlling the parameters in certain conditions. To meet these aims, many
parameters should be taken into consider. The most effective ones are pH,
temperature, dissolved oxygen (DO), carbon to nitrogen (C/N) ratio, carbon and
nitrogen sources, trace elements, and fermentation mode.
10.3.1 pH Effects
pH is one of the most important parameters that can highly affect the cell growth
of the microorganisms. First, at a suitable range of pH, certain enzymes in the
microorganisms are activated (Figure 10-1). The microorganisms absorb sub-
strates fast at the suitable range of pH; thus, the cell division is enhanced, and more
cells of microorganisms could be obtained in a shorter time. Second, pH can affect
the form of ammonia and phosphorous [Equations (10-1) through (10-4)].
The major form of ammonia is NH3·H2O at high pH and NH4+ at low pH
[Equation (10-1)]. There are always four forms of inorganic phosphorous: H3PO4,
H2PO4−, HPO42− and PO43−, and the composition of them varies with pH. Because
microorganisms need mostly NH4+, H2PO4−, and HPO42− to grow, the pH
condition is very important to make sure these three forms are in the right
proportion. Third, pH can affect the concentration of heavy metals. Heavy metals
exit normally as ions in the water under the acidic pH condition and as
precipitates under the alkaline pH condition. Low concentration of heavy metals
may enhance the cell growth of the microorganisms, but high concentration of
heavy metals could cause inhibition. Alkaline pretreatment is a common method
to eliminate the overload of heavy metals. Moreover, pH can affect the proton
pumping rate. Accompanying the cell growth, a lot of protons are generated in the
cells, and the cells need to pump the protons out of the cells; this process
contributes most of the energy generated (80%) by the cell growth. When pH
is low, the electrical potential difference between the two sides of the cell (inside
the cell and outside the cell) is little, and the proton pumping process consumes
Table 10-6. Residential Wastes for Lipid Production with Oleaginous Microorganisms.
Lipid productivity
Time Lipid content (g/g wastes or
Residential wastes Pretreatment Microbes (h) (% w/w) L wastewater) References
Municipal solid Acid hydrolysis Cryptococcus aerius 144 30.6 0.17 Ghanavati et al. (2015)
waste followed by
enzymatic
hydrolysis
Wastewater — Rhodosporidium 72 8 — Xue et al. (2008)
toruloides
Primary and — Chlorella vulgaris 168 33 — Ebrahimian et al.
secondary (2014)
municipal
wastewater
Wastewater — Chlorella — 11 — Chiu et al. (2015)
Sewage sludge — Chlorella vulgaris 264 20.1 0.05 Cho et al. (2015)
Domestic effluent Physical removal of Chlorella vulgaris 432 27 — Cabanelas et al.
large particles (2013)
and fat materials
Food waste Enzyme hydrolysis Chlorella vulgaris 192 33.3 — Lau et al. (2014)
Waste Cooking oil Filtration and Starmerella 240 — — Maddikeri et al. (2015)
washing bombicola
245
Optimum pH
Enzyme activity
pH
Figure 10-1. Enzyme activity versus pH (Grasso et al. 2015).
more energy; otherwise this process consumes less energy. Overall, it shows that
pH can significantly affect the cell growth of microorganisms.
NH4 þ þ OH− — NH3 · H2 O (10-1)
H3 PO4 þ OH− — H2 PO−4 þ H2 O (10-2)
H2 PO−4 þ OH− — HPO2−

4 þ H2 O (10-3)
−
4 þ OH — HPO4 þ H2 O
3−
HPO2− (10-4)
10.3.2 Temperature Effects

Temperature can highly affect the cell growth of the microorganisms. As shown in
Figure 10-2, all microorganisms have their own optimal growth temperature. The
suitable temperature varies between different types of microbes. There are five
classifications on microorganisms according to their optimal growth temperature:
• Hyperthermophile (optimal growth temperature 80 °C and up to 120 °C);
• Thermophile (optimal growth temperature between 50 °C and 85 °C);
• Mesophile (optimal growth temperature between 30 °C and 40 °C);
• Psychrotroph (optimal growth temperature between 15 °C and 30 °C); and
• Psychrophile (optimal growth temperature between 10 °C and 15 °C).
Regardless of the enzyme activity under optimal growth temperature, the
different membrane structures are another main reason why microorganisms have
different optimal growth temperature. The membrane of most microorganisms is
Optimum Temperature
Growth rate
Minimun Maximum
Temperature
Figure 10-2. Growth rate at different temperatures (Hatfield and Prueger 2015).
composed of either saturated or unsaturated fatty acid. Normally, saturated fatty

acid is in a solid phase, whereas unsaturated fatty acid is in a liquid phase at room
temperature (20 to 25 °C). Thus, temperature can affect the fluidity of the
membrane, which highly affects its function. For hyperthermophiles that can
undertake very high temperature, the structure of its membrane is unique, mainly
composed of C5 compound, phytane, and isoprenoid substance. These substances
can remain stable at very high temperature.
Temperature can affect dissolved oxygen concentration. When temperature
goes up, the solubility of most airs such as oxygen goes down (Figure 10-3). Under
higher temperature, the available dissolved oxygen is lower. The further discussion
of this effect is posed in the next section on DO effect.
Temperature can affect the solubility of heavy metallic salts. In most cases, the
solubility of heavy metallic salts has positive correlation with the temperature.
Higher temperature provides higher solubility of most heavy metallic salts.
DO
DO(ppm)
Temperature (°C)
Figure 10-3. DO under different temperatures in standard atmospheric pressure.
Overall, temperature is a very important parameter that should be taken into

consideration in cell growth and lipid accumulation in microbes.
10.3.3 DO Agitation and Aeration Effects

DO is the parameter that is most affected by the temperature of water (tempera-

ture goes up, DO goes down), pressure of the air (pressure goes up, DO goes up
also), salinity of water (higher salinity in the water causes less DO in the water),
and the microorganism activities (more aerobic microorganisms could cause less
DO). To determine the DO effect on the microorganisms, two more parameters
are involved: oxygen uptake rate (OUR) and oxygen transfer rate (OTR). The
OUR is used to describe the activities of aerobic microorganisms. Normally the
more aerobic microorganisms there are in the water, the faster OUR occurs. OUR
and cell mass concentration of aerobic microorganisms are in linear correlation:
OUR = QO2X (QO2 is the specific rate of oxygen consumption, and X is the cell
mass concentration). OTR describes the oxygen transferring from air to water,
and the transportation only occurs at the interface between air and water:
OTR = KLa × (C − C), where KLa is the volumetric oxygen transfer coefficient
(h−1), which is determined by the interfacial resistance. C and C are saturated and
actual oxygen concentration in the water, respectively. The actual concentration of
oxygen is also the current oxygen that is available for the activity of aerobic
microorganisms. The high activity of aerobic microorganisms requires that the
actual concentration of oxygen remains at a high level. The relationship between
the actual concentration of oxygen C and other parameters (all defined previously)
can be described by Equation (10-5) (Sahlmann et al. 2004)
C = C þ OTR − OUR (10.5)

From Equation (10-5), it is obvious that there are three methods to increase the
actual concentration of oxygen: (1) increase the saturated concentration of oxygen
in the water; (2) increase the OTR; and (3) decrease the OUR. First, the saturated
concentration of oxygen is determined by the surrounding environmental con-
ditions; it cannot be changed without the modification of environmental condi-
tions. Second, the main aim is to let the aerobic microorganisms grow as much as
possible, so the OUR increases gradually during the growth phase. It is impossible
to increase the cells of aerobic microorganisms and to decrease the OUR.
Therefore, the only way to increase the actual concentration of oxygen is to
increase the OTR.
To increase the OTR, there are three functional parameters: KLa, C , and C.

C and C are fixed at each instant moment. The only parameter that can be
modified is KLa. Based on the two-film theory, the oxygen is transferred from air
phase to interface (the supposed phase that is between air phase and water phase)
in the first step and then transferred from interface to water phase. It is recognized
that the concentration of oxygen in the interface is stable and invariable. The
crucial step is the mass transfer from air phase to interface. There are several
approaches to enhancing this step: (1) by enlarging the interface between air phase
and interface so that more oxygen in the air phase can contact a larger interface,
and some aeration models (such as flat aeration and jet aeration) have been
designed based on this consideration; (2) by enhancing the concentration of
oxygen in the air, for example, pure oxygen or compressed air in the air phase can
lead to faster mass transfer to the interface; (3) by increasing the agitation rate to
reduce the viscosity coefficient of water, and make the interface between air and
water thinner, and thus the transferring rate is enhanced; or (4) by increasing the
temperature. This is a special method. Higher temperature could enhance
the OTR—as temperature goes up, the water irons are activated—and thus, the
viscosity coefficient of water goes down, which then enhances the transferring rate.
But temperature can affect not only the viscosity coefficient of water but also
solubility of dissolved oxygen, which is also a determinate parameter of oxygen
transferring rate.
10.3.4 C/N Ratio Effects

With the increase in C/N ratio, different metabolic behaviors happen in the
oleaginous cells. Under carbon limitation, lipid accumulated in the cells could be
digested to generate energy for supporting cell activities. Under the balanced
carbon furnishing condition, oleaginous microorganisms can rapidly generate
cells (cell numbers increase) rather than lipid accumulation. In the carbon excess
condition, part of the carbon is directly used toward lipid accumulation. This is the
ideal carbon condition to be used to generate the lipid by oleaginous micro-
organisms. With overdose of carbon, inhibition occurs, and in addition the
oleaginous microorganisms use the carbon to generate high amounts of organic
acid (citric acid) and only part of lipids in the cells (Azim et al. 2008, Abu Bakar
et al. 2015).
Heldal et al. (1996) have reported that a C/N ratio between 5 and 10 is
superior for the cell growth. It was discovered that the C/N ratio of the dry cell is
around 5 (153:33) at the exponential growth phase (Heldal et al. 1996), which
explains why a C/N ratio of 5 to 10 enhances cell division. Similar C/N ratios
(5 to 10) are commonly used to cultivate the oleaginous microorganisms during
the cell exponential phase. However, the optimal C/N ratio for lipid accumulation
is very different in different studies. Normally, the carbon sources that are easier to
be used by the cell have a smaller C/N ratio. For example, the widely used C/N
ratio for lipid accumulation by feeding glycerol as carbon source is between 30
and 60 (Liang et al. 2010, Duarte et al. 2013b), whereas it is 120 or even more if
lignocellulosic waste is used as a carbon source (Ruan et al. 2012, Cheirsilp and
Kitcha 2015, Patel et al. 2015).
Among all carbon sources that have been used as feedstock for lipid
accumulation in the oleaginous microorganisms, glucose, glycerol, and molasses
are the best carbon sources for cell growth and lipid accumulation (Gautam et al.
2013). It is also observed that carbon sources with short carbon chains are superior
to those with long carbon chains. Pretreatments (thermal, acid thermal, alkaline
thermal, and enzyme hydrolysis) have been often performed to break the long
carbon chain to short carbon chain.
Campos et al. (2014) utilized five nitrogen sources, NH4Cl, NH4OH, NaNO3,
CH4N2O, and a mixture of all these sources, to evaluate the biomass yield and the
lipid content by the oleaginous microalgae Nannochloropsis salina. The highest
biomass yield was observed by using NaNO3 or the mixture; this was also observed
by Wu et al. (2013). The highest lipid content was observed by using the mixture
as nitrogen sources.
In addition to the optimal C/N ratio, the components of carbon sources and the
nitrogen sources are also important factors affecting biomass and the lipid
productivity of oleaginous microorganisms. For certain oleaginous microorganisms,
the optimal C/N ratio can vary in a large rang due to different feeding substrates.
10.3.5 Trace Elements Effect

Trace elements are essential micronutrients that the microorganisms require in
minute quantity for their normal functions, mainly including chromium, cobalt,
copper, fluorine, iodine, iron, manganese, molybdenum, selenium, and zinc
(Strachan 2010). These trace elements are normally essential to the cell growth
of microorganisms, but some of them are also toxic to the microorganisms. For
instance, copper and manganese are essential for microorganisms but they are
toxic as well (Knežević et al. 2014). In general, the constitution of each of these
trace elements is less than 0.01% of body mass.
Of these trace elements, iron and zinc are particularly important for obtaining
the optimum growth of the strain. The strain cultivated with the addition of trace
elements grows more and faster than one without trace elements (Zhang and
Jahng 2012, Nagano et al. 2013). Even though trace elements such as iron, zinc,
and magnesium are needed for cell composition, an overdose of them can also
cause inhibition, because the activity of enzymes is repelled or even stopped in the
high concentration of trace elements (Knežević et al. 2014). It is important to
control them under proper concentrations. Alkaline precipitation is a promising
method to remove overdosed trace elements.
10.3.6 Fermentation Mode Effects

There are three fermentation modes: batch, fed batch, and continuous fermenta-
tion. In the conventional batch process, all the nutrient substrates are filled into
the reactor at the beginning, and the microorganism is inoculated into the
fermenter. During fermentation, nothing except oxygen, antifoam agent, and
acid or base (used to control the pH) are added to the reactors. When product
(lipid) productivity reaches the highest, the fermentation will be stopped. Micro-
organisms will be harvested for further processing. The conventional fermentation
process is easy to be prepared and performed, but the culture age during the
fermentation is not able to be changed. The strain in the batch mode goes through
all the four phases: lag phase, log phase, stationary phase, and death phase. The
strain grows fast in the log phase, but the production mainly happens in the
stationary phase. The concentration of nutrient substrates has a big difference for
the growth of strain and the production. In general, high production needs a high
concentration of nutrient substrates; however, a high concentration of substrates
can inhibit the growth of the strain. To achieve high cell division and high lipid
accumulation in the batch process, an eclectic concentration of nutrient substrates
is used. However, this eclectic concentration can neither grow the maximum
number of cells nor accumulate maximum product. Thus, the productivity of the
strain is usually not efficient in the batch culture.

To prevent nutrient depletion, to achieve high cell density, and to produce
high metabolite of the microorganisms, fed batch is introduced to meet these
goals. Fed-batch culture is the biotechnological process in which two or more
nutrient substrates are fed to the reactor during the cultivation. Normally, a basic
medium is added to the reactor at the beginning to support the cell growth,
followed by the addition of nutrient substrates to maintain the concentration of
nutrient in a desired level. Fed-batch culture is superior to conventional batch
culture when the nutrient concentration can highly affect the productivity (in this
case lipid accumulation in the cells).
For both batch culture and fed-batch culture, the culture ends at the point of
harvesting. The desired product cannot be generated continuously. Despite that
another run follows the harvesting, the strain in the reactor needs to go through
one more round of the four growth phases. The lag phase and log phase are time
consuming. In continuous fermentation, the strain in the stationary phase should
be maintained to continuously produce the product. The nutrient substrates are
continuously fed into the reactor, and the broth in the reactor is withdrawn in the
same flow rate. The medium in the reactor maintains a stable volume, and the
product is continuously generated and harvested. This process is rarely used in
lipid production because the process of lipid production is critical, and continuous
batch culture can be forced to stop due to the contamination. Compared to batch
mode, fed batch and continuous batch usually have higher productivity, but they
also have a longer fermentation time than batch mode.
10.4 CASE STUDIES
Based on the limited studies, the first and only commercial single cell oil production
factory was carried out in a pilot plant located in Austria in 1985. The oleaginous
fungus Mucor circinelloides had been inoculated in stirred-tank fermenters of
220 m3. Harvesting and drying of the biomass had been followed by oil extraction,
refinement, and purification. This factory was operated for about 6 years and finally
was forced to be shut down as there was no more benefit thereafter. As the price of
feedstock oil is the essential factor in biodiesel generation, reviewing an international
statistical yearbook of these years explained why the plant had to be stopped: in
1979, Organization of the Petroleum Exporting Countries (OPEC) cut the produc-
tion of crude oil, which directly pushed the price of crude oil to go up sharply. In less
than 3 years (1979 to 1981), the price of crude oil went from around USD 40/barrel
to around USD 100/barrel. The high price of crude oil made the production of single
cell oil to be cost-effective, and thus, the first factory appeared due to demand in
1985. However, 1 year after the biodiesel production plant appearance, the OPEC
chattel collapsed in 1986, which made the price of crude oil go down immediately to
less than USD 30/barrel. The single cell oil production company had to close the
company in 1991 because the cost of biodiesel production was no more attractive
than petro-diesel.
Since the first single cell oil production company was closed, no more
biodiesel production from the oleaginous microorganism company has been
taken in practice. The production using oleaginous microorganisms has been
limited to the laboratory. Nevertheless, researchers have never given up the
opportunity to bring them to industrial practice. They have become more
cautious. Cost and technical evaluations have been used to guide the further
research and the possibility of industrial utilization.
Among the evaluation reports, the most effective report was made by
Koutinas et al. (2014). The assumptions used in the study were the following:
production capacity was set at 10,000 t/year; the microbe was Rhodosporidium
toruloides; medium included glucose, peptone and yeast extract; the fermentation
was conducted for 134 h with fed-batch mode; lipid content, biomass yield, lipid
yield, and productivity were 67.5 (w/w), 0.35 g/g glucose, 0.23 g/g glucose, and
0.54 g/L-h, respectively. It was assumed that the optimal condition could be
realized in real practice. This evaluation aimed to discover the potential of the
lowest production price of lipid in the industrial practice. The research revealed
three great factors that can highly affect the microbial lipid price: (1) the price of
feedstock, showing that the microbial lipid price is USD 11.3/gal. with cost-free
feedstock compared to USD 18.3/gal. with feedstock cost of USD 400/t; (2) the
lipid productivity, meaning that microbial lipid price can be reduced from USD
11.3/gal. to USD 5.33/gal. (with feedstock cost free) with the lipid productivity
increased five times; and (3) fermenter cost because the costliest instrument of the
process is the fermentation reactor, which contributes 74% of the total instru-
ments of the lipid production process and the operation of the reactor contributes
49% of the total operation cost.
The study indicates that there are three approaches to reducing the microbial
oil production price: by replacing the nutrients with cost-free substrates; by
enhancing the lipid productivity; and by reducing the fermentation reactor
investment (Koutinas et al. 2014). However, the study did not revise the
fermentation time which affects the microbial oil price. In fact, fermentation
time can affect the fermentation reactor volume (shorter fermentation time
requires a smaller fermentation reactor). Thus, fermentation reactor can be an
important factor on the final lipid production cost.
10.5 CHALLENGES AND FUTURE PERSPECTIVES
Because the price of biodiesel produced from oleaginous microorganisms is the

main obstacle to prevent it from industrial practices, the current work should
focus on reducing the production price. The feasible methods would be to find
cost-free substrate replacement, enhance lipid productivity, and reduce fermen-
tation reactor volume. If lipid productivity and fermenter volume reduction could
be done at the same time, it would reduce the lipid production cost. To achieve
this, the operation conditions and the microorganism growth conditions should
be studied and controlled in the optimal range, so that the microorganisms may
accumulate high lipid in a short time. Simplifying the operation condition may
also reduce the cost in some parts. Further research is needed on eliminating the
sterilization and on performing in situ lipid extract.
10.6 SUMMARY
The major obstacle of biodiesel production from microorganism lipid is the high
cost. Utilization of organic wastes generated in agriculture, industries, and human
activities to grow oleaginous microorganisms is a promising strategy of reducing
biodiesel production cost. Compared to agriculture wastes, industrial and resi-
dential wastes are more suitable substrates and/or nutrients for oleaginous
microbe production as they usually can be directly used without pretreatment.
Enhancing lipid productivity is another way of reducing biodiesel (from lipid)
production cost. By controlling the cultivation conditions including pH, DO, C/N
ratio, temperature, trace elements, and fermentation modes, lipid productivity can
be improved. The combination of using organic wastes and cultivating oleaginous
microorganisms under suitable conditions would bring the biodiesel production
from microbial lipid to the industrial-scale practice.
10.7 ACKNOWLEDGMENTS
Sincere thanks are due to the Natural Sciences and Engineering Research Council
of Canada (Grant A 4984, Strategic Grant- STPGP 412994-11 and Canada
Research Chair) for their financial support. The views and opinions expressed
in this chapter are those of the authors.
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CHAPTER 11
Oil Extraction Using
Wastewater Sludge
L. R. Kumar
S. K. Yellapu
L. Kumar
R. D. Tyagi
11.1 INTRODUCTION
Current biodiesel feedstock like agriculture crops, fossils, and wood cause
environmental concerns (e.g., global warming, greenhouse gas emissions) and
lead to depletion of natural resources (e.g., deforestation). The soaring price of
edible oil leads to unaffordable biodiesel production. There is a need for more
cost-effective feedstock for biodiesel production. Today, municipal sludge is
gaining attention because it can meet the requirements of a renewable energy
source owing to its high carbon content (Kwon et al. 2012). Sludge is a waste
formed during wastewater treatment that needs specific treatment before disposal.
Sludge treatment requires a major cost in wastewater treatment plants. Based on
the treatment train, sludge can be divided into primary, secondary, and scum
sludge. Primary sludge is obtained after mechanical treatments like sedimentation,
screening, and flocculation. It has high volatile solids, fats, and grease content.
Secondary sludge is generated in biological treatment processes; it contains many
microbial cells but with fewer solids and less oil concentration compared with
primary sludge because microbes consume them at secondary treatment. Scum is a
skilled material that floats on the surface of primary and secondary settling tanks
in wastewater treatment plants. It is mainly composed of animal fat, vegetable oil,
food wastes, plastic material, soaps, waxes, and many other impurities discharged
from restaurants, households, and other facilities (Bi et al. 2015).
Recently, sludge generated in wastewater treatment plants has been used as
feedstock for biodiesel production. Figure 11-1 shows oil extraction strategies for
different types of sludge. This chapter overviews current methods of sludge
disposal, sludge characterization, different methods for oil extraction from sludge,
263
Sludge
Primary and
Secondary Scum
sludge
Thermo- Mechanical Solvent Mechanical

chemical methods extraction treatment
Solvent Solvent
Oil Oil
extraction extraction
Oil Oil
Figure 11-1. Oil-extraction techniques reported on different types of sludge.
properties of oil extracted from sludge, techno-economic studies, and recent

developments in the field.
11.2 METHODS OF SLUDGE DISPOSAL
Annual sludge production in Canada, the United States, and Europe is depicted in
Table 11-1. Annual sludge production is rising as a result of the increase in many
industries and treatment plants, making sludge handling and disposal methods an
area of great concern.
In general, sludge is treated in wastewater plants by many different methods.
After treatment, sludge is disposed of in different ways (Table 11-2). This section
describes different sludge disposal methods.
Table 11-1. Annual Sludge Production.
Continent/country Canada United States Europe
Sludge volume (million tons/year) 0.8 5.4 6.1

OIL EXTRACTION USING WASTEWATER SLUDGE 265
Table 11-2. Sludge Disposal Method Used in the

European Union.
Sludge disposal method Use (%)

Agriculture 37
Incineration 11
Landfill 40
Others 12
11.2.1 Agricultural Reuse

Because wastewater sludge contains phosphorus and nitrogen, it can be used as a
fertilizer because N and P in sludge are essential for plant growth (Metcalf and
Eddy 1991). However, reuse of sewage sludge for agricultural use faces many
obstacles owing to the presence of heavy metals in sludge and the strict
regulationsfor meeting the specific criteria regarding requirements of substances
present in sludge, which are often not met (Davis 1996).
11.2.2 Incineration
Incineration can play a role in long-term sludge disposal because agricultural
reuse, land filling, and sea disposal are no longer an outlet because of legal
limitations (Malerius and Werther 2003). Modern fluidized-bed incinerators are
more and more attractive regarding capital as well as operating costs compared
with conventional incinerators (Mininni et al. 1997). Moreover, the technology of
incineration has recently been improved by incorporating the use of process
engineering and energy efficiency. The advantages of incineration are summarized
as follows:
• Substantial reduction of sludge volume,
• Thermal destruction of toxic organic compounds,
• Recovery of energy content because calorific value of sewage sludge is equal to
brown coal, and
• Reduction in odor generation.
One of the major disadvantages for the use of incineration is harmful
emissions during incineration that are undesirable. The amount of sludge being
incinerated from sludge produced has increased to 24% in Denmark, 20% in
France, 15% in Belgium, 14% in Germany, but in the United States and Japan, the
percentage is 25% and 55%, respectively (Lundin et al. 2004).
11.2.3 Scum Disposal

Scum sludge is mainly composed of fats, oil, and grease, which are waste products
rich in free fatty acids (FFAs) and with high oil content (Bi et al. 2015). Scum
sludge is discarded into garbage cans as municipal trash and directly disposed of in
landfills. The disposal scum landfills not only increases the cost of treatment
facilities but also causes many environmental problems (Sangaletti-Gerhard et al.
2015). Landfill leachate could be a potential source of groundwater pollution.
Treatment plants must pay for the disposal of scum in landfills. For instance, the
Metro plant spends $200,000 a year in transporting and burying scum. Therefore,
it is important to develop a technology for energy recovery and beneficial use of
scum (Bi et al. 2015).
11.3 SLUDGE CHARACTERIZATION
As shown in Table 11-3, primary sludge contains 93% to 99.5% water with a high
ratio of suspended and organic solvents. Waste-activated sludge, or secondary
sludge, has a total suspended solids concentration between 0.8% and 1.2%,
depending on the type of biological treatment processes used. Scum contains
around 56% by weight of oil of total scum that can be extracted to convert into
fatty acid methyl esters (FAMEs), because biodiesel is a mixture of fatty acid alkyl
esters and solid sludge, containing 8% to 20% fats and grease. Besides, sludge also
contains cellular membranes of microbes that are mainly composed of phospho-
lipids, which also can serve as feedstock for biodiesel production.
11.4 VALUABLE PRODUCTS FROM WASTEWATER SLUDGE
Several relevant products can be produced using wastewater sludge, including

biogas, fuel gas, electricity generation, biofertilizers, biopesticides, bioplastics,
hydrolytic enzymes, biosorbents, among others, depending on the technique used
(Table 11-4).
The problems associated with some of the processes shown in Table 11-4
(e.g., wet air oxidation and supercritical water oxidation) are high maintenance
and operating cost, the requirement of safety systems for pure O2, production of
ammonia that requires further purification, and high metal concentration in
products (Tyagi and Lo 2013).
11.5 RECENT STUDIES ON OIL EXTRACTION USING WASTEWATER

SLUDGE
11.5.1 Thermochemical: Pyrolysis

Pyrolysis is the thermal decomposition of organic substances under anaerobic
circumstances. Oils (organic liquids and tars), gasses, char, and reaction water are
the primary products formed by pyrolysis. Temperatures between 275 °C and 500 °C
has been used to produce oil from wastewater sludge. It has been a topic of interest
Table 11-3. Characteristics of Primary and Activated Sludge and Scum.

Parameter Primary sludge Activated sludge Scum Composition
Total dry solids (TS) % 5–9 0.8–1.2 15.8 Water, varying to more than 95%
Volatile solids (VS) (% TS) 60–80 58–68 55 Nontoxic organic carbon compounds
(60% on dry weight basis)
Water content (%) 90–95 98–99 28.2
Nitrogen (% TS) 1.5–4 2.4–5 — Toxic pollutants: heavy metals (Zn, Pb, Cu, Cr,
Phosphorus (% TS) 0.8–2.8 0.5–0.7 — Ni, Cd, Hg, As)
Potash (K2O % TS) 0–1 0.5–0.7 —
Cellulose (% TS) 8–15 7–9.7 —
Iron (Fe g/kg) 2–4 — —
Silica (% TS) 15–20 — — Inorganic compounds: Aluminates, calcium,
pH 5–8 6.5–8 — silicates, and Mg-containing compounds
Grease and Fats (% TS) 7–35 5–12 40–55
Protein (% TS) 20–30 32–41
Alkalinity (mg/L) 500–5,000 580–1,100 — Pathogens and other microbiological
Organic acids 200–2,000 1,100–1,700 pollutants
OIL EXTRACTION USING WASTEWATER SLUDGE
—
Energy content (kJ/ kg TS) 23,000–29,000 19,000–23,000 —
267
Table 11-4. Various Products Made from Wastewater Sludge Using Different
Techniques/Processes.
Technique Process Products developed

Biochemical Anaerobic and aerobic Biogas, fuel gas, electricity

digestion, microbial generation, bioplastics,
fuel cell biopesticides, and
others
Thermochemical Incineration and CO2, H2O, Syngas, fuel gas,
co-incineration, bio-oil, ash, char,
gasification, pyrolysis, biosorbents, heat,
wet air oxidation, building material,
hydrothermal fertilizer
treatment and
supercritical water
oxidation
Mechanical-chemical Ultrasonication and Biodiesel, H2 gas, proteins,
homogenization and enzymes
for researchers because oil with low NO2, and SO2 emissions are produced. The
primary product formed in the process is pyrolysis oil, or bio-oil, which can be
used as a fuel. The choice of reactor configuration is necessary to maximize
pyrolysis oil yield. Three main technologies have been used for fast pyrolysis:
ablative, fluid bed, and vacuum. The most common configuration for pyrolysis
reaction is fluid beds owing to their ease of operation and fast scale-up. Only fluid
beds are used on a commercial scale (Bridgwater et al. 1999). Process temperature,
reaction time, operating pressure, and sludge characteristics are important factors
that decide the oil yield (Khiari et al. 2004). The heating value of biomass pyrolysis
oil is around 17 MJ kg−1, which is around 40% to 50% of that exhibited by
hydrocarbon-based fuels (Czernik and Bridgwater 2004). Schematic of a labora-
tory-scale pyrolysis reactor is shown in Figure 11-2.
The pyrolysis reactor is wrapped with a temperature-controlled heating tape
to add heat to achieve pyrolysis temperature. Gas, such as N2, H2, and CO2, is used
to maintain an oxygen-free environment. The flow from a gas cylinder is
controlled by a flow meter, and gas is heated using a heating coil inside the
chamber. The oil and gasses formed as products are transferred to a separator
where oil is collected in a vial, and gasses pass through a condenser above the
separator (Kim and Parker 2008).
Primarily, studies have been conducted on pyrolysis without the use of
catalysts. However, some studies have reported use of catalysts in the pyrolysis
reaction (Kim and Parker 2008), who wanted to see the impact of pyrolysis
conditions and use of pretreatment of sludge on oil yield. In their paper, the
pyrolysis was performed on primary, activated, and digested sludge and operating
Figure 11-2. Laboratory-scale pyrolysis reactor.
temperature ranged from 250 °C to 500 °C, and gas phase residence time main-
tained was 20 min. The study found that temperature and volatile solids are
important factors determining oil yield along with sludge type. The maximum oil
yield was achieved with 500 °C on primary sludge, and there was a steep increase
in oil yield between 250 °C and 400 °C because volatile solids (VS) decomposition
was maximum in this range. Although VS was not varied in the study, it could be
said that higher temperature is necessary for higher VS decomposition. The
pretreatment of sludge with acid or chemical catalyst (zeolite) did not enhance the
oil yield.
The parameters that have affected the oil yield in other studies during
pyrolysis are (1) temperature, (2) gas residence time, (3) solid feed rate,
(4) sewage sludge particle size, (5) reaction atmosphere composition, (6) sewage
sludge composition, and (7) use of catalyst. Pyrolysis products are of various types
like aliphatic hydrocarbons including fatty acids, aromatic hydrocarbons, steroids,
nitrogen-containing compounds, dioxins, and polycyclic aromatic compounds
(PAHs). The following factors also affect the composition of pyrolysis oil.
11.5.1.1 Influence of Temperature

Most studies indicated that maximum oil yield was obtained in the temperature
range of 450° to 550 °C. Initially, an increase in temperature promotes breaking
down of organic matter as energy is available to break the strong organic bonds.
The decrease in oil yield above optimal temperature results from secondary
reactions of volatile solids, such as thermal cracking, which becomes significant at
temperatures above 500 °C or 550 °C. Table 11-5 summarizes various pyrolysis
studies conducted. The disparity between the studies regarding operating tem-
perature is because oil yield is dependent on temperature and other operational
variables. One of the studies indicated that an increase in temperature from
Table 11-5. Different Studies Conducted on Sludge Pyrolysis for Oil Extraction.
270
Oil yield
Moisture Oil yield (percent by
content Ash content (percent weight as
(percent (percent by based on ash fed Operational
Type of sludge by weight) weight) SS feed) basis) conditions References
Secondary (dry) 6.23 42.48 28.32 52.65 450 °C Piskorz et al.

Gas residence (1986)
time: 0.55 s
Mechanically treated 5.1 40.7 16.26 30.0 500 °C Stammbach et al.

anaerobically digested (1989)
(dry)
Anaerobically digested 5 29.6 40 61.2 650 °C Inguanzo et al.
(2002)
Secondary sludge (dry) 3 22.6 20.1 30 525 °C Shen and Zhang
Gas residence (2003)
time: 1.5 s
Primary sludge 6 46 30 62.5 550 °C Jindarom et al.
CO2 atmosphere (2007)
Anaerobically digested (dry) 7.1 41 Oil: 24.2 Oil: 46.6 550 °C Fonts et al. (2008)
Water: 16.5 Water: 18.1 gas residence
time: 2 s
solid feed rate:
0.18 kg/h
Secondary (dry) and 1.5 25 31 51 500 °C Pokorna et al.
anaerobically digested (2009)
(dry)
Anaerobically digested 6.8 32.4 Oil: 13 Organic 450 °C Sánchez et al.
Water: 18 phase 21.4 (2009)
Aqueous
phase 18.4
Secondary 5.1 26.9 50.4 66.8 (oil and 450 °C Park et al. (2010)
(dry) water) Gas flow rate:
5 L/min
SS particle size:
0.7 mm
Solid feed rate:
0.3 kg/hr
OIL EXTRACTION USING WASTEWATER SLUDGE
271
450 °C to 650 °C resulted in a decrease in oxygen-containing aliphatic compounds

(Fonts et al. 2009). Maximum aliphatic hydrocarbons are formed around 550 °C.
The increase in temperature tends to form more aromatic and polycyclic aromatic
compounds and more nitrogen-containing aromatic compounds, but a decrease in
steroids and oxygen-containing aliphatic compounds. Similar results were

obtained when an increase in temperature was seen (Sánchez et al. 2009).
11.5.1.2 Influence of Gas Residence Time

According to pyrolysis studies, lower gas residence time (less than 2 s) results in
higher oil yield. Some studies reported an increase in oil yield with an increase in
gas residence time. This is because of the increase in water content when there is
an increase in gas residence times between 0.3 s and 0.55 s (Piskorz et al. 1986).
This can be explained by an increase in condensation reactions with water as a
byproduct. Gas residence time is inversely proportional to gas flow rate and
therefore proportional to the ratio between fluidization velocity and minimum
fluidization velocity (uf/umf) (Fonts et al. 2008). Very low residence times indicate
that some particles move out of the bed without reaching the total conversion rate.
Therefore, an optimum flow rate/gas residence time should used for high pyrolysis
oil yield. At high gas residence time and high temperature, compounds formed
directly from devolatilization disappear, resulting in formation of aromatic
compounds (Fonts et al. 2008).
11.5.1.3 Solid Feed Rate

Higher solid feed rate results in lower gas residence time and ultimately higher
oil yield because lower gas residence time avoids secondary cracking reactions
(Park et al. 2010). One of the studies indicated higher feed rates have resulted in
lower oil yield, which can be accounted by a reduction in char conversion,
resulting in a decrease in production of vapors and gasses (Fonts et al. 2008).
11.5.1.4 Sewage Sludge Particle Size

The particle size also influences oil yield because smaller particles heat up faster,
reaching a higher temperature and taking less time to devolatilize. However,
particles <0.3 mm results in overheating of the particle, followed by conversion
of vapors into gas. Maximum oil yield was obtained for particles of size 0.7 mm
(Park et al. 2010).
11.5.1.5 Reaction Atmosphere Composition

Pyrolysis has been conducted under the influence of various gasses like N2,
methane, CO2, and H2. In a study, CO2 and N2 gas were compared for oil yield,
and it was found that under a temperature range of 350°−650 °C, CO2 resulted in
higher oil yield compared with N2. It may be because CO2 is more reactive than N2
and tends to form more oil and gas (Jindarom et al. 2007). A similar result was
obtained when CO, CO2, or H2 influenced oil yield at a temperature of 450 °C
(Park et al. 2010).
11.5.1.6 Sewage Sludge Composition

Pyrolysis has been performed on three types of sludge: primary, secondary, and
anaerobically digested. Oil yields obtained from three different sludges were
compared (Karayildirim et al. 2006), were in the following order: primary >
secondary > anaerobically digested. This can be attributed to more volatile solids
present in primary sludge, which are consumed in secondary and digested sludge.
Catalytic pyrolysis studies indicated a decrease in yield when the catalyst is
used in the pyrolysis system. This is because of the increase in catalytic cracking
reactions of tar, leading to the formation of secondary products. To obtain a high
oil yield from pyrolysis reaction, primary sludge should be used with an optimum
temperature (400° to 450 °C), gas residence time (0.3 to 0.4 s), and optimum solid
feed rate. In all the studies conducted, the temperature was the most influential
factor in deciding oil yield and oil composition.
11.5.2 Mechanical-Chemical
11.5.2.1 Oil Extraction Using Ultrasonication and Mechanical

Homogenization
Use of ultrasonication pretreatment and acid hydrolysis on oil extraction
from wastewater sludge obtained after partial thickening has been investigated
(Figure 11-3) (Olkiewicz et al. 2015). Current problems associated with ultra-
sonication and mechanical disintegration are that they are energy-intensive
processes with high operational requirements.
To improve the extraction efficiency, the pretreatment methods were inves-
tigated along with the duration of these treatments. The extraction experiments
were carried out in a Soxhlet apparatus using hexane as a solvent on untreated,
ultrasonically disintegrated and mechanically disintegrated, blended, and
Wastewater
Sludge
Ultrasonication Oil
Hexane
Acidification
Extraction
Figure 11-3. Oil extraction from sludge using ultrasonication and mechanical
disintegration.
secondary sludge with and without acidification. Pretreatments before solvent

extraction can be important to secondary sludge because they can release the oils
from other macromolecules that otherwise are not available to solvent. (Solvents
can extract the oils only from the cell membrane, not from macromolecules
present inside the cell.) Therefore, the utilization of the sludge pretreatments can
improve the efficiency of extraction. Problems associated with mechanical treat-
ment for oil extraction are that they are highly energy-intensive processes and
result in low extraction efficiency.
Primary sludge gave higher extraction when compared to secondary and
blended because primary sludge contains organic matter such as floating grease
and solids. On the other hand, secondary sludge consists mainly of microbial cells
and suspended solids generated during aerobic biological treatment of primary
treated wastewater. Blended sludge is a mixture of primary and secondary sludge,
and its oil extraction yield is slightly lower than that of primary. Acidified treatment
did not have much impact on oil extraction yield. The oils extracted had undergone
transesterification to produce biodiesel. However, oil yields reported were nearly
similar for primary (19%), secondary (14%), and blended sludge (14%) after
treatments. FAMEs analyzed after gas chromatography indicated similarities
between fatty acid composition, and all the sludge mainly contained palmitic acid
(C16:0), stearic acid (C18:0), and oleic acid (C18:1), which are desirable for biodiesel
production. Time for pretreatments was also optimized, and it was observed that
10 min pretreatment resulted in enhanced disruption or integration of flocs and lysis
of bacterial cells. Thus, more organic matter is available to the solvent. The
laboratory-scale study showed that pretreatments did not result in high oil extrac-
tion from wastewater sludge. However, production of biodiesel from sewage sludge
can reach a maximum of 19% based on dry weight, but in the study yield of biodiesel
obtained was 10% based on dry sludge.
One of the laboratory studies was conducted using solvent extraction from
scum sludge, and oil yield obtained was compared with oils extracted from
primary and secondary sludge (Wang et al. 2016). Sludge was subjected to a
homogenizer after freeze drying, and then solvent extraction was done using a
mixture of hexane, methanol, and ethanol in different ratios. The methanol:
hexane:acetone ratio of 20:60:20 resulted in the highest extraction yield of 32%
from scum sludge. Irrespective of the ratio of solvents, scum sludge resulted in the
highest yield (27% to 32%) when compared to primary (19% to 25%) and
secondary (11%to 16%) sludge. The study showed that scum sludge has a high
potential for biodiesel production owing to its high oil content. In a later section,
fatty acid profiles, calorific values, and environmental impacts of oils obtained
from different sludge will be considered.
11.5.2.2 Oil Extraction Using Solvents

Zhu et al. (2014) used three methods to extract oils from dry sludge in a
laboratory-scale study. The concentration of oils in sewage sludge was about
12%. The oils extracted from the Soxhlet, hydrolysis, and water bath shaking
method were 6%, 4.8%, and 5.1%. The study also showed that polar solvents
extracted more oils (6.4%) than nonpolar solvents (3.4%). Oil extracted by mixed
solvents are similar to those obtained by polar solvents. Therefore, it can be
concluded that in sewage sludge, polar componds are more than nonpolar
compounds, which could be caused by the presence of microbial cells in sludge
containing polar phospholipids in their cell walls.

A similar study reported oils extracted from municipal sludge for production
of biodiesel using polar and nonpolar solvents (Dufreche et al. 2007). As shown in
Figure 11-4, secondary sludge was pretreated with centrifugation or pressure
filtration, and then a solvent extraction system was operated for 1 h at 100 °C using
the following solvent mixtures or pure solvent:
• 60% hexane/20% methanol/20% acetone (HMA),
• Methanol followed by hexane,
• Pure hexane (one-time extraction),
• Pure methanol (one-time extraction), and
• Supercritical CO2 (45 °C and 32.5 MPa).
The oil yield (per 100 g of dry sludge) and FAME yield (per 100 g of dry sludge)
obtained through different extraction methods are compared in Table 11-6.
The difference in extraction yield by solvent can be attributed to using the
Hildebrand solubility parameter (δ), which is a combination of London dispersion
forces, dipole moment contribution, and hydrogen-bonding contribution. HMA
Figure 11-4. Pretreatment with centrifugation and pressure filtration followed by

oil extraction.
Table 11-6. Extraction and Transesterification Yield of Waste-Activated Sludge.
Extraction medium Oil yield (%) FAME yield (%)
100% hexane 1.94 0.38

100% methanol 19.39 2.76
Extraction 1: HMA 21.2 3.44
Extraction 1: 100% methanol 19.39 2.76
Extraction 2: 100% hexane 2.57 0.31
SC-CO2 3.55 0.28
SC-CO2 w/1.96% MeOH 4.19 1.12
SC-CO2 w/13.04% by weight MeOH 13.56 2.31
In situ transesterification — 6.23
showed the highest oil extraction yield, which is attributed to the presence of polar
cosolvents, methanol, and acetone besides hexane. Secondary sludge mainly
consists of microorganisms with cell membranes containing phospholipids.
Methanol/acetone mix would expose phospholipids with high values for polarity
and hydrogen bonding. It can be hypothesized that the cell membrane is bonded
through hydrophobic interactions and protected by polar groups, and solvent
mixture tends to disrupt the membrane. In situ transesterification was performed
using methanol and 1% sulfuric acid. Because the reagents have access to all lipids
in the sludge instead of just what was extracted, the yield of in situ transester-
ification biodiesel was higher than with the other methods. Acid profiles obtained
after various extraction methods showed that sewage sludge has a much higher
concentration of saturated fatty acids when compared to a soybean sample.
Although saturated fatty acids pose the problem of gel formation during winter,
they give better burning biodiesel (Ritz and Croudace 2003). CO2 provided
maximum polyunsaturated fatty acids. The study showed that both polar and
nonpolar solvents are required to extract oils from secondary sludge.
Another study used both chloroform and toluene for oil extraction from dried
raw sludge (Boocock et al. 1992). Soxhlet extraction and boiling extraction were
used for extraction. Soxhlet extraction extracted 12% oil, whereas boiling extrac-
tion resulted in 17% to 18% oil. Although the similar elemental analysis was
obtained after both types of extraction, both methods produced oils with low
nitrogen and sulfur content. Toluene was the preferred solvent based on cost and
ability to extract oils, and any residue of chloroform left can cause environmental
problems. Free fatty acids were the primary component of extracted oils from
sewage sludge, and the fatty acid fraction mainly contained palmitic, stearic, oleic
and linoleic acids, which are significant for biodiesel production.
In one study, an approach for extracting fatty acids from sewage sludge
using solvents like toluene, hexane, ethanol, and methanol has been discussed
(Pokoo-Aikins et al. 2010). As shown in Figure 11-5, in the first extraction step,
sludge and solvent are mixed, and the mixture is pumped to a filter where solids
Figure 11-5. Oil extraction from sewage sludge using solvents.

are removed. Permeate obtained from the filtration process is then distilled to
obtain triglycerides (oils), and most of the solvent is recovered in the distillation
process (>98% by weight). The solvent is separated from the triglycerides in a
decanter after cooling of the streams. In the decanter the heavier oils settle to the
bottom, are recovered, and stored in a tank. The recovered solvent is recycled to
the process. The fatty acid–rich stream from the bottom of the distillation process
includes some residual ash and some valuable triglycerides. Three flash operations
are used to remove ash and obtain FFAs. Ash is removed in the first flash
operation. The fatty acid–rich stream from the top of the flash operation is cooled
and enters a decanter where the remainder of the triglyceride is recovered. In
extraction processes in which methanol and ethanol are used as the solvents, an
additional decanter is required to remove residual xylose and obtain the trigly-
cerides (oils). The triglycerides (oils) obtained from the latter decantation
processes are stored in the same tank as the triglycerides from the first decantation
process. Two flash operations are used to separate the FFAs from the remaining
oils in the stream. The FFAs obtained are cooled and stored for further use.
A case study is presented to demonstrate the insights and usefulness of the
developed approach. For all extraction processes, the maximum yield of 3.4% was
obtained for the triglyceride stream. The yield of fatty acids for extraction using
toluene and hexane was 24.8% and 24.9%, respectively. Extraction utilizing metha-
nol and ethanol resulted in a slightly higher fatty acid yield of 25.5% for either
process. Moreover, methanol [$0.23lb ($0.50/kg)] and ethanol [$0.27lb ($0.60/kg)]
are cheaper than toluene [$0.3lb ($0.66/kg)] and hexane [$0.45/lb ($0.99/kg)], and
methanol and ethanol are safer than hexane. Techno-economic evaulation for using
different solvents are discussed in later sections.
11.6 ADVANTAGES AND DISADVANTAGES OF DIFFERENT OIL

EXTRACTION PROCESSES
Table 11-7 shows the advantages and disadvantages of the processes used for oil
extraction with wastewater. From Table 11-7, it is revealed that high extraction
yields are obtained using solvent extractions but it is an expensive process at large
scale. Mechanical methods are energy intensive but not that effective because further
treatment with solvents is also required. Thermochemical methods release heat
during oil extraction, which can be used for power and steam generation. However,
because thermochemical methods are operated at high temperatures, safety is one of
the concerns along with char production and the low economic value of oil.
11.7 COMPARISON OF SLUDGE-BASED OIL WITH OTHER OILS
One study compared fatty acid profiles of oil obtained from primary, secondary,
and scum sludge with vegetable oil (Wang et al. 2016). The major fatty acids
present in oil from different types of sludge were palmitic acid (C16:0), stearic acid
278
Table 11-7. Advantages and Disadvantages of Processes Used for Oil Extraction Using Wastewater Sludge.
Process Advantages Disadvantages
Thermochemical Heat generation that can be used for steam and Low economic value of oil
power generation
Production of stable residues for easy recycling Safety issues at high temperature
Lower generation of flue gas and NOx emissions Production of char that requires further
disposal
Synthetic gas generation that can be used for Complex processing
power generation Cost and operating data on current studies not

available
Mechanical methods Capacity to disintegrate sludge leading to Not that effective; most of the studies required
homogenized sample solvent extraction after mechanical
treatments
Particle size reduction Energy-intensive process
Solvent extraction High extraction yields Distillation and recovery step required to
separate solvent from oil
Less energy intensive compared to mechanical When used at large scale, high amounts of
methods extraction volume and time lead to increase
in production costs
Use of toxic solvents such as toluene reported
in some of the studies
Table 11-8. Comparison of Fatty Acid Profile of Soybean Oil with Oil from Different
Types of Sludge.
Scum Primary Secondary

Fatty Acid Soybean sludge sludge sludge
Palmitic acid 16:0 7–11 25–30 44–48 37.5

Palmitoleic acid 16:1 0–1 3–5 3–5 15
Stearic acid 18:0 3–6 5–8 10–15 11.75
Oleic acid 18:1 22–24 40–42 20–25 20
Linoleic acid 18:2 50–60 18–22 5–8 6.5
Linolenic acid 18:3 2–10 — — —
(C18:0), oleic acid (C18:1), and linoleic acid (C18:2), which are also found in
vegetable oil. However linolenic acid (C18:3) is present in vegetable oil, but not in
oil from any type of sludge. Scum oil contained more of oleic acid than palmitic
acid, whereas primary and secondary sludge contained more of palmitic acid than
oleic acid. Table 11-8 compares fatty acid profiles obtained from oil of three
different types of sludge along with soybean oil.
The variation in fatty acid profile among sludge oils arises because of the
variation of the ratio of cosolvents (methanol:hexane:acetone) used in the
extraction system. It was observed that unsaturated fatty acids like 18:2, 18:1
increased when the concentration of hexane was more than ethanol in the
extraction system. Unsaturated fatty acids in scum oil ranged from 57.5% to
64.1%, comparable to 70% in soybean oil, whereas the range was 30% to 40% only
for primary and secondary sludge. Scum-produced biodiesel oil, when compared
to that conventionally produced from soybean or vegetable oil, has less environ-
mental impacts as shown in Table 11-9. Scum is a waste and does not assign any
upstream impacts, but soybean and vegetable oil produce upstream impacts.
As shown in Table 11-10, scum oil has a high heating value compared with
that of the primary and secondary sludge because of the presence of high oil and
greasy content in scum. Moreover, scum oil has a high C content compared with
oil derived from primary and secondary sludge, making it a good renewable
energy source.
11.8 TECHNO-ECONOMIC EVALUATION
11.8.1 Thermochemical
Economic value of pyrolysis oil would be calculated on the basis of difference in
price of oil produced during pyrolysis (V1) and cost of energy input required for
pyrolysis (V2). Total energy consumption for pyrolysis reaction is a summation of
(1) energy required for drying of sludge after dewatering, (2) energy required to
280
Table 11-9. Life Cycle Impacts of Scum-Derived Oil and Other Oils.
Fossil fuel use GHG emissions Acidification Eutrophication

Biodiesel oil (MJ) (kg CO2 eq.) (kg SO2 eq.) (kg N eq.) Reference
Scum 7.58 0.316 0.00278 0.000569 Mu et al. (2016)

Soybean 11.4 1.85 — — Sheehan et al. (1998)
Vegetable oil 14.6 2.42 0.0124 0.032 Mu et al. (2016)
Table 11-10. Elemental Analysis for Oil Content from Different Types of Sludge.
Element Scum Primary Secondary
C (%) 73.2 62–70 63–65

N (%) 0.0561 9.5–9.9 6.8–8.5

O (%) 15.1 8–22 17–20
H (%) 11.6 2.7–8.5 9.4–9.7
Heat value (MJ/kg) 22.3 11–15 9–12
Table 11-11. Economic Oil Value Obtained from Pyrolysis of Primary and
Secondary Sludge at Different Temperatures.
Oil Energy
Temperature Yield (%) Cost Required Cost Economic
(°C) (kg-oil/kg-ds) (₵/kg-ds) (kJ/kg-ds) (₵/kg-ds) oil value
Primary sludge
300 26 8.3 2,555 3.1 5.2
400 39 12.5 2,750 3.3 9.2
500 42 13.4 2,945 3.5 9.9
Secondary sludge
300 18 5.8 2,900 3.5 2.3
400 33 10.6 3,095 3.7 6.9
500 32 10.2 3,290 3.9 6.3
take heat-dried sludge to the target temperature, and (3) heat of endothermic
reaction during pyrolysis. In the study reported by Kim and Parker (2008), energy
cost was calculated by multiplying cost of electricity and total energy consumed
during the process. Economic value of bio-oil for primary and secondary sludge
are compared in Table 11-11 at different temperatures.
It is evident that the economic oil value is greater for primary sludge
compared with secondary sludge owing to the high oily and greasy content in
primary sludge. Economic oil values increase with temperature because of the
breakdown of volatile organic matter. Moisture content present in sludge also
decides the economic value of oil, because higher moisture content requires higher
energy input for drying of sludge, and energy for drying contributes 75% of total
energy input during pyrolysis (Kim and Parker 2008).
11.8.2 Mechanical-Chemical
In one of the studies, oil extraction was performed on wastewater sludge by four
different solvents: toluene, hexane, methanol, and ethanol (Pokoo-Aikins et al.
Table 11-12. Safety Index and Cost for Solvents Used in the Extraction Process.
Biodiesel cost Biodiesel cost

Solvent Safety index (USD/gallon) (USD/L)
Ethanol 18 3.39 0.90

Hexane 22 2.89 0.76
Methanol 18 3.37 0.87
Toluene 45 2.79 0.74
2010). Economic analysis of the process was performed using a software, Aspen
Icarus. A new safety matrix, known as safety index, was calculated by the software
to compare various solvent extraction processes. Based on the matrix, it was
estimated that methanol and ethanol are safer solvent options, but biodiesel
production costs are economical in the case of hexane and toluene (Table 11-12).
Recommendation of a particular solvent is dependent on a company’s goals.
Safety and cost are both high priorities for a company, but there are trade-offs that
a company must make when making decisions about security and cost. The trade-
off for selecting ethanol or methanol, which were characterized as the safest, would
be an increased cost. The cost penalty would 20% increase, but the safety index will
be enhanced by 60%. Although toluene is the most economical for biodiesel
production, looking at the trade-offs between cost and safety, the penalty for safety
would be higher than the penalty for the cost. Because methanol is used in the
pretreatment and biodiesel steps of the process and methanol is the one of the
safest of the solvents evaluated, it may be of benefit to use methanol as the solvent
and modify the process to reduce costs. Safety analysis should be done at the
design stages along with an analysis of other criteria to obtain a comprehensive
evaluation of a process.
Another study on oil extraction from primary sludge using solvents has been
simulated by AspenHysys V8 (Olkiewicz et al. 2016). The most optimized process
in the study was when they eliminated the drying step before oil extraction, which
is a very expensive process in scale-up. Primary sludge taken directly from the
settling tank was acidified to pH 2 and went through oil extraction, in which
sludge and solvents were fed in countercurrent directions in mixers where oil and
hexane were separated in the distillation column. This optimized process was also
compared with the process with drying included. However, the conventional dry
process ($2,014/t) was much more expensive than the wet process ($1,232/t). The
biodiesel price from the optimized process using liquid primary sludge was
comparatively low compared with the current fossil diesel price ($1376/t).
Sensitivity analysis showed that oil extraction yield was a most significant factor
in deciding unit cost production price. The operational variables such as extrac-
tion time and the amount of solvent influenced the cost. The study showed that
biodiesel production using municipal sludge is cost-effective because it eliminates
the cost of biomass production, which is 65% of the overall cost in biodiesel
manufacturing using microalgae (Ríos et al. 2013).
11.9 RECENT ADVANCEMENTS AND FUTURE PERSPECTIVES
11.9.1 In Situ Transesterification

Usually, oils are first extracted from dried sludge and then undergo transester-
ification. In situ transesterifcation can be done directly using dried sludge in the
presence of catalysts and solvent. One study reported in situ transesterification of
municipal and secondary sludge for biodiesel production (Mondala et al. 2009).
The sludge was freeze dried and subjected to an acid-catalyzed in situ transester-
ification. In the study, the effect of temperature, sulfuric acid concentration, and
the mass ratio of methanol to sludge were investigated, and they were optimized to
yield 14.5% and 2.5% FAME based on primary and secondary sludge. Fatty acid
composition was nearly the same for both primary and secondary sludge. Cost
analysis of the whole process revealed that biodiesel obtained from these feed-
stocks was estimated to be $3.23gal. ($0.85/L), which was lower than the average
price of petroleum diesel fuels [$4.8/gal.($1.27/L)] and soybean biodiesel [around
$4.25/gal. ($1.12/L)]. However, the cost for freeze-drying and centrifugation was
not incorporated into the cost estimation for biodiesel production. The study
showed that municipal wastewater sludge has a high potential for biodiesel
production because it is cost-competitive and abundantly available.
Another study reported scum sludge used as potential feedstock for biodiesel
production. Both in situ and ex situ transesterification were performed on scum
sludge and compared with primary and secondary sludge (Wang et al. 2016). The oil
yield obtained after in situ transesterification was 22.7% from scum. Maximum yield
from scum sludge, primary, and secondary sludge was 22.7%, 9.0%, and 1.9%
respectively, which was higher than the corresponding yield gained through ex situ
transesterification. Besides, the ratio of solvents used for extraction of oils is also
important. Maximum yield was obtained when methanol:hexane:acetone was
20:60:20. Fatty acid composition was higher in palmitic acid and oleic acid,
irrespective of sludge type and solvent used. Oleic acid was dominant in FAME
derived from scum sludge, whereas palmitic acid was dominant in FAME obtained
from scum sludge. Unsaturated fatty acid ester in FAMEs from scum sludge
composed of 57.5% to 64.1%, higher than obtained from primary and secondary
sludge. The study showed scum sludge is a potential feedstock for biodiesel
production. More work needs to be done to gain its advantage as biodiesel obtained
from scum sludge had higher calorific value than primary and secondary sludge.
Another study reported in situ transesterification of activated sludge for
biodiesel production (Revellame et al. 2010). In the study, the response surface
model was to describe the three-way integration of temperature, methanol-to-
sludge ratio, and sulfuric acid concentration. Numeric optimization showed that
an optimum biodiesel yield of 4.88% can be obtained at 55 °C, methanol-to-sludge
ratio of 25%, and 4% sulfuric acid volume after 120 treatment combinations of
simulation. Later experiments were conducted, and optimized experimental yield
was found to be 4.79%. Acid-catalyzed transesterification above 60 °C decreased
biodiesel yield significantly. The fatty acid profile obtained mainly consisted of
significant amounts of methyl esters of palmitic, palmitoleic, stearic, oleic, and

linoleic acids, indicating that activated sludge can be used as potential feedstock
for biodiesel production.
11.9.2 Nanoparticles for Oil Extraction

Oil yield obtained during oil extraction directly affects biodiesel production cost;
therefore, methods for improving oil extraction yield should be worked on.
Functionalized nanoparticles can be used for oil extraction from sludge. Nano-
particles functionalized with specific functional groups and pore size can be
helpful in adsorbing fatty acids or greasy material from sludge. No such study has
yet been conducted, and nanoparticles can be helpful for oil extraction from sludge
because they also have good recyclability (Wang et al. 2011, Tran et al. 2012).
However, their high fabrication costs along with poor reliability are concerns.
11.10 CONCLUSIONS
1. Of scum, primary, secondary, and anaerobically digested sludge, scum

sludge resulted in high oil yield owing to the presence of high oil and
greasy content present in it. Based on the elemental analysis, scum sludge
has the highest C content, making it a good renewable source of energy.
2. Thermochemical treatments for oil extraction require a huge amount of
energy, decreasing the economic value of the oil. At high temperatures,
secondary reactions can occur, decreasing oil yield and formation of more
stable aromatic and cyclic compounds along with water. Selection of
optimum temperature and gas composition affects oil yield. Temperature
around 450° to 500 °C was found optimum for high oil yields. Moisture
content in sludge directly affects the economic value of oil because the
energy required for sludge drying was reported to be two to three times
higher than the energy required for pyrolysis and attaining temperature.
3. No study has been reported for oil extraction by mechanical methods only.
Studies reported were pretreatment of sludge with mechanical methods
before solvent extraction. However, pretreatments with mechanical methods
did not affect oil yield significantly. The separate study should be conducted
for oil extraction from scum with mechanical methods and compared with
solvent extraction methods.
4. Most of the studies were conducted using solvent extraction methods. Polar
solvents were effective with secondary sludge because it mainly consists of
microbial cells. Nonpolar solvents can be effective for primary sludge

because they do not have microbial cells. However, the cosolvent extraction
system with hexane, methanol, and acetone resulted in higher oil yields
compared with the single solvent system. The HMA ratio of 60:20:20 was
effective in enhancing oil yields from scum, primary, and secondary sludge
as demonstrated by a few studies. However, oil yield can be further enhanced
by optimizing temperature, pressure, and duration during the solvent
extraction process. At the industrial scale, factors that influence the cost
of production during solvent extraction are the amount of solvent used and
time duration.
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CHAPTER 12
Biodiesel Production Using
Fermented Wastewater
Sludge–Derived Lipids
B. Bhadana
L. R. Kumar
R. D. Tyagi
12.1 INTRODUCTION
As biodiesel demand increases, the need for lipid feedstocks (e.g., rapeseed,
sunflower, soybean, palm, canola, and coconut oils) is also increasing. The current
feedstocks (pure vegetable or seed oils) are expensive because they constitute about
70% to 80% of total biodiesel production cost (Haas and Foglia 2005, Kargbo
2010). Thus, researchers are looking for nonedible feedstocks including jatropha,
animal fats, and waste cooking oil. Recently, wastewater sludge is gaining attention
as a lipid feedstock for biodiesel production because of the following advantages:
(1) sludge contains significant amounts of mono-, di-, and triglycerides, phos-
pholipids, and free fatty acids (Nelson and Cox 2005, Dufreche et al. 2007,
Rittmann and McCarty 2012); (2) wastewater sludge is readily available in large
quantities; and (3) using sludge as feedstock would reduce the burden of sludge
management and treatment, which are formidable challenges. It is well known that
harmful ingredients (e.g., odor, heavy metals, and contaminants of emerging
concerns) have made it difficult to use sludge as a fertilizer for agricultural
purposes, and incineration of wastewater sludge may lead to emissions containing
dioxins and heavy metals. Hence, using sludge for biodiesel production will help to
solve both environmental and energy problems.
Although Chapter 11 focuses on oil extraction from wastewater sludge
directly using thermal, chemical, and mechanical methods because sludge con-
tains oil and grease content, this chapter focuses on using wastewater sludge as a
substrate for lipid production during microbial fermentation. The microbial lipids
289
accumulated inside the cells can be extracted and converted into biodiesel/fatty
acid methyl ester (FAME) through a transesterification reaction. Various oleagi-
nous microorganisms can utilize sludge as a culture medium for their growth, such
as Lipomyces starkeyi, Cryptococcus curvatus, Acidithiobacillus ferrooxidans,
Bacillus thuringiensis, Sinorhizobium meliloti, Trichosporon oleaginosus ATCC

20509, Pichia amethionina sp. SLY, Galactomyces sp. SOF (Picher et al. 2002,
Vidyarthi et al. 2002, Angerbauer et al. 2008, Zhao et al. 2009, Zhang et al. 2017).
Thus, microbial oil can be produced in a sustainable manner using wastewater
sludge, which can reduce the cost of lipid production and biodiesel production.
This will also mitigate the pressure on sludge treatment and disposal.
This chapter mainly overviews the feasibility of using wastewater sludge as a
raw material to produce lipids and their subsequent conversion into biodiesel,
along with sludge characteristics, challenges to use sludge as feedstock at a
commercial level, and future perspectives.
12.2 CHARACTERISTICS OF WASTEWATER SLUDGE
Processing of wastewater is done by physical, chemical, and biological methods

(Madigan et al. 1997; Manara and Zabaniotou 2012). Different types of sludge
are produced on the basis of the treatment stage, such as (1) primary sludge,
which is produced where removal of solids is done by sedimentation, and the
other lighter solids, oil, and grease float to the surface of the wastewater (Horan
1989); (2) biological sludge, which is produced in secondary (biological)
treatment of wastewater (Horan 1989, Metcalf and Eddy 2003, Mantzavinos
and Kalogerakis 2005, Morillo et al. 2009); (3) mixed sludge, which is the
mixture of the biological and primary sludge; and (4) tertiary sludge, which is
produced in advanced wastewater treatment processes. Sludge produced in the
wastewater treatment plant (WWTP) undergoes sludge treatment (via thicken-
ing, organic matter stabilization, dewatering, and disinfection) and then final
disposal (or reuse).
Table 12-1 shows the typical characteristics of the sludge. Depending on
the treatment method, sludge obtained from WWTPs consists of a complex
mixture of organic and inorganic materials. Some of the ingredients in the sludge
are valuable components with agricultural value, including organic matter,
phosphorus, potassium, nitrogen, calcium, magnesium, and sulphur. Besides,
some inorganic components are also present, for example, aluminates and
sulfates. Wastewater sludge also shows high calorific value (Horan 1989,
Andersen 2001). Chemically, the sludge consists of bacterial constituents, their
decay products (including nucleic acids, carbohydrates, proteins, and lipids),
cellulose, inorganic material, as well as many functional groups (e.g., carboxylic,
amine, aliphatic, aromatic, amide, and methylene groups or even silicon,
halogens, and phosphorus) (Fonts et al. 2009).
BIODIESEL PRODUCTION USING FERMENTED WASTEWATER 291
Table 12-1. Typical Characteristics of the Sludge.
Primary Biological Mixed

Characteristic Units sludge sludge sludge
Dry matter (DM) g/L 12 7 10

Volatile matter (VM) Percent DM 65 77 72
pH — 6 7 6.5
C Percent VM 51.5 53 51
H Percent VM 7 6.7 7.4
O Percent VM 35.5 33 33
N Percent VM 4.5 6.3 7.1
S Percent VM 1.5 1 1.5
C/N — 11.4 8.7 7.2
P Percent DM 2 2 2
Cl Percent DM 0.8 0.8 0.8
K Percent DM 0.3 0.3 0.3
Al Percent DM 0.2 0.2 0.2
Ca Percent DM 10 10 10
Fe Percent DM 2 2 2
Mg Percent DM 0.6 0.6 0.6
Fat Percent DM 18 10 14
Protein Percent DM 24 34 30
Fibers Percent DM 16 10 13
Calorific value KWh/t DM 4,200 4,800 4,600
12.3 BIODIESEL FROM WASTEWATER SLUDGE–DERIVED LIPIDS
There are two major processes for biodiesel production from sludge-derived lipids:
(1) direct lipid extraction from the sludge and conversion of the extracted lipids to
biodiesel (see Chapter 11); and (2) using wastewater sludge as a source of carbon
and other nutrients to grow microorganisms and subsequent extraction of lipids
from them for biodiesel/FAME production, as shown in Figure 12-1.
To start this process, wastewater sludge initially is allowed to settle at 4 °C for
24 h to maintain appropriate suspended solids (SS) concentration. This can also be
done by centrifugation or filtration (Mondala et al. 2009). Then the pH of the
culture medium (wastewater sludge) is set at 12 with 4 M NaOH for hydrolysis of
the sludge during sterilization at 121 °C for 15 min. This pretreatment increases the
carbon availability in the culture medium for oleaginous microorganisms. Before
inoculating the medium with oleaginous microorganisms, the pH is reduced to 6.5
with 4 M H2SO4, that is, to the optimum pH for their growth, and thereby, leading
to their highest lipid production ability. After inoculation, the culture medium
is incubated at 25 °C, 300 to 400 rpm for intracellular lipid accumulation.
Lipid extraction
Wastewater Trans -
(discussed in
sludge esterification
chapter 11)
Overnight Fermentation
settling followed using oleaginous FAME analysis
by centrifugation microbes
Suspended solids pH adjustment to

measurement 6.5
Sterilization to
pH adjustment
increase carbon
with 4 M NaOH
availability
Figure 12-1. Schematic diagram for biodiesel/FAME production using wastewater

sludge as feedstock.
The produced lipids are extracted by the methods discussed in the previous chapter
of this book. The lipid content is determined by the standard chloroform-methanol
extraction method (Figure 12-2) (Folch et al. 1957, Vicente et al. 2009).
Final weight (Y) Lipid content (%):

Fermented broth
after evaporation (Y-X)/200 *100
Filter the solvent in Store lipids in dark

Centrifugation for
preweighed glass at 4oC for further
15 min
vial (X) studies
Washing by Incubation at 60oC

distilled water for 4 h
200 mg sample +
Discard 10 mL solvent
supernatant and
drying (2:1 (v/v)
chloroform:methanol)
Figure 12-2. Chloroform–methanol extraction method for lipid content

determination.
Dissolve extracted Washing with 2%

Collection of hexane
lipids in hexane (20 mL sodium bicarbonate (20
phase (upper layer)
hexane/g lipid) mL/g lipid)
1% H2SO4 and
Washing with hexane Oven drying of top
methanol (40 mL (100 mL/g lipid) layer at 60oC
methanol/g lipid)
FAME analysis by
Incubation at 50oC for Addtion of 5% NaCl GC-MS after dissolving
12 h (100 mL/g lipid) in hexane
Figure 12-3. Transesterification of sludge-derived lipids.
Finally, the extracted lipids undergo transesterification by acid catalysis

(Figure 12-3) because wastewater sludge and the cultivated microorganisms
contain less than 2% free fatty acid (FFA) content (Zhang et al. 2017). If alkaline
catalysis is performed with the lipid feedstock containing more than 0.5% FFA, it
leads to soap formation and hence reduced biodiesel yield (Berrios et al. 2007).
The produced FAME is analyzed by gas chromatography–mass spectroscopy
(GC–MS) to determine yield.
12.4 FACTORS AFFECTING LIPID PRODUCTION USING

WASTEWATER SLUDGE
Major factors affecting lipid production during fermentation conducted with

wastewater sludge as feedstock, include the following:
• Wastewater sludge characteristics: As discussed in Section 12.2, wastewater
sludge consists of essential nutrients (nitrogen, phosphorus, and other trace
elements) and organic and inorganic materials required for the growth of
oleaginous microorganisms (Horan 1989, Andersen 2001, Fonts et al. 2009,
Zhuang et al. 2011, Su et al. 2012, Zhang et al. 2017).
• Sludge pretreatment: The nutrients in the sludge are available as large molecules
which are difficult to be utilized by microorganisms (Angerbauer et al. 2008,
Su et al. 2012, Zhang et al. 2017). Thus, the pretreatment (acidic or alkaline-
thermal) of the sludge is required to convert the large nutrient molecules into
smaller ones, so that they can be assimilated by oleaginous microorganisms
(Brar et al. 2004, Yezza et al. 2005, Zhang et al. 2017). Pretreatment is
done either at high or low pH by subjecting the sludge to high temperature
(121° to 130 °C) for a certain time to hydrolyze it. This enhances the micro-
bial growth, such as Bacillus sp., Methanosarcina sp., Methanosaeta sp., and
Clostridium sp., Trichosporon oleaginosus, and Pichia amethionina sp., as
investigated in many studies with pretreated sludge culture media (Yezza
et al. 2005; Tommasi et al. 2008; Zhang et al. 2010, 2017). With acidic-thermal
pretreatment of municipal sludge at 30 g/L SS concentration, Zhang et al.
(2014a) observed 37.1% w/w lipid content by Trichosporon oleaginosus, whereas
for alkaline-thermal-pretreated and thermal-pretreated sludge, they observed
38.8% and 35.2% w/w lipid content, respectively. Alkaline-thermal pretreatment
was most effective in converting bigger molecules present in sludge to smaller
ones resulting in high lipid content during fermentation.
• Type of sludge and treatment process: A comparative study has been reported
in which four types of sludge—primary municipal, secondary municipal,
mixed municipal, and pulp and paper secondary sludge—were compared in
terms of initial lipid content and final lipid content obtained after 48 h
fermentation cultivated with T. oleaginosus (Zhang et al. 2014a). As shown in
Table 12-2, the high initial lipid content in pulp and paper sludge (PPS) is
caused by the high C/N ratio (C/N = 50) in pulp and paper wastewater
compared with municipal wastewater (C/N = 5). High lipid content was
observed for secondary pulp and paper and municipal sludge during fermen-
tation because secondary sludge do not inhibit the microbial growth, and the
lipid present in the raw sludge was not utilized by the microorganisms. Trace
elements present in the secondary sludge also enhance lipid accumulation.
• Microorganisms used for lipid production: In a study by Zhang et al.
(2014b), different sludge media are compared with the synthetic medium
for lipid accumulation by three different strains (Pichia amethionina sp.,
Galactomyces sp., and Trichosporon oleaginosus). With the synthetic medium
having glucose, P. amethionina sp. SLY (58.56% w/w) accumulated
higher lipid content than Galactomyces sp. SOF (53.26% w/w). However,
Galactomyces sp. SOF gave higher lipid content in sludge medium (31.19% in
PPS) than P. amethionina sp. SLY (28.55% in PPS). It indicates that
Galactomyces sp. SOF is more suitable for lipid accumulation with sludge
Table 12-2. Comparison of Lipid Contents Using Different Types of Sludge.
Municipal Municipal Pulp and paper

primary secondary Municipal secondary
Sludge type sludge sludge mixed sludge sludge
Lipid content 6.81 5.33 6.42 10.95

at 0 h
Lipid content 16.48 35.99 24.16 33.29
at 48 h
fermentation
as medium, and it has also been proven with Biolog results that
Galactomyces sp. SOF could better use complex carbon sources than
P. amethionina sp. SLY. Wastewater sludge has been used as solo or part
of culture medium for cultivation of various oleaginous microorganisms,
for example, Trichosporon oleaginosus, Pichia amethionina sp. SLY,

Galactomyces sp. SOF, Lipomyces starkeyi, Cryptococcus curvatus, Acidithio-
bacillus ferrooxidans, Bacillus thuringiensis, and Sinorhizobium meliloti (Chen
et al. 2012; Leiva-Candia et al. 2014; Zhang et al. 2014a, 2014b, 2017).
• Sludge concentration: With an increase in SS concentration in the medium to
a certain value, the amount of available nutrients also increases, thereby
leading to increased lipid accumulation. A further increase in SS concentra-
tion shows inhibitory effects on lipid accumulation in oleaginous microbes.
This may be caused by the increasing amount of FFA in the medium with the
increase in SS concentration. FFA may cause inactivation of the enzymes,
disruption of electron transport chain, and oxidative phosphorylation in the
microbial cell membrane, and hence, gives a negative impact on microbial
growth (Shin et al. 2007, Desbois and Smith 2010, Zhang et al. 2017).
SS concentration of 25 to 30 g/L was reported to be the optimum concentra-
tion for lipid production (Zhang et al. 2014b).
• The C/N ratio: The major parameter affecting microbial lipid accumulation
is the C/N ratio (Karatay and Dönmez 2010, Kraisintu et al. 2010). A higher
C/N ratio in the culture medium leads to higher accumulation of microbial
lipid content (Isleten-Hosoglu et al. 2012). In a recent study by Zhang et al.
(2017), higher lipid content has been observed in secondary PPS than
in municipal secondary wastewater sludge (MSWS) because of the higher
C/N ratio (C/N = 50) in PPS than MSWS (C/N = 5). Similar results have
been observed in other studies (Dobrzynska et al. 2004; Pokhrel and
Viraraghavan 2004). However, the C/N ratio varies from one organism to
another for optimal lipid production. For T. oleaginosus, the C/N ratio
affected the low SS concentration medium (10 to 30 g/L), but no impact was
observed on high SS concentration for the C/N ratio of 50 and 100. The
greater impact on low SS concentration than in the high one indicates that
certain substances in sludge inhibit the lipid accumulation, and the inhibi-
tion was more profound than the enhancement of the C/N ratio on lipid
accumulation.
• Degradability of sludge: The sludge contains biodegradable suspended par-
ticles and nonbiodegradable inert substances. The higher the biodegradability
of sludge, the higher the lipid content obtained during fermentation. Also, the
inert substances present in the sludge require the use of a larger volume of
solvents during lipid extraction, increasing the biodiesel production cost. The
nonbiodegradable suspended solids in the sludge can be estimated by the
following:
M non þ M RB = SSf (12-1)

LRB × M RB þ L0 × SS0 = Lf × SSf (12-2)

where Mnon = nondegradable suspended solids concentration (g/L);
MRB = real biomass obtained by microorganism (g/L); SSf = total suspended
solid concentration at the end of fermentation (g/L); LRB = final lipid content
accumulated by microorganism (%); L0 = initial lipid content in sludge (%);
SS0 = suspended solid concentration at 0 h (g/L); and Lf = total lipid content at
the end of fermentation (%).
• FFA content: Selection of transesterification catalyst depends on the amount
of FFA in the lipid. If the available FFA content is more than 0.5%, then
transesterification with alkaline catalyst leads to reduced FAME yield result-
ing from soap formation (Berrios et al. 2007). Because the raw wastewater
sludge and the cultivating microorganisms contribute to a high FFA (more
than 2%), using acidic catalytic transesterification for significant FAME yield
is suggested (Zhang et al. 2017).
• FAME profile of sludge-derived lipids: FAME composition has a significant
role on biodiesel quality, that is, viscosity, oxidation stability, flashpoint,
calorific value, cold flow properties, and biodiesel density. Viscosity is directly
related to the combustion process of fuel. Higher viscosity causes early
injection, and thus, increases the temperature of the combustion chamber.
This may be caused by the increased chain length and fatty acid saturation
level in the fuel (Graboski and McCormick 1998). The cold flow properties
become poorer with a higher saturation level in the feedstock oil (Chapagain
and Wiesman 2009, Ramos et al. 2009). Higher saturated fatty acid compo-
sition in biodiesel gives better oxidation stability as well as a higher calorific
value (Karmakar et al. 2010), whereas the density of the fuel is directly
proportional to the polyunsaturation level (Karmakar et al. 2010). When
compared with soybean oil with a higher unsaturated fatty acid fraction,
Zhang et al. (2017) observed that sludge-derived oil is rich in saturated fatty
acids. Hence, sludge-derived FAME showed greater density and oxidation
stability than soybean FAME (Table 12-3). Hence, sludge is a promising
feedstock for biodiesel production.
12.5 LIPID ACCUMULATION IN OLEAGINOUS MICROORGANISMS

FED WITH SLUDGE
Lipid accumulation by the cultivating microorganisms depends on the availability

of nutrients in the culture medium. Suspended solids is the sum total of the
microbial biomass and sludge suspended solids. During fermentation, SS concen-
tration first increases and then starts decreasing. The rapid utilization of soluble
nutrients by the microbes results in an increase in SS concentration as a result of
increased biomass. Once the soluble nutrients are exhausted, the microbes shift
toward the complex carbon source present in the sludge culture medium. At this
Table 12-3. FAME Composition from Different Oil.

Oil C14:0 C15:0 C16:0 C16:1 C17:0 C18:0 C18:1 C18:2 C18:3 References
Trichosporon 3.1 1.8 14.6 11.9 1.4 18.6 24.4 18.8 4.2 Zhang et al. (2017)
oleaginosus
(acidic- thermal
pretreatment)
Trichosporon 1.8 1.4 13.1 5.7 1.5 20.0 15.4 11.7 21.9 Zhang et al. (2017)
oleaginosus
(alkaline- thermal
pretreatment)
Palm 1.1 0 42.5 0.2 0.1 4.2 41.3 9.5 0.3 Marker (2005)
Sunflower 0.1 0 6.4 0.1 0.1 3.6 21.7 66.3 1.5 Hu et al. (2008);
Pereyra-Irujo
et al. (2009)
Canola 0 0 4.2 0.3 0.1 2.0 60.4 21.2 9.6 Pereyra-Irujo
et al. (2009)
Soybean 0.1 0 11.6 0.2 0.1 3.9 23.7 53.8 5.9 Xu et al. (2006)
BIODIESEL PRODUCTION USING FERMENTED WASTEWATER
297
stage, microbial biomass increases slowly with respect to nutrient utilization from
the sludge, because they use these nutrients for maintenance for their cellular
activities. Thus, lipid accumulation increases in the initial stage because of easy
availability of nutrients and then decreases as previously discussed (Girault et al.
2012, Zhang et al. 2017).

In a recent study by Zhang et al. (2017), Trichosporon oleaginosus (an
oleaginous yeast) was used for lipid accumulation using municipal sludge as the
substrate in a 15 L fermenter. The municipal sludge was fortified with crude
glycerol at four different concentrations of 25, 50, 100, and 150 g/L that
corresponded to maximum lipid concentration of 9.35 (32%), 10.13 (33.6%),
9.13 (33.3%), and 9.03 g/L (33.1% w/w), respectively, at 30 g/L SS concentration of
municipal sludge. Growth inhibition was observed when glycerol was supple-
mented in higher concentrations, 100 and 150 g/L. The optimal concentration of
crude glycerol for maximum lipid production was found to be 40 g/L. Thereafter,
they supplemented the sludge-glycerol medium with nitrogen sources like peptone
and urea and obtained the enhanced lipid concentration of 16.4 and 11.39 g/L,
respectively, which is because nitrogen is involved in the synthesis of important
cell components, including protein and nucleic acids, enzymes, peptone, and urea
assisted in cell growth phase. They observed that the energy conversion efficiency
(energy output/net energy input) of the control (with no nitrogen), with additional
1 g/L urea and additional 3.7 g/L peptone was greater than 1 indicating an
energetically favorable process.
Zhang et al. (2017) also performed cost estimation of biodiesel production; it
was nearly $0.75/L assuming crude glycerol free of cost. Although it was only
$1.25/L, when the price of crude glycerol was considered to be $0.1/kg while
sludge was considered free of cost assuming biodiesel production plant was near to
municipal wastewater treatment plant. The biodiesel unit production using waste
substrates was comparable to that of vegetable oil–based biodiesel, $1.15/L
(Apostolakou et al. 2009). The techno-economic studies reveal that sludge can
be a potential and economical source for lipid production, which can be extracted
from cells and later converted into biodiesel.
12.6 CHALLENGES AND FUTURE PERSPECTIVES
Using sewage sludge as a raw material for biodiesel production at the commercial
level poses many challenges, including (1) variability of sludge characteristics,
(2) soap formation and product separation, (3) screenings of oleaginous microbes,
and (4) impurities present in sludge (Kargbo 2010). These challenges are briefly
explained in this section.
• Variability in sludge characteristics: Currently, primary and secondary
sludges are under intense research for biodiesel production. Primary sludge
is composed of floating grease and solids that are collected after screening and
grit removal, whereas activated sludge or secondary sludge is a combination of

microbial cells and suspended solids. Variability in sludge characteristics like
the presence of inert materials, biodegradability, initial lipid content, and
others, affects the final lipid concentration obtained during fermentation. The
synergistic effects of treatment conditions in the WWTP, concentration of

acid catalyst, and the mass ratio of methanol to sludge affect the final FAME
yield significantly. Although the FAME yield by activated sludge is signifi-
cantly affected by these factors independently, delivering a similar FAME
yield with variability in sludge characteristics is a challenge.
• Soap formation and product separation: Because the sewage sludge consists of
a high concentration of FFA, it may lead to soap formation and hence is
difficult in product separation. At ambient temperature, the resulting soap
forms the gel, making the entire product a semisolid mass instead of biodiesel.
Soaps also interfere with the glycerol separation and washing. Water also
interferes in transesterification because it results in the hydrolysis of fats to
FFA, thereby soap formation. Hence, for a feedstock with greater than 1%
FFA, esterification (acid catalysis) followed by transesterification (basis
catalysis) is recommended. However, an additional esterification step can
increase biodiesel production cost.
• Screening of oleaginous microbes: WWTPs are equipped with the bioreactors
that contain microorganisms to use the pollutants as a food source to
maintain their metabolism and reproduction. The challenge comes in the
screening of high lipid-accumulating microorganisms to boost biodiesel
production from wastewater sludge because sludge contains many inhibitory
compounds like lignin and hemicellulose. Hence, potential microbes that can
use these complex carbon sugars should be isolated.
○ T. oleaginosus has been used for lipid accumulation using sludge. Other
oleaginous microbes should be tested at laboratory scale for lipid accu-
mulation using sludge.
• Impurities present in sludge: Wastewater sludge also contains emerging
contaminants, for example, the fatty acids that affect lipid accumulation
during fermentation. The traces of the organic chemicals adsorb on the
surface of the sludge, and subsequently need effective pretreatment to increase
nutrient availability to microbes during lipid fermentation. The presence of
inert materials in sludge also requires a high volume of solvents during lipid
extraction and transesterification process for high conversion efficiency.
As compared in many studies, biodiesel produced from sludge shows similar
fatty acid profile with that of the vegetable oils or current commercial biodiesel.
Because the FAME from sludge is rich in saturated fatty acids, it gives a better
oxidation stability to the biodiesel. This indicates that wastewater sludge is being
proved to be a potential candidate for biodiesel production. Also, the wastewater
sludge is a cheap raw material and easily available. Hence, biodiesel production
from sludge could also help in reduction of excess sludge.
12.7 SUMMARY
Biodiesel has occupied its prominent position among the renewable liquid fuels. In
comparison to diesel fuel, biodiesel shows many benefits, but its higher production
cost makes it uncompetitive with petro-diesel. Current raw materials (vegetable

oils and animal fats) used for biodiesel production create a food-versus-fuel
problem, thereby increasing biodiesel production cost. To overcome this, waste-
water sludge can be used as a potential source for biodiesel production because it is
cheap and readily available. However, using sludge as feedstock also poses many
challenges, including (1) pretreatment of raw sludge involves the collection of
sludge from the site, its dewatering, concentration, and thermal pretreatment
to make it suitable for further process, which adds the cost to the process;
(2) expensive extraction of lipids from sludge or sludge-derived microbial oil
resulting from the requirement of a large volume of organic solvents; and
(3) biodiesel production methods and downstream processing are very expensive
for sludge-derived FAME. Hence, extensive research work is required for opti-
mizing the downstream process to make wastewater sludge a more profitable
candidate compared with other sources.
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CHAPTER 13
Conversion of Crude Glycerol
to Lipid and Biodiesel

L. R. Kumar
R. D. Tyagi
13.1 INTRODUCTION
Biodiesel production is increasing worldwide because it is made from renewable

biological sources and it does not pose environmental concerns (Xie et al. 2006).
As a byproduct of the transesterification step of biodiesel manufacturing, the
quantity of glycerol in the market has increased. Although crude glycerol is mainly
produced from five industries—soap, fatty acids, biodiesel, fatty alcohol, and
synthetic production—Table 13-1 clearly indicates that the percentage supply of
crude glycerol has shifted from fatty acid in 1999 to biodiesel in 2009. Currently,
the glycerol price is determined by demand and production of biodiesel.
Glycerol production has increased substantially in the last few years as clearly
shown in Figure 13-1. It has been reported that 87,000 tons (1.87 × 108 kg) of
crude glycerin were created in 2007 from biodiesel manufacturers (Ayoub and
Abdullah 2012). By 2020, 5.87 billion lbs (2.66 × 109 kg) of crude glycerol would be
produced (Figure 13-1). The estimated demand for biodiesel production in 2020 is
8 billion gal. (3.03 × 1010 L). Although glycerol has applications in several indus-
tries (Table 13-2), its overproduction is a serious problem, and alternate ways of
disposal or use are sought.
Commercial substrates for lipid fermentation are expensive, and the cost for
raw material acquisition is a major contributing factor in biodiesel production cost
(Koutinas et al. 2014). Thus, alternative substrates for lipid production should be
found. Recently, considerable research has been conducted on utilizing crude
glycerol as and economical carbon source for cultivation of oleaginous micro-
organisms for lipid production. Through transesterification, crude glycerol can be
directly used for biodiesel production. Crude glycerol contains glycerol along with
other nutrients that can enhance biomass and lipid accumulation during
fermentation.
305
Table 13-1. Change in Trend for Glycerol Supply.
Industry Supply in 1999 (%) Supply in 2009 (%)
Biodiesel 9 64
Fatty acid 47 21
Soap industry 24 6
Fatty alcohols 12 8
Synthetic production 6 0
Others 2 1
Crude glycerol production with time

3.00
2.50
Quantity (Billion kg)
2.00
1.50
1.00
0.50
0.00
2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020
Year
Figure 13-1. Estimated production of crude glycerol.
This chapter overviews lipid production studies using oleaginous microbes,

metabolism of glycerol utilization by oleaginous microbes, effect of impurities in
crude glycerol on lipid production, optimization of fermentation parameters for
Table 13-2. Distributions of Crude Glycerol Applications to Various Worldwide

Outlets in 2006 (Adapted from Ayoub and Abdullah 2012).
Product Value (%)
Food 11
Detergents 2
Tobacco products 6
Polyether 14
Pharmaceuticals and personal care 34
Alkyd resin 8
Other products 11
Triacetin 10
Explosives 2
Cellophane 2
CONVERSION OF CRUDE GLYCEROL TO LIPID AND BIODIESEL 307
enhanced lipid production, and kinetic studies for lipid production by different
microbes.
13.2 CHARACTERISTICS AND COMPOSITION OF CRUDE GLYCEROL
Glycerol is a material of choice because its physical and chemical properties make
it versatile, easy to handle, and compatible with other substances. It is nontoxic to
human health and the environment. Physically, it is an odorless, colorless, viscous
liquid with high melting point. Glycerol has three hydroxyl groups making it
soluble and hygroscopic in nature. The high value of carbon content (52.77%)
makes it a renewable source of energy. The major elements of crude glycerol from
the biodiesel industry include C (52.77% w/w), O (36.15%), and H (11.8%), with
N < 0.00001% (Ayoub and Abdullah 2012).
Table 13-3 compares quality parameters for crude glycerol, purified glycerol,
and commercial glycerol, which is also known as glycerin. Glycerol content varies
even if crude glycerol is taken from same industry. Table 13-4 shows the
composition of crude glycerol taken from different sources.
Table 13-3. Quality Parameters of Different Categories of Glycerol.
Crude Purified Refined/commercial

Parameter Units glycerol glycerol glycerol
Glycerol % 60–80 99.1–99.8 99.2–99.98

content
Moisture % 1.5–6.5 0.11–0.8 0.14–0.29
content
Ash % 1.5–2.5 0.054 <0.002
Soap % 3.0–5.0 0.56 N/A
pH — 0.7–1.3 0.1–0.16 0.04–0.07
Table 13-4. Composition in Various Crude Glycerol Sources.
Soap Stearin
Source/content production (%) Biodiesel (%) production (%)
Glycerol content 80 50–87 42

Nitrogen content 0.041 0.014–0.078 0.136
NaCl 7.59 0.2–5.47 1.23
Ash 8.76 0.93–6.34 1.35
Water 3.60 8.16–43.42 55.3
Studies have been conducted to evaluate the impact of glycerol concentration

on lipid production, and it was found that a glycerol concentration of 10% to 15%
w/v along with an optimized C/N ratio results in higher lipid productivities
(Dobrowolski et al. 2016). However, glycerol concentration is not the only
governing factor for deciding lipid productivity by a particular strain; operating

parameters like pH, aeration rate, and temperature, among others, need to be
optimized, along with the concentration of crude glycerol and suitable nitrogen
source for enhanced lipid productivity. A study indicated that pure glycerol
resulted in higher biomass and lipid productivity under the same cultivation
conditions (Dobrowolski et al. 2016). However, it is not always true, because other
studies indicated that crude glycerol resulted in higher lipid productivities
compared with pure glycerol due to the presence of additional nutrients available
in the media (Chen and Walker 2011). These studies are discussed in detail in
Section 13.5.
13.3 RATIONALE FOR USE OF CRUDE GLYCEROL IN LIPID

PRODUCTION
Biodiesel and lipid manufactured using oleaginous microbes are more sustainable
and biodegradable compared with conventional biodiesel processes. Also, micro-
bial lipid-based biodiesel does not pose environmental concerns like deforestation,
global warming, and greenhouse gas emissions. For biodiesel production, the
feedstock cost and operational expense are the two major costs. The cost of
feedstock accounts for 60% to 70% of the total cost of the biodiesel (Huang et al.
2010). During heterotrophic algae cultivation, usually glucose is used as a carbon
source, which can contribute to 80% of the total medium cost (Li et al. 2007).
Therefore, an inexpensive substitute for glucose is required. Crude glycerol can be
used as a carbon substrate during heterotrophic cultivation of microalgae for lipid
production because carbon content in crude glycerol is around 52% as discussed in
Section 13.2.
In transesterification processes, alcohols are mixed with triacylglycerols
(TAGs) in the presence of catalysts to produce fatty acids esters along with crude
glycerol as a primary byproduct. In general, glycerol produced during transester-
ification is 10% (by weight) of biodiesel produced. Figure 13-2 clearly shows
glycerol is produced with biodiesel at a ratio of 1:10, that is, for every 45.3 kg of
biodiesel, 4.53 kg of crude glycerol is produced. The crude glycerol possesses
purity of 80% to 88% and needs further purification steps to meet the purity of
industrial-grade glycerol (99% purity).
Because crude glycerol was continuously produced as a byproduct over
the last decade, a substantial decrease in crude glycerol (80% purity) price, from
USD 0.25/lb (USD 0.55/ kg) to USD 0.025/lb (USD 0.05/kg), between 2004 and
2005 was seen (Yazdani and Gonzalez 2007). Besides, the cost of purification
of crude glycerol to commercial pharmaceutical-grade is high (Chi et al. 2007).
Figure 13-2. Crude glycerol formation during transesterification process.
The purification process is costly for both small- and medium-scale biodiesel plants
(Haas et al. 2006). However, at lower prices of crude glycerol, the cost becomes very
competitive with sugars, such as glucose, that can be used as substrate for cultivation
of biomass and lipid production by oleaginous microorganisms. In addition,
converting crude glycerol to value-added products, including lipids, not only
provides an alternative for crude glycerol disposal but also poses a solution to
replacement of expensive glucose as a carbon source in fermentation medium.
13.4 METABOLISM FOR GLYCEROL UPTAKE
A variety of microorganisms can accumulate a substantial amount of lipid

(>20%, w/w) on dry weight basis when environmental conditions are appropri-
ate. These microorganisms are called oleaginous and are characterized by their
ability to use citric acid as an acetyl-CoA donor in the anabolic pathway of fatty
acid synthesis. Isocitrate dehydrogenase (ICDH), the enzyme responsible for the
transformation of iso-citric to α-ketoglutaric acid, loses its activity since it is
allosterically activated by intracellular adenosine monophosphate (AMP),
resulting in the accumulation of citric acid inside the mitochondrion. When
the concentration of citric acid becomes higher than a critical value, citric acid is
secreted into the cytosol. In the case of oleaginous yeasts, cytosolic citric acid is
cleaved by ATP-citrate lyase (ACL), the key enzyme of lipid accumulation
process, into acetyl-CoA and oxaloacetate, where acetyl-CoA is converted to
cellular fatty acids (Papanikolaou and Aggelis 2009). The presence of ACL does
not make any organism completely oleaginous. Even though certain organisms
possess ACL, they accumulate less than 20% lipid of total dry cell mass, Hence,
ACL can be called as a precursor provider for lipogenesis (acetyl-CoA). Lipid
biosynthesis begins when acetyl-CoA is reduced to malonyl-CoA by fatty acid
synthesis. The metabolic pathway for lipid production from glycerol is described
in Figure 13-3.
Figure 13-3. Pathways for glycerol catabolism and intracellular lipid synthesis.
It has been reported that oleaginous yeasts like Y. lipolytica have three types of
metabolism. The first type of metabolism happens when lipid production occurs
in nitrogen exhaust condition (Fontanille et al. 2012). The second type is when
lipid is stored inside cells but afterward, it starts disintegrating and citric
production starts (Makri et al. 2010). The third type is when citric acid is
produced outside the cells while lipid is simultaneously accumulated inside yeast
cells without degradation of lipids (André et al. 2009). The problem of citric acid
production can be solved by genetic manipulation and directing the metabolic flux
for lipid synthesis instead of citric production.
Microorganisms responsible for lipid production using glycerol as substrate
are oleaginous yeasts and algae. Oleaginous yeasts like C. curvatus and Y. lipolytica
have been used for lipid production because they are capable of accumulating high
lipid content and can grow on a variety of substrates (Liang et al. 2010). Algae
species like Chlorella protothecoides and Schizochytrium limacinum are effective
oil producers. Studies have reported their use for lipid production with different
carbon substrates (Chen and Walker 2011, Miao and Wu 2006). Two critical
regulatory enzymes, ACL and nicotinamide adenine dinucleotide phosphate
(NADPH)-malic enzyme, affect lipid accumulation. A strong correlation between
the presence of ACL activity and the ability to accumulate lipid in yeast, fungi,
and other oleaginous microorganisms has been reported (Ratledge 2002).
Malic enzyme is the second key enzyme in lipogenesis. The two-carbon compound
(acetyl-CoA) is added to the fatty acid chain by fatty acid synthase. This requires
two molecules of NADPH as a reducing power for the growth of TAG, and
NADPH is supplied mainly by malic enzyme (ME) as shown in Figure 13-4.
Although there are several pathways that lead to the generation of NADPH in
oleaginous and nonoleaginous organisms, ME has been reported as the key
enzyme that supplies NADPH toward lipid biosynthesis (Ratledge and Wynn
2002). ME is crucial for lipid synthesis because the complete inactivation or
inhibition of ME totally arrests lipid synthesis.
Besides malic enzyme and ACL, acetyl-CoA carboxylase (ACC) plays a key
role in the synthesis and degradation of fatty acids. ACC is a multi-subunit
enzyme in prokaryotes, but in eukaryotes it is a multidomain enzyme. ACC is a
biotin-dependent enzyme and catalyzes the first carboxylation of acetyl-CoA to
generate malonyl-CoA, which is regarded as the primary step in synthesis of long-
chain fatty acids. The carboxylation activity of ACC depends on the control of
three different signals in eukaryotes, including glucagon, epinephrine, and insulin.
Among these, insulin activates the carboxylase and favors the fatty acid synthesis,
whereas the other two have a negative impact on fatty acid synthesis (Berg et al.
2002).
The pattern of lipid production is expressed in Figure 13-5. At the beginning
of nitrogen limitation, lipid production begins with the activation of lipogenic
enzymes. The initial growth phase lasts for about 20 h, and then the organism
undergoes a transition phase during which excess carbon can be translated into
Figure 13-4. Reaction for NADPH production using malic enzyme.
Figure 13-5. Pattern for lipid accumulation.

312
Table 13-5. Comparison of Biomass and Lipid Production for Different Oleaginous Species Grown under Crude Glycerol.
Biomass Glycerol uptake Lipid content
Species DW (g/L) rate (g/L-day) (%) Strategy References
C. protothecoides 23.5 NA 62 Batch Chen and Walker (2011)

C. protothecoides 45.1 NA 54 Fed-batch Chen and Walker (2011)
C. curvatus 31.2 19.2 44.6 Fed-batch Liang et al. (2010)
C. curvatus 32.9 28.3 52.9 Improved Fed-batch Liang et al. (2010)
S. limacinum SR21 11.5 8.3 50.4 Batch Pyle et al. (2008)
S. limacinum SR21 18 5.9 50.5 Batch Chi et al. (2007)
M. isabellina 8.5 3.1 51.7 Batch Papanikolaou et al. (2008)
Y. lipolytica 8.1 NA 43 Continuous Papanikolaou and Aggelis (2002)
citrate, which then results in lipid synthesis. Media composition and C/N ratio
during fermentation decide the citric acid production. The fermentation can shift
from citric acid to lipid production or only citric acid production or vice versa,
depending on the media composition and cultivation condition. Nitrogen content
is more desirable in lipogenic phase as higher carbon concentration inhibits ACL,

which results in citrate accumulation instead of being diverted to TAG synthesis
(Beopoulos et al. 2009).
Table 13-5 summarizes growth characteristics along with lipid production
capabilities of various species. However, the difference in biomass and lipid
productivities arises because of variances in basal media composition, inoculum
size, and cultivation conditions. A comparative study on all the reported opti-
mized lipid producing technologies is described in Section 13.6.
13.5 RECENT STUDIES ON LIPID PRODUCTION FROM CRUDE

GLYCEROL
Several successful laboratory-scale studies have been conducted on utilizing crude

glycerol as substrate for lipid production. Some of the studies are described in the
following sections.
13.5.1 Selection of Strains Utilizing Crude Glycerol

In one study, 899 yeast strains were isolated to test whether they can grow using
glycerol as carbon source (Kitcha and Cheirsilp 2011). They were identified based
on the 26S rDNA sequence, and obtained sequences were BLAST searched against
the National Center for Biotechnology Information (NCBI) database. Of them,
only 23 strains were identified as potential lipid producers or oleaginous yeast like
Kodamaea ohmeri and Trichosporonoides spathulata using Sudan Black B tests. In
this study, influence of crude glycerol and inorganic nitrogen sources (ammonium
sulphate) were also investigated on biomass and lipid production. The optimal
condition for both strains was found to be 0.5% ammonium sulphate and 10%
crude glycerol (C/N ratio of 17). The study showed that bioconversion of crude
glycerol to oils by oleaginous yeast is a promising method to produce biodiesel and
may even help in reduction of accumulated carbon in the atmosphere and disposal
of crude glycerol. However, the paper does not highlight fatty acid composition of
lipids produced and comparison with vegetable oils. Figure 13-6 represents
important steps mentioned in this study for lipid production using crude glycerol
as a carbon source.
In another study, 113 different species of yeast with potential biotechnological
applications were screened for their ability to utilize crude glycerol as carbon
source (Taccari et al. 2012). Yeast strains were screened based on specific growth
rates (h−1), biomass production, and yields. Response surface methodology (RSM)
was used to optimize temperature and crude glycerol concentration during
fermentation for enhanced biomass production and yield by Yarrowia lipolytica,
Screening of yeast
Identification of lipid Selection of nitrogen
strains utilising glycerol
producing yeasts source
as carbon source
Optimization of C/N
Biodiesel production High lipid productivity
ratio
Figure 13-6. Methodology of biodiesel production starting with selection of

glycerol-utilizing strains.
Metschnikowia sp., Debaryomyces sp., and Rhodotorula mucilaginosa. The

study described crude glycerol valorization by yeast species for value-added
products and potential use of crude glycerol as carbon source in yeast cultivation.
Figure 13-7 represents important steps mentioned in this study for lipid produc-
tion using crude glycerol as a carbon source.
13.5.2 Fermentation Cultivation Mode

Biodiesel-derived crude glycerol (made by Southeast Biodiesel) has been studied
for biomass and lipid production during heterotrophic cultivation of algae
C. protothecoides (Chen and Walker 2011). C. protothecoides was chosen as an
oil producer because high biomass and lipid production was obtained with
C. protothecoides using different carbon substrates in the past (Miao and Wu
2006). Chen and Walker (2011) drew a comparison of biomass and lipid
production based on different substrates and a comparison between different
Primary screening of yeast

Secondary screening using
strains using pure glycerol
crude glycerol medium
medium
Optimization of temperature
Enhanced lipid and biomass
and crude glycerol
production
concentration using RSM
Figure 13-7. Methodology for enhanced lipid production starting from screening
of yeast strains.
Table 13-6. Biomass and Lipid Productivity under Different Carbon Sources and
Fermentation Modes.
Biomass Lipid
productivity productivity
Carbon source (g/L day) (g/L day)
Results of batch mode

Glucose 3.1 1.5
Pure glycerol 3.2 1.6
Crude glycerol 3.8–4 2.4
Results of fed-batch fermentation
Pure glycerol 5.2 2.8
Crude glycerol 5.5 3
fermentation strategies—fed batch and batch. As shown in Table 13-6, crude

glycerol resulted in higher cell biomass and lipid productivity. This can be
attributed to the presence of additional nutrients like nitrogen source in crude
glycerol medium because a small amount of impurities present in crude glycerol
medium may have beneficial effects on microalgae growth (Chi et al. 2007). Lipid
production and cell growth were higher in the fed-batch strategy because the effect
of methanol inhibition is reduced by regular feeding of the carbon source.
However, the study did not mention the lipid composition produced because
lipid composition would reveal the percentage of polyunsaturated fatty acids
present in lipids which are highly desirable for biodiesel production. This
technology can be highly economical for biodiesel production because it uses
low-cost substrate, crude glycerol as feedstock source, and the problem of disposal
of crude glycerol produced in transesterification is also eliminated.
In another study, crude glycerol produced during biodiesel production
was used to grow oleaginous yeast, Cryptococcus curvatus (Liang et al. 2010).
C. curvatus was chosen because it requires minimum nutrients for growth, is
capable of accumulating up to 60% of its dry weight with intracellular lipid
(Hassan et al. 1996), the accumulated oil resembles plant seed oils (Davies 1988),
and it can grow on a wide range of substrates (Daniel et al. 1999). Crude glycerol
was generated from biodiesel production using yellow grease as feedstock. Liang
et al. (2010) draws a comparison between two-stage fed-batch culture operation
(glycerol source was fed at different points of time, and nitrogen source was
discontinued mid-way of the operation) and single-stage fed-batch culture
operation (nitrogen and glycerol source were fed intermittently). It is evident
from Table 13-7 that lipid accumulation was higher in the two-stage fed-batch
process when nitrogen content was limited, which is in accordance to studies
in literature (Hassan et al. 1996). Table 13-8 summarizes fatty acid composition
from the study conducted by Liang et al. (2010) and other vegetable oils where
C. curvatus produced monounsaturated fatty acids in the similar range compared
Table 13-7. Comparison of Two-Strategies FOR Lipid Production Using Oleaginous

Yeast.
Strategy Biomass concentration (g/L) Lipid content (%)

Fed-batch single-stage 31.2 44.2

Fed-batch two-stage 32.9 52
Table 13-8. Comparison between Yeast and Other Oil Feedstocks for Fatty Acids.
Jatropha Soybean Palm C. curvatus Liang et al.

Fatty acid (%) (%) oil (%) (%) (2010) (%)
Palmitoleic 0.3–0.5 0–1 0 0 0.9

acid 16:1
Oleic 41–49 22–34 40–52 45–50 39.6
acid 18:1
Monounsaturated 41–49 22–35 40–52 45–50 40.5
with natural oils. C. curvatus is capable of uptake of methanol, which is considered

inhibitory for microalgal growth because methanol concentration decreased in the
medium with respect to time. Lipid produced in this study has high concentration
of monosaturated fatty acid, making it an excellent source for biodiesel produc-
tion. The paper provides the crude glycerol-to-lipid culture model that will
provide oil feedstock for biodiesel production while eliminating the problem of
crude glycerol disposal.
Lipid has been produced by Y. lipolytica utilizing crude glycerol as substrate
(Papanikolaou and Aggelis 2002). An alternate way of glycerol valorization is
biotransformation to single cell oil (SCO) by oleaginous yeasts and molds.
Previous studies showed that this yeast presented high lipid accumulation (around
40% of dry weight) when cultivated on industrial wastes (Papanikolaou et al.
2001). It was found that glycerol can be a good carbon source for oleaginous
microorganisms because growth was not inhibited by large concentrations of
glycerol. Unpurified industrial glycerol was obtained from the biodiesel produc-
tion unit and fat saponification unit. Papanikolaou and Aggelis 2002 drew a
comparison between highly aerated single-stage continuous culture and nitrogen-
limited flasks; in the latter case, lipid productivity was low and citric acid
production was more. Hence, continuous culture was used for lipid production.
The authors also observed that lipid yield was mainly influenced by dilution
rate (Table 13-9). Lower dilution rates resulted in high lipid production rates
(Table 13-9), whereas higher dilution rates resulted in nonlipid material synthesis.
Oleic (45%) and linoleic acids (15%) are dominant fatty acids produced in the
study conducted by Papanikolaou and Aggelis 2002, which are monosaturated
Table 13-9. Steady-State Parameters for Single-Stage Continuous Fermentation

for Lipid Production.
Dilution rate (h−1) Biomass concentration (g/ L) Lipid concentration (g/L)

0.03 8.1 3.5

0.04 7.1 3.0
0.08 4.9 1.1
0.085 4.0 0.4
0.130 3.8 0.3
fatty acids, useful for biodiesel production. However, no effect of dilution rate or
initial glycerol concentration was seen on fatty acid composition. The above study
displayed production of lipids using oleaginous yeast, Y. lipolytica, which can be
metabolically engineered to produce larger quantities of monounsaturated fatty
acids, useful for biodiesel production.
In Yang et al. (2014), a two-stage lipid production process has been developed
for monounsaturated fatty acid (MUFA) production. In the first step, cells are
cultivated in a nutrient-rich medium for growth; in the second step, cells are
resuspended in a medium having carbon source with auxiliary nutrients for lipid
production.
Two media were prepared—synthetic crude glycerol media (obtained after
transesterification in biodiesel company) after dilution with water and pH
adjustment (used for lipid production) and pure glycerol/glucose media with
yeast extract and peptone (used for cell growth) (Yang et al. 2014). The preculture
media were centrifuged after 48 h of cultivation, and harvested cells were used as
an inoculum for lipid production. Oleaginous yeast Rhodosporidium toruloides
showed lipid yield of 0.22 (with respect to glycerol) using two-stage lipid
production strategy. Presence of impurities like K2SO4 in lipid production media
resulted in higher lipid content and a higher percentage of MUFA compared with
the control. Initial pH of lipid production media also affected lipid content because
lipid content decreased on increasing pH from 5.5 to 11. Fatty acid compositional
profiles were similar to those of vegetable oils, suggesting that microbial lipids
were potential feedstock for biodiesel production. This unique technology of
two-stage lipid production is an attractive route for biodiesel production with
better economic viability (Figure 13-8).
Y. lipolytica ACA-DC 50109 was cultivated in a highly agitated and aerated
bioreactor under crude glycerol, and lipid content observed to be 43% to 44% on
weight basis (Papanikolaou and Aggelis 2009). Experiments were conducted in
nitrogen-limited continuous culture and fed-batch aerated systems by varying
dilution rates and glycerol concentration. Lipid content was found to be 25% at
low dilution rates in 55 h. The lipids produced were mainly oils having polyun-
saturated fatty acids like γ-linolenic acid. SCO produced could be directly
converted into biodiesel. This paper also presented that Y. lipolytica is also
Figure 13-8. Microbial lipids produced from the conventional process (Path A) and
the two-stage (Path B) process.
capable of producing citric acid in addition to lipids, which has applications in

food, pharmaceutical, and chemical industries since citric acid is considered as safe
and has pleasant acidic taste, high water solubility, and chelating and buffering
capacities. Although Papanikolaou and Aggelis (2009) highlighted the potential of
Y. lipolytica for both lipid and citric acid production, metabolic engineering can
assist in regulating metabolic flux for lipid production.
T. spathulata, a high lipid-accumulating yeast was cultivated using crude
glycerol as a sole carbon source (Kitcha and Cheirsilp 2013). The process was first
optimized in a 5 L bioreactor equipped with a pH control and aeration system.
Suitable nitrogen source was first found using an optimized C/N ratio of
8.6—yeast extract, peptone, urea, (NH4)2SO4, and NH4NO3. Lipid content of
44.3% was obtained using 10% (w/v) of crude glycerol supplemented with 0.5%
(w/v) ammonium sulphate (single feed strategy). Two fed-batch studies were used
to optimize lipid production and two-stage fed-batch feeding resulted in higher
lipid production (7.78 g/L) and lipid content (56.4%) compared with single-stage
fed-batch feeding strategy. In this study, crude glycerol–based medium with
addition of ammonium sulphate was used in the first stage, and only crude
glycerol was fed in the second stage. Fatty acid composition obtained was similar
to that obtained using glucose as carbon source and was similar to that of plant
and vegetable oil. T. spathulata has a high potential for lipid production, serving as
biodiesel feedstock, and lipid production is achieved using cheap substrates while
reducing carbon dioxide content in atmosphere.
13.5.3 Optimization of C/N Ratio, Media Components, and

Fermentation Parameters
Oleaginous red yeast Rhodotorula glutinis has been used for bioconversion of
crude glycerol generated by a biodiesel plant to lipids and carotenoids (Saenge
et al. 2011). Rhodotorula glutinis was chosen because it has high growth rate and is
capable of rapidly accumulating both lipids and carotenes. In Saenge et al. (2011),
a suitable surfactant Tween 20 was also screened for enhanced production of
lipids. Parameters like the C/N ratio (85, ammonium sulphate was used as
N source) and glycerol concentration (9.5%) in fermentation medium were
optimized by three-level response surface analysis to give optimum concentration
of lipids. The production of lipids was further improved in a stirred tank
bioreactor with optimizing pH (6) and aeration rate (2 vvm). A comparison was
Table 13-10. Comparison of Batch and Fed-Batch Fermentation for Lipid

Production.
Parameter Batch Fed-batch

Biomass (g/L) 8.17 10.05

Lipid content (%) 52.91 60.70
Productivity of lipids (g/L/h) 0.06 0.09
Total lipid concentration (g/L) 4.33 6.10
drawn between batch cultivation and fed-batch cultivation (Table 13-10). Effect of
surfactant on biomass, lipid content, and glycerol consumption was also investi-
gated (Table 13-10). Because of the high lipid content and glycerol uptake,
Tween 20 was selected as a suitable surfactant. The yeast lipids have shown
favorable properties of a feedstock in biodiesel production owing to a similar
composition of fatty acids with that of vegetable oil. The highest fatty acid content
was oleic acid (45.75%), which is monounsaturated fatty acid useful for biodiesel
production (Table 13-11). This study showed that optimized parameters like pH,
aeration rate, C/N ratio, glycerol concentration, along with suitable surfactant in
fed-batch mode can lead to enhanced lipid production using crude glycerol as
substrate.
In Dobrowolski et al. (2016), conversion of crude glycerol from various
industrial wastes into lipids by yeast Yarrawia lipolytica was reported. Glycerol
sources from five different waste products from industries (soap, biodiesel, and
stearin production), each containing different concentrations of glycerol
(Tables 13-12 and 13-13), were compared. Growth rates and lipid productivities
were also investigated on different C/N ratios from 20 to 100, but they were found
maximum when the C/N ratio was 75 or 100. It was found that crude glycerol
derived from soap production showed faster cell growth and higher lipid
productivities. Moreover, this substrate did not require further purification and
yeast supplementation, resulting in lower production costs. The C/N ratio also
affected fatty acid profile, but total monounsaturated fatty acids produced were
Table 13-11. Impact of Nitrogen Source and Surfactant on Lipid Production.
Biomass Lipid Glycerol

Culture condition (g/ L) content (%) consumption (%)
Control 3.06 16.12 30.55

(NH4)2SO4 4.53 23.05 40.55
(NH4)2SO4 + Tween 20 5.47 35.22 48.21
(NH4)2SO4 + Tween 80 5.26 33.33 46.50
(NH4)2SO4 + gum arabic 5.22 31.18 44.24
320
Table 13-12. Composition in Various Crude Glycerol Sources.

Soap Biodiesel Biodiesel Biodiesel Stearin Pure

Source/Content production (%) (Source-1) (%) (Source-2) (%) (Source-3) (%) production (%) glycerol (%)
Glycerol content 80 50 87 80 42 100

Nitrogen content 0.041 0.078 0.023 0.014 0.136 0
NaCl 7.59 3.04 0.20 5.47 1.23 0
Ash 8.76 3.62 0.93 6.34 1.35 0
Water 3.60 43.42 11.85 8.16 55.30 0
Table 13-13. Fermentation Parameters for Different Crude Glycerol Sources.

C/N 75 C/N 100
Biomass Lipid Lipid Biomass Lipid Lipid
Glycerol source (g/L) production (g/L) content (%) (g/L) production (g/L) content (%)
Soap production 7.8 1.30 16.6 6.87 1.70 25

Biodiesel (Source-1) 6.07 1.10 18 3.67 0.65 18
Biodiesel (Source-2) 2.87 0.17 5.9 2.4 0.27 11.20
Biodiesel (Source-3) 6.63 1.32 19.8 3.83 0.93 24.30
Stearin production 3 0.15 5.10 2.47 0.68 28
Pure glycerol 8.47 2.30 27.30 8 2.06 26
CONVERSION OF CRUDE GLYCEROL TO LIPID AND BIODIESEL
321
74% of total lipid pool in all cases, which is significant and desirable for biodiesel
production. Along with lipids, citric acid was also produced during fermentation.
The problem of citric acid production can be solved by genetic manipulation and
directing the metabolic flux for FFA synthesis instead of citric production. This
technology demonstrates the feasibility of the lipid production process using

cheap substrate as carbon source without purification and yeast supplementation.
Engineered Y. lipolytica strain with enhanced lipid metabolism and modification
of fatty acid composition can yield a promising breakthrough in low-cost and
efficient synthesis of lipids.
Tchakouteu et al. (2015) studied lipid production by yeasts on biodiesel-
derived crude glycerol, in which strain was selected and impact of substrate
concentration was studied on fermentation efficiency. Microorganisms like
Y. lipolytica, C. curvatus, R. toruloides, and L. starkeyi were grown in nitrogen-
limited flask cultures. Y. lipolytica mainly produced mannitol and citric acid, but
L. starkeyi DSM 70296 and R. toruloides NRRL Y-27012 showed potential for lipid
production. They were cultivated at constant nitrogen concentration and increas-
ing initial glycerol concentration. Even with high initial glycerol concentration,
faster growth rates and high lipid productivities were observed. High lipid content
was observed (35% to 40%) for both L. starkeyi and R. toruloides, which was quite
high compared with values obtained in literature (Zhao et al. 2008) (Li et al. 2008).
The yeast lipids mainly produced palmitic acid (saturated fatty acids) and oleic
acid (monounsaturated fatty acids). Oleic acid is a good source of biodiesel
production, and palmitic acid has applications in nutraceutical and pharmaceuti-
cal industries.
Table 13-14 summarizes the effects of glycerol concentration and growth
phase on composition of fatty acids for R. toruloides NRRL Y-27012 and
Table 13-14. Fatty Acid Composition of Cellular Lipids Produced by Yeast Strains
obtained from Tchakouteu et al. (2015).
R. toruloides L. starkeyi
NRRL Y-27012 DSM 70296
Growth
Glycerol (g/L) phase Oleic acid Oleic acid
30 Early 55.5 46.1

Late 50.2 52.1
50 Early 53.9 52.3
Late 48.7 57.3
95 Early 54.1 —
Late 50.1
120 Early — 53.1
Late 57
180 Early 47 —
Late 44
Lipomyces starkeyi DSM 70296. Higher glycerol concentration resulted in higher

content of monosaturated fatty acids (MUFA) in Lipomyces starkeyi DSM 70296,
whereas the opposite effect of glycerol concentration was seen on R. toruloides
NRRL Y-27012. More MUFA was produced in late growth phase for Lipomyces
starkeyi DSM 70296; for R. toruloides NRRL Y-27012, more MUFA was produced
in early growth phase. Tchakouteu et al. (2015) showed that L. starkeyi and
R. toruloides are capable of growing on economical substrate crude glycerol while
producing lipids, which can further undergo transesterification for biodiesel
production. This was one of the few studies that indicated lipid/SCO production
by R. tortuloides and L. starkeyi growing on crude glycerol.
13.5.4 Pretreatment of Crude Glycerol

Use of microalgae Schizochytrium limacinum SR21 for lipid production using
crude glycerol has been reported (Liang et al. 2010). Schizochytrium limacinum
SR21, a marine microalgae, has shown great potential in converting glycerol to
lipids (Chi et al. 2007). The crude glycerol used in the study was derived from two
biodiesel manufacturers—Source M1 without methanol recovery and Source
M2 with methanol recovery (Table 13-15). Optimal glycerol concentration was
determined in untreated and treated crude glycerol (FFA removal after pH
adjustment) in batch culture, 25 and 35 g/L, respectively, and 35g/L glycerol
concentration (treated crude glycerol) resulted in 73.3% lipid content. The paper
by Liang et al. (2010) is unique from other papers because it compares fermenta-
tion using crude glycerol as substrate with and without treatment to see the effect
of glycerol treatment on lipid productivity (Tables 13-15 and 13-16).
As shown in Table 13-16, treated glycerol resulted in higher lipid productiv-
ities compared with untreated glycerol because removal of FFAs from crude
glycerol promoted algal growth and it has additional nutrients compared with
pure glycerol. Along with 65% to 70% lipid production, 10% to 15% each of
carbohydrate and proteins were formed. However, methanol recovery from crude
glycerol is imperative to avoid growth inhibition because untreated glycerol
without methanol recovery resulted in lower biomass dry weight and lipid
productivity (Table 13-16). Methanol was not consumed by S. limacinum cells
throughout fermentation. However, if methanol concentration after dilution was
Table 13-15. Characterization of Crude Glycerol from Two Sources.
Source-1 with Source-2 without

Composition (% w/w) Methanol Methanol
Glycerol 48.7 42.3

Soap 3 NA
Methanol 22.7 0
Water 25.6 9.6
Other impurities NA 48.7
Table 13-16. Parameters Related to Biomass and Lipid Production under Different
Conditions.
55 g/L Pure Source-1 Source-2

Parameter glycerol 23 g/L (UT) 26 g/L (T)
Biomass dry weight (g/L) 12.5 6.96 11.10

Substrate uptake rate (g/L-day) 7.57 3.77 6.13
Lipid productivity (g/L-day) 1.71 0.74 1.33
Lipid content (%) Not reported 65.80 62.90
Note: UT=untreated; T = treated.
less than 10 g/L, it would not cause an inhibitory effect. Higher concentrations of
glycerol resulted in slower cell growth due to substrate inhibition and methanol
presence. High methanol content can be removed by a 10 times dilution of the
crude glycerol sample with water and then passing through a 0.2 μm filter. Excess
NaOH or soap can be eliminated by adjusting pH to 1, followed by centrifugation
and then removing the top layer (reddish in color) having FFAs. However,
pretreatment methods like pH adjustment, centrifugation, filtration, and others
increase the production cost, proving a disadvantage of using crude glycerol as a
substrate for lipid production.
13.5.5 Coculture System for Lipid Production

In Cheirsilp et al. (2012), coculture of a microalgae Chlorella vulgaris and an
oleaginous yeast Rhodotorula glutinis enhanced biomass and lipid production
from crude glycerol. Microalgae can function as an oxygen producer in culture
and assist in yeast growth. The coculture system was grown on crude glycerol for
biomass and lipid production after optimizing the C/N ratio (urea was used as
cheap nitrogen source) to 32. The lipid produced consisted mainly of palmitic acid
(40.52%) and oleic acid (21.3%) and was similar in composition when compared
to natural oils. Although lipid production reported in this study was not high as
reported in previous works, this coculture system can be applied to other yeast
species for enhanced biomass and lipid production.
13.5.6 Other Studies

Valorization of biodiesel-derived crude glycerol by Y. lipolytica strains was
reported by André et al. (2009), in which crude glycerol was used as carbon
source for three natural Y. lipolytica strains (LFMB 19, LFMB 20, and ACA-YC
5033) in nitrogen-limited submerged shake-flask experiments. Strains LFMB 19
and LFMB 20 cultivation conditions favored more citric acid secretion and,
mannitol was the principal metabolic product synthesized. Strain ACA-YC-5033
produced higher lipid and citric acid content with constant nitrogen and
increasing glycerol concentration. Lipid content increased with an increase in
glycerol concentration and was accumulated inside the cells. Cellular lipids mainly
contained phospholipid fraction compared with neutral lipids, and microbial lipid
was found as saturated fatty acids in early growth steps. However, monosaturated
fatty acids were produced relatively more in the stationary phase compared with
the early growth phase. The technology showed how the appropriate glycerol and
nitrogen concentration in media can prove beneficial for biodiesel production and
also eliminate the problem of crude glycerol disposal.
In another study (Yen et al. 2012), crude glycerol derived from a biodiesel
production company (carbon source) was mixed with waste solution from a
brewing company (thin silage as nitrogen source), and Rhodotorula glutinis was
cultivated in the medium for production of microbial lipids. The two-component
mixture could result in higher lipid production compared with using just crude
glycerol or another expensive nitrogen source like yeast extract. Combinations of
cheap carbon and nitrogen sources for enhanced lipid productivity using oleagi-
nous yeasts could optimize economics for biodiesel production.
13.6 ANALYSIS OF STUDIES REPORTED IN LITERATURE
The aforementioned studies used different oleaginous microorganisms and culti-

vation modes that resulted in high lipid productivity and content. Table 13-17
compares important fermentation parameters used in recent laboratory-scale
studies that showed best results after optimization of media components and
operating parameters.
13.6.1 Fermentation Profiles for Reported Studies

Studies done using R. glutinis and C. protothecoides indicate that fed-batch
cultivation has high lipid yield (YP/X) and specific product formation rate (qP)
compared with batch cultivation (Chen and Walker 2011, Saenge et al. 2011).
Figure 13-9, (b) and (g), clearly indicates fed-batch strategy leads to higher
lipid and biomass production compared with batch strategy. Moreover, methanol
inhibition due to the presence of methanol in crude glycerol is also reduced
by constant feeding of the carbon source during fed batch. As shown in
Figure 13-9(a), crude glycerol has a high potential for fermentation substrate
for oleaginous microorganisms because higher biomass and lipid concentration
were observed during cultivation using crude glycerol compared with pure
glycerol; this is because crude glycerol can have additional nutrients that assist
in heterotrophic algae growth. On the basis of lipid yield (YP/X) and specific
product formation rate (qP), it can be concluded that R. glutinis TISTR 5159 and
S. limacinum have high potential for lipid production compared with other studies
when crude glycerol is used as carbon source. R. glutinis also has higher
μmax, 0.083 h−1, compared with other studies. Apart from R. glutinis and
S. limacinum, C. protothecoides also has high potential for lipid production. In
all previously mentioned studies, methanol present in crude glycerol source was
around 1%−22%; after being diluted with nitrogen source, the final methanol
Table 13-17. Comparison and Summary of Parameters and Operating Conditions Used in Various Lipid-Producing Technologies Utilizing
326
Crude Glycerol as Substrate.

Cultivation
mode/scale/ Medium and cultivation Biomass Lipid qP μmax
Microorganism time (h) conditions (g/L) (g/L) (h−1) (h−1) YP/X References
Chlorella Batch/ Media: 30 g/L (100% Glycerol), 19.2 9.8 0.0035 0.041 0.510 Chen and
protothecoides 200 mL/ 4 g/L yeast extract; Initial pH: Walker (2011)
144 h 6.8; Agitation: 200 rpm;
Aeration rate: 10% v/v; Temp:
28 °C, dark conditions
Chlorella Batch/ Media: 30 g/L (Glycerol: 62%, 23.5 14.6 0.0043 0.041 0.621 Chen and
protothecoides 200 mL/ Methanol: 22% + others), 4 g/L Walker (2011)
144 h yeast extract; Initial pH: 6.8;
Agitation: 200 rpm; Aeration
rate: 10% v/v; Temp: 28 °C, dark
conditions
Chlorella Fed-batch/ Initial media: 30 g/L (Glycerol: 45.2 24.6 0.0029 0.041 0.563 Chen and
protothecoides 2L/196.8 h 62%, Methanol: 22% + others), Walker (2011)
4 g/L yeast extract; Feed
media: 30 g/L (Glycerol: 62%,
Methanol: 22% + impurities),
4 g/L yeast extract; Feeding
strategy: started at 2 h at
interval of 24 h;
pH control: 6.8,Agitation:
200 rpm; Aeration rate: 30 L/h;
Temperature: 28 °C, dark
conditions
R. glutinis TISTR Batch/ 1 L/ Crude Glycerol: 50% glycerol, 7.8 4 0.0071 0.083 0.513 Saenge
5159 72 h 1%–3% methanol, 12% salts et al. (2011)
and organic matter and 36%
water; Media composition:
Glycerol concentration 9.5%,
Nitrogen source: Ammonium
sulphate, yeast extract and
peptone; C/N: 85 and Tween
20 as surfactant; Temperature:
30 °C,pH control: 6; Aeration
rate: 2 vvm;Agitation: 200 rpm
R. glutinis TISTR Fed-batch/ Crude Glycerol: 50% glycerol, 10 6 0.0083 0.083 0.60 Saenge
5159 1 L/72 h 1%–3% methanol, 12% salts et al. (2011)
and organic matter and 36%
water; Media composition:
Glycerol concentration: 9.5%,
Nitrogen source: Ammonium
sulphate, yeast extract and
peptone; C/N: 85 and Tween
20 as surfactant; Feeding
started at 12 h at interval of
12 h; Temperature: 30 °C; pH
control: 6; Aeration rate: 2 vvm;
Agitation: 200 rpm
(Continued)
327
328
Crude Glycerol as Substrate. (Continued)

Cultivation
Yarrawia lipolytica Batch/ Crude Glycerol: 25 5 0.0026 0.0417 0.20 Dobrowolski
A101 2 L/ 96 h 80% glycerol, no methanol and et al. (2016)
20% others; Media: Crude
glycerol from soap industry
with ammonium sulphate with
C/N: 100; Temperature: 28 °C;

pH control: 6; Aeration rate:
0.6 L/min; Agitation: 800 rpm
Trichosporonoides Two-stage Glycerol source: 40% glycerol, 13.5 7.78 0.004 0.042 0.564 Kitcha and
spathulate Fed-batch/ 1–3% methanol, salts and 12% Cheirsilp (2013)
2 L/144 h organic matter and 36–45%
water; Initial Media: 10% crude
glycerol (w/v) with 0.5%
ammonium sulphate; Feed
media: 12% crude glycerol.
Started at 60 h at interval of 12;
pH control: 6; Aeration rate:
3 vvm; Agitation: 140 rpm;
Room Temperature
Rhodotorula Co-culture Glycerol source: 50% glycerol, 1.5 0.9 0.005 — 0.600 Cheirsilp
glutinis batch/ potassium and sodium salts et al. (2012)
TISTR 5159 90 mL/ (4–5%), Methanol (1–3%),
(yeast) and 120 h nonglycerol organic matter
Chlorella vulgaris (1.6–7.5%) and water (36%);
TISTR 8261 Media: 3% pure glycerol and
C/N ratio of 32 with
ammonium sulphate as N
source; Initial pH: 6.0;
Temperature: 30 °C; Agitation:
140 rpm; 16h:8h light: dark
intensity
Rhodosporidium Batch/ Glycerol source: 91–93% (w/w) 30 12 0.001 0.02 0.40 Tchakouteu
toruloides 250 mL/ Glycerol, 2–4% water, 2–4% et al. (2015)
NRRL Y-27012 400 h potassium salts and 1% FFA;
Initial glycerol concentration:
120 g/L under nitrogen limited
condition; Initial pH: 6; pH
Control 5.2: 6.0; Incubation
temperature 28 °C; Agitation:
185 RPM; DO > 20% (v/v)
Lipomyces Batch/ Glycerol source: 91–93% (w/w) 23 8 0.001 0.02 0.348 Tchakouteu
starkeyi 250 mL/ Glycerol, 2–4% water, 2–4% et al. (2015)
DSM 70296 350 h potassium salts and 1% FFA;
Initial glycerol concentration:
120 g/L under nitrogen limited

condition; Initial pH: 6; pH
Control 5.2: 6.0; Incubation
temperature 28 °C; Agitation:
185 RPM; DO > 20% (v/v)
329
(Continued)
330
Crude Glycerol as Substrate. (Continued)

Cultivation
K. ohmeri Batch/ Media: 10% crude glycerol 10.5 3.2 0.0025 0.041 0.305 Kitcha and
50 mL/ (0.13 M of carbon) was used as Cheirsilp (2011)
120 h a sole carbon and 0.5% (w/v)
ammonium sulphate was used
as a nitrogen source;
C/N ratio: 17; pH: 6; Agitation:

140 rpm; Room temperature
T. spathulata Batch/ Media: 10% crude glycerol 10.4 4 0.0056 0.042 0.381 Kitcha and
50 mL/ (0.13 M of carbon) was used as Cheirsilp (2011)
120 h a sole carbon and 0.5% (w/v)
ammonium sulphate was used
as a nitrogen source;
C/N ratio: 17; pH: 6; Agitation:
140 rpm; Room temperature
S. limacinum Batch/ Media: Treated crude glycerol 16.03 11.76 0.0044 NA 0.734 Liang
SR21 100 mL/ with 35 g/L glycerol; Agitation: et al. (2010)
168 h 170 rpm; Initial pH: 7; Room
Temperature
S. limacinum SR21 Batch/ Media: Untreated crude glycerol 7.91 5.18 0.0039 NA 0.654 Liang
100 mL/ with 25g/L glycerol, 11.65 g/L et al. (2010)
168 h methanol (after dilution);
Agitation: 170 rpm; Initial pH: 7;
Room Temperature
concentration was around 1%−10% and is not considered inhibitory for the cells.
Methanol concentration greater than 10 g/L can be inhibitory for cells (Liang et al.
2010). Another important observation was that lipogenic phase observed (rapid
increase in lipid concentration) for most of the organisms was between 35 and 60 h
as mentioned in literature (Kuttiraja et al. 2015). However, for C. protothecoides,

R. toruloides, and L. starkeyi, lipogenic phase was attained between 96 and 120, 250
Figure 13-9. Fermentation profile of lipid-producing studies reported in literature

using crude glycerol: (a) pure and crude glycerol study using Chlorella
protothecoides; (b) batch and fed-batch strategy from crude glycerol using
R. glutinis; (c) batch study using Y. lipolytica; (d) batch study using Lipomyces
starkeyi; (e) batch study using R. toruloides; (f) batch study using K. ohmeri; and
(g) batch and fed-batch study using T. spathulate.
332

and 300, and 250 and 300, respectively. Lipogenic phase varies from organism to
organism and is dependent on several stress conditions like C/N ratio, dissolved
oxygen (DO), and pH. It can also be said that lipid is growth-associated in most of
the cases except Y. lipolytica, because its lipid curve follows the same pattern as that
of biomass. Studies using R. glutinis, S. limacinum, and C. protothecoides should be

conducted at the pilot scale to take advantage of industrial demand for lipid
production utilizing crude glycerol as a carbon source.
13.7 MAJOR FINDINGS FROM PREVIOUS STUDIES AND FUTURE

PERSPECTIVES
Figure 13-10 shows a schematic diagram describing the important steps/factors

that affect lipid productivity and fatty acid composition, that is, treatment of crude
glycerol and optimization of concentration of suitable media components besides
crude glycerol. The findings after reviewing the existing literature can be summa-
rized in the subsequent sections.
Figure 13-10. Important steps for enhanced lipid productivity.

13.7.1 Treatment of Crude Glycerol

There are several impurities present in crude glycerol that can inhibit cell growth
and ultimately lipid synthesis if they are present in high concentrations—
methanol, metals, soap, salts, free fatty acids, and others. Apart from these,
glycerol itself can be inhibitory if present in a high concentration depending on the

tolerance level of microbes (Wynn and Ratledge 2005). In one of the studies, 1%
methanol concentration increased overall lipid productivity by 1.2% in Chlor-
ococcum sp. (Liang et al. 2010). In fact, the cell utilizes methanol for its
proliferation as shown from one study in which methanol concentration reduced
from its initial value after fermentation (Choi et al. 2011). Methanol concentration
greater than 10 g/L can be inhibitory for cells (Liang et al. 2010). NaOH seems to
have assisted rather than inhibited cell growth. Normally, concentration of salts in
crude glycerol varies between 5% and 7%, and it aids in microbial growth (Xu et al.
2012). NaCL improved the lipid yield from 4−16 to 12−40 g/L (Xu et al. 2012).
Fatty acid methyl esters (FAMEs) are also present in crude glycerol, and methyl
oleate is most commonly found in biodiesel-derived crude glycerol. Methyl oleate
is reported to have improved lipid accumulation in R. toruloides (Xu et al. 2012).
Soap is also present in crude glycerol and can affect lipid production. The
inhibition or noninhibition of microbial growth in presence of soap depends on
the interaction of soap with the cell wall membrane. Inhibition may occur as a
result of the complex reaction of surfactant (soap) with the cell membrane,
whereas the positive effect may be caused by the enhancement of flow of nutrition
from the surroundings into the cell interior owing to the modification of its cell
wall permeability.
One of the studies was conducted to see the effect of treatment of crude
glycerol on biomass production and lipid productivity. It was observed that
treating crude glycerol with pH adjustment, removal of methanol, and FFAs can
be beneficial because higher lipid productivity and lipid yield were attained with
treatment of crude glycerol (Liang et al. 2010). High methanol content can be
removed by 10 times dilution of crude glycerol sample with water and then
passing through a 0.2 μm filter. Excess NaOH or soap can be eliminated by
adjusting pH to 1 followed by centrifugation and then removing the top reddish
layer containing FFAs. However, these pretreatment methods like pH adjustment,
centrifugation, filtration, and others increase the production cost, proving a
disadvantage of using crude glycerol as a substrate for lipid production.
13.7.2 Selection of Cultivation Mode

Analysis done in Section 13.6 clearly indicated that fed-batch strategy was more
effective than batch strategy for lipid production because of the higher qP and YP/X
observed in the comparative study. If continuous mode is used, it is important to
keep dilution rates to provide high lipid productivities (Papanikolaou and Aggelis
2002). One unique technology was to use a two-stage lipid production process for
higher lipid production on R. toruloides; the first stage was used for cell growth,
and the second stage with limited nitrogen concentration was used for lipid
synthesis (Yang et al. 2014). This could be an effective strategy for higher lipid
synthesis because it provides segregation between growth and lipid production
phase. Cheirsilp et al. (2012) mentioned use of a coculture strategy in which algae
acts as an oxygen producer and assists in yeast growth. Although the lipid content
reported was comparable to other reported studies, specific product formation rate
was not that high. More coculture studies should be conducted with R. glutinis and
other oleaginous algae for enhanced lipid content.
13.7.3 Optimization of Media Components

C/N ratio varies for organism to organism, and it is an important parameter for
enhanced lipid productivity. ACL, a key enzyme for lipid synthesis, requires
ammonium ions for its activation. A high C/N ratio is desirable during lipogenic
phase for enhanced lipid production compared with nitrogen-free media. How-
ever, an increase in the C/N ratio beyond a certain value results in deficiency of
nitrogen source during lipogenic phase, leading to ACL inhibition and citrate
accumulation (Beopoulos et al. 2009). Therefore, optimizing C/N ratio during
growth and lipogenic phase is imperative. C/N ratio has been optimized in most of
the studies along with selection of suitable nitrogen sources to obtain high lipid
productivities.
Further, effect of glycerol concentration on fatty acid composition was also
investigated using R. toruloides NRRL Y-27012 and L. starkeyi DSM 70296
(Tchakouteu et al. 2015). More studies should be conducted to manifest the
effect of C/N ratio and crude glycerol concentration on fatty acid composition on
other microbes to obtain maximum MUFAs for biodiesel production.
13.7.4 Optimization of Fermentation Parameters

Optimization of fermentation parameters like pH, aeration rate, temperature, and
others is most imperative for enhanced lipid production because DO concentra-
tion and pH affect lipid synthesis and microbial growth. It has been reported that a
low level of DO (15%) improved the total lipid accumulation in Mucor sp. RRl001
and Candida lipolytica (Ahmed et al. 2009). Meanwhile, a high level of DO (80%)
leads to citric acid production (Bati et al. 1984). Normally, growth phase requires
high DO, and lipogenic phase requires low DO. Most of the aforementioned
studies were carried out under pH of 6 and controlled conditions, which is why the
pH controlled strategy resulted in 6% higher lipid content when compared to pH
uncontrolled strategy (Saenge et al. 2011). The study involving R. glutinis, which
showed the highest qP, involved optimization of fermentation parameters using
response surface analysis.
One study showed impact of initial pH during fermentation on fatty acid
composition (Yang et al. 2014). More studies should be conducted to discover the
exact relationships between fermentation parameters and fatty acid composition
so that maximum of MUFAs are produced, which are desirable for biodiesel
production. Besides, the successful studies previously mentioned should be
conducted at the pilot scale to determine the feasibility of lipid production

utilizing crude glycerol on a large scale.
13.7.5 Genetic and Metabolic Engineering of Selected Strains

Several studies were focused on selecting suitable oleaginous microorganisms for

lipid production that can utilize crude glycerol. The species with reported high
lipid productivities are R. glutinis, C. protothecoides, and S. limacinum SR21.
However, few studies indicated metabolic engineering for enhanced lipid produc-
tion. As mentioned, oleaginous yeasts produce citric acid besides fatty acids. The
role of metabolic engineering is concerned with directing the flux toward fatty acid
production. Lipid content in the mentioned studies was 40% to 60%, and it can be
enhanced using genetically modified strains. Although studies have been con-
ducted to increase the activity of ACC (a key enzyme for lipid synthesis) through
metabolic and genetic engineering, not much success was attained with the step
(Radakovits et al. 2010). However, one study reported that knocking out a gene
product involved in β-oxidation of fatty acids along with overexpression of ACC
and thioesterases increased the fatty acid deposition in Escherichia coli 20-fold
(Lu et al. 2008). Such studies should be conducted with other oleaginous
microorganisms to see the effect of metabolic engineering on lipid productivity.
Biotin acts as the prosthetic group for ACC, which converts acetyl-CoA to
malonyl-CoA following lipid synthesis. Studies should be conducted in which
biotin can be added in medium during lipogenic phase that would enhance the
activity of key enzymes and affect lipid productivity. Although genetically
engineered strains have worse stability and growth compared with wild strains,
metabolic and genetic engineering can assist in changing fatty acids composition
and chain length as more MUFAs are desirable for biodiesel production.
13.8 SUMMARY
Crude glycerol is an inexpensive substitute to glucose as a carbon source for

cultivation of oleaginous microorganisms for lipid production. Moreover, crude
glycerol produced during transesterification as a byproduct is currently disposed
of with high purification costs. Crude glycerol can be utilized as an economical
carbon source for cultivation of oleaginous microorganisms. Moreover, the fatty
acid composition obtained is comparable to nature or vegetable oils and lipids
obtained using glucose as carbon source. However, there are several governing
factors that should be optimized for enhanced lipid and biomass production.
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CHAPTER 14
Lignocellulosic Biomass: The
Future Renewable Low-Cost

Carbon Source for Microbial
Lipid Production
M. Kuttiraja
R. D. Tyagi
14.1 INTRODUCTION
Today, basic academic research on biomass to biofuels has been taken into the
next level at the pilot scale. Some companies (e.g., Enerkem in Alberta, Canada,
and Neste Biofuels in Finland) have been testing the second-generation biofuel
production at the pilot scale. On the other hand, the ethical concerns about the use
of food as fuel have encouraged research efforts to be more focused on the
potential of inedible feedstocks. In the last decades, research has focused on
converting used cooking oils, animal fats, and plant-derived oils for the produc-
tion of biodiesel. Because of its limited supply and high cost for converting them
into biodiesel, this process was insufficient to meet the growing demand; hence,
microbial oil production was focused in several countries. Microbial oils are lipids
having the similar composition of plant-derived oils. Many oils accumulating
organisms have been identified as potential oil producers for biofuel applications.
The organisms that are capable of accumulating lipids from 20% to 80% of its dry
weight are known as oleaginous microbes. There are several bacteria, filamentous
fungi, and yeast strains have also been identified as potential lipid producers
(Pérez-Guerra et al. 2002).
From the energy and environmental perspective, second-generation fuels
from lignocellulosic biomass hold the promise because lignocellulosic biomass
offers a greater possibility of producing various platform chemicals, food, fodder,
and fuels (Faraco and Hadar 2011). Lignocellulosic materials are a substantial
renewable substrate for biofuel applications and do not compete with the food
341
production and animal feed. Moreover, lignocellulosic biomass could be supplied

in a large quantity with low cost (Akhtar et al. 1998). The most common
lignocellulosic biomass used for biofuel generation is agro-residues, agro- and
food-processing industrial waste, forestry waste, and municipal waste.
At present, the most promising and abundant cellulosic feedstock is derived

from plant residues. More than 90% of the global production of plant biomass is
lignocellulose, which is about 200 × l09 tons per year, with about 8 to 20 × l09 tons
of the primary biomass being potentially accessible (Kuhad and Singh 1993). Of
that accessible biomass, 11% was used for biofuel production and 7% for
biomaterials (Wirsenius 2007).
Lignocellulosic biomass is mainly composed of three major subunits:
(1) cellulosic polymers, which are the backbone of any lignocellulosic biomass,
and these are the repetitive units of glucose molecules linked with β-1,4 linkage;
(2) hemicellulose moieties, which include C5 and C6 sugars such as xylose and
arabinose; galactose mannose is the other C6 group in the hemicellulose and is
cross-linked with the cellulose by 1,6 linkage; and (3) lignin, which is made up of
polyphenolic compounds such as p-coumaryl alcohol and p-coniferyl alcohol to
protect the plant from the insects and to give the mechanical strength of the plant
(Tsigie et al. 2012).
Biofuels such as bioethanol, biobutanol, syngas, and electricity were already
demonstrated in commercial levels. However, biodiesel from lipids produced from
lignocellulosic fermentation is a new endeavor and is under active research, with
the primary goals to lower the production cost of the process. The major hurdle in
the lignocellulose to biofuel (biodiesel) production is the degradation or the
extraction of available sugars for fermentation. Because of its recalcitrant nature,
lignocellulosic materials are very limited to access by any catalytic molecules such
as enzymes and other chemicals. Research on lignocellulose has a long tradition to
develop various biomass conversion technologies for converting lignocellulosic
biomass free for accessing by chemical or by other biocatalysts, but the research is
limited to the laboratory-scale because of the technical and economical barriers.
This chapter overviews the lipid production from different lignocellulosic
biomass using single cell organisms. The advancement in lignocellulosic biomass
pretreatment and hydrolysis method are discussed; followed by in-depth descrip-
tions about different microbes reported to grow and produce lipid from lignocel-
lulose hydrolysate, cofermentation, simultaneous hydrolysis, and lipid production;
and tolerance against different fermentation inhibitors.
14.2 LIPID PRODUCTION FROM LIGNOCELLULOSIC BIOMASS
Lignocellulosic biomass is recalcitrant in nature. To make use of these materials, it

has to be preprocessed. The classical way of utilizing the lignocellulosic biomass
starts from the collection of biomasfollowed by size reduction/milling. In the second
stage, the biomass will be pretreated with dilute acid or alkali in controlled
LIGNOCELLULOSIC BIOMASS: THE FUTURE RENEWABLE LOW-COST CARBON 343
temperature and pressure. In the third stage, the pretreated biomass will be
hydrolyzed with chemicals or enzymes to release fermentable sugars. The final
stage is the fermentation to produce biofuels (Figure 14-1). Several techniques are
available for lipid production by fermentation of lignocellulosic biomass as follows:
• Direct utilization of lignocellulosic biomass: Naturally, some of the organisms

could produce a wide range of biomass hydrolyzing enzymes, including
cellulolytic and lignolytic enzymes. However, the hydrolysis and the lipid
production are slow under direct biomass utilization.
Figure 14-1. Biodiesel production from lignocellulosic biomass.

• Simultaneous saccharification and fermentation: Organisms are capable to

hydrolyze the pretreated biomass and accumulate lipids on its biomass with
the addition of a very little amount of externally added enzymes.
• Lipid accumulation in hydrolysates of lignocellulosic biomass: The sugars that
are released due to the action of enzyme/chemical hydrolysis are utilized for
the production of lipids by oleaginous microbes.
• Co-utilization of C5 and C6 sugars: Organisms can accumulate lipids by
utilizing cellulose and a hemicellulosic portion of lignocellulosic biomass
hydrolysate, and they are the better candidates for this process.
14.2.1 Lipids from Lignocellulose-Derived Sugars

Lignocellulose to bioethanol production has a long tradition; however, current
research attempts single cell oil from lignocellulosic sugars. The work was
segmented into three major steps: first, lignocellulosic biomass is converted into
fermentable sugars; second, the sugars are converted into microbial lipids by
oleaginous microorganisms; and third, microbial lipids are translated into bio-
diesel (Burke and Cairney 1997).
14.2.2 Fractionation of Lignocellulosic Biomass

The ultimate source for the production of renewable energy is from lignocellulose-
derived sugars. Among lignocellulosic materials, agro-residues are a major
lignocellulosic biomass available for renewable energy applications. Bioenergy
from agro-residues has several advantages like environmental benefits that far
exceed its economic benefits. Moreover, lignocellulose is outside the human food
chain and is inexpensive and available through the year. Based on the data
available, 14% of the total world energy is produced from biomass, which plays a
crucial role in the world economy (Kim et al. 1985, Hui et al. 2010).
The process of converting lignocellulosic biomass into its fuel components
starts from the pretreatment of the biomass. Pretreatment of the lignocellulosic
biomass (LCB) is the primary step to depolymerize the biomass components into
simplified form, and it could be achieved by two different principle ways.
The first way is to remove the hemicellulose and lignin components by
treating the biomass with dilute acids and alkali. The most commonly used acid
for the biomass pretreatment is dilute H2SO4. Upon dilute acid pretreatment, the
ester linkage between hemicellulose and lignin molecules will get depolymerized.
The depolymerization results in increased pore size of the biomass. At the same
time, the hemicellulose portion is solubilized owing to the pretreatment, and it will
be collected as the acid pretreatment liquor (APL). The pretreatment liquor is
further used as a carbon source for other fermentations such as amino acid,
organic acids production, and so forth.
The second way of pretreatment is based on the solubilization of lignin from
the biomass at elevated temperature, a method adopted from the kraft pulping
method used in the paper and pulp industry. Upon the alkali treatment, the
carbon and carbon bonds between the lignin, ester bonds between lignin and
hemicellulose, as well as ether bonds between cellulose-lignin-hemicellulose are

broken, and the lignin molecules become small molecular weight phenolics and
come out in the liquid portion, named black liquor. The chemical reaction is
reported elsewhere because a alkaline hydrolysis mechanism is based on saponi-
fication of intermolecular ester bonds crosslinking xylan, hemicelluloses, and

other components such as lignin.
There are several other types of pretreatment methods that are practiced based
on the need and the application. However, dilute H2SO4 and dilute NaOH pretreat-
ments are the most commonly practiced chemical pretreatment methods for LCB.
14.2.3 Saccharification of Cellulose and Hemicellulose Polymer

The bottleneck in LCB to generation of any kind of biofuel or platform chemicals
lies on the efficiency of the saccharification reaction. Because of the high cost of the
hydrolytic enzymes, the whole process is very costly. Considerable research has
been done to bring down the cost and reduce the use of enzymes.
The enzymatic saccharification of cellulose to glucose involves three different
enzymes, and they act synergistically on cellulosic materials and generate the
fermentable sugars. These enzymes are (1) endo-β-1,4-glucanase (EC 3.2.1.4),
(2) exo-cellobiohydrolase (EC 3.2.1.91), and (3) β-glucosidase (β-D-glucosidic
glucohydrolase; EC 3.2.1.21). Endoglucanase and exo-cellobiohydrolase act
synergistically on cellulose to produce cellobiose, which is then cleaved by
β-glucosidase to glucose. The enzymatic saccharification of the biomass is basically
affected by the efficiency of the pretreatment, availability of free cellulose
molecules, and the amount of free enzyme in the reaction mixture. Different
kinds of commercial enzymes are available today for different applications.
A complete hydrolysis of lignocellulosic biomass results in a mixture of sugar
monomers, which includes hexoses (glucose and mannose) and pentoses (xylose
and arabinoses), and the ratio of hexoses to pentoses typically ranges from 1.5:1 to
3:1. Because the cellulosic polymer is the first major component in the LCB, the
concentration of glucose is usually higher, and this amount is followed by that of
xylose (from hardwood and agricultural residues) (Huang et al. 2012). Because of
the different nature of LCB hydrolysates, the microbes that are capable of utilizing
both sugars are limited, and this could be overcome by screening potent organisms
which could utilize both hexoses and pentoses from natural resources either by
mutagenic approaches or genetic manipulations.
14.3 FERMENTATION
Very few oleaginous organisms are identified as potent utilizers of LCB hydrolysates.
Henceforth, the urge for the identification of efficient oleaginous strains having high
lipid yield on lignocellulosic biomass hydrolysates is obligatory. Studies are very
limited in lipid fermentation using LCB hydrolysates as the carbon source. Apart
from other kinds of fermentation, LCB hydrolysates consist of different sugars such
as cellobiose, glucose, xylose, and other C5 and C6 sugars. Preferably the oleaginous
microbes assimilate the glucose, followed by xylose, and so forth. In the meantime,
most of the other forms of sugars are unavailable or unutilized by the microbes.
Because of this reason, a selection of suitable microbe and fermentation processes is
essential. There are two different types of fermentation techniques that are practiced
to accumulate lipid solid-state and liquid-state fermentation. Considering the
growth, recovery, and other economic factors, submerged fermentation is identified
as the economically feasible method for oil fermentation.
14.3.1 Organisms that Can Grow on LCB Hydrolysates

Because of several years of research on microbial lipid production, numerous
bacterial, fungal, and yeast strains have been isolated and studied. Among them,
three groups of fungal species have gained more attention owing to the following
characteristics (Dai et al. 2007, Li et al. 2007, Liu and Zhao 2007, Papanikolaou
et al. 2007, Angerbauer et al. 2008):
• They can utilize a wide range of substrates;
• They have the capability of producing high titers of lignolytic and cellulolytic
enzymes;
• They have a fast growth rate on LCB hydrolysates;
• They have high lipid accumulation ratio;
• They can grow with proper morphology to facilitate the downstream proces-
sing; and
• They have tolerance against different growth inhibitors present in the biomass
hydrolysate.
The major fungal species that have been reported to grow on LCB hydro-
lysates and produce lipids include: Yarrowia lipolytica (Papanikolaou and Aggelis
2002), Lipomyces starkeyi (Angerbauer et al. 2008, Zhao et al. 2008), Rhodotorula
glutinis (Xue et al. 2008, Easterling et al. 2009), Rhodosporidium toruloides (Akhtar
et al. 1998, Li et al. 2007) Aspergillus niger, Aspergillus terreus, Chaetomium
globosum, Cunninghamella elegans, Mortierella isabellina, Mortierella vinacea,
Mucor circinelloides, Neosartorya fischeri, Rhizopus oryzae, Mucor plumbeus, and
Thermomyces lanuginosus. Table 14-1 represents the microbes, biomass, and lipid
production in LCB hydrolysates. Some of the filamentous fungi are reported to
utilize the acetic and formic acid produced during the pretreatment, and these
organisms are able to use acetic and formic acid along with glucose (co-utiliza-
tion). Conversely, the acetic acid in the medium enhances the cell pelletization and
thus reduces the downstream processing. Moreover, it may contribute to the
accumulation of lipids in certain molds. There were studies conducted with and
without acetic acid to prove the preceding statement. Results shows that lipid
production in the acid hydrolysate was equivalent to that found in the synthetic
medium (4.78 versus 4.82 g/L, respectively), which was also the maximum
concentration found in all media.
Table 14-1. Lipid Production by Oleaginous Organisms in LCB Hydrolysate and Their Yield.
Lipid Lipid
Carbon source Organism Biomass (g/L) content (%) concentration (g/L) References
Defatted rice bran Yarrowia lipolytica Po1g 10.75 48.02 5.16 Tsigie et al. (2012)
hydrolysate
Sugarcane bagasse Yarrowia lipolytica Po1g 11.42 58.56 6.68 Tsigie et al. (2012)
hydrolysate
Rice straw Trichosporon fermentans 28.6 40.1 11.5 Huang et al. (2012)
Rice straw Rhodotorula glutinis 3.58 5.74 0.21 Dai et al. (2007)
Corn stalk Rhodotorula glutinis 17.04 11.78 2.01 Dai et al. (2007)
Wheat straw Mortierella isabellina 5.79 40.87 2.36 Zheng et al. (2012)
hydrolysate
LIGNOCELLULOSIC BIOMASS: THE FUTURE RENEWABLE LOW-COST CARBON
347
14.4 TYPES OF FERMENTATION FOR LIPID PRODUCTION
14.4.1 Submerged and Solid-State Fermentation

Submerged/liquid fermentation and solid-state fermentation are the two major
fermentation modes studied mainly for the biotechnological production of single

cell oil from lignocellulosic residues. Submerged fermentation (SMF) requires
prior sugar extraction from agro-biomass to the bulk liquid, by hydrolyzing the
materials with costly enzymes. On the other hand, in solid-state fermentation
(SSF), microorganisms grow on moist solid materials in the absence of free-
flowing water, and limited lipid accumulation can be obtained (Economou et al.
2011). However, apart from low lipid yield, SSF has many advantages over SMF,
for example, simpler technique, smaller bioreactor or volume, reduced down-
stream processing cost, reduced energy requirement, and low wastewater output
(Kim et al. 1985, Burke and Cairney 1997, Pérez-Guerra et al. 2002). Although it
has several advantages over SMF, the lipid extraction is tedious in SSF. In
addition, SSF requires organisms having both cellulolytic and lipid accumulation
properties.
In the case of SSF experiments conducted by Hui et al. (2010), the steam-
exploded wheat straw was used as the substrate. The solid-state medium was
inoculated with 5 mL spore suspension (106 spores/mL) of a 7 day old culture
that is precultured in a pattato dextrose agar (PDA) plate. The SSF medium was
incubated at 30 °C for a period of 10 days, and the cellulose and oil production
were estimated to determine the efficiency of the process. During the initial
stage, the oil production was very low due to the lesser concentration of sugar
availability in the medium. As time progressed the cellulolytic enzyme secretion
was increased, and the lipid accumulation in the medium also progressed to
42 milligrams/grams of dry solid (mg/gds) after 10 days. The increase in the
cellulase activity was noted from Day 4 to Day 9 of incubation. From Day 10
onward, the lipid yield was noted to decrease slowly in the media. This
phenomenon was explained as follows: in the case of Microsphaeropsis sp.,
once the sugar in the media is depleted, the stored lipid will be consumed as the
carbon source in carbon limited condition. Microorganisms consume their
accumulated lipids mainly through the glyoxylate bypass pathway; more spe-
cifically, different microbes might preferentially consume different kinds of fatty
acids to maintain their growth. The second reason is the limitation in the free
substrate (glucose) in the medium. It was noted that under the best growth
condition the highest yields of lipid are 22 g/100 g glucose used (Ratledge and
Wynn 2002). This study demonstrates that in the case of SSF, cellulose is the
major limitation for achieving higher oil yield.
In submerged culture, mycelia of different ages intertwine to form either
pellets or mats or both, depending on culture conditions. Therefore, lipid analysis
during growth in submerging culture involves an inherent error caused by the
inevitable agglomeration of young and aged mycelia.
14.4.2 Fed-Batch and Continuous Fermentation

In the present scenario, the lipid production from lignocellulosic biomass hydroly-
sate is carried out using the fed-batch or continuous mode. In the fed-batch mode,
the substrate will be fed at sequential or continuously, depending on the rate of
substrate assimilation. It helps to reduce the substrate-associated growth inhibition

and helps to achieve high cell density. Fei et al (2016) reported a fed-batch
cultivation of R. toruloides in corn stover hydrolysate. The study revealed that,
compared to the batch mode, the yield of lipid production in the fed-batch mode
improved from 0.19 g/g to 0. 29g/g with 0.4g/L/h lipid productivity; in the
meantime, the biomass reached 54 g/L (Fei et al. 2016). In another case, L. starkeyi
was cultivated in a hemicellulosic fraction with a media composition of xylose
45.1 g/L, acetic acid 13.1 g/L, and furfural 4.7 g/L under a pH-regulated fed-batch
mode. The maximum lipid concentration of 8 g/L (51.3% w/w) with a lipid yield of
0.1 g/g was attained (Brandenburg et al. 2016).
In the case of synthetic medium, the inhibition of initial sugar concentration
is higher, which will inhibit the cell growth and lipid accumulation. Thus, the fed-
batch fermentation helps to overcome these issues. Using the fed-batch fermen-
tation, the biomass and lipid yield by oleaginous microbes were reported at 100
and 60 g/L. Meanwhile, the recent advancement in LCB to lipid production under
fed-batch mode showed competitive results with respect to batch mode. It is
believed that incorporating fed-batch/continuous fermentation mode on LCB
hydrolysate could improve cell density and higher productivity in the case of lipid
fermentation, and this will undoubtedly benefit the industrialization of lipid
production. However, the fed-batch fermentation performance is highly depen-
dent on the feeding strategy applied during the cultivation. Several control
methods for substrate feeding methods were developed. Among them, pH- and
dissolved oxygen (DO)-based feedings were practiced because of their simple and
inexpensive nature. On the other hand, the direct feeding depends on substrate
concentration. The online monitoring of substrate concentration helps to control
the flow of substrate concentration, thus resulting in high cell density and
productivity.
14.4.3 Carbon Source Assimilation and Lipid Accumulation by

M. isabellina in Corn Fiber Hydrolysate
The lignocellulosic biomass hydrolysate is mainly composed of C5 and C6 sugars.
Because of the mixed nature of the hydrolysate, the fermentation process in
LCB hydrolysate-based medium requires proper strain selection. Preferably the
microbes assimilate the C5 and C6 sugars sequentially. The batch culture
experiment contacted with M. isabellina in hydrolysate from the corn fiber
includes the glucose 32.14%, xylose 41.91%, mannose, and arabinose. The
fermentation was conducted for 144 h, and the substrate assimilation profile was
evaluated. From the total reducing sugar analysis, it was confirmed that 90% of the
reducing sugars were consumed by the organism within 6 d of fermentation. From
the results, it was evident that M. isabellina not only used glucose for its growth
and accumulation of lipid but also assimilated the other sugars in the medium.
However, in the case of Saccharomyces cerevisiae, it cannot utilize the xylose
fraction for ethanol production. This gives an idea about the biochemical pathway
required to assimilate the pentose sugars in the case of certain oleaginous

microbes, especially in M. isabellina. In all cases, the lipid contents and lipid
yields exceeded 50% and 5.0 g/L, respectively, and when glucose was used as the
sole carbon source the biomass and lipid content reached 10.49 g/L and 65.98%,
respectively. Therefore, glucose appeared to be the optimum carbon source (of
those tested) for growth and lipid accumulation by M. isabellina M2, and the
results clearly demonstrate the potential of utilizing biomass hydrolysates for
producing microbial oils.
Similarly, a cofermentation study in cellobiose and xylose from LCB hydro-
lysate was conducted by Gong et al. (2012). The mixed sugar analysis was carried
out using Dionex ion exchange chromatography coupled with a CarboPac PA20
column and an ED50 electrochemical detector. Interestingly, the study confirms
the utilization of cellobiose and xylose simultaneously by L. starkeyi. The sugar
consumption rate shows (g/L/h) 0.68 cellobiose, 0.60 glucose, and 0.41 xylose were
utilized. Among the three, cellobiose was consumed at a higher rate than other
forms of sugars. However, the lipid conversion was noted to be higher in the case
of xylose compared to the other sugars (Gong et al. 2012).
14.4.4 Simultaneous Saccharification and Fermentation Using

Filamentous Fungus
Apart from the normal fermentation technology, today industries are looking
forward to simultaneous saccharification and fermentation for higher productivity
and lower use of hydrolytic enzymes. Meanwhile, it reduces the end product
inhibition. Although the process of simultaneous saccharification and fermenta-
tion has more advantages in the case of lipid fermentation, it was not reported;
instead, cofermentation works were reported by some authors.
Simultaneous saccharification and fermentation has a certain adverse effect
on the lipid fermentation owing to the nature of the process. In the case of lipid
fermentation, the productivity is lower, perhaps caused by the lower concentration
of fermentable sugars in the media. This directly reduces the biomass generation
and eventually leads to lower productivity. The simultaneous saccharification and
fermentation process is mainly designed for ethanol production; at the same time,
the optimum activities of the hydrolysis enzymes are slightly higher than the
fermentation temperature. Integrating the whole process for lipid production
needs more effort in research and development.
At present, there are several organisms screened for lipid production from
LCB with the capability to secrete cellulolytic and hemicellulolytic enzymes along
with the production of lipids. Attempts were made to produce lipids by simulta-
neous saccharification and fermentation using a wheat straw with a cellulolytic
strain of Aspergillus oryzae (Hui et al. 2010). Endophytic fungi from the oleaginous
plants were screened. These strains belonged to the genera of Microsphaeropsis,
Phomopsis, Cephalosporium, Sclerocystis, and Nigrospora that simultaneously

accumulated lipids (21.3% to 35.0% of dry weight) and produced cellulase (Peng
and Chen 2007). In contrast, the temperature is an important factor in regulating
lipid accumulation and composition in oleaginous fungi. Screening thermophilic
oleaginous fungi holds the future of simultaneous saccharification and fermenta-

tion using LCB as a sole organic carbon source. Simultaneous saccharification and
fermentation has been carried out using high cell density inoculum ranging from
50 to 200 g/L, and in most cases it will be maintained at least at 100 g/L. Usually,
the growth period for simultaneous saccharification and fermentation is 48 to
96 h. Based on the growth of the microbe, the incubation time may be reduced
(e.g., to 72 h).
Xiaochen et al. (2013) used avicel cellulose as the substrate with three different
solid loadings (1%, 3%, 5% w/v) and four different enzyme loadings (5, 15, 30, 60
filter paper units (FPU)/g LCB). The cellulose preparation was formed with
novozymes, and the saccharification was conducted using phosphate buffer at a
pH of 5 in a 250 mL culture flask. The culture was maintained at 28 °C with an
agitation of 180 rpm for a defined period. From the results of this experiment, it
was assumed that the main limiting factor for the simultaneous saccharification
and fermentation process was enzyme loading. When the enzyme loading was
lower, the lipid accumulation was lower because the available free enzyme for the
hydrolysis was less, and the proportion of lipid accumulation increased with an
increase in the solid loading. Enzyme loading of 15 FPU was concluded as
optimum for the lipid production using M. isabellina. A similar kind of experi-
ment was carried out with Cryptococcus curvatus, and a comparable result was
observed (Yu et al. 2011).
14.4.5 Co-utilization of Fermentable Sugars in Lignocellulosic

Biomass Hydrolysate
The major sugars in hydrolysates of lignocellulosic biomass are glucose and xylose.
Most microorganisms prefer glucose over other monomeric sugars and do not
assimilate other sugars until glucose is consumed (Gancedo 1998, Stülke and
Hillen 1999, Berlin et al. 2007). Thus, simultaneous utilization of glucose and other
sugars is rarely observed for most of the microbes. However, simultaneous and
rapid utilization of sugar mixtures is considered essential for the economically
feasible production of biofuel and fuel commodity and biochemicals from biomass
hydrolysates (Kim et al. 2010).
To add more evidence for bioconversion of LCB to lipids, studies were
conducted by L. starkeyi in SMF. There were three different combinations tested
for lipid production by co- utilization of sugars by Gong et al. In the first
experiment, cellobiose alone was used to examine whether L. starkeyi can
assimilate cellobiose into lipids, and a positive result was found. The condition
used in this study was to load 70 g/L cellobiose without the addition of
cellobiohydrolases, and then let the fermentation occur for a period of 103 h.
The biomass and lipid content reached up to 27.9 g/L and 50%, respectively. Next,
fermentation was conducted under the identical culture conditions except for
using glucose or xylose as the carbon source. The results show cellobiose was
comparable to glucose but better than xylose as a carbon source for lipid
production. The overall sugar consumption rates for cellobiose, glucose, and
xylose were 0.68, 0.60, and 0.41 g/L-h, respectively (Gong et al., 2012).
Because the commercially available cellulase is known to have low

β-glucosidase activity, it is necessary to study the performance of L. starkeyi in
a mixture of cellobiose, glucose, and xylose. The study shows that sugar con-
sumption started with the medium containing 40 g/L cellobiose, 20 g/L xylose, and
10 g/L glucose. On the initial stage (24 h), glucose was consumed at the rate of
4.6 g/L-h while both cellobiose and xylose concentrations remained same. The
three sugars started decreasing in the last stage (from 24 to 36 h) of the
fermentation. These results suggested that the presence of glucose did not change
the tendency of simultaneous utilization of cellobiose and xylose. As a result, when
the culture was terminated after 108 h, cell mass, lipid content, lipid coefficient,
and total sugar consumption rate were 25.5 g/L, 52%, 0.20 g/g, and 0.60 g/L-h,
respectively. These data are comparable to those using a mixture of cellobiose and
xylose, suggesting that L. starkeyi could also effectively use a sugar mixture
containing cellobiose, xylose, and a small amount of glucose. This feature is highly
relevant in terms of developing new strategies to use lignocellulosic biomass and to
produce microbial lipid more cost-effectively on a large scale. It is known that the
commonly used cellulase from fungi has high endoglucanase and exoglucanase
activities, but low β-glucosidase activity. Thus, extra β-glucosidase is necessary to
facilitate glucose production (Stockton et al. 1991, Berlin et al. 2007, Kumar and
Wyman 2009). Because the lipid production by cofermenting cellobiose and
xylose requires no glucose formation, the hydrolysis of biomass can potentially be
performed in the absence of extra β-glucosidase, thus reducing enzyme costs for
biomass hydrolysis (Zhou et al. 2009).
Apart from lignocellulosic biomass, food waste such as tomato peel, apple and
pear pomace, and other forestry wastes were tested for biomass and lipid
production. Table 14-2 represents the alternative carbon sources that can be
useful for lipid production.
14.5 PRETREATMENT INHIBITORS AND THEIR EFFECT ON

MICROBIAL GROWTH AND LIPID ACCUMULATION
Breakdown of lignocellulose by acid or alkali generates several byproducts along

with fermentable sugars. The formation of byproducts are the following:
• If the lignin is degraded under the acidic condition, the ether bond is
converted into hydroxyl and then converted to carbonyl or carboxyl group;
finally, it will be fragmented into C3 or C2 molecules;
• Subsequent to hemicellulose hydrolysis, pentose sugar monomers may dehy-
drate to form the inhibitor furfural;
Table 14-2. Reported Agro/Agro-Based Industrial Residues for Lipid Production.

Residual material Pretreatment method Fermentation mode Oleaginous microbe References
Wheat straw and bran Steam explosion Solid state Microsphaeropsis sp. Peng and Chen (2007);
Sun et al. (2008)
Pitch pine Organosolv Liquid Lipomyces starkeyi Park et al. (2010)
Pear pomace Without treatment Solid state Mortierella isabellina Fakas et al. (2008)
Sweet sorghum Without treatment Semisolid state Mortierella isabellina Economou et al. (2011)
Tomato waste Acid hydrolysis Liquid Cunninghamella echinulata Fakas et al. (2008)
Cornstalk Acid hydrolysis Liquid Rhodotorula glutinis Dai et al. (2007)
Rice straw Acid hydrolysis Liquid Trichosporon fermentans Huang et al. (2009)
Tree (Populus Acid hydrolysis Liquid Rhodotorula glutinis Dai et al. (2007)
euramevicana)
leaves
LIGNOCELLULOSIC BIOMASS: THE FUTURE RENEWABLE LOW-COST CARBON
353
• Similarly, hexose sugars (e.g., glucose) may degrade to the toxic hydroxy-
methylfurfural (HMF);
• In the case of alkaline pretreatment, the lignin will be depolymerized and then
converted into small molecular weight phenolics; and
• Phenolic acids/aldehydes in the hydrolysate are mainly derived from the para-
hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units during the decon-
struction of the lignin complex. It is believed that the toxicity of phenolic
compounds was determined by the position of the substituent (hydroxyl or
methoxyl) rather than the identity of the substituent (hydroxyl or methoxyl).
Moreover, aldehydes are proven more toxic than the corresponding carbox-
ylic acids and alcohols (Larsson et al. 2000).
In most of the fermentation processes, acetic acid was usually considered as
an inhibitor to the organisms. However as an organic acid, it can be used as the
carbon source to support the growth of oleaginous yeast. Because of the negative
inhibition of pretreatment byproducts on the fermentation process, the biomass
hydrolysate is usually detoxified with costly materials such as ionic resins
(Amberlite -XAD resin, silica, activated charcoal, and so forth) and overliming.
Detoxification by overliming process removes 92.8% of furfural and 58.0% of
HMF. Although this could retain the acetic acid in the media, excess detoxifica-
tion will lead to a loss of 22.2% sugars. HMF is considered less toxic than
furfural, and its concentration in (hemi)cellulose hydrolysates is usually low.
There were reports showing some of the fungal species have the ability to
degrade the furfural by furfural reductase; the organisms must process the
representative genes for furfural reductase (Yubin et al. 2012). However,
NAD(P)H was used as the electron donor in this reduction, which made the
furfural act as a redox sink, oxidizing NAD(P)H formed in biosynthesis
(Gutiérrez et al. 2002), which may cause the reduction in the biomass accumu-
lation, and thus directly leads to the lesser lipid accumulation. On the other
hand, the hydrolysate from the alkali-pretreated biomass shows comparatively
low lipid production, presumbly due to the inhibitory effects of phenols and
carboxylic acids present in the media.
When comparing the hydrolysate from acid and alkali treatment, the
acid-based process shows a higher lipid production over the alkaline process.
This was assumed that the alkaline-treated hydrolysate attributes greater amounts
of phenolic inhibitors and also shows the presence of excess sodium salts in the
media. The composition of these two different hydrolysates makes a huge
difference in the supply of carbon sources for the fermentation. The acid process
degrades the cellulose and solubilizes the hemicellulose at a very high ratio; along
with this, it generates organic acid that is specific for the oleaginous microbial
growth. There are studies on the production of lipids using both detoxified and
non-detoxified LCB hydrolysates (Ruan et. al., 2012). Both the media were
fermented with M. isabellina, and a synthetic media was used as a control to
monitor the cell growth and lipid accumulation. Interestingly, results obtained
from these three conditions were comparable. This clearly indicates the prospects
of using an already cheap and abundant carbon source for filamentous fungal lipid
production, which is even more attractive owing to the high cost associated with
detoxification of inhibition products. Further investigation is needed on the
detailed impact of inhibitors such as acetic acid, formic acid, and phenolic
compounds on lipid accumulation of this filamentous fungus.
14.6 SUMMARY
The world is moving toward a crisis of petroleum-based fuels. Finding a better

alternative for petroleum-based fuel is under active research; among these, lipids
from lignocellulosic biomass plays an important role because it is a cheap,
renewable, and sustainable natural resource. A few laboratory-scale studies
already exist for the production of lipids from LCB. The major concern in this
field is that biomass conversion technologies have not been developed as seen in
the case of ethanol fermentation from lignocellulose. Still, the bottlenecks in this
research are associated with lignocellulosic biomass proceses, including pre-
treatment, cost-effective cellulolytic enzymes, suitable lipid fermentation tech-
nologies, and efficient downstream processing of lipids from biomass. Although
the technical process parameters are similar in lipid and ethanol fermentation
from lignocellulose, lipid fermentation follows a completely different biochemi-
cal process. In the case of lipid fermentation, the whole lignocellulosic biomass
could be converted to lipids because most of the fermentation inhibitors from
the medium enhance the lipid accumulation. The other fermentation inhibitors
show the very low impact on cell growth and lipid accumulation. Co-utilization
of cellobiose, glucose, and xylose prove the potentiality of these oleaginous
microbes to assimilate the carbon sources in the lignocellulosic hydrolysates.
The results of Ratledge (1988) showed that the theoretical yield of lipids on
glucose and xylose is merely 33% and 34%, respectively, which indicates the
presence of both the biochemical pathways in certain oleaginous microbes. To
make LCB-based lipids a potential resource for biodiesel production, in-depth
understanding about the lignocellulosic biomass and high throughput technol-
ogies are necessary.
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CHAPTER 15
Lipid Extraction Technologies
X. L. Zhang
S. Yan
R. D. Tyagi
R. Y. Surampalli
15.1 INTRODUCTION
Currently, biodiesel is converted from oil or fat contained in plant seeds,

microorganisms, or animals. Therefore, extraction of the oil and fat from oil-
bearing materials is an essential step of biodiesel production. The extraction
methods should be rapid, efficient, and preserve the originality of the oil/fat.
Several methods have been established to achieve the extraction. Mechanical
pressing was the leading technology before the 1900s. It is still applied today
because it requires low cost and provides high-quality products (oil and residual
cakes). However, the extraction efficiency (50% to 80%) is undesirable, especially
for the substances with low oil/fat content (<20%). To enhance the efficiency,
mechanical pressing followed by solvent extraction has been established and
widely used in oil extraction from oilseeds. The process could achieve a 98% oil
recovery (Amalia Kartika et al. 2010). The application of solvents has significantly
enhanced the efficiency of oil extraction. Therefore, mechanical pressing has been
slowly replaced by solvent extraction. Organic solvents can dissolve oil and be
readily evaporated. Methanol-chloroform, hexane, and hexane-isopropanol are
normally utilized solvents (Cheng et al. 2011, Boyd et al. 2012). Currently, solvent
extraction is the most often applied method in the industry. However, using
organic solvents has raised health and environmental concerns because of their
flammability and toxicity.
Technologies with less threat to the human population and environment are
demanded. Therefore, to lower the amount or eliminate toxic solvent use
becomes the key solution of the problem. Ultrasonification and microwave in
oil extraction avoid the large amount of solvent use and enhance the oil yield
(Ranjan et al. 2010, Araujo et al. 2013). Fatty acid methyl ester, which is
nontoxic, renewable, and biodegradable, has been studied in oil extraction from
359
sunflower seeds and showed comparable performance to the conventional

solvents (hexane, chloroform, methanol, and isopropanol) (Amalia Kartika
et al. 2010). This chapter reviews the technologies of oil extraction and discusses
their advantages and disadvantages.
15.2 CELL DISRUPTION
Biological products synthesized by cells are intracellular and extracellular. Extra-

cellular products are easily separated from the cells by filtration or centrifugation.
Intracellular products are either in the cytoplasm or as inclusion bodies such as
lipid. Lipid is mainly present in the cell membrane (to form the bilayer) and
cytoplasm (in the form of lipid droplets). To obtain the desired intracellular
products, cell disruption has to be conducted to release these products before
further separation is carried out. Therefore, cell disruption is a critical step of lipid
separation from cells.
Blade homogenizer, bead milling, liquid homogenization, sonication, and
freezing/thawing are the most utilized physical approaches (Prabakaran and
Ravindran 2011, Dhanani et al. 2013). Blade homogenizer uses rotating blades to
grind cells and achieve the disruption. In general, a higher energy input provides
higher disruption efficiency. Bead milling is normally used along with agitation,
and the cell disruption efficiency is determined by bead size and agitation speed
(Klimek-Ochab et al. 2011). Liquid homogenizer is widely applied in the
disruption of microorganism cells. It lyses the cells by forcing the cell suspension
to pass through a narrow space and then shearing the cells (Zheng et al. 2011).
Sonication uses sound waves to form microscopic vapor bubbles and then obtain
the disruption. This method is efficient and suitable for the disruption of small-
sized materials such as bacteria, spores, cells, and finely diced tissues (Choonia
and Lele 2011). Freezing/thawing completes the disruption by freezing the cells
to cause the swell, and then contraction during thawing ultimately breaks the
cells (Shin et al. 1994, Schwede et al. 2011). The physical methods show
disadvantages to product quality because the methods tend to increase the local
temperature and lead to oxidation and denaturization.
Chemical methods of cell disruption are the processes with the addition of
chemicals such as solvents, detergents, and enzymes. Detergents such as triton-X
series and tween series are capable of solubilizing phospholipid and thus causing
the cell membrane disruption. However, a pretreatment to weaken the cell wall is
required before detergent can act (Northcote and Horne 1952). Organic solvent
cell disruption works in a similar way as detergent to solubilize the cell membrane.
Normally the solvent can disrupt the cell wall; therefore, pretreatment is not
demanded (Klimek-Ochab et al. 2011). An enzyme such as lysozyme has the
ability to disrupt the cell wall, but cannot break the cell membrane; hence, it is
usually used with detergent (Jin et al. 2012). Some of the cell disruption methods
are presented in Table 15-1.
Table 15-1. Approaches of Cell Disruption Comparison.

Methods Description Advantages Disadvantages
Blade A blender, using cutting blades to Easy to operate Not efficient for disrupting
homogenizers reduce size of substances microorganisms
Bead milling Using glass, ceramic, or steel bead Clean and suitable on cell Not so efficient as high pressure
to crush the cells as they collide disruption of spores, yeast, and and ultrasonification, heat
with agitation or stirring fungi; the process is cheap generation
Pressure Using pressure to produce shear Suitable for large-scale production High requirement on design
to break the cells
Ultrasonic Forming microbubbles to vibrate Efficient Nonspecific cell wall disruption,
the cells high heat generation, long
operation time, generation of
harmful free radicals
Freeze/thaw Forming of ice crystals to break the Easy to operate Requiring several cycles, slow and
cells high cost
Pressing Compressing the cells and Easy to operate and cheap Low efficiency
ultimately breaking the cells
Osmosis stock Utilizing osmosis pressure Cheap Pretreatment to weaken the cell
resulting from the wall for further disruption
concentration difference
between inside and outside of
LIPID EXTRACTION TECHNOLOGIES
the cell membrane to break the

cells
(Continued)
361
Table 15-1. Approaches of Cell Disruption Comparison. (Continued)

362
Methods Description Advantages Disadvantages
Detergent Using detergents to solubilize the Preserving the properties of the Pretreatment to weaken the cell
phospholipid and disrupting the products wall for further disruption,
cells requires a subsequent process
to remove the detergent
Solvent Using solvents to solubilize the Efficient Requires a subsequent process to
phospholipid and disrupting the remove the detergent
cells
Enzyme Using lysozyme to disrupt cells Selective Needs the addition of detergents
to complete the disruption
LIPID EXTRACTION TECHNOLOGIES 363
15.3 PHYSICAL TECHNOLOGIES OF LIPID SEPARATION
15.3.1 Expeller Pressing

Expeller pressing is a mechanical method for separating lipid from raw materials
such as nuts. Different types of expellers, such as hand bridge press, hydraulic
press, ram press, and screw press, have been used. The structures of the expellers
vary from one type to another. The principle of expeller lipid extraction is that the
target materials fed between two heavy metal plates is ground, crushed, and
pressed as the plates rotate toward each other driven manually or powered
electrically, which results in the lipid separation from the oleaginous materials.
The pressure generated by the driving force (manual, motor, or engine) is the
main factor of extraction efficiency because it is the main cause of cell disruption.
Expeller pressing lipid extraction is clean and cost-efficient. However, there
are two major disadvantages—namely, low efficiency and oil flavor change. The
general lipid recovery from expeller pressing is normally less than 70% w/w (more
than 90% w/w for solvent extraction) (Bamgboye and Adejumo 2007). To recover
more lipids from the raw materials, solvent extraction has to be performed after
the pressing. The other concern about the pressing method is the high temperature
generation during the pressing. The temperature increase depends on the hardness
of the raw materials. The harder the material is, the higher the temperature reaches
in the process. The lipid extraction with a temperature-controlled expeller is called
cold pressing in which the temperature will not rise above 50 °C. Cold pressing is
usually used to obtain lipid from delicate materials such as olives. The expeller
pressing efficiency on different raw materials is shown in Table 15-2.
Expeller pressing is suitable for lipid extraction from any type of oleaginous
material. It is specially used for lipid extraction from soybean, sunflower seed, and
nuts in farms and small-scale rural industries. To date, expeller pressing for lipid
extraction from microorganisms has not been reported. Because no special
requirement on raw material is demanded for lipid extraction, expeller pressing
could be used in lipid separation from oleaginous microorganisms.
15.3.2 Thermal Extraction

Hot water floatation is the simplest and oldest method of lipid extraction. Raw
materials are immersed in boiling water and kept simmering for a certain period
(normally several hours). As temperature goes down, the raw material becomes a
paste. Lipid floats to the surface and then can be skimmed off. In general, in this
process, the lipid is required to be reheated to 100 °C to drive off the trace amount
of water. The extraction efficiency of the method depends on the lipid content of
the materials and the lipid property (liquid or solid form at room temperature).
The extraction efficiency is high when the oil-bearing substance has high lipid
content and lipid is in solid form at room temperature. Thermal extraction is
normally applied in lipid separation from animal fat and fish, and salt could be
added to enhance the separation (Bimbo 2012). Thermal extraction has also been
applied in lipid extraction from groundnuts. With vegetable oils, the method is
364
Table 15-2. Oil Separation with Expeller Pressing.

Separation
Technologies Raw material Lipid content efficiency (%) References
Expeller Peanut 50 92 Sivakumaran et al. (1985)

Ram press Sunflower seed 25–40 50–56 Bachmann (2001)
Mechanical expression rig Shea kernel 34–44 58.5 Olaniyan and Oje (2007)
Expeller Sunflower seed 25–40 70 Bamgboye and Adejumo (2007)
Screw press Groundnut 35–50 75 Olaniyan (2010)
Mechanical expression rig Shea kernel 34–44 58.63 Olaniyan and Oje (2011)
Screw press Palm kernel 46–57 22.79 Adesoji et al. (2012)
Screw press Soybean 19–23 36.55 Adesoji et al. (2012)
undesirable because of the formation of oil–water emulsions, which makes the

separation of floating oil from the water difficult. Microorganism lipid content
could reach 80% w/w, and the lipid is usually in solid form at room temperature.
Hence, the method could be utilized for lipid separation from microorganisms.
15.3.3 Ultrasonication
Ultrasonication provides cavitation phenomena. Microscopic bubbles at various
nucleation sites in fluid are formed during ultrasonication, which has two
phases—namely, rarefaction and compression phase. The bubbles grow during
the rarefaction and are compressed during the compression phase, which cause
the collapse of the bubbles. A violent shock wave is formed by the collapse of the
bubbles, and then tremendous heat, pressure, and shear are generated.
Ultrasonication has been widely applied in industry and is grabbing more and
more attention because it has accomplished protein extraction, chemical synthesis,
disinfection, and cell disruption with reducing or eliminating chemical addition,
which is considered to be green chemistry. The application of ultrasonication in
cell disruption for the intracellular products recovery is not new. The method has
been widely used in protein (especially enzymes such as β-galactosidase) and
lactase releasing from cells (Becerra et al. 2001, Benov and Al-Ibraheem 2002,
Choonia and Lele 2011). In general, cells harvested from fermentation will have to
be washed before being subjected to ultrasonication to avoid product contamina-
tion. Filtration and centrifugation are performed after ultrasonication to separate
the products from the impurities.
Ultrasonication was used for lipid extraction from Nannochloropsis oculata
(Adam et al. 2012). Response surface methodology was used to obtain the optimal
condition. Parameters including extraction time (10 to 30 min), biomass concen-
tration (10 to 50 g/L), and ultrasound power (450 to 1000 W) were varied.
The optimal condition was found at the power of 1,000 W for 30 min with a
biomass concentration of 50 g/L. Afterward, the extraction salt was added to
enhance the separation of lipid from the solution. The highest lipid yield was
0.21% w/w, which is considerably lower than solvent (chloroform and methanol)
extraction yield (5.47% w/w). More effort is required to increase lipid recovery
with ultrasonication.
15.4 CHEMICAL TECHNOLOGIES OF LIPID SEPARATION
15.4.1 Organic Solvent Extraction
15.4.1.1 Organic Solvent Extraction Development

So far, many methods can be found on lipid extraction from various
materials such as animal and plant tissues, and microorganism cells. The first
popular lipid extraction is described in 1879 by Franz von Soxhlet, who invented a
special apparatus called Soxhlet Apparatus to extract lipid from solid materials
(Soxhlet 1879). The extraction is accomplished by boiling solvent to generate the

vapor that constantly flows over the solid to extract the lipid. At the end, solvents
containing lipid are collected, and then lipid will be obtained after evaporating the
solvents. The method is simple, but there is a risk of lipid oxidation caused by the
high temperature.
In fact, the method is often used in the extraction of pesticides and poly-
chlorinated biphenyls (PCBs) rather than in lipid extraction (Zhou et al. 2008).
So far, the most cited two lipid methods are reported by Folch et al. (1957) and
Bligh and Dyer (1959). The common points of the two methods are the use of
chloroform and methanol aiming to estimate the total lipid. Both methods have
been well established. The method of Folch et al. (1957) is known for its simplicity
(one step extraction), and the one of Bligh and Dyer (1959) is considered to be a
rapid method (no requirement on predrying). Both methods have an adverse
effect on the environment because of the use of chloroform.
Therefore, the mixture of hexane and isopropanol, which are less toxic and
cheaper than chloroform and methanol, was studied on lipid extraction (Hara and
Radin 1978). However, it was observed that the method could not extract
gangliosides. In fact, ganglioside is just a minor fraction of the total lipid; hence,
the method is still widely used and recommended by the USEPA for field studies.
Another method with a similar procedure as the method of Bligh and Dyer (1959)
has also been reported, but a mixture of isopropanol and cyclohexane was used
instead of chloroform and methanol (Smedes 1999). However, the method was
found unsuitable for specific tissues such as liver because of the possibility of
emulsion formation. Another halogenate-free solvent extraction using 2-propanol
(Sree et al. 2009), diethyl ether (DEE), and n-hexane completes the extraction
with (1) 2-propanol, commonly called isopropanol (IPR) and DEE extraction,
(2) n-hexane/DEE and IPR, and (3) n-hexane/DEE (Jensen et al. 2003). The
advantages of the method are that there is no requirement for heating, and it is
easy to handle. The method is normally used for large samples (>10 g), and the
suitability to small samples is not studied.
Accelerated solvent extraction is similar as Soxhlet extraction but the method
applies both high temperature and high pressure to keep the solvent in liquid
phase (Richter et al. 1996). The method is time saving but expensive; in general, it
is used for extracting environmental contaminants such as PCBs, dioxins, and
pesticides. Current organic solvent extraction is either the previously mentioned
original method or the modification. Modification is mainly embodied on the
combination of the aforementioned methods with the assistance of cell disruption
treatment such as bead milling, ultrasonication, microwave, and so on.
15.4.1.2 Effect of Solvent Type on Extraction

Solvent extraction efficiency is up to 96% (Ferraz et al. 2004, Dufreche et al. 2007).
The most used solvents include alcohols (mainly referring to methanol), chloro-
form, hexane, petroleum ether, and diethyl ether. The solvent selection is critical
because of impacts on the extraction efficiency, lipid property preservation, and
solvent recovery. To extract lipid from tissues, it is necessary to create enough
force to break the cell membrane and lipoprotein to release the lipids. In addition,
the solvents would not react chemically with the lipids.
The cell membrane has a double lipid layer. Each lipid layer is composed of
polar head and nonpolar tail. The tail is oriented inward and the head faces outward
(toward the aqueous cytosol of the cell or the outside environment). These tails or
heads are grouped together to form the bilayer. The structure of the cell membrane
determines that a nonpolar solvent cannot perform the extraction because it cannot
approach and pull out the lipid from the cell membrane, and hence cannot rupture
the membrane. However, if cell disruption is performed prior to nonpolar solvent
extraction, the extraction will be possible to complete. Polar solvent (such as water)
could approach the membrane, but if the polarity of the solvent is lower, then the
solvent cannot pull out the lipid because the tails are tightly bonding (hydrophobic
interaction) together. Therefore, a mixture of polar and nonpolar solvent is required.
The polar solvent interacts and pulls apart the cell membrane, and the nonpolar
solvent accesses to the nonpolar tail and dissolves the lipid. So far, many solvent
extractions have been used (Table 15-3). The mixture of polar and nonpolar solvents
provides high extraction efficiency (around 95%) at the mild condition (around
25 °C). For the extraction with a single polar or nonpolar solvent, the extraction
efficiency is normally low (less than 75%).
The lipid droplets, also called lipid bodies, in oil-bearing tissues are mainly
triglycerides (TAGs) which are a nonpolar substance. They are soluble in hexane,
cyclohexane, diethyl ether, and chloroform. Therefore, when the focus is on TAG
extraction, nonpolar solvent should be used. However, it is necessary to break the
cell first to allow nonpolar solvent to access TAGs. Therefore, either a polar
solvent should be added along with the nonpolar solvent, or other cell disruption
methods such as milling and ultrasonification should be used.
15.4.1.3 Effect of Oil-Bearing Substances on Lipid Extraction

Types of oleaginous substances also have an effect on the extraction (Table 15-3).
The structure differences in the cells of plants, animals, and microorganisms
are the main cause of the difference in extraction efficiency. Unlike plant and
microorganisms, animal cells have no cell wall, making their lipid extraction
easier (such as short extraction time and high efficiency) (Ferraz et al. 2004,
Vicente et al. 2009). The cell wall of plants is formed by a cellulose-hemicellulose
network with embedded pectin matrix. For fungus, the cell wall consists largely
of β(1-3) and β(1-6)-D-glucans, chitin, and protein. The linkage between β(1-3),
β(1-6)-D-glucans, and chitin forms the cell wall, and protein is normally embed-
ded. The solubility of the cell wall in the solvent or solvent mixture determines the
extraction efficiency. Lipid extraction with n-hexane from fungus is easier
compared with soybean as the extraction time (1 h for fungus and 2.5 h for
soybean) was shorter and the required temperature was lower (25 °C for fungus
and 70 °C for soybean) (Nikolić et al. 2009; Vicente et al. 2009). In addition, the
extraction efficiency is higher for fungus (70.7%) than for soybean (68.7%),
because the cell wall of soybean is harder to be broken by hexane than that of
the fungus.
Table 15-3. Lipid Extraction with Different Organic Solvents.

368
Extraction Extraction
Solvent Oleaginous substance Character conditions efficiency (%) References
Chloroform: Animal tissue Nonpolar and 24 h; 25 °C 96 Folch et al. (1957)

methanol (2:1) polar and
Chloroform: Human serum Nonpolar and 11 min; 20 °C 96 Ferraz et al. (2004)
methanol (2:1) polar and
Chloroform: Mucor circinelloides Nonpolar and 1 h; 25 °C 94 Vicente et al. (2009)
methanol (2:1) (fungus) polar and
Chloroform: Rhodotorula glutinis Nonpolar and 1 h; 25 °C 95 Cheirsilp et al. (2011)

methanol (2:1) (yeast) and Chlorella polar and
vulgaris (microalga)
Chloroform: Fish Nonpolar and A few minutes; 94 Bligh and Dyer (1959)
methanol: polar and 25 °C
water (2:2:1)
Chloroform: Mucor circinelloides Nonpolar and 1 h; 25 °C 89.6 Vicente et al. (2009)
methanol: (fungus) polar and
water (2:2:1)
Hexane: Serum Nonpolar and 11 min; 20 °C 88 Ferraz et al. (2004)
isopropanol (2:1) polar and
Hexane: Rhodotorula Nonpolar and 1 h; 25 °C 95 Galafassi et al. (2012)
isopropanol (3:2) graminis (yeast) polar and
Hexane: Sludge Nonpolar and 1 h; 100 °C 97 Dufreche et al. (2007)
methanol:acetone polar
(3:1:1)
Methanol followed Sludge Polar and 1 h; 100 °C 78 Dufreche et al. (2007)

by hexane Nonpolar
Hexane Serum Nonpolar 11 min; 20 °C 18 Ferraz et al. (2004)
Hexane Food-grade sorghum Nonpolar 30 min; 65 °C 10 Christiansen et al. (2008)
Hexane Sludge Nonpolar 1h; 100 °C 6.92 Dufreche et al. (2007)
n-Hexane Mucor circinelloides Nonpolar 1 h; 25 °C 70.71 Vicente et al. (2009)
(fungus)
n-Hexane Soybean Nonpolar 2.5 h; 70 °C 68.7 Nikolić et al. (2009)
Chloroform Soybean Nonpolar 150 min; 61.2 °C; 75.7 Nikolić et al. (2009)
Methanol Sludge Polar 1 h; 100 °C 69 Dufreche et al. (2007)
Methanol Mucor circinelloides Polar 30 min, 25 °C 35.72 Mitra et al. (2012)
(fungus)
369
15.4.1.4 Effect of Pretreatment on Extraction

Except for simple organic solvent extraction, pretreatment in combination with
other technologies such as bead milling, grinding, and ultrasonication has also
been reported (Table 15-4). The addition of other technologies aims to disrupt the
cells and enhance the extraction. The extraction can be performed in two
separated steps, that is, cell disruption followed by solvent extraction, or in one
combined step—simultaneous cell disruption and solvent extraction.
• Bead milling-assisted solvent extraction: Bead milling is the process in which
beads mix with a cell suspension at high-speed agitation. The mixing provides
the contact and shearing between cells and beads, and thus achieves the
disruption. After the disruption, solvent is usually used to recover the oil.
Therefore, the milling is also considered as a pretreatment of solvent
extraction. Size and shape of the bead, agitation, the strength of the cell
wall, and cell concentration of the suspension have great effects on the degree
of the disruption (Klimek-Ochab et al. 2011).
Lipid extraction with chloroform and methanol (2:1 v/v) from Chlorella sp.
biomass with or without bead milling showed significantly different results
(Prabakaran and Ravindran 2011). Higher lipid was obtained from milling
(0.15 g lipid/0.5 g dry biomass) than without milling (0.08 g lipid/0.5 g dry
biomass). Similar results were obtained with the study on Botryococcus sp.,
Chlorella vulgaris, Scenedesmus sp., Nostoc sp., and Tolypothrix sp. (Lee et al.
2010). Apart from beads, sand has also been used in cell wall disruption
(Somashekar et al. 2001). However, sand self-breaking during homogeniza-
tion in pestle and mortar is a great concern.
• Ultrasonication-assisted solvent extraction: Similar to bead milling, ultrasoni-
cation is performed to disrupt the cell wall. The assistance with cell disruption
means extraction can be completed in a few minutes instead of a few hours in
conventional solvent extraction with high reproducibility (Wei et al. 2008).
Several parameters including extraction time, solvent, and ultrasonication
power, have been associated with extraction efficiency (Metherel et al. 2009,
Araujo et al. 2013). It was found that high power led to high lipid extraction
efficiency. Normally, to obtain a similar extent of lipid recovery, ultrasonica-
tion-assisted lipid extraction required 15 min, but traditional chloroform and
methanol extraction needs several hours (Metherel et al. 2009).
• High-pressure homogenization assisted solvent extraction: Applying high
pressure in the cell induces high shear stress inside the orifice and creates a
large pressure drop at the outlet, which results in the cell disruption. Study has
showed that high-pressure homogenization (8 Mpa 35 °C) could finish lipid
extraction from microalga, Scenedesmus sp. within 30 min, whereas the
traditional chloroform and methanol extraction demanded 5 h (Cho et al.
2012).
• Microwave assisted solvent extraction: Microwave irradiation rapidly gen-
erates high heat and pressure in the extraction system and forces cell
Table 15-4. Organic Solvent Extraction with Assistance.

Assistance Solvents and conditions Extraction
Oleaginous substances technology of the extraction efficiency (%) References
Botryococcus sp. Bead milling Chloroform–methanol 94.2 Lee et al. (2010)

(1:1 v/v); 5 min, 25 °C
Chlorella vulgaris Chloroform–methanol 25.8 Lee et al. (2010)
(1:1 v/v); 5 min, 25 °C
Chlorella vulgaris Chloroform–methanol 48.6 Zheng et al. (2011)
(1:1 v/v); 10 min, 30 °C
Chlorella sp. Chloroform–methanol 83.2 Prabakaran and
(2:1 v/v); 10 min; 30 °C Ravindran (2011)
Scenedesmus sp. Chloroform–methanol 34.8% Lee et al. (2010)
(1:1 v/v); 5 min, 25 °C
(2:1 v/v) 5 min, 25 °C Ravindran (2011)
Nostoc sp. Chloroform–methanol 97.1 Prabakaran and
Tolypothrix sp. Chloroform–methanol 98.0 Prabakaran and
Mucor rouxii Sand milling Chloroform–methanol 96.23 Somashekar et al. (2001)
(2:1 v/v)
Mucor hiemales Chloroform–methanol 92.6 Somashekar et al. (2001)

(2:1 v/v)
(1:1 v/v); 10 min, 30 °C
371
(Continued)
Table 15-4. Organic Solvent Extraction with Assistance. (Continued)

372
Assistance Solvents and conditions Extraction

Oleaginous substances technology of the extraction efficiency (%) References
Scenedesmus sp. High-pressure Chloroform–methanol 0.21 Cho et al. (2012)

homogenization (2:1 v/v); 30 min; 35 °C
Scenedesmus sp. Ultrasonication Chloroform–methanol 0.16 Cho et al. (2012)
(2:1 v/v); 30 min; 35 °C
(2:1 v/v); 10 min; 30 °C
Scenedesmus sp. Microwave Chloroform–methanol 0.12 Zheng et al. (2011)

(2:1 v/v); 30 min; 35 °C
Saccharomyces Chloroform–methanol 0.09 Khoomrung et al. (2013)
cerevisiae (2:1 v/v); 16 min; 60 °C
(2:1 v/v); 10 min; 30 °C
Chlorella sp. Ultrasonication Chloroform–methanol 98.1 Prabakaran and

Rhodosporidium Enzyme Chloroform; 60 min; 30°C 96.6 Jin et al. (2012)
toruloides
Chlorella vulgaris Hexane-methanol 93.4 Zheng et al. (2012a)
(1:2 v/v); 30 min; 30 °C
Chlorella vulgaris Hexane-methanol 96.2 Zheng et al. (2012b)
(1:2 v/v); 30 min; 30 °C
Chlorella vulgaris Enzyme+sonication Water; 10 min; 95 °C 49.82 Liang et al. (2012)
Scenedesmus Water; 10 min; 95 °C 46.81 Liang et al. (2012)
dimorphus
Nannochloropsis sp. Water; 10 min; 95 °C 11.73 Liang et al. (2012)
373
disruption. Pretreating the wet microorganism with microwaves achieves

water reduction as well as breaking the cell, which indicates that it would
eliminate the dewatering process. It was reported that temperature has an
essential effect on the microwave extraction (Boldor et al. 2010). Every 10 °C
increase in temperature from 50° to 70 °C could obtain around 6% (w/w)

higher lipid recovery.
• Enzyme lysis–assisted solvent extraction: It has the potential to partially or
fully disrupt cells with minimal damage to lipid. Appropriate enzyme
selection is critical because the composition of cells largely varies (Mercer
and Armenta 2011). Enzyme-assisted lipid extraction is not widely practiced
primarily because of the high cost of enzyme production and the difficulty in
recovering and recycling the enzyme. Enzyme is normally combined with
other cell disruption methods such as microwave and ultrasonication as
pretreatment (Jin et al. 2012, Liang et al. 2012). Recombinant plMAN5C was
used in lipid extraction from wet yeast Rhodosporidium toruloides, and 94% of
total lipid was obtained with enzyme dosage of 3 g/kg cells at 30 °C and pH 4.5
for 1.5 h (Jin et al. 2012).
Several studies have compared the different pretreatment effects on lipid
extraction, and their performance varied from one to another (Lee et al. 2010,
Cho et al. 2012). There is no consistent report of optimal pretreatment for
lipid extraction from microorganisms. Lee et al. (2010) observed that the
optimal pretreatment for lipid extraction with chloroform and methanol for
Botryococcus sp., Chlorella vulgaris, and Scenedesmus sp. were bead milling
and microwave, heating, and microwave. Cho et al. (2010) addressed that
high-pressure homogenization performed the best on lipid extraction from
Scenedesmus sp. compared to microwave and ultrasonication. It was reported
that ultrasonication was best for Chlorella vulgaris lipid extraction (Zheng et al.
2011). This diversity could be a result of the differences in solvent selection, time,
temperature, and so on. The assisting technologies of lipid extraction could be
used as pretreatment of solvent extraction as well as used during solvent
extraction. However, safety issues are the main concern regarding simultaneous
solvent extraction and cell disruption by ultrasonication, microwave, or high-
pressure homogenization.
15.4.2 Supercritical Fluid Lipid Extraction

In recent years, supercritical fluid extraction (SFE) has gained considerable
attention because of several advantages: it preserves the originality of the product,
lacks harmful solvent residues, separation is easy, and the process is environmen-
tally friendly (Sahena et al. 2009, Mercer and Armenta 2011). SFE achieves the
lipid extraction by manipulating the chemicals, which behave as both a liquid and
a gas in their critical temperature and pressure. In the critical stage, the solvating
power of the compound using SFE is increased, and then it plays as a solvent to
extract the product from the cells. The most remarkable point is that SFE is highly
selective in extracting triglycerides (Cheng et al. 2011).
Several parameters such as viscosity, diffusivity, and critical temperature and

pressure are considered on the selection of the chemical. Carbon dioxide is the
most commonly used because of the low viscosity (<100 μPa·s), high diffusivity
(<0.1 mm2/s), and suitable critical temperature (31.1 °C) and pressure [72.8 atm
(7.35 × 106 Pa)]. In an extraction vessel, oil-bearing substances contact with

supercritical carbon dioxide for a certain time period (several hours). During
the process, oil will be solubilized in CO2 and extracted. Oil-containing CO2 is
then collected and depressurized to allow the escape of CO2, and finally, oil is
obtained.
Temperature, pressure, carbon dioxide flow rate, and moisture of the sample
are significant factors in the extraction (Andrich et al. 2006, Spence et al. 2009).
The impact of the factors is complicated. Low temperature leads to high density of
supercritical fluid, which results in the low mass transfer (Lou et al. 1995) and thus
low lipid extraction efficiency. Increasing the temperature increases the diffusion
rate and hence lifts the extraction rate. Raising the temperature from 50 to 200 °C
could enhance the extraction efficiency from 66% to 99% (Langenfeld et al. 1993).
High pressure provides a high diffusion rate, but when the pressure reaches a
certain level, the extraction efficiency becomes constant, and to increase the
efficiency requires the assistance of temperature. High carbon dioxide flow rate
increases extraction efficiency as fresh flow enhances mass transfer. The moisture
of samples influences the extraction because it determines the contact time of
supercritical fluid and lipid. Samples tend to keep their thickness consistent, and
moisture will be the barrier of the diffusion of any intruder (here refers to CO2) to
the inside of the cells and of the diffusion of intercellular products (here refers to
lipid) out of the cells (Mercer and Armenta 2011). Another significant consider-
ation of extraction is the pretreatment process, including the technologies dis-
cussed in organic solvent extraction. Normally, SFE requires the assistance of
pretreatment or addition of a cosolvent such as ethanol and methanol; otherwise
the extraction time would be too long (more than 20 h) (Mouahid et al. 2012).
The application of CO2 SFE from microorganisms has been extensively
reported. The factors were evaluated in terms of the extraction efficiency
for particular microorganisms. Some of the applications are summarized in
Table 15-5. It was observed that high temperature provided high efficiency, and
pressure had a low impact on the extraction. Large variation on carbon dioxide
flow rate (from 0.2 to 10 kg/h) has slight influence on the extraction efficiency
(Table 15-5). The extraction time of SFE is usually similar to that used in
traditional organic solvent extraction; hence, it suggests that SFE with carbon
dioxide is not prior to solvent extraction in terms of time.
15.4.3 Other Technologies for Lipid Extraction

New technologies, such as switchable solvent extraction, for lipid extraction have
also been developed recently. In fact, these methods also count on solvents to
dissolve the lipid and achieve the extraction. Their advantage is to utilize greener
chemicals such as 1,8-diazabicyclo-[5.4.0]-undec-7-ene (DBU) (French et al.
1976) and ethanol. A study of lipid extraction with a mixture of DBU and
376
Table 15-5. Supercritical CO2 Lipid Extraction from Microorganisms.

Temperature Pressure CO2 flow Water Time Extraction
Microbes (°C) (atm) rate (kg/h) content (%) (h) efficiency (%) References
Chlorella 50 346 0.05 5 3 80 Chen and

protothecoides Walker (2012)
Chlorella vulgaris 40 197 10 5 9 98.1 Dejoye et al. (2011)
Chlorella sp. 40 296 0.9 5 3 92.2 Char et al. (2011)

Mix culture 77.6 233 NA 8 0.25 95.0 Hanif et al. (2012)
(digested sludge)
Nannochloropsis sp. 55 690 10 5 6 82.5 Andrich et al. (2005)
Pavlova sp. 60 300 NA NA 6 98.7 Cheng et al. (2011)
Pseudomonas 60 500 4 5 3 43 Hampson and
resinovorans Ashby (1999)
Pythium irregulare 60 271 NA 30 6 NA Walker et al. (1999)
Scenedesmus 100 400 0.2 NA 1 98.5 Soh and
dimorphus Zimmerman (2011)
Spirulina (Arthrospira) 55 690 10 5 4 90.3 Andrich et al. (2006)
platensis
Tetraselmis chui 60 246 NA NA 1 5 Grierson et al. (2011)
ethanol has successfully separated lipid from soy flakes to gain similar extraction
efficiency as organic solvent extraction (Phan et al. 2009). The research took
advantage of the polarity change in the presence and absence of carbon dioxide.
The switch from low polarity to high polarity as environment altering makes the
switchable solvent play the similar role as chloroform and methanol mixture.
Nanomaterial lipid extraction has also been reported. It is stated that modified
nanomaterial accomplished lipid extraction from live microalgae without harm to
cells (Lin 2009). Nanomaterials considered as a great carrier of immobilization due
to the high surface area have been extensively applied. If organic solvent-like
chemicals were immobilized onto solid nanomaterials, it would achieve lipid
extraction as well as avoid contamination of the solvent on lipid. Rare research has
been conducted in this aspect, and the effort is demanded.
15.5 SUMMARY
Physical and chemical technologies have been developed in lipid extraction.

Physical extraction breaks the cells to release the lipid by providing force, whereas
chemical extraction uses a solvent to pull out lipid from cells. Each method has its
advantage and disadvantage. Physical methods are clean but have low lipid
extraction efficiency (about 70% of total lipid) and high energy consumption.
Chemical extraction has a high possibility of contamination of the lipid due to the
presence of the residual solvents when toxic organic compounds are used. A clean
solvent such as supercritical CO2 gives high-quality lipid, but it normally depends
on pretreatment or a cosolvent addition to achieve high extraction efficiency. New
technologies such as switchable solvent and nanomaterial extraction have also
been reported, but detailed information is not available because it is still in the
infant research stage.
We sincerely thank the Natural Sciences and Engineering Research Council of

Canada (Grant A 4984, Canada Research Chair) for their financial support. The
views and opinions expressed in this chapter are those of the authors.
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CHAPTER 16
Milking of Lipids from
Oleaginous Microorganisms
B. Bhadana
R. D. Tyagi
16.1 INTRODUCTION
Usually, oil crops (e.g., sunflower, rapeseed, soybean), animal fat, or waste
cooking oil (WCO) are used to prepare biodiesel by transesterification. However,
the use of oil crops comes under the food-versus-fuel controversy because of
their long growth cycle that contributes to high production costs. Similarly, WCO
and animal fat do not have equal quality, and they are hard to collect
(Wahlen et al. 2012). Because of these limitations, research has started to
investigate microbial sources that lead to the production of advanced high-value
molecules (HVM) and lipids with economical nutritional requirements
(Peralta-Yahya et al. 2012). Several potential production hosts have been
investigated, including yeast, algae, cyanobacteria, and bacteria (Peralta-Yahya
et al. 2012). Thus, lipid biofuels (biodiesel) are a promising alternative that can
replace fossil fuels (Liu et al. 2008).
Each microorganism has the capacity to synthesize lipids that play an
important role in its membranous structure. Some microorganisms can accumu-
late lipids (>20% dry cell weight). Such microorganisms are called oleaginous
microorganisms (Ratledge and Wynn 2002). Under nutrient starvation conditions
(limitation of nitrogen, phosphorus, sulfur, and so forth), lipid accumulation can
reach up to 70% of microbial biomass. The lipids are stored as triacylglycerol
(TAG) in oil globules (or lipid bodies) of these microbes (Sibi et al. 2016). Thus,
oleaginous microbes are promising candidates for sustainable biofuel production
[also called single cell oil (SCO)] because of their unique capability to reach high
lipid productivities and the short production cycle (Christophe et al. 2012).
Currently, at the commercial level, carotenoids are being produced by micro-
algae, such as astaxanthin from Haematococcus pluvialis and β-carotene from
Dunaliella salina. Dunaliella can withstand higher than 50 °C temperature for up
383
to 8 h under stressed conditions and produces several pigments, such as

canthaxanthin, lutein, and astaxanthin. These oleaginous microbes also contrib-
ute to nutritionally important fatty acids, including docosahexaenoic acid
(DHA) and eicosapentaenoic acid (EPA) (Sahu et al. 2013). They also synthesize
bioactive compounds for biomedical, neutraceutical, and pharmaceutical

prospects, the so-called HVMs (Veena et al. 2007). Thus, the oleaginous
microorganisms play an important role in both biodiesel and HVM production
(Hu et al. 2013).
In a study by Gendy and El-Temtamy (2013), biofuel cost varies from USD
9/gal. to USD 40/gal., which does not meet the economic requirement. The
technoeconomic analysis is the key factor in the development of a biorefinery
process. According to Brownbridge et al. (2014), microbial biodiesel or the third-
generation renewable biofuel cannot be commercially feasible as a primary product
without extraction of other HVM products. The major hindrance in the commer-
cialization of microbial biofuel is the energy and costs invested in the downstream
processing, that is, during harvest and extraction of fuel from microbial cells. In
current commercialized processes, after microbial biomass production, lipid and
HVMs are harvested and processed via dewatering, drying, grinding, extraction,
and purification steps (Bahadar and Khan 2013). Grinding and extraction
(Greenwell et al. 2009, Zhang et al. 2011) are widely used steps to separate
microbial HVM (Figure 16-1). Drying and grinding need energy inputs and result
in killing of microbial biomass. Thus, it is necessary to regrow microbial culture each
time, which can take several hours to weeks. In addition, extraction and purification
steps lead to organic waste generation (Bahadar and Khan 2013). Hence, down-
stream processing poses many technical and economic challenges (Bahadar and
Khan 2013, Montagne et al. 2013). It is estimated that downstream processing costs
represent 50% to 80% of total production cost (Ahmed et al. 2012, Richardson
et al. 2012). Thus, there is a need to develop alternate downstream processes.
Figure 16-1. Current commercialized process to extract microbial lipids/HVM.

MILKING OF LIPIDS FROM OLEAGINOUS MICROORGANISMS 385
Figure 16-2. Milking of HVM.
Milking is one of the best possibilities that was introduced by Frenz and
collaborators in 1989 for extraction of hydrocarbons from Botryococcus braunii
(green algae) via exposing cells to hexane for a short time (Frenz et al. 1989).
Milking specifies that microbes remain alive during HVM extraction and thus,
can be reused repeatedly; milking is closure to a closed system (Figure 16-2).
There are various extraction processes that are potentially applicable for
milking of microorganisms, including the use of biocompatible organic sol-
vents, pulsed electric field (PEF), spontaneous oozing, and membrane transport
proteins. Hence, if milking comes to the commercial level, it would be more
economical and environmentally friendly. This chapter critically discusses the
potential microbes for lipid production and various milking methods for their
extraction.
16.2 MICROBIAL POTENTIAL FOR LIPID PRODUCTION
Oleaginous microorganisms have proven to be the promising candidates for

biodiesel production because they have high lipid productivities. Presence of
excess carbon source and a limiting element (e.g., nitrogen) in the growth medium
induces lipid accumulation in oleaginous microbes (Beopoulos et al. 2009).
Under nitrogen-limiting conditions, cells stop proliferating while the carbon
source is continuously assimilated by the microbes. This causes diversion of the
carbon flux for lipid synthesis, thereby resulting in TAG accumulation within lipid
bodies of the cells (Beopoulos et al. 2009).
Microalgae synthesize certain secondary metabolites (or HVM) (Gordon and
Seckbach 2012, Heydarizadeh et al. 2013, Lemoine and Schoefs 2010) that play
an important role in biomedical, nutraceutical, and pharmaceutical projects.
Table 16-1 represents major HVMs produced by some microalgae.
Yeasts have also proven to be potential microbes for lipid production.
Of 600 known strains of yeasts, only 30 strains are able to accumulate greater
than 20% of biomass weight as lipids (Ratledge and Wynn 2002). Table 16-2
presents the major oleaginous strains with the accumulated lipid percentage using
different carbon sources in the growth media.
Table 16-1. Major HVM and Oil Percentage of Different Microalgae.

386
Oil content
Alga (% dry weight) HVM References
Chlorella vulgaris 58 Neutral lipids Illman et al. (2000)

Chlorella emersonii 34 Neutral lipids Scragg et al. (2002)
Nitzschia laevis 28–69 EPA Wen and Chen (2003)
Dunaliella tertiolecta 36–42 Carotenoid, β-carotene, mycosporin-like Li et al. (2008)
amino acids
Crypthecodinium cohnii 20 DHA, starch Deschamps et al. (2008)
Chlorella protothecoides 15–55 EPA, ascorbic acid Liang et al. (2009)

Dunaliella salina 10 Carotenoid, β-carotene, mycosporin-like Mojaat et al. (2008)
amino acids, sporopollenin
Botryococcus braunii 29–75 Isobotryococcene, botryococcene, Ge et al. (2011)
triterpenes
Chlorella minutissima 57 C16- and C18- lipids Li et al. (2011)
Schizochytrium limacinum 50–77 DHA Ethier et al. (2011)
Nannochloropsis sp. 46–68 EPA, TAG, ω-3. Pal et al. (2011)
Ankistrodesmus sp. 28–40 Mycosporin-like amino acids, Priyadarshani and Rath (2012)
Polysaccharides
Cyclotella sp. 42 Neutral lipids Sharma et al. (2012)
Neochloris oleoabundans 35–65 Fatty acids, starch Garibay-Hernández et al. (2013)
Tetraselmis suecica 15–32 Carotenoids, chlorophyll, tocopherol, Pérez-López et al. (2014)
lipids
Table 16-2. Lipid Accumulation by Different Oleaginous Yeast Strains.
Substrate Strains Lipid (wt.%) References
Glycerol Yarrowia 17.9 Mathiazhakan

lipolytica et al. (2016)

SKY7
Sucrose Trichosporon 63 Zhu et al. (2008)
Xylose fermentans 58
Lactose 50
Fructose 41
Molasse 37
Glycerol Rhodotorula 22.3 Makri et al. (2010)
glacialis
Glucose Rhodosporidium 71 Wu et al. (2011)
Glucose toruloides 71 Zhao et al. (2011)
Glucose Rhodotorula 30–40 Xue et al. (2010)
and starch glutinis
wastewater
16.3 MILKING METHODS TO EXTRACT LIPIDS
Table 16-3 summarizes various extraction processes for milking. This section
describes some of these milking processes.
16.3.1 Biocompatible Organic Solvent

Biocompatible organic solvents have been used for lipid milking from microalgae.
Here, the microbial biomass is exposed to a biocompatible hydrophobic solvent
that is absorbed by the cells. The solvent creates pores and openings in the cell
membrane and results in the secretion of lipids (inside cytosol) from the cells
(Hejazi et al. 2004). The partition coefficient of the biocompatible solvent should
be high (log P > 5) to obtain highly efficient extraction and also to maintain a
separation between the extracellular chemicals and the aqueous cytosolic
content so that cell culture can be prevented from being contaminated (Mojaat
et al. 2008). The high partition coefficient prevents irreversible membranous
damages (such as uncontrolled cracks and holes), and thereby extends the cell life
for milking.
The concept of algal milking using this process has been demonstrated to
extract β-carotene from D. salina culture using a biphasic reactor (consisting
of two phases: an aqueous phase and a biocompatible organic solvent phase)
(Hejazi et al. 2002, 2004). In this method, first the cells were grown under normal
growth conditions and then stressed by applying excess light to induce high
β-carotene production. Then a biocompatible organic solvent (dodecane) was
Table 16-3. Extraction Processes for Milking.

388
Process Microorganism Comments References
Biocompatible organic Microalgae 1. Positive effect on growth Zhang et al. (2011, 2013)
solvents (n-hexane 2. Improve lipid production
to n-dodecane)
3. Cells remain alive when hydrophobic
solvents are used
Pulsed electric field (PEF) Microalgae, yeast, 1. High extraction yield Coustets et al. (2013, 2015),
cyanobacteria 2. Not an energy-intensive process Sheng et al. (2011),
Stirke et al. (2014)

3. Cells remain alive, but depends
on PEF parameters
4. Effect of PEF is size dependent
Spontaneous oozing Bacteria, 1. Not an energy-intensive process Liu et al. (2011), Vinayak
cyanobacteria, 2. Cells remain alive because it is et al. (2014)
microalgae a natural mechanism
3. Results in slow oozing of HVM
Resistance-nodulation-cell Escherichia coli 1. Excretion of limonene is increased Dunlop et al. (2011)
division (RND) family 2. Large tripartite protein complex
ATP-binding cassette (ABC) Escherichia coli 1. Import or export molecules Doshi et al. (2013)
protein family (carotenoids, squalene, botryococcene)
or ions across
cell membranes
added, and β-carotene was harvested by continuous recirculation of dodecane

through aqueous phase containing the cells. Because the cells continuously
produce β-carotene, thereby replacing the extracted product by newly produced
molecules, the cells can be reused continuously and do not need to be grown
again. D. salina accumulates β-carotene in oil globules inside the chloroplasts

(Hejazi et al. 2004). The dodecane dissolves into the cell membrane, thus creates
pores in the cell membrane, thereby allowing the secretion of β-carotene out of the
cell. Some globules are extracted from the chloroplast to the space between
chloroplast and cell membrane, and subsequently to the outside of the cell.
β-Carotene extraction efficiency in this system was greater than 55%, and produc-
tivity was 2.45 mg m−2 day−1. In another study, the concept of milking was
demonstrated in the case of Nannochloropsis sp. in which an extensive screening
of biocompatible solvents has been done (Zhang et al. 2011). The selected solvent
(hexadecane) showed enhanced lipid production. Marchal et al. (2013) reported the
biocompatible extraction of 65% β-carotene using ethyl oleate with 5% CH2Cl2.
The mixing efficiency in a biphasic reactor had been improved by cycling
between two phases (An et al. 2004), or using stirred tanks (Martín et al. 2008) and
static mixers (Fang and Lee 2001). However, these methods pose certain problems,
including high mechanical stress because of fluctuating forces in the flow field and
wide solvent droplet size distribution (Zhang et al. 2010). To overcome this
drawback, a novel membrane dispersion technique was developed so that uni-
form-sized (in micrometer) droplets can be produced by dispersing one phase into
another through a microporous membrane (Chen et al. 2004). Development of a
dispersion extractor using a ceramic microfiltration membrane and evaluation of
its performance in a 30% tributylphosphate (TBP)-nitric acid-H2O system have
been done. The major advantage of this technique is that it produces the droplets
of a defined size with a narrow size distribution that increases the contact area
between two phases and thereby shortens the extract time (Chen et al. 2004).
In a study done by Zhang et al. (2013), a poly (ether sulfones) (PES) hollow
fiber microfiltration membrane was used as dispersion medium for lipid extrac-
tion from B. braunii FACHB 357 by using tetradecane as organic solvent
(Figure 16-3). They achieved a high level of lipid yield of 50.15% after 96 h.
Thus, the membrane dispersion medium improves the algal lipid recovery
efficiency in biphasic systems.
16.3.2 Pulsed Electric Field

Electroporation is another technique for lipid extraction from yeast, cyanobac-
teria, diatoms, and other microalgae (Coustets et al. 2013, 2015; Sheng et al. 2011;
Stirke et al. 2014). In this system, two electrodes (anode and cathode) are
connected with an electrical power supply in a PEF treatment chamber, and
then the aqueous (culture) medium is passed between the electrodes by applying a
voltage of 0.5 V to 50 kV. The electrical power is pulsed at a frequency range
of 1 Hz to 50 kHz. Pulsation results in the fracture of the cell wall, thereby
releasing oil content. PEF does not lead to cell flocculation, and hence no cellular
components come out of the cell. The fractured cells then undergo healing, and
Oil containing liquid and

biomass separation
through hollow fiber
Continuous membrane
transfer of oiled
biomass for
milking reactor
Mixture of
water and oil
Algae/Yeast
Milking
cultivation
reactor
reactor
Continuous transfer of milked

biomass to cultivation reactor Water-oil separation
Figure 16-3. Continuous bench scale milking reactor with hollow fiber membrane.
thereby remain viable. Thus, this same microbial batch can be reused for further
HVM extraction (Reep and Green 2012).
Table 16-4 represents recent studies based on PEF for lipid extraction.
Flisar et al. (2014) investigated the effect of PEF for lipid extraction from Chlorella
vulgaris in a continuous flow system. C. vulgaris consists of 50% to 58% lipid
content based on dry biomass weight. In this study, the PEF treatment chamber
was fabricated with stainless steel as electrodes with a gap of 15 mm between them.
They obtained 50% lipid yield (percent by weight), when an electric field strength
of 2.7 kV/cm was applied for 21 pulses in 100 μs. Eing et al. (2013) also used
stainless steel as electrodes in the PEF treatment chamber with a 4 mm gap
between them. Here, an electric field strength of 35 kV/cm was applied on the
Table 16-4. Lipid Extraction from Oleaginous Microbes Using PEF Approach.
Electric field
Lipid (percent strength
Microbial strain by weight) (kV/cm) References
Chlorella vulgaris 50 2.7 Flisar et al. (2014)

Auxenochlorella 22 35 Eing et al. (2013)
protothecoides
Ankistrodesmus 6.1 mg/L 45 Zbinden et al. (2013)
falcatus
Synechocystis 25–75 > 35 kWh/m3 Sheng et al. (2011)
PCC 6803
target sample (Auxenochlorella protothecoides) for 1 μs duration. This resulted in

22% lipid extraction. In another study (Zbinden et al. 2013), lipid was been
extracted from Ankistrodesmus falcatus using this approach. At an electric field
strength of 45 kV/cm for 100 ms, PES resulted in the electroporation of 90% of
algal cells and thus led to 6.1 mg/L lipid yield. Sheng et al. (2011) reported
electroporation of 87% of the cells of Synechocystis PCC 6803 by applying an
electric field strength of 35 kWh/m3 that resulted in 25% to 75% lipid recovery.
Thus, PES has high potential to be used in a large scale because of its low energy
consumption, which makes it economical.
16.3.3 Spontaneous Oozing

Some bacteria, green algae, and genetically engineered cyanobacteria can
secrete lipids from their cytoplasm to the external environment (Liu et al. 2011).
Recently, spontaneous oozing of the oils has been demonstrated in the case of
diatoms. Vinayak et al. (2014) investigated a diatom strain Diadesmis confervaceae
having 14 to 18 μm length and 6 to 7 μm width. Lipid droplets secreted into the
culture medium was observed to lead to the production of 14.6% lipid content
(Vinayak et al. 2014). By the end of the 10th day of cultivation, 50% of the cultured
diatoms oozed out their oil content, but by the end of the month, 80% of the
cultivated cells had oozed out. When subcultured on a fresh medium, the cells
resumed their growth and followed the similar manner of oozing again. The exact
mechanisms for spontaneous oozing are not yet determined, but it can result in
cost reduction in the biofuel production, which is otherwise very expensive
because of the downstream processing cost of current commercialized methods.
16.3.4 Membrane Transport Proteins

New biosynthetic pathways are being developed for HVM production.
In Dunlop et al. (2011), membrane transport proteins were used for biofuel
secretion. In this study, the transporters of resistance-nodulation-cell division
(RND) family were expressed in bacteria (Escherichia coli), which resulted in a
1.5-fold increase in limonene production (Dunlop et al. 2011). However, the large
tripartite protein complexes of these transporters limit their translation to other
economical hosts (algae and yeasts) (Nikaido and Takatsuka 2009). Another
family of transporters, ATP-binding cassette (ABC) protein family, is present in all
five kingdoms of life. ABC transporters can recognize certain biofuel molecules,
thereby leading to their export (Rees et al. 2009). In Doshi et al. (2013), isoprenoid
compounds (zeaxanthin, canthaxanthin, β-carotenoids, lycopene) have been
extracted using an E. coli BL21 model system. These isoprenoid compounds were
selected as model biofuel compounds owing to various reasons: (1) they can be
readily detected and quantified by the colorimetric method; and (2) they resemble
squalene and botryococcene (validated biofuel compounds) in their chemical
composition. They used the biphasic culture system for secretion of hydrophobic
biofuel compounds from E. coli BL21 liquid cultures. Decane was used as the
biocompatible organic solvent in this study. In comparison to the control, the
expression of ABC exporter MsbA in E. coli led to a 4.4-fold increase in
Figure 16-4. Milking effect of ABC transporters.

Note: IM = inner membrane of bacterial cell, OM = outer membrane of bacterial cell,
PS = periplasmic space.
canthaxanthin and β-carotene production. The functioning of bacterial ABC

transporters can be understood by a schematic representation (Figure 16-4).
Figure 16-4 represents that the lipid A is exported from the inner leaflet to the
outer leaflet of the inner membrane (IM) of the bacterial cell by the multidrug
ABC exporter, MsbA in E. coli. Then, different transporters and lipid transfer
proteins come together to move lipid A through periplasmic space (PS) to the outer
membrane (OM) of the cell, which is then collected in the organic phase. Similarly,
biosynthesized carotenoids are exported from the bacterial cells in the same
manner as that of lipid A. Carotenoids and other biofuel molecules are also
accessible to ABC transporter MsbA for secretion. This is how ABC transporters
help in easy extracellular recovery and sustained intracellular production of biofuel
molecules that could eliminate the need for harvest, extraction, and refining.
If this approach is taken to the commercial level, it would add significant value.
16.4 SUMMARY AND FUTURE PROSPECTS
The study of milking of lipids/HVM from oleaginous microorganisms is still in the

phase of its infancy and needs to be taken to the commercial level. To maximize
the benefits of this concept, the economic and environmentally friendly biofuel
production processes must be designed.
However, it is difficult to follow two or more functions simultaneously in
biorefinery without any compromise. One of the solutions can be the continuous
milking of any HVM products and lipids with the oleaginous microbes remaining
in the growth phase. Although the studies demonstrating the milking paradigm
are limited, they are convincing in the concept. However, the mass transfer of the
lipids from oleaginous microbes to their outer space is the area of future
investigation to understand and enhance extraction efficiency of lipids.
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CHAPTER 17
Application of
Nanotechnology in Biodiesel
Production
L. R. Kumar
S. K. Ram
R. D. Tyagi
17.1 INTRODUCTION
Nanotechnology is self-assembly of atoms, molecules, or molecular groups into

nanostructures or nanocomposites aimed at creating materials and devices with
novel properties and functions. This new technology deals with matter of length
between 1 and 100 nm (1 nm = 10−9 m). Hence, nanotechnology focuses on
characterization, fabrication, and manipulation of materials, tools, and products
with structures less than 100 nm. Products made from nanotechnology mainly
comprise nanomaterials (nanoparticles, nanotubes, nanostructured materials, and
nanocomposites), nanotools (nanolithography tools and scanning probe micro-
scopes), and nanodevices (nanosensors and nanoelectronics). These products are
safe, cheap, long-lasting, and better equipped with applications in communication,
agriculture, nutraceutical, and pharmaceutical industry (Moston 2008). According
to BCC Research (Business Communications Company Inc.), global nanotech-
nology market should reach USD 90.5 billion by 2021 from USD 39.2 billion in
2016 at a compound annual growth rate of 18.2% (BCC Research 2019).
Current biodiesel production methods are expensive, energy intensive, and
time-consuming. Burning fuels derived from agricultural and wood feedstocks
poses environmental concerns (such as global warming and greenhouse gas
emissions) and results in depletion of existing resources (e.g., deforestation).
Nanobiotechnology has emerged as one of the most innovative technologies in the
last few decades. Nanomaterials are biocompatible, possess enhanced specific
surface area, and are resistant to breakage through mechanical shear, which make
them suitable for multiple uses, and their application in biodiesel manufacturing is
397
apt. There are potential applications of nanotechnology in bioenergy and

biosensor, which encouraged researchers in recent years to investigate new
nanostructures to build robust nanobiocatalytic systems that can be helpful in
biodiesel manufacturing process. Figure 17-1 displays the process flow chart of
biodiesel production using cultivation of oleaginous microbes along with potential

applications of nanotechnology at specific process steps. Nanotechnology has
application at every process step of biodiesel manufacturing.
This chapter reviews implementation of nanotechnology at various process
steps of biodiesel production, such as the cultivation of microbes, lipid extraction,
purification of hydrocarbons from oil, and transesterification. The chapter also
discusses advantages and disadvantages of using nanotechnology at various
process steps.
17.2 LIPID PRODUCTION USING NANOTECHNOLOGY
Major sources for biodiesel manufacturing are plant oils, animal fats, or lipids
from oleaginous microbes like microalgae. The faster growth rate, higher lipid
content, and tolerance to high CO2 content by microalgae are advantages of
microalgae cultivation for biodiesel production over other technologies. Research-
ers have been developing novel methods for biodiesel production using microalgae
instead of plant oils. Autotrophic algae are heavily light and energy dependent,
with lower efficiency of lipid production. Heterotrophic algae are more flexible
under cultivation conditions, can grow in light-free conditions, and are capable
of producing higher lipid contents in cells (Miao and Wu 2006). It has been
observed that cultivations conditions such as low temperature (lower than 20 °C),
Figure 17-1. Process flow chart for biodiesel manufacturing.

APPLICATION OF NANOTECHNOLOGY IN BIODIESEL PRODUCTION 399
Table 17-1. Lipid/Biomass Productivity under Different Conditions for

Scenedesmus sp.
Temperature (°C) 10 20 25 30
Biomass [g biomass (gP)−1] 194–206 307–320 325–355 334–346

Lipid productivity 60–65 94–130 80–90 70–80
[g lipid (gP)−1]
nutrient scarcity, and high metal concentration have triggered lipid accumulation
(Xin et al. 2011). Table 17-1 reveals the effect of temperature on lipid production.
Nanomaterials were observed to enhance microbial activities in a previous
study in which palladium nanoparticles enhanced catalytic activities of bacteria
(Windt et al. 2005). Adding nanomaterials in heterotrophic algae cultivation
medium can enhance lipid accumulation as a result of the increase in sheer
between cell and nanoparticle. In this particular situation, the cell senses nano-
particle as a foreign object in cultivation medium and considers it as competitor of
nutrients, which forces cells to uptake more nutrients than usual and results in
more lipid formation inside the cells (Zhang et al. 2013). Moreover, studies have
shown that addition of nanoparticles have no negative impact on growth and
activity of living cells (Jin et al. 2005, Williams et al. 2006).
Lipid production in cultivation medium and lipid extraction contribute to
70% of biodiesel production cost. Although lipid accumulation can be enhanced
by appropriate selection of nanoparticles, exact interactions between algae cells
and nanoparticles are not clear and should be investigated for enhancing lipid
accumulation inside cells. Because not much literature is available on lipid
production using nanomaterials, lipid production in heterotrophic cultivation
should be further studied with use of nanoparticles along with other cultivation
parameters like temperature, pH, metal ion concentration, agitation rate, and
carbon-to-nitrogen ratio.
17.3 LIPID EXTRACTION USING NANOTECHNOLOGY
Lipid extraction is one of major steps in deciding the cost of biodiesel production
from microalgae. The commonly used method is solvent extraction, in which
organic solvents such as chloroform, hexane, methanol, or the combinations of the
solvents are used to wash the wet or dried algae biomass repeatedly to obtain
lipids. The extraction yield of lipids varied largely while using different solvents;
methanol and chloroform resulted in 20% w/w lipid yield, but use of hexane
resulted in 15% w/w lipid yield (Vicente et al. 2009). Extraction of lipids can be
facilitated by using ultrasonication, grinding, or a combination of these methods
(Pernet and Tremblay 2003). Because these are energy-intensive techniques, they
can result in increased extraction costs.
The problems with current lipid extraction methods include use of toxic
solvents; energy-intensive methods like sonication, homogenization, bead-mill-
ing, or radiation; expensive solvent-lipid separation step (using distillation); and
low extraction efficiency of lipids. Because nanomaterials are biocompatible,
less toxic, and less energy intensive, their use in lipid extraction is suitable.
Furthermore, nanoparticles can be functionalized with lysozyme or functional
groups that can play critical roles for lipid extraction. Recently, researchers in the
US Department of Energy’s Ames Laboratory at the Iowa State University
developed nanofarming technology to harvest biofuel oils without harming
algae. They used mesoporous nanoparticles with functional groups for dissolving
lipids to extract oil from algae, and the algae could be continuously used for
lipid accumulation (Lin 2009). The advantage of this technology is that extrac-
tion can be accomplished from live cells, which would be used for lipid
accumulation again, and hence, the process avoids recultivation. Another study
was reported using an innovative photo-bioreactor for cultivation of microalgae,
and nanomaterials were synthesized for most efficient combinations for lipid
extraction (Lu 2009).
The first class of nanomaterials was based on the following components:
glycerol, xylitol, sorbitol, among others, and the other class was synthesized based
on imidazole chemistry. Their efficiency was later compared with hexane extrac-
tion. Nanomaterials have potential in cell disruption and lipid extraction. How-
ever, because not much literature is available on lipid extraction from cells, more
efforts should be made to investigate synthesis of functionalized nanomaterials to
assist in cell disruption and dissolving lipids.
17.4 NANOTECHNOLOGY IN SELECTIVE PURIFICATION OF

HYDROCARBONS FROM OIL
A variety of high-value-added hydrocarbons and polyunsaturated free fatty acids

(PUFAs) are produced in the lipid extraction step. These compounds need to be
separated from algal oil because they have several applications in the pharma-
ceutical, cosmetic, and food industries and because they have negative effects on
conversion of oil to biodiesel (Metzger and Largeau 2005). Nanotechnology used
to selectively purify these compounds is described subsequently.
17.4.1 Selective Extraction of Free Fatty Acids from Oil

An example of a high-value hydrocarbon found in algae is free fatty acids (FFAs),
which have a substantial value in the pharmaceutical, cosmetic, and nutraceutical
industries and pose a negative effect on conversion of oil to biodiesel. Biodiesel is
produced from very low FFA percentage (<1%) oils by converting triglycerides
through catalyzed transesterification (Valenstein 2012). Thus, there is a need to
develop technology for separation of algae-produced hydrocarbons from oil.
Current separation methods for organic hydrocarbons like distillation and
extraction using organic solvents or supercritical fluid are time-consuming, energy

intensive, and include use of toxic organic solvents.
Preparation of mesoporous silica nanoparticles (MSNs) has been reported
with high surface area and pore volume for sequestration of fatty acids from
oil (Valenstein 2012). In this study, the surface of the nanoparticle was
functionalized with organic moieties for the sequestration purpose of specific
biomolecules. Impact of pore size on sequestration capacity was observed by
synthesizing two MSNs with different pore sizes: MSN-2.5 (with 2.5 nm pore
size) and MSN-10 (with 10 nm pore size). The appropriate functional group for
sequestration of fatty acids was also determined by mixing MSN-10 (pore size
10 nm) materials with hexane solution of FFAs. Results showed that the
3-aminopropyl group gave a maximum fatty acid absorbance among the
functional groups of benzyls, hexadecyl, 1-propyl-3-methyl imidazolium chlo-
ride, and 3-mercaptopropyl.
Adsorption was performed using palmitic acid (free fatty acid) to investigate
recyclability of functionalized nanoparticles. Adsorption was performed using
unfunctionalized ASN-10 and AP-ASN-10 (silica nanoparticles functionalized by
amino propyl) with analytes commonly found in microalgal oil, palmitic acid
(FFA), glyceryl tristearate (triacylglyceride), squalene (terpene), and ergosterol
(sterol). As expected, the maximum adsorption capacity was displayed by AP-
ASN-10 for palmitic acid which is a FFA. Adsorption using AP-ASN-10 was
performed on commercially available microalgal oil. The ability to reduce the FFA
content of microalgal oil to less than 1% was displayed by AP-MSN-10, making it
more favorable to be used for FFA sequestration.
17.4.2 Purification of Polyunsaturated Free Fatty Acids

Oil substrate for transesterification reaction contains some PUFAs, which are
undesired components for transesterification reaction because the degree of
unsaturation of fatty acid methyl esters (FAMEs) is not important in biodiesel.
Because PUFAs also have applications in cardiovascular disease, cancer, and
arthritis and play a crucial role in brain function, they need to be separated from
substrate oil. Commonly used separation techniques at this stage are supercritical
fluid extraction, selective esterification (Schmitt-Rozieres et al. 2000), and urea
complexation (Pham et al. 2009). These methods have low selectivity for desired
molecules, and some of the adsorbents have low recyclability. Functionalized
MSNs have been reported to separate PUFAs from the mixture having saturated
and monosaturated FFAs (Valenstein 2012). In this study, different functional
groups were investigated to observe their interaction with FFAs. Table 17-2
summarizes the results of using 3 nm pore size MSN to sequestrate different types
of fatty acids. The functional group amine has the best ability to selectively extract
PUFAs from a mixture of FFAs commonly found in algal oil because of relatively
simple synthesis and because it has comparable selectivity to silver dicarboxylate.
MSNs functionalized with amine groups and with pore sizes of 3, 5, and
10 nm were prepared and were tested for selectivity. Table 17-3 summarizes
results of percent sequestration from different pore size MSNs. With decreased
Table 17-2. Sequestration Percent of MSN with Different Functional Groups.
Succinic Silver
Analyte (fatty acid) Naked acid Amine dicarboxylate
Saturated (%) 65 80 29 32
Monounsaturated (%) 73 83 31 30
PUFA (%) 100 100 100 100
Table 17-3. Sequestration Percent from Different Pore-Sized MSN.
Analyte 3 nm 5 nm 10 nm
Saturated FFA (%) 30 95 100

Monosaturated FFA (%) 31 95 100
PUFA (%) 100 83 100
pore size, selectivity increased as PUFA has a smaller hydrodynamic radius

compared with saturated and monosaturated FFA because of unsaturation.
Silica nanoparticles (amino functionalized) were investigated to selectively
adsorb from solution containing saturated, unsaturated, and PUFA similar to
microalgal oil. Nanomaterial functionalized with amine sequestrated 86% of
PUFA, 11% and 28% of saturated and unsaturated FFAs, respectively, which
was very much according to expectations. The technology described previously
exploited use of mesoporous nanomaterials for sequestration of specific biomo-
lecules produced by microorganisms. Results indicated that appropriate selection
of the pore size and functional group of nanoparticles can assist in selective
purification of specific biomolecules from microalgal oil. Incorporating the
aforementioned technology at the industrial scale can be beneficial for the biofuel
industry because it would avoid the use of toxic solvents, prove more economical,
and is less energy intensive.
17.5 NANOTECHNOLOGY IN THE TRANSESTERIFICATION

REACTION
Transesterification is the most critical process step in biodiesel manufacturing. It is

a process for oils derived from animals, plants, or oleaginous microorganisms to
react with alcohol (mainly methanol) for synthesizing FAMEs (biodiesel) and
byproducts such as glycerol (Figure 17-2).
The main concerns for traditional transesterification methods are the long
process time, soap formation in the reaction, lower mass-transfer rates, and lower
Figure 17-2. Reaction displaying lipase-based transesterification.

yields of biodiesel. Base-catalyzed transesterification of vegetable oil with alcohol

usually imparts impurities in the medium by forming excess soap, which takes a
long time to settle, increases the purification period, and steps, and thus, increases
the cost of biodiesel production. Nanotechnology is not only biocompatible but
also safe to use because it does not impart impurities in the reaction medium.
Numerous applications of nanotechnology have been reported at this process step.
Transesterification has been performed using a microreactor having microchan-
nels because transesterification occurs faster in the microreactor owing to reduced
residence time and higher mass and heat-transfer rates owing to the large specific
surface area available in channels of the microreactor. Transesterification can also
be attained through nanoparticle-supported immobilized enzyme because of their
high efficiency, larger specific surface area, high enzyme loading capacity, and
higher mass-transfer rates. Moreover, nanomaterials can be easily recovered from
liquid phase using filtration or centrifugation methods. Transesterification using
nanotechnology also addresses issues of soap formation, reducing further purifi-
cation steps, and cost reduction.
17.5.1 Transesterification in a Microreactor

Individual streams of alcohol (100% NaOH catalyst is dissolved in alcohol) and oil
can react with each other to produce biodiesel along with the byproduct, glycerin,
but it has a long process time. With the aid of nanotechnology, biodiesel
production can become more economical and faster than traditional methods
of production. The latest technology has developed a microreactor smaller than a
thick credit card (Deepak and Bharath 2013). A series of parallel microchannels
about the width of human hair are present in a microreactor. Alcohol and oil feed
are injected into each microchannel where the chemical reaction for conversion of
oil into biodiesel is almost instantaneous at such a small scale (Figure 17-3).
Biodiesel produced in this method is faster than reactors used in large refineries
Figure 17-3. Top view of a microreactor for biodiesel production.

owing to the reduced residence time in a microreactor and higher mass-transfer

rates resulting from the large specific surface area. Thousands of microchannels
can be stacked side-by-side to create a microreactor in the size of a suitcase to
produce biodiesel at the industrial scale. However, no information about reaction
conditions and factors affecting reaction rates and yields has been reported in this
technology.
Another study has been reported for carrying out transesterification reaction
(99% yield) in a microreactor with unrefined rapeseed oil and cottonseed oil as
raw materials (Sun et al. 2008). In this study, reaction conditions and factors
affecting reaction rates and biodiesel yield were also attributed. Reaction mixture
was made by dissolving KOH as catalyst in methanol and oil under stirring. The
reaction mixture was injected into the capillary channels of a microreactor (with
an inner diameter of 0.25 or 0.53 and 3 m in length) at a constant flow rate by a
high pressure liquid chromatography pump. Batch reaction also occurred in shake
flasks with similar conditions, but the residence time in the microreactor was
greatly reduced for high methyl ester yields. The parameters affecting biodiesel
yield in a microreactor were (1) reaction temperature, (2) residence time, (3)
methanol-to-oil ratio, and (4) channel size. Temperature should be controlled at
the boiling point of alcohol for faster reaction rates. Because temperature further
increases saponification starts, higher residence times results in a saponification
problem. Thus, residence time should be controlled precisely in a microreactor.
The methanol-to-oil ratios act as emulsifiers that can cause the glycerol and
biodiesel phase to merge. With a smaller channel size, a larger specific surface area
is available for reaction, resulting in intensified mass transfer and higher biodiesel
yields.
Advantages of the microreactor technology include excellent performance in
liquid-liquid phase reaction, higher mass-transfer and heat-transfer rates, large
surface area, short reaction time, among others, but the high fabrication cost of the
microreactor is a disadvantage. Also, problems of meeting the industrial demand
and quality of biodiesel are foreseen for this technology. More studies should be
conducted to see whether industrial grade biodiesel could be produced with this
technology and whether it can cater to industrial-scale demand for biodiesel
manufacturing. Lack of information available on microreactors should also be
addressed.
17.5.2 Transesterification by Enzyme Immobilization

Enzyme immobilization has been studied in the last few decades, and their
commercial applications have been explored as they are highly efficient.
Table 17-4 summarizes advantages of enzyme immobilization techniques over
free enzyme.
17.5.2.1 Methods of Enzyme Immobilization

Enzyme immobilization can be attained through four major methods
(Figure 17-4) based on binding of enzyme with support structure:
Table 17-4. Characteristics Comparison of Free Enzyme and Immobilized Enzyme.
Characteristics Free enzyme Immobilized enzyme

Price High Low

Efficiency Low High
Reusability and recovery Not possible Possible
Activity Unstable Stable
Tolerance to pH and temperature Low High
Separation from substrate Difficult Easy
Separation from product Difficult Easy
Figure 17-4. Diagrammatic representation of various methods of enzyme

immobilization.
• Physical adsorption: This process is based on Van der Waals forces, ionic and
hydrogen bonding, and hydrophobic interactions, which are very weak forces,
but in large numbers, they impart sufficient binding strength (Rao et al. 1998).
• Covalent binding: Covalent immobilization involves the formation of cova-
lent bonds between the enzyme and the support matrix. The functional
groups present in the enzymes form covalent bonds with matrix because these
functional groups are not responsible for the catalytic activity (D’Souza 1999).
• Entrapment: It is defined as the restricted movement of enzymes in a porous
gel, but keeping them as free molecules in solution (Grosová et al. 2008).
• Cross-linking: This method involves attachment of biocatalysts to each other
by bi- or multifunctional reagents or ligands (Datta et al. 2013).
These enzyme immobilization methods have been performed on lipase.
Table 17-5 compares merits and demerits of various methods to immobilize
enzymes.
Table 17-5. Merits and Demerits of Various Immobilization Techniques.
Method Advantages Disadvantages
Adsorption Can work in mild Weak interactions between

condition; easy to carrier and lipase; enzyme is

perform and sensitive to pH, ionic
economical; carrier strength and temperature,
can be regenerated and so forth; adsorption
for re-use capacity is low
Covalent bond Enzyme is thermally Laborious preparation can
stable make enzyme lose its
activity
Cross-linking Strong interaction Cross-linking conditions are
between lipase intense and mechanical
and carrier strength of immobilized
enzyme is low
Entrapment Fast, economical, and Not effective for high
easy to perform molecular weight substrates
because it has mass transfer
restriction in catalytic
reaction
17.5.2.2 Nanoparticles as Immobilization Support Matrix

Nanoparticles can act as suitable support material in enzyme immobilization
because they provide ideal characteristics for determining biocatalyst efficiency
like specific surface area, mass-transfer resistance, and effective enzyme loading.
It has been reported that enzymes immobilized through nanomaterials have
higher half-lives compared with free enzymes (Wang 2006). Immobilization of
enzymes to the nanoparticles reduces protein unfolding and improves stability
and performance of enzymes. Nanocatalytic systems are highly stable and
efficient, capable of self-targeting, and can function as molecular machines to
catalyze multiple reactions. Table 17-6 summarizes advantages and disadvantages
of nanoparticles as an immobilization matrix (Ahmad and Sardar 2015).
Magnetic nanoparticles overcome the problem of separation of reaction medium
because their responses to the external magnetic field is an additional advantage.
There are four main approaches to linking enzymes to nanoparticles (Figure 17-5):
• Electrostatic adsorption: It is the simplest and widely used approach in which
the interaction can be modulated by pH or changing ionic strength of the
medium. An adsorption method was reported in which colloidal gold was
successfully adsorbed to proteins for indirect detection of specific cell surface
molecules (Geoghegan and Ackerman 1977). The study displayed that protein
immobilization on gold is quite rapid and reproducible because colloidal gold
Table 17-6. Merits and Demerits of Nanoparticles as Enzyme Immobilization

Matrix.
Merits Demerits
High surface area Large scale purification

Mass transfer resistance Cost of fabrication process
High mechanical stress Separation of reaction medium
High enzyme loading capacity
Reduction in diffusional problems
Multiple times use
Figure 17-5. Diagrammatic approaches to linking enzymes to nanoparticles:

(a) electrostatic adsorption; (b) covalent attachment to nanoparticle ligand;
(c) conjugation using specific affinity of protein; and (d) direct conjugation to the
nanoparticle surface.
is inexpensive and adsorption is dependent on protein concentration and

solution pH.
• Covalent attachment to the nanoparticle ligand: Nanoparticle ligand can be
covalently linked to protein. Surface chemistry of nanoparticles plays an
important role in covalent attachment to nanoparticle ligand. A variety of
organic functional groups can be introduced to the nanoparticle surface using
mild conditions (Aubin-Tam and Hamad-Schifferli 2008). For example,
nanoparticles labeled with N-Hydroxy succinimide (NHS) esters can form

covalent bonds with primary amine of lysine on a protein. Oxide nanopar-
ticles can be easily modified using amino groups, which are used as adsorbing
or linking various proteins.
• Conjugation using specific affinity of the protein: Nanoparticle–protein

conjugation can also be achieved by using specific labeling strategies. For
example, antibody-coated nanoparticles can bind selectively to a specific
protein, and streptavidin-coated nanoparticles can bind selectively to biotin-
labeled proteins (Di Marco et al. 2010).
• Direct conjugation to the nanoparticle surface: Ag and Au nanoparticles can
be attached directly to the protein/enzyme through a Ag–S or Au–S bond
after incubation of protein and nanoparticle together. Direct linkages can be
achieved by poly-histidine (His) tags present on protein, which can attach
directly to atoms of nanoparticles Zn, Ni, Cu, Co, Fe, and Mn (Ahmad and
Sardar 2015).
Successful immobilization of Candida antarctica lipase B (Cal B) on polysty-
rene (PS) nanoparticles has been reported (Miletić et al. 2010). PS nanoparticles
were prepared by the nanoprecipitation process. It was found that lipase adsorp-
tion to PS nanoparticles was independent of pH (same enzymatic loading at
different pH). It further indicated that hydrophobic interactions were the driving
force of the adsorption process. Hydrolysis reaction using immobilized lipase was
performed in organic media. When results of the assay were compared with
Novozyme 435 and free enzyme, it was found that activity of the enzyme was
improved on immobilization (Table 17-7).
Enzyme (laccase) immobilization has been achieved on multiwalled carbon
nanotubes (MWCNTs) successfully (Xu et al. 2015). Enzymes chemically or
physically adsorbed on carbon nanotubes maintain their effectiveness. The
structure shown in Figure 17-6 is suitable for enzyme immobilization because
of high tensile strength and bulk modulus of carbon nanotubes.
17.5.2.3 Nano-Biocatalysis-Assisted Biodiesel Production

Enzyme denaturation, enzyme inactivation by substrates/byproducts, high cost of
enzymes, and scale-up at the reactor level are the main concerns in enzyme-
assisted biodiesel production. Magnetic nanobiocatalytic systems are the best
Table 17-7. Hydrolytic Catalyst Activity of Cal-B.
Activity [nmol/(minimum
Type mg catalyst)]
PS nanoparticles 4,400
Novozyme 435 3,750
Crude lipase B 2,400
Figure 17-6. Enzyme immobilization through MWCNTs.
choice because of increased thermostability, reusability, ease of separation from

reaction mixture, and process economics consideration. Packed-bed reactors
overcome the problem of disruption of enzyme carriers by shear stress caused
from stirring (Wang et al. 2011b).
Nanobiocatalytic systems have been successfully used in the transesterification
process for biodiesel production (Figure 17-7). Significant use of nanotechnology
applications has been studied on lipase immobilization. Table 17-8 summarizes
different nanomaterials and their use and effect on conversion rates and reusability
of enzyme/lipase in the transesterification process.
Figure 17-7. Lipase-catalyzed transesterification process in a bioreactor for

Table 17-8. Nanomaterial Application in Enzyme Immobilization during Transesterification.

410
Ratio of Ratio of
initial rate conversion
Remaining to free rate to free
Lipase Source Nano materials activity (%) lipase lipase Reusability References
Candida rugosa Carbon nanotubes 97 2.2–1.4 4.44 — Shah et al. (2007)

Candida rugosa Nanogel 85 — 7.67 — Ge et al. (2009)
Candida rugosa Fe3O4 nanoparticles 80 110 20.5 4 Solanki and
Gupta (2011)
Candida rugosa ZrO2 214 — 3.3 8 Chen et al. (2008)

Candida rugosa γ-Fe3O4 <100 — — — Dyal et al. (2003)
Candida Antarctica Fe3O4 nanoparticles 200 — — 4 Netto et al. (2009)
Candida Antarctica Polystyrene 204 — — — Miletić et al. (2010)
nanoparticles
Pseudomonas cepacia ZrO2 — — 3.6 — Chen et al. (2008)
Thermomyces lanuginosus Nanosized silica 93 — — — Kwon et al. (2007)
Thermomyces lanuginosus Fe3O4 nanoparticles 70 — 1.05 4 Xie and Ma (2009)
Apart from biocatalyst (lipase), nanosized heterogeneous catalysts have also

been used in the transesterification process in biodiesel production (Table 17-9).
Some of the successful examples of nanocatalysis in biodiesel production are
introduced as follows:
• Lipase immobilization by simple and efficient adsorption of Pseudomonas

cepacia lipase on a nanoporous gold support resulted in higher conversion of
biodiesel production (90%) from soybean oil than free lipase (74%) in 24 h
(Wang et al. 2011b). The authors of this paper described catalytic perfor-
mance of enzyme-nanoporous gold (NPG) biocomposite under different
reaction conditions, which separates it from other research papers on enzyme
immobilization. They compared catalytic activities of immobilized lipase with
that of a free lipase in the presence of organic solvents and heat treatment.
They conducted experiments for recovery and reusability of enzyme NPG-
biocomposite and found that immobilized lipase could still produce 80% yield
after reusing 20 times. The authors presented a simple immobilization
method in which the lipase solution was mixed with nanoporous gold, and
the mixture was incubated at temperature of 4 °C, after which the supernatant
was removed to give an enzyme biocomposite. Further, there are several
advantages of the technology over other technologies, such as excellent
performance of enzyme biocomposite, wide range of operational stability,
ease of use, and high recovery and reusability. The approach provides an
efficient strategy for encapsulating functional biomolecules into pores of
nanostructured material. The technology not only would be helpful in
biodiesel production but also holds applications in biosensing, molecular
electronics, and controlled drug delivery.
• Lipase bound on alkyl-grafted silica nanoparticles was reported to give a
high conversion yield for biodiesel production (>95%) at a high water content
and methanol-to-oil ratio (Tran et al. 2012). The authors of this paper
presented two approaches for the transesterification process and compared
their yield. In the first approach (Figure 17-8), microalgal oil extracted
through sonication and use of organic solvents was used for transesterifica-
tion; in the second approach, transesterification was performed directly on
disrupted microalgal biomass without dewatering and oil extraction. Using
the second approach (Figure 17-9), immobilized lipase can give high biodiesel
yield under the conditions of high-water content (71% by weight for algal
biomass) and a high methanol-to-oil ratio (68%). The authors also conducted
experiments to show recyclability of immobilized lipase, which could work
efficiently up to six cycles without much loss in activity. Advantages of this
technology are a good range of operational stability, high recovery, and
reusability. The second approach gives higher biodiesel yields eliminating
solvent extraction strategies. This approach can be implemented at the
industrial scale. However, cost of the complex method of preparation of
alkyl-grafted Fe3O4–SiO2 should be looked on, along with consistency and
uniformity of nanoparticles used in enzyme immobilization.
Table 17-9. Nanocatalyst Applications in Transesterification.

412
Heterogeneous Catalyst-to-oil Reaction Yield

nanocatalyst Oil ratio (% w/w) time (h) (%) References
CaO Poultry fat 0.6 12 99 Venkat Reddy et al. (2006)

CaO Soybean oil 16 6 93.5 Luz Martínez et al. (2010)
Cs2Mg(CO3)2 Butter — 3 100 Montero et al. (2010)
KF/Al2O3 Soybean oil 3 8 99.84 Boz et al. (2009)
KF/CaO-Fe3O4 Plant oil 4 3 95 Hu et al. (2011)
KF/CaO-CaO Rapeseed oil — — 95 Wang et al. (2009)
KF/CaO Tallow seed oil — — 96.8 Wen et al. (2010)

K2O/γ-Al2O3 Rapeseed oil 3 3 94 Han and Guan (2009)
K2CO3/CaO Soybean oil 3 1 99 Zhang et al. (2011)
Li-CaO Karanja and jatropha 5 1 100 Kaur and Ali (2011)
seeds
MgO Soybean oil 2 17 99 Wang and Yang (2007)
MgO Sunflower oil and 1.5 6 90 Verziu et al. (2008)
rapeseed oil
MgO Palm oil 0.5 4 51.3 Yacob et al. (2009)
Zn1.2H0.6PW12O40 Waste cooking oil 2.5 12 97.2 Li et al. (2009)
Figure 17-8. Transesterification after oil extraction with organic solvents.
Figure 17-9. Ultrasonication-assisted in situ evaporation and transesterification

using wet algal biomass.
• A four-packed-bed-reactor system for repeated and highly efficient use of

lipase was designed using an effective nanobiocatalytic system of lipase–Fe3O4
nanoparticle for biodiesel production. Higher conversion rates and stability
were displayed using the four packed-bed reactors than using a single packed-
bed reactor (Figure 17-10). The conversion of biodiesel was maintained at a
high rate of more than 88% for 192 h, and it slightly dropped to 75% after
240 h of reaction (Wang et al. 2011). A simple and effective immobilization
procedure was described with high enzymatic activity and efficient reusability.
Flow rate was optimized to 0.25 mL min−1 because it is an important
parameter in the packed-bed reactor. In a single packed-bed reactor, conver-
sion was 45%, whereas in the four packed-bed reactors, conversion was 88%
in 192 h. The advantages are longer residence time of reaction mixture and
less inhibition of catalyst byproducts in the four packed-bed reactors. An
additional advantage can be efficient recycling of enzyme catalysts, contrib-
uting to low biodiesel production cost. These results have a great potential for
improving the design and operation of large-scale enzymatic systems for
continuous production of biodiesel.
Figure 17-10. A four-packed-bed-reactor system for biodiesel production.
• Thermomyces lanuginosus lipase amino-functionalized nanoparticles have

shown a 90% conversion of biodiesel production from soybean oil and
methanol (Xie and Ma 2009). This paper presented enzymatic transester-
ification of vegetable oil, which offers an alternate source for biodiesel
production. Lipase was covalently bound to amino-functionalized Fe3O4
nanoparticles by the coupling reagent glutaraldehyde. In this work, various
factors affecting lipase immobilization were investigated, such as immobili-
zation time, concentration of glutaraldehyde, ratio of lipase to magnetic
carrier, and temperature. Immobilized enzyme was also tested for pH
tolerance, thermostability, recovery, and reuse. The immobilized enzyme
displayed reusability for four cycles without much decrease in activity.
• A nanomagnetic catalyst KF/CaO–Fe3O4 resulted in more than 95% biodiesel
production under optimal conditions (Hu et al. 2011). In this paper,
nanomagnetic solid catalyst was developed using an impregnation method
in which a Fe3O4 magnetic core and a mixture of MgO, CaO, and SrO was
thoroughly mixed and dipped in aqueous solution of KF with different mass
ranges. The authors also described methods of characterization of nanomag-
netic catalyst with X-ray diffraction and transmission electron microscopy.
The catalyst had a unique porous structure with an average particle diameter
of 50 nm and a ferromagnetic property that was studied by vibrating sampling
magnetometry. The factors affecting biodiesel yield were also investigated,
such as calcination temperature during synthesis of catalyst, KF mass ratio in
catalyst, and mass ratio of water in the reaction mixture. These parameters
were optimized to give more than 95% biodiesel yields in 3 h. According to
experimental studies, the nanomagnetic catalyst was able to be reused up to
14 times without much deterioration in its activity.
• A novel solid acid catalyst, sulfonated-multiwalled carbon nanotubes
(s-MWCNTs), has been used in production of biodiesel with 95% conversion
by weight from methanol and oleic acid at 135 °C in a reaction time of 1.5 h
(Shu et al. 2009). In this work, a new process was described for production of
biodiesel from methanol and oleic acid that coupled reaction and separation.
A fluidized-bed reactor was used for synthesis of MWCNTs, which were
dipped in solution of concentrated H2SO4 to obtain s-MWCNTs. Fourier
transform infrared spectroscopy (FTIR) analysis and scanning electron
microscopy were used for characterization of catalysts, and it was confirmed

that the catalyst had a stable structure, and the structure of MWCNT did not
change after sulfonation. The reaction occurred in a 250 mL autoclave, where
parameters like catalyst:oleic acid mass ratio, methanol:oleic acid molar ratio,
and reaction temperature were optimized to obtain a maximum conversion of
95% by weight in reaction time of 1.5 h. Methanol phase was recycled from
the reaction mixture to separate water produced, which eventually increased
transesterification conversion rates and decreased acidity of the product.
By removing water from the reaction mixture through methanol recycling,
the conversion increased to 99% after 1 h. The catalyst could be easily
recovered, making the process better performing and more environmentally
friendly than a liquid acid catalyst. This technology combines both reaction
and separation, and thus holds a tremendous potential for production of
biodiesel on a large scale.
• Nanocatalysts have been used for production of biodiesel from butchery waste
(Hussain et al. 2011), specifically for production of hydrocarbon gases and
biodiesel. In the first set of experiments, animal fat was heated at high
temperatures with Co and Ni nanoparticles in a furnace to yield vapors of
gases CH4, C2H2 (analyzed by gas chromatography–mass spectrometry),
hydrocarbons, and low-melting point oil. Cracked oil can also be produced
with nanophotocatalyst TiO2 in the presence of sunlight under continuous
stirring, also resulting in hydrocarbons. In the second experiment, cracked oil
was transesterified in the presence of catalyst NaOH to yield biodiesel at 25 °C
and 1 atm (101325 Pa). There are several advantages of this technology over
other technologies, such as biodiesel can be produced from cheap raw
material like butchery waste, waste gases produced in the process are
environmentally friendly because they have low sulfur and nitrogen content,
cheap and reusable catalyst with low energy input can be used in the biodiesel
process, and high-value hydrocarbons are also produced during the process.
• Immobilized Pseudomans cepacia lipase (PCL) on a polyacrylonitrile nano-
fiber (PAN) has displayed efficient biocatalytic activity for biodiesel produc-
tion from soybean oil. The biodiesel conversion rate achieved was 91% of the
original rate even after 10 cycles (Li et al. 2011). In this work, polyacrylonitrile
nanofibrous membrane was fabricated by the electrospinning method and
was immobilized on PCL by an amidination reaction. It was characterized
using FTIR and the enzyme was immobilized in membranes through covalent
bonding. The parameters such as methanol concentration, the methanol-to-
water ratio, and reaction temperature were optimized to give a biodiesel
conversion of 90% after 24 h. The immobilized lipase maintained its
catalytic activity after 10 cycles. In addition, Wang et al. (2009) reported
that immobilized PCL using magnetic Fe3O4 nanoparticle yielded 100%

conversion of biodiesel production.
These aforementioned laboratory-scale studies show that nanomaterials have
tremendous applications at the transesterification stage owing to their high
mechanical strength, reusability, enhanced tolerance to organic solvents, high

enzymatic stability and activity, among other factors. Biodiesel yields reported
from use of nanobiocatalytic systems were high, and waste gases produced during
the process had low sulfur and nitrogen content. Future studies should be
conducted at the pilot scale increase knowledge for large-scale applications of
nanotechnology for biodiesel production and to address challenges associated with
high preparation/fabrication costs, reliability, and the nonuniformity of nano-
particles (Ramsurn and Gupta 2013).
17.6 CONCLUSIONS
Biodiesel manufacturing using traditional approaches is a costly, complex, and

time-consuming process. The growing price of edible oil leads to making biodiesel
production unaffordable. The present feedstock sources (forestry, agricultural, and
aquatic) for fuel production results in depletion of natural resources (e.g.,
deforestation) and can also lead to serious environmental concerns like global
warming and greenhouse gas emissions. Nanotechnology application in biodiesel
production could significantly impact the biodiesel market because nanomaterials
are less energy-demanding, easy to recover, and highly reusable. An appropriate
selection of nanomaterials does not affect the reaction or cultivation medium
adversely. Nanotechnology has potential in lipid accumulation during heterotro-
phic algae cultivation; hence, further research needs to be conducted on nano-
technology in stimulating lipid accumulation during heterotrophic algae cultiva-
tion. Nanotechnology also has application in cell disruption and lipid extraction.
Efforts should be made toward synthesis of functionalized nanomaterials that can
assist in cell breakage and dissolving lipids. Study of functionalized silica nano-
particles in selective extraction of hydrocarbons and polyunsaturated fatty acids
from microalgal oil has been performed at the laboratory scale. Considerable
research has been conducted at the transesterification stage with nanomaterial-
immobilized catalysts because they have advantages over traditional base-
catalyzed transesterification, such as no soapy substance formed (which eliminates
extra purification steps), high reaction rates, and high enzymatic stability, activity,
and reusability. Studies incorporating use of nanotechnology in transesterification
and selective extraction of value-added products from oil (just before the
transesterification step) should be conducted on a large scale. Challenges like
high fabrication cost of nanomaterials, reliability, and nonuniformity of nano-
particles need to be addressed. As nanotechnology application in biodiesel
production develops, biodiesel production using oleaginous microbes will be
more sustainable than the current biodiesel production method.
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CHAPTER 18
Genetic/Metabolic
Engineering and Synthetic

Biology Applications to
Improve Single Cell Oil
Accumulation
S. K. Ram
B. Tiwari
B. Sellamuthu
R. D. Tyagi
18.1 INTRODUCTION
The dire effects of a rapidly increasing global population on the balance between
natural resource usage and regeneration cannot be overemphasized. Almost all
environmental scientists worldwide have arrived at a universal agreement that the
naturally available nonrenewable resources are going to be exhausted at the current
rate of consumption. The increased demands are a consequence of our booming
population and a global trend leaning toward industrialization, which causes the
inevitable need to cater the global population’s energy demands. The bright side of
manufacturing and globalization is the improvement in overall living standard
indexes accounting for every sphere of life (education and health). On the darker side
of this development, the limitations of our natural resources have been neglected.
Our disregard and negligence toward the negative impacts of abusive consumption of
energy resources have led to severe environmental issues like global warming,
pollution, and its consequences. Thus, there is an urgent need to seek renewable
energy resources that are cheaper, easy to produce and environmentally benign.
Biotechnology has been providing an alternate form of renewable fuels,
termed biofuels. The realm of biofuels comprises various fuels like ethanol,
421
hydrogen gas, methane, and hydrocarbons. The biofuels underwent a series of

technical advancements to improve their performance and net utility.
To date, biofuel productions have evolved into four generations. Unlike
previous generations in which plant oil crops (first generation), animal fats and
commercial crops (second generation), and microalgae oil (third generation) were
used as feedstock for fuel production, the fourth generation of biodiesel utilizes
microbial fatty acids and lipids as a substrate for the transesterification process to
generate biodiesel. The fourth generation of biodiesel is considered one of the
most promising biofuels owing to their fuel-like properties and the requirement
for minimal or no change of engineering design for their manufacturing.
Recently, scientists started focusing on developing single cell oil (SCO) produc-
tion using microbes to obtain improved biofuel yield. The biofuel research has been
significantly advanced, but the growth rate of oil-producing (oleaginous) micro-
organisms are insufficient to meet the required amount of feasible and economical
biofuel production. Hence, there is a definite need to improve the oil production
capabilities of oleaginous microbes regarding their growth rate and oil productivity.
Genetic engineering, in which genetic information is transferred from one
organism to another organism to achieve improvement in genotype and pheno-
type of the organism, has been in practice since 1972 (Nicholl 2008). Genetic
engineering is believed to provide advanced and rapid solutions for the current
and future energy requirements. The genetically modified organisms (GMOs)
evidently have higher productivity compared with their wild-type strains. Micro-
bial genome modification is conducted using various tools and methods to achieve
desired genotypic variation. The choice of the gene to be modified depends on
several factors like strain type, methods, and tools available for the genome
modification, along with the availability of complete genome sequencing data.
Indeed, various advantages of genetic engineering of the microorganisms comes
with specific technical challenges that are necessary to be addressed to enhance oil
production. This chapter introduces the required gene editing tools for developing a
genetically modified strain. It also discusses the various strategies adopted to
improve the overall biodiesel production process and the accompanying challenges
in different oleaginous microorganisms including yeast, algae, and bacteria.
18.2 BASIC GENE TRANSFER MECHANISMS
Foreign DNA can be transferred into the targeted host by three processes: (1) gene
transfer via a vector; (2) physical gene transfer; and (3) chemical gene transfer
(Brown 2016).
18.2.1 Gene Transfer via Vectors

The vector is itself a DNA molecule that carries foreign DNA into the host cell and
ensures its proper integration and expression within the cell. The vector possesses
two crucial functions: (1) it prevents the degradation of foreign DNA from the
GENETIC/METABOLIC ENGINEERING AND SYNTHETIC BIOLOGY APPLICATIONS 423
enzyme (nucleases) secreted by the host cell; and (2) it enters the host cell and
replicates and transcribes the large DNA (insert) molecule. For successful gene
transfer, the vector should meet the specific criteria so that it should not evoke the
immune response in the host cell; it should be capable of transcribing and
regulating gene expression. It must be easily cultivable, stable, and should not
require a high cost during commercial production. The commonly used vectors in
genetic engineering for cloning are the plasmid, viral vector, cosmids, phasmids,
and artificial chromosome vectors. Plasmids are extrachromosomal DNA mole-
cules consisting of an origin of replication (Ori), which provides its characteristic
feature of self-replication within the host cell. The host range of plasmid vectors is
based on the ori site of the plasmid. For instance, Col E1 plasmid can replicate only
in enteric bacteria, whereas promiscuous plasmid has a broad host range for
replication in the host cell (Ibraheem et al. 2014, Das et al. 2015). The low
molecular weight, the presence of the selectable marker, ori site, and a single site
for a large number of restriction endonuclease make plasmid an efficient candidate
for transformation.
Viruses are used as vectors or carriers owing to their inherent function of
infecting and expressing their genetic material in the host cell. In viral vectors, the
virulence region of the gene is replaced by targeted foreign DNA sequence, and the
gene sequence region responsible for penetration and transcription in the host cell
is retained. The DNA insert size in viral vectors varies from species to species such
as 40 to 50 kb for herpes simplex virus and 4 to 5 kb in adenovirus (Ibraheem et al.
2014). Plasmid and viral vectors can transform DNA size of up to 50 kb. The
transformation of large fragments of DNA is carried out by the hybrid vectors
such as cosmids and phasmids. They are designed by combining the plasmid DNA
with phage DNA. The artificial chromosome vectors are created in vitro for stable
transformation of large DNA fragments. The insert capacity of artificial chromo-
some vectors ranges from 100 to 300 in the bacterial artificial chromosome (BAC)
and from 100 to 500 kb in yeast artificial chromosome (YAC) (Brown 2016).
These highly sophisticated and sensitive laboratory practices possess a probability
of immunogenic response in the host owing to the presence of the viral DNA
molecule. Hence, the development of physical and chemical gene delivery systems
emerged. For detailed information on gene transfer mechanisms via a vector can
be found in Brown (2016).
18.2.2 Physical Gene Transfer Mechanisms

A physical gene delivery mechanism uses physical force that allows intracellular
gene transfer by increasing permeability of the cell membrane. The various methods
used for a physical gene transfer mechanism are electroporation, microinjection,
hydroporation, gene gun, laser irradiation, sonoporation, magnetofection, and
impalefection. Among these techniques, the commonly used and recent approach
of gene delivery via physical methods is described in detail following.
Electroporation is the method of physical gene transfer in which controlled
electric field is applied for transfection or transformation. The application of
electric field increases the permeability of the cell membrane and facilitates the
uptake of DNA in the host cell. The principle of the electroporation technique is
disruption of phospholipid bilayer of the membrane by the electric field, which
results in the formation of aqueous pores for DNA transfer. The efficiency of gene
transfer in electroporation depends on physical and biological factors such as
electrical pulse duration, electric field intensity, cell size, cell shape, and DNA
concentration. Electroporation is a quick, simple, and low-cost method. However,
it requires extensive laboratory practices for regeneration of transformed cells.
Gene gun or ballistic gene transfer mechanism is a microparticle bombard-
ment technique, first used for transfer of genes in plants. In the gene gun method,
biocompatible inert heavy metal, such as gold or tungsten, is coated with DNA.
The coated carrier then is accelerated by vaporization under high-voltage electric
shock or by helium for gene delivery into the host cell. The gene gun method is
highly efficient in gene delivery by reducing dependence on the characteristic of
the target cell and by providing high-level and long-lasting gene expression.
Sonoporation involves the use of ultrasonic waves that create acoustic cavitation,
or pores, temporarily for gene delivery into the host cell (Ibraheem et al. 2014, Das
et al. 2015). Sonoporation is a noninvasive, safe technique, but its transformation
efficiency is low.
Magnetofection is the technique of gene transfer using magnetic nanopar-
ticles and cationic molecules. In this method, nucleic acids form a complex with a
magnetic nanoparticle (iron oxide) and are targeted to the host cell under the
influence of a magnetic field (Das et al. 2015). Magnetofection keeps the cell
structure intact and provides high transfection efficiency. The impalefection
technique is the outcome of the combination of nanotechnology with synthetic
biology. It involves the use of nanomaterials such as carbon nanotubes, nanofibers,
and nanowires. It consists of the fabrication of a needlelike nanostructure. DNA is
immobilized on the surface of the nanostructure and pressed against cell tissue,
which delivers the gene into the target cell. This technique provides simultaneous
delivery of the gene into many cells at the same time (Das et al. 2015).
18.2.3 Gene Delivery through Chemical Carriers

Chemical methods of gene delivery involve the use of inorganic, synthetic, or
natural biodegradable compounds. The advantages of the chemical gene delivery
system over other methods include safety, ease of preparation, and the ability to
transfer a large-size gene. Cationic lipid and polymers such as natural peptides,
polyethyleneimines, chitosan, dendrimers, polymethacrylates, are commonly
utilized for the gene delivery (Wang et al. 2013).
The electrostatic interaction between cationic lipid and negatively charged
DNA molecule forms a complex referred as lipoplex. The positively charged
lipoplex interacts with glycoprotein and proteoglycans of the cell membrane and
facilitates cellular uptake. The efficiency of lipoplex for gene delivery depends on
the nature of the lipid anchor, linker bondage, the number of charged ions, and an
overall geometric shape. Polyplexes improve transformation efficiency by com-
pressing DNA molecules into relatively small sizes. However, the insolubility
under physiological pH, toxicity, polymer polydispersity, and low transcription
efficiency (low dissociation efficiency of complexes) caused by the polyplexes and

lipoplexes is the major limitation. For the efficient DNA delivery into the host
nucleus, peptide-based chemical carriers are designed and used. The attachment of
short peptide sequence of viral origin to lipoplexes and polyplexes facilitates the
nuclear localization (Ramamoorth and Narvekar 2015).

The transfer of the gene into the host cell via cationic complexes is facilitated by
electrostatic interaction. However, the release of DNA from these compounds is
incomplete, which prevents expression of the gene. Another strategy is encapsula-
tion, which ensures DNA delivery without enzymatic degradation and provides
gene expression with control over DNA release. In the encapsulation process, DNA
is enclosed within the polyelectrolyte capsule or polymeric microparticles. In this
process, various techniques such as layer by layer (LBL), inversion emulsion, and gel
coacervation are utilized for encapsulation. LBL is used for preparing a polyelec-
trolyte capsule. In LBL, DNA is encapsulated within the capsule, which is formed by
the deposition of the polymer via electrostatic, covalent, or hydrogen bonding
(Zelikin et al. 2007). Inversion emulsion technique mainly utilized for delivery of the
therapeutic gene into the human body was discussed by Ibraheem et al. (2014). In
coacervation technique, hydrogels are prepared using chitosan and gelatin and
added to DNA solution, which forms two separate liquid phases because of colloidal
dispersion, and DNA encapsulated nanoparticles are formed. This technique has an
excellent advantage for delivery of a fragile DNA molecule; however, the strong
electrolytic interaction between hydrogels and nucleic acid presents an obstacle in
DNA delivery (Ibraheem et al. 2014).
18.3 ADVANCES IN GENETIC ENGINEERING TOOLS
Every day new tools and technology are developed in molecular biology to manage
and control the biological system by engineering their essential fundamental
functions. Recent techniques broadly used in genetic engineering are discussed
next.
18.3.1 Transformation Techniques

Transformation of the chromosomal genome is one of the most efficient methods to
bring about any desired metabolic alteration in an organism. Microalgae have been
the primary target of many metabolic modifications that researchers have been
studying to improve SCO production. To perform such an alteration, the complete
characterization of the genome is essential. Genomes of only a few microalgae like
Chlamydomonas reinhardtii, Volvox carteri, and Chlorella ellipsoidea have been
completely characterized (Scaife et al. 2015). Organelle genome is a step farther in
the case of genetic manipulation owing to targeting and localization problems of the
site of modification (Boynton et al. 1988, Scaife et al. 2015). A detailed list of
transformation technologies can be found in the literature (Kornberg and Baker
1980, Birch 1997, Balasubramani and Ramesh 2017).
18.3.2 Heterologous Protein Expression

Heterologous nuclear genes have been difficult to express because of localization and
targeting of the gene. Various strategies like codon optimization, the inclusion of
introns, and use of a mutant strain have made overexpression of genes relatively
easier. Recently scientists have demonstrated that heterologous protein can be

successfully expressed with a cotranscriptional system (Donnelly et al. 2001). Kong
et al. (2015) successfully demonstrated the overexpression of heterologous protein
(squalene synthaselike protein) in Chlamydomonas when the protein was linked via a
2A self-cleaving peptide. The 2A peptide method has high potential, and researchers
are now trying to express a series of multiple proteins using the same system.
18.3.3 Site-Specific Genome Editing

Genome editing is a prevalent practice in molecular biology to bring changes in
genes like insertion, mutation, gene silencing, or deletion. Three striking tech-
niques for genome editing exist: zinc finger nucleases (ZFNs), transcription
activator-like effector nucleases (TALENs), and clustered regularly interspaced
short palindromic repeats (CRISPRs) (Gaj et al. 2013).
ZFNs comprise restriction enzymes (DNA-cleaving enzymes) with multiple
zinc finger domains that recognize particular sites in the genome. The ZFNs for each
specifically targeted edit needs to be designed and expressed. Recently, ZFNs were
used to alter C. reinhardtii channel rhodopsin genes (COP3) that were present in the
intragenic spaces of the selectable marker gene. The study demonstrated that ZFNs
could selectively edit the genome. The successfully edited transformant displayed
the expression of the functional marker gene (Sizova et al. 2013). ZFNs solve, to a
great extent, the problem of specificity and targeting a gene in the large genome.
Similar to ZFNs, TALENs are also nucleases containing two domains. One
domain is for specific DNA site recognition and binding, whereas the second one
is for nuclease activity. TALENs are used to perform nonhomologous end joining
and high-efficiency homologous recombination to alter the gene. TALENs are
very frequently used with microalgae and diatoms for enhancing lipid production
(Gao et al. 2014).
Contrary to the two aforementioned techniques, CRISPR is relatively easy to
target the genome. It uses a Cas9 endonuclease to edit the gene. The targeting is
achieved by guide RNA, which is perfectly homologous to the target sequence.
Unlike ZFNs or TALENs, one single enzyme can be used with many targets, and it
need not be redesigned every time. This technology is relatively new and has been
widely applied (Jiang et al. 2014).
18.4 MULTIPLE KNOWLEDGE NEEDED FOR PLANNING THE

GENETIC ENGINEERING
To achieve high lipid accumulation, researchers propose to incorporate, delete,

suppress, overexpress, and replace various responsible genes related to lipid
metabolism. The strategy to genetically modify the given microorganisms requires

a complete knowledge of the microbial metabolism and all the interlinking factors.
The choice of the desired gene to be altered is essential knowledge. Using advanced
molecular biology, technology researchers have successfully revealed the under-
lining mechanism of lipid production in various representative microorganisms

like microalgae (autotropic microbe) and yeast (heterotopic microbes). The
details of the genes and enzymes involved in lipid metabolism are illustrated in
Figure 18-1.
The mechanism of lipid production is genotypically and biochemically
different for the autotrophic and heterotrophic microorganism. Therefore,
depending on the biochemical pathways, different strategies are developed for
improving lipid production of the microorganism. There are many technical
shortcomings and knowledge gaps that challenge genetic engineering develop-
ment. Knowledge from various domains can help to find an optimal and efficient
engineering strategy to produce the desired strain.
18.4.1 Genomic, Proteomic, and Transcriptome Profiles

Redox balance of a cell is a crucial factor. Prior knowledge of redox balance of the
cell as a consequence of the intended genetic engineering can help in determining
Figure 18-1. Metabolic pathways and genes involved in lipid production in a

typical microalga.
the feasibility of genetic engineering modifications. High redox balance is in

general required for the production of energy-containing (fuel) biomolecules like
ethanol or lipids, which are relatively reduced products. To increase the yield of
the fuel molecules, cofactor alteration has been considered as one of the vital tools
in metabolic engineering. Researchers have overexpressed NAD+-dependent Fdh

gene, which doubled the nicotinamide adenine dinucleotide (NADH) yields from
2 to 4 mol/glucose consumed and ultimately resulted in high biomass density
(Berríos-Rivera et al. 2002).
To understand the complete regulatory mechanism, one must consider the
metabolism, genetic regulations, and the signaling networks involved in microbial
oil production. With currently available advanced sequencing tools it has been
much easier than before to have a fully characterized and well-annotated microbial
genome profiling. A complete genome profile of an organism allows a more
comprehensive understanding of functional genes and regulatory genes in the
genome, which can assist in planning for the gene insertion, gene deletion, or any
other alteration to up-regulate or down-regulate the genotypic trait to have the
desired metabolic phenotype. In fact, a complete genome profile can be used to
compare the genome with other strains having the desired phenotype, which will
further assist in the genetic engineering development (Lee et al. 2005).
Transcriptome profiling using DNA microarrays allows a genome-wide exam-
ination of mRNA expression levels that can vary with genetic and environmental
conditions. Similarly, proteome profiling of the microbe permits knowledge expres-
sions of every protein encoded in the genome. This information is more meaningful
than having the genome profiling because it represents the metabolic state of the
organism. Proteome profiling is often done using two-dimensional gel electropho-
resis. Genes present in two different strains can be expressed (translated) at two
different levels, causing them to show two different phenotypic behaviors. Thus,
proteomic profiling allows better understand and development of a better genetic
engineering strategy to obtain the desired phenotypic trait in the strain.
18.4.2 Metabolomics and Silico Models

The collective profiles of genome, transcriptome, and proteome allowed research-
ers to chalk out the complete transcriptional genetic architecture of various strains
(Cho et al. 2010). These algorithmically integrated pieces of information are
incorporated into various silico metabolic models and network designs (Gowen
and Fong 2010). All the individual metabolites studied together fall within the
realms of metabolomics. In metabolomics, the substrates, products, intermediates,
and the byproducts of metabolic reactions can be profiled. The metabolic profile of
a strain represents the metabolic status of the cell and thus can elucidate the cause-
effect relationship between genetic and environmental changes on the metabolic
responses of the cell. Fluxomics (studies on metabolic flux) are considered to be
the most reliable phenotypic responses of a cell, which are closely related to the
overall performance of the cell to perform the desired metabolism (like lipid
production). Fluxomics are performed for a cell using advanced C13 isotope-
labeling techniques. Computational models and algorithmic optimizations can be
coupled with fluxomics data to predict substrate uptake and product production
rates. This modeling has to take account of various mass balances, side reactions,
and metabolite conversion yield factors, conversion rates, and reversible equilib-
rium constants. Silico genome-scale models make it easier to devise a strategy to
engineer the strain for desired metabolite production [Figure 18-2(a)]. Genome-
scale silico profiling and modeling have been done only in a few microbes (Duarte
et al. 2007).
Various silico genome-wide metabolic models represent every possible
metabolic state of the cell. To have meaningful use of these models, it is necessary
to incorporate all the necessary cell regulatory mechanisms and constraints to
narrow down the model solutions (Sauer 2006). Information like cell normal
physiological limits, thermodynamics, regulatory controls at transcription and
translation levels, and metabolic constraints incorporation into the models
requires real laboratory experimental data (Henry et al. 2007, Covert et al.
2008). These data are essential to simulate and strengthen the model to make
it more accurate (Park et al. 2010). There are some models like MOMA
Figure 18-2. (a) Different knowledge domains required to decide the target gene to
perform the genetic modification for enhanced production of lipid; (b) different
strategies applied by researchers for enhancement of lipid production; and (c)
objectives of performing genetic engineering in oleaginous microorganisms in the
ambit of oil production.
(minimization of metabolic adjustment), OptKnock (gene knockout algorithm for

metabolite overproduction), OptReg (algorithm for gene regulation), OptForce
(algorithm for identifying gene manipulation for overexpression of target product),
and FSEOF (gene target selection framework) (Choi et al. 2010) available
for description of the metabolic networks of the various organisms. However,

in the realms of lipid production, many high-lipid-producing microbes remain
unraveled.
18.5 GENETIC ENGINEERING STRATEGIES FOR LIPID PRODUCTION
Researchers have been trying to exploit the tools of genome editing to develop
strains for maximum SCO production since the beginning of the 1990s. The first
genetic engineering attempt for increasing fatty acid production in microalgae was
made in 1996 on Cyclotella cryptica (Gimpel et al. 2015). Acetyl-CoA carboxylase
(ACCase) gene synthesizes ACCase protein (enzyme), which converts acetyl-CoA
into malonyl-CoA as the first committed step in fatty acid (FA) synthesis. A two-
to threefold increase in ACCase activity was observed, but there was no increase in
fatty acid content. As discussed, to have a positive outcome with increasing lipids
and fatty acid synthesis, a more holistic approach to genetic engineering was
required to develop feasible strategies [Figure 18-2(b)]. Recently, researchers
working in genetic engineering for SCO production have developed several
strategies (Table 18-1) to enhance the lipid titers. These studies are briefly
classified subsequently.
18.5.1 Blocking the Competing Pathways

The most obvious strategy to increase the metabolic carbon flux for lipid synthesis
is to prevent sharing of the carbon flux from the cytosolic carbon pool. There are
many competing metabolic pathways that either use substrates or product of lipids
(or fatty acid) synthesis pathways like beta-oxidation and central metabolism.
Mostly the competing flux entry or product consumption is prevented by deleting
the key enzymes in the metabolic network (Chen et al. 2014). To prevent product
(FA) consumption, removal of enzymes involved in beta-oxidation may lead to
penultimate platform molecules (Valle-Rodríguez et al. 2014) for other metabolite
production like polyhydroxyalkanoate (PHA).
Almost all the carbon source utilized as carbon feedstock ends up as acetyl-
CoA in the common cytosolic pool of the microbe. This acetyl-CoA is the familiar
starting point for diverse metabolic activities like the tricarboxylic acid (TCA)
cycle, acetate secretion, or biosynthesis of lipid and amino acids. Deletion of
enzymes for acetate formation and other fermentation pathways has shown a
positive impact on common acetyl-CoA pool (Goh et al. 2014), which in turn
increases the subsequent metabolite production. For example, Chen et al. (2010)
showed that by overexpressing the two acetyl-CoA synthases, ACS1 and ACS2, the
intracellular acetyl-CoA increased two- and fivefold, respectively.
Table 18-1. Recent Studies to Engineer Oleaginous Organisms to Produce Lipids.

Transformation
Host strain Transgene source Gene Strategy method Lipid (%) Result Remark References
Yarrowia lipolytica Escherichia coli, UdhA, PntAB, GAP- Redox Balance Plasmid construct 67 25% Lipid Highest Qiao et al. (2017)
(mutant) Clostridium dehydrogenase, electroporation enhancement productivity
acetobutylicum, malic enzyme 1.3 g/L/h
Mucor
circinelloides
Phaeodactylum Haematococcus Oil globule Light inducible Biolistic 30 17% Improvement Localization of lipid Shemesh et al.
tricornutum pluvialis protein-GFP, promoter bombardment: droplets (2016)
DGAT1(N- gold
limitation
inducible)
Cornybacterium N/Ap atf1, atf2 (dGAT); Delete lipases Electroporation 17.8 3.7fold improvement Increase of acetyl- Plassmeier et al.
glutamicum pgpBtadA genes; delete in lipid content CoA pool and (2016)
(plasmid transcription removal of
construct) regulator byproduct
(delete fasR formation
gene);
overexpress
fadD and tesA;
remove
byproduct
formation
genes
Fistulifera solaris Indigenous Frustulin-GFP Cell surface Biolistic: He particle — — Cell harvesting Maeda et al. (2016)
expression
Trichosporon 3GP-Dehydro- Isochrysis galbana, Heterologous A. tumefaciens 57 Modified FFA Carbon source Görner et al. (2016)
oleaginosus genase, Fusarium expression of composition in type affects the
ATCC 20509 elongase moniliforme, elongases, and proved in novel FFA
IgASE2, Propioni- Fm1 FFA composition of
GENETIC/METABOLIC ENGINEERING AND SYNTHETIC BIOLOGY APPLICATIONS
desaturase, bacterium desaturase mutant

Fm1, and acnes expression
isomerase
(Continued)
431
Table 18-1. Recent Studies to Engineer Oleaginous Organisms to Produce Lipids. (Continued)
432
Transformation
Chlamydomonas GPAT L. incisa Overexpression Glass bead method — 50% Increase in TAG Elevated oleic acid Iskandarov et al.
reinhardtii (2016)
Nannochloropsis ▴12 saturase Endogenous genes Overexpression of Electroporation Increased LC-PUFA biosynthesis N starvation Kaye et al. (2015)
oceanica (NoD12) the gene production of alteration induced
PUFA promoter
P. tricornutum PNPLA3 P. tricornutum Overexpression Electroporation 0.41 g/L 70% Increase neutral Elevated C20:4 by Wang et al. (2015)
lipid disease
causing gene
Chlorella NADH kinase Arabidopsis Overexpression Electroporation — 110.4% Increase in No effect on Fan et al. (2015)
pyrenoidosa thaliana lipid content growth
Y. lipolytica Succinate — — Electroporation 86.9% lipid, 45 g/L 55% Lipid 0.56 g/L/h Liu et al. (2015)
semialdehyde biomass improvement productivity

dehydrogenase
deletion
Chlamydomonas Diacylglycerol Brassica napus Heterologous Electroporation 18.76% 12% Increase linolenic — Ahmad et al. (2015)
reinhardtii acyltransferase expression acid
(bndgat2)
Saccharomyces Betaoxidation E. coli Multiple — — mCFAEEs production Platform organism Lian and Zhao
cerevisiae enzymes heterologous for fuel (2014)
genes production
expressed
S. cerevisiae ACC1, FAS1, FAS2 Indigenous Replace promoters LiAc/SS DNA carrier 17.3% 302% Improvement DGA in high copy Runguphan and
BY4742 and couple number Keasling (2014)
with DGA1 plasmid
overexpression
S. cerevisiae ACOT5 acyl-CoA Mus musculus Replace the acetyl- electroporation 500 μg/L 6.43-fold higher Overproduction of Chen et al. (2014)
(double thioesterase CoA (extracellular) extracellular FFA FFA was
deletion synthetase 42% UFA demonstrated
mutant) were deleted.
And Acyl-CoA
thioesterase
was encoded
C. reinhardti CrFAB2- stearoyl Indigenous Overexpression of Glass bead method 28% improved 2.4-fold increase in Effect of Fab2 on Hwangbo et al.
acyl carrier the gene fatty acid Oleic acid fatty acid (2014)
protein content production
desaturase was studied
C. reinhardtii PEPC 1; — Gene silencing by — — 20% Improved TAG PEPC1 gene had Deng et al. (2014)
Phosphoenol- RNA level negative effect
pyyruvate interference
carboxylase
isoform 1
C. reinhardtii DOF type — Overexpression Agrobacterium — Twofold increase in — Ibáñez-Salazar
inscription TAG et al. (2014)
factor
P. tricornutum DGAT2 Endogenous genes Overexpression electroporation 35% increase in 76.2% EPA increase 6.5-fold increase in Niu et al. (2013)
neutral lipid m-RNA
content
C. reinhardtii Citrate synthase (Cr — Gene silencing by — — 169.5% Increase in TAG CrCIS gene Deng et al. (2013)
CIS) RNA decrease lipid
interference
E. coli U. californica,M. BTE, FadD, MAACR Production of — — 1.6 g/L Fatty alcohol Yields were Youngquist et al.
aquaeolei VT8 primary with yield of over improved by (2013)
alcohols with 0.13 g balancing
chain lengths expression
of 12 to 14 levels of each
carbons gene
Thalassiosira Indigenous Thaps3_264297 Knockdown of lipid — — 4.1- and 3.2-fold higher Trentacoste et al.
pseudonana catabolism lipid content (2013)
strains 1A6 and
1B1
S. cerevisiae Indigenous idh1 and idh2 Gene disruption — — 92% Increase in C16:1 Overexpression of Tang et al. (2013)
strategy and 77% increase ATP-citrate
in C18:1 lyase
Y. lipolytica Indigenous ACC1 and DGA1 Intron-enhanced — 61.7% lipid content Fourfold increase in — Tai and
co- lipid production Stephanopoulos
GENETIC/METABOLIC ENGINEERING AND SYNTHETIC BIOLOGY APPLICATIONS
overexpression (2013)
Crematogaster S. cerevisiae INVSC1, G3PDH, GPAT, Multiple-gene — — Twofold increase in — Hsieh et al. (2012)
minutissima Y. lipolytica LPAAT, PAP, transfer storage lipid
UTEX 2219 DGAT, PDAT, approach content
(Continued)
433
Table 18-1. Recent Studies to Engineer Oleaginous Organisms to Produce Lipids. (Continued)
434
Transformation
Synechocystis sp. Indigenous tesA — — — Fatty acid secretion Fatty acid Liu et al. (2011)
PCC6803 strain yield increase to uncoupling
SD100 197 ± 14 mg/L strategy was
performed
P. tricornutum Thioesterase C14- Cinnamomum Heterologous Biolistic 8.1% (mol/mol) Short-chain fatty acids Increased levels of Radakovits et al.
TE, C12-TE camphora, expression of transformation Lauric acid improved myristic and (2011)
Umbellularia thioesterases increase Lauric acid
californica
E. coli Indigenous accA, accB, accC, Overproduction of P1 phage- — Sixfold increase in the — Davis et al. (2000)
and accD the enzyme mediated rate of fatty acid
Acetyl-coA transduction synthesis.
carboxylase
Starch is also one of the energy storage molecules produced by microalgae.

Many studies have confirmed that blocking starch synthesis provides an excess
diversion of precursor intermediates for lipid accumulation. An increase in the
triacylglycerol (TAG) was reported after deletion of glucose-phosphate adenylyl
transferase (AGPase) gene. Similarly, many starchless mutants show elevated lipid
production (Ramazanov and Ramazanov 2006).
Lipid molecule gets consumed by the competing beta-oxidation pathway. The
pathway involved many lipase enzymes in degrading lipid molecules. In an
experiment (Trentacoste et al. 2013), an antisense construct was prepared and
used to knock down multiple lipase enzyme units in the diatom Thalassiosira
pseudonana. The modified strain resulted in a 3.3-fold higher lipid content than in
wild type.
It is assumed that removing all the competing pathways and overexpression
of lipid production genes will enhance the lipid production, but it will not be
irrational to expect that these modifications might impede the cell proliferation.
Lipid production under nutrient stress conditions might result in lower biomass.
This problem can be resolved by using inducible promoters for the free fatty acid
genes. After the proliferation of biomass, these gene expressions can be elicited by
providing the induction element. A few recent studies have already demonstrated
the feasibility of this technique in C. reinhardtii (Quinn and Merchant 1995).
18.5.2 Deregulation of Fatty Acid Synthesis

Various metabolic pathways in a cell are controlled and regulated by various
mechanisms like feedback inhibition and feed-forward inhibition, in which the
product or reactants (signal molecule) will act on the functional sites of the
enzymes involved in the critical step (irreversible, slow step, or regulatory step) of
the biochemical pathway. Manipulations with vital regulatory enzymes inhibitions
have led to significant improvement in oil production.
Because each microorganism differs regarding its regulatory mechanism, one
genetic manipulation strategy might not work for every microorganism. For
example, in some cases, overexpression of regulatory proteins leads to a higher flux
of fatty acids. High fatty acid flux was due to the consequent increase in fatty acid
synthesis enzyme levels, whereas overexpression of FadR in Escherichia coli leads
to increased levels of FabB, FabF, FabH, FabI, and AccAD enzymes in addition to
fatty acids (Goh et al. 2014). Again, the microbial regulatory system responding to
each genetic manipulation remains significant impedance to designing strategies
for overproduction of fatty acids. In various oleaginous organisms, ACCase is
considered as a rate-limiting enzyme, but overexpression of ACCase would yield
different results. In E. coli, overexpression of ACC enzyme resulted in a sixfold
increase in the rate of fatty acid synthesis (Davis et al. 2000). In Yarrowia lipolytica
the increase was just twofold (Tai and Stephanopoulos 2013). On the other hand,
no improvement in fatty acid synthesis was observed by overexpression of ACC1
enzyme from a plasmid.
Fatty acid synthesis (FAS) is tightly regulated at both transcriptional and
translational levels in oleaginous microorganisms. There exist many yeast
transcription factors (like RAP1, ABF1, and REB1) that could activate the
expression (Schüller et al. 1994). Two transcriptional factors SNF1 protein and
regulators of INO1 have been studied in detail because they down-regulate ACC
enzyme (Feng et al. 2015). Simple overexpression of ACC1 strategies has failed
because ACC enzyme is also regulated at the posttranslational level. SNF1 is one
such regulator that represses ACC1 enzyme and simultaneously stimulates fatty
acid oxidation. Alteration in the SNF1 gene increases ACC1 enzyme activity
threefold, resulting in higher titers of malonyl-CoA (Shi et al. 2014).
18.5.3 Optimization of Enzyme Expression Levels

Protein (or enzyme) synthesis in a cell demands high energy resource. An
optimized and balanced overexpression (regulation) of enzymes can only facilitate
the desired enhancement in fatty acid production. Synthetic biology provides
various manipulation tools to control gene expressions like variation in DNA copy
number, promoter strength, inducible promoters (induction agents), and transla-
tion initiation rate. Often the maximum fatty acid production levels are related to
intermediate values of this parameter, and any deviation leads to decrease in
productivity. For example, intermediate level of acyl-CoA synthetase expression
by replacement of stronger promoter, PTRC (45× higher mRNA titer) gave higher
productivities of dodecanoic acids compared with plasmid mode overexpression
(giving 1,000× higher mRNA titer) and wild-type strain (Youngquist et al. 2013).
Thus, even when the type of genetic engineering is chosen, the method of
executing the strategy (like overexpression of a gene) is important to have the
optimum level of gene expressions.
18.5.4 Balancing Cofactor Generation and Utilization

Electrons or energy in a cell is carried over from one part to another part by the
help of cofactors like NADH, nicotinamide adenine dinucleotide phosphate
(NADPH), and flavin adenine dinucleotide (FADH). A sophisticated balance is
maintained by the cell to manage all the necessary metabolic reactions at the
required rate. NADPH is required explicitly in anabolic reactions like the
synthesis of various biomolecules (fatty acids, DNA, proteins), whereas NADH
is required more often in catabolic reactions to obtain energy. Typically, a cell
has more catabolic reactions than anabolic reactions, resulting in more NADH
and causing an imbalance in NADH/NADPH. Researchers use genome-level
metabolic models to identify genetic manipulations to force carbon flux via the
Entner-Doudoroff (ED) pathway and generate required levels of NADPH to
favor fatty acid synthesis (Ranganathan et al. 2012). To further enhance
NADPH, glyceraldehyde-3P dehydrogenase (GAPN) enzyme, which converts
glyceraldehyde-3P and NADP+ into NADPH and 3-phosphoglycerate was
heterologously expressed, which improved the NADPH availability (Guo et al.
2011). Researchers have also explored the possibility of enhancing NADPH by
overexpressing NADP-dependent malic enzyme. The malic enzyme converts
merely malate and NADP+ into pyruvate and NADPH. A marginal increase of
14% in final fatty acid titer was observed (Runguphan and Keasling 2014). A very
recent study developed 13 strains of Y. lipolytica having synthetic pathways to
convert NADH obtained from substrate catabolism to NADPH and acetyl-CoA.
The best-engineered strain showed 25% improvement in lipid production
over all the previously developed mutant strains so far with lipid productivity
of 1.2 g/L-h (Qiao et al. 2017).
18.5.5 Availing Excess Precursors for Fatty Acid Synthesis

In many oleaginous yeasts, like Saccharomyces cerevisiae, the fatty acid synthesis
takes place in the cytoplasm (majorly) and mitochondria. Acetyl-CoA is the
primary molecule for fatty acid synthesis. Acetyl-CoA is produced in various
compartments like nucleus, mitochondria, cytosol, and peroxisomes. Because
acetyl-CoA is mostly used in central metabolism, the available acetyl-CoA pool is
limited in the cytosol. To increase the acetyl-CoA pool in the cytosol, it is essential
to impair the central metabolism by genetic engineering methods to direct the flux
to fatty acids synthesis. It has been found that deleting major (ADH) dehydro-
genases results in a twofold increase in acetyl-CoA (Li et al. 2014 Lian and Zhao
2015). Inactivating competing pathways like glyoxylate shunts, which consume
cytosolic acetyl-CoA, revealed an increase in the precursor levels for fatty acid
synthesis. With the similar approach, Ma et al. (2014) used an antisense codon and
silenced pyruvate carboxylase kinase (PDK) enzyme. PDK enzyme deactivates
pyruvate dehydrogenase enzyme (PDH) that facilitates the conversion of pyruvate
into acetyl-CoA. The deletion of PDK led to an increase in the flux of acetyl-CoA
and thus increase in lipid content by 82%.
Malonyl-CoA is another essential precursor for fatty acid synthesis. ACC
converts acetyl-CoA into malonyl-CoA. The simplest approach is to overexpress
the ACC and FAS genes to increase fatty acid production. Significant improve-
ment in fatty acids titer has been observed when FAS and ACC enzymes were
overexpressed in S. cerevisiae (Runguphan and Keasling 2014). Malonyl-CoA can
also be synthesized from malonate by malonyl-CoA synthetase (MCS) enzyme.
Researchers have expressed heterologous genes (from plants) in S. cerevisiae to
obtain 2.4-fold improvements in lipid production (Wang et al. 2014).
18.6 GENETIC ENGINEERING IN ALGAE
Autotrophic microalgae and many other autotropic prokaryotes can harness

the light energy and carbon dioxide to produce various valuable metabolites
(Figure 18-3). Microalgae have been exploited as a promising source of feedstock
oil production for third-generation biofuels because of their rapid growth kinetics
and high photosynthetic efficiency (Blatti et al. 2013). In the past, microalgae have
been a significant source of interest for biodiesel feedstock oil production; thus,
they deserve to be discussed separately. Most of the microalgae have been reported
to produce lipids under stressed conditions, which can hinder the average growth
Figure 18-3. Autotrophic microalgae metabolic pathways for biofuel production.
and biomass production of the algae. Metabolic engineering approaches have been
applied to microalgae to improve the lipid productivity and yield. There are
various aspects of microalgae that have been the primary concern for their
metabolic engineering.
18.6.1 Genome Characterization

The complete genome sequence of the microalgae is available from genbank
databases for academic (research) and industrial purposes. Many nuclear
genome projects have been completed recently for various microalgae, such as
Chlamydomonas reinhardtii, Phaeodactylum tricornutum, Thalassiosira pseudo-
nana, Cyanidioschyzon merolae, Ostreococcus lucimarinus, Ostreococcus tauri, and
Micromonas pusilla (Radakovits et al. 2010). Currently, there are numerous
ongoing microalgae genome sequencing projects, including Fragilariopsis
cylindrus, Pseudo-nitzschia, Thalassiosira rotula, Botryococcus braunii, Chlorella
vulgaris, Dunaliella salina, Micromonas pusilla, Galdieria sulphuraria, Porphyra
purpurea, Volvox carteri, and Aureococcus anophagefferens (Liolios et al. 2008).
The sequencing projects generate valuable genome profiles to further advance the
know-how of genetic engineering the microalgae for lipid production.
18.6.2 Transformation
To date, more than 30 microalgae have been successfully transformed for
overproduction of microbial oil, resulting in stable expression of the transgenes
originated from the nucleus or the plasmid. Transformation methods developed
using C. reinhardtii designated that proper codon usage, strong endogenous

promoters, and the inclusion of intron sequences could radically improve the
stability of the transformants. The efficiency of the transformation is one of the
essential factors, which differs from species to species. Therefore, the transforma-
tion methods should be chosen carefully. Transformation in microalgae was
conducted differently including agitation in the presence of glass beads or carbide
whiskers (Te and Miller 1998), electroporation (Maruyama et al. 1994), biolistic
microparticles bombardment (Zaslavskaia et al. 2001), and Agrobacterium tume-
faciens-mediated gene transfer (Kumar et al. 2004).
The selection of transformants (modified strain) from wild type can be
done using selection markers (antibiotic resistance or fluorescence genes).
Genes with different antibiotic resistances are used, including bleomycin, specti-
nomycin, streptomycin, paromomycin, nourseothricin, G418, hygromycin, and
chloramphenicol. Biochemical or fluorescence markers include luciferase,
β-glucuronidase, β-galactosidase and green fluorescent protein (GFP).
Gene expression in the cell organelle was a significant challenge in genetic
engineering. Many studies attempted gene expressions in the chloroplast of the
microalgae with a low transformation efficiency. Genes targeted to nuclear
transformation often resulted in random integration. Homologous recombination
methods have been used to alter the organelle genome (Minoda et al. 2004).
Another successful way to modify the genome is the RNA-silencing method,
which can knock down gene expressions (Moellering and Benning 2010). Recent-
ly, artificial microRNA has been developed to transform C. reinhardtii, which were
found to be more stable and specific than those conventional transformation
methods (Molnar et al. 2009).
18.6.3 Photo-Inhibition
Autotrophic microalgae have light harvesting complexes (LHC) to absorb maxi-
mum light possible as an adaptation to deep aquatic environments where light
penetration is weak (Figure 18-3). These algae, when grown in artificial conditions,
receive excess light energy than required. This excess light is eventually dissipated
and causes photodamage to the cell. Excess residual light generates reactive
oxidants that inhibit the growth of algae (photo-inhibition). To overcome the
photo-inhibition problem, researchers used RNAi construct to silence the LHC
proteins isoforms in C. reinhardtii (Mussgnug et al. 2007). As a result, the cells
have 0.1% to 26% of LHC mRNA compared with wild type. These cells grew faster
under high light conditions without any photo-inhibition, resulting in high
growth rate, although the cell concentration remained unchanged.
Light also damages the multiprotein complex Photosystem II (PS-II). Nota-
bly, the D1 subunit of PS-II is mostly affected. Rea et al. (2011) used error-prone
polymerase chain reaction (PCR) to mutate the D1 protein. The resulting strains
performed worse under laboratory light conditions compared with the control.
The reason for failure for the strategy could not be deciphered. However, Gimpel
and Mayfield (2013) used advanced metabolic engineering tools and showed that
heterologous genes are not the best way to engineer microalgae, but it is feasible
when heterologous genes are accompanied by endogenous regulatory regions.
Using this approach when D1 genes from Synechococcus sp. (cyanobacteria) was
expressed in C. reinhardtii, the resulting strain had 11% more dry weight.
18.6.4 Carbon Assimilation

Atmospheric carbon dioxide is assimilated by microalgae in the form of Ribulose
1,5-bisphosphate (RuBP) to yield 3-phosphoglycerate. The assimilation reaction is
catalyzed by RuBP carboxylase/oxygenase (RuBisCO). Many metabolic studies
claim that RuBisCO enzyme activity is the bottleneck of the carbon assimilation
process (Ducat and Silver 2012). RuBisCO enzyme is made up of two protein
subunits, large (rbcL) and small (rbcS). The enzyme has an affinity toward CO2
and O2, which makes it essential to have a large amount of this enzyme for
sustainable carboxylation rate. Researchers expressed rbcS from Arabidopsis and
sunflower plants in Chlamydomonas while leaving rbcL intact. This strategy
improved the CO2/O2 specificity factor up to 11% without compromising the
rate of carboxylation (Gimpel and Mayfield 2013). This approach hints toward
the fact that genetic traits can be transferred from one organism to another to have
the desired physiological characteristics.
18.7 IMPROVEMENT IN BIODIESEL PRODUCTION BY GENETIC

ENGINEERING
Genetic engineering techniques have been in practice for a long time by molecular
biologists and engineers to develop modified stains to overproduce microbial oil.
The geneticist target different aspects of microbial oil synthesis process to enhance
the SCO production. Broadly the efforts have been directed to increasing the lipid
quantity, lipid quality, in situ biodiesel production, and facilitating downstream
extraction of microbial lipid bodies. These goals have been discussed in the
following subsections.
18.7.1 Lipid Quantity

Lipid quantity or productivity is the first scale of efficiency to measure a microbe
for its potential as an oleaginous organism. Researchers have done many
homologous and heterologous gene transfers and mutations to achieve higher
productivities. Mostly, it has been believed that lipid accumulation occurs in
growth-limiting stress conditions, but it is more desirable to have lipid accumu-
lation in the typical exponential growth phase without any nutrient stress or
altered process conditions. Several genetic engineering studies have focused on
ACC enzyme, which is involved in the rate-limiting step to lipid formation

(Roesler et al. 1997). Conversely and despite having similar properties, in vitro
studies of two ACC enzymes from diatoms and plants showed that heterologous
gene transfer from plant to diatom did not always give an improvement in lipid
yield and titer levels (Sheehan et al. 1998). The contradictory case-specific results
hint toward the absolute requirement of complete understanding of biochemistry
and regulatory metabolism studies.
18.7.2 Lipid Quality

Genetic engineering approach can be used to improve various aspects of
the feedstock oil (lipids) to enhance its fuel properties. Primary efforts have been
made to increase fuel yield, but in some cases, the fatty acid composition is unlike
the desired fuel composition. In those cases, it requires some alteration to have the
desired fuel properties. Biodiesel is made up of methyl fatty acids, ideally with
shorter chain length (C8–C14).
Thioesterase enzyme (TE) is the most prominent target for researchers to
alter the chain length of fatty acids. Plant-sourced fatty acid-thioesterases (FA-TE)
are highly substrate specific. FatA TEs hydrolyze C18:1-ACP and FatB TEs work
on a range of acyl-ACPs (acyl carrier protein). Mexican cigar shrub (Cuphea
hookeriana) produces up to 75% caprylate (C8:0) and caprate (C10:0) in its seed
storage. Use of TE to manipulate chain length of fatty acids was first demonstrated
using plant (Umbellularia californica) FatB TE in Arabidopsis thaliana, achieving
24% improvement in C12:0 fatty acids (Voelker et al. 1992). In another example,
the same gene expressed from U. californica into transgenic Brassica napus
achieved a 58% improvement in C12:0 in the storage oil (Voelker et al. 1996).
Unlike plants, the thioesterase in microalgae are hybrid of FatA/B TE and
show different specificity toward fat acid chains. The nuclear genome of
C. reinhardtii contains one single type of TE, which produces chain length
ranging between C14 and C18 with varying degrees of unsaturation (James
et al. 2011). Scientists also have performed homologous overexpression of TE
enzyme from one cell organelle (nucleus) to another (chloroplast), resulting in an
imbalance of enzyme and acyl-ACPs, and thereby, increasing short-chain fatty
acids due to prematuration (Blatti et al. 2012). Much research is still in progress for
improving the fatty acid composition of the lipid feedstock oil to obtain superior
fuel properties.
18.7.3 In Situ Biodiesel Production

In practice, biodiesel is produced by first producing and extracting lipid feedstock
oil obtained from fermentation of carbon-rich substrates, and then performing a
chemical transesterification to convert the fatty acids to biodiesel (methyl esters of
fatty acids). However, it is possible to produce the fatty acid methyl esters
(FAMEs) directly within the organism, thereby skipping many steps of biodiesel
production. E. coli has been engineered to produce FAMEs in situ by heterologous
expression of fatty acid O-methyl transferase (FAMT) obtained from Mycobacte-
rium maximum. The enzyme works more specifically on C8–C12 chain length and
takes methyl group from S-adenosylmethionine (SAM). Researchers enhanced the

biodiesel formation by implementing overexpression of SAM genes. To do so,
researchers deleted the regulatory gene metJ that acts on the SAM gene by
feedback inhibition (Nawabi et al. 2011). The research on in situ biodiesel
production is still at the developmental stage but has high potential.
18.7.4 Engineering the Downstream Processes

Downstream processing remains the expensive step of any bioprocess. Solid-liquid
separation usually is obtained by energy-intensive centrifugation (Uduman et al.
2010). Flocculation can be used as an alternative for easy and economical separation
of biomass. Microbial cell surface (hydrophobicity), chemical characteristics, and
surface charge predominantly determine the flocculation ability of the biomass. In
practice, many expensive chemicals are used to enhance the flocculation of biomass.
To respond to this problem, researchers have developed a genetically modified
oleaginous organism with enhanced flocculation activity. The cell surface of
T. pseudonana was modified to display silica affinity protein on the cell surface
by using frustulins (cell surface anchoring proteins) obtained from the diatom
Fistulifera solaris (Nemoto et al. 2014). The efficiency of the cell surface display was
evaluated by complexing the cell surface protein display with GFP. Ultimately the
cells with outward expression of silica affinity proteins were flocculated using silica
beads (Maeda et al. 2016). This study provides an alternative technology with vast
potential for cell harvesting and downstream extraction.
Extracellular lipids production has been attempted. Random mutagenesis
studies in yeast produced a high secretion of TAGs and fatty acids (Nojima et al.
1999). Few studies have been conducted to excrete free fatty acids (FFAs) by
overproducing intracellular FFAs. Michinaka et al. (2003) inactivated FAA1 and
FAA4 genes with acetyl-CoA oxidase and obtained a high accumulation of
intracellular FFA levels, and eventual secretion in S. cerevisiae was observed.
Unfortunately, this high FFA buildup was also accompanied by compromised cell
proliferation. The mechanism of lipid excretion has not been entirely known yet.
Researchers have studied secretion of FFAs to a certain extent and proposed
various pathways for the secretion (Tietge et al. 1999, 2000).
18.8 IMPORTANCE OF GENE CONSTRUCTS AND SYNERGY OF

OPERONS
Although implementing new genetic engineering strategy also requires consider-

ing all the repercussions of the manipulation on each aspect of the cell metabolism
(Hsieh et al. 2012). The overall display of physiological trait is a function of genetic
manipulation related to regulatory reactions, cofactors balance, and optimized
expression levels.
At the beginning, geneticists had a very straightforward approach, that is,
overexpression of a gene will efficiently give an excess of the functional translation of
the gene’s protein. As reported by Dunahay et al. (1995), when ACCase was
overexpressed in C. cryptica, the ACC enzyme titer increased threefold, but the lipid
content remained unchanged. As evident from this example, an ensemble approach
is required to have high lipid production. In this regard, scientists have confirmed
that a combined construct of multiple gene manipulations can work better than the
one-gene-at-a-time approach. Tai and Stephanopoulos (2013) first devised a
construct with an intron-containing translation elongation factor 1-α (TEF). When
this platform was used to overexpress diacylglycerol acyltransferase (DGA1), a
fourfold increase in lipid content was observed. Further overexpression of ACC gene
enhanced lipid production twofold. When the two genes were combined in tandem
for simultaneous expression of ACC1 and DGA1 gene, 17-fold enhancement in lipid
content was observed. These studies have established the importance of the
synergistic effect of expression of the tandem of genes in a construct.
A similar observation was observed when ACC1 was overexpressed by
plasmids (58% improvement in lipid content) by Runguphan and Keasling
(2014). The total lipid content improvement found in algae was lower than that
in E. coli. Furthermore, as a second approach researchers overexpressed fatty acid
synthase genes FAS1 and FAS2 using plasmid. By this strategy, 30% increase in
lipid content was observed. Finally, using the plasmid overexpression method,
diacylglecerol aceyl O-transferase (DGAT) gene was overexpressed, resulting in a
150% increase in lipid content.
Going by the same logic, Runguphan and Keasling (2014) attempted to use the
synergy of simultaneous overexpression of multiple genes. All the genes (ACC1,
FAS1, and FAS2 enzyme) were overexpressed in S. cerevisiae chromosome along
with a TEF1 (constitutive) promoter. Moreover, promoter of DGA1 gene was
replaced with the TEF1 promoter and obtained 142% improvement in lipid content.
In observance of a lower increase in lipid accumulation, ultimately ACC1, FAS1, and
FAS2 were expressed in the genome with TEF1 promoter, whereas DGA1 was
expressed using plasmid with a high copy number. In this case, the lipid content was
improved by 302%. Thus, Runguphan and Keasling (2014) confirm that multiple
gene constructs give better results compared with single gene overexpression.
18.9 CHALLENGES IN DESIGNING METABOLIC PATHWAYS
Certain classes of molecules can only be made by biosynthetic pathways rather

than by synthetic chemistry methods. The difference lies in the biological
intelligence incorporated by the enzymes and their mode of highly specific
actions. The selectivity of the biological systems gives us an exceptional control
over the features of the final product. Genetic engineering tools are one way for
users to communicate with the biological system (microbes) about the desired
modifications and features in the final product. In this context, the first and
foremost objective should be where exactly the change (gene) has to be made by
genetic engineering.
18.9.1 Gene Discovery and Pathway Construction

Development of computational methods by biochemical reaction databases
and genome mining has revolutionized the genetic engineering process. Next-
generation sequencing (NGS) methods and bioinformatics tools have facilitated
vast data mining and gene discovery. The functional information about the gene is
still limited. Experimental validations of different bioinformatics predictions are
limited by their cost-intensive nature and size of iterative attempts. Once the gene
is found, it remains difficult to discover the correct genotypic version of the gene
that will give the maximum product by the model organism.
Pathway design has become more straightforward because of the advanced
NGS technologies, cheaper DNA synthesis, standardized expression vectors, and
genome integration. Traditional methods are usually iterative and more directed
to the product construct. Combinatorial techniques allow development of vast
libraries with the collection of open reading frames (ORFs) and protein expression
variants within the genetic context of the host organism. These libraries for
different pathway constructs can help researchers to select different variants.
Strain construction requires resources for library construction, experimental
design, and massive amounts of the data analysis.
18.9.2 Characterizing Enzyme Expression by Proteomics

Simple introduction of a gene in the host genome has not been working with
certainty (Dunahay et al. 1995). To diagnose the failure, one has to verify the levels
of gene expression. Real time polymerase chain reaction (RT-PCR) or RNA
sequencing can ascertain the levels of transcription in the host, but any defects
during translation or at posttranslational stages requires a protein level analysis.
Mass-spectroscopy techniques have revolutionized the studies related to protein
quantification and identification within a large-scale proteome.
Current technologies use extended chromatographic separation techniques
with long separation times (ranging from minutes to hours). The chromatograph-
ic methods are susceptible to methods of sample preparation as well. Therefore,
new technologies should be suitable for high throughput analysis.
18.9.3 Optimization of Expression Levels of Protein

Higher lipid yields can be achieved by tuning the exogenous pathways to
maximize the carbon flux (C-flux). Although, it is not explicit to know in advance
which pathway could lead to maximum C-flux and lipid yield. The approach is
challenged by lack of enough data for enzymatic activity and substrate affinities, in
vivo protein expression, and effects of inhibitors/activators.
Researchers have been proposing various kinetic models for metabolic net-
works (pathways) by heuristic statistical approaches to maximize product yields.
These models use a small set of actual experimental results to predict extrapolated
results for new metabolic pathway designs. Each metabolic pathway network
results in a certain degree of expression of the target protein (or a set of proteins).
Even if we know how to maximize the product at a particular expression level of
the protein, designing the actual metabolic pathway in the host (in vivo) system is
yet another challenge to get that level of gene (and protein) expression.
Optimization of protein expression is essential but often cannot accurately
predict the expression level. Overexpression of any protein creates a metabolic
burden on the cell machinery, causes toxic effects, and interrupts the normal cell
function. For example, the overexpression of membrane transport protein can be
toxic to the cell because it changes the membrane fluidity and other functional
properties (Wagner et al. 2007).
18.10 SUMMARY AND FUTURE PROSPECTS
The discussion in this chapter shows how genetic engineering has tremendous
potential to enhance the lipid productivity of oleaginous organisms. More likely,
genetic engineering is the key to achieving sustainable solutions to the current
energy requirements. The technology and scientific advancements in the field of
genome editing and molecular biology, in general, have boosted the progress of
knowledge advancement and provided solutions to challenging technical pro-
blems, which made genetic engineering near to impossible in earlier times.
Advancement in gene sequencing, molecular imaging, and metagenomic data
analysis has made the genome-wide characterization and profiling of different
oleaginous microbes convenient. Although many industrial strains for lipid
production have been thoroughly characterized (genetically), a vast majority of
oleaginous microorganisms remain unraveled. Gene sequencing techniques are
rapid but expensive, and researchers need to deploy costly sequencing projects for
complete characterization of a single organism. Although the economic imped-
ance in full genomic annotation and profiling of an organism is discussed, other
technical challenges remain, like availability of efficient transformation methods,
editing tools, expression systems, specificity, and statistical analysis to ascertain
the knowledge generated by sequencing projects.
In spite of being costly, genome sequencing projects have very high potential
to serve as a database of information regarding proteomic, transcriptional, and
genomic profiles of the organism. The knowledge of omics to profile an organism
facilitates easy planning and in silico evaluation of different genome editing
strategies to create new genotypes with high lipid production capacities. Before
actual execution of any genetic modification in the laboratory, it is essential to
perform the metabolomic analysis using various software tools and metabolic
models. Detailed flux balance analysis, cofactor balance analysis, study of regula-
tory mechanisms, and in silico kinetic modeling should be done to predict the
possible outcomes [Figure 18-2(a)]. The challenge is that not many microbes have
been studied, and very few metabolic models are available that can be applied to
the different microorganism.
As discussed in this chapter, a combined approach considering different genes
together in a construct is more likely to give better results than standalone
overexpression of a gene. Determining the combination of these genes and how

they interact at regulatory levels is vital to the success of the construct. To date, it
remains challenging to be sure about one construct, let alone comparing one
construct combination with another. In fact, not just the set of genes, but how and
where genome expression occurs (e.g., genome or plasmid expression) are also
very important. More dedicated research is required to identify each of different
combinations of genes and methods of overexpression to have a justified strategy
for developing more productive strains.
Various advanced genetic engineering tools are used to execute given genome
editing strategies. Although most of the industrial strains are studied in depth, new
strains with higher lipid production capacities bring along new challenges to
transformation techniques (e.g., editing their genomes with higher efficiency and
specificity). A straightforward approach is to extrapolate and try existing technolo-
gies on newly discovered strains, hoping that the techniques work efficiently and
give stable transformants (or GMOs). Assay of genetic stability of the new genotype
developed is essential to evaluate its potential as an industrial strain. New strains
might be susceptible to lose the newly integrated gene in due course of replication
and regeneration; such stability studies should be done to assure its utility.
Once a genetically modified organism with stable integration of the desired
gene is made, it is essential to evaluate the growth kinetics of the new strain. Most
of the strategies in lipid enhancement like blocking the competing pathways,
deregulation of metabolism, and increasing precursor metabolites were aimed. It is
possible that in trade-off (genetic manipulations), the growth characteristics of the
microbe may be lost. Such postmodification studies for all the strains should be
conducted to characterize the kinetics of the new organism.
Although most of the studies are focused on improving the lipid yield of the
microbes, it is also worth focusing on the profile of the produced feedstock oil.
Every genetically modified organism should be further studied to manipulate and
control the composition of the feedstock oil [Figure 18-2(c)].
Ultimately to fit in the broader view, it is essential to consider various genetic
engineering strategies to answer questions arising from the techno-economic
perspective of biodiesel production process. How can we utilize cheaper (or waste)
resources as carbon sources? How can we entirely avoid downstream transester-
ification (in situ biodiesel formation)? How can we have an economical downstream
operation (surface modification for cheaper solid-liquid separations)? There are
various elements of process development in the biodiesel industry, and genetic
engineering can contribute to each of these aspects to reduce the cost of the process.
Although the primary focus of genetic design is in the development of microbe (or
the biocatalyst) for the bioprocess, it is possible to produce strains with better
downstream abilities and enzyme overproduction for enzymatic transesterification.
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CHAPTER 19
Recovery and Purification
Technologies of Biodiesel
J. S. S. Yadav
L. Kumar
S. Pilli
S. Yan
T. T. More
R. D. Tyagi
R. Y. Surampalli
19.1 INTRODUCTION
Transesterification involves the catalytic transformation of triglycerides and alcohols

into fatty acid alkyl esters-FAAEs) (fatty acid methyl ester-FAME or ethyl esters of
fatty acid-FAEE), that is, biodiesel with the generation of glycerol as a main
byproduct along with some other impurities and minor byproducts (Vasudevan
and Briggs 2008). The different impurities/intermediates generated during the
reaction are shown in Figure 19-1. The produced biodiesel must be purified to
meet the required standards set by ASTM D6751 or EN 14214. To meet the
standard requirement, the postreaction processes (separation and purification steps)
used are ester/glycerol separation, ester washing, and ester drying (to remove the
water used for washing). Of all these separation and purification steps, purification is
the most critical and challenging in-process step of the biodiesel production.
After purification of biodiesel, it is necessary to evaluate its quality for market
and end-user acceptance. In general, chromatography and spectrophotometric
analytical methods are the most commonly used techniques for biodiesel charac-
terization (Monteiro et al. 2008). Furthermore, it is also necessary to evaluate the
stability of the produced biodiesel. Figure 19-2 presents a schematic overview of
steps, that is, separation, purification, quality control, and stability.
This chapter overviews the techniques and methods that are used for the
separation and purification of biodiesel. The traditional method (gravitational
453
Figure 19-1. General chemical reactions of biodiesel synthesis via base catalysis
involving different steps with the generation of various intermediates.
settling) and new techniques (centrifugation and coalescing technologies) of

biodiesel separation are discussed. After that biodiesel purification techniques
are elucidated, including the conventional biodiesel purification techniques and
modern evolving technology (i.e., membrane technologies). The analytical tech-
niques used for characterization of biodiesel and the factors that should to be
considered to prolong the stability of biodiesel are also covered and discussed in
RECOVERY AND PURIFICATION TECHNOLOGIES OF BIODIESEL 455
Figure 19-2. Overview of separation and purification steps for obtaining biodiesel
for end users.
the present chapter. Finally, the challenges and future perspectives of the new
approaches to developing a robust and economic purification technology are
discussed.
19.2 BIODIESEL SEPARATION TECHNIQUES
Separation of biodiesel starts after completion of the transesterification reaction by

using the conventional phase separation method. In general, the first separated
component from the crude biodiesel is the major byproduct glycerol. Separation of
biodiesel and glycerol is based on the differences in their polarity and density. The
glycerol (a trihydric alcohol with dielectric constant of 42.5) is highly polar
compared to biodiesel (mono alkyl ester with dielectric constant of approximately
3.0). Biodiesel and glycerol are immiscible owing to the differences in their
dielectric constant. The second important factor, which accelerates the phase
separation, is the glycerol density. The density of biodiesel and glycerol are 0.88
and more than 1.05 g/cm3, respectively (Nazir et al. 2009). In addition, glycerol
density also depends on the amount of methanol, water, and catalyst in the
glycerol. However, this density difference is sufficient for simple gravity separation
technique for the two phases. The following processes/approaches are commonly
used to complete the separation process.
19.2.1 Gravitational Settling

The content of water, catalyst, and alcohols (methanol and ethanol) in crude
biodiesel mixture affect the density of glycerol. However, it is sufficient enough for
phase separation of biodiesel and the glycerol layer (Gerpen et al. 2004). The
higher soap content interferes with the phase separation of biodiesel and glycerol
because of formation of semisolid and solid substances (Balat and Balat 2008). In
general, 12 h or overnight is required for settling of two phases. Kaewkannetra
et al. (2010) evaluated the effect of oil type (e.g., used cooking oil, palm oil,
rice bran oil, soybean oil, used vegetable oil, and used animal fat), alcohol
(e.g., methanol, ethanol, and their mixtures), and alcohol concentration on the
settling behavior of glycerol. The biodiesel was produced using 0.58% of NaOH as
catalyst at 60 °C and 20 min of reaction time. The produced biodiesel was separated
by gravitational settling. The biodiesel produced from fresh oils (palm, rice bran,
and soya oil) settled faster compared with the mixture of used vegetable oil and used
animal fat. Individual alcohol has no effect on settling behavior. The settling of
glycerol can be enhanced by using a mixture of alcohol (i.e., methanol and ethanol).
Furthermore, the use of some accelerators has been reported for phase
separation. Mohammad et al. (2013) reported for acceleration of glycerol settling
using NaCl. The study was conducted on biodiesel produced from the mixture of
oils (canola oil and sunflower) and methanol using KOH as a catalyst. After
transesterification, the mixture was kept for 48 h for phase separation. Then 100
mL biodiesel and glycerol (1:1) with different quantities of NaCl (0, 0.5, 1.0, 3.0,
5.0, and 10.0 g) were well mixed using a magnetic stirrer and transferred into a
100 mL graduated cylinder for gravitational separation. It was concluded that the
addition of NaCl salt had decreased the glycerol settling time by more than five
times. However, it is reported that addition of NaCl more than 3.0 g to the mixture
resulted in impurity in methyl ester (biodiesel) caused by the occurrence of the a
mini-emulsion phenomenon. A mixture with 1 g NaCl/100 mL biodiesel was
reported to be optimum for accelerating the separation process by 100% and
maintaining the purity equivalent to the purity without salt addition. Gravitational
phase separation technique is a time-consuming process, but still it is the more
applicable method because of the simple and inexpensive process. Today, empha-
sis has been on the eradication of the phase separation problem by using the
heterogeneous catalyst.
19.2.2 Centrifugation
The use of settling tanks is a well-known method for the storage and physical
treatment of produced biodiesel fluid. Although effective, settling tanks demand
labor costs, long incubation time, space and operating costs, besides chemicals and
production costs. On the other hand, a biodiesel production process could be
integrated with one or several technologies in the production process to separate
biodiesel from the reaction mixture. Further, biodiesel production methodology
can be changed from batch mode to continuous mode, and this could be achieved
through the use of centrifuge technology. Because centrifuges are established
technology, they are safe technological substitutes for the optimization of biodiesel
production depending on the production methods used. Centrifuges may be used
to improve the clarifier and separation process in biodiesel production (i.e., as a
clarifier centrifuge and as a separator centrifuge). Clarification is used to separate
solids from a liquid. Centrifuge as a clarifier is designed to discharge suspended

solids from a liquid by using centrifugal force. The clarifier centrifuges enhance
the settling process, otherwise it takes more time in the settling tank by a simple
phase separation process in raw biodiesel. The separation centrifuges are used to
define the outcome of centrifugal force acting on one liquid and separating it into
two or more liquids. After complete separation, the two phases of liquid with
different densities exit the centrifuge through separate ports. This is essentially a
liquid–liquid separation process (Wang et al. 2007).
The advantages of centrifuges are (1) reduction in time versus product output;
(2) reduction in labor cost versus product output; (3) batch operation versus
continuous operation; and (4) centrifuge can separate liquid with moderately high
viscosity and can also be used for relatively high solid loading. Despite these
advantages, an initial high investment and its maintenance costs are the main
barriers. Moreover, centrifugation is an energy intensive process. However, today
different types of centrifuges are designed especially for biodiesel industries and
the investment cost could be reduced in the near future (Wang et al. 2007). In
addition to glycerol separation, centrifuges are also used to remove the impurities
after water washing (i.e., during purification steps). The heavy phase is differen-
tially separated to the outer surface of the centrifuge, while the fatty acid methyl
ester (FAME) remains in the inner surface. A sufficient residence time is required
for oil to float from the top of the water surface, and this process is less effective
for the separation of the FAME–water emulsion. Sometimes, emulsion of
FAME–water is not separated even after days (Wang et al. 2007).
Coalescing technology is another technology used for liquid–liquid separa-
tion. Coalescing technology is a multistage separation scheme to remove solution
gas and free floating and dispersed oil from water. This technology has the ability
of performing multiphase separation by combining the physical principle of
adsorption, coalescence, desorption, and gravity separation to recover the pro-
cessed oil and gas from the water. Two immiscible liquids can form an emulsion or
colloidal suspension. In both cases, the dispersed liquid resulted in the droplet
forms in the continuous phase. Droplet size in the emulsion or in suspension is
less than 1 μm in diameter, and the liquid cannot readily be separated with normal
separation technologies. To separate the small-size droplets (typically in the size
range of 1 to 50 μm) requires the use of special equipment. High-efficiency liquid–
liquid coalesces have been developed to break these emulsions and provide
improved separation. Liquid–liquid coalesce are used to accelerate the merging
of many droplets to form larger droplets. Settling of the larger droplets down-
stream of the coalesce element thus requires substantially less residence time. In
general, three-step processes are used for coalescing separation: (1) collection of
individual droplets; (2) combining of numerous small droplets into larger ones;
and (3) the upsurge (or drop) of the enlarged droplets by gravity.
Coalescers are usually manufactured in different forms such as pads or

cartridge filters, which are designed specifically to combine small droplets in an
emulsion and convert them into large drops that are separated more easily. The
designed coalescing process accelerated over natural coalescing owing to the
presence of fiber in the coalescing media, which enhanced the interaction of small
droplets and resulted in acceleration of the coalescing process. Factors affecting the
coalescing separation process are interfacial tension, viscosity, density, and temper-
ature. Liquid–liquid coalescers are fabricated from polymer and fluoro-polymer
ingredients, which have been optimized to separate the most challenging emulsions
with interfacial tensions as low as 0.0005 N/m. This coalescer can be used with a
broad range of applications. It can process aggressive chemicals and handle
demanding operating conditions while providing the highest level of performance
(KLM Technology Group 2012 Anez-Lingerfelt 2009). The advantages of coalescers
are low capital and operational cost, lower maintenance, and lower energy
consumption. Currently, the company Pall Corporation (Anez-Lingerfelt 2011) is
fabricating coalescence technology available for the separation of biodiesel over
centrifuge technology. This technology is looking attractive; however, further pilot-
to-large-scale studies are required to accept the technology universally at the
commercial level.
The residual methanol/ethanol present after glycerol separation is usually
removed before applying the purification steps through distillation. Alternative-
ly, distillation can be applied prior to recover alcohol followed by glycerol
and ester separation. The recovered alcohol is reused in the process (Atadashi
et al. 2014).
19.3 BIODIESEL PURIFICATION TECHNIQUES
19.3.1 Conventional Biodiesel Purification Techniques

After separation of the major fraction of byproduct (glycerol), further purification
steps are required to remove the trace impurities such as glycerol, soap, residual
alcohol, glycerides, and catalyst. Glycerol exists as impurities in two forms, that is,
free glycerol and bound glycerol in the form of triglycerides (TG), diglycerides
(DG), and monoglycerides (MG) (Figure 19-1, Shahbaz et al. 2010). There are
various approaches for purification such as water washing (wet washing) and
nonwater washing (dry washing). The wet washing processes are the more
commonly applied when biodiesel is produced through homogeneous catalysis
processes (Schultz 2010).
19.3.1.1 Wet Washing

Wet washing is used for purification of the crude biodiesel (FAME) phase.
The crude biodiesel phase mainly contains soap, catalyst, methanol and
trace glycerol, MG, DG, and TG. This technology is carried out through the
following ways.
Washing with purified water. This is carried out to remove soap, catalyst,
methanol, glycerol, and other impurities. There are various studies on the washing
of biodiesel with water and mainly applied for the crude biodiesel washing when it
is produced through the acid transesterification process. Rahayu and Mindaryani
(2007) optimized the biodiesel purification condition through water washing. The
biodiesel was prepared from castor oil using KOH as the catalyst. The steps used to
purify the biodiesel are shown in Figure 19-3. The used water washing process
reduced the glycerol from initial 0.93% w/w to the final value of less than 0.05% w/w.
During the washing process, pH decreased from 9 to neutral (pH 7.3). The authors
suggested that washing should be carried out in multiple stages to remove the
glycerol below 0.02% w/w. Chongkhong et al. (2009) conducted water washing with
10.24% w/w NaOH (3 M)-water solution to neutralize the free fatty acid (FFA) in
crude biodiesel. Then, soap formation was removed using 2.00% by weight of
sodium chloride. The water washing was carried out at 60–80 °C and allowed to
settle the water phase. After phase separation of water, biodiesel was heated to
evaporate the residual water.
Acid washing. Acid washing is used to neutralize catalyst and to decompose
the soap formed (especially when transesterification is carried out by base catalyst
such as NaOH and KOH). The processes of acid washing include the water
Figure 19-3. Steps for purification of biodiesel via washing with water.
washing to remove the impurities (e.g., trace free glycerol, catalyst, methanol, and
soap) from biodiesel. The 10% H3PO4 has been used to wash the biodiesel after
glycerol separation using the bubble wash method, followed by additional
purification by an aquarium stone for 24 h. After, acid washed biodiesel was
treated with hot purified water to eliminate residual impurities such as catalyst and
alcohol (Sharma and Singh 2009). Silica gel was used to remove the catalyst from
the biodiesel product. The difficulty during the separation of glycerol and FAME
has been reported when a higher alcohol-to-oil ratio was used (Meher et al. 2006).
The use of TG with low content of FFA has been recommended to avoid the soap
formation. The products formed were separated using sedimentation. Ester phase
(FAME) was distilled at 80 °C to remove the excess methanol, followed by several
washes with purified water, and then treatment with Na2SO4, and finally by
filtration (Meher et al. 2006). Kywe and Oo (2009) reported the production of
biodiesel from jatropha oil in a pilot plant (in a 45 gal. or 170.34 litres stainless
steel reaction tank) using methanol and ethanol and using the base catalysts
NaOH and KOH, respectively. The crude biodiesel was first neutralized by adding
phosphoric acid, followed by washing with warm water. The warm water was
added and gently agitated for 30 min. The gentle agitation was carried to avoid the
formation of emulsions. Washings were repeated until pH of biodiesel reached
neutral. The washed biodiesel layer was sent to sand filtration, which was carried
out in a 15 gal. (56.78 L) stainless steel filtration tank. The size of sand mesh was
−20 to + 60 mesh; kept over a 100 mesh size stainless screen. After filtration over
sand mesh, the color of purified biodiesel was clear amber yellow. The physical
properties of methyl ester and ethyl ester were reported to be in the range of
ASTM standard limits. However, total glycerol, free glycerol, and bound glycerol
were reported to be slightly higher than ASTM specified limits.
Washing with organic solvents. Organic solvents have been used to purify
the crude biodiesel. The most commonly used organic solvent is petroleum ether.
Karaosmanoglu et al. (1996) evaluated the purification of crude biodiesel using
petroleum ether. The biodiesel was produced from rapeseed oil using base catalyst.
After transesterification, the reaction mixture was cooled to room temperature
and transferred to a separating funnel for phase separation. Following the
separation of the methyl ester (biodiesel) and glycerol layers, methanol was
removed by a vacuum rotatory evaporator. The obtained methyl ester (biodiesel)
was mixed with equal volume of petroleum ether and twice the volume of distilled
water, followed by adjustment of pH 7 using acetic acid. The washing process with
water was repeated three times. The methyl ester phase (biodiesel) was left over
heated sodium sulfate overnight and then filtered. The solvent (petroleum ether)
was removed by a vacuum rotatory evaporator. The refining yield obtained was
82.60%. Although refining with petroleum ether is efficient, it is less popular owing
to use of organic solvent. In another study, the crude biodiesel has been washed
with petroleum ether followed by adjustment of pH 7 using glacial acetic acid.
After that, purification was achieved by washing with water three times. Then the
product was dried over anhydrous magnesium sulfate and filtered. The solvent
was removed by evaporation (Fangrui et al. 1998).
19.3.1.2 Dry Washing

Dry washing is applied to reduce the effluent water (wastewater) produced during
the water washing (wet washing); it is economical compared with other more
expensive operations (like centrifugation) that have the main drawback of wet
washing (Vera et al. 2011). Dry washing is easier, less time consuming, and
reduces the water content in biodiesel. The dry washing is carried out by using
silicates, ion-exchange resins, cellulosic, activated clay, activated carbon, among
others, to remove the impurities and meet the standard quality compliance
(Bertram et al. 2005). The detail of agents used for dry washing is described next.
Silicate. Adsorption is a basic mechanism used for refining (purification) of
biodiesel using different adsorbents. Adsorption is usually performed in columns
packed with adsorbent material. Moreover, it can also be applied in the agitated
tanks with adsorbent in suspension. The enhanced separating efficiency that
resulted in the packed column is a result of the chromatographic effect and is a
distinctive advantage of adsorption compared with other separation processes.
Adsorption is preferably suitable for purification applications and problematic
separation. The adsorptive separation is achieved by one of three mechanisms:
adsorption equilibrium, steric effect, and kinetic effect. However, in the processes,
especially with both solid and liquid phase involved, it works on the principle of
adsorption equilibrium, and is called equilibrium separation processes. In the case
of biodiesel, a product with the low elution rates in the packed column makes the
process dynamic separation (kinetic effect) of no use for a practical separation. In
the case of steric effect, this is expected to work fine for molecules differing widely
in size, and this could be the case for molecules of the organic and polar phases
normally found at the outlet of the transesterification reactors. TG, DG, MG, FFA,
and FAME have high molecular weights and long acyl chains; they are the main
components of the organic phase. On the other hand, glycerol, water, and
methanol have small molecular size and could be retained in a packed bed
containing suitable adsorbents. Because of their relative high vapor pressure, water
and methanol need a relatively few number of theoretical plates to be separated
from the organic phase by distillation/evaporation, and this indeed is the preferred
method of water and methanol removal. Removal of glycerol from biodiesel using
adsorbent has been tried. The use of the steric effect in the adsorption of water on
zeolite has, however, been proposed for the drying of the methanol to be recycled
to the biodiesel process (Vera et al. 2011).
Washing using synthetic magnesium silicates (Magnesol) has been carried out
to purify the crude biodiesel (FAME) derived from crude soybean, yellow grease,
and rapeseed feed stocks by Bertram et al. (2005). The synthetic magnesium
silicates are polar in nature and adsorbed the residual impurities (such as free
glycerol, mono- and diglycerides, free fatty acids, and soap) from the crude
biodiesel (Figure 19-4). Commercially available Magnesol is the most commonly
used silicate. Mazzieri et al. (2008) evaluated the adsorptive property of silica gel
for biodiesel refining. The transesterified mixture of biodiesel was first purified
from glycerol and glycerides by repeated cycles of washing using distilled water,
Figure 19-4. Steps involved for purification of biodiesel via dry washing.
followed by centrifugation until glycerol content reached less than 0.004%. Then,
in 100 mL of purified biodiesel, measured quantities of impurities (glycerol,
methanol, monoglycerides, and soap) were added followed by mixing. After that,
different amounts of silica (0.5 to 3.0 g) were added in solution, and the formed
slurry was agitated for 2 h. Then solid phase was left, and the liquid phase was
decanted. The obtained result reported that glycerol has more affinity for silica and
selectively adsorbed from the biodiesel mixture. The adsorption of glycerol from
biodiesel mixture not affected by presence of soap. However, dissolved methanol
reduced the adsorption of glycerol owing to the competition for the adsorption site
on silica. Presence of monoglycerides in mixture reduced the saturation capacity
from co-adsorption. The result indicates that conventional refining, which is
performed by decanting/washing or centrifugation/washing, can be suitably
replaced by simple decanting and silica adsorption, which will prevent the issue
of generation of excess effluent wastewater.
Manique et al. (2012) evaluated the Magnesol and rice husk ash for
purification efficiency of biodiesel. The step of purification involved the heating
of crude biodiesel sample at 65 °C for 20 min with 1% (w/w) and 4% (w/w) of
Magnesol and rice husk ash, respectively. The treated samples were filtered
to remove the adsorbent. Results showed that 1% (w/w) Magnesol as well as
4% (w/w) rice husk ash was effective for biodiesel purification. The purified
biodiesel was in compliance with required standards. Saengprachum et al. (2013)
also prepared a silica extract from rice husk ash and evaluated its efficiency for
removal of glycerin. The extracted silica was efficient in the removal of glycerin
with compliance to European standard EN 14214. Moreover, extracted silica was
reported to be efficient for color removal of biodiesel. Although the silicates are
effective, more studies are required to scale-up the process and commercial
applications.
Washing with ion-exchange resins. Chemically, organic resins are derived
from an active functional group to adsorb, via exchange of charge, the impurities
from crude biodiesel, while leaving the biodiesel (methyl alkyl ester). There are
various resins that are commercially available and being used in pilot industries,
such as Amberlite BD10 DRY, Purolite PD 206, Indion BF 170, and Lewatit
(Faccini et al. 2011). Berrios and Skelton (2008) used ion-exchange resin Purolite
PD 206 to purify biodiesel. The resin was filled in a glass tube and flow was
controlled through a meter pump. The crude biodiesel sample was passed through
the column and analyzed after 2 h for methanol and glycerol content. The resin
was able to remove the glycerol as required by EN 14214 and substantially the soap
content. However, methanol removal was relatively low (about 20 L biodiesel/kg
resin). The less soap removal is a limitation for high soap containing feed. The ion-
exchange resins are economical and offer good performance for removal of
glycerin, water, salts, soap, and catalyst without use of water. However, methanol
removal is a limitation of this process (Atadashi et al. 2011a).
Other washing agents. Activated carbon is commonly used for dry washing
of biodiesel to remove the excess color. Thus, for effective dry washing of crude
biodiesel, the adsorbent is channeled into a paddle-type mixing tank and
thoroughly agitated. Different activated fibers, activated carbon, activated clay,
and acid clay were used to purify the crude biodiesel. It has been reported that clay,
especially acid clay treated with sulfuric acid is superior and better in the aspect of
the de-alkaline effect, deodorant effect, and decoloring effect (Savaliya et al. 2014).
The clay grain size ranging from 0.1 to 1.5 mm is more suitable for efficient
biodiesel purification. The clay with smaller grain size offers better purification,
but their separation after purification process is difficult. Conversely, the larger
clay grain size is easier to remove after the process, but the purification process is
inferior (Atadashi et al. 2011a). The rice grain process produces a waste byproduct
—rice husk. The rice husk is utilized in power generation through burning and
converted into rice husk ash. The rice husk ash, which is produced by incineration
at a temperature range of 500 to 600 °C, is rich in amorphous silica. Manique et al.
(2012) evaluated the efficiency of rice husk ash from the industry Cotrisel of Sao
Sepe (RS, Brazil) for the purification of biodiesel produced from waste frying oil
using KOH as catalyst. After phase separation of glycerol, crude biodiesel was
purified with different concentrations (1%, 2%, 3%, and 4% w/w) of husk rice ash
and purified biodiesel was filtered. The authors concluded that 4% w/w of rice
husk ash was efficient to purify the crude biodiesel that fulfilled the standards
normal requirement.
Fadhil et al. (2012) used spent tea waste to produce activated carbon with an
electrical tubular furnace at temperature of 600 °C. Biodiesel was produced from
waste cooking oil by transesterification using methanol and KOH as catalyst. Once
the reaction was completed, the glycerol and FAME were separated in a separating
funnel. Further, FAME was distilled under vacuum to remove extra methanol.
Finally, crude FAME was used for purification using activated carbon. The spent
tea waste–activated carbon purification yield and efficiency was compared with
silica gel and water washing. The authors reported that purified biodiesel using
spent tea waste–activated carbon resulted in higher yield and better fuel property
compared with biodiesel purified through silica gel and water washing. The
purified biodiesel also meets the standard specification. Furthermore, spent
activated carbon was regenerated and reused, which was effective.
Ionic liquids (ILs) are the group of new organic salts, which exist as liquid at
low temperature (below 100 °C) and have been accepted as the new green chemical
revolution. The new chemical group can reduce the use of hazardous and polluting
organic solvents owing to their unique characteristics as well as taking part in
various new syntheses (Abbott et al. 2007, Hayyan et al. 2010). The development
of low-cost ILs termed deep eutectic solvents (DESs) have recently received intense
interest because of their environmentally friendly solvent and their solvation
properties. DES is a mixture of two or more compounds with their melting points
lower than either of their components. Examples of DES are the mixture of
organic halide salts, such as choline chloride with hydrogen bond donor (HBD)
organic compound glycerol. The quaternary ammonium salt–glycerin based DES
has been evaluated by Hayyan et al. (2010) for removal of total glycerin from the
palm oil–based biodiesel. The biodiesel was prepared using methanol and
potassium hydroxide and left the mixture of biodiesel for phase separation for
overnight. The DES was prepared using choline chloride with glycerin in different
choline chloride-to-glycerin mole ratios (1:1, 1:1.25, 1:1.5, 1:2, and 1:3). The
prepared DES was added to biodiesel phase in different biodiesel:DES molar ratios
(1:1, 1:1.5, and 1:2). The samples were agitated in a rotatory shaker at 170 rpm for
1 h and finally left for settling for 2 h. The content of glycerol in purified and
unpurified biodiesel was measured by high performance liquid chromatography
(HPLC). The result reported that maximum removal (51.25%) of glycerin can be
achieved with a DES:biodiesel molar ratio of 1:1 and DES molar composition of
1:1 (salt:glycerin). The purified biodiesel fulfilled the ASTM D 6751 and EN 14214
standard specifications. Further, the used solvent can be recovered by crystallizing
the ammonium salt. Shahbaz et al. (2010) have also evaluated the DES for removal
of glycerol from palm oil–based biodiesel. The researcher prepared two DES by
combining choline chloride:ethylene glycol and choline chloride:2,2,2-triflurace-
tamide. Both the DES solvents were effective for removal of free glycerol from
crude biodiesel with an optimum DESs:biodiesel ratio of 1:1.
19.3.2 New Biodiesel Purification Technology (Membrane

Technology)
The membrane technologies have been shown to play an important role in the
separation and purification in biorefining and bioenergy product processes. The
separation and purification of biodiesel by membrane technology appeared as an
attractive option. Application of membranes for separation and purification has

several advantages over the conventional processes such as reduced capital and
production costs as well as being environmentally friendly (He et al. 2012).
Membranes applied for biodiesel refining are usually made from inorganic
microporous ceramics or polymeric membranes and are utilized in various

bioprocessing applications. Membrane separation works on the basis of a selective
barrier to regulate the transport of substances, such as gases, vapors, and liquid at
different mass transfer rates (He et al. 2012, Shuit et al. 2012, Abels et al. 2013).
The membrane module configuration comprises hollow fiber, tubular, flat plate,
spiral wound, and rotating devices. Two common ways of operating are dead-end
and cross-flow configuration (Figure 19-5). In the cross-flow method, the fluid to
be filtered flows parallel to the membrane surface, and permeates pass through the
membrane because of a pressure difference. The cross-flow reduces the formation
of filter cake to keep it at a low level (Charcosset 2006). The mass transfer rates of
substances are controlled by the permeability barrier toward the feed components.
In the case of biodiesel production processes, the membranes play an important
role by removing the glycerol from the product biodiesel stream or by retaining
the unreacted triglycerides within the membrane. The process of biodiesel
production and purification by applying membrane technology is based on the
oil or fat droplet size or catalytic membrane (Shuit et al. 2012). The most
commonly used membranes for the separation and purification are separative
ceramic, polymeric membranes, and membrane bioreactors.
19.3.2.1 Different Membranes

Organic membrane. The organic membranes used in separation processes are
pressure driven, including microfiltration (MF), ultrafiltration (UF) ,and nanofil-
tration (NF). In the beginning, most of the membranes were cellulosic in nature.
However, these are now being replaced by polyamide, polysulphone, polycarbon-
ate, polyacrylonitrile, and some other improved polymers. The new synthetic
polymers have improved chemical stability and better resistance toward microbial
degradation (Atadashi et al. 2011c). Moreover, polyacrylonitrile (PAN) a porus
Feed
Feed
Retentate
Retentate
Membrane Membrane
Permeate Permeate
(a) (b)
Figure 19-5. Difference between (a) dead-end, and (b) cross-flow membrane
filtration.
and asymmetric membrane has been reported, which combines high selectivity
with the permeation rate (Salahi et al. 2010).
He et al. (2006) evaluated the purification efficiency of biodiesel using two
different hollow fiber membranes made of polysulphone (hydrophilic membrane)
and polyacrylonitrile (hydrophobic membrane) in comparison to water and solvent

purification methods. Crude biodiesel from soybean oil was used for purification
and passed through a hollow fiber membrane at a flow rate of 0.5 mL/min and
operating pressure of 0.1 MPa. Then, purified biodiesel was dried using 10% w/v
Na2SO4 for 12 h and filtered. Both of the membranes were effective for the
purification of biodiesel (FAME) without formation of emulsion during filtration
and product loss. Among the two membranes tested, polysulfone membrane
(hydrophilic membrane) was better compared to polyacrylonitrile (hydrophobic
membrane) in terms of water content in biodiesel. Polymeric membranes are
effective in biodiesel purification; however, in the presence of organic solvents,
polymeric membranes may swell, resulting in instant and/long-term pore-size
changes. Therefore, polymeric membranes in solvent application have a shorter
operating life time (Atkinson 2005); thus, more attention is focused on the use of
ceramic membranes.
Othman et al. (2012) evaluated the purification efficiency of different
polymeric solvent resistant nanofiltration (SRNF) membranes, which were made
of polyamide, polydimethylsiloxane (PDMS), polyimide, and silicon-type poly-
mers. The biodiesel was prepared from refined, bleached and deodorized (RBD)
palm olein, methanol, and sodium hydroxide. After transesterification, the
alkalinity of the product was modified by adding H3PO4. The produced Na3PO4
can be utilized as fertilizer. The pH value after addition of acid came down from
12.43 to 8.86, which was the suitable range for the polymeric membrane separa-
tion process. The permeation experiments were carried out through the polymeric
SRNF membranes at a constant separation temperature (40 °C) and operating
pressure of 600 to 2,200 kPa. The results reported that Solsep-030705 (hydro-
phobic, silicon-type) membrane gave the highest permeate flux, and the highest
rejection for TG 99.8%, DG 97.16%, MG 40.65%, and 75.24% for free glycerin at
constant separation temperature 40 °C and a pressure of 1,000 kPa within 30 min
of separation time. The maximum methanol rejection was obtained at 30 min of
separation time with rejection of 74.98%. Although the SRNF membranes were
effective for separation and purification, further scale-up and pilot-scale studies
are warranted for its potential application.
Ceramic membranes. Ceramic membranes, a class of inorganic membranes
(porous membrane such as Al2O3, TiO2, ZrO2, and SiC) have received much
attention because of their thermal, chemical, and mechanical stability; high
porosity, high flux, and long durability; resistance to microbial degradation; less
fouling problem; and availability in various pore sizes (Atadashi et al. 2011b).
Separation of soap and free glycerol through the membrane is based on the
immiscibility of free glycerol and biodiesel. Further, the soap surface activity
resulted in the formation of large-size reversed micelles, which is almost similar to
the existence of phospholipids in hexane micelles. The free glycerol in crude
biodiesel forms droplets with residual alkali catalyst. The free droplets of glycerol
bonded with the hydrophilic end of the soap, while the hydrophobic end of the
soap immersed in crude biodiesel. The mean size of the formed reversed micelles
of soap and glycerol molecules was reported to be 2.21 μm, larger than the
biodiesel (FAME) molecule, and thus were removed by membrane separation

(Wang et al. 2009). The crude biodiesel produced from refined palm oil by base
catalysis processes has been evaluated by Wang et al. (2009) for purification
efficiency using ceramic membrane (Pall Corporation). The different pore sizes
(0.1, 0.2, and 0.6 μm) of the membranes have been tested. The obtained result
reported that membrane with pore size 0.1 μm at the transmembrane pressure of
0.15 MPa and temperature of 60 °C was efficient for purification, and the purified
biodiesel met the specification set by EN 14214.
Atadashi et al. (2012a) evaluated the purification of biodiesel using low pore
size (0.02 μm) multichannel tubular-type Al2O3/TiO2 and filtration area of
0.031 m2. The process operating parameters tested were temperature (30 to
50 °C), transmembrane pressure (TMP) (1–3 bar/100–300 kPa) and cross-flow
rate (60 to 150 L/min). The biodiesel was prepared using palm oil, methanol, and
potassium hydroxide as catalyst. After, the reaction byproduct glycerol was first
separated by gravitational settling followed by residual methanol recovery via
rotatory evaporation. The obtained crude biodiesel was purified through a
membrane with and without adding acidified water. The results reported that
ceramic membrane of pore size 0.02 μm was efficient in separation of biodiesel
from different contaminants. The addition of acidified water improved the
retention of glycerol and catalyst (potassium). The optimum process parameters
were TMP 2 bar (200 kPa), temperature 40 °C, and feed flow rate (150 L/min) with
the corresponding flux of 9.08 (kg/m2/h). The purified biodiesel met the standards
requirement of both ASTM D6751 and EN 14214. Some of the other studies and
conditions used by various researchers are presented in Table 19-1. To date, most
of the studies for separation and purification are of laboratory scale. Thus, more
research and time will be needed to apply the membrane separation and
purification technology commercially.
19.3.2.2 Membrane Reactor

A membrane reactor is a device that combines the reaction and separation process
in a single unit and is also known as a membrane-based reactive separator. The
classification of a membrane reactor is usually based on four concepts: the reactor
design (extractor, distributor, or contractor), the type of membrane used (organic,
inorganic porous or dense membrane), inert or catalytic membrane reactor, and
the type of reaction that happens in the membrane reactor (Shuit et al. 2012).
A schematic diagram combining reactor and membrane separator is presented in
Figure 19-6.
Membrane reactors are designed based on the membrane separation by using
a membrane as a selective barrier to control the passage of constituents such as
gases, vapors, and liquids at different mass transfer rates. Biodiesel production and
separation via a membrane reactor is accomplished by removing the glycerol from
Table 19-1. Summary of Membrane Purification of Biodiesel Production.

Membrane characteristics Operating conditions
Raw material Pore size Temp TMP

(percent by weight) Scale Manufacturer Classification (μm)/kDa Material Configuration Mode (°C) (bar) Performance References
Oil-biodiesel-glycerol L CFTRI UF 40 kDa Cellulase Flat sheet ME — — Glycerol removal: >80% Belafi-Bako et al.
(Glycerol:5.0) acetate (2002)
Crude biodiesel L — — — Polysulfone Hollow fiber ME 20 1.0 Biodiesel purity: 99%, ester He et al. (2006)
loss: 8.1%, H2O content
Crude biodiesel L Pall Corporation MF 0.1/0.2/0.6 Ceramic Plate and R 60 1.5 Initial flux: 360/480/675 L·m2· Wang et al. (2009)
(Glycerol:0.261) frame h−1, rejection of
glycerol: >90/>90/>90,
permeate glycerol content
(percent by weight):
0.0152/0.0257/0.0276
Oil-FAME-MeOH (20:30:50) L TAMI Industries MF 0.14 μm/300 ZrO2/C T R 20 600 Flux: ~400 kg·m2·h−1, MeOH, Cheng et al. (2009)
kDa FAME and oil content
(percent by weight) in
permeate: 85, 15 and 0
Biodiesel-glycerol-ethanol L Shumacher MF 0.2 α-Al2O3 T C 60 4.0 Flux: 59.5–78.4 kg.m2.h−1, Gomes et al. (2010)
(70–85:10:5–20) GmbH glycerol rejection:
(Ti 01070) 98.1–99.6%, permeate
glycerol content (percent
by weight): 0.04–0.19,
methanol = 1.0:0.0036:
1.4:5.0:92.4:00.14
FAME:glycerol: L Sterlitech UF 100 kDa Polyacrylonitrile Flat sheet C 25 5.52 Flux: ~10 L.m−2.h−1, glycerol Saleh et al. (2010)
water = 99.76:0.04:0.2 Corporation rejection: 59–71%,
(Ultrafilic) permeate glycerol
(percent by weight): 0.013
Crude biodiesel L Jiangsu Jivwu UF 0.05 μm Al2O3/TiO2 T R 40 2.0 Permeate flux: 22.7 kg.m2.h−1 Atadashi et al.
Hitech Co. (2015c)
FAME:ethanol: L Laboratory made UF 7.0 kDa PVDF Flat sheet C 30 5.0 Permeate flux: 9.5 L·m2·h−1, Torres et al. (2017)
glycerol = 87:7.8:1.74 glycerol rejection: 67%
Note: TMP = transmembrane pressure, PVDF = polyvinylidene fluoride, CFTRI = Central Food Technological Research Institute, L = laboratory scale, UF = ultrafiltration,
MF = microfiltration, ME = membrane extraction, R = recycling, C = concentration, T = tubular.
Figure 19-6. Schematic of a rector combined with the membrane module for the
simultaneous production and purification of biodiesel.
the product (biodiesel) or retaining the unreacted triglycerides within the reactor
(Dube et al. 2007, Saleh et al. 2010, Baroutian et al. 2011). Biodiesel production
and further separation in a membrane reactor operates on the basis of the
membrane separation principle and depends on oil droplet size or by catalytic
membrane (Table 19-2). Membrane separation that works on the basis of oil
droplet size involves a microporous membrane, which is usually a ceramic
membrane or carbon membrane. Because of differences in polarity, methanol
is immiscible with oil and lipids; thus, a mixture of methanol and lipid will exist in
a two phase system or as an emulsion of lipid droplets suspended in a methanol-
rich phase. The lipid and methanol immiscibility is the key basis of the mass
transfer limitation in the transesterification reaction, but this emulsified system is
favored for operation of a membrane reactor. In the emulsified system, the
transesterification reaction is believed to occur at the interface between lipid
droplet and the continuous methanol phase in which they are dispersed. Biodiesel,
glycerol, and catalyst are soluble in methanol. Thus, the unreacted lipids remain
suspended and dispersed in a mixture of methanol, biodiesel, glycerol, and catalyst
on the membrane retentate side. Because of the smaller molecular size, methanol
and other soluble components, such as biodiesel, glycerol, and catalyst are able to
pass through the microporous membrane into the permeate stream when
membrane TMP is increased. Meanwhile the emulsified lipid droplets with larger
molecular size are trapped within the membrane to be continuously converted
into biodiesel (Figure 19-6, Saleh 2011, Shuit et al. 2012).
Both organic and inorganic membranes are used to design the membrane
reactors for biodiesel synthesis and purification. The membrane reactor provides
several advantages over conventional reactors. The advantages are (1) a combi-
nation of reaction and separation into a single process that results in reducing
Table 19-2. Summary of the Membrane Reactors Used for Production and Purification of Biodiesel.
470
FAME content in
Catalyst Type of Operating permeate
Feedstock (molar (percent membrane Temp pressure Oil to FAME (percent by
ratio) by weight) Scale Membrane characteristics reactor (°C ) (kPa) conversion (%) weight) References
Soybean oil: Solid acid L PVS/SSA, flat sheet C 60 101 — 0.7 Guerreiro et al.
methanol (1:5) (2006)
Canola oil/methanol H2SO4 L Koch, carbon, tubular, I 60–70 138 25–92/35—64 — Dube et al.
pore size: 0.05 μm (2007)
Canola oil/methanol NaOH L Koch, carbon, composite, I 55 207 88.98.7 20–50 Cao et al.
(1:11–46) tubular, pore size: (2007)
0.05–1.4 μm
Canola oil/methanol NaOH L TAMI, composite ceramic I 65 ~275 — 55–60 Cao et al.
(1:23.93) (TiO2), tubular, MWCO: (2008)
300 kD
Canola/methanol NaOH L TAMI, composite ceramic I 65 32.3–46.3 90—100 28.4–59.8 Tremblay et al.
(1:1) (TiO2), tubular, MWCO: (2008)
300 kD
Canola/methanol NaOH L TAMI, composite ceramic I 65 32/42/46 32.7/95.5/98 0.5/1.6/1.6 Cao et al.
(1:1) (TiO2), (2009)
Palm oil/methanol KOH/AC L Atech Innovation Gmbh, I/PB 70 — 94 — Baroutian et al.
(1:1) TiO2/Al2O3, tubular, (2011)
pore size 0.05
Triolein/methanol/ Microencapsulated L Carbosep (Novasep, Inc., C 30 6 — — Badenes et al.
ethanol/butanol cutinase Boothwyn), tubular (2011)
ceramic membrane,
NMWCO 15 kDa
Note: PVS/SSA = polyvinyl alcohol/ sulfo succinic acid, TAMI = TAMI Industries, L = laboratory, C = catalytic membrane, I = inert, I/PB = inert/packed bed, NMWCO = nominal
molecular weight cutoff.
separation costs and recycling necessities; (2) an improvement of thermodynami-

cally restricted or product-inhibited reactions resulting in higher transformation
per pass; (3) a precise interaction of incompatible reactants; and (4) a removal of
undesired side reactions (Dube et al. 2007).
Dube et al. (2007) designed a membrane reactor of 300 mL volume to

produce biodiesel using a carbon membrane (Koch Membrane Systems, Wil-
mington, Delaware). The pore size of the carbon membrane used was 0.05 μm.
The inside and outside diameters of the membranes were 6.0 and 8.0 mm,
respectively. The length of the carbon membrane tube was 1,200 mm giving a
surface area of 0.022 m2 for the entire membrane. The synthesis experiments
were carried out using canola oil (100 g) at 60, 65, and 70 °C in the reactor
containing a methanol premixed mixture for 6 h with 0.5, 2, 4, and 6% by weight
concentrations of sulfuric acid as catalyst. They controlled the pressure at 138
kPa between the reaction side and permeation side. The authors reported that
microporous carbon membrane reactors can selectively permeate FAME,
methanol, and glycerol during transesterification from the reaction zone. The
molecule of canola oil is trapped in droplet forming and emulsion. The droplets
cannot pass through the pores of the membrane because their size is larger than
the pore size of the carbon membrane. Results reported that during the reaction,
canola oil did not appear on the permeate side.
Baroutian et al. (2011) used a packed bed continuous membrane reactor
comprising a catalyst supported through activated carbon to avoid the permeation
of catalyst through the membrane. The activated carbon ranging from 550 to 810
μm in size was oven dried to activate at 100 °C for a day, and activated carbon was
added into the potassium hydroxide solution. The mixture was subsequently
agitated at a temperature of 25 °C for 24 h. The total adsorbed potassium
hydroxide on activated carbon was 30.31% w/w. Subsequently, the catalysts were
packed inside the tubular TiO2/Al2O3 ceramic membrane reactor. The biodiesel
was produced by using the palm oil in a membrane reactor. The highest oil-to-
FAME conversion for this packed bed membrane reactor was 93.5% (Baroutian
et al. 2011). The conversion achieved by the membrane reactor was comparable
with addition of H2SO4 or KOH catalysts. Moreover, it was reported that high-
quality biodiesel was produced from such a reactor without washing or purifica-
tion steps (Baroutian et al. 2011).
Another approach of applying the membrane bioreactor is through a catalytic
membrane involved as nonporous dense membrane, such as poly(vinyl alcohol).
The operation of this type of membrane works on the basis of interaction between
the target component and the polymer functional groups of the membrane.
During the synthesis process, catalytic membrane, glycerol, and methanol are able
to form hydrogen bonds with OH groups in the polymeric membrane. Therefore,
glycerol and methanol are continuously removed from the mixture during the
reaction. Meanwhile, the unreacted lipids and the produced biodiesel are retained
within the membrane because of their difference in chemical properties with the
polymer group of the membrane. In this case separation occurred under
atmospheric pressure (Shuit et al. 2012). The advancement in the catalytic

membrane development is continuing. Shi et al. (2010) prepared two organic–
inorganic hybrid catalytic membranes {Zr(SO4)2/sulphonated poly(vinyl alcohol)
(SPVA) and Zr(SO4)2/poly(vinyl alcohol) (PVA)} and evaluated their catalytic
efficiency. The researchers reported that the Zr(SO4)2/SVPA hybrid membrane

showed higher catalytic (esterification) efficiency (94.5%) and stability compared
with Zr(SO4)2/PVA hybrid membrane, which showed 81.2% of conversion during
biodiesel production from free fatty acid in acidified conditions.
Pervaporation technique has also arisen as an attractive process in biodiesel
production and purification, which is based on the separation of components on
relative rates of permeation through a membrane. Pervaporation is different from
normal membrane separation in that it combines the permeation and evaporation
in a single module. A phase change of molecules occurs that permeates through
the membrane in the direction of the downstream side (Shuit et al. 2012, Abels
et al. 2013). Separation occurs when a liquid stream that contains two or more
components is placed in contact with one side of a nonporous polymeric
membrane or molecularly porous inorganic membrane, and the components
sorb into/onto the membrane. The penetrated molecules diffused through the
membrane and evaporate owing to the chemical potential difference across the
membrane induced by vacuum or gas purge. The pervapoation process is shown in
Figure 19-7 (Jaimes et al. 2014). The separation of the different components is
achieved when some of the components preferentially diffuse across the mem-
brane (Jiang et al. 2009). Moreover, the advancement of membranes is continuing
to replace polymeric and microporous membranes by synthesizing carbon
molecular sieve membranes (CMSMs). CMSMs are highly porous and possess
small selective pores in the range of 3 to 6 Å (Tin et al. 2011).
Figure 19-7. (A) Vacuum pervaporation, and (B) purge gas pervaporation.
19.3.3 Advantages and Disadvantages of Purification

Technologies
The primary objective of crude biodiesel purification is to achieve the purity and
quality of biodiesel according to the standards set by EN 14214 and ASTM D6751
for diesel engine use. Purification of the crude biodiesel is a main factor for its
commercial production and utilization. Thus, the continuous development of the
purification technologies to purify biodiesel has raised expectation for industrial
production of biodiesel and practical usability. Moreover, attainment of high-
quality biodiesel fuel could offer benefits such as reduction in elastomeric seal
failure; decrease in fuel injector blockage and corrosion resulting from the absence
of glycerol, catalyst, and soap; reduced degradation of engine oil; better lubricating
properties; and improved quality exhaust emissions. Furthermore, high-quality
biodiesel could also decrease the chances of fuel tank corrosion, suppression of
bacterial growths, and congestion of fuel lines and filters as well as the extinction
of pump seizers originating owing to higher viscosity at low temperature (Berrios
and Skelton 2008, Atadashi et al. 2011c). The merits and demerits of the different
purification methods are summarized in Table 19-3.
19.4 QUALITY TESTING OF BIODIESEL
The purified biodiesel (FAME) quality must be evaluated to meet the international
standards (as discussed in Chapter 1). In general, biodiesel standards identify the
parameters that pure biodiesel must meet before being used as a pure fuel or being
blended with distillate fuel (Monteiro et al. 2008, Leung et al. 2010). To meet the
standard quality, assessment of biodiesel is carried out by determining some
chemical characteristics such as the acid value, saponification value, iodine value,
calorific value, cetane number, flash point, ash content, refractive index, viscosity,
specific gravity, fatty acid composition, among others. Moreover, mono alkyl
esters, glycerol, alcohol, catalyst, FFA, TG, DG, and MG composition of the final
biodiesel are also important. The presence of these components in biodiesel is
responsible for severe operational and environmental problems. Thus, quality
control of biodiesel is very important for its commercialization and market
acceptance (Monteiro et al. 2008, Weiksner et al. 2008).
To determine the specific quality, the preferred analytical methods are
chromatographic and spectroscopic methods. A suitable analytical method would
be able to reliably quantify all the specific values even at trace levels with
experimental ease (Ibeto et al. 2011). Among the chromatographic methods gas
chromatography (GC) is the most robust instrument to quantify the FAME as well
as TG, DG, and MG after derivatization. The most commonly used derivative
agents are N,O-bis(trimethylsilyl)trifluoracetamide (BSTFA) and N-methyl-N-
trimethylsilyltrifluoroacetamide (MSTFA). Columns used for separation are (5%-
phenyl)-methylpolysiloxane capillary column, capillary column of 95% dimethyl-
Table 19-3. Merits and Demerits of Different Purification Technologies.

474
Method Merits Demerits References
Wet washing • Excellent in methanol removal • Emulsion formation, wastewater Berrios and Skelton (2008)
treatment, no effect on glycerides
and drying of final product
• Can reduce methanol, soap, and free • Consumption of water and Na2SO4 Van Gerpen (2008), Canakci and
glycerol level below the amount result in high biodiesel cost Van Gerpen (2001)
needed by EN14214, and biodiesel • Considerable loss in product due to
purity up to 99% formation of soap and emulsion,
and treatment further demands

high energy cost
Dry washing • Magnesol can remove free and • — Faccini et al. (2011), Berrios and
bonded glycerol, soap, and • Chemical composition of the resin is Skelton (2008)
potassium difficult to predict and little effects
• Magnesol has the potential to on methanol
replace water washing • Involve many consumables that
• Can effectively remove residual demands high cost, require
methanol, catalyst traces significant resources, larger size of
• Can augments the stability of powder grains making them
biodiesel in the oxidation process, exceedingly difficult to remove that
moreover, it has the ability to cause an abrasive contaminated fuel
remove sulfur
• Can save time, lower energy, save Bertram et al. (2005)
capital cost and lead significant
disposal cost reduction
Membrane • Provides good yield, high purity and • Organic membranes are less He et al. (2006)
purification quality products that are stable and easily get swollen in
comparable to the conventional organic solvent
diesel fuels, simple, demands less • No large-scale industrial
energy consumption, applications
environmental friendly and energy
savings
• Reduction in separation and Saleh et al. (2010)
purification costs, improved fuel
quality, and high recovery of
valuable products
• Zero waster washing with no waste
discharges, and provide biodiesel
with less glycerol content
Membrane • Membrane reactor permits the • Required more research to Dube et al. (2007), Oh et al. (2012)
reactors reaction and separation to occur commercialise the process on an
within a compartment industrial scale
• Membrane reactor ensures the • Membrane fouling due to soap
reversible reaction proceeds fast formation
towards forward directions due to
removal of product simultaneously
• Membrane reactor makes the
RECOVERY AND PURIFICATION TECHNOLOGIES OF BIODIESEL
process less time consuming,

energy saving and less wastewater
generation during the purification
process
475
5% diphenyl polysiloxane, HP-1 wide-bore column, and polar capillary column

(Agilent J&W). The detection of separated components through GC is carried out
either by using flame ionization detector (FID) or mass spectrometric detector
(MSD). MSD is preferred because it eliminates the uncertainties about the nature
of the eluting components due to mass spectra unique to individual compounds

(Wawrzyniak et al. 2005, Monteiro et al. 2008, Wawrzyniak and Wasiak 2008,
Pauls 2011). For determination of free glycerol and methanol simultaneously in
biodiesel, Mittelbach et al. (1996) reported on the GC-FID or GC-MS methods.
The method used the fused silica 60 × 0.25 mm, 0.25 μm DB 5MS (Agilent J&W)
capillary column for separation, followed by detection with FID or MS detector.
High performance liquid chromatography is used to quantify the components
of biodiesel such as FAME, FFA, TG, DG, MG, and methanol. GC is fast compared
with liquid chromatography. However, GC requires more time for derivatization.
The columns used for separation in HPLC are cyano-modified silica columns
coupled with gel permeation chromatography (GPC), normal phase as well as
reverse phase columns. The commonly used detectors in the HPLC system are
evaporative light scattering detector (ELSD), ultraviolet (UV) detector (205 and
210 nm), refractive index (RI) detector, and pulsed amperometric detector (PAD).
The liquid chromatography mass spectrometry (LC-MS) is also used to quantify
the components of biodiesel. However, it is more expensive and complicated for
routine analysis (Tyagi et al. 2010, Ibeto et al. 2011, Pauls 2011).
The commonly used spectrometric methods are nuclear magnetic resonance
(NMR) and near-infrared (NIR) spectroscopy and mainly analyze biodiesel during
the transesterification reaction. Both spectroscopic methods assure peaks char-
acteristics for TG and FAME in the spectra and show how much conversion of TG
to FAME has progressed. The spectroscopic methods are fast to carry out without
derivatization. Scherer and Kosman (2007) evaluated the Thermo Scientific
Antaris II Fourier transform near-infrared (FT-NIR) analyzer to quantify the
concentration of biodiesel impurities (e.g., water, FFA, TG, DG, MG, methanol,
free glycerol, and total glycerides). The Antaris II FT-NIR was accurately
quantifying the concentration of impurities from biodiesel within a very short
time. The researchers concluded that this instrument can be used for fast analysis.
Moreover, this instrument can also be applied for online monitoring of impurities
during a production process.
Inductive coupled plasma optical emission spectrometry (ICP-OES) has been
used for multielement analysis in biodiesel. However, elemental analysis in
biodiesel by ICP instrumentation requires some specification to get good results.
The specifications required are good organic capabilities, high resolution optics for
peak separation, high saturation resistance of the detector to resist blooming from
high carbon and diatomic carbon emissions, as well as rapid multielement analysis
(Tyagi et al. 2010, Ibeto et al. 2011). Pauls (2011) reviewed the application of
chromatographic methods for the characterization of biodiesel and its blends, and
reported all the chromatographic protocols in detail for quantitative analysis of
FAME composition and the content of impurities such as methanol, glycerol, TG,
DG, MG, and sterol glucosides until now.
19.5 STORAGE, STABILITY, AND TRANSPORTATION
Biodiesel should confirm the particular standard, if it is stored for a long time.
There are many vital factors that need to be considered for the stability of
biodiesel, such as exposure temperature, oxidative stability, fuel solvency, and

material compatibility. The storage temperature for biodiesel should be controlled
between 7 °C and 10 °C to avoid the formation of crystals, which are responsible
for plugging the fuel lines and fuel filters. In extremely cold climates, underground
storage of pure biodiesel is needed, with further support of the external heating
system to avoid crystal formation (Leung et al. 2010).
Besides the storage temperature, the stability during storage of biodiesel also
depends on the factors such as degree of unsaturation of vegetable oils used,
content of residual glycerol, triglycerides, free fatty acids, water, and metals. The
unsaturation of vegetable oil derivatives is susceptible to thermal and oxidative
polymerization. The polymerization may lead to the formation of insoluble
product that triggers the problem within the fuel system. Feedstocks with a larger
proportion of saturated fatty acids are more stable than those having a higher
proportion of unsaturated fatty acids, but a higher proportion of saturated fatty
acids lowers cloud and pour points. Hence a major drawback of biodiesel lies in its
compromise between the level of saturation of biodiesel and its cold flow property
(Banga and Varshney 2010).
Biodiesel produced from plant oils and other feedstocks are more susceptible to
oxidation compared with petroleum diesel unless treated with additive or modified.
Biodiesel prepared from commonly used feedstocks such as oils from soy, rapeseed,
palm, lard, and tallow contains the fatty acid carbon chains of mainly 16 or 18 atoms
with 0 to 3 double bonds represented as C18:0-3 in the chain. The fatty acid ester
containing higher double bonds has a relatively high rate of oxidation (C18:3 >
C18:2 > C18:1) because the tri or di-unsaturated fatty acids contain the most
reactive sites for initiation of the auto-oxidation chain reaction. The oxidation is
initiated due to presence of peroxide radical, which is formed in the presence of air
or other pro-oxidizing condition even at ambient temperature during storage of
biodiesel (Bouaid et al. 2007, McCormick and Westbrook 2010). The FAME is
cleaved by peroxide radicals into acids and aldehyde, or they can react with another
fatty acid chain to form a dimer. The oligomer formation leads to the formation of
insoluble material (sediment). Besides oxidation and dimer formation, some other
factors are also responsible for molecular weight increment and insoluble material
formation through aldol condensation of forming aldehydes (McCormick and
Westbrook 2010). Further, degradation is accelerated by the presence of oxygen,
water, and impurities (glycerol, free fatty acids, and metals). Degradation of
biodiesel may form corrosive products such as acids that may affect negatively
fuel injection equipment and form insoluble gums and sediments, which can plug
fuel filters. The reaction rate in autoxidation schemes is dependent on hydrocarbon
structure, heteroatom concentrations, heteroatoms speciation, oxygen concentra-
tion, and temperature (Banga and Varshney 2010). The unreacted TG may be
oxidized or dissociate over time, which results in the formation of free glycerol.
Furthermore, free glycerol could polymerize or be oxidized (Banga and Varshney
2010). Presence of free fatty acids causes instability in vegetable oils. FFA may
degrade or cause corrosion and thermal instability and can undergo oxidation and
break down into aldehydes, ketones, epoxides, and alcohols. Presence of metals
catalyzes oxidation and polymerization reaction of hydrocarbons. Heavy metals
promote auto-oxidation. Transition metals such as Cu and Fe may be leached if
corrosion occurs during the manufacturing process. Alkali metals (Na, K, Ca, and
Mg) can form sediments and cause injector failures. Alkali metals could also form
soaps and contribute to insoluble or water haze in diesel blends (Banga and
Varshney 2010).
The stability of biodiesel is usually evaluated by determining the different
parameters such as the acid value (AV), peroxide value (PV), viscosity, iodine
value (IV), and insoluble impurities (sediment). The increase in PV, AV, viscositys
and insoluble impurities (II) with a decrease in IV is an indication of degradation
of biodiesel (Bouaid et al. 2007). The long-term storage test such as ASTM D4625
has been used to evaluate oxidation stability. Aging effect in long-term storage test
can be measured by change in viscosity, which arises in relation to stability of B100
biodiesel fuel, initiated by polymerization reactions that occur because of degra-
dation of B100 (Banga and Varshney 2010). Water content in biodiesel increases
biodiesel degradation owing to hydrolysis. The condensed water from long-term
storage can support the microbial growth, which may lead to formation of biofilm
or slime. The biofilm or slime can be detached from tank walls, form sediment,
and clog the fuel filters. Furthermore, ice crystals formed caused by the presence of
nucleation sites would accelerate the gelling of the biodiesel (Fernando et al. 2007,
Atadashi et al. 2012b).
The stability of biodiesel during storage has been enhanced by the presence of
natural and synthetic antioxidants. The natural antioxidants include tocopherols
(α, β, and γ) and sterols, and synthetic antioxidants are tert-butyhydroquinone
(TBHQ) and pyrogallol (Pyro). Among the three tocopherols, γ-tocopherol has
been reported to be more effective (BIOSTAB 2003; Bondioli et al. 2003, Bouaid et
al. 2007). Mushrush et al. (2011) evaluated the storage tank stability of soybean
oil–derived biodiesel by ASTM D5304, which defines instability as the formation
of 2 mg of sediment/100 mL of fuel. The authors concluded that pure soybean–
derived biodiesel failed the air-water test by ASTM D5304 while stable in a blend
of 5% to 20% v/v with petroleum.
19.6 FUTURE WORK AND PROSPECTS
The advancement in the separation and purification technologies is continuing

and proving new technologies are better than conventional technologies. Trans-
esterification step is a main step of biodiesel synthesis process and plays a key role
in selection of a biodiesel purification technique. Improvement in the separation
process is continuing with the application of an external additive like sodium

chloride. Thus, research and development work should continue to enhance the
current settling process of crude biodiesel and glycerol. Moreover, current
advancement in centrifuges also needs to be implemented to enhance the rate
of glycerol and biodiesel separation. In addition, material used for development

and fabrication of centrifuges should be rust resistant.
Washing steps applied for the current purification of the crude biodiesel need
to be replaced with dry washing procedures, which will reduce the generation of
wastewater. Moreover, the drying materials from the waste material such as rice
husk ash need to be explored. The use of waste material will reduce the cost of
currently applying resins in the process. New technology such as coalesce
technology has proven the potential to fasten the process in combination with
the centrifugation process. The new technologies like heterogeneous transester-
ification and transesterification via enzymatic have proven much easier for
separation and purification processes. The heterogenous catalyst should be
explored further to reduce the separation and purification cost applied in the
current industrial practice such as homogeneous catalysis processes.
Further, the membrane technologies have proven to be more efficient and
eco-friendly and should be explored further. Membrane technology or membrane
reactors have the potential to be applied for simultaneous biodiesel production
and purification process. Moreover, the membrane reactors with hybrid catalytic
membranes have shown the potential to replace the use of external catalyst.
Pervaporation technique has also arisen as potential membrane technology. These
membranes, although a little expensive, have not yet been commercialized. The
cost of the membranes could be brought down with more research and develop-
ment work. In addition, use of membrane technology has the potential to reduce
the generation of wastewater.
The analytical equipment and techniques for the quality evaluation of the
final product should be explored further to minimize the lead time and cost.
Further, research should be conducted on the integrations of online equipment for
measurement or monitoring the quality of the product during production and
purification steps. This will help to reduce the separation quality time as well as the
monitoring of the process separately.
The stability of the final product could be improved with the use of synthetic or
natural antioxidant additives; therefore, investigation work should be conducted on
the new stabilizing material to improve stability. Further, extraction of natural
stabilizers from other natural sources or some biotechnological approaches could be
applied to enhance the production of natural stabilizers. The technology like plant
tissue culture or plant cell culture could be of potential interest.
19.7 SUMMARY
In summary, separation and purification methods are the key steps for post-
biodiesel synthesis reaction. The efficiency and economy could be improved by
using the new technology of separation and purification methods. Both conven-
tional and new methodology have their advantages and disadvantages, although
homogeneous catalyst such as sodium and potassium hydroxide indicates faster
rates at commercial scale biodiesel production. The transesterification reaction
involving these catalysts generates soap, leading to great difficulties in the

separation and purification of the biodiesel from product mixture.
The development of lower cost heterogeneous catalysts for the production of
biodiesel should be encouraged to overcome the effects of soap formation. These
immensely contribute in lowering the cost of biodiesel separation and purification
processes. It is observed that application of water for the purification of biodiesel
leads to generation of a large quantity of wastewater, energy and time consumption,
and low biodiesel yields. The use of the higher oil-to-methanol ratios in the
transesterification reaction will contribute to the high cost of biodiesel synthesis
and purification. The use of raw materials with water content above the standard
specification would result in the deactivation of the catalyst in some cases and
promotes soap formation. The use of higher fatty acid vegetables oils and animal fats
was observed to promote saponified products and thereby contributed greatly to the
difficulties encountered in the separation and purification of biodiesel from
transesterified products. The introduction of membrane technology has minimized
the difficulties encountered in the separation and purification of biodiesel. The
potential applications of this technology need to be determined for separation and
purification of biodiesel product mixture. Scale-up of membrane separation and
purification for commercial application is important. Using new and advanced
analytical equipment is also helpful for fast quality determination of biodiesel.
The authors thank the Natural Sciences and Engineering Research Council of
Canada (Grant A4984, RDCPJ379601-08, and Canada Research Chair) for their
financial support.
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CHAPTER 20
Purification of Biodiesel Using
Resins and Adsorbents

L. R. Kumar
S. K. Yellapu
R. D. Tyagi
20.1 INTRODUCTION
Besides biodiesel/fatty acid methyl esters (FAMEs), and glycerol, several impuri-
ties are formed in the transesterification process (Banga and Varshney 2010).
The impurities are mainly residual catalyst, an excess of alcohol and metals,
unconverted fat, soap, water, and acidity. The presence of these impurities affects
the performance of biodiesel (Banga and Varshney 2010). The presence of soap
contributes to corrosion problems in the engine, and the presence of metal ions
can catalyze oxidation and polymerization reactions (Banga and Varshney 2010).
The presence of water in biodiesel reduces heat of combustion, promotes gel
formation in fuel, and encourages bacteriological growth that causes blockage of
filters (Atadashi et al. 2011). The final biodiesel obtained after purification should
meet ASTM standard specifications (Banga et al. 2014) for good biodiesel
performance. Table 20-1 summarizes the effects of several impurities on biodiesel
performance.
Several purification methods have been used for biodiesel purification like
gravitational settling, centrifugation, dry washing, membrane purification, and
membrane bioreactor (Table 20-2). Problems associated with these methods are
their incompatibility with continuous large-scale purification processes because
they are time-consuming, expensive, or energy intensive. Alternative methods
should be considered.
Recently, several resins and adsorbents have been reported to purify biodiesel
from soap and metal contaminants. LEWATIT GF202 has been reported for
purification of biodiesel from waste cooking oils (Berrios et al. 2011). Another
resin, PUROLITE PD 206, has been reported for removing impurities like soap,
free and bonded glycerol, and potassium from the biodiesel (Banga et al. 2014).
485
Table 20-1. Effects of Contaminants on Biodiesel Performance.
Impurities Effects on performance
Glycerol • Fouling of injectors

• Biodiesel storage problem

• Settling problems
• Emission of aldehydes and acrolein
• Deposits at bottom of fuel tank
• Destruction of rubber seals and gaskets
Catalyst/soap • Filter plugging
• Weakening of engines
• Damage of injectors
• Corrosion problems in engines
Free fatty • Corrosion problems in engines
acids (FFAs) • Less oxidation stability
Glycerides • Results in turbidity
• Crystallization
• Higher viscosities of biodiesel
• Deposits formation at pistons, valves, and injection
nozzles
Water • Reduces heat of combustion
• Corrosion of system components (such as fuel
tubes and injector pumps)
• Failure of fuel pump
• Hydrolysis (FFAs formation)
• Formation of ice crystals resulting in gelling of
residual fuel
• Bacteriological growth causing blockage of filters
Methanol • Lower flash points (problems in storage,
transport, and utilization)
• Lower viscosity and density values
• Corrosion of components having Al and Zn
• Deterioration of natural rubber seals and gaskets
• Lower flash points (problems in storage, transport,
and utilization)
• Lower viscosity and density values
• Corrosion of pieces of Al and Zn
ALX Enterprises DW-R10 is a dry ion exchange resin designed to remove salts,
soap, catalyst, glycerin, and water from crude biodiesel and is currently being used
in industries. The organic resin Amberlite BD10 DRY is used in pilot-scale tests in
which biodiesel is purified after passing through a column filled with resin. Besides
ion-exchange resins, inorganic adsorbents like Magnesol and silica have been
PURIFICATION OF BIODIESEL USING RESINS AND ADSORBENTS 487
Table 20-2. Conventional Methods for Biodiesel Purification and Their Associated
Problems.
Method Problems
Gravitational Time-consuming process and not favorable for

settling continuous operations
Centrifugation High investments and maintenance cost; energy
intensive process
Wet washing Requires two cycles of washing and one cycle of
with water centrifugation, time-consuming process and not
feasible in continuous process; the loss in the
product as a result of soap and emulsion formation;
high cost for further treatment of soap and
emulsion; requires drying of final product that
increases production costs and time, not feasible
in continuous operation; and a huge amount
of wastewater is generated that increases
treatment cost
Washing with Use of toxic and expensive solvents that requires
organic solvents further centrifugation step resulting in increased
production costs and time
Membrane Membrane fouling caused by soap formation; stability
technology issues with membrane because they swell in the
presence of organic solvents
reported to remove acidity and water content along with soap and metal
contaminants (Faccini et al. 2011). The inorganic matrix Magnesol is a synthetic
adsorbent composed of magnesium silicate and anhydrous sodium sulfate. It can
be used to remove contaminants such as water, soap, free glycerol, and glycerides
from biodiesel (Yori et al. 2007).
This chapter overviews recent studies reported on applications of ion-
exchange resins and adsorbents in biodiesel purification. The chapter explores
ion-exchange resins, commercial adsorbents, agricultural and industrial wastes in
biodiesel purification, advantages and disadvantages of each, process parameters,
comparison with wet-washing techniques, and improvements that can be done for
biodiesel purification using resins.
20.2 RECENT CASE STUDIES
20.2.1 Silica as Adsorbent for Biodiesel Purification

A single-step biodiesel purification has been attained with silica as an adsorbent by
optimization of reaction temperature, contact time, and adsorbent concentration
(Manuale et al. 2014), in which 100 cm3 of biodiesel was put in contact with silica
Trisyl 3000 (1 and 3 g) at different temperatures varying between 50 and 90 °C for
different contact times, 15 to 100 min. The vacuum condition of 20 kPa was
maintained for the treatment. After the treatment, treated biodiesel was filtered,
and the liquid obtained was analyzed for impurities. Adsorption capacity was first
optimized using different temperatures, reaction times, and the adsorbent
concentrations. It was found that a reaction time of 90 min, reaction temperature
of 90 °C, adsorbent concentration of 1.1%, and a vacuum pressure of 20 kPa gave
maximum adsorption capacity toward methanol, soap, water, and other impurities
(Table 20-3). The optimized conditions were used to purify crude biodiesel, and
the results obtained were compared with other dry-washing methods reported in
the literature.
Table 20-4 shows that silica trisyl 3000 under vacuum conditions and high
temperature (90 °C) displayed higher adsorption capacity when compared to
treatment conditions under atmospheric pressure (101325 Pa) and 65 °C. The
surface of silica is covered by one or more layers of adsorbed water, leaving no free
sites available for adsorbing impurities when the treatment is performed
at atmospheric pressure (101325 Pa). The retention of impurities occurs by
H-bonding through adsorbed water molecules. Under vacuum and high-
temperature conditions, the water is removed from the surface sites of silica, which
then are occupied by other impurities, leading to high adsorption of impurities
present in crude biodiesel. The technology described is advantageous because it is a
single step purification with no pretreatment to remove excess methanol. Moreover,
silica retains 33% of its weight when performed at atmospheric pressure (101325 Pa)
and nearly 235% of its weight when performed under vacuum conditions. Thus, it is
a simple process with enhanced utilization of adsorbent, eliminating high opera-
tional costs and use of large amounts of solvents/water in the wet-washing process.
In this study, silica displayed high capacity of adsorption and nonselectivity under
optimal process conditions, making it an excellent adsorbent to remove various
types of impurities in biodiesel.
20.2.2 Purification of Biodiesel Using Ion-Exchange Resins

Simultaneous production of high-quality biodiesel and glycerin from Jatropha
oil using ion-exchange resins as catalysts and adsorbents has been reported
(Shibasaki-Kitakawa et al. 2013). The cation-exchange resin Diaion PK208LH
and the anion-exchange resin Diaion PA306S were used in the process. The
temperature of each resin was maintained at 50 °C by hot water circulation
through the jacket. The solution mixture of oil and methanol was fed to the
bottom of the first column, and elute coming from the final column was analyzed
for reactants, FFAs, triglycerides, products, and fatty acid methyl ester (Figure 20-1).
The reaction was stopped when the FAME concentration in the elute from the final
column started decreasing owing to loss of the anion exchange resin’s catalytic
activity. The operating conditions were first optimized with different flow rates and
reaction times. It was found that a flow rate of 0.233 dm3/h with reaction time
Table 20-3. Adsorbtion Capacity of Silica with Optimized Reaction Conditions.

Time Soaps Water MeOH Free Monoglycerides Diglycerides Triglycerides
T (°C) (min) (%) (%) (%) glycerides (%) (%) (%) (%) FFA (%)
Acceptable 0.07 0.05 0.2 0.02 0.8 0.2 0.2 0.5

values
Crude 0 0.25 0.1 5 0.2 2.41 0.08 0.04 0.27
biodiesel
90 90 Nd 0.03 Nd 0.019 0.32 Nd Nd 0.16
Note: Nd = not detectable.
PURIFICATION OF BIODIESEL USING RESINS AND ADSORBENTS
489
490
Table 20-4. Comparison of Adsorption Capacity of Various Biodiesel Treatment Processes.

Adsorption capacity (%)
Adsorbent Cads (%) FG MG DG TG Soaps FFA Qtotal (%)
Pretreatment: The crude biodiesel was heated at 90 °C for 10 min to remove excess methanol
Treatment with adsorbents: T = 65 °C, P = 101325 Pa contact time = 20 min, slow stirring
Magnesol 1 24 5 6 8 16 5.9 64.9

Silica 2 11.5 2.0 2.5 4.5 8 4.7 33.2
Amberlite 1 — — — — 15.2 8.5 23.7
2 — — — — 2 4.7 6.7
Purolite 1 — — — — 14.5 9 23.5
2 — — — — 1 3 4
Pretreatment: None
Treatment with adsorbents: T = 90°C, P = 20 kPa, contact time = 90 min, stirring at 250 rpm
Trysil 3000 1.1 15.6 180.1 6.9 3.4 21.6 7.8 235.4
Source: Adapted from Manuale et al. (2014).
Note: Cads = adsorbent concentration, FG = free glycerol, MG = monoglycerides, DG = diglycerides, TG = triglycerides, FFA = free fatty acids, Qtotal = total adsorption
capacity.
Figure 20-1. Simultaneous production and purification of biodiesel using ion-

exchange resins.
Source: Adapted from Shibasaki-Kitakawa et al. (2013).
between 4 and 16 h would give a maximum FAME yield. Table 20-5 displays the
results of the analysis for various impurities and FAME yield.
The biodiesel purified using anion exchange resin Diaion PA306S met the EN
14214 standard values of impurities and FAME content. The process has many
advantages such as no requirement of upstream processing for refining the crude
biodiesel, and glycerin adsorbed by anion-exchange resin can be easily recovered
by supplying methanol. There was no decrease in catalytic activity of cation
Table 20-5. Analysis of Purified Biodiesel Obtained from Column Chromatography.
Content/parameter Product biodiesel EN 14214
FAME (% w/w) 99.3 ± 0.283 ≥96.5

Monoglyceride (percent by weight) 0.03 ± 0.014 <0.8
Diglyceride (percent by weight) 0.015 ± 0.007 <0.2
Triglyceride (percent by weight) 0.13 ± 0.099 <0.2
Acid value (FFA, mg/g) 0.05 ± 0.028 <0.5
Methanol (percent by weight) 0.09 ± 0.028 <0.2
Water content (mg/kg) 280 ± 28.3 <500
Free glycerine (percent by weight) 0.005 ± 0.007 <0.02
Total glycerine (percent by weight) 0.03 ± 0.014 <0.25
Group 1 metals (Na, K) (mg/kg) <2 ± 0 <5
Group II metals (Ca, Mg) (mg/kg) <2 ± 0 <5
Sulfur content (mg/kg) <5 ± 0 <10
Phosphorus content (mg/kg) <1 ± 0 <4
Total contamination (mg/kg) 8.5 ± 0.707 <24
exchange resin, whereas the decrease in catalytic activity of anion exchange resin
was observed. However, catalytic activity of anion exchange resin can be regen-
erated by sequential pass of (1) methanol to recover glycerin; (2) acetic acid in
methanol to displace fatty acid ion from resin; (3) NaOH aqueous solution to
displace acetic acid ion; (4) deionized water to remove NaOH solution; and
(5) methanol to restore the resin. The process could be helpful at continuous large-
scale operation, because scale-up of column operation can be easily performed
based on FAME productivity per hour per the anion-exchange resin’s weight
(dm3/h/kg-resin weight). However, a large number of solvents required to
regenerate the anion-exchange resin is a disadvantage of the process.
20.2.3 Wastes as an Adsorbent for Purifying Biodiesel

One of the studies reported use of rice husk ash as an adsorbent in biodiesel
purification, and the results were compared with two other purification methods
such as Magnesol (1% by weight) and traditional acid solution of aqueous H3PO4
(1% by weight) (Manique et al. 2012). Rice husk is a waste product of the
processing of grain. It is used in power generation through combustion, and its
burning generates another residue, the rice husk ash. By incineration (500° to
700 °C), rice husk can produce about 20% of weight in ash rich in amorphous
silica. In this study, biodiesel obtained after glycerin separation was preheated
at 65 °C and treated with different concentrations of rice husk ash (RHA; no
pretreatment, 1% to 5% w/w) for 20 min under slow stirring conditions. A similar
procedure was used for Magnesol treatment. For wet-washing treatment, biodiesel
was transferred to a separating funnel and treated with 1% H3PO4 at 55 °C. After
removal of the aqueous layer, the sample of biodiesel was washed twice with hot
water and the washed biodiesel was later subjected to heat treatment at 90 °C for
30 min to eliminate water. The samples were analyzed for impurities (Table 20-6).
Table 20-6 indicates that RHA with 4% (w/w) concentration produced similar
results when compared with the Magnesol treatment. Moreover, it displayed
efficiency in removing organic and inorganic impurities from biodiesel, making it
an effective alternative adsorbent for biodiesel purification. The process has
advantages such as the use of an agro-industrial waste, rice husk, which after
the process can be used as a soil corrective owing to its high biodegradable organic
content (biodiesel, glycerin, and residual oil) and potassium (catalyst).
In one of the studies (Santos et al. 2017), solid waste from the ceramic
industry (chamotte clay) was used as glycerol adsorbent for biodiesel purification
by dry-washing methods. In the study, a face centered composite design was used
to analyze the combined effect of chamotte concentration (varied between 2% and
8% w/v) and temperature (varied between 30 and 50 °C) on glycerol removal.
Based on graphical optimization models, the optimum glycerol concentration
(2.4% by weight) and temperature (45 °C) were found. Glycerol removal reached
1,282 mg/g within 30 min of adsorption time. Biodiesel obtained using biological
(immobilized lipase) and chemical catalysts (niobium oxide impregnated with
sodium) was purified using chamotte clay, and free glycerol was removed per
ASTM standards (<0.02% by weight). The high adsorption capacity can be related
Table 20-6. Analysis of Purified Biodiesel with Different Methods.

Acid number Methanol Free Total Water Potassium
Method (mg/g) (%) glycerine (%) glycerine (%) (mg/kg) (mg/kg)
Unpurified 0.33 ± 0.01 0.76 ± 0.02 0.00792 0.69 ± 0.02 2265.74 0.48 ± 0.03
Acid solution 0.18 ± 0.01 0.018 ± 0.01 0.00204 0.53 ± 0.01 3610.41 0.21 ± 0.03
Magnesol 1% 0.29 ± 0.01 <0.01 0.0023 0.45 ± 0.01 2023.48 <0.1
RHA 4% 0.13 ± 0.02 <0.01 0.0042 0.46 ± 0.01 1292.13 <0.1
EN 141214 <0.5 <0.2 <0.02 <0.25 <500 <5
Note: EN 14214 represents quality standards for purified biodiesel.
493
to the high silica (56% w/w) and alumina content (36% w/w) with porous
structure and large surface area. However, chamotte clay was unable to be
regenerated with organic solvents at 50 °C. In addition, chamotte–glycerol com-
posite can be reused in a brick formulation. Chamotte clay is a low-cost substrate
with good adsorption capacity, which can be a promising adsorbent in biodiesel

purification. However, non-regeneration of the adsorbent is the main concern for
its use in industrial processes. Therefore, studies need to be conducted for its
regeneration at high temperatures.
Raw sugarcane bagasse has been used for biodiesel purification using a dry-
washing technique (Alves et al. 2016). Raw sugarcane bagasse was first cleaned
with distilled water and dried at 80 °C for 24 h. Next, 100 mL of crude biodiesel
was treated with adsorbent loading concentration ranging from 0.1% to 3% (w/v)
at 120 rpm and 30 °C for 120 min. It was found that 0.5% w/v sugarcane ash
resulted in 40% glycerol removal in crude glycerol for bringing glycerol content
lower than 0.02% by weight. The results for biodiesel purification were comparable
to that of Magnesol. However, sugarcane bagasse ash (obtained by heating raw
sugarcane bagasse at 700 °C for 4 h) performed poorly compared with raw
sugarcane bagasse because of its low content of cellulosic material. From the
adsorption kinetics, it was discovered that with the addition of 3% by weight of
sugarcane bagasse, the necessary glycerol removal was achieved after only 10 min of
adsorption process. The process has many advantages like low-cost adsorbent with
lower process time compared with the wet-washing technique. However, there are
disadvantages of the process in that it was unable to remove water per ASTM
standards, and regeneration studies have not been performed on the adsorbent.
20.3 COMPARATIVE STUDIES AMONG ADSORBENTS,

ION-EXCHANGE RESINS, AND WET-WASHING METHODS
A comparative study has been reported for use of ion-exchange resins and
adsorbents in biodiesel purification, and the results obtained were compared
with the wet-washing method (Faccini et al. 2011). The crude biodiesel obtained
after transesterification reaction was heated at 90 ºC for 10 min to evaporate the
excess of methanol. The crude biodiesel obtained after methanol evaporation was
heated at 65 °C under slow-stirring conditions. Then, 1% to 2% (w/w) of
adsorbents like silica, Magnesol, PUROLITE PD206, and Amberlite BD10 DRY
were added to preheated biodiesel, and the reaction occurred for 20 min at the
same temperature under slow-stirring conditions, followed by filtration to remove
the adsorbents. Acid number, soap, potassium content, water, and methanol were
analyzed for unpurified and purified biodiesel (Tables 20-7 and 20-8).
Tables 20-7 and 20-8 show that inorganic matrixes like Magnesol and silica
were effective in removal of glycerol, methanol, soap, potassium, water, and acidity
from crude biodiesel, whereas Amberlite was effective in removing potassium,
soap, and acidity but was not effective against methanol and water. Acid water
Table 20-7. Comparative Analysis of Unpurified and Purified Biodiesel Using

Different Methods.
Acid number Water Methanol
Method (mg/g) Soap (ppm) K (mg/kg) (mg/kg) (%)
Not purified 0.33 ± 0.01 1670.05 ± 7.3 23.2 ± 0.2 1300 ± 15 2.13 ± 0.1
Acid water wash 0.22 ± 0.01 158.12 ± 3.01 19.9 ± 1.6 1400 ± 15 0.18 ± 0.01
Magnesol 1% 0.17 ± 0.01 60.84 ± 2.1 1.1 ± 0.1 500 ± 6 0.19 ± 0.1
Silica 2% 0.14 ± 0.01 60.80 ± 2.5 Nd 500 ± 5 0.22 ± 0.1
Amberlite 2% 0.14 ± 0.01 182.62 ± 5.03 2.6 ± 0.3 900 ± 9 0.39 ± 0.01
Purolite 1% 0.15 ± 0.02 212.87 ± 5.3 10.5 ± 1.5 1200 ± 12 0.44 ± 0.1
EN 14214 0.5 — 5.0 500 ± 5 0.2
maximum
Note: EN 14214 represents quality standards for purified biodiesel, Nd = not detectable.
Table 20-8. Analysis of Free and Bonded Glycerol in Unpurified and Purified
Glycerol.
Purified biodiesel
Unpurified Acid water
Impurity biodiesel wash Magnesol 1% Silica 2%
Free glycerol (%) 0.26 0.01 0.02 0.03

Monoglycerides (%) 0.14 0.15 0.09 0.1
Diglycerides (%) 0.12 0.07 0.06 0.07
Triglycerides (%) 0.19 0.13 0.11 0.1
Note: Bold denotes maximum removal of impurity from biodiesel.
wash was effective in removing methanol content only. The main advantages of
using dry purifications are the reduction of aqueous effluents, making the process
environmentally friendly, and the substantial reduction in the total time of
production, because water washing requires the use of two cycles of washing
and one stage of centrifugation, which are time-intensive processes.
In another study, the efficiency of removing several impurities in biodiesel
was investigated using three different methods: (1) adsorption using magnesium
silicate and bentonite; (2) liquid–liquid extraction using distilled water, tap water,
and glycerol; and (3) cation-exchange resin (Berrios et al. 2011). The experiments
for adsorption were conducted at room temperature with 100 mL crude biodiesel
using different adsorbent concentrations (0.5%, 0.75%, and 1% w/w) and different
agitation rates (200, 400, and 600 rpm) for 15 min in a batch reactor. After
adsorption, the final product was separated by centrifugation at 4,000 rpm for
10 min, and samples were analyzed for impurities. Liquid–liquid extraction was
performed using three different agents: distilled water, tap water, and glycerol at
room temperature. Extraction was performed in single or multiple steps with
different concentrations (5%, 10%, and 15% by weight). The mixture was
vigorously shaken, followed by settling for 10 min and a centrifugation step for 10
min to obtain the final product. Sample analysis was performed using purified
biodiesel. A column (1.5 cm diameter) filled with Lewatit GF202 resin was
operated at room temperature with a flow rate of 1.5 to 2 bed volume h−1.
Lewatit GF202 is a sodium-based resin that does not impart H+ ions like other
resins and can be regenerated several times by flushing with methanol. The resin
was first equilibrated by washing with 3 to 4 bed volumes of methanol, and then
crude biodiesel was transferred into the resin. Samples from the elute were
analyzed after every 10 h of operation until the resin saturation was detected
at 54 h. Table 20-9 summarizes the results obtained from the study.
The results indicated that glycerol with 15% concentration (w/w) was
successful in eliminating all the impurities and meeting the standards even for
water content. However, it requires two cycles of washing and an additional
centrifugation step to get purified biodiesel. On the other hand, adsorbents like
Magnesol and bentonite were able to remove most of the impurities except water.
Similar results were displayed by ion-exchange resin Lewatit, which was effective
in removing most of the impurities except water. Because adsorbents would
require a holding tank during its operation, their feasibility in a continuous
purification process is less likely compared with ion-exchange resins, for which no
holding tanks are required and continuous purification can be achieved using two
columns in series for complete removal of impurities, resulting in purified
biodiesel complying with the final standard values.
In another study, biodiesel purification was done using three different
procedures: (1) wet-washing with distilled water; (2) dry washing with Magnesol;
and (3) ion-exchange resins, and results were compared (Sabudak and Yildiz
2010). Crude biodiesel obtained after two-step acid-base transesterification using
waste frying oils was subjected to glycerin and methanol separation. Thereafter, it
was treated with 1% Magnesol and the resulting mixture was heated at 70 to 80 °C
for 1 h followed by filtration. Ion-exchange resin (PD-206) was filled in a column,
and the crude biodiesel with 1.5 to 2 times bed volume was passed through the
column for 1 h. Wet-washing was carried out using soft hot water (100% w/w) at a
temperature of 50 to 60 °C, followed by settling for 10 h for separation of water from
biodiesel. The washed biodiesel was further heated at 110 °C for 20 min to remove
traces of water content. Table 20-10 compares the results from different procedures.
As shown in Table 20-10, ion-exchange resin was most effective in removing
impurities from biodiesel, but purification ability of Magnesol was comparable to
the ion-exchange resin. However, toxic waste produced from filtered Magnesol
requires further treatment, which would increase the process cost. Biodiesel
purified using ion-exchange resin met the ASTM standards.
In another study, purification of Jatrophas curcas–based biodiesel was
achieved using organic adsorbents like Amberlite BD10 DRY, Purolite PD206,
and Tulison T-45BD, and results were also compared with wet-washing methods
(Banga et al. 2014). The crude biodiesel (obtained after transesterification) was
preheated at 65 °C, 3% (w/w) of each adsorbent was added under slow stirring
conditions, and the reaction occurred for 25 min. Another set of purification was
Table 20-9. Analysis of Impurities in Purified Biodiesel with Different Methods.

Distilled Tap water, Glycerol, Magnesol, Bentonite,
water, 10% 10% by 15% by 600 rpm, 1% 200 rpm, 1% LEWATIT
by weight weight weight by weight by weight Resin
Ion EN 14214
Parameters Liquid extraction (two steps) Adsorption exchange maximum
Acid value (mg/g) 0.46 0.43 0.38 0.27 0.22 0.35 0.5
Density at 25 °C 0.861 0.864 0.866 0.875 0.863 0.88 0.9
(g/cm3)
Soap (g/g) 2.37 × 10−5 7.94 × 10−5 6.42 × 10−5 3.38 × 10−5 2.64 × 10−4 3.25 × 10−4 —
FAME content 92.1 91.4 91.8 90.9 89.9 91.4 —
(% by weight)
Water content 1,603 1,284 253 803 809 741 500
(mg/kg)
Methanol (% by 0.05 0.04 0.08 0.2 0.27 0.14 0.2
weight)
Free glycerol content 0.002 0.002 0.002 0.005 0.008 0.008 0.02
(% by weight)
Triglyceride content 0.02 0.04 0.06 0.04 0.05 0.03 0.2
(% by weight)
Diglyceride content 0.36 0.33 0.35 0.35 0.34 0.35 0.2
(% by weight)
Monoglyceride 0.49 0.5 0.51 0.8
content (% by
weight)
497

Table 20-10. Analysis of Purified Biodiesel Using Different Methods.
Maximum Washing Dry washing Ion-exchange

Parameter limits with water by Magnesol resin Purolite
Density (kg/m3) 900 885 882 882

Viscosity (mm2/s) 5 4.92 4.74 4.63
Flash point (°C) — 148 146 151
Water content 500 422 487 372
(mg/kg)
Fatty acid 0.5 0.38 0.26 0.23
number (mg/g)
Methyl ester (%) — 95.6 96.9 97.2
Note: Bold represents maximum limits for quality standards.
conducted with 10% (w/w) of adsorbent at room temperature. The purification

step was followed by filtration to remove adsorbents. Samples from purified
biodiesel were analyzed for impurities. Ten percent (v/v) of phosphoric acid was
added to distilled waster to prepare acid water for wet washing. The crude
biodiesel was treated with 10% acid water for 5 min, followed by settling in a
separating funnel. The biodiesel obtained was further washed with 10% (v/v)
water. The upper layer, containing the purified biodiesel was analyzed for
impurities. Table 20-11 summarizes the results obtained after analysis.
As shown in Table 20-11, crude biodiesel treatment with Amberlite at 3%
concentration and the temperature of 65 °C for 25 min was most effective in
removing soap, potassium, and methanol, but the treated biodiesel did not meet
ASTM standards for water and acid value. Similar values were obtained after
treatment with Purolite at 3% concentration. However, organic adsorbents were
more effective than the wet-washing technique. Amberlite was also effective in
removing free glycerol and bonded glycerol. Table 20-12 summarizes results for
bonded and free glycerol after treatment with Amberlite.
With all the organic adsorbents (at 3% w/w concentration with stirring time
25 min at 65 °C), the values for free potassium, residual methanol, and free and
bonded glycerol are found within the EN 14214 maximum limits. At the industrial
scale, the organic adsorbents can be used in the form of ion-exchange resins
packed in a column for continuous biodiesel purification processes.
20.4 RESEARCH ON IMPROVEMENT OF ION-EXCHANGERS AND

ADSORBENTS FOR BIODIESEL PURIFICATION
Following are the research gaps that were found in the literature for biodiesel
purification using ion-exchangers and adsorbents:
Table 20-11. Analysis of Biodiesel Purified by Ion-Exchangers.

Samples Density pH Soap (mg/kg) Acid value (mg/g) Water (mg/kg) K (mg/kg) Methanol (%)
Unpurified 0.87 10.03 1920 0.7 2010 12 5

EN 14214 Maximum — — — 0.5 500 5 0.2
Acid water washing 0.872 7.01 224 1.12 2290 2.8 0.05
Treatment: 3% (w/w) adsorbent at 65 °C min for 25 min
Amberlite 0.8717 6.61 192 0.84 860 3 0.1
Purolite 0.8713 7.3 192 0.98 860 2.2 0.12
Tulison T-45 BD 0.8716 8.8 288 0.98 860 2.6 0.07
Treatment: 10% (w/w) adsorbent at room temperature for 1 h
Amberlite 0.8710 6.5 256 1.26 860
Purolite 0.8710 6.88 224 1.12 570
Tulison T-45 BD 0.8706 8.1 192 1.12 1140
499
Table 20-12. Comparision of Bonded and Free Glycerol after Treatment with
Amberlite.
Free Monoglycerides Diglycerides Triglycerides

Sample glycerol (%) (%) (%) (%)
Unpurified 0.068 1.48 1.06 0.570

Amberlite: 3% 0.0074 0.071 0.094 0.035
at 65 °C for
25 min
• Using the industrial wastes reported in the aforementioned studies as

adsorbents was not effective to remove water and acid content per quality
standards. More research should be conducted in identifying low-cost
adsorbents that can remove all sorts of impurities.
• Most of the studies reported for use of wastes as adsorbents in biodiesel
purification were conducted at the laboratory scale. Pilot-scale studies should
be conducted for their industrial feasibility along with techno-economic
evaluation. Techno-economic studies for biodiesel purification are described
in Chapter 22.
• Regeneration studies for use of adsorbents and resins in dry-washing biodiesel
purification technique were missing. Regeneration studies should be con-
ducted with different solvents and temperature to avoid frequent replacement
of resins and adsorbents on a large scale because frequent replacement at the
industrial scale leads to increased process time and labor costs.
A strategy has been described for regeneration of ion-exchange resins and
adsorbents for their frequent use in biodiesel purification. Adsorbent of optimized
concentration will be treated with crude biodiesel for optimized reaction time
under continuous stirring. During adsorption, adsorption isotherms can be
described with the Freundlich model as follows:
y = kCn (20-1)
where
y = Impurity adsorbed per gm of adsorbent,
c = Concentration of impurity in biodiesel, and
n = Order of reaction, and k = rate constant.
Once adsorption is complete, the adsorbent/resin will be filtered and regen-
erated using methanol or by heating at 100 °C (methanol for resin and heating for
adsorbent). Methanol will dissolve the bound impurities in it and heating at high
temperatures can weaken the interactions between the adsorbent and bound
impurities. Desorption isotherms can be drawn during the regeneration process by
the following:
R = rN x (20-2)
where
R = Rate of desorption.
N = Concentration of adsorbed material, and

x = Kinetic order of desorption, and r = rate constant.
After the resin/adsorbent is regenerated, it will be treated with crude biodiesel
again under optimal conditions. Adsorption kinetics and isotherms will be drawn
after every regeneration cycle, and one can estimate numbers of regeneration
cycles with minimum reduction in adsorption capacity of resin/adsorbent. Once
this is optimized via batch studies, the regeneration studies can be performed at
the continuous scale to optimize the methanol volume required for regeneration.
20.5 CONCLUSIONS
Dry-washing methods such as ion-exchange resins and inorganic adsorbents were

more effective in removing residual catalyst, methanol, glycerol, acidity, and water
content when compared with wet-washing methods, because large amounts of
water and an additional centrifugation step is involved in the wet-washing process.
Wet-washing was not able to remove water and acidity per ASTM standards. Also,
wet-washing results in soap or emulsion formation and a large amount of
wastewater is produced which needs further treatment.
Magnesol and silica displayed good adsorption capacity for all types of
impurities. At high temperatures (90 °C) and under vacuum conditions, silica
acts as a nonselective adsorbent because the adsorption capacity of silica is very
high. For biodiesel purification, silica and Magnesol are good choices as adsor-
bents because the crude biodiesel treated with them met the ASTM standards. For
continuous biodiesel purification process, inorganic adsorbents can be added
along with ion-exchange resins in the column to prolong the life of the resin and
increase the removal efficiency.
Industrial and agricultural wastes can be used as low-cost adsorbents for biodiesel
purification, but they were unable to remove water and acidity from the crude
biodiesel per quality standards. Their non-regeneration is also a major concern.
Most of the ion-exchange resins reported in the literature were able to removal
glycerol, soap, K, and Na, but only a few were able to remove water and acid content
per ASTM standards. Because most of the cation-exchange resins are imported with
H+ ions, and they impart acidity (H+) in the biodiesel. Ion-exchange resins can be
operated as two columns in sequence for enhanced removal of impurities.
References
Alves, M. J., Í. V. Cavalcanti, M. M. de Resende, V. L. Cardoso, and M. H. Reis. 2016.
“Biodiesel dry purification with sugarcane bagasse.” Ind. Crops Prod. 89: 119–127.
Atadashi, I., M. Aroua, A. A. Aziz, and N. Sulaiman. 2011. “Refining technologies for the
purification of crude biodiesel.” Appl. Energy 88 (12): 4239–4251.
Banga, S., P. Varshney, N. Kumar, and M. Pal. 2014. “Optimization of parameters
for purification of jatropha curcas based biodiesel using organic adsorbents.” Int. J.
Renew. Energy Res. (IJRER) 4 (3): 598–603.

Banga, S., and P. K. Varshney. 2010. “Effect of impurities on performance of biodiesel:
A review.” J. Sci. Ind. Res. 69, 575–579.
Faccini, C. S., M. E. D. Cunha, M. S. A. Moraes, L. C. Krause, M. C. Manique, M. R. A.
56–61.
Manuale, D. L., E. Greco, A. Clementz, G. C. Torres, C. R. Vera, and J. C. Yori. 2014.
“Biodiesel purification in one single stage using silica as adsorbent.” Chem. Eng. J. 256:
372–379.
Sabudak, T., and M. Yildiz. 2010. “Biodiesel production from waste frying oils and its
quality control.” Waste Manage. 30 (5): 799–803.
Santos, F. D., L. R. V. da Conceição, A. Ceron, and H. F. de Castro. 2017. “Chamotte clay as
potential low cost adsorbent to be used in the palm kernel biodiesel purification.” Appl.
Clay Sci. 149: 41–50.
Shibasaki-Kitakawa, N., K. Kanagawa, K. Nakashima, and T. Yonemoto. 2013.
“Simultaneous production of high quality biodiesel and glycerin from Jatropha oil using
ion-exchange resins as catalysts and adsorbent.” Bioresour. Technol. 142: 732–736.
Yori, J., S. D’ippolito, C. Pieck, and C. Vera. 2007. “Deglycerolization of biodiesel streams by
adsorption over silica beds.” Energy Fuels 21 (1): 347–353.
CHAPTER 21
Management of Coproducts
from Biodiesel Production

T. T. More
R. P. John
G. S. Anisha
R. D. Tyagi
R. Y. Surampalli
21.1 INTRODUCTION
The last few decades witnessed a continuous search for alternative fuels to replace
the unsustainable fossil fuels by the production of biofuels. A large area of
cultivable land is currently being used for the cultivation of oil- or sugar-yielding
plants for the production of biodiesel. However, biodiesel production from
agricultural crops such as oilseeds generates conflict with the traditional use of
agricultural land for food production. Because of food security issues, the
alternative sources of biofuels such as micro- and macroalgae were introduced.
Oily or soapy washing water is generated during purification of raw biodiesel. The
production of biofuels creates another environmental problem because of the
generation of phytobiomass such as crop residues, oil cakes and algal biomass,
microbial biomass, and coproducts formed during the processes of biodiesel
production. Managing these wastes should be accomplished in an eco-friendly
manner to avoid further issues regarding environmental pollution. Biotechnolog-
ical conversion and/or value additions in an economical way are the most
attractive solutions for managing these coproducts. With an increasing need for
biodiesels and expanding markets for coproducts, the biodiesel production
approach can only become economically sustainable if the coproducts of biodiesel
production are fully utilized or managed. This chapter discusses the management
of the coproduct during biodiesel production and covers a wide array of
coproducts from biodiesel production and the current state of knowledge and
its management.
503
21.2 BIORESIDUE GENERATED AFTER HARVEST AND EXTRACTION

OF OIL FROM AGRICULTURAL BIOMASS
The cultivation of food and cash crops produces a huge amount of biomass and is
usually an issue of management. Around 200 billion tons of organic matter per
year are produced globally through the photosynthetic process. Most of these
organic matters are not directly edible by higher animals and cause environmental
problems (Philippoussis 2009). Organic matter including straw, cobs, and leaves
are general wastes produced during harvest. Oil cakes, algal biomass residues,
microbial biomass, and other wastes are generated during oil extraction. In
general, these wastes can be treated biotechnologically for value-added products.
21.2.1 Crop Residues during Harvest

Crop residues such as straw and other plant materials contain high amounts of
waste compared with what is generated from grains or seeds. Annual plants
(soybeans and cotton, among others) generate whole-plant waste such as roots,
stems, leaves, and fruit (such as pods) at every harvest. Tree species such as
Jatropha, Pongamia, and Madhuca are perennial plants, and harvests mean only
plucking beans or fruits. Thus, the biomass generated during harvest is mainly
pods and old leaves, and falling branches are considered as residue during
cultivation.
21.2.2 Coproducts Generated after Extraction of Oil from

Agricultural Biomass
Agricultural biomass residues, including oil cakes, are usually produced during oil
extraction. One liter of biodiesel from soybean generates 3.835kilograms soymeal,
and that from rapeseed generates 1.234 kilograms rapeseed meal (Popp et al.
2016). Oil cakes, algal biomass, and microbial biomass are generated during oil
extraction.
Oil cakes. Oil cakes are produced during the extraction of oil from seeds of
land plants. There are many edible or nonedible seeds used for the production of
biodiesel. Nonedible plants such as Jatropha curcus, Pongamia pinnata, Mahua
(Madhuca indica), Undi (Calophyllum inophyllum), Castor (Ricinus communis),
Simarouba glauca, cotton and edible vegetable oils yielding plants such as oil palm,
soybean, sunflower, and rapeseed are the major plants used for the production of
biodiesel (Ghadge and Raheman 2005, Bobade and Khyade 2012, Dalvi et al. 2012,
Mishra et al. 2012). Ten liters of biodiesel from soybean generates 38.3 kg of
soybean meal, and rapeseed generates 12.3 kg rapeseed meal (Taheripour et al.
2010). Jatropha is one of the major tropical nonfood crops used for the production
of high-grade biodiesel. Jatropha curcus is a major species cultivated for its oil.
Although the toxic oil is not edible (it contains poisonous substances including
curcin), it contains nearly 80% unsaturated oil. The seeds of this plant contain 25%
to 30% oil. Jatropha oil cakes are rich in macro-elements such as nitrogen,
phosphorus, and potassium (NPK) and are used as organic manure. The oil
MANAGEMENT OF COPRODUCTS FROM BIODIESEL PRODUCTION 505
extracted after pyrolysis of these cakes can be used for furnace fuel (Raja et al.
2011). Pongamia pinnata is another tropical tree producing high-grade oil seeds
for biodiesel (Bobade and Khyade 2012). Pongamia seeds contain 30% to 35% oil,
but the oil has high viscosity and needs preheating for engine work (Acharya et al.
2011). Mahua is a large deciduous tree found in India and Sri Lanka. The fruit
contains one to four seeds, and 20% to 50% of the seed kernel is oil. In general, the
oil cake is used as a fertilizer or animal feed (Puhan et al. 2005). Calophyllum
(undi) is also known as Indian laurel or Alexandrian laurel. It is a broad-leaved
evergreen tree usually found as a costal tree or rarely in inland. Most of its oil, 60%
to 70%, is found in the kernel of its fruit (Dalvi et al. 2012). Simarouba belongs to
the family Simaroubaceae. The seed of this plant contains about 40% kernel, 55%
to 65% of which is oil. Because of the high mineral content, the oil cake can be used
as organic manure (Mishra et al. 2012). Castor (Ricinus communis) is a plant able
to grow in subtropical and tropical regions. Castor seed contains 40% to 60% oil.
Algal biomass. Oleaginous algae, especially microalgae, are used to produce
oil. Oil from microalgae is considered to be a single cell oil (Yousuf 2012).
Microalgal biomass production helps in CO2 sequestration and wastewater
treatment along with biofuel production. Botryococcus braunii, Chlorella spp.,
Cylindrotheca sp., Dunaliella spp., Crypthecodinium cohnii, Isochrysis sp.,
Monallan thus salina, Nannochloris sp., Nannochloropsis sp., Neochloris
oleoabundans, Nitzschia sp., Phaeodactylum tricornutum, Schizochytrium sp.,
Tetraselmis suecica, and others are major microalgae, producing a high amount
of oil (Chisti 2007). Microalgae contain approximately 50% carbon by dry weight,
and this carbon is fixed by photosynthesis from carbon dioxide (183 tons of CO2
used to produce 100 tons of algal biomass) (Chisti 2007). Different microalgae
need different levels of nutrients. Even marine algal culture needs supplementa-
tion of micro- and macro-elements. Certain growth promoting bacteria can be
used for better performance of microalgae as co-inoculation. Microalgal growth
promoting bacterium such as Azospirillum brasilense can be used to produce
Chlorella vulgaris or C. sorokiniana (de-Bashan et al. 2004). Annual microalgal
biomass yield is much higher than that of land plants. Biomass production from
Nannochloropsis is 23,000 to 34,000 L/ha/y, Chlorella vulgaris is 8,200 L/ha/y,
jatropha is 2,700, and soya is 544 L/ha/y (Li-Beisson 2012).
Microbial biomass. The replacement of edible vegetable oil for the produc-
tion of biodiesel can be achieved by single cell organisms or microorganism
culturing. Besides microalgae, other microorganisms including bacteria and fungi
can be effectively used to produce oil. Yeast species including Rhodosporidium sp.,
Rhodotorula sp., and Lipomyces sp. accumulate lipids in their body as high as 70%
of their biomass dry weight. Crptococcus curvatus produces oil greater than 60%
on a dry weight basis (Meng et al. 2009). Oleaginous microorganisms are fast
growing and accumulate a high quantity of lipid (as efficient as vegetable oil). They
are less lipid reutilizing microbes and can culture easily throughout the year,
giving agro-industrial biomass as substrates (Yousuf 2012). Waste materials from
biodiesel plants also can be used for the cultivation of oleaginous microorganisms.
Glycerol from biodiesel plants and stillage from breweries are used for the
cultivation of Rhodotorula glutinis (Yen et al. 2012). Some of the microorganisms

used to produce oil are listed in Table 21-1. Kalscheuer et al. (2006) engineered
Escherichia coli by heterologous expression of pyruvate decarboxylase, alcohol
dehydrogenase, and acyltransferase. Ethanol formed is used for the esterification
of fatty acid. All these microbial productions of oil generate a large amount of
microbial biomass as a waste. Most of these microbial biomasses can be used as a
protein source for to produce value-added products for human and animal
consumption (Ahmed et al. 2010). The fungal biomass can be utilized to produce
enzymes such as chitinase or for N-acetyl glucosamine.
21.2.3 Crude Glycerol

Nearly one litre of crude glycerol is formed as a byproduct for every 10 liters of
biodiesel produced. It is estimated that about 4 billion gallons of crude glycerol will
be produced by 2016 (Fan et al. 2010). Crude glycerol generated by homogeneous
base-catalyzed transesterification contains 50% to 60% of glycerol, and the
rest includes alkalies, methyl esters, methanol, water, and further components
(Kolesarova et al. 2011). The properties and constituents of crude glycerol are
varied owing to the change in processes. Glycerol shows very low value because of
its impurity. However, Bournay et al. (2005) reported that a novel method of
esterification of vegetable oil with heterogeneous catalyst and the crude glycerol
originated was with high purity of about 98%. In general, there was no inorganic
impurity in crude glycerol, and major impurities include other organic
compounds and water. However, as the global biodiesel production increases
exponentially, the crude glycerol produced is extensively high and creates
environmental and/or economic issues related to its safe disposal or effective
utilization. To utilize crude glycerol for high-end applications, it needs to be
upgraded through various purification stages such as bleaching, deodorizing, and
ion exchange. In general, up-gradation of crude glycerol is not efficient and almost
not viable to biodiesel industries because of technological limitations. In this
context, extensive research is currently being done by researchers to investigate the
value-added uses of crude glycerol in various applications. The glycerol generated
as a byproduct of biodiesel production can be effectively utilized to produce other
value-added products or used for many other applications. Glycerol is conven-
tionally used to produce products such as food, paint, pharmaceuticals, cosmetics,
soaps, and toothpaste (Fan et al. 2010). However, because of the surplus increase
in production, other commodity chemicals such as citric acid, lactic acid, hydro-
gen, and ethanol can be produced.
21.2.4 Wastewater
In general, wastewater is generated as a result of washing, cooling, and boiling
operations. Discharged wastewater has an alkaline pH and high hexane-extracted
oil and a negligible amount of nutrients (Suehara et al. 2005). Suehara et al. (2005)
used an oil-degrading contaminant, Rhodotorula mucilaginosa, for the biological
treatment of wastewater discharged from a biodiesel production plant. A large
Table 21-1. Some of the Microorganisms Used for the Production of Oil.
Microorganism Substrate used Yield (lipid) References
Trichosporon fermentans Sulfuric acid–treated sugar cane bagasse 15.8 g/L Huang et al.
hydrolysate (2012)
Trichosporon fermentans Sulfuric acid–treated rice straw 10.4 g/L Huang et al.
hydrolysate (2009)
Cryptococcus curvatus, Rhodotorula glutinis, Hydrolysate from the dilute sulfuric acid 5.8 g/L (highest, Yu et al. (2011)
Rhodosporidium toruloides, Lipomyces pretreatment of wheat straw C. curvatus)
starkeyi, and Yarrowia lipolytica
Trichosporon cutaneum Sulfuric acid–treated corncob ~8 g/L Chen et al.
hydrolysate (2012)
Yarrowia lipolytica Detoxified defatted rice bran ~5.2 g/L Tsigie et al.
hydrolysate (2012)
Rhodosporidium toruloides Y4 Lignocellulosic biomass hydrolysate — Hu et al. (2009)
Aspergillus niger, Aspergillus terreus, Hydrolysate from dilute sulfuric acid 39.4% of biomass Zheng et al.
Chaetomium globosum, Cunninghamella pretreatment of wheat straw (highest, (2012)
elegans, Mortierella isabellina, Mortierella Mortierella
vinacea, Mucor circinelloides, Neosartorya isabellina)
fischeri, Rhizopus oryzae, Mucor plumbeus,
Thermomyces lanuginosus
Mortierella isabellina Crusted sweet sorghum 11 g/100 g dry Economou et al.
weight of (2010)
substrate
MANAGEMENT OF COPRODUCTS FROM BIODIESEL PRODUCTION
Escherichia coli Glucose and oleic acid Fatty acid methyl Kalscheuer et al.
esters and fatty (2009)
acid ethyl esters
(26% of the
507
cellular dry mass)

quantity of pure water and other nutrients are needed to make the wastewater
suitable for biological treatment. A coupled photo-Fenton/aerobic sequential
batch reactor system is superior to the biological system for the treatment of
wastewater from the biodiesel plant (Ramírez et al. 2012). The biological system
needs double the time to reduce chemical oxygen demand (COD) up to 90%
compared with the photo-Fenton/aerobic sequential batch reactor system. Jaruwat
et al. (2010) developed a two-stage process of chemical recovery and electro-
chemical treatment to manage wastewater from the biodiesel plant. The combined
treatment completely removed COD and achieved 95% reduction of biochemical
oxygen demand (BOD). Ngamlerdpokin et al. (2011) compared chemical and
electrochemical techniques to treat wastewater from the biodiesel plant. Chemical
coagulation is considered to be best in the case of process cost, whereas
electrochemical processes are best in the case of quality of treated wastewater.
21.3 COPRODUCT MANAGEMENT
The coproducts and biomass can be converted to many other value-added products
by physical–chemical processes or by biotechnological ways (Table 21-2). Con-
verting these residues into other fuel sources or industrially valuable commodity
materials and chemicals are ways of management and are discussed subsequently.
Table 21-2. Major Coproducts and the Areas of Their Use.
Coproduct Utilization
Crop residues Thermal degradation, heat energy

Oil cakes Substrate for methane production, fertilizers, thermal
degradation, heat energy
Algal biomass Production of methane, hydrogen, ethanol; animal food,
chemicals
Crude glycerol Production of hydrogen, ethanol, omega-3 polyunsaturated
fatty acids, poly (hydroxyalkanoates), 1, 3-propanediol,
organic acids, lipids, enzymes, antibiotics, natural colors,
methanol synthesis, pharmaceuticals; use for livestock,
pigs, beef cattle, fish, dairy, poultry, glycerol as drench
and supplement for dairy cattle
Soybean meal Biomaterial applications: polymeric biocomposites, food
packaging
Camelina meal Use for livestock, poultry (broilers and layers); camelina
meal, which derives from member of the brassica family
that grows on marginal land, no irrigation needed, and
meal is rich in amino acids and antioxidants
Kernel meal Feedstock for fish, turkeys, and pigs
21.3.1 Anaerobic Digestion and Biogas Production

Anaerobic digestion of organic compounds yields value-added products. Anaero-
bic degradation of glycerol was carried out using single or mixed culture of
organisms (Qatibi et al. 1991, 1998). Plant biomass including oil cakes can be used
as a substrate for biomethanization. Ali et al. (2010) reported that Jatropha curcas
defatted oil cake can be used as a substrate for biogas production. This substrate
contains a poisonous substance called curcin and is not suitable for cattle feed.
During methanization the nitrogen (N), phosphorus (P), and potassium (K) or
NPK content increased and the oil content decreased. Thus, the slurry can be used
as a biofertilizer. Kolesarova et al. (2011) reviewed the generation of biogas from
biodiesel byproducts. The byproduct from biodiesel production such as g-phase
can be anaerobically digested to methane. Many microbial species can be used for
the anaerobic digestion including Citrobacter sp., Klebsiella sp., Clostridium spp.,
Enterobacter spp., and Lactobacillus sp. The biomass sludge production is low
compared with other treatments, and energy utilization is very low. The anaerobic
digestibility of sunflower oil cakes was tested in different conditions, and high
stability of the process under mesophilic conditions was demonstrated (Raposo
et al. 2008, 2009; De La Rubia et al. 2009). Algal biomass can be converted to
biogas by anaerobic digestion (John and Anisha 2011). The first step of biogas
production is the acid phase or hydrolytic phase in which organic biomass is
converted into organic acids and gases. Later, mixed population of methanogenic
bacteria produce methane and carbon dioxide from the hydrolytic products. The
conversion of algal biomass into methane could recover as much energy as
obtained from the extraction of cell lipids (Sialve 2009). Moreover, the leftover
nutrient-rich products can be recycled into a new algal growth medium (Brennan
and Owende 2010). Microalgae can have a high proportion of proteins that result
in low C/N ratios that can affect the performance of the anaerobic digester. In such
cases, the codigestion of biomass with a high C/N ratio product (e.g., waste paper)
can be adopted. Yen and Brune (2007) reported enhanced production of methane
gas by the addition of waste paper to algal biomass. The methane production rate
was doubled from 50:50 waste paper:algal biomass blend as compared with
anaerobic digestion of pure algal biomass. Higher ammonium production attrib-
uted to high protein content in the algae can negatively affect the anaerobic
digestion. Salt-adapted microorganisms also can be used for the anaerobic
digestion of marine algae biomass.
21.3.2 Production of Other Fuels

Besides biogas production, biomass and other coproducts generated during
biodiesel production can be utilized to produce other forms of fuels (e.g., ethanol,
hydrogen). According to Brady et al. (2008) the waste glycerol can be mixed with
biomass to produce combustible pellets as an alternative energy source for coal.
Residues of wheat and corn can be managed along with the glycerol, and the pellet
produced has an energy yield of approximately 16 kJ/g. Ethanol is an alternative
fuel for petrol or it can be blend with petrol at different proportions. Ethanol can
be produced by the fermentation of hydrolyzed biomass or coproducts. Hydrogen

is a potential fuel produced from the byproducts.
Ethanol production. The biomass can be hydrolyzed and used for the
fermentation of ethanol using desirable organisms. The byproduct such as glycerol
can be biotechnologically converted to ethanol. Use of micro- and macroalgal

biomass for the production of ethanol was reviewed by John et al. (2011). The
starch or other complex sugars from the algal biomass can be converted to ethanol.
The degraded carbohydrate can be converted to ethanol by ethanol-producing
organisms. After extraction of oil, the biomass can be hydrolyzed by chemical or
enzymatic methods. Microalgae such as Chlorella, Chlamydomonas, and others
are known to contain a large amount (>50% of the dry weight) of easily digestible
carbohydrate and useful as raw materials for ethanol production (John et al. 2011).
Algae biomass requires additional processing before fermentation. Moreover, a
purification process such as distillation is required to remove the water and other
impurities in the diluted ethanol product (10% to 15% ethanol). The concentrated
ethanol (95%) is drawn off and condensed into liquid form, which can be used as a
supplement or substitute for petrol in cars (Brennan and Owende 2010). Micro-
algae such as C. vulgaris are a good source of ethanol owing to the high starch
content and for which up to 65% ethanol conversion efficiency has been recorded
(Brennan and Owende 2010). Ueno et al. (1998) also reportedly produced ethanol
from microalgae via dark fermentation process at high rate. Jarvis et al. (1997)
reported that a red deer rumen bacterium Klebsiella planticola can produce
ethanol by glycerol fermentation. Glycerol dissimilation by K. planticola yielded
ethanol of 30 mmol L − 1 along with 30 mmol L−1 of formate. Escherichia coli SS1
produced 1.0 mol ethanol from 1.0 mol glycerol (Suhaimi et al. 2012). Chaudhary
et al. (2011) reported that a maximum ethanol yield (0.40 g/g glycerol) was
obtained with a glycerol concentration of 10 g/L during anaerobic fermentation
using E. coli. The coproduct generation during glycerol fermentation is insignifi-
cant compared with the fermentation of other sugars. Fermentation of sugars such
as glucose generates coproducts such as succinate and propanodiols. Ethanol
fermentation using glycerol is less expensive than aerobic fermentation and is
preferable for the management of glycerol (Chaudhary et al. 2011). E. coli cannot
ferment glycerol because of the lack of the dha regulon encoding glycerol
dehydratase (dhaB) and 1,3-propanediol oxidoreductase (dhaT) (Trinh and Srienc
2009). However, it was possible by the introduction of this pathway from Klebsiella
pneumoniae. Trinh and Srienc (2009) reported that ethanol fermentation from
glycerol was enhanced by nine gene knockout mutations of E. coli strain. More
than 500 times reduction in other glycerol utilizing metabolic pathways were
observed in the mutant, and there was a yield of 90% theoretical production from
40 g/L of glycerol to ethanol in 48 h.
Hydrogen production. Hydrogen is a naturally occurring molecule snf
considered to be a future fuel because of its renewable and harmless nature; in
other words, it does not generate GHG such as CO2 during combustion. Both
physical–chemical methods and biotechnological methods are adopted to produce
hydrogen from glycerol. Steam reforming of glycerol is a potential way for making
hydrogen. Faudzi and Irsyad (2010) reported that best conditions for producing
hydrogen by steam reforming is at a temperature greater than 699.85 °C and a
water:glycerol molar ratio of 5:1. An alternative way of utilizing organic com-
pounds for biohydrogen production is fermentation using suitable organisms.
Chaudhary et al. (2011) reported during E. coli fermentation that the maximum
production of hydrogen was obtained with an initial glycerol concentration of
25 g/L at a final concentration of hydrogen of 32.15 mmol/L. Soares et al. (2006)
demonstrated the generation of synthesis gas or syngas (hydrogen and carbon)
monoxide from glycerol at very low temperature between 225° and 300 °C by
using a Pd-based catalyst. They also suggest this process for the effective utilization
of various crude glycerol feedstocks for the fabrication of high-value fuels/
chemicals.
Microalgae possess the necessary genetic, metabolic, and enzymatic char-
acteristics to photoproduce hydrogen gas (Ghirardi et al. 2000). Under anaerobic
conditions, hydrogen is produced from eukaryotic microalgae either as an electron
donor in the CO2 fixation process or evolved in both light and dark (Ueno et al.
1998). During photosynthesis, microalgae convert water molecules into hydrogen
ions and oxygen; the hydrogen ions are then subsequently converted by hydroge-
nase enzymes into hydrogen under anaerobic conditions (Cantrell et al. 2008).
Because of reversibility of the reaction, hydrogen is either produced or consumed
by the simple conversion of protons to hydrogen. Photosynthetic oxygen produc-
tion causes rapid inhibition to the key enzyme, hydrogenase, and the photosyn-
thetic hydrogen production process is impeded (Miura et al. 1995, Melis and
Happe 2001, Akkerman et al. 2002, Melis 2002, Cantrell et al. 2008). Conse-
quently, microalgae cultures for hydrogen production must be subjected to
anaerobic conditions. There are two fundamental approaches for photosynthetic
hydrogen production from water. The first hydrogen production process is a two-
stage photosynthesis process in which photosynthetic oxygen production and
hydrogen gas generation are spatially separated (Ghirardi et al. 2000). In the first
stage, algae are grown photosynthetically in normal conditions. In the second
stage, the algae are deprived of sulfur, thereby inducing anaerobic conditions and
stimulating consistent hydrogen production (Melis and Happe 2001). This
production process becomes limited with time, because hydrogen yield will begin
to level off after 60 h of production. The use of this production system does not
generate toxic or environmentally harmful products but could give value-added
products as a result of biomass cultivation (Melis 2002). The second approach
involves the simultaneous production of photosynthetic oxygen and hydrogen gas.
In this approach, electrons that are released on photosynthetic H2O oxidation are
fed directly into the hydrogenase-mediated hydrogen-evolution process (Ghirardi
et al. 2000). The hydrogen productivity is theoretically superior to the two-stage
photosynthetic process, but the simultaneous production process suffers.
21.3.3 Animal Feed

The use of feedstocks derived from agricultural commodities such as oilseeds for
biodiesel production yields the coproduction of animal feed. The major biodiesel
feedstocks are vegetable oils that are usually produced by crushing oil seeds and
leaving a significant quantity of proteinous meals as coproducts. Many of these
coproducts can be used as feed for ruminant animals when used properly.
Ruminants can turn materials that are not useful for humans into animal-origin
products of high biological value, because of the microbial fermentative process

that takes place in their gastrointestinal tract. The use of these products can also
reduce animal feeding costs. Soybean meal is traditionally used as filler in animal
feed including poultry, swine, beef, dairy, pet, and other animals because of its
concentrated protein content. Soybean meal has been demonstrated the best
nutritional composition in the study by Carrera et al. (2011), who also suggested
that other products such as macaúba seed cake, cottonseed meal, and peanut and
turnip cakes also had equally nutritional composition. This was attributed to the
good quality of protein that was retained in the rumen-undegradable protein
portion.
Oil cakes are solid residues obtained after oil extraction, and their composi-
tion widely varies based on the types of nuts, cultivation varieties, cultivation
conditions, and extraction methods (Kolesarova et al. 2011). Some of the oil cakes
are edible, but certain others contain poisonous substances. Edible oil cakes have a
protein content ranging from 15% to 50%. Glycerol can be used as a feed for
ruminant and nonruminant animals because they can be converted easily to
glucose in the livers of animals by the enzyme glycerol kinase (Yang et al. 2012).
The level of glycerol in the feed must be optimized because it can influence the
physiological process of animals. Many workers studied the effect of glycerol as a
supplement of corn or other nutrient materials and its effect on the animals
(Cerrate et al. 2006, Hampy et al. 2008, Donkin 2008, Donkin et al. 2009, Gunn
et al. 2010). The toxicity of the chemicals such as methanol or potassium must be
taken in account while supplying crude glycerol as an additive. Testing feedstuffs
during in vivo evaluations is highly recommended to establish the possibility of
using these feedstuffs and if so, in which amounts. Algal biomass can be used for
preparation of animal feed supplements after careful evaluation. The main
applications for algal biomass in aquaculture are fish feed including larval
nutrition for mollusks or penaeid shrimp, coloring for farmed salmonids, stabili-
zation, and improvement of quality of culture medium, inducement of essential
biological activities in bred aquatic species, and enhancement of the immune
systems of fish (Brennan and Owende 2010). However, prolonged feeding at high
concentrations could be detrimental.
21.3.4 Chemical Feedstock from Crude Glycerol and Other

Residual Biomass
Omega-3 polyunsaturated fatty acids. Docosahexaenoic acid (DHA), an impor-
tant omega-3 polyunsaturated fatty acid is a therapeutic agent used against
cardiovascular diseases, cancers, schizophrenia, and Alzheimers (Chi et al.
2007). Ethier (2010) used crude glycerol as a substrate for the fermentation of
the microalgae Schizochytrium limacinum to produce omega-3 polyunsaturated
fatty acids. S. limacinum produce DHA, and the highest DHA productivity
(0.52 g/L per day) was obtained at a dilution rate 0.3 per day and feed glycerol
concentration 90 g/L. Athalye et al. (2009) reported that eicosapentaenoic
acid (EPA) yield and productivity reached 90 and 14.9 mg/L/day, respectively,
when a fungus Pythium irregulare was grown in medium containing 30 g/L crude
glycerol and 10 g/L yeast extract. Ciriminna et al. (2006) reported that DHA
could be obtained by electrocatalytic oxidation of glycerol using a small electric
potential applied to a glycerol solution at pH 9.1 with the help of oxidizing agent
2,2,6,6-tetramethylpiperidine-1-oxyl. Chi et al. (2007) cultivated S. limacinum in
crude glycerol medium and found the highest DHA yield was 4.91 g/L with 22.1 g/L
cell dry weight.
Poly(hydroxyalkanoates). Poly(hydroxyalkanoates) (PHA) is a bacterial natu-
ral polymer and is a good substitute for the nondegradable artificial polymer. PHA
produced using glycerol as substrate by Paracoccus denitrificans and Cupriavidus
necator was as good as PHA produced using glucose as the substrate. Usually the
presence of salts in the crude glycerol can negatively affect the production of PHA in
small- or large-scale fermentation (Ashby et al. 2004, Mothes et al. 2007, Cavalheiro
et al. 2009). One of the strains of Zobellella denitrificans could produce high
amounts of PHA even with the high concentration of glycerol and in the presence of
salt (Ibrahim and Steinbüchel 2009). Besides single culture, some microbial
consortia can also be used to produce PHA (Dobroth et al. 2011). Mothes et al.
(2007) reported the synthesis of poly(3-hydroxybutyrate) (PHB) using crude
glycerol (rapeseed oil based) as the feedstock via the biotechnological process using
P. denitrificans and C. necator microbes. Mothes et al. (2007) compared the
properties of the synthesized PHBs from two different feedstocks and found that
the properties were very similar.
1,3-propanediol. 1,3-propanediol is a versatile organic compound used in
industries such as of polymers, cosmetics, foods, and medicines. Mu et al. (2006)
demonstrated that Klebsiella pneumonia can produce 1,3-propanediol from crude
glycerol. They used the crude glycerol obtained during soybean oil–based biodiesel
production using alkali catalysis. They ultimately compared the product of
1,3-propanediol obtained from pure glycerol and found that they were similar to
each other. Maximum 1,3-propanediol production was 13.8 g/L from K. pneumonia
(Oh et al. 2008) and 56 g/L from K. pneumoniae ATCC 15380 (Hiremath et al.
2011). Under nonsterile culture conditions, Clostridium butyricum VPI 1718 could
produce 67.9 g 1,3-propanediol/L (Chatzifragkou et al. 2011b).
Organic acids. In general, organic acids are used as a preservative or food
additive. They are used as pH regulators or substrate to produce other chemicals.
Organic acids such as lactic acid or acetic acid can be used as a monomer to
produce biodegradable polymers. The biomass hydrolysate and byproduct such as
glycerol can be used as a substrate for fermentation and can be biotechnologically
produced from organic acids. Scholten et al. (2009) used a bacterium Basfia
succiniciproducens to produce succinic acid with a low-cost culture medium such
as glycerol. The yield of succinic acid in a continuous cultivation was 1.02 g/g
glycerol. Yarrowia lipolytica was used to produce citric acid from crude glycerol
and showed an equal yield to conventional sugar-based medium (Papanikolaou

and Aggelis 2003). Rywińska et al. (2009) used an acetate mutant of Y. lipolytica
Wratislavia AWG7 strain and found the final concentration of citric acid was
131.5 g/L and was 139 g/L from pure glycerol. Hong et al. (2009) tested bacterial
isolates for the production of lactic acid from glycerol, and E. coli strain could
produce 85.8 g/L of lactic acid with a productivity of 0.97 g/L/h. Zhou et al. (2008)
reviewed the chemoselective oxidation of crude glycerol into various products
such as glyceric acid, hydroxypyruvic acid, and mesoxalic acid, which can be used
as precursors for various fine chemicals and polymeric materials.
Lipids. Saenge et al. (2011) used R. glutinis TISTR 5159 to produce lipids and
carotenoids from crude glycerol. The 10.05 g/L lipid and 6.10 g/L carotenoids were
the maximum production. Chlorella protothecoides could produce 0.31 g lipids/g
substrate from crude glycerol (O’Grady and Morgan 2011). Maximum lipid
productivity was 3 g/L per day from crude glycerol in a fed batch operation
using C. protothecoides (Chen and Walker 2011). Many fungi were tested for the
production of lipid using crude glycerol as substrate (Chatzifragkou et al. 2011a).
Xu et al. (2012) used Rhodosporidium toruloides for microbial conversion of crude
glycerol to triacylglycerols (microbial lipid) by one-stage batch fermentation.
Higher biomass concentration and lipid yield were obtained with crude glycerol
than with glucose and refined glycerol. The highest biomass concentration was
26.7 g/L with an intracellular lipid content of 70%.
Enzymes. Many agro-industrial residues, especially biofuel crop residues,
contain many complex carbon and nitrogen sources. These residues and algal
biomass obtained after oil extraction can be used for the cultivation of micro-
organisms to get desired enzymes. Cellulases, amylases, proteases, and
hemicellulases are some of the example for these commercial enzymes. Biotech-
nological application of oil seeds was studied by many researchers for the
production of enzymes such as alpha amylase, phytase, and tannase, among
others. (Ebune et al. 1995a, b; Ramachandran et al. 2004, 2005, 2007; Sabu et al.
2005, 2006). Thanapimmetha et al. (2012) tested jatropha de-oiled cakes for the
production of protease. Aspergillus oryzae cultivated on a mixture of de-oiled
jatropha oil cake and cassava bagasse yielded 14,273 units protease g(dry matter,
DM)−1. Joshi and Khare (2011) used a thermophilic Scytalidium thermophilum to
produce xylanases using de-oiled J. curcas seed cake. Ebune et al. (1995a, b) used
canola meal to produce phytase enzymes using Aspergillus ficuum.
Miscellaneous products. The biomass and byproducts of the biodiesel
industry can be used for the production of many other products. Antibiotics
such as Cephamycin and Clavulanic acid were produced by species of Streptomy-
ces using oil cakes as substrate (Sarada and Sridhar 1998, Sircar et al. 1998, Kota
and Sridhar 1999). Shashirekha et al. (2002) used oil cake for the enhancement of
bioconversion efficiency of the mushroom, Pleurotus sajor-caju. Tuli et al. (1985)
supplemented mustard oil cake for enhanced production of lactic acid from whey
permeate by immobilized Lactobacillus casei. Many organisms including Asper-
gillus sp., Blakeslea trispora, Monascus purpureus, Helminthosporium catenarium,
Hypericum gramineum, Rhodotorula sp., and others, can be used for the
production of natural colors by the fermentation of agro-residues. Soares et al.

(2006) suggested that the crude glycerol can be effectively used for methanol
synthesis.
21.3.5 Biomaterial Applications

Soybean meal is the main proteinase coproduct of biodiesel production. Soy flour
(48% protein), soy protein concentrate (64% protein), and soy protein isolate (92%
protein) are the three important protein types that can be extracted from soybean
meal after oil extraction of soybean powder with hexane (Kumar et al. 2002). Soy
flour, soy protein concentrate, and soy protein isolate can be plasticized to produce
films and formable thermoplastics. There are several reports on biomaterial
application of the proteins (Shukla and Cheryan 2001, Reddy et al. 2010). The
biomaterial applications of the meals in the form of plasticized meals as well as
reinforcing fillers used in polymeric biocomposites have been extensively studied
recently. Soybean meal has been characterized for its chemical composition,
moisture content, thermal behavior, and infrared spectrum. Soybean’s potential
as particular filler in value-added biocomposites has been evaluated by com-
pounding with polycaprolactone (Diebel et al. 2012). Diebel et al. (2012) prepared
the composite of soybean meal (30%) and polycaprolactone (70%) by extrusion
and injection molding and then tested it for mechanical properties such as tensile,
flexural, and impact. It was observed that the composite of soybean meal (30%)
and polycaprolactone (70%) has higher tensile/flexural modulus, but lower
strength, elongation, and impact strength compared with polycaprolactone. The
composite had relatively less cost than polycaprolactone itself. The study demon-
strated that soymeal addition to polycaprolactone increases the rigidity; however,
the particle-matrix adhesion needs to be improved.
Soymeal has been plasticized and blended with other polymers such as
polycaprolactone, poly(butylenes succinate), poly(butylene adipate terephthal-
ate) (Reddy et al. 2012a, b), and natural rubber (Wu et al. 2007, Mohanty et al.
2010). Reddy et al. (2012a) had demonstrated that soymeal can be plasticized
and destructurized using glycerol and urea in a twin screw extruder and then
blended with biodegradable polyesters, polycaprolactone and poly(butylenes
succinate). Mechanical properties of the protein-based blends can be improved
by the destructurization. Soymeal can be plasticized using glycerol in the
presence of two different denaturants (destructurizers) (Reddy et al. 2012b).
The produced thermoplastic soymeal can be blended with different polyesters,
polycaprolactone, poly(butylenes succinate), and poly(butylene adipate tere-
phthalate). They adopted Taguchi experimental design in their study to investi-
gate the effect of each constituent on the tensile properties of the final blend. In
the study conducted by Wu et al. (2007), a vulcanized blend of natural rubber
and soymeal was produced in which the rubber phase was embedded by the
soymeal matrix. The interaction between these two phases was suggested by the
increase in the glass transition temperature of the rubber phase. The produced
blend in the study by Wu et al. (2007) demonstrated good elasticity and water
resistance.
Kim et al. (2004, 2005) reported that defatted soymeal can potentially be used
to produce edible films for their applications in food packaging. In their study,
soymeal was fermented in a soybean meal solution (15 g/100 mL of water) by
inoculation with Bacillus subtilis bacteria fermented under optimum conditions of
33 °C and pH 7.0 to 7.5 for 33 h. Then, the fermented soybean solution was heated
for 20 min at 75 °C with 2 to 3 mL glycerol added to the solution to overcome film
brittleness. The filtered solution was finally cast in a petri dish to produce films.
The increasing amount of plasticizer in the fermented film led to a decrease in
tensile strength and an increase in percentage elongation of the film compared
with the ordinary soybean film. Moreover, the soymeal-based film exhibited
higher water vapor permeability. However, experiments showed that growth
inhibition of the produced soymeal-based film in the agar media containing
E. coli was much higher than the ordinary soy protein film. These results suggested
that the fermented soymeal-based films can be used as a new packaging material to
extend the shelf life of foods. However, extensive scientific research to improve
mechanical and physical properties of soymeal-based films is necessary to
establish their industrial applications.
21.3.6 Thermal Degradation of Residual Mass after Extraction

of Oil
Biomass after oil extraction is usually less valuable and a hurdle for the industrial
sector, because their management is too difficult. Thermochemical conversion
methods including combustion, pyrolysis, and gasification are alternative ways to
value add the biomass. Combustion is an exothermic chemical reaction accom-
panied by high heat and light generation. Pyrolysis is the process of degradation of
biomass to gases, liquids, and solids with the help of heat and pressure.
Hydrothermal gasification is the treatment of biomass in hot compressed water,
usually above 350 °C at high pressure to obtain combustible gas. González et al.
(2011) tested the thermal degradation of glycerin. The mass spectrometry analyses
on the emission of sulfur dioxide and dimethyl sulfide allowed identifying the
release of these compounds around the temperature of 370 °C.
21.3.7 Microbial Cultivation on Residual Mass Generated during

Microbial strains such as yeasts or filamentous fungi and probiotic bacteria can be
cultured on the leftovers after oil extraction from algal biomass and oil seeds.
Single cell protein or other protein fortification can be done on low-valuable
residues by the growth of these substrates. Solid agro-industrial residues are
heterogeneous substances having macromolecular structure such as cellulose,
hemicelluloses, pectin, and lignin. These substances can be easily used to produce
mushrooms. An aroma compound is a chemical compound that has a smell or
odor. Aroma compounds can be produced biotechnologically by the culturing of
microorganisms in suitable substrate. Geotrichum candidum is a fungus that can

produce fatty acid esters (having fruity aroma). This strain can be cultivated on oil
cakes or other oleaginous materials for the products.
21.3.8 Nutraceutical and Bioactive Compound Extraction from

Residue
Agro-industrial residues have many bioactive compounds as their primary or
secondary metabolites. These metabolites have many pharmaceutical applications.
Some are poisonous to living organisms and can be used as biocontrol agents.
Omega-3 polyunsaturated fatty acid is one of the main parts of algal oil.
Oleaginous algae contain biopigments, and chlorophyll/carotenoid like pigments
can be used as nutraceutical agents. Antimicrobial substances from the residue can
be used for the control of unwanted microorganisms. Fungal biomass after oil
extraction can be used to produce glucosamine.
21.3.9 Fertilizers
Oil cakes including mustard or canola meals can be used for the beneficial growth
of plants and to control soil pathogens such as nematodes and pathogenic fungi
(Ramachandran et al. 2007). Oil cakes combined with Bradyrhizobium sp. and
Paecilomyces lilacinus were used against mung bean pathogen to control root rot
(Ehteshamul-Haque et al. 1995). Algal biomass after extraction of oil contains
nitrogenous compounds and other micro- and macronutrients. These residues can
be used as manure for cultivating plants. Conversion technologies such as
pyrolysis, can be applied to algal biomass to produce the biochar that has potential
agricultural applications as a biofertilizer and for carbon sequestration (Brennan
and Owende 2010).
21.4 BIOREFINERY CONCEPT
The production of bioenergy and chemicals from biomass can be defined as

biorefinery by the integration of bioprocessing and appropriate low environmental
impact chemical technologies in a cost-effective and environmentally sustainable
manner (Chisti 2007). A biorefinery-based production strategy is superior to
single product production strategy in terms of economics when considering the
feasible commercialization of bioenergy (John and Anisha 2011). The algal
biomass or oil seeds can be used to produce other fuels such as biomethane or
bioethanol from the hydrolyzed de-oiled cakes or algal biomass. In addition to
biofuels, many types of products comprising nutraceuticals, animal feed, phar-
maceuticals, biofertilizers, among others, can be obtained from the waste biomass
such as solid algal waste and oil cakes.
21.5 CONCLUSIONS
The enormous growth of the biodiesel industry in the last decade has caused the
generation of a huge amount of undervalued coproducts such as protein-rich
meals and crude glycerol. To claim biodiesel production as the sustainable

technology of the future, it is necessary to create added value for these products.
Adding value to biodiesel coproducts provides a solution to the emerging issues on
the environmental impact of the accumulation of biodiesel coproducts. The
technological development related to adding value to biodiesel coproducts has
huge potential to create novel eco-friendly products, strengthen the economy, and
create new job opportunities.
The industrial applications of crude glycerol in different sections are not yet
well established. However, crude glycerol coproduced in the biodiesel industry has
huge opportunities in biomaterial applications including chemicals/monomers,
plasticizers, hydrogen generation, carbon sources for bacterial growth, and
polyester production. Crude glycerol has been used to produce various products
including 1,3/1,2 propanediol, dihydroxyacetones, polyesters, hydrogen, and
methanol. Crude glycerol compositions vary with their feedstocks; thus, it is
challenging to adopt a universal protocol to fabricate value-added products from
crude glycerol from various feedstocks. The utilization of algal or agricultural
biomass for biofuel production is certainly a sustainable and eco-friendly
approach. Oil cakes and algal residues are rich in fiber, protein, and carbohydrates.
These biomass and byproducts including glycerol can be used as substrate to
produce different biochemicals. The single and mixed substrates can be used to
improve yield and should be evaluated before commercialization. The main hurdle
in this sector is process economy and can be solved by the efficient process
development and use of the organisms or biomass in a zero-waste manner. The
most suitable option is biorefinery strategy and by exploiting the substrate for
gaining maximum value-added products. Novel innovative research in using
biodiesel coproducts to generate different value-added products is expected to
create new avenues for utilizing renewable resources as feedstocks for high-value
applications.
We sincerely thank the Killam Trusts for providing the Killam Postdoctoral
Fellowship to T. T. More at the University of Calgary, Schulich School of
Engineering, Calgary, Alberta, Canada. We also thank the Natural Sciences and
Engineering Research Council of Canada (Grant A 4984, Canada Research Chair)
for their financial support. Views and opinions expressed in this chapter are those
of the authors.
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CHAPTER 22
Economic Evaluation of
Processes
S. K. Ram
L. R. Kumar
S. K. Yellapu
R. Kaur
R. D. Tyagi
22.1 HISTORICAL ACCOUNT OF BIODIESEL INDUSTRY

DEVELOPMENT
Around 4000 BCE, Sumerians discovered the process of fermentation, and around
the tenth century BCE, Assyrians started using biogas to heat water for bathing
purposes (Prajapati and Nair 2008). The science of fermentation further pro-
gressed, and in the seventeenth century, Helmont observed the combustible nature
of organic matter emitting flammable gases (Kumar et al. 2018). Davy in 1808
discovered the production of methane to be used as biofuel coming out as a
byproduct of anaerobic digestion (Kumar et al. 2018). The science of biofuel
production advanced lately, and in the mid-1800s, people started using plant oils
in transesterification to distill glycerin while producing soap. In 1853, E. Duffy and
J. Patrick conducted the transesterification of a vegetable oil. In the years of 1958
to 1964, French biologist A. Bechamp concluded that the fermented products
contain living organisms and they can be controlled. It was during this year that
Louis Pasteur described the whole fermentation process scientifically (Gal 2008).
The late 1800s was a revolutionary period when industrialization started to
occur with the discovery of modern internal combustion engine during 1976 to
1980. During the last decade of the nineteenth century, Rudolf Diesel (1893) filed
his patent for the working methods and design for combustion engines. The first
practical diesel engine worked with an efficiency of 75% using vegetable oil. In the
527
very early twentieth century, Henry Ford’s T-Model was designed to run on
ethanol. World War II caused a huge shortage of fuel oil and resulted in many
discoveries in Germany to use gasoline with ethanol. Because of geopolitical
events, a huge shortage and increase in oil prices have rekindled the interest in
biodiesel. On August 31, 1937, Belgian scientist G. Chavanne got his patent for the
process of transformation of vegetable oil to be used as fuel. The year 1940 marked
the first US ethanol plant. During the same decade, the extensive use of biogas to
replace gasoline took place. Toward the late twentieth century, ethanol producing
plants started increasing in number. During this period, the first bioethanol plant
began to switch energy resources from coal to natural gas. The US government
also subsidized farmers for ethanol production. International events during 1973
to 1974 in Saudi Arabia and the Iranian revolution in 1978 to 1979, along with
falling domestic oil production caused the price of oil to go up. According to the
US Department of Energy, during this period the United States limited its import
of crude petroleum oil, and the world witnessed a radical change in the price of
crude oil from $14/barrel to a whopping $35/barrel during 1979 to 1981. The
prices took another 3 to 4 years to stabilize back to $29/barrel. Close to the end of
the twentieth century, 3 million US cars and light trucks were running on the road
with E85 (a blend of 85% ethanol and 15% gasoline). However, few gas stations
invested in the project, and the E85 fuel was sporadically available.
During the research with transesterified sunflower oil, the first standards for
diesel fuel were developed in South Africa in 1979. Within the next 4 years, the
complete standards for manufacturing and quality of biodiesel were available to
the world. Gaskoks, an Australian company, bought the technology from South
Africa and set up the first biodiesel plant in 1987. By 1990, the Clean Air Act
became more stringent. The required fuels must have a higher oxygen content to
lower monoxide emissions. The Environmental Protection Agency (EPA) passed
the Energy Policy Act in 1992. This pact was targeted to increase alternate fuels in
US transportation. The pact was amended in 1998 to include biodiesel as an
alternative fuel to diesel vehicles.
Environmentalists by this point started putting particular emphasis on issues
such as climate change and greenhouse gas emissions. During 2004 owing to
international political events, crude oil prices surged by 80%, gasoline prices rose
by 30%, and diesel prices hiked by nearly 50% globally. In the United States,
225,000 barrels of ethanol was produced per day. E85 unit prices were lower than
gasoline prices, resulting in greater than 4 million hybrid fuel vehicles on the road
that were being served by more than 400 filling stations across the United States.
Despite all efforts, ethanol did not stand out to be a competitive fuel source
because tax credits on ethanol fuel were imposed —four to five times that of
gasoline fuel oil. In 2006, the Renewable Fuel Standards (RFS) program promoted
the use of biofuels (ethanol and biodiesel) with the goal set to double the use by
2012. The Energy Independence and Security Act of 2007 mandated the incor-
poration of 56.78 billion L of ethanol into the fuel supply by 2015 and 136.27
billion L by 2022.
ECONOMIC EVALUATION OF BIODIESEL PRODUCTION PROCESSES 529
22.2 NECESSITY OF TECHNO-ECONOMIC EVALUATIONS
Because of stringent environmental laws and mandates from the United States and
the European Union (EU), research for alternative green fuel has gained speed. An
enormous amount of research is going on, and new technologies are published
every day to produce biofuels. However, are these studies going to serve the
ultimate purpose? A detailed and honest techno-economic analysis can be the fine
line of difference between a failed and successful biodiesel microeconomy. In an
academic perspective, a techno-economic analysis looks the least attractive
research activity for a scientist or to researchers, even in an applied research
setting where the researcher is busy increasing the efficiency, yield, and through-
put of the process for rapid technology transfer. The first opportunity to
appreciate the importance of techno-economic analysis comes to a researcher
when the industry inquiries about the feasibility of the process on a large scale.
These academic–industry interactions are seldom observed in most of the research
facilities in many developing and developed countries. Dedicated technology
incubation centers provide a platform for such interactions, but government
policies are not usually inclined to invest in these incubation centers. Such
technology incubation centers have made their appearances lately in developed
economies, and they are yet to come in developing economies.
Techno-economic studies of newly developed processes evaluate each and
every small component of the technology in relation to the economic feasibility
and market prices of all the requisites. Techno-economic studies are the right
avenues for the discussion, such as what kind of solid-liquid separation technology
should be chosen, whether or not the excess solvent can be recycled, or should the
byproduct stream be extracted and sold as a secondary economic return (revenue).
The techno-economic studies evaluate the trade-off between energy, labor, and
other resource investment in performing any activity in the production line and
concludes if there is any economic motivation to do the activity in an economically
sustainable manner for an industry or not. During the exercise of techno-
economic evaluation, the study reveals the actual economic impedances and
bottlenecks that should be improved by the researcher for making the process
(technology) economically feasible.
Results obtained by academic researchers for the techno-economic analysis
using various simulation software for any technology vary from one study to
another. Even the use of different software by the same researcher for the same
process brings out variation in results. The variation in the results across various
investigators, software, or different technology is evidently attributable to different
assumptions, logistics, calculation algorithms, and sometimes databases of infor-
mation. Because no strict guideline is available to researchers to follow while
performing techno-economic evaluations, the studies are prone to make vague and
unrealistic assumptions, which can give misleading results. It is the responsibility of
the researcher to do a comprehensive and realistic study of current market prices for
various elements of the process like equipment freight on board (FOB) cost, labor
cost, utility cost, and the cheapest available cost for feedstock (raw material). There
is no proper database available for researchers to use in their cost estimations for the
simulations of their technology. Therefore, it is essential to have a common
guideline to avoid unrealistic assumptions and misleading results in these studies.
Big enterprises have their own economic evaluations for the business
development to account for various factors like market entry, market competi-
tions, and consumer demographic distributions, which are beyond the scope of
techno-economic evaluations to arrive at their business decisions. This fact not
only dwarfs the importance of techno-economic studies done by academic
researchers but also makes them obsolete to a quick observation. Although it
may seem irrelevant at the business development level for a researcher, the techno-
economic evaluation is the first window to reality. These studies open up a
discussion platform to whether or not the research done in an Erlenmeyer flask is
relevant to society or what kind of changes are required to bring the innovation
into use to the society, which is the primary goal of any scientific discovery or
invention. The techno-economic evaluation studies are tools of real-life simulation
of proposed technology and its various different scenarios and configurations to
discover the one with the maximum potential to become a reality.
Usually, policy makers and social or political leaders are not scientists and
researchers, and it is less likely that they can understand the scientific jargons. For
a researcher, techno-economic evaluation studies are the mode of communication
to translate the scientific technology into an understandable economic language
for the policy makers to understand. These studies after proper validation can
serve as the basis for policy design and appropriate governance.
22.2.1 Economic Fallout of Biodiesel Industry

In 2016 one of the biggest biodiesel company in Australia (Australian Renewable
Fuels) shut down because of changes in government policies (Farrell 2018). They
revealed that the demise occurred because of the withdrawal of the Fuel Grant
Scheme by the government, which was running the plant with subsidies. Accord-
ing to The Globe and Mail (February 22, 2013), the Canadian government stopped
its controversial biodiesel subsidy ecoEnergy program, which failed to accomplish
the overambitious targets as projected by its techno-economic analysis (Mccarthy
2013). Ottawa paid $672 million to various biodiesel companies to manufacture
production units to produce biodiesel from grains. Biodiesel companies in Canada
failed to reach the target and standards of biofuel produced after refining.
The global biodiesel industry witnessed rapid growth between 2003 and 2010,
but suddenly in subsequent years, the industry faced a severe pitfall with
the statistics plummeting. The primary reasons for the pitfall of the biodiesel
industry, despite having government support programs, is rising unit production
cost, waning consumer interest in biodiesel, wavering government assistance, and
taxes. According to cost–benefit analysis, the biodiesel programs seem to be a
riskier investment to financial institutions that are reluctant to invest or some-
times withdraw existing support. Europe is the biggest market for biodiesel
consumption, where the biodiesel blend is mandated in a decentralized manner
allowing each of the individual states to have their own targets. However, owing
to tighter budgets and scarcity of farming land in Europe, it has to import most of
its biodiesel to agree with the mandate. Europe is forced to invest in other
alternative energies that are cheaper and economically competitive like wind
and solar energy. Details about various mandates, energy acts, and supporting
biofuel (biodiesel) subsidy programs can be found elsewhere (Hertel et al. 2010,
Lutterbach and Galvão 2010).

Thus, it is essential that while the researchers try to develop and further the
technology of biodiesel production, they do so with the global vision of reduction
in the cost of production to make it competitive. The knowledge of economic
competence of their developed technology can only be supplemented with honest
and genuine techno-economic analysis. In the subsequent sections of this chapter,
the techno-economic studies performed with vegetable oil, algal oil, and single cell
oil (SCO) are examined, and the technological aspects that affect the economy of
the process of biodiesel production are revealed.
22.3 EVALUATIONS WITH VEGETABLE OIL AND WASTE COOKING

OIL
Lipid, or simply oil, is the primary feedstock for biodiesel production. The source
of oil has varied over generations. Initially, plants and animal fat were the main
sources of oil for biodiesel production, but increasing population and the
subsequent food crisis raised the food-versus-fuel debate. The biodiesel industry
switched from sugar-based oil to cellulosic and microalgae oil. Once again, the
algae growth and cellulosic biomass still occupy a significant arable land, which
can otherwise be used for agriculture. Therefore, the biodiesel industry made a
paradigm shift toward microbial oil. Although microbial SCO is in its stage of
infancy, it appears to be a desirable alternative. It might seem that the transition
from agricultural-based oil to microbial oil is a result of the moral debate, but it is
the result of more than just the food-versus-fuel debate. The shift in technology is
also driven by the question of economy.
22.3.1 Vegetable Oil for Biodiesel Production

Initially, the techno-economic analysis performed by researchers to produce
biodiesel directly from vegetable oil had a different concern. Because the source
of feedstock is oil yielding plants, the techno-economic evaluation cannot be
compared with other technologies of biodiesel production like microalgal biodiesel
or SCO biodiesel. Techno-economic analysis performed by Apostolakou et al.
(2009) and Haas et al. (2006) had a broad range of conclusions over the unit cost of
production of biodiesel using vegetable oil. Apostolakou et al. (2009) used
rapeseed oil, and Haas et al. (2006) developed a cost estimation model on soy
oil. The final unit production cost for the Apostolakou et al. (2009) study was
$1.15/L of biodiesel, whereas Haas et al. (2006) reported $0.53/L of biodiesel.
The major differences in the two studies are summarized in Table 22-1. Both
studies followed an almost similar process for the production of biodiesel. The
vegetable oil was transesterified using a base catalyst. The reactor had a residence
Table 22-1. Comparison between Two Techno-Economic Analysis Studies from Vegetable Oils.
532
Technical assumptions Haas et al. (2006) Apostolakou et al. (2009)
Basis of production 37,854 t/yr (1 million gal.) 50,000 t/year (1.6 million gal.)
Methanol:oil 6:1 6:1
Catalyst Sodium methoxide (1.78% w/w) Sodium methoxide (1% w/w)
Conversion efficiency 90% in each tank (sequentially) to 90% in each reservoir (sequentially) to
have overall 99% have overall 99%
Oil separation Centrifugation Centrifugation
Biodiesel cleaning Vacuum-dried removal of water Vacuum-dried removal of water
Methanol recovery By distillation By distillation

Software of simulation ASPEN Plus 2001 HySYS 2009
FCI $11.3 million $9.4 million
Price of oil $0.052/kg ($0.236/gal.) $1.1/kg
Methanol price $0.286/kg $0.3/kg
Raw material cost $19.022 million $57.9 million
Labor wage rate $12.5/h $13/h
Electricity prices $0.05/kW-h $0.15/kW-h
Unit production cost $0.53/L biodiesel $1.15/L biodiesel
Note: FCI = fixed capital investment.
time of 1 h, and the reaction was carried out at 60 °C. For the reaction, 100% excess
methanol was used, and the reaction was performed in two sequential (reactors)
steps to improve the efficiency of transesterification. Transesterified fatty acid
methyl esters (FAMEs) were separated by centrifugation, neutralized by acid, and
separated from free fatty acids (FFAs) by centrifugation. The trace water was
cleaned by vacuum evaporation of water. The waste stream was treated to obtain
and recycle methanol. Byproduct crude glycerol was finally obtained to be sold as
credit (Haas et al. 2006), but Apostolakou et al. (2009) did not consider the
glycerol credits.
The major difference in the cost of production between the two studies with a
similar configuration of process setup resulted primarily from the difference in
their prices taken from the raw material (or feedstock) oil. The price of raw
feedstock oil (rapeseed oil and soy oil) is compared in Figure 22-1 over a span of
15 years (2001 to 2015). The prices of the feedstock vegetable oil have been
fluctuating across a wide range of values, which eventually affect the techno-
economic analysis. Many factors may influence the price of feedstock oil,
including political issues, international events, government policies, climatic
variation, and others. Thus, the most important conclusion is that if the biodiesel
industry is dependent on feedstock vegetable oil, the microeconomy of the process
plant or the whole biodiesel industry will be at the mercy of suitable conditions
(including climate) for the prices of the oil crops to be low and stable.
Figure 22-1. Temporal variation of prices of virgin rapeseed oil and soybean oil in
bulk market for 15 years (2001–2015).
22.3.2 Waste Cooking Oil for Biodiesel Production

Along similar lines, these days waste cooking oil has also become an attractive
candidate as feedstock for biodiesel production. Because the feedstock oil cost is
the primary cost when using virgin vegetable oil for biodiesel, the incorporation of
waste cooking oil (WCO) will reduce the cost of production significantly because
the cost of WCO is three times lower than virgin oil. Techno-economic analysis
(TEA) study performed by Zhang et al. (2003) revealed that the transesterification
cost accounts for 50% of the direct cost of production. For a plant with a capacity
of 8,000 tons/yr using WCO produced from base-catalyzed transesterification, the
direct cost of production (excluding capital cost) represents 75% of the total cost of
production. The study compared the use of virgin oil with WCO (base- and acid-
catalyzed) for biodiesel production.
In the case of base-catalyzed WCO, the waste cooking oil was esterified with
methanol in the presence of sulfuric acid as pretreatment to remove FFAs. After
this pretreatment the effluent is washed with glycerin to remove the hydrophilic
impurities from oil, and the resulting refined oil is base-transesterified in the next
reactor.
In the case of acid-catalyzed WCO, the waste cooking oil was acid catalyzed
with a high methanol:oil ratio (100:1) in the presence of acid. The results obtained
(Zhang et al. 2003) concluded that the reactors account for a significant fraction of
the capital investment (Table 22-2). The capital investment for equipment in the
acid-catalyzed process is high owing to the large volume of methanol required
(because of the high methanol:oil ratio). Even in the WCO base case, the use of
acid in pretreatment requires the material of construction to be stainless steel,
which increases the capital cost. Therefore, the total capital investment in the
WCO case was higher than for virgin oil. The direct manufacturing cost accounted
for 63% to 75% of the total manufacturing cost. Of the total manufacturing cost,
for virgin oil, the raw material accounts for 61% of total cost of production,
whereas it is a mere 29.25% and 34.29% in WCO base and WCO acid catalysts,
respectively.
Table 22-2. Economic Comparison of Virgin Oil versus Waste Cooking Oil for
Biodiesel Production.
Cost/investment (million dollars) Virgin oil WCO: base WCO: acid
Equipment cost 0.81 1.64 1.57

Total capital investment 1.34 2.68 2.55
Raw material cost 4.69 2.27 2.03
Credits from glycerol 0.73 0.68 0.77
Direct manufacturing cost 5.14 4.75 3.28
Indirect manufacturing cost 0.46 0.83 0.71
Total cost of production 7.59 7.76 5.92
Using virgin oil, the capital cost for plant setup is minimum, whereas in the
case of base-catalyzed WCO, pretreatment is required before proceeding to
transesterification, which requires some additional capital cost. The cost savings
incurred by using WCO is large enough to compensate for the capital cost
requirement for setting up additional equipment. The requirement of additional

raw material in pretreatment equalizes the overall difference. In the case of acid-
catalyzed WCO, the absence of pretreatment step is beneficial and all the excess
methanol (100:1) is recovered by distillation. Therefore, the direct cost of the
production of acid-catalyzed WCO is the lowest, thereby giving the lowest
total cost for biodiesel production. Sincere efforts are required to improve the
transesterification technology to utilize the WCO as replacement of virgin oil for
biodiesel.
Today, researchers are convinced that use of plant-based agricultural
resources for biodiesel production is not going to be a sustainable approach,
and new alternates for feedstock oil must be discovered.
22.4 TECHNO-ECONOMIC STUDIES WITH ALGAL OIL
Microalgae were supposed to answer the food-versus-fuel debate and dependence

on crop quality and weather. Microalgal biofuel has been studied very exhaustively
with a series of technological advancement aiming to finally reduce the overall cost
of production of biodiesel. Although the microalgae production industry is
considered the favorite source of feedstock oil, it has already been used for many
different products like high protein animal feed, platform chemical molecules, and
so forth. Many researchers have performed TEA to produce biodiesel using
microalgae, and they claimed the possibility of obtaining energy molecules from
algae, but the economic feasibility remains in question. The basic motivation of
any fuel should be convenience, low price, scalability, and reproducibility. All
these features should be evaluated for biodiesel from microalgae to compete
with petrochemical fuels, which are becoming costlier and are imported from
politically volatile regions. In this section, representative TEA performed with
algae oil–based biodiesel production process is considered.
The very first step to produce biodiesel is the de novo production of microalgae
biomass with intracellular lipid inclusions. The process requires a reaction site,
which can be either as basic as classical raceway ponds or a sophisticated photo-
bioreactor and green wall panels. The change in the growth systems is going to affect
the fixed cost investment of the process. Microalgae are microorganisms that can
survive in the marine or freshwater environment naturally. They harness the solar
energy and fix carbon dioxide from their ambient surrounding. The autotrophic
microalgae require a continuous feed of CO2. There are also some microalgae that
can utilize flue gases surviving the toxic NOx and SOx. The other macronutrients
required by algae are N and P which can be procured from wastewater or cheap
chemical fertilizers. As discussed previously, that cost of feedstock oil has a very
significant impact on overall cost of the biodiesel. The cost of production of oil can
be affected by various factors, among which the system for producing the biomass
holds high significance.
When the biomass is produced in open raceway pond, it still takes up a
significant amount of land because it needs to be shallow to accommodate the

limitation of light penetration and mixing problem. This issue can be solved by
using a photobioreactor, but photobioreactors (PBR) themselves are expensive to
make, presenting a trade-off between the cost of land and cost of PBR. Table 22-3
demonstrates that growing biomass is cheaper in PBRs compared with open
raceway ponds (Chisti 2007). Although Alabi et al. (2009) reported cost of
production of algal biodiesel in PBR with the highest cost ($7.32/kg in Study 6
of Table 22-3), a deeper scrutiny of the study revealed that the author used the
higher range prices for the PBR development ($150/m2), whereas the cost in
literature varies between $90 and $100/m2. The highest contribution to the cost of
algal fuel was from the capital investment to set up the facility. The study also
assumed 25% oil content for PBR and 50% oil content in fermenters. In certain
cases, cost in PBR of biomass is very high. Stir tank reactors or fermenters provide
alternate avenues for microalgal growth. Alabi et al. (2009) compared the three
configurations—open ponds, PBRs, and fermenters—for oil production using
photoautotrophic, mixotrophic, and heterotrophic, respectively, and found that
operating a fermenter saves an enormous amount of money on land owing to the
compact design of this equipment. The well-agitated design of this equipment can
give high productivity and oil content in the microalgae biomass. In general, the
cost of biodiesel production (> $1/kg), in the case of microalgae, is greater than
that of direct vegetable oil (or plants) transesterification. The reason can be as
simple as that plant oil shares a food interest, so humans have all the economic and
motivation to mass produce seed crops. As the scale of production increases, the
cost of production decreases, which is why plant oil can be as cheap as $0.50/L.
The cost of land is another serious concern in microalgae growth and can
range anywhere between $0.12/m2 (Xin et al. 2016) to $0.91/m2 (Nagarajan et al.
2013). The cost of land can make the cost of biomass generation expensive. New
design systems are available for algal growth to reduce the price of biomass
growth. The algal turf scrubber (ATS) system is one such system recently reported
to reduce the cost of biomass growth. Hoffman (2016) studied and compared the
biomass production in an open raceway pond (ORP) system with the ATS system.
The study concluded that for the same biomass productivity rate, biomass
production cost was $510/ton for ATS and $673/ton for ORP. The biomass
productivity has a direct impact on the cost of biodiesel. Nagarajan et al. (2013)
concluded that doubling the biomass productivity reduces the cost of biodiesel by
40.7% to 42.3%. Results from a study that implemented a simultaneous oil
extraction and transesterification (SOET) strategy indicate that if flue gas replaces
the pure CO2, the cost of biodiesel drops by 24.8% to 26.8%. The study also
suggests that for biomass harvesting, use of polyelectrolytes improves flocculation,
reducing the harvesting cost by 85.6%. Nagarajan et al. (2013) estimated the unit
cost of biodiesel production to be around $0.68 to $1.26/L. Keeping in mind the
Table 22-3. Comparison of Various Techno-Economic Studies on Microalgae Oil for Biodiesel Production.
Study
Parameters 1 2 3 4 5 6 7 8
Oil content (% w/w) NA NA 20 30 44 48 15 25 50 25 50

Yield (g/m2/d) 25 33 27 72 32 12.8 15.5 9.38 15.3 50 25 1.25 kg/m3/d 60
Biodiesel cost ($/kg) NA NA 1.06 1.41 1.81 2.4 14.44 24.6 2.6 2.25 4.78 0.42
Biomass cost ($/kg) 8–15 0.1 0.29 0.47 0.60 NA 2.6 7.32 1.54 — — NA
Production system Open Open Open PBR Open FMT Open PBR FMT Open PBR Open
CO2 source NA Free Free Free NA Free Paid Paid Free
Note: NA = not available, PBR = photobioreactor, FMT = fermenter.
Study: 1 = Lee (2001), 2 = Benemann et al. (2003), 3 = Van Harmelen and Oonk (2006), 4 = Chisti (2007), 5 = Li et al. (2007), 6 = Alabi et al. (2009), 7 = Davis et al. (2011),
8 = Nagarajan et al. (2013).
ECONOMIC EVALUATION OF BIODIESEL PRODUCTION PROCESSES
537
rising petroleum prices, single-step extraction and transesterification can become

a commercial reality.
To make biodiesel further economical, a more integrated approach with
preexisting industry has also been applied. The wastewater-based microalgae
biodiesel production system was evaluated by Xin et al. (2016). The study used
multilayered PBR, which is why 47.7% of the total investment was attributed to
PBR. Capital investment for land was lowest in this scenario. To operate the plant,
the flocculant cost was highest (33.8%), followed by equipment depreciation
(22.8%) and then labor cost (19%). In this scenario, significant improvement in
biomass production cost was achieved ($0.33/kg) because a significant amount of
nutrient was provided from wastewater. The study further demonstrated that
improvement in harvesting technology could significantly improve the biodiesel
production cost (Xin et al. 2016). The implementation of electrocoagulation
technology improved the internal rate of return from 5.41% to 10.1%.
In summary, many different TEA studies conducted on biodiesel from
microalgae expressed mutual concerns. It has been recommended that improving
the strain of microalgae to produce higher oil content will drastically improve
the economy. Microalgal growth is also limited by carbon assimilation (fixation)
rate, specific biomass growth rate, and photosynthetic efficiency. New and
improved engineered species of algae should be made to improve the economy
of biodiesel production. Improvement in biomass harvesting technology will
enhance the economic revenues from biodiesel production. Although from
recent studies (Nagarajan et al. 2013, Xin et al. 2016) the microalgae biodiesel
seems a very suitable alternate fuel, it is still far from being competitive with
petroleum fuels.
22.5 ADVENT OF SINGLE CELL OIL AND TECHNO-ECONOMIC

SHORTCOMINGS
Oil production from oleaginous microbes grown in a bioreactor started making

sense when researchers discovered microbes with as high as 80% (w/w) lipid
content of their cell dry weight. With high lipid content, the productivity achieved
by microbial oil production can be at par with microalgae. However, because of
the light (subsequent geographical) dependency of microalgae and slower (com-
pared with bacteria and fungus) growth rate, microalgae still have a massive land-
use footprint. The land usage can be minimized by using oleaginous microorgan-
isms to produce SCO. These organisms are pluripotent and can metabolize
different types of waste substrates. The research on SCO is motivated by the
integrated concept of waste valorization, independence from climatic conditions,
and high throughput of lipid production. Because the SCO production is very
recent, an insufficient number of TEA studies are available to highlight the issues
pertaining to technological improvement of the process for economic feasibility.
In this section, the best-reported TEA for microalgae is compared with SCO TEA
studies and the economic enhancement and direction of the feasibility of the
technology is analyzed.
The technological difference between the two technologies in comparison
(algae versus SCO) is that in the case of algae, Nagarajan et al. (2013) used SOET
by sonication thereby eliminating the need for solvents like hexane or chloroform.
Conversely, in the case of SCO, Koutinas et al. (2014) used conventional lipid
extraction (using hexane) followed by transesterification using purified microbial
oil. Koutinas et al. (2014) compared the direct transesterification of dry microbial
biomass using 20 times methanol for 20 h at 70 °C. This variant of the process was
prohibitively uneconomical because of the very high direct capital investment on
the order of $100 million, which will take very long to recover.
When comparing the two TEA studies, it was observed that Nagarajan et al.
(2013) reported the lowest ever reported price for biodiesel production from
microalgae. A closer scrutiny reveals some overambitious assumption used by the
study. The study claims to have transesterification efficiency of 90% using
microalgae biomass slurry without drying right after flocculation and settling.
The slurry was directly subjected to transesterification using a 4:1 (methanol:oil)
ratio of methanol which is lower than those reported in all the other studies (6:1
for 100% excess). Lower methanol concentration can have a reversible conversion
of FAMEs into FFAs. Further study is using wet biomass with base-catalyzed
transesterification, which is reported to be less efficient resulting in prolonged
reaction rates and formation of soap. Thus, the assumptions by the study
(Nagarajan et al. 2013) is apparently unrealistic. Similarly, TEA performed by
national renewable energy laboratory, NREL (Benemann and Oswald 1996)
assumed 100% extraction efficiency of algal oil, which is unrealistic. Likewise,
owing to an incorrect assumption, many other TEAs can give misleading results.
Therefore, all the assumptions for each TEA should be carefully examined before
adopting.
Nonetheless, Table 22-4 compares the two proposed processes, and it is quite
apparent that the land required by the algae-based process is higher than the
microbial oil–based process. Land cost is a paramount consideration while setting
up a microalgal-based biodiesel process, and the locality and value of the land will
decide the price of the land. Land cost can vary anywhere between $0.36/m2
(Benemann and Oswald 1996) and $13.78/m2 (Norsker et al. 2011).
The algae-based process has an advantage in that it can use flue gases
(concentrated source of CO2) for free, whereas the microbes need to use
(heterotrophic) fixed carbon sources, either pure substrate like glucose or waste
streams (whey), which come along with some processing cost. In the present
scenario, the cost of carbon (substrate) is around 80% of the raw material cost. The
microalgae-based process in the discussion has three times higher productivity of
biodiesel compared with the microbial counterpart, but the capital investment
required to set up the microbial oil process is 1.5 times higher. The reason quite
obviously falls on the expensive fermenters (majorly) and vessels used to carry out
the process. Although the process does save on the vast area of land that is now
Table 22-4. Economic Comparison between the Algae Oil–Based and Microbial Oil–Based Biodiesel Production Processes.
540
Koutinas et al.
Parameter Nagarajan et al. (2013) (2014) Remarks
Productivity 60 g/m2/d 10,000 t/yr Algae capacity 29.56 × 103 t/year is greater
than microbial plant
Land used 4 × 106 m2 (400 ha) NA Significantly less land required
Land cost $0.91/m2 NA —
Carbon source Flue gas/pure CO2 Glucose Algae are autotrophic and they fix
(Yx/s = 0.35 and atmospheric CO2
YL/s = 0.23)
Biomass Microalgae R. toruloides Fast growing
Oil content of 50% 67.5% High lipid content
biomass
Extraction 75% NA 99% (homogenizer) in hexane (25% w/w)
efficiency for SCO
Transesterification 90% 90% Two steps final 99% efficiency
efficiency slurry + methanol + sonicate
Biomass harvesting Flocculation Centrifugation The biomass dried in fluidized bed up to
1% moisture for SCO extraction
Flocculants used Sod. CMC NA —
Flocculants cost $0.55/kg NA —
Growth system Open raceway pond Bioreactors Expensive reactors
Methanol cost $0.239/kg $0.42/kg —
Methanol:oil 4:1 6:1 In algae the methanol used is very low;
implausible assumption
Catalyst NaOH (0.3% w/w in methanol) NaOH (1% w/w) —

Cost of catalyst $5.432/kg $2.6/kg Price taken by Nagarajan et al. is higher
than Koutinas et al.
Material of SS316 steel SS304 —
construction
Electricity cost $0.0989/kW-h $0.08/kW-h —
Contingency 5% 1.5% —
Engineering cost 5% NA —
Insurance NA 1.5% —
Labor wage rate $19.42/h 25000/yr Low wages assigned in Koutinas et al.
Biodiesel cost $0.42/L $5.89/kg —
Capital cost comparison: algae versus SCO
Parameters Nagarajan et al. (2013) Koutinas et al. Remarks
(2014)
Site, equipment, $0.123 million/ha or $49.2 million $74 million The process using SCO require expensive
supply systems bioreactors and other equipment
with 10%
contingency and
engineering cost
541
available for other economic (or agricultural) activities, but such factor cannot be
incorporated in TEA.
Evidently, the microalgal process (in the discussion) is less power consuming
(Table 22-5) compared to the SCO process. It requires only an investment of
$2.2 million compared with $9 million for the SCO process. This is because in the
case of bioreactors the process has to be maintained sterile, and an enormous
amount of heat is required to sterilize the media and control the temperature
during the microbial fermentation. Open raceway ponds do not have such a high
utility requirement, and therefore they are energetically more beneficial to operate.
Ultimately the final cost of biomass production in algae is on the order of $0.16/kg,
whereas the biomass production cost is very high in the microbial lipid process
($3.41/kg). The final biodiesel price for algae is far away for comparison ($0.42/kg)
with SCO-based biodiesel ($5.89/kg). In general, the SCO-based biodiesel remains
expensive when the prices are compared with the one mentioned in other TEA
studies (Table 22-5).
Although the prices for SCO-based biodiesel production are demotivating,
SCO-based technology may still represent future direction to solve the possible
food crisis problem of the world because the microalgae-based industry requires a
large area of arable land that can be used for agricultural purpose. Secondarily, the
photosynthetic microalgae are light dependent, and therefore the microalgae-
based technology is not suitable for all geographical locations on earth. Some
countries witness insufficient amounts of sunlight therefore algal biodiesel tech-
nology is inefficient for these countries. Keeping the aforementioned points in
mind, it is essential to develop more compact and independent processes for
biodiesel production. In fact, one may argue about the photobioreactors, but as
mentioned previously, the cost of photobioreactors is still very high, and they are
easily out of the competition when the prices are compared with petroleum fuel.
Hence, the present need is to develop new compact and independent biorefineries
for (alternate fuel) biodiesel production using SCO.
To make the SCO-based biodiesel industry competitive to petroleum industry
the research should be directed toward developing new strains with high biomass
growth rate, attaining very high biomass concentration with high biomass yield
and oil content. The new strains should be pluripotent to metabolize all the
different kinds of substrates available (such as cellulosic) and can withstand the
hostile environment of various waste streams. Such work is possible in the ambit
of genetic engineering. Basic science experimentations should be conducted to
isolate naturally adapted strains from extreme natural conditions to develop
processes where sterilization is not required, and the process can be used in
nonsterile mode. This kind of modifications can dramatically change the whole
cost scenario. Simultaneous production of value-added products with biodiesel is
another very fruitful direction to make the process economical. Some researchers
have reported oleaginous strains to fix atmospheric CO2 in the form of energy
molecules (lipids) (Wang et al. 2008, Taher et al. 2015, Kumar et al. 2017).
Further contributions from different disciplines are required to make the
technology cheap and economical. New and inexpensive composite material needs
Table 22-5. Comparison of Operating Cost for Biodiesel Production Using Algal Oil and Single Cell Oil.
Operating cost comparison: algae versus SCO
Parameters Nagarajan et al. (2013) Cost (%) Koutinas et al. (2014) Cost (%)
Power (electricity, utilities) $5,536/ha/yr or $2.24 million 34.5 Production $7.1 million; extraction —
$1.3 million; transesterification
$0.7 million
Nutrients $6,639/ha/yr 41.4 Yeast extract $3.5 million 16.46
CO2 Free (flue gas) NA Glucose ($0.4/kg) $16.8 million 79.28
Methanol $20,971/ha/yr 130.8 $0.456 million 2.14
NaOH catalyst $4,660/ha/yr 29.1 $0.26 million 1.22
Flocculants $416/ha/yr 2.6 NA —
Labor $4,331/ha/yr or $1.7 million 27 Production $0.53 million; extraction —
$0.125 million; transesterification
$0.35 million
Waste $1,854/ha/yr or $0.7 million 11.6 — —
Credits $33,857 −211 NA —
Maintenance, tax, insurance $5,487 34.2 — —
(5% on capital cost)
Biomass cost $0.157/kg — $3.41/kg
Lipid cost — — $5.48/kg
Biodiesel cost $0.42/L — 5.89/kg
543
to be discovered that can replace stainless steel (SS316 or SS304) to bring down the
capital cost of process setup. New reactor configurations are required from
mechanical engineering perspectives to manufacture large vessels at low cost.
Process engineering should play a fundamental role in designing processes, which
are efficient and have very high productivities. Fermentation technology can
improve the fed-batch or continuous mode of fermentation to exploit the
maximum potential of the microbes. In the field of thermodynamics, if the
cheaper technology of production of heat carriers, heat sinks, and coolants can
be discovered, then it can reduce the cost of production drastically. There is always
some scope in thermodynamics to improve the heat transfer coefficient by
designing the heat transfer systems and flow configurations.
22.6 COST CONSIDERATIONS IN DOWNSTREAM PROCESSING
The downstream section of the process is one of the most cost intensive
sections and requires an enormous amount of resources for the conversion of
oil-containing biomass into biodiesel. In a typical biodiesel production process,
the downstream processing section consists of harvesting (oil seeds or biomass),
oil extraction, transesterification, and post-transesterification processes to get the
final product, biodiesel (B100). The downstream process contributes 60% of the
total production cost. Therefore, it is necessary to reduce the total combined cost.
Researchers have already been working on in situ transesterification using wet
biomass, direct transesterification of oil without extraction and purification of oil
(Koutinas et al. 2014, Yellapu et al. 2016). Some studies are claiming to produce
extracellular lipids, which will completely remove the barrier of downstream
extraction from the picture (Bharti et al. 2014). In this section economic
consideration regarding the downstream processing of oleaginous microbes to
obtain pure microbial oil has been discussed.
22.6.1 Biomass Harvesting

The harvesting process will vary depending on the feedstocks. Plant seeds are
harvested using a swatching machine, and microalgae and oleaginous hetero-
trophs are harvested by using flocculation, centrifugation, and filtration process.
Depending on the process used for harvesting, the cost and energy utilization will
vary from 10% to 30% of overall biodiesel production cost (Barros et al. 2015).
There are several approaches of harvesting that have been improvised in labora-
tory-scale studies. As apparent in Table 22-6, the method of harvesting will be
directly affecting the energy investment and cost of oil extraction. In a US
international trace commission (USITC) report (Reeder 2003), the cost for
harvesting soybeans using a swatching machine was $4.30 per bushels (43 bushels
planted per acre). Harvesting technology for microalgae, oleaginous yeast, fungi,
and bacteria are comparatively expensive owing to their smaller sizes. The
efficiency of harvesting will increase requiring a proportional amount of energy
investment thereby increasing the overall harvesting cost.
Table 22-6. Effect of Cost and Energy Investment by Using Different Harvesting Methods.
Harvesting Energy investment
Feedstock Method of harvesting efficiency (%) (kWh/m3) Cost References
Oil seeds Swatching machine-Korvan — — $184.9/ac Reeder (2003)

9240
Microalgae Centrifugation 94 0.80 $0.864/L oil Dassey and
Theegala (2013)
Oleaginous yeast Centrifugation 96 8 — Uduman et al. (2010)
Oleaginous fungi Filtration — 0.4 — Koutinas et al. (2014)
545
Microalgae are a good alternative for biodiesel production, but the biomass
harvesting is one of the major problems. In the last decade, various improvements
in harvesting technologies were developed such as centrifugation, flocculation,
sedimentation, tangential flow filtration, and electrolytic flocculation. Among all
the technologies, centrifugation is a most efficient technique to recover maximum

biomass, but the energy consumption and capital cost associated are unappealing.
Flocculation is a well-established approach with maximum harvesting efficiency
above 95%, and it has been immensely studied in wastewater treatment systems.
However, there is no well-reviewed techno-economic research in this area
comparing the cost and energy investments involved in each of these harvesting
methods separately. Oleaginous yeast and fungi are well studied in the last 5 years
(2012 to 2017), and yet again the harvesting of oleaginous biomass has been done
mostly using centrifugation and filtration.
22.6.2 Economics of Lipid Extraction and Separation

The lipid extraction/separation is one of the major contributors to enhance overall
biodiesel production cost. The moisture content in the oil seeds is very crucial for
high oil extraction. There are several extraction methods from oil seeds, and they
are commercialized based on the economic importance and feasibility. However,
after cell wall disruption to separate oil, organic solvents are necessary for some
feedstocks such as microalgae, yeast, and fungi to enhance lipid recovery. Oil
extraction from oil seeds by a mechanically driven screw press is a traditional
technique with lowest cost but the major disadvantage is in the filtration and
degumming, which are necessary steps to remove remaining kernels and fibers in
the extracted oil. The other oil extraction methods such as solvent oil extraction
and pressurized solvent extraction are efficient but expensive. Further, these
techniques will cause solvent contamination in oil and a safety problem for the
environment as well as the biodiesel industry.
The autotrophic and heterotrophic oil-producing microorganisms such as
microalgae, yeast, and fungi are very tiny in size, and they possess thick and
rigid layer cell wall, which is composed of carbohydrates and glycoproteins.
Therefore, mechanical cell wall disruption such as bead milling and ultrasonication
(Table 22-7) are very effective for cell wall disruption, but they are energy
consuming processes. According to Koutinas et al. (2014), by using the mechanical
cell wall disruption process, the cost of microbial oil extraction is $3.4/kg. Enzyme-
based cell wall disruption is a very efficient method to enhance lipid yield. Although
enzymatic hydrolysis is a scalable approach, its economic viability owing to the high
cost of enzymes impedes the implementation in biodiesel production.
Bligh and Dyer (1959) developed total lipid extraction from animal tissue using
chloroform and methanol. It is a combination of nonpolar and polar solvent to
extract total lipids. In the present scenario, the modified folch method is well studied
for lipid extraction using organic solvents by heating up to its boiling point with
biomass and extract total lipid. However, it is an energy consuming process, and it
will affect the final product (biodiesel) cost. Therefore, in a recent study (Yellapu et al.
2016), wet oleaginous biomass lysis with N-lauryl sarcosine (N-LS) was used for cell
wall disruption. The maximum oil extraction efficiency recorded was 98% (w/w), but
Table 22-7. Oil Extraction Methods for Different Feedstocks.
Feedstock Oil extraction method Cost References
Oil seeds Engine driven screw press Low Gonfa Keneni and Mario
Solvent oil extraction High Marchetti (2017)

Pressurized solvent High
extraction
Enzymatic oil extraction High
Microalgae Milling High Kim et al. (2013)
Ultrasonication High
Solvent oil extraction High
Enzymatic oil extraction High
Yeast Bead milling High Dong et al. (2016)
Ultrasonication High
Solvent oil extraction High
Fungi Solvent oil extraction High Dong et al. (2016)
techno-economic studies are not yet conducted. In the study, 40-g N-LS was used for
1 kg oil content biomass (50% oil content), which means 40-g N-LS is necessary to
extract approximately 0.5 L microbial oil (Yellapu et al. 2016).
22.7 COST CONSIDERATION IN THE TRANSESTERIFICATION

PROCESS
Transesterification process is a simple chemical method to convert triacylglycer-

ides to FAMEs (biodiesel). There are several factors that need to be considered
while discussing the cost of biodiesel production such as (1) transesterification
reaction system, (2) catalyst, (3) alcohols, and (4) cosolvents.
22.7.1 Comparison of Cost between Transesterification Reactors

Transesterification reaction can be conducted in two different reactors such as (1)
stirred tank reactor with heating, and (2) cavitation reactor. In a mechanically
agitated reactor, the reactants incubation time can be changed according to the
type of reactants such as the nature of the catalyst, alcohols, and percentage of
water and free fatty acid content in the oil. Researchers have been continuously
working to reduce the energy requirement in transesterification reaction. Chuah
et al. (2017) compared the classical mechanical stirring with hydrodynamic
cavitation and ultrasonic cavitation and found that using optimized conditions
of hydrodynamic cavitation (1:6 oil:methanol ratio; 60 °C, 1% NaOH) the maxi-
mum efficiency of 98% was achieved in only 15 min (Table 22-8). Hydrodynamic
cavitation (HC) methods follow first-order reaction kinetics (same as mechanical
stirring), but the reaction kinetics is 7.7 (600% shorter reaction time) times higher
Table 22-8. Comparison of Transesterification Reactors for Different Feedstocks.
Hydrodynamic Mechanical Ultrasonication

Parameters cavitation stirring cavitation
Temperature 60 60 40
Time (min.) 15 90 —
Reaction order 98 99 50 (L)
Rate constant (I order) 0.238 0.031 II order
Oil:methanol 1:6 1:6 1:6
Catalyst (%) 1 1 1
Yield efficiency (g/J) 1.25 0.25 —
References Chuah et al. Chuah Mostafaei
(2017) et al. (2017) et al. (2015)
compared with mechanical stirring. The HC method was 1.8-fold more energy
efficient, and 4.6-fold lower feedstock was used per unit of product produced.
These improvements can lead to very significant cost reduction and should be
studied further in cost estimation simulations.
22.7.2 Reactants in Transesterification

The reactants in transesterification reaction such as catalyst (NaOH, H2SO4, or
enzyme) and short-chain alcohols (methanol and ethanol) will react together to
convert oil [triacylglycerides (TAGs)] into fatty acid alkyl esters (biodiesel).
Evidently in Table 22-9, the unit production cost ($/L) of biodiesel varies with
the catalyst used in each case study. The sodium methoxide is cheaper than other
reactants, but it can only be used essentially with feedstock oils having minimal
Table 22-9. Production Cost of Biodiesel Using Different Feedstocks and Reactants.
Unit production
Conversion cost ($/L
Feed stock Catalyst/alcohol efficiency (%) biodiesel) References
Spent oil Sodium methoxide >98 0.5 Marchetti et al.

Spent oil Acidified methanol >98 0.51 (2008)
Spent oil Enzyme-packed >98 0.52
bed reactor
Castor oil Sodium methoxide 100 1.56 Santana et al.
(2010)
Microbial Sodium methoxide 92 5.9 Koutinas et al.
oil (2014)
free fatty acid and water content. If excess of free fatty acid is present, it will
cause soap formation, which can directly affect the conversion efficiency percent-
age (w/w). Marchetti et al. (2008) compared three configurations (acid-catalyzed,
base-catalyzed, and resin-based transesterification) of biodiesel production
using used oil, the respective unit production cost ($0.5, $0.51, and $0.52/L
biodiesel) did not vary significantly. This indicates that although the change
in esterification catalyst might change internal distribution of cost, overall the
final cost of biodiesel production remains unchanged. The observations in
this study agrees with our previous discussion about results obtained by Zhang
et al. (2003).
Previous studies (Apostolakou et al. 2009, Marchetti et al. 2008, Zhang et al.
2003) using oil or virgin oil revealed that feedstock oil cost still weighs significantly
(60% to 80%) in economics of biodiesel production. Santana et al. (2010) explored
the variation of direct operating cost of various components (raw materials, energy,
maintenance, and labor) when the raw material price was varied. When the
castor bean cost varied from $1.15 to $0.526/kg, the price of biodiesel reduced
from $1.52/L to $0.92/L. The contribution of feedstock oil in direct capital cost
changed from 72.29% to 54.26%. Furthermore, the study simulated the effect of
glycerin purification on biodiesel cost (Santana et al. 2010). Results showed that in
absence of glycerin purification the cost of biodiesel was $1.56/L compared with
$1.52/L with glycerin purification. This decrease is the trade-off between the cost
procured to purify the glycerin waste stream and the credit recovered from the waste
stream. Because the purification of glycerin is in economic favor of the process one
should always treat the waste stream instead of throwing away the carbon for
wastewater treatment units to do the end-of-pipeline cleanup. This waste stream will
certainly technically affect the wastewater treatment plant down the line.
There are very limited techno-economic studies on biodiesel production
using microbial oil. Koutinas et al. (2014) analyzed economic studies using direct
transesterification and pure microbial oil. The estimated unit production cost of
purified microbial oil is 3.4$/kg by using mechanical cell wall disruption
(homogenizer), and hexane is an organic solvent to separate microbial lipid. The
study tried to simulate the cost associated with direct transesterification of
microbial biomass for biodiesel production, but the study abandoned the case
because the capital investment required in the direct transesterification of the
microbial mass was 1.5-fold higher than conventional extraction and transester-
ification of the purified microbial oil. The reason for this was a very high biomass-
to-methanol ratio (1:20) required to have technically feasible transesterification of
microbial oil at convenient rate. The high volume of methanol invested requires an
enormous amount of heat energy to recover by distillation. Furthermore, the size
of transesterification reactor required needs to be very large, which makes it
economically unreasonable.
Ultimately, the whole product line is worth nothing if the final product does
not meet the standard of application (or intended use). The biodiesel produced
after transesterification contains various impurities that further need to be
addressed.
22.7.3 Effect of Impurities on Oil for Biodiesel Conversion

There are different impurities in oil obtained after the process of harvesting, and it
will hinder the biodiesel efficiency. Such impurities are moisture content present
in oil seeds. After mechanical crushing of oil seeds, traces of water will be present
in the oil. To remove all insoluble impurities, Jain and Sharma (2010) heated oil at
100 °C for 10 min to remove all moisture content. In the same way, a high
concentration of free fatty acid (7% to 20% w/w) present in WCO and animal fats
will cause saponification process in the presence of base catalyst. Therefore,
another esterification step is necessary to convert free fatty acid to fatty acid esters.
The pretreatment of oil to remove impurities increases the production cost of
biodiesel.
22.8 POST-TRANSESTERIFICATION PURIFICATION COST

CONSIDERATION
The performance of biodiesel in an internal combustion engine depends on its

purity. It is preferred to have no or minimum impurities. The possible impurities
in biodiesel are glycerol, methanol, catalyst, acylglycerol, soap, and salts. The
presence of these impurities will decrease the fuel performance and might even
negatively affect the combustion system (Berrios and Skelton 2008). For example,
if methanol is present in the biodiesel, it will cause reduced viscosity and lowered
flash point of the fuel. Similarly, the presence of water reduces the heat of
combustion, hydrolyzes part of the methyl esters, and might lead to biomass
development. Water can easily form ice crystals at low temperatures and corrode
fuel tubes and injector pumps. Water can also cause pitting damage in the piston.
If soap is present in biodiesel, it can damage the injectors and result in a corrosion
problem in the engine and blockage of filters, which will make engine weak. The
presence of FFAs causes the fuel to have low oxidation stability. Acylglycerol can
result in an increase in the turbidity and viscosity of the fuel oil. The presence of
residual glycerol can lead to several problems, such as storage problems, settling
problems, deposits at the bottom of the fuel tank, and injector fouling.
Once the feedstock oil is transesterified, the produced FAME biodiesel needs
to be separated from the hydrophilic (water, glycerol, catalyst, and methanol)
fraction. After the separation by settling, the crude biodiesel further needs refining
to remove any trace contaminants or remnants to meet the standard specifications
(EN14214 or ASTM D6751) by environmental authorities. The crude biodiesel is
therefore further cleaned using various methods like wet washing, dry washing,
membrane extraction, or using ion liquids. The purification of biodiesel can be
performed by two different methods: (1) wet washing, and (2) dry washing. These
methods are briefly described in subsequent sections, followed by their cost
analysis for the process configuration variants designed with these purification
methods.
22.8.1 Wet Washing

Because the impurities present in the biodiesel are water soluble in nature, washing
of biodiesel is effectively used to remove methanol and glycerol from biodiesel.
This method also removes sodium compounds and soap molecules (if initial FFA
was high). Formation of soap is the most undesirable event because it causes the
emulsification of FAMEs, thereby causing problems with oil–water separation.
When compared to ethanol and methanol, use of methanol gives low soap
formation. A higher presence of soap will require a large amount of water
(Yellapu et al. 2016).
22.8.1.1 Wet Washing with Deionized Water

To make the biodiesel cleaner, wet washing is often performed with deionized
water. It is a classical technique of crude biodiesel purification after the removal of
glycerol. Hot deionized water at 50° to 60 °C is mixed well with the crude biodiesel
at different proportions to dissolve and remove the water-soluble impurities. Balat
and Balat (2010) indicated that 5.5% (v/v) addition of water resulted in efficient
removal of impurities within 5 min of stirring. Water washing has been studied by
many researchers (Gonzalo et al. 2010, Coêlho et al. 2011). The water washing is
considered to be complete when the pH of the water is 7.0 after washing. Washed
water was removed by centrifugation for 20 min to get purified biodiesel.
22.8.1.2 Wet Washing with a Water–Organic Solvent Mixture

To extract pure biodiesel, Siler-Marinkovic and Tomasevic (1998) treated a FAME
and glycerol mixture obtained after acidic transesterification of sunflower oil with
petroleum diesel and water. The mixture was filtered and washed with petroleum
ether and then four times with water. The obtained methyl ester (biodiesel) was
mixed with an equal volume of petroleum ether and twice the volume of distilled
water, followed by adjustment of pH 7 using acetic acid. The washing process with
water was repeated three times. The FAMEs and petroleum ether fraction were
further dried using sodium sulfate followed by filtration. After drying with sodium
sulfate, the petroleum ether fraction was evaporated at a low boiling point of
petroleum ether. Although by this method the product quality is superior, a need
of solvent addition (petroleum ether) and removal makes this process expensive.
22.8.2 Dry Washing

To purify the crude biodiesel, dry washing has also been in use. In this method, the
crude biodiesel is passed through a bed of adsorbents or an ion-exchange resin.
Various adsorbent materials can be used for crude biodiesel purification like
magnesium silicate (Magnesol), Ca-Mg silicate, and cheap biosorbents (Faccini et
al. 2011). Optimization of the operating conditions for the dry washing of crude
glycerol is usually performed by factorial designs and iterative trial-and-error
methods. Also, various ion-exchange resins used for the purpose of crude biodiesel
purification are mainly small styrene beads with polar functional groups. The
suppliers recommend not to recycle the material for quality control reasons
because the resin irreversibly loses its functional properties and thereby its
efficiency. It leads to further disposal problems in a dry-washing process. Insoluble
impurities present in crude biodiesel can be removed by surface (tangential) or
depth filtration. It should be noted that the amount of methanol significantly
affects the solubility of soap and glycerol in biodiesel. The chemical or physical
mechanism can remove soluble impurities. In the case of glycerol, the glycerol-
saturated ion-exchange resins can be regenerated by washing with methanol.
22.8.2.1 Adsorption-Based Dry-Washing Techniques

Different researchers have tried different materials as adsorbents to remove the
impurities. Özgül-Yücel and Türkay (2003) experimented with rice husk and
compared it with silica gel to purify FAMEs in atmospheric conditions. Although
both the adsorbents reduced the FFA content from biodiesel, the silica showed
better performance with lesser loss of FAME recovery. The increase in silica
dosage increases the removal of impurities. Using silica gel only, Dmytryshyn et al.
(2004) purified crude biodiesel obtained from canola oil; first methanol was
vacuum evaporated, then the biodiesel was stirred with silica for 20 min, and last
the biodiesel was passed through sodium sulfate solution to remove water traces.
The final biodiesel has properties equivalent to commercial diesel.
22.8.2.2 Ion Exchange–Based Dry-Washing Techniques

Ion-exchange resins are developed chemically, and they show their activity owing
to the presence of functional groups on their surface. Commercially, there are
many different types of resins available, which are in use at the pilot scale in
industries. Resins like Amberlite BD10 DRY, Purolite PD 206, Indion BF 170 and
Lewatit (Faccini et al. 2011) are already in use.
22.8.2.3 Other Dry-Washing Techniques

Dry washing can be performed by many other adsorbents like activated carbon. By
virtue of its natural ability, activated carbon can perform decolonization and
purification of crude biodiesel. Other adsorbents include activated fibers; activated
clay, especially acidified clay, is reported to have multiple effects to improve crude
biodiesel. Unconventional waste products are also used to make adsorbents. For
example, rice husk was used to make amorphous silica particles at 500° to 600 °C
(Manique et al. 2012). Spent tea waste was used to make activated charcoal (Fadhil
et al. 2012) by burning it at 600 °C. Crude biodiesel obtained after transesterifica-
tion was passed through the column filled with activated carbon for biodiesel
purification. The unconventional adsorbents produced from waste have a similar
efficiency of biodiesel purification as silica.
22.8.2.4 Advanced Techniques for Biodiesel Purification

Membrane technology is a very well-known technology in the downstream
processing industry. Different types of membranes have been used for biodiesel
purification, including organic membrane (polymer-based membranes), ceramic
Table 22-10. Biodiesel Purification Methods Reported in Literature.
Technique References
Wet washing using acid Faccini et al. (2011)

Wet washing using glycerol Berrios et al. (2011)

Wet washing using distilled water Berrios et al. (2011)
Dry-washing technique using ion-exchange resin Berrios et al. (2011)
Membrane technology (0.1 μm hollow fiber He et al. (2006)
membrane)
Dry-washing technique using Magnesol Faccini et al. (2011)
membrane (made of inorganic materials), among others. Although some

studies have been reported for biodiesel purification using membrane technology,
issues like swelling and fouling of membranes have been reported. So far in the
literature, no comprehensive study compares the economics of all post-
transesterification processes to clean the biodiesel. Therefore, in the subsequent
sections, the economics of existing conventional methods (six of them as shown in
Table 22-10) for cleaning the produced biodiesel have been compared. The
standard simulation software SuperPro Designer has been used to compare
different variations of the process.
22.8.3 Techno-Economic Comparison for Biodiesel Purification

Techniques
Many biodiesel purification techniques have been reported in the literature. The
following six purification techniques (Table 22-10) have been compared regarding
their economics. The basis for calculations and assumptions is described by the
following steps:
1. SuperPro Designer v8.5 was used for simulations for techno-economic
evaluation. Simulations were performed to result in 10,000 L purified biodiesel
per batch for each technique. Unit production cost ($/L) was calculated based
on production cost for obtaining 10,000 L purified biodiesel.
2. Total operating cost = facility-dependent cost + cost of raw materials pur-
chase + waste disposal cost + operating labor cost + utilities cost.
3. Facility-dependent cost (maintenance and repair of the facility) was calcu-
lated based on 10% of fixed capital investment. Fixed capital investment was
assumed to be 4.74 × equipment purchase cost (Kolmetz 2008). The cost of
equipment used in the process was considered from the database of
SuperPro Designer.
4. The cost of raw materials used during the process was taken from the bulk
wholesale vendor website (Alibaba 2017).
5. The waste disposal cost has been examined from costwater website (Cost-
Water 2017). Operating labor cost was calculated based on hours of
operation and labor cost per hour, which was deemed to be $15.45/h based
on average salary of the operator in the United States (database of the
software). Total operating labor cost/h = [1 + 0.15 (supervision factor) + 0.15
(laboratory factor)] × 15.45.
6. The cost of utilities for running the facility, heating, and cooling purpose was
considered from the database of the software.
22.8.3.1 Wet Washing Using Acid

The process of biodiesel purification using acid washing was simulated in
SuperPro Designer to obtain 10,000 L purified biodiesel (Figures 22-2 and
22-3). The removal efficiency for each component is tabulated in Table 22-11. The
cost to purify 10,000 L biodiesel and unit production is tabulated in Table 22-12. As
shown, the unit production cost for purifying biodiesel using acid washing is
only $0.03/L, where 40.2% of the cost is contributed by raw materials—phosphoric
acid and water. Acid water mainly contributes to 90.44% of the raw material cost.
Acid water is prepared by adding 2% (v/v) phosphoric acid in distilled water. The
1. Treatment of crude
6. Upper phase-
biodiesel with 10% (v/v)
Purified Biodiesel
phosphoric acid water
2. Reaction at 55°C for 5. Phase separation

5 min under continuous using differential
stirring centrifuge
3. Phase separation 4. Upper layer was

using differential treated with hot water
centrifuge for 30 min (55°C)
Figure 22-2. Process flow schematics for biodiesel purification using acid washing.
Figure 22-3. Biodiesel purification using acid washing.

Table 22-11. Removal Efficiency of Various Components from Crude Biodiesel Using Acid Washing.
Type of biodiesel Acid number (mg/g) Soap (ppm) Total glycerol (%) Water (mg/kg) Methanol (%)
Not purified 0.33 ± 0.01 1,670.05 ± 7.3 0.71 1,300 ± 15 2.13 ± 0.1
Purified 0.22 ± 0.01 158.12 ± 3.01 0.36 1,400 ± 15 0.18 ± 0.01
Removal 33 90.54 49.3 0 91.55
efficiency (%)
555
Table 22-12. Cost Distribution for Purifying Biodiesel Using Acid Washing.
Component’s
Cost for Unit purification Contribution
Component 10,000 L ($) cost ($/L) to cost (%)
Raw materials (phosphoric 108 1.1 × 10−2 40.2

acid and water)
Labor 70.3 7 × 10−3 26.1
Facility-dependent cost 89.1 9 × 10−3 33.2
Waste disposal (waste acid 0.5 5 × 10−5 0.20
and wastewater)
Utilities 0.8 8 × 10−5 0.3
Total operating cost ($) 268.8 0.03 100
acid water price primarily results from the high price of phosphoric acid ($850/T),
and 33.2% of the cost is facility-dependent cost. Time to process one batch cycle
is 2.6 h.
22.8.3.2 Wet Washing Using Glycerol

The process of biodiesel purification using glycerol was simulated in SuperPro
Designer to obtain 10,000 L purified biodiesel (Figures 22-4 and 22-5). The
removal efficiency for each component is tabulated in Table 22-13. The cost to
purify 10,000 L biodiesel and unit production is tabulated in Table 22-14. The unit
1. Treatment of
crude biodiesel with 6. Upper phase -
15% (weight) pure Purified Biodiesel
glycerol
2. Reaction at room 5. Phase separation

temperature for 10 using differential
min under centrifuge for 30
continuous stirring min
4. Upper layer was

3. Phase separation
treated with 15%
using differential
(weight) glycerol at
centrifuge for 30
room temperature
min
for 10 min
Figure 22-4. Biodiesel purification with wet washing using glycerol.

Figure 22-5. Biodiesel purification using acid glycerol.
Table 22-13. Removal Efficiency of Various Components from Crude Biodiesel

Using Wet Washing with Glycerol.
Crude Glycerol washed Removal

Parameters biodiesel biodiesel efficiency (%)
Acid value (mg/g) 0.29 0.38 0

Density at 25 °C (g/cm3) 0.872 0.866 —
Soap (g/g) 4.4 × 10−4 6.42 × 10−5 85.41
FAME content 88.2 91.8 0
(% by weight)
Water content (mg/kg) 771 253 67.19
Methanol (% by weight) 15.16 0.08 99.28
Total glycerol content 0.9 0.43 51.66
(% by weight)
Table 22-14. Cost Distribution for Purifying Biodiesel Using Washing with Glycerol.
Component’s
Cost for Unit purification contribution
Raw materials (Glycerol) 1,464.5 0.15 92.1

Labor 39.105 4 × 10−3 2.5
Facility-dependent cost 86.3 9 × 10−9 5.4
Waste disposal 0.4 4 × 10−5 0.03
(waste glycerol)
Utilities 0.6 5.8 × 10−05 0.04
Total cost ($) 1,590.8 0.16 100
production cost for purifying biodiesel using glycerol is $0.16/L for which 92.1% of
the operating cost is contributed by the high purchase cost of glycerol ($500/T),
which is used as 15% weight of biodiesel, twice in each batch. Time to process one
batch cycle is 1.6 h only.
22.8.3.3 Wet Washing Using Water

The process of biodiesel purification using distilled water was simulated in
SuperPro Designer to obtain 10,000 L purified biodiesel (Figures 22-6 and
22-7). The removal efficiency for each component is tabulated in Table 22-15. The
cost to purify 10,000 L biodiesel and unit production is tabulated in Table 22-16. The
unit production cost for purifying biodiesel using distilled water is $0.01/L, for which
Figure 22-6. Biodiesel purification with the wet-washing technique using distilled
water.
Figure 22-7. Biodiesel purification using distilled water.

Using Distilled Water.
Crude Water washed Efficiency

Parameters biodiesel biodiesel (%)

Density at 25 °C (g/cm3) 0.872 0.861
Soap (g/g) 4.4 × 10−4 2.37 × 10−5 94.61
FAME content (% by weight) 88.2 92.1 0
Water content (mg/kg) 771 1603 0
Total glycerol content (% by weight) 0.9 0.38 59.57
Table 22-16. Cost Distribution for Purifying Biodiesel Using Distilled Water.
Component’s
Raw materials (water) 5.9 0.0006 4.4

Labor 39.2 0.0039 29.2
Facility-dependent cost 87.9 0.0088 65.6
Waste disposal (wastewater) 0.4 0.00003 0.3
Utilities 0.6 0.00005 0.4
Total cost ($) 134 0.01 100
65.6% of the operating cost is facility-dependent cost for maintenance of two reactors
and two centrifugation units. Time to process one batch is 1.6 h only.
22.8.3.4 Dry-Washing Technique Using Ion-Exchange Resin

The process of biodiesel purification using ion-exchange resin was simulated in
SuperPro Designer to obtain 10,000 L purified biodiesel (Figures 22-8 and 22-9).
The removal efficiency of various impurities using ion-exchange resin is tabulated
in Table 22-17. The binding capacity of resin was assumed to be 400 g/L
considering 40% weight of the resin (40% of total biodiesel weight) is used in
the column (Dias et al. 2014). The unit cost of purification using ion-exchange
Regenerate a column
filled with LEWATIT Pass the crude biodiesel
GF202 at a flow-rate of into the column at a Purified Biodiesel
2 BV h-1 with 3 Bed flow-rate of 2 BV/h at
volume (BV) of room temperature
methanol
Figure 22-8. Biodiesel purification with the dry-washing technique using ion-
exchange resin.
Figure 22-9. Process for the dry-washing technique using ion-exchange resin.

Using Ion-Exchange Resin.
Purified biodiesel Removal

Crude obtained after efficiency

Parameters biodiesel dry washing (%)

Density at 25 °C (g/cm3) 0.872 0.88
Soap (g/g) 4.4 × 10−4 3.25 × 10−4 18.75
Water content (mg/kg) 771 741 3.89
(% by weight)
Table 22-18. Cost Distribution for Purifying Biodiesel Using Ion-Exchange Resin.
Component’s
Raw materials (methanol) 4,537.7 0.45 98.5

Consumables (resin) 0.8 1 × 10−4 0.02
Labor 41.1 4 × 10−3 0.9
Facility-dependent cost 24.9 2.5 × 10−3 0.5
Waste disposal 0.9 0 0
(waste methanol)
Utilities 0.1 0 0
Total cost ($) 4,605.8 0.46 100
resin is $0.46/L (Table 22-18), which is comparatively high compared with wet-
washing techniques, and 98.5% of operating cost is contributed by the high
purchase cost of methanol ($700/ton) and the large amount of methanol (three
bed volumes) used in the process. Regeneration of resin has been considered after
every batch processing. In practical situations, regeneration of resin depends on a
number of impurities present in crude biodiesel. If regeneration of resin is done
after two to three batches, unit purification cost can be reduced. Unit production
cost can also be reduced by using methanol obtained after regeneration in the
transesterification process. Time to process one batch cycle is 2 h.
22.8.3.5 Dry-Washing Technique Using Magnesol

In the laboratory scale, dry-washing technique is performed in a reactor using 1%
to 2% Magnesol (w/w) with heated biodiesel, and adsorption occurs for 15 to
Regenerate a column
filled with Magnesol at a Pass the crude biodiesel
flow-rate of 2 BV h-1 into the column at a Purified Biodiesel
with 3 Bed Volume (BV) flow-rate of 2BV/h
of methanol
Figure 22-10. Biodiesel purification with the dry-washing technique using

Magnesol.
20 min under continuous stirring conditions. Adsorption is followed by centrifu-

gation/filtration to remove Magnesol from purified biodiesel (Banga et al. 2014).
However, at the industrial scale, the dry-washing technique will be performed in
an adsorption column filled with Magnesol, and after every batch cycle, the
adsorbent is regenerated using methanol (Figure 22-10).
The process of biodiesel purification using adsorbent was simulated in
SuperPro Designer to obtain 10,000 L purified biodiesel (Figure 22-11).
The removal efficiency of various components using Magensol is tabulated in
Table 22-19. The adsorption capacity of Magnesol for simulation was considered
Figure 22-11. Process for the dry-washing technique using Magnesol as adsorbent.

Using Magnesol as Adsorbent.
Purified biodiesel
Crude obtained after Removal
Parameters biodiesel adsorption efficiency (%)
Acid value (mg/g) 0.29 0.27 6.19

Density at 25 °C (g/cm3) 0.872 0.875 —
Soap (g/g) 4.4 × 10−4 3.38 × 10−5 91.55
Water content (mg/kg) 771 803 0
(% by weight)
Table 22-20. Cost Distribution for Purifying Biodiesel Using Magnesol as

Adsorbent.
Component’s
Cost for Unit purification contribution to

Component 10,000 L ($) cost ($/L) cost (%)
Raw materials (methanol) 4,456.8 0.45 98.6

Consumables (adsorbent) 0.1 0.0000 0.00
Labor 39.1 0.0039 0.9
Waste disposal 0.9 0.0001 0.02
(waste methanol)
Utilities 0.1 0.0000 0
Total cost ($) 4,520.9 0.45 100
to be 64% (Manuale et al. 2014). The unit cost of purification using adsorbent is
$0.45/L (Table 22-20), and 98.6% of operating cost is contributed by the high
purchase cost of methanol ($700/ton) and a large amount of methanol (three bed
volumes) used in the process. Regeneration of resin has been considered after
every batch processing. In practical situations, regeneration of resin depends on a
number of impurities present in crude biodiesel and the breakthrough point of the
resin. If regeneration of resin is done after two to three batches, the unit
purification cost can be reduced. Time to process one batch using dry-washing
technique is 1.9 h only.
Although the unit purification cost using dry-washing techniques is higher
compared with wet-washing technologies, the removal efficiency of impurities is
higher in dry-washing technologies compared to all the other technologies. The
studies have reported that dry-washing technique with Magnesol/silica alone can
purify biodiesel according to ASTM standards (Banga et al. 2014, Manuale et al.
2014).
22.8.3.6 Membrane Technology

One of the studies has reported where biodiesel was purified to 99% purity using
hollow fiber membrane (He et al. 2006). The study was performed at the
laboratory-scale in which hollow fiber membrane was placed in a 200 mL beaker
filled with water. The process for biodiesel purification using membrane technol-
ogy is described in Figure 22-12.
The biodiesel purification using microfiltration (MF) unit was simulated to
produce 10,000 L purified biodiesel in a batch (Figure 22-13). The unit cost of
purification using MF is $0.058/L (Table 22-21), and 42.83% of the cost is
contributed by hollow fiber membrane purchase cost ($400/m2), which needs to
be replaced after the 1,000 h operation. Although unit purification using mem-
brane technology is low compared with dry-washing techniques, the time to purify
Crude biodiesel was

Permeate was collected
passed through MF unit Membrane cleaning with
as refined biodiesel while
with 0.1 µm 0.5 M NaOH (supplied
retentate/ concentrate
Polysulphone membrane @ 60°C) for 1 h
was discarded as waste
with flux-rate 20 L/m2-h
Figure 22-12. Biodiesel purification using membrane technology.
Figure 22-13. Biodiesel purification using a microfiltration unit.
Table 22-21. Cost Distribution for Purifying Biodiesel Using Membrane Technology.
Unit Component’s
Cost for purification contribution
Component 10,000 L ($) cost ($/L) to cost
Raw materials (0.5 M NaOH) 78 0.008 13.4

Consumables 250 0.025 42.8
(polysulphone membrane)
Labor 140.6 0.0141 24.1
Waste disposal (waste caustic) 0.8 0.0001 0.1
Utilities 12.5 0.0013 2.1
Total cost ($) 583.6 0.06 100
biodiesel using membrane technology is high—5 h for one batch. For the faster
operation, a greater number of membrane modules/filtration units are required.
The cost analysis for all the purification methods have been summarized in
Table 22-22. The following conclusions can be drawn based on the techno-
economic study for various purification methods:
1. In wet-washing techniques, the major unit cost is facility dependent, which
rises because of the equipment used in the process (two reactors and two
centrifugation units). Moreover, wet-washing techniques have large capital
investments ($4.3 to $5 million) compared with other techniques. Wet-
washing technique using glycerol has better removal efficiency for impurities
than acid and water washing, but the method is relatively costly when
compared to other wet-washing techniques, which is caused by the high
purchase cost of glycerol ($500/ton). Wet washing using water is the most
Table 22-22. Cost Analysis for All Purification Methods (10,000 L per Batch) Reported in Literature.
564
Fixed capital Unit

investments purification Time for
Method (million $) cost ($/L) Major cost contributing factors operation (h)
Acid washing 4.7 0.03 Raw material (40%), facility-dependent 2.6

cost (33%)
Wet washing using glycerol 5 0.16 Glycerol as raw material (92%) 1.6
Wet washing using water 4.3 0.01 Facility-dependent (65%) 1.6
Dry washing using 1 0.46 Methanol for regeneration (98.5%) 2
ion-exchange resin
Dry washing 0.98 0.45 Methanol for regeneration (98.5%) 1.9
using Magnesol
Membrane technology 1.6 0.06 Membrane purchase cost (42.83%); labor 5
cost (24.09%)
economical purification technique, but wet-washing techniques are unable

to purify biodiesel (moisture content especially) according to ASTM stan-
dards (Banga et al. 2014, Berrios et al. 2011).
2. The membrane technology is the most time-intensifying technique among
all. The membrane purchase cost is a major contributing factor in the unit
purification cost because it needs to be replaced after 1,000 h operation. Also
fouling of membrane can occur easily because of high soap content in crude
biodiesel. Labor cost is another contributing factor that results from the
extended hours of operation for microfiltration.
3. Dry-washing techniques using ion-exchange resin and Magnesol have the
same purification cost—$0.45 to $0.46/L—but Magnesol is more efficient in
removing all sorts of impurities because of its inorganic nature, and it does
not impart acidity into the biodiesel, unlike ion-exchange resins (Banga et al.
2014, Faccini et al. 2011). Of operating cost in dry-washing techniques,
98.5% is contributed by the high amount of methanol (three bed volumes)
used in the process. In the simulation, regeneration of resin has been
considered after every batch cycle processing. In practical situations, regen-
eration of resin depends on a number of impurities present in crude
biodiesel and breakthrough point of the resin. If regeneration of the resin
is done after —two to three batches or 50% methanol solution is used for
regeneration, unit purification cost can be reduced. In spite of its high unit
purification cost, it has small capital investments (one-fourth of that
required for wet-washing techniques), less processing time, and is more
efficient than other purification techniques.
After all the analysis and techno-economic studies, the results that intrigue or
interest a nonresearcher the most is the economic feasibility. In fact, indirectly the
discussion in this chapter has tried to answer the question of whether or not the
proposed process is economically feasible. The policy makers or industry in real life
understand a different language; the process is viewed in a different vision—one
called profits and revenues. In the next section, the chapter elaborates on how to
summarize the whole techno-economic study in different parameters of economic
feasibility and evaluates which variant of the process is economically better.
22.9 EVALUATION OF ECONOMIC FEASIBILITY
22.9.1 Return on Investment

Return on investment (ROI) is used to evaluate the efficiency of an investment. In
purely economic terms, it is a way of relating profits to capital invested.
Mathematically, ROI is calculated as
ROI = Annual net profit∕Total investment × 100 (22-1)

ROI is dependent on the net profit generated by the company, which is

dependent on the difference between annual revenues produced by the enterprise
(through selling the commodity) and annual operating cost. The increase in ROI
indicates that the biodiesel producer can recover its investment cost in a shortest
possible time. Negative ROI means that annual operating cost is greater than the
revenues generated, or in other words, unit production cost is higher than selling
price of the commodity. In such a case, the production plant can never recover its
investment cost. Because it is dependent on the unit production cost, ROI is
dependent on the choice of feedstock and the process used for biodiesel produc-
tion. ROI for various biodiesel production processes has been compared in
Table 22-23.
Because microbial oil production using glucose and lipid extraction from
primary sludge (dry route) have high annual operating expenses compared with
revenues, ROI for these processes is in the negative, indicating the operations are
economically not feasible at the industrial scale. ROI for vegetable oil is highest,
21.59% because of its low annual operating cost and high revenues generated
annually. ROI for primary sludge using the dry route is 4.7% only, but it is
economically feasible. The process is very much dependent on the lipid content
present in the primary municipal sludge and the extraction efficiency of the
process. Because the unit production cost is dependent on the scale of operation,
ROI is also reliant on the scale of operation. One of the techno-economic studies
for biodiesel production using vegetable oils indicated that ROI increases loga-
rithmically with the scale of operation as shown in Figure 22-14 (Apostolakou
et al. 2009).
22.9.2 Cash Flow Analysis

A discounted cash flow (DCF) is an evaluation method used to estimate the
attractiveness of an investment opportunity. DCF analysis uses future free cash
flow projections and discounts them to arrive at a present value estimate, which is
used to evaluate the potential for investment.
Net present value (NPV) is estimated as the difference between cash inflow
and cash outflow. It is used to assess a project’s future returns at a particular
period. NPV is 0 at the break-even point at which the investment cost incurred by
the company is completely recovered. NPV is calculated as
X
NPV = C∕ð1 þ rÞt − Co (22-2)
where:
C = Net cash inflow during the period t,
Co = Total investment costs (cash outflow),
r = Discount rate, and
t = Number of years.
The internal rate of return (IRR) is a discount rate that makes the NPV of all
cash flows from a particular project equal to zero. IRR is calculated from the
Table 22-23. Comparison of ROI for Different Biodiesel Production Processes.

Microbial oil production Biodiesel from Extraction from Extraction from
Process/economic parameter using glucose vegetable oil wet primary sludge dried primary sludge
Total investment (million $) 70.2 9.4 7.45 17.25

Scale of operation 10 kT/year 50 kT/year 3.55 kT/year 4.7 kT/year
Biodiesel selling price ($/T) 1,376 1,376 1,376 1,376
Revenues (million $) 13.76 68.8 4.88 6.47
Annual operating cost (million $) 65.9 65.9 4.38 9.49
Annual profit generated (million $) −52.14 2.9 0.5 −3.02
30% tax (million $) 0 0.87 0.15 0
Annual net profit (million $) −52.14 2.03 0.35 −3.02
ROI (%) Unable to recover its 21.59 4.7 Unable to recover its
investment investment cost
References Koutinas et al. (2014) Apostolakou Olkiewicz Olkiewicz et al. (2016)
et al. (2009) et al. (2016)
567
Figure 22-14. ROI as a function of scale of operation.
formulas of NPV putting NPV = 0, and the discount rate is calculated as IRR. It is
also a valuable tool in analyzing profitability of an investment. One of the techno-
economic studies for biodiesel production using vegetable oils indicates that
discounted cash flow return (DCFR) increases logarithmically with the scale of
operation as shown in Figure 22-15.
22.9.3 Investments, Profits, Revenues, and Credits

Economic feasibility of an industrial process is also dependent on investments
incurred by the company and annual profits generated by the manufacturing
plant. The higher the investments, the more time is required to recover the cost.
The investments incurred by the firm is comprised of two components: (1) capital
investment to set up the new plant, including contingency and fees, and so forth;
and (2) working capital that is assumed to be 10% to 15% of capital investment
(Olkiewicz et al. 2016). The capital investment incurred by the biodiesel producer
is dependent on the scale of operation, the process used, and the number and type
of equipment used in the process. Table 22-24 compares capital investment of
various biodiesel processes.
Figure 22-15. Discounted cash flow return (DCFR) as a function of scale of

operation.
Table 22-24. Comparison of Capital Investments for Different Biodiesel Production Processes.
Biodiesel from waste Biodiesel from waste
Process/Economic Microbial oil cooking oil (acid cooking oil Biodiesel from Extraction from wet Extraction from dried
parameter production catalyzed) (base catalyzed) vegetable oil primary sludge primary sludge
Capital Investment 70.2 2.55 2.68 9.4 7.45 17.25

(million $)
Scale of operation 10 kT/year 8 kT/year 8 kT/year 50 kT/year 3.55 kT/year 4.7 kT/year
Major investment Fermenter (250 m3) Transesterification Esterification and Transesterification Equipment for Equipment for
factors reactors transesterification reactors (2 × 9 m3) storage of centrifugation and
reactors solvents, drying
methanol,
catalyst, base, and
so forth
Process highlights 1. Fermentation 1. Transesterification 1. Pretreatment of 1. No pretreatment 1. Extraction and 1. Drying
2. Cell harvest 2. Neutralization of acid waste cooking oil 2. No extraction recovery 2. Extraction and
3. Extraction and 3. Distillation 2. Separation of 3. Transesterification 2. Transesterification recovery
Recovery refined cooking oil 4. Purification/ 3. Purification 3. Transesterification
4. Transesterification 3. Transesterification distillation 4. Purification
5. Purification 4. Distillation
References Koutinas et al. (2014) Zhang et al. (2003) Zhang et al. (2003) Apostolakou et al. Olkiewicz et al. Olkiewicz et al. (2016)
(2009) (2016)
It is evident from Table 22-24 that for microbial oil production, high capital
investment is a result of the high capacity fermenter, which has a large FOB and
installation cost. However, for biodiesel production from vegetable oil, even for the
scale of 50,000 tons/yr, the capital investment is $9.4 million only, which is due to
the simple process that requires less equipment and time to process a single batch,
resulting in more batches annually. Biodiesel from WCO (base-catalyzed) has
large capital investments compared with vegetable oils because of the additional
equipment required for pretreatment. Acid-catalyzed biodiesel production from
WCO has small capital investments compared with the base-catalyzed method
because no pretreatment is required in acid-catalyzed biodiesel production.
Comparing two methods using primary sludge, capital investment is higher for
the dry method compared with the wet method because the major cost results
from the purchase of equipment for centrifugation and drying.
Profits generated by the manufacturing facility affect the economics of the
industrial process. Profit making is dependent on the difference between revenues
generated annually (through selling the commodity) and the annual operating
cost. Biodiesel producing plants can earn profits by selling the biodiesel, or they
can also earn by selling byproducts like crude glycerol. Additional revenues/credits
increase the profits made by the company; in other words, additional income
reduces the annual operating cost of the facility.
Some cases have been reported in which waste products produced during
biodiesel manufacturing are purified or recycled. Four scenarios have been
compared in which crude glycerol produced during transesterification is disposed
as waste (Scenario 0) or purified to 80% purity through distillation (Scenario 1), to
95% purity (Scenario 2), or used as a carbon source for producing succinic acid
via fermentation (Scenario 3) (Vlysidis et al. 2011). Crude glycerol of purity 80%
(sold at $77/ton) and purity 95% (sold at $392/ton) as well used for producing
succinic acid (sold at €4,311/t, $4828/ton) are additional revenue streams in
Scenarios 1, 2, and 3, respectively, besides biodiesel and rapeseed meal (generated
during oil extraction from rapeseed). Unit production and income earned for
all four cases are presented in Table 22-25. Refining/recycling the crude
glycerol increases the unit production cost because additional cost is required
for utilities, facility maintenance, and plant overhead owing to the additional
equipment (distillation, fermentation, and so forth). However, annual profits also
increase if purified glycerol and succinic acid are sold in the market along with the
biodiesel.
22.9.4 Cost of Unit Production

The cost of unit production is defined as the investment to produce 1 L or 1 kg of
any commodity, and is evaluated as
Unit production cost ð$∕LÞ = annual operating cost ð$Þ∕

biodiesel produced annually (22-3)
Table 22-25. Production Costs and Profits Earned for Different Scenarios.
Crude glycerol
Crude glycerol is purified and 80% Crude glycerol purified and Crude glycerol is used for
Economic parameter generated as waste pure glycerol sold 95% pure glycerol sold succinic acid production
Unit production cost- 0.898 (1.00) 0.903 (1.011) 0.904 (1.012) 1.014 (1.14)
7€/L (US $/L)
Annual profits million 1.085 (1.22) 1.129 (1.27) 1.274 (1.43) 1.95 (2.18)
€ (US $)
Note: Scale of operation = 7.92 kT/year.
571
For economic feasibility of a process, the selling price of a commodity must be

greater than the unit production cost ($/L), otherwise the manufacturing company
will never be able to recover its investments. The selling price of a commodity is
influenced by consumer demand, number of consumers to be targeted, competi-
tion between the industries producing the same product, and the market forces.
The unit production is comprised of raw material purchase cost, utility usage cost,
facility-dependent cost, labor cost, plant overhead cost, and waste treatment cost.
The unit production cost of biodiesel varies, depending on the choice of feed-
stocks, the process itself, number and type of equipment used in the process
(influencing utility, labor, and facility-dependent cost), the quantity of solvents in
the process affecting raw material purchase cost and the waste disposal cost.
Table 22-26 compares the unit production cost of biodiesel produced from
different feedstock and the processes used.
Table 22-26 shows that biodiesel from primary sludge using the dry route is
more expensive than the wet route because additional equipment is required for
centrifugation and drying of sludge, increasing the facility-dependent and utility
cost. The unit production cost is least for WCO because they are more economical
than vegetable oils. Acid-catalyzed biodiesel production using WCO is more
economical than base-catalyzed because additional pretreatment steps are re-
quired in the base-catalyzed reaction, increasing the production cost. For vegetable
oil–based biodiesel production, the simple manufacturing process is used with less
equipment and auxiliary services, but it is highest for microbial oil production
using glucose and yeast extract for which the high purchase cost of glucose is a
major contributing factor. The unit production cost in the case of vegetable oils is
primarily a result of raw material acquisition cost. Also, the unit production cost
using wet sludge is comparable to that using vegetable oils, indicating that waste
sources such as municipal sludge have high potential as substrates for biodiesel
production.
The unit production cost is also dependent on the scale of operation. The unit
production price of any commodity decreases with an increase in the scale of
operation and reaches a constant value (Figure 22-16). One of the techno-
economic studies using vegetable oils revealed that the unit production cost for
5,000 tons/yr capacity was $1.32/L; as the production capacity increased to
140,000 tons/yr, the unit cost approached $1.16/L (Apostolakou et al. 2009).
Because the selling price of biodiesel was $1.19/L during that time, the minimum
scale of the operation was estimated to be 50,000 tons/yr for which the unit
production cost of biodiesel was $1.18/L.
22.9.5 Payback Period

Payback time is defined as the period in which the company can recover all its
investment costs through the profits earned by selling biodiesel. Mathematically,
the payback period is evaluated as
Payback period = total investment∕annual net profit (22-4)

Table 22-26. Comparison of Biodiesel Unit Production Cost for the Different Feedstocks.
Extraction from Extraction from Microbial oil
Process/Economic Transesterifying Biodiesel from WCO Biodiesel from WCO primary sludge (wet primary sludge (dry production using
parameter vegetable oils (acid catalyzed) (base catalyzed) route) route) glucose
Unit production $1.15/L $ 0.74/kg $ 0.97/kg $1.23/kg $2.014/kg $5.48/kg

cost
Process highlights 1.No pretreatment 1. Transesterification 1. Pretreatment of 1. Extraction and 1. Drying (major cost 1. Fermentation
2.No extraction 2. Neutralization of waste cooking oil Recovery (major factor) (major cost factor)
3.Transesterification acid 2. Separation of cost factor) 2. Extraction and 2. Cell harvest
4.Purification/ 3. Distillation refined cooking 2. Transesterification Recovery 3. Extraction and
distillation oil 3. Purification 3. Transesterification Recovery
3. Transesterification 4. Purification 4. Transesterification
4. Distillation 5. Purification
Main contributing Raw materials Raw materials Raw materials Plant overhead Plant overhead Glucose (35)
factors (%) (87.86) (29.25) (34.29) cost (61.9) cost (54.5)
References Apostolakou et al. Zhang et al. (2003) Zhang et al. (2003) Olkiewicz et al. Olkiewicz et al. Koutinas et al. (2014)
(2009) (2016) (2016)
573
Unit production cost ($/ gallon)

Scale of operaion (KT/ year)

Figure 22-16 Unit production cost as a function of scale of operation.
Because the payback period is inversely proportional to net profit, annual net
profits earned should be higher so that the plant can recover its capital investment
in the shortest time. Table 22-27 compares the payback period of different
industrial biodiesel processes.
It is evident from Table 22-27 that the payback period is 4.6 years only for
vegetable oil as a result of the comparatively small capital investments (simple
process with less equipment) and high net profit (low annual operating cost and
more batches produced annually). Biodiesel from sludge and microbial oil
production has high annual operating expenses compared with revenues earned
annually, and hence these processes are economically not feasible at the industrial
scale. Although researchers are looking for alternatives for expensive substrates
such as glucose for microbial oil production, obtaining a high lipid content and
lipid concentration using waste sources such as crude glycerol and municipal
sludge is a challenge that needs to be addressed.
22.10 GLOBAL BIODIESEL ECONOMY AND ROADMAP AHEAD
Understanding the sheer importance of biodiesel and clean alternate fuels, many
nations worldwide started taking part in global biodiesel production. Because of
the gravity of carbon footprints and global warming, active governments in recent
decades started showing special interest in biodiesel production by allocating
budget heads to renewable energy. New subsidies and support programs have been
launched by the various governments to encourage the biodiesel industry which is
currently economically unprofitable. The United States and the European Union
Table 22-27. Comparison of Payback Period for Various Biodiesel Production Processes.
Microbial oil Extraction from Extraction from
production using Biodiesel from wet primary dried primary
Process/economic parameter glucose vegetable oil sludge sludge
Total investment (million $) 70.2 9.4 7.45 17.25

Scale of operation 10 kT year 50 kT/year 3.55 kT/year 4.7 kT/year
Biodiesel selling price ($/T) 1,376 1,376 1,376 1,376
Revenues (million $) 13.76 68.8 4.88 6.47
Annual operating cost (million $) 65.9 65.9 4.38 9.49
Annual profit generated (million $) −52.14 2.9 0.5 −3.02
30% tax (million $) 0 0.87 0.15 0
Annual net profit (million $) −52.14 2.03 0.35 −3.02
Payback period (years) Unable to 4.6 21.2 Unable to
recover its recover its
investment investment
cost cost
575
have been very active in investing in alternate fuel programs. The United States
started a biofuel support program to target 60 billion L of second-generation
biofuel by the end of 2022. Similarly, the European Union started the renewable
fuel campaign to target 10% renewable energy in the transport sector by 2020.
Mandates in each country play a fundamental role in driving the global biofuel
markets. According to the International Energy Agency, the United States and the
European Union (EU) have been falling short of their domestic production
capacity to meet the requirements of the mandates. Therefore, they are heavily
dependent on import of biofuels.
Although many TEAs were unable to show profitability in biodiesel produc-
tion using microalgae, few venture capitalists have been investing in the algal
biofuel programs. In this context, the US DOE on May 2010 published informa-
tion on US policy trends in National Algal Biofuels Technology Roadmap (Ferrell
and Sarisky-Reed 2010). This document actually summarized the techno-eco-
nomic challenges that need to be overcome for making algal biofuel successful
(Ragauskas et al. 2006). Following the recommendation from the Enivronmental
Protection Agency, the National Renewable Fuel Standards program was revised.
The revision brought up new volumetric targets for cellulosic biofuel, biodiesel,
and total renewable fuel for transport per year. Although cellulosic ethanol made
significant progress, other biofuels including biodiesel, significantly contributed to
reaching the 21 billion gal. goal from the 2007 American Energy Independence
and Security Act. On May 5, 2009, the US government announced an investment
of $800 million for biofuel research, according to the American Recovery and
Renewal Act (ARRA). The funding was mandated to be invested in research for
biodiesel development programs. Meanwhile, to encourage the renewable energy
use, the EU passed a directive, 2009/28/EC, which established a framework for
biofuel development. Toward late 2010, the European Union launched the
Strategic Energy Technology plan across various sectors. European program
listed many technologies that will work together for a clean decarbonized
environment.
Globally, the United States is the leading producer of biodiesel with 5.5 billion L
of production, followed by Brazil, owing to its high sugarcane productivity, to
produce 3.8 billion L of biodiesel (Figure 22-17). Germany, Indonesia, and
Argentina produce equally, 0.5 billion L of biodiesel per year. In the global
production index, Canada remains at the 11th position behind France, Thailand,
Spain, Belgium, and Colombia. On July 5, 2007, Canada launched its Eco Energy
program for biofuels. The project targeted to invest $1.5 billion in nine years.
The program provides incentives to research and development projects and new
industries for developing clean alternative fuels. The biofuel sector in Canada
was small until 2005, but owing to different support programs today, Canada
produces 124 million L of biodiesel (2013 data). In total, nine big biorefineries
are currently operating in Canada (Table 22-28); understandably, none of the
biodiesel production units is based on algae, cellulosic, or SCO because of
economic reasons.
Figure 22-17. Global leaders in biodiesel production (2017).

Source: Data derived from www.statista.com
Table 22-28. Biodiesel Companies Operational in Canada.
Capacity
(Million
Company Site Feedstock L/year)
Archer Daniels Lloydminster Canola oil 265

Midland
Atlantic Biodiesel Dain City Canola/soy oil 170
Corporation
Biox Corporation Hamilton Animal fats 67
Cowichan Biodiesel Duncan Waste vegetable oil 0.2
Co-op
Evoleum Saint-Jean-sur- Multiple feedstocks 19
Richelieu
Innoltek, Inc. Thetford Mines Multiple feedstocks 6
Milligan Biofuels, Foam Lake Canola oil 14
Inc.
Noroxel Energy, Ltd. Springfield 5
Rothsay Biodiesel Ville Ste. Animal fats 45
LLC Catherine
Total 591
22.11 CONCLUSIONS
Although the researchers have been doing a huge amount of work to improve the
existing know-how and engineering science behind the biodiesel production, a
very small fraction of the generated knowledge gets assimilated in the industry,
imparting real socioeconomic impacts. Ideally every new process modification

should undergo techno-economic evaluation, and the results from these TEAs
should feed back into the loop to keep the research directions on the right
track so that technology moves faster from laboratory benches to industrial
production reactors. The applied science and basic science of biodiesel produc-
tion should work in harmony and develop a confluence of technologies, which
can sustainably uplift not only the life of mankind but also the life on earth at
large.
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CHAPTER 23
Biodiesel Impact on
Environment and Health

T. T. More
S. Yan
J. P. Hettiaratchi
R. D. Tyagi
R. Y. Surampalli
23.1 INTRODUCTION
Biodiesel is a diesel fuel substitute that can be produced by transesterification of a

variety of biological products such as vegetable oils (from crops like rapeseed,
soybean, jatropha, sunflower, and palm) and animal fats (e.g., recycled cooking
greases) (Lin et al. 2011, Silalertruksa and Gheewala 2012). Being a potentially
alternative energy source, biodiesel has been paid a great deal of attention in recent
years. Biodiesel can reduce dependence on fossil oils because feedstock can be
grown and refined. As a renewable energy, biodiesel is important owing to
continuous rising petroleum prices, decreasing fossil fuels, and increased envi-
ronmental concerns. Biodiesel is commonly considered as a dual solution to the
problems of dependence on fossil fuels and climate change, but their impacts are
not confined to these two areas. Biodiesel production and use is one of the most
intensive enterprises, with many economic and societal benefits, as well as
unintended societal and environmental drawbacks. Any policy that seeks to
significantly alter the form and method of energy exploitation raises the specter
of unintended or misjudged consequences. Despite multiple potential benefits,
biodiesel can also have disadvantages, and the net environmental benefit depends
greatly on how it is produced. Although the biodiesel is from a renewable source, it
should be produced in an efficient and economic process in which the impacts and
emissions are minimized or eliminated according to precepts of cleaner produc-
tion (Bonilla et al. 2010). Research has been performed during the last several
years to assess the environmental effects of biodiesel production, use, and
produced wastes. Several research papers, government and nongovernment
583
reports, and documents have been published about the specific effects associated
with the industrial and agricultural aspects of the biodiesel industry (Phalan 2009,
Lin et al. 2011, Kaercher et al. 2013.
Biodiesel is a credible source of low-carbon energy that delivers lower
amounts of greenhouse gases (GHGs) compared with fossil fuels, and the impact
of biodiesel on environment widely varies, that is, it may not necessarily be
positive, or as positive as is often initially assumed (Lin et al. 2011). Biodiesel is not
always offering emissions reductions when compared with fossil fuels. Apart from
the aforementioned problems, the development of the biodiesel industry may
directly or indirectly cause other negative effects on the environment (Searchinger
et al. 2008, Sims et al. 2010). The goal of GHG savings was challenged after indirect
land-use change resulting from displaced food and feed production was included
in the estimated emissions. Although estimation of indirect land-use change
attributed to biodiesel policies continues to be debated in the literature, such
attention to unintended consequences of biodiesel production and use has
stimulated discussion of other potential environmental impacts—food security,
biodiversity, and health.
Despite rapid growth in biodiesel production worldwide, it is uncertain
whether decision makers possess sufficient information to fully evaluate the
impacts of the biodiesel industry and avoid unintended consequences on the
environment and human health. The energy and input balance are helpful in
the industrial processes for the environmental assessment. It is important for
evaluating the sustainability of the biodiesel industry. This assessment must
consider all aspects such as pre-, during, and post-biodiesel production stages
and usage, recycling, and end-of-life management of the products. The accurate
assessment requires rigorous peer-reviewed data and analyses across the entire
range of direct and indirect effects of biodiesel on the environment and human
health. In that context, this chapter addresses recent scientific and industrial
information on the biodiesel industry impact on biodiesel-related topics such as
water resources, emissions, soil pollution, land use, biodiversity, greenhouse gases,
and human well-being.
23.2 EFFECT ON AIR QUALITY
One of the prime issues when operating a diesel engine on a conventional

petroleum fuel is that of exhaust emissions. Exhaust emissions from conventional
petroleum fuels in the form of carbon monoxide (CO), carbon dioxide (CO2),
oxides of nitrogen (NOx), sulfur dioxides (SO2), volatile organic carbons (VOCs),
hydrocarbons (HC), and particulate matter (PM) are of high concern because they
are mainly responsible for reducing the air quality. The VOCs emitted from
gasoline-fueled vehicles arise from uncombusted or partially combusted fuel and
typically include cyclohexane, octanes, and aromatics. The NOx and VOCs
emissions react in the presence of sunlight by way of a series of photochemical
BIODIESEL IMPACT ON ENVIRONMENT AND HEALTH 585
reactions involving hydroxyl, peroxy, and alkoxy radicals to form the secondary
pollutant ozone (Phillips 2001). Biodiesel feedstocks include agricultural and food
processing wastes, trees, and various grasses that are converted to biodiesel.
Biodiesel is considered to be carbon neutral because all the CO2 released during
consumption had been sequestered from the atmosphere for the growth of
vegetable oil crops (Barnwal and Sharma 2005). Air pollution emissions from
fuel combustion in transport vehicles related to biodiesel feedstocks and fuels are
not materially different than those associated with ethanol. Significant evaporative
emissions are not expected from storage and transport of biodiesel fuel because the
containers are usually airtight (Brown 2008).
The effects of biodiesel usage on vehicle emissions have been extensively
investigated (Hoekman and Robbins 2012). Xue et al. (2011) surveyed the
literature reports of the pure biodiesel use on engine performance and exhaust
emissions. There is a substantial body of evidence showing that burning biodiesel
(and biodiesel blends) has a mixed, although usually positive, impact on air
pollution. Compared to conventional diesel fuel, use of biodiesel is usually found
to reduce emissions of HC, CO, and PM (Robbins et al. 2011). One of the most
important differences between biodiesel and conventional diesel is the oxygen
content. Biodiesel has 10%–12% more oxygen than petroleum-based diesel, and
that means lower CO, PM, and non-methane volatile organic compounds
(NMVOC) emissions, but higher NOx emissions and ozone-forming potential
as well (Xue et al. 2011). However, there are variable observations about NOx
emissions from biodiesel combustion. Moreover, biodiesel is better than diesel
regarding sulfur content, flash point, aromatic content, and biodegradability
(Gupta and Demirbas 2010). The emission variations are widely dependent on
both fuel blends and vehicle technologies (Gaffney and Marley 2009). Low blends
of biodiesel on diesel can help in improving air quality and reduce the requirement
of conventional diesel fuels without significantly sacrificing engine power and
economy (McCormick et al. 2006). The benefit of using biodiesel is proportionate
to the blend level of biodiesel used. Substituting B100 for petroleum diesel reduces
life cycle petroleum consumption by 95%. B20 blends reduce life cycle petroleum
consumption by 19% (Brown 2008). Per the report prepared by Brown (2008),
biodiesel yields 3.2 units of fuel product energy for every unit of fossil energy
consumed in its life cycle. B20 production yields 0.98 units of fuel product energy
for energy unit of fossil energy consumed. In contrast, petroleum diesel uses
1.2 units of fossil resources to produce one unit of petroleum diesel. Moreover,
Brown (2008) also observed that biodiesel reduces net CO2, emissions by 78.45%
compared with petroleum diesel. For B20, CO2, total particulate, tailpipe particu-
late (<10 μm), and SOx, emissions from urban buses fall by 15.66%, 35%, 68%,
and 8%, respectively, compared with petroleum diesel. However, for B20, NOx
emissions were increased by 13.35%. Most of this increase in NOx emissions
results from increased tailpipe emissions. Life cycle HC emissions are 35% higher,
but tailpipe emissions are 37% lower. All these emissions effects are proportional
to the biodiesel blend; B20 blends emissions will be one-fifth that of B100.
Table 23-1. Reduction in Pollution Emission with Different Percentages of Biodiesel

Blending.
Emissions reduction Emission (g/km)

Pollutant B100 B20 Diesel B10 B15
Particulate matter (PM) −30 −20 0.129 0.093 0.080

Oxides of nitrogen (NOx) +13 +2 0.79 0.83 0.89
Carbon monoxide (CO) −50 −20 0.77 0.65 0.62
Unburned hydrocarbons −93 −30 0.37 0.22 0.16
Sulfur dioxide (SO2) −100 −20 − − −
Note: (−) and (+) = Less and more percentage of pollutant emission from biodiesel in comparison to
100% diesel.
Reduction in pollution emission with different percentages of biodiesel blending is

shown in Table 23-1.
23.2.1 Emissions from Biodiesel Combustion
23.2.1.1 Carbon Monoxide

In general, combustion of biodiesel has lower carbon monoxide (CO) emissions
than diesel fuel (Nabi et al. 2006, Rao et al. 2007, Öner and Altun 2009). This is
because biodiesel has a higher oxygen content and a lower carbon-to-nitrogen
ratio compared with that of diesel (Kumar et al. 2013). Excess oxygen of biodiesel
ensures the complete combustion of the fuel as well as supplies the necessary
oxygen to convert CO to CO2 (Nabi et al. 2009). Krahl et al. (2003) obtained about
50% reduction in CO emissions for biodiesel from rapeseed oil compared with low
and ultra-low sulfur diesel. Raheman and Phadatare (2004) observed about 73% to
94% reduction in CO emissions from the karanja methyl ester (B100) and its
blends (B20, B40, B60, and B80) compared with that of diesel. Canakci (2007),
Öner and Altun (2009), and Nabi et al. (2006) found 18.4%, 14.5%, and 4%
reductions in CO emissions, respectively, when the engine was fueled with B100.
Ozsezen et al. (2009) reported 86.89% and 72.68% reduction in CO emissions for
waste (frying) palm oil methyl ester (WPOME) and canola oil methyl ester
(COME), respectively. With an increase in the pure biodiesel (in blends), there is
reduction in CO emissions (Xue et al. 2011). CO emissions from biodiesel also are
affected by the type of feedstock and biodiesel properties such as cetane number.
Metal-based additives, methanol, and ethanol also reduce the CO emissions from
biodiesel combustion. Engine load has been proven to have a significant impact on
CO emissions. There is a largely unanimous conclusion about the effect of engine
speed on CO emissions, that is, CO emissions for biodiesel decrease with an
increase in engine speed. An oxidative catalytic converter, which is designed for
diesel engine, plays an important role on CO emissions for biodiesel, but its
conversion efficiency may become weak.
23.2.1.2 Carbon Dioxide

Of the total GHG emissions, traffic (combustion of conventional diesel) con-
tributes up to 23% of carbon dioxide (CO2) emissions (UNFCCC 2006). There are
several reports that CO2 emissions from biodiesel combustion are higher than that
of diesel fuel (Labeckas and Slavinskas 2006, Mbarawa 2008, Chauhan et al. 2012).
The presence of oxygen in biodiesel and the relatively lower content of carbon in
biodiesel for the same volume of fuel consumed are considered to be the reasons
for higher emission of CO2 (Labeckas and Slavinskas 2006). However, it is argued
that increased CO2 emissions from biodiesel combustion can be compensated by
biodiesel crops. The studies on effect of biodiesel on global GHG emissions
through the life cycle of CO2 emissions, have suggested that biodiesel will cause
50% to 80% reduction in CO2 emission compared with petroleum diesel (Xue et al.
2011). Some researchers reported lower CO2 emissions from biodiesel combustion
than that of diesel (Labeckas and Slavinskas 2006, Mani et al. 2009). Biodiesel is a
low-carbon fuel and therefore, the reduction in CO2 emissions from biodiesel
combustion is attributed to the lower carbon-to-hydrogen ratio (Xue et al. 2011).
Moreover, the high viscosity of biodiesel reduces cone angle, which leads to
reduction of the amount of air entrainment in the spray, resulting in hindrance to
incomplete combustion (Gumus 2008, Mani et al. 2009).
23.2.1.3 Hydrocarbons
Most of the literature has reported that biodiesel combustion had lower hydrocar-
bon (HC) emissions than that of petroleum diesel (Xue et al. 2011). Wu et al. (2009)
observed that the five different biodiesels reduced HC emission by 45% to 67%
compared with that of diesel fuel. The HC reduction amounts vary in the study data
(Kalam et al. 2003, Mbarawa 2008, Rehman et al. 2011). HC reduction of 63%
(Puhan et al. 2005b), 60% (Alam et al. 2004), and 22.47% to 33.15% (Lin et al. 2009)
were observed for biodiesel combustion than that of diesel combustion. Reduction
in HC emissions by 20.73%, 20.64%, and 6.75% were observed for pure biodiesel of
jatropha, karanja, and polanga, respectively (Sahoo et al. 2007). Kalligeros et al.
(2003) reported that the addition of methyl esters contributed to a faster evaporation
and more stable combustion, and hence, a decrease in HC in comparison to diesel.
The oxygen content and the higher cetane number of biodiesel, along with advanced
injection and combustion timing, significantly reduce HC emission for biodiesel
(Shi et al. 2005). As discussed, oxygen provides cleaner combustion and advance in
injection provides more time for oxidation of HC.
Several reports stated that there was no significant difference between
biodiesel and diesel, and others reported the increase in HC emissions from
biodiesel (Xue et al. 2011). A 10% increase in HC emissions was observed for
methyl ester of jatropha oil with regard to diesel by Kumar et al. (2013).
Furthermore, HC emission was reported to be a function of load. At higher
loads, HC emission increases because of the higher fumigation rate and non-
availability of oxygen relative to diesel (Shi et al. 2005). Several factors affect the
HC emissions from biodiesel combustion such as content of biodiesel, feedstock
and properties of biodiesel, engine and operating conditions, and additives used in
biodiesel (Xue et al. 2011). Most of the literature showed that HC emissions for
biodiesel reduce with an increase in biodiesel content. The feedstock of biodiesel
and its properties influence HC emissions, especially for the difference in
injection, and combustion of biodiesel favors the lower HC emissions. There are
inconsistent conclusions about effects of engine load on HC emissions for
biodiesel. Although an oxidative catalytic converter has a positive impact on HC
emissions for biodiesel, its function seems to be weakened. Metal-based additives
have less efficiency to improve HC emissions for biodiesel than the other
emissions. A small proportion of ethanol and methanol added into biodiesel and
its blends with diesel may be advantageous to HC emissions.
23.2.1.4 Particulate Matter

Particulate matter (PM) is composed mainly of three components: dry soot (Shipp
et al. 2006), sulfate, and soluble organic fraction (Chen et al. 2007, Chakrabarti
et al. 2012). Compared to conventional diesel fuel, combustion of biodiesel is
usually found to reduce PM emissions (Xue et al. 2011). PM emissions are 12% less
for B20, and 48% less for B100 (Robbins et al. 2011). The maximum increases and
decreases in PM2.5 and PM10 mass concentrations are extremely small. The 100%
B20 biodiesel fuel scenario is estimated to reduce exposure to annual and 24 h
emissions limitation exceedances of the PM10 standard of the USEPA by 4% and
7%, respectively. The results for the 50% B20 penetration are approximately half
those of the 100% B20 penetration scenario. Abu-Jrai et al. (2011) and Puhan et al.
(2005a) show that PM emissions were usually reduced with the use of biodiesel
when compared with diesel, owing to the oxygen contained in the biodiesel
molecules, the low levels of sulfur content, and higher cetane number. Particulate
matters were formed in the locally rich regions of the heterogeneous mixture of
fuel and air during combustion in the combustion chamber. Further mixing of air
and fuel resulted in burning of particulates at the boundary of diffusive flame as a
result of the high temperature and available oxygen at the region. The increase of
oxygen content in the biodiesel, which contributes to a complete fuel oxidation
even in locally rich zones, led to a significant decrease in PM and smoke (Lapuerta
et al. 2008).
Higher cetane number of biodiesel compared to diesel resulted in shorter
ignition delay and longer combustion duration and hence low particulate emis-
sions (Nabi et al. 2009). Diesel contains sulfur that results in sulfates that are
absorbed on soot particles and increase the PM emitted from diesel engines.
Biodiesel is free from sulfur, and it has an advantage over diesel (Kalligeros et al.
2003). In addition, utilization of biodiesel substantially reduced the larger size
particulates, which strongly contributed to the volume and weight of particulates.
At the same time, biodiesel produced lower concentrations of particulates (Kim
et al. 2008). The low C/H ratio also lowered the smoke emission (Tree and
Svensson 2007). If the applied fuel is partially oxygenated, locally over-rich regions
can be reduced and primary smoke formation can be limited (Nabi et al. 2009).
Finally, because the requirement of the stoichiometric air for biodiesel is lower
(Kouzu and Hidaka 2012), it reduces the formation of fuel-rich regions in the
heterogeneous mixture and PM emission (Lapurta et al. 2002). However, the
soluble organic fraction (Chakrabarti et al. 2012) of the PM was significantly
higher with the biodiesel (Knothe et al. 2006).
23.2.1.5 Oxides of Nitrogen

The literature contains a rich amount of information regarding the effects of
biodiesel on oxides of nitrogen (NOx) emissions. However, contradictions re-
garding emissions of NOx were also found. Some of the literature reported less
NOx emission with the utilization of biodiesel (Sahoo et al. 2007, Kumar et al.
2013). Öner et al. (2009) reported 38.5% reduction, whereas Sahoo et al. (2007)
reported 4% reduction. The explanations given are higher cetane number and
lower flash point of biodiesel compared with diesel. Increasing cetane number
reduces the size of the premixed combustion by reducing the ignition delay, and
hence lower NOx formation rate, because the combustion pressure rises more
slowly, giving more time for cooling through heat transfer and dilution and
leading to lower localized gas temperatures (Öner et al. 2009). Absence of aromatic
and polyaromatic hydrocarbons in biodiesel lowers the flame temperature, ergo,
less NOx emission. Also, shorter ignition delay from the higher cetane number
would allow less time for the air/fuel mixing before the premixed burning phase.
Consequently, a weaker mixture would be generated and burned during the
premixed phase, resulting in relatively reduced NOx formation. Saturation in the
fatty compounds causes decrease in NOx emissions (Kumar et al. 2013). Wyatt
et al. (2005) reported that all the animal fat biodiesels, including beef tallow, lard,
and chicken, had lower NOx levels compared to the soy-oil-based biodiesel
because of the higher saturation level and cetane number. Quantification of the
NOx effect is relatively more difficult than the CO, HC, and PM effects because the
magnitude of the NOx effect is much less. Recent studies with B20 have showed
varying effects on NOx. The NOx emissions arising from B0 and B20 test fuels
across a large number of test cycles and engines are not significantly different
(Yanowitz and McCormick 2009).
Although as suggested earlier, biodiesel or biodiesel blends decrease NOx
emissions in comparison with petroleum diesel fuel, some studies have reported
that the burning biodiesel yields higher NOx emissions than petroleum diesel.
NOx emissions are 2% higher for B20 blend and 10% higher for B100 blend
(EPA 2002). Approximately 10% increase in NOx emission was realized with
30% biodiesel mixtures (Kumar et al. 2009). This increase in NOx by biodiesel
combustion is referred to as the biodiesel NOx effect (Hoekman and Robbins
2012).
The assessment on the biodiesel NOx effect was reported by the USEPA a
decade ago, and the data from some engine types now considered obsolete were
included (Hoekman and Robbins 2012). There is no single factor that is
responsible for the NOx emissions by biodiesel combustion. Rather, there are
numerous factors (such as engine type and configuration, duty cycle, fuel injection
strategy, emissions control strategy, and other factors) that are responsible for
NOx emissions by biodiesel combustion (Hoekman and Robbins 2012). Several
theories have been developed to understand these factors and to explain the
predominance of test data that show increased NOx with biodiesel combustion.
These theories include (1) theory of speed of sound and bulk modulus of
compressibility (Tat et al. 2000, Monyem et al. 2001, Szybist and Boehman
2003, Tat and Gerpen 2003, Boehman et al. 2004, Szybist et al. 2007), (2) theory of
decreased radiative heat loss (Cheng et al. 2006, Mueller et al. 2009a, Schönborn
et al. 2009), (3) theory of higher adiabatic flame temperature (Ban-Weiss et al.
2007, Mueller et al. 2009b), (4) combustion phasing theories (Hoekman et al.
2012), and (5) engine control/calibration theories (Eckerle et al. 2008). These
theories relate primarily to biodiesel’s effect on injection timing, ignition delay,
adiabatic flame temperature, radiative heat loss, and other combustion phenom-
ena. The extent to which these theories apply also varies with engine technology
and operating condition. For example, older engines having mechanical fuel
injection systems exhibit an inadvertent advance in fuel injection timing, attrib-
utable to the higher bulk modulus of compressibility of biodiesel. Modern
electronically controlled diesel engines no longer experience this effect, but they
can exhibit other NOx effects (both positive and negative), depending on how the
engine control system responds to fuel changes, such as the lower energy content
of biodiesel compared with conventional diesel.
Recent laboratory experimental work has demonstrated that with modern
engines, use of biodiesel typically results in a more advanced and faster overall
combustion event, which leads to elevated cylinder temperatures and increased
NOx formation. A likely contributing factor is the oxygen content of biodiesel,
resulting in a charge gas composition that is closer to stoichiometric (less rich) in
the flame auto ignition zone. Several approaches for mitigating the biodiesel NOx
effect have been investigated. The most effective engine modifications involve
retarded injection timing and increased use of exhaust gas recirculation (EGR).
Modern engines, with sophisticated computer control systems, offer the possibility
of other mitigation strategies, including a process called low-temperature com-
bustion (LTC). Fuel modifications including reduced aromatics content and use of
cetane improvers have been shown to mitigate the biodiesel NOx effect in some
situations. However, it appears that with modern engines and fuels ultra-low-
sulfur diesel (ULSD), the benefits of such fuel modifications are quite limited.
Exhaust after-treatment systems required to meet the US 2010 heavy duty engine
standards have only been in use for a short time. Consequently, there is little real-
world operational experience with them. However, based on the information
currently available, it appears that use of biodiesel—especially at blend levels of
B20 and below—does not seriously affect the short-term performance of either
NOx adsorber catalyst (NAC)- or selective catalyst reduction (SCR)-based after-
treatment systems. However, there are some indications that long-term usage of
biodiesel and/or use of poor-quality fuel could result in increased NOx emissions.
Further long-term study is warranted.
23.2.1.6 Other Nonregulated Emissions

Nonregulated emissions include emissions in aromatic and polyaromatic com-
pounds and carbonyl compounds. Emissions from formaldehyde, acetaldehyde,
1,3-butadiene, benzene, toluene, and xylene are of concern because they are air
toxics. There is increasing interest on the nonregulated emissions when biodiesel is

fueled instead of diesel.
Aromatic Polyaromatic Compounds. Aromatic compounds and derivatives
are toxic, mutagenic, and carcinogenic. Most reports state that there is decreased
aromatic and polyaromatic emissions with biodiesel combustion. Sharp et al.
(2000) found that polycyclic aromatic hydrocarbons (PAH) and nitro-PAH
emission reduced by 50% to 75% on three different engines fueled by biodiesel.
Lin et al. (2006) tested pure palm oil biodiesel and the blend fuel with 20%
biodiesel and ultra-low-sulfur (ULS) diesel on the NA2.84 L engine. They
observed that PAH reduced 43% for the blend fuel and 90% for pure biodiesel.
He et al. (2010) carried out an experimental study on a diesel engine (DI) and non-
turbo (TU) diesel engine, and found that, compared with diesel, using B100 and
B20 could greatly reduce the total PAHs emissions by 19.4% and 13.1%,
respectively. In a USEPA (2002) report, it was inferred that some aromatic
emissions with biodiesel (such as ethylbenzene, naphthalene, and xylene) had
consistent reduction, whereas others (such as styrene, benzene, and toluene) had
different results. The National Biodiesel Board (NBB 2007) estimated that PAH
and nitro-PAH reduced to about 80% and 90%, respectively (Xue et al. 2011). The
reduction in PAH emission was attributed to the enhanced absorption of PM to
PAH components (Pinto et al. 2005). Corrêa and Arbilla (2006) pointed out that
biodiesel had lower benzene emissions than Euro V diesel fuel owing to the
characteristics of non-light-aromatics. Agarwal (2007) agreed that the lack of
aromatic hydrocarbon (benzene, toluene, among others) in biodiesel reduces
nonregulated emissions like ketone, benzene, and so forth. Overall, most research-
ers showed that aromatic and polyaromatic compounds emissions for biodiesel
reduce with regard to diesel, and it depends on engine operating conditions (load,
cycle mode, and so forth).
According to some studies, there was no significant differences in PAH
emissions between diesel and biodiesel, whereas according to other studies, there is
a slight increase in aromatic emissions. Turrio-Baldassarri et al. (2004) reported
the reduction in toluene, rather than any of the analyzed PAH or nitro-PAH.
Munack et al. (2001) verified that the increase of rapeseed biodiesel content in the
blend fuel caused the increase in benzene emissions on a single-cylinder 4.2 kW
engine. They also tested on a 52-kW engine and observed that aromatic emissions
increased slightly. Engine operating conditions (load, cycle, and so forth) play a
noticeable role in aromatic and polyaromatic emissions of biodiesel. Cheung et al.
(2009) measured benzene, toluene, and xylene (BTX) emissions on a diesel engine
under five engine loads at a steady speed of 1,800 rpm with Euro V diesel fuel, pure
biodiesel, and biodiesel blends with 5%, 10% and 15% methanol. Compared with
the diesel fuel, the BTX emissions of biodiesel were lower, and with an increase of
methanol in the blends, benzene emissions decreased owing to an increase of

oxygen in the biodiesel that improves combustion and promotes the degradation
of benzene. They also observed that the BTX emissions decreased with engine
load. Similarly, Di et al. (2009) found higher benzene emissions at lower
engine loads. Takada et al. (2003) also showed higher benzene emissions at lower
engine loads and lower exhaust gas temperatures and concluded that benzene
could be easily degraded at high exhaust gas temperature. Ballesteros et al. (2008)
reported that there are significant reductions in aromatic and oxygenated aromatic
emissions with the use of biodiesel in the urban mode. In the extra-urban mode,
the amount of aromatics emitted with biodiesel fuel blends was negligible.
Carbonyl compounds. Carbonyl compounds in the emissions were increased
when using pure biodiesel or its blends (Xue et al. 2011). He et al. (2009) analyzed
14 carbonyl compounds emissions, mainly including formaldehyde, acetaldehyde,
acrolein and acetone, among others, and found that a biodiesel-fueled engine
almost had triple carbonyls emissions of diesel-fueled engine and emitted a
comparatively high content of propionaldehyde and methacrolein. Fontaras et al.
(2009) identified 13 carbonyl compounds in the exhaust gases and measured their
concentrations over the various driving cycles with B100 biodiesel and the
petroleum diesel. The experimental results demonstrated a significant increase
in carbonyl emissions with the use of pure biodiesel, probably caused by the
oxygen atoms in the ester molecule. Turrio-Baldassarri et al. (2004) found that
when the 20% blend fuel from rapeseed oil biodiesel was tested in a six-cylinder
engine, there existed a marked increase in formaldehyde, but no significant
differences in acrolein, acetaldehyde, and propionaldehyde. Corrêa and Arbilla
(2008) reported that all seven carbonyls, except benzaldehyde which showed a
reduction on the emission (−3.4% for B2, −5.3% for B5, −5.7% for B10, and
−6.9% for B20), showed a significant increase: 2.6%, 7.3%, 17.6%, and 35.5% for
formaldehyde; 1.4%, 2.5%, 5.4%, and 15.8% for acetaldehyde; 2.1%, 5.4%, 11.1%,
and 22.0% for acrolein + acetone; 0.8%, 2.7%, 4.6%, and 10.0% for propionalde-
hyde; and 3.3%, 7.8%, 16.0%, and 26.0% for butyraldehyde. Discordant results
were reported by Sharp et al. (2000), who found a substantial reduction in
carbonyl emissions with pure biodiesel and a smaller reduction with B20 blend.
Krahl et al. (2003) tested diesel and soybean oil biodiesel in three engines
(119, 205, and 276 kW). They concluded that aldehydes and ketones reduced
approximately 0 to 30% for biodiesel. Peng et al. (2008) found that B20 (20% waste
cooking oil biodiesel and 80% diesel) generated slightly less emissions in total
aldehyde compounds than that of diesel. Formaldehyde and acetaldehyde emis-
sions showed a slight decrease of approximately 10% when using pure biodiesels.
It is widely accepted that biodiesel increases these emissions because of higher
oxygen content (Xue et al. 2011). In addition, Fontaras et al. (2009) found that the
quality of biodiesel regarding the fatty acid profile, iodine number, and purity level
played a role on the formation of certain carbonyl emissions.
Some authors reported that acrolein concentration in the emissions was
strongly related to the higher glycerin content of biodiesel used (Xue et al. 2011).
Arapaki et al. (2007) found that acetaldehyde emission increased sharply with
biodiesel–diesel blend, as compared with Euro V diesel fuel, and concluded that
the acetaldehyde emissions could be caused by a higher free glycerol or total
glycerol content of the methyl ester. Corrêa and Arbilla (2008) however reported
an increase in formaldehyde emission when 2%, 5%, 10%, and 15% of biodiesel
made from waste cooking oil were tested. Formaldehyde was formed during the
frying or cooking process and the esterification process; many short chain
chemicals in biodiesel also favored the formation of formaldehyde during
combustion.
Corrêa and Arbilla (2008) found that all carbonyl emissions exhibited a
strong correlation (correlation coefficients better than 0.96) with the biodiesel
content, which indicates that carbonyl emissions are strongly influenced by the
biodiesel content and that the biodiesel ester molecules are probably the source of
these carbonyls. However, Liu et al. (2009) reported that the total concentration of
emitted carbonyls did not increase with the biodiesel content. Engine operating
conditions and engine types also influenced these emissions. Liu et al. (2009)
elucidates the carbonyl compound emissions increased when the engine was run
on biodiesel and its blends (10%, 30%, 50%, 75%, and 100% of biodiesel by
volume) at idling, 10%, 33%, and 55% loads. Cheung et al. (2009) reported that
formaldehyde emissions increased when the engine load was increased from
0.08 to 0.38 MPa but decreased when the engine load was increased from 0.38 to
0.70 MPa. Formaldehyde emissions attained the peak value at medium engine
load, similar to that of the total hydrocarbon emissions. Cheung et al. (2009)
obtained continuous increases in formaldehyde emissions with the engine load.
Zhang et al. (2008) concluded that both engine speed and engine load affect
formaldehyde emissions and showed that formaldehyde emissions increased
with engine load under medium and high engine loads at the engine speed of
1,200 rpm, but decreased with engine load under medium and high engine loads at
the engine speed of 1,400 rpm. Fontaras et al. (2009) observed that some of the
carbonyl compounds were also influenced by the driving cycle. Comparing the
new European driving cycle (NEDC) and the Artemis urban, most carbonyls were
found in higher concentrations than over the Artemis road and motorway.
However, acrolein was significantly increased especially over Artemis urban and
road cycles.
Munack et al. (2001) tested two different engines using five operation modes
from an agricultural cycle. In one engine fueled with pure diesel and rapeseed oil
biodiesel, biodiesel had higher formaldehyde emissions than diesel. In another
engine, 40% blend fuel was added to test; the emissions for pure biodiesel were
lower than that of diesel, but the emissions for 40% blend fuel was the highest.
Some researchers showed the effect of alcohol (methanol content). Cheung et al.
(2009) found that the formaldehyde and acetaldehyde emissions increased with an
increase in methanol fraction in the biodiesel blend fuel compared with diesel fuel.
Zervas et al. (2002) reported that the formaldehyde emissions increased with the
methanol fraction and concluded that exhaust formaldehyde was mainly pro-
duced from methanol. However, they reported that acetaldehyde emission was
produced from ethanol or straight chain hydrocarbons, and methanol had no
significant effect on its emission. Chao et al. (2000) observed that the formalde-
hyde emissions increased when methanol increased from 5% to 15%. Meanwhile,
they showed an increase in acetaldehyde emissions with 8%, 10%, and 15%
methanol in the blended fuel, but a decrease with 5% methanol. Arapaki et al.
(2007) pointed out that although methanol itself has no effect on acetaldehyde
emissions, the addition of methanol in biodiesel could increase the oxygen content
leading to the increase of acetaldehyde emissions, and simultaneously the contents
of straight chain hydrocarbons will change after the addition of methanol. Thus,
methanol tends to increase acetaldehyde emissions.
Overall, carbonyl compounds emissions have discordant results for biodiesel,
but it is widely accepted that biodiesel increases these emissions because of higher
oxygen content. In addition, engine operating conditions (load and cycle mode)
and engine types also impact these emissions, and methanol tends to increase
acetaldehyde emissions.
Increase or decrease in air pollutants caused by combustion of different
biodiesel fuels compared with that of conventional diesel fuel is shown in
Table 23-2, and potential effects of biodiesel production on air quality and its
mitigation is shown in Table 23-3.
23.2.2 Agrochemicals in the Atmosphere

Spray application is the major source of atmospheric contamination by pesticides
because of drift and vaporization. Figure 23-1 demonstrates the amounts and
types of herbicides applied to soybean agriculture as reported by the National
Renewable Energy Laboratory (NREL) (2009). Studies have found that large
amounts of aerially applied pesticides miss their agricultural targets. Drift loss
depends on factors such as wind, size, and height at which the material is released.
Most soybean acreage receives herbicides by ground broadcast (90%), and
integrated pest management (IPM) application timing recommendations helps
to minimize drift. Only about 2% is applied by aerial broadcast and about 8% by
band treatment (ERS 1997). Banded treatment is applying pesticides over, or next
to, each row of plants in a field. The drift from the aircraft applications ranged
from 1% to 31%. Pesticide residues can be transported into the atmosphere by
attaching to dust particles generated during tilling operations. This represents a
secondary form of off-target pesticide drift that takes place over a much longer
period. No till or minimum till can help minimize soil disturbances.
Volatilization from treated agricultural soils and plant foliage is also the major
source of atmospheric contamination by pesticides. Volatilization occurs when
herbicides reach the plant or soil surface and are degraded or transformed by
chemical, biological, and photochemical processes and enter the atmosphere.
Volatilization is a major cause of disappearance of herbicides from target areas,
particularly where they are surface applied. The rate of this loss can exceed that of
chemical degradation, depending on the moisture content of the soil, temperature,
the relative humidity, wind velocity, and soil type. Volatilization rate is variable, but it
is a continuous process. High volatilization occurs during the application phase,
following a rain event, or during irrigation. When spray application is used, loss from
Table 23-2. Increase or Decrease in Air Pollutants due to Combustion of Different Biodiesel Fuels Compared with Conventional Diesel Fuel.
Feedstock Type of biodiesel CO CO2 NOx Smoke HC References
Jatropha Jatropha blends — Increase — — — Forson et al. (2004)

Jatropha methyl ester Decrease Increase Increase — Decrease Chauhan et al.
and its blends (2012)
Jatropha methyl ester Decrease — Increase — Decrease Rao et al. (2007)
and its blends
Palm oil Palm methyl ester Increase Increase Increase — Increase
Preheated palm oil Decrease Decrease Increase Decrease — Kalam et al. (2003)
Emulsified palm oil Increase Increase Decrease Increase —
Blends of palm oil Increase — Decrease — —
Preheated palm oil Increase — Increase — — Bari et al. (2002)
Karanja oil Karanja oil and lower Decrease — Decrease — Both Agarwal (2007)
blends preheated
and unheated
Karanja methyl ester Increase — Increase — Increase Srivastava et al.
(2008)
Methyl and ethyl esters Increase — Decrease Decrease — Baiju et al. (2009)
of Karanja oil
Soybean oil Crude soybean oil Decrease Increase — Decrease Decrease Pryor et al. (1983)
Soybean methyl ester Decrease — Decrease Decrease Decrease Qi et al. (2009)
BIODIESEL IMPACT ON ENVIRONMENT AND HEALTH
Soybean biodiesel B20 Decrease Increase Decrease Decrease Canakci (2007)

Soybean biodiesel Decrease Increase Increase Decrease Decrease Canakci (2007)
B100
(Continued)
595
Table 23-2. Increase or Decrease in Air Pollutants due to Combustion of Different Biodiesel Fuels Compared with Conventional Diesel
596
Fuel. (Continued)
Feedstock Type of biodiesel CO CO2 NOx Smoke HC References
Mahua oil Methyl ester (Mahua Decrease Increase Increase — Decrease Puhan et al. (2005a)
oil)
Ethyl ester (Mahua oil) Decrease Increase Decrease — Decrease Puhan et al. (2005a)
Butyl ester (Mahua oil) Decrease Increase Decrease — Decrease Puhan et al. (2005a)
Methyl ester of Mahua Decrease — Increase — Decrease Godiganur et al.
oil and its blends (2009)
(except B20)
Mahua oil ethyl ester Decrease Increase Decrease Decrease Decrease Puhan et al. (2005)
Mahua oil methyl ester Decrease — Increase Decrease — Raheman and
and its blends Ghadge (2007)
(except B20)
Rapeseed oil Preheated rapeseed oil Decrease — Decrease Decrease — Hazar and Aydin
with 20% and 50% (2010)
blends
Rapeseed methyl ester Both Increase Decrease — Decrease Nwafor (2004)
and its blends
Rapeseed methyl ester Decrease — Increase — Decrease Tsolakis et al. (2007)
and its blends
Waste Waste cooking oil — Increase Increase Decrease Decrease Abu-Jrai et al. (2011)
cooking oil biodiesel (B50)
Waste cooking oil Decrease — — — Decrease Ghobadian et al.
biodiesel (B10–B50) (2009)
Preheated waste Increase — Decrease Increase — Pugazhvadivua and
cooking oil (30 °C, Jeyachandran
75 °C, 135 °C) (2005)
Sunflower oil Preheated crude Increase Decrease — Decrease Decrease Ozsezen et al. (2009)
sunflower oil
Sunflower and Decrease — Increase Decrease — Rakopoulos et al.
cottonseed methyl (2008)
esters
Cottonseed Preheated cottonseed Decrease — Increase — Decrease Karabektas et al.
oil oil methyl ester (2008)
Cottonseed oil methyl Decrease — — Decrease Decrease Nabi et al. (2009)
ester
Cottonseed oil methyl Decrease — Decrease Both — Aydin and Bayindir
ester and blends (2010)
597
Table 23-3. Potential Effects of Biodiesel Production on Air Quality and Its Mitigation.
Characteristics and
magnitude (without Residual
Activity Potential effects mitigation) Mitigation Effects References
Land conversion Increase in Effects and impacts with • Selecting suitable crops and using Medium Weibe et al.
to biodiesel greenhouse climate change and efficient systems for greenhouse (2008)
crop gas emissions greenhouse gas reductions
production • Nature: negative • Fuel efficiency
• Magnitude: medium • Forest conversions and restorations
• Spatial: regional/global
• Timing: continuous
• Duration: short/long-term
• Reversibility: high
• Likelihood: medium
Fertilizers use for Increase in Effects and impacts with • Improvements in farming techniques Medium Weibe et al.
biodiesel greenhouse climate change and • Improved management practices such (2008)
crops, gas emissions greenhouse gas as conversion tillage, crop rotations
producing the • Nature: negative • Growing perennials such as palm,
fertilizers, • Magnitude: medium short-rotation coppice, sugarcane, or
pesticides • Spatial: regional/local switch grass instead of annual crops
• Timing: construction and can improve soil quality by increasing
operation soil cover and organic carbon levels
•
• Duration: short/long-term In combination with no-tillage and
• Reversibility: high reduced fertilizer and pesticide inputs,
• Likelihood: medium positive impacts on biodiversity can be
obtained.
Fuel used in Increase in Effects and impacts with • Fuel efficiency Medium Weibe et al.
farming, greenhouse gas climate change and • Improved farming techniques to low (2008)
during emissions greenhouse gas • Using efficient systems for greenhouse
chemical • Nature: negative reductions
processing, • Magnitude: medium/low
transport and • Spatial: local/regional
distribution, • Timing: construction and
up to final use operation
• Duration: short term
• Likelihood: low
Construction Fugitive dust from Effects and impacts with Use dust mitigation techniques where Minimal AAFC (2011)
work construction good air quality/existing possible (e.g., water spray) to low
operations airborne pollutants
• Nature: negative
• Magnitude: medium/low
• Spatial: local
• Timing: construction
• Duration: short term
• Likelihood: low
(Continued)
Table 23-3. Potential Effects of Biodiesel Production on Air Quality and Its Mitigation. (Continued)
Characteristics and
Activity Potential effects mitigation) Mitigation Effects References
Methane Effects include Effects and impacts with • Build vapor recovery systems into Minimal AAFC (2011)
evaporation escape of vapors good air quality/Existing biodiesel production processes
from reaction into the airborne pollutants • Store methanol in sealed containers
vessels or atmosphere • Nature: negative • Store glycerine in closed space with
glycerine • Magnitude: medium adequate vapor recovery equipment
• Spatial: regional
• Timing: operation
• Duration: intermittent
• Likelihood: low
Shipping and • Increased Effects and impacts with • Ensure transport vehicles are well Low to AAFC (2011)
receiving of greenhouse gas good air quality/existing maintained and have working medium
raw materials emissions from airborne pollutants emissions control equipment
and final the burning of • Nature: negative • Use dust control measures on unpaved
products fossil fuels for • Magnitude: medium roadways and parking areas
transportation • Spatial: regional/local
purposes • Timing: operation
• Increased • Duration: continuous
particulate matter • Reversibility: medium/high
released into the • Likelihood: high
atmosphere due
to increased traffic
Figure 23-1. Amounts and types of herbicides applied to soybean agriculture.

Note: Total amounts of herbicides equals 28.7 million kgs of active ingredients/acre.
volatilization ranges from 3% to 25% for most pesticides, but for some pesticides it
may be as great as 20% to 90%, depending on weather conditions. In contrast,
pesticide losses from soil-incorporated application methods are usually much lower.
23.3 EFFECT ON WATER RESOURCES
Water plays a crucial role in all stages of biodiesel production—from cultivation of

feedstock through its conversion into biodiesel (GAO 2009). The concerns
regarding water are very critical at this stage of time because clean water and
energy demands are increasing rapidly along with the world population. Ensuring
inexpensive and clean water is a global challenge that will likely be intensified by
the increasing demand for biofuels for transportation (Dominguez-Faus et al.
2009). Biofuel production has unintended consequences on water quality and
quantity in Mississippi (Welch et al. 2010). As demand for water from various
sectors increases and places additional stress on already constrained supplies, the
effects of expanded biofuel production may need to be considered (GAO 2009).
Mitigating global water crisis along with balancing future biofuels demands is a
very critical challenge that exists for two primary reasons: first, large quantities of
water are needed to grow the biofuel crops; and second, agricultural drainage
containing fertilizers, pesticides, and sediments increases the water pollution
(Dominguez-Faus et al. 2009). That means, in today’s economies biofuels and
water are tightly linked. Therefore, availability of adequate water supply has a
profound impact on the development of biofuels, and biofuel production activities
affect the availability and quality of water sources.
23.3.1 Water Availability

Among two leading biofuel sources, biodiesel production requires much less water
than ethanol production. There are two major consumers of water—biodiesel
crops and biorefineries. The amount of water used in the biorefining process is
modest compared to the water used to grow bioenergy crops. However, because
water use in biorefineries is concentrated into a smaller area, the effects of such
facilities can be substantial locally. In biorefineries, the main use of water is
associated with washing and evaporative processes. Water use is variable but is
usually less than 1 gal. (3.78 L) of water required for each gallon of biodiesel
produced (EPA 2010). However, overall water use may be up to 3 litre of water for
each gallon of biodiesel produced (Pate et al. 2007). This water consumption is
relatively low compared with that of crude oil production. The production of a
barrel [31.5 gal. (119 L)] of crude oil requires 1,851 gal. (7,006 L) of fresh water.
Recycling wash water with various degrees of treatment reduces overall water
consumption in biodiesel production. Still lower water usage may be possible in
the future with new technologies, which include the possibility of using recycled
wastewater with various degrees of treatment. Larger well-designed facilities use
water more sparingly, but smaller producers tend to use more water per
production volume. New technologies that improve water efficiency will help
mitigate water quantity impacts. Recent plant designs have included either
waterless processes or water recycling (NRC 2008). The water use facts listed
in Table 23-4 will put the amount of water that biodiesel production uses into
perspective (CRFA 2010).
Although relatively moderate water is consumed by biorefineries to produce
each gallon of biodiesel as shown in Table 23-4, total global water consumption
Table 23-4. Water Use for Different Products and Activities.
Gallons of
Product/activity Amount/units/quantity water required
Hamburger One quarter pound 1

Biodiesel production One gallon 1–3
Automatic dishwasher One-time wash 9–12
Shower/bath Five minutes 25–50
Average personal use (adult) Per day 50
Beer One barrel 1,500
Crude oil One barrel 1,851
Tires Four 2,072
New car (including tires) One 39,090
Steel One ton 62,600
Residence use Per year 107,000
Golf course irrigation 75 acres 51,300,000
900
Thousand gallons per year

800 Global biodiesel production
700
Water use by biorefineries
600
500
400
300
200
100
0
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Global biodiesel production and projected water use
Figure 23-2. Global biodiesel production and projected water use by biorefineries.
has continuously increased over the last decade, and it will rapidly increase in the
near future (Figure 23-2). Therefore, biodiesel biorefineries are likely to add extra
pressure on the freshwater resources.
The extent to which increased biodiesel production will affect the water
resources depends on the feedstock selected and how and where it is grown. The
choice of feedstock is based on local availability, cost, government support, and
performance of a fuel (Haas et al. 2006). Furthermore, processing feedstocks into
biofuels can use large quantities of water, mainly for washing plants and seeds and
for evaporative cooling. It is important to consider the regional variability of water
resources when choosing which feedstock to grow and how and where to expand
their production. However, it is irrigated production of these key biofuel feed-
stocks that will have the greatest impact on the balance of local water resources.
Many crops currently are used for biodiesel production, such as rapeseed,
soybean, sunflower, and oil palm. Rapeseed oil and sunflower oil are the
predominant feedstocks used for biodiesel in Europe, whereas soya oil is the
predominant feedstock in the United States. Even perennial plants such as
jatropha and pongamia that can be grown in semiarid areas on marginal or
degraded lands may require some irrigation during hot and dry summers. Both
palm oil and jatropha are being used in Africa to produce biodiesel. Other oil crops
under both experimental and commercial cultivation are cotton seed, groundnut,
soybean, caster bean, and sunflower (Malaviya and Ravindranath 2012). Among
these, cultivation of jatropha is being promoted in many African countries because
of its ability to grow relatively well in marginal areas, reclaim degraded lands, and
contain soil erosion. Moreover, jatropha can be cultivated with other annual crops
in agroforestry systems. It is perennial and drought resistant and can be grown on
nutrient-poor soils. Many countries grow jatropha, such as Togo, Ghana, Mali,
Tanzania, and Niger. Another aspect of biodiesel crop is the yield, which is
important from the water consumption point of view. Among the oil crops used
for producing biodiesel, oil palm has the highest yield potential per hectare
(5,950 L/ha) with large areas under oil palm cultivation in Angola, Democratic
Republic of Congo, Nigeria, Ghana, and Tanzania (Jumbe et al. 2009). About 70%
of freshwater withdrawn worldwide is used for agricultural purposes. Water
resources for agriculture are becoming increasingly scarce in many countries as
a result of increased competition with domestic or industrial uses. Moreover, the

expected impacts of climate change in terms of reduced rainfall and runoff in some
key producer regions (including the near East, North Africa, and South Asia) will
place further pressure on already scarce resources. Biofuels currently account for
about 100 km3 (or 1%) of all water transpired by crops worldwide, and about
44 km3 (or 2%) of all irrigation water withdrawals (Fraiture et al. 2008).
Expansion of irrigated areas is likely to happen in some areas based on water
resources and land, but the actual scope for increased biodiesel production under
irrigated conditions on existing or new irrigated lands is limited by infrastructural
requirements and by land-tenure systems. Moreover, this expansion is also
constrained by higher marginal costs of water storage and land acquisition.
Irrigation expansion of agriculture into dry western areas has the potential to
dramatically affect water availability (Brown 2008). In the next 5 to 10 years,
increased agricultural production for biofuels will probably not alter the national
aggregate view of water use. However, there are likely to be significant regional and
local impacts where water resources are already stressed. The potential for growth
of irrigated areas for the near East and North Africa region is reaching its limit.
Although there remains an abundance of water resources in South Asia and East
and Southeast Asia, there is very little land available for extra irrigated agriculture.
Most potential for expansion is limited to Latin America and sub-Saharan Africa.
However, in the latter region it is expected that the current low levels of irrigation,
water withdrawals will increase only slowly.
Encouraging biodiesel production by growing more oil crops without con-
sidering the quality and availability of water by region could put a significant strain
on water resources in some parts of the world, especially in developing countries
(NRC 2008). There is very high demand for access to water for irrigation, cooking,
and drinking. Because the production of biodiesel is very water intensive,
agricultural shifts to growing biodiesel crops could change the availability of
clean water and greatly increase pressure on water resources in those areas. As a
result, those areas will face serious challenges to meet the predicted increase in
demand for food produce, let alone sustain any further growth prompted by
expanding biodiesel production (Fraiture et al. 2008, Lin et al. 2011).
Depending on what crops are grown, where the crops are grown, and whether
there is an increase in overall agricultural production, significant acceleration of
biofuel production could cause much greater water quantity problems than are
currently experienced. The use of certain agricultural practices and technological
innovations can mitigate the effects of biodiesel production on water resources,
but there are some barriers to their widespread adoption. Agricultural conserva-
tion practices can reduce water use, but their economic feasibility must be checked
(GAO 2009). Similarly, alternative water sources, such as brackish water, may be
viable for some aspects of the biodiesel conversion process and can help reduce
biorefinery reliance on freshwater resources. However, the high cost of retrofitting

plants to use these water sources may be a barrier, according to experts and
officials. Dry cooling systems and thermochemical processes have the potential to
reduce the amount of water used by biorefineries, but many of these innovations
are not currently economically feasible or remain untested at the commercial scale
(GAO 2009).
23.3.2 Water Pollution

In 2009, the US government reported that growing US production of biodiesel
could increase water pollution (GAO 2009). The report highlighted that biodiesel
refineries release pollutants such as glycerin, which disrupts the microbial cleaning
processes used in wastewater treatment. In addition to water use in the washing
and evaporation processes, sources of wastewater include steam condensate;
process water softening and treatment to eliminate calcium and magnesium salts,
iron, and copper; and wastewater from the glycerin refining process. In a joint
study conducted by the US Department of Energy (DOE) and Department of
Agriculture (USDA), it was estimated that consumptive water use at a biodiesel
refinery accounts for approximately one-third of the total water use, or about 0.32
gal. (1.21 L) of water per gallon of biodiesel produced (Sheehan et al. 1998). New
technologies have reduced the amount of wastewater generated at facilities.
Process wastewater disposal practices include direct discharges (to waters of the
United States), indirect discharges (to wastewater treatment plants), septic tanks,
land application, and recycling.
Most biodiesel manufacturing processes result in the generation of process
wastewater with free fatty acids (as soap) glycerin (a major coproduct of biodiesel
production); however, the quantity of wastewater will be significantly reduced
for facilities with waterless processes or water recycling. Despite the existing
commercial market for glycerin, the rapid development of the biodiesel industry
caused a glut of glycerin production, which resulted in many facilities disposing of
glycerin. Glycerin disposal may be regulated under several EPA programs,
depending on the practice. Glycerin can be marketed as a feedstock following
methanol recovery and additional refining. Significant research on alternative
beneficial uses for glycerin is ongoing. Some potential options exist for the
catalytic and biological conversion of glycerin into value-added products. Soybean
production has negative environmental impacts through movement of agrichem-
icals, especially nitrogen (N), phosphorus (P), and pesticides from farms to other
habitats and aquifers. Agricultural N and P are transported by leaching and
surface flow to surface, ground, and coastal waters causing eutrophication, loss of
biodiversity, and elevated nitrate and nitrite in drinking water wells. Pesticides can
move by similar processes. Biodiesel refining requires much less water per unit of
energy produced than bioethanol. Data on agrichemical inputs for corn and
soybeans and on efficiencies of net energy production from each feedstock reveal,
after partitioning these inputs between the energy product and coproducts, that
biodiesel uses (per unit of energy gained) 1.0% of the N, 8.3% of the P, and 13% of
the pesticide (by weight) used for corn grain ethanol. The markedly greater
releases of N, P, and pesticides from corn, per unit of energy gain, have
substantial environmental consequences, including being a major source of the
N inputs leading to the dead zone in the Gulf of Mexico and to nitrate, nitrite,
and pesticide residues in well water. Moreover, pesticides used in corn produc-
tion tend to be more environmentally harmful and persistent than those used to
grow soybeans.
Producing more biodiesel crops will affect water quality as well as quantity.
Converting pastures or woodlands into biodiesel crops may exacerbate problems
such as soil erosion, sedimentation, and excess nutrient (nitrogen and phospho-
rous) runoff into surface waters, and infiltration into groundwater from increased
fertilizer application. Excess nitrogen in the Mississippi river system is a major
cause of the oxygen-starved dead zone in the Gulf of Mexico, where many forms of
marine life cannot survive. Biodiesel production results in organically contami-
nated wastewater that if released untreated, could increase eutrophication of
surface water bodies. However, existing wastewater treatment technologies can
deal effectively with organic pollutants and wastes. Fermentation systems can
reduce the biological oxygen demand of wastewater by more than 90%, so that
water can be reused for processing and methane can be captured in the treatment
system and used for power generation. Regarding the distribution and storage
phases of the cycle, because biodiesel is biodegradable, the potential for negative
impacts on soil and water from leakage and spills is reduced compared with that of
fossil fuels. Pesticides and other chemicals can wash into water bodies, negatively
affecting water quality. Biodiesel feedstock differs markedly in their fertilizer and
pesticide requirements.
To understand the potential effects of biodiesel production, GAO was asked
to examine (1) the known water resource effects of biodiesel production in the
United States, (2) agricultural conservation practices and technological innova-
tions that could address these effects and any barriers to their adoption, and
(3) key research needs regarding the effects of water resources on biodiesel
production. As discussed earlier, the use of certain agricultural practices, alterna-
tive water sources, and technological innovations can mitigate the effects of
biodiesel production on water resources, but there are some barriers to their
widespread adoption. According to experts and officials, agricultural conservation
practices can reduce nutrient runoff, but they are often costly to implement.
Similarly, alternative water sources, such as brackish water, may be viable for some
aspects of the biodiesel conversion process (GAO 2009). There is great need for
additional extensive research in the area of water resources. In that context, further
research in discovering improved crop varieties could help to reduce water and
fertilizer needs. Moreover, cultivation of algae to increase commercial-scale
biodiesel production and to control potential water quality problems also is a
key research area. The optimization of conversion technologies that could help to
ensure water efficiency need to be studied. The information about water resources
in local aquifers and surface water bodies would aid in decisions about where to
cultivate feedstocks and locate biorefineries. Potential effects of biodiesel produc-
tion on surface water quality and its mitigation is shown in Table 23-5.
Table 23-5. Potential Effects of Biodiesel Production on Surface Water Quality and Its Mitigation.
Characteristics and
Activity Potential effects mitigation) Mitigation effects References
• Irrigation and • Freshwater scarcity Effects and impacts • Selection of the suitable crops Medium Wiebe et al.
other agricultural • Climate change: dominantly exist in some according to the water (2008)
purposes reduced rainfall key biofuel producer availability in the region
• Land conversion and runoff in East, regions • Improvements in farming and
to biodiesel North Africa, South • Nature: negative irrigation techniques
crops from Asia • Magnitude: medium • Improved management
undisturbed • Soil erosion • Spatial: regional/local practices such as conversion
natural • Sedimentation and • Timing: construction and tillage, crop rotations
vegetation excess nutrient operation • In combination with
(nitrogen and • Duration: continuous no-tillage and reduced fertilizer
phosphorous) • Reversibility: medium/ and pesticide inputs
runoff into surface high
waters, and • Likelihood: medium
infiltration into
groundwater from
increased fertilizer
application
(Continued)
607
Table 23-5. Potential Effects of Biodiesel Production on Surface Water Quality and Its Mitigation. (Continued)
608
Characteristics and
• Construction and Effects include Effects and impacts only • Place/construct impermeable Low AAFC (2011)
operation: contamination of exist if surface water is pans/pads
• Spills of process water bodies and/ present near the site at loading/transfer points
or machine or destruction of • Nature: negative • Construct secondary
fluids, including aquatic habitats containment berms around
• Magnitude: medium
fuels, hydraulic • Spatial: regional storage vessels
fluids, canola oil, • Timing: construction and • Undertake regular inspection of
methanol, operation equipment and storage vessels
biodiesel, • Duration: intermittent for leaks or wear
potassium • Reversibility: low • Ensure appropriate spill
hydroxide, • Likelihood: medium response plan, training and
phosphoric acid. equipment is available onsite
and near transfer and storage
points
• Excavate contaminated soil
promptly if spills occur, and
dispose of it appropriately
• Contain used or contaminated
fluids and dispose of them at
appropriate disposal facilities
• Excavation, • Contamination of Effects and impacts only • Use existing trails or roads where Minimal AAFC (2011)
vegetation water bodies with exist if surface water is possible
clearing and sediment from present near the site • Keep disturbance within
dewatering of excavation • Nature: negative development area(s)
excavations dewatering • Magnitude: medium • When working in areas with
• Contamination • Spatial: local native vegetation, seed
with surface runoff • Timing: construction disturbed areas with native seed
due to ground • Duration: short term mixture (certified weed free)
disturbance and/or • Reversibility: high • Seed other disturbed areas with
defoliation • Likelihood: high fast growing seed mix (certified
weed free) to minimize erosion
in disturbed areas
• Ensure dewatering water flows
into well vegetated area or
settling basin
• Use appropriate erosion control
measures including silt fences,
hay bales, geotextiles and
temporary settling ponds
(Continued)
609
Table 23-5. Potential Effects of Biodiesel Production on Surface Water Quality and Its Mitigation. (Continued)
610
Characteristics and
• Utility Drilling fluids Effects and impacts only • Use water rather than mud as Low AAFC (2011)
installation via entering water exist if surface water is drilling fluid
directional bodies via a present near the site • Drill during times of low
drilling fracture in the drill • Nature: negative groundwater flow as identified
bore (frac-out) • Magnitude: medium during Groundwater analysis
• Spatial: local • Use appropriate monitoring and
• Timing: construction shutdown procedures in case of

• Duration: short term frac-out
• Reversibility: medium • Ensure availability of cleanup
• Likelihood: medium and containment equipment
• Wastewater Contamination of Effects and impacts only • Use appropriate treatment Low Wiebe et al.
disposal water bodies exist if surface water is system to render wastewater (2008);
present near the site safe (e.g., nondeleterious to fish) AAFC
• Nature: negative for release to surface water at (2011)
• Magnitude: medium the point of discharge, or to
• Spatial: immediate municipal WWT system as
• Timing: operation applicable
• Duration: intermittent • Verify the adequacy of capacity
• Reversibility: medium and capability, and ensure
• Likelihood: low appropriate disposal
• Cum. Effects: minimal agreements are in place with the
WWT system operator if
wastewater is to be disposed of
in a municipal WWT system
• Monitor treatment efficacy

where onsite treatment is used
to ensure wastewater meets
disposal standards (municipal or
federal and provincial surface
water quality, as appropriate)
• Monitor wastewater quality
where onsite water treatment is
not required to ensure that off-
spec water is not entering the
municipal WWT system.
• Ensure appropriate spill
response plan, training and
equipment is available onsite
and near transfer and storage
points
• Construction, • Effects include Effects and impacts only • Work only during acceptable Low AAFC (2011)
modification, damage to fish exist if surface water is work windows for the species
and use of water habitat or present present in the water body
crossings spawning grounds • Nature: negative • Avoid in stream work whenever
• Magnitude: medium possible
• Spatial: local • Use appropriate erosion control
• Timing: construction and measures including silt fences,
operation hay bales, and geotextiles

• Duration: intermittent • Grade approach roads so as to
• Reversibility: high limit runoff to water body
• Likelihood: low • Do not use structures which may
611
present a barrier to fish passage

23.4 EFFECT ON SOIL
23.4.1 Soil Productivity

There are increasing concerns regarding the effect of biodiesel crops on the soil.
The effects of removing agricultural residues on soil organic matter, soil erosion,
or crop yields may vary considerably because of site-specific conditions like
climate, soil type, and crop management (Cherubini et al. 2009). The removal of
agricultural residues may reduce the input of organic matter to soil, which in turn
would reduce soil fertility and plant productivity if nutrient loss is not replaced
through fertilizers. However, because residues in conventional crop (and forest)
management systems in developing countries are often burned after harvest,
biomass removal may not change carbon inputs significantly in these systems.
Even in systems in which the residues are not burned but rather are retained on
the soil surface, impacts may be low because above-ground litter that decays on the
surface often does not enter the soil carbon pool.
In general, it must be considered that the amount of biomass removed at
harvest represents only a fraction of total biomass produced; the major inputs to
soil carbon are fine roots and leaf litter. Losses in soil carbon caused by the
removal of agricultural residues are thus considered to be low in crop systems
(Cowie 2006). Where forestry residues or whole tree cutting of short rotation
coppices are used for second-generation biofuel production, the levels of soil
organic matter and soil carbon may be reduced. Considering the high concentra-
tion of nutrients in leaves, branches, and bark, as well as the high proportion of
nutrient-rich biomass in young trees, short rotation whole tree-harvesting systems
would remove greater amounts of nutrients than stem-only harvesting. This
impact can be reduced if trees are harvested during winter, when leaves have
already fallen (although this is not possible in evergreen tropical regions). In
general, the effects of second-generation biofuel production from woody biomass
on soil nutrient levels are strongly dependent on site-specific factors, such as the
forest management system. As such, a general assessment on the chemical-
biological impacts on soil is very difficult. The use of machinery in bioenergy
production systems may increase soil bulk density (compaction) and create deep
ruts, thereby decreasing the amount of aeration, water infiltration, and root
growth in the soil. However, although tree growth will decrease on most soils (e.g.,
clay), the water holding capacity of sandy soils may improve and help increase tree
growth (Lattimore et al. 2009). In cases for which dedicated energy plantations for
second-generation biofuels replace other production systems, the impact on soil
will depend on the features of the bioenergy system and the system being replaced.
Substitution of cropland through short rotation bioenergy crops (i.e., perennial
grasses, willow, and eucalyptus) may increase soil carbon owing to reduced
frequencies of harvest and soil disturbance. If short rotation bioenergy crops are
substituting pastures, soil carbon balance will depend on the relative balance of
organic inputs and the decomposition rate of the old and new land use; again, in
such cases, a general assessment is difficult (Cowie 2006). Regardless, in areas
subject to strong erosion, like the loess plateau in China, perennial cultivation can
significantly decrease the level of erosion and help to preserve fertile land from
degradation.
The amount of chemicals and fuels used on the farm and their associated
emissions, as well as manufacturing, packaging, and processing of inputs used to

grow the biodiesel crops are the important factors affecting the soil. Biodiesel
crops such as soybeans remove essential minerals from the soil, which must be
replaced to maintain soil productivity. Soybeans remove about 408 g of phospho-
rus per bushel of beans harvested. Soybeans also use large amounts of potassium,
removing about 545 g of potassium from the soil for each bushel of beans
harvested. Nitrogen fertilizer is seldom required for successful soybean production
because, like all members of the legume family, soybeans fix atmospheric nitrogen
if the proper strain of Rhizobium bacteria is present in the soil or if the seed is
properly inoculated. Thus, only small amounts of nitrogen fertilizers are used on
US soybeans. Potential effects of biodiesel production on soil and its mitigation are
shown in Table 23-6.
23.4.2 Soil Erosion

Soil erosion not only moves large amounts of soil away from the field, but also
removes unused farm chemicals and natural nutrients. Soil erosion data are
available from USDA’s National Resources Inventory (NRI) (Cavalett and Ortega
2010). The NRI is an inventory of land use and soil erosion on prime farmland,
wetlands, and other nonfederal rural land in the United States. The NRI erosion
trends are reported by state and land use, for example, cultivated and uncultivated
croplands, pastureland, and range land. Inventories are conducted every 5 years by
the National Resources Conservation Service (NRCS; formerly, the Soil Conser-
vation Service). Data for 1992 were collected for more than 800,000 locations. NRI
data are statistically reliable for national, regional, state, and sub-state analysis
(NRC 2008). To obtain erosion estimates for the 14 soybean states, the 1992 NRI
data set was downloaded into a statistical analysis software file. Erosion estimates
were averaged for land where soybeans were produced during the 1992 growing
season. Missouri has the highest soil loss of 8.1 tons/ac of soybeans, and South
Dakota has the lowest, 2.8 tons/ac. Alabama had the highest soil erosion overall,
but Missouri had the highest soil erosion specifically associated with soybean
production. Sheet erosion occurs when there is a little water at the soil surface, and
it runs over the soil as a thin sheet. Rill erosion is caused by the energy of water
that has concentrated and is moving downslope. As water concentrates, it forms
small channels called rills. Water collected into small rills coalesces to form large
channels and produce gully erosion (Foth 1984). Only seven of the 14 states
studied reported wind erosion, which varied from 4.2 tons/acin Minnesota to
0.20 tons/acin Ohio. The universal soil loss equation (USLE) is used to predict
water erosion on agricultural land. It was designed to predict the long-term
erosion rates on farmland so that management systems could be devised that
result in acceptable levels of erosion. It is a complex equation that uses informa-
tion on the quantity and intensity of rainfall, soil erodibility, slope length, slope
Table 23-6. Potential Effects of Biodiesel Production on Soil and Its Mitigation.
614
Characteristics and
Magnitude (without Residual
Activity Potential Effects mitigation) Mitigation Effects References
• Land-use change • Reduction in soil Effects and impacts on • Improvements in farming Minimal to Wiebe et al.
• Intensification of organic matter virgin soil/previously techniques medium (2008)
agricultural • Soil erosion disturbed soil • Improved management
production on • Nature: negative practices such as conversion
existing tillage, crop rotations
• Magnitude: medium/
croplands low • Growing perennials such as
• Spatial: local palm, short-rotation coppice,
• Timing: construction sugarcane, or switch grass
and operation instead of annual crops can
• Reversibility: improve soil quality by
medium/high increasing soil cover and
• Likelihood: medium/ organic carbon levels
low • In combination with no-tillage
and reduced fertilizer and
pesticide inputs, positive
impacts on biodiversity can be
obtained.
• Spills of process Soil contamination Effects and impacts on • Place/construct impermeable Minimal AAFC (2011)
• Spills of machine virgin soil/previously containment pans/pads at to low
fluids disturbed soil loading/transfer points
Spills including • Nature: negative • Construct secondary
containment berms around
○ Fuels • Magnitude: medium/
low storage vessels
○ Hydraulic
• Spatial: local/ • Undertake regular inspection of
fluids equipment and storage vessels
○ Canola oil immediate
for leaks or wear
○ Methanol • Timing: construction
and operation • Ensure appropriate spill
○ Biodiesel response plan, training and
○ • Duration: intermittent
Potassium equipment is available onsite
hydroxide • Reversibility:
medium/high and near transfer and storage
○ Phosphoric
• Likelihood: medium/ points
acid • Excavate contaminated soil
low
promptly if spills occur, and
dispose of it appropriately
• Contain used or contaminated
fluids and dispose of them at
appropriate disposal facilities
(Continued)
615
Table 23-6. Potential Effects of Biodiesel Production on Soil and Its Mitigation. (Continued)
616
Characteristics and
Activity Potential Effects mitigation) Mitigation Effects References
• Excavation • Soil erosion Effects and impacts on • Avoid working in excessively Minimal to AAFC (2011)
• Vegetation • Soil compaction virgin soil/previously wet or muddy conditions medium
clearing disturbed soil • Use appropriate erosion control
• Nature: negative measures including silt fences,
hay bales, and geotextiles
• Magnitude: medium/
low • Work in dry season or winter if
• Spatial: local possible to minimize
• Timing: construction compaction and rutting
• Duration: short term • Use low impact equipment if
• Reversibility: low/ operating in sensitive areas
medium
gradient, cropping management practices, and erosion control practices. The

cropping management factor looks at vegetative cover, types of tillage (moldboard
plow, minimum till, or no till), and crop rotation. Information is collected on past
years because crop rotation sequences, tilling practices, and other factors in
previous years have a direct effect on current erosion rates. Thus, although this
project focuses on a single growing season for soybeans, the soil loss estimates for a
given year depend on factors that occur in previous years. Erosion control factors
include contour tillage, strip cropping on the contour, and terrace systems. Since
the 1920s, federal legislation and government programs, such as USDA’s Con-
servation Reserve Program (CRP), have provided incentives for farmers to use
conservation practices. Although soil loss is still considered a major problem, it
has been declining in recent years. Soil loss from erosion on cropland dropped
from a total of 3.1 billion tons on 421 million acres in 1982 to 2.1 tons on
382 million acres in 1992 (NRC 2008).
23.4.3 Fuel Spillage

Biodiesel production considered a nonpolluting fuel because of its biodegradabil-
ity and reduction in exhaust emissions. However, the assessments of biodiesel
pollution in water and soil are still limited. There are few studies on the biodiesel
effects on the soil. Experiments evaluating toxicity of biodiesel carried out by
Peterson and Reece (1994) showed that it was 15 times less harmful for Daphnia
magna than diesel oil. In another study (Randall and Point 1999), LC50 for the
early developmental stage of Menidia beryllina was 578 ppm for biodiesel when
compared to 27 ppm for diesel oil. Biodiesel is nontoxic up to a concentration
of 12% (Lapinskiene and Martinkus 2006), whereas diesel oil exhibits toxic
properties just above 3% of its soil content. Soil microorganisms are very sensitive
to any ecosystem perturbation (Andreoni et al. 2004), and measurement of
microbial parameters (soil microbial biomass or enzyme activities) may serve as
indicators of soil pollution and soil health (Eibes et al. 2006, Labud et al. 2007).
Hawrot-Paw and Martynus (2011) investigated the effect of conventional diesel
fuel modification with biodiesel addition on the activity of soil microbiota. In this
study, diesel fuel, biodiesel, and their mixture in a concentration of 5% (w/w) were
introduced into soils—light loamy silty sand and light silty loam. Reduction in the
content of live microbial biomass was found, irrespective of the soil type and
contamination. In all the examined treatments and throughout the entire incu-
bation, inhibition of microorganisms was observed when biodiesel was introduced
into soils. In the case of using rapeseed oil methyl ester (RME), the emission of
hydrocarbons, carbon monoxide, and particulate soils is respectively lower by
20%, 13%, and 16% in relation to biodiesel fuel (Hawrot-Paw and Martynus 2011).
The soil contaminated with biodiesel and their diesel blends promoted
genotoxic/mutagenic effects by inducing chromosomal aberration (CA) and
base-pair substitution mutations (Leme et al. 2012). These effects were associated
with the biodiesel contamination; these effects can be related to both fatty acid
methyl esters (FAMEs), that is, the biodiesel itself, and impurities (e.g., free sterols)
from the feedstock used in the biofuel production, all of which are present in the
marketed biodiesel. Soybean, one of the main sources of biodiesel production, is
known for its high concentration of phytoestrogens and phytosterols (Canakci and
Sanli 2008, Leme et al. 2012). Yang et al. (2011) showed that several phytosterols
are present in biodiesel, and their concentrations in biofuel are highly dependent
on carboxylic acid sources. The harmful effects of phytosterols, plant sterols
structurally quite similar to cholesterol, have been intensively investigated by food
administration studies. In general, these agents raised no obvious concerns when it
comes to human safety so far. Owing to their chemical structures, phytosterols can
oxidize and produce a wide variety of oxidation products that have controversial
biological effects (Leme et al. 2012). Oxidation of phytosterols is accelerated by
high temperatures, exposure to ionizing radiation, light, among other conditions.
Fuel spillages in soil are influenced by several environmental factors, which can
promote changes in the chemical structures of pollutants. The spill simulations
have shown that the exposure of biodiesel to high temperatures and solar radiation
may induce some reactions, such as the photooxidation of phytosterols present in
the soy-based biodiesel used (Leme et al. 2012).
More research should be carried out in integrated pest management and
organic agricultural fields. Promotion of land stewardship, changing taxes, and
zoning laws to favor small family farms is necessary to make possible the social,
economic, and environmental diversity for achieving agricultural and ecosystem
stability (Opie 2000). More research on pests and disease control with diverse
crops, crop rotations, and so forth are necessary. Integrating livestock into the
crop rotation and limiting the construction of homes and businesses on prime
farmland are some of the necessary steps to be taken.
23.5 EFFECT ON BIODIVERSITY
Although biodiesel is less hazardous than petrochemical fuels, any material

that is volatile enough to power an engine or fire up a furnace will be harmful to
the plants and wildlife if spilled into rivers or streams. Large-scale cultivation
of food crops often involves heavy use of pesticide, herbicide, and fertilizers,
and their effects could extend far away from the actual plantations. In Brazil,
evidence suggests that widespread pesticide use in soya bean farms is threat-
ening the downstream Pantanal wetland area (WWF 2003). This area is one of
the world’s largest and most important wetlands and provides refuge to
hundreds of bird species, including the endangered hyacinth macaws
(Anodorhynchus hyacinthinus), Jabiru mycteria, and some healthy nesting
sites for wood storks (Mycteria americana). Pantanal is rich in mammals and
reptiles as well, including jaguars, alligators, giant otters, iguanas, anacondas,
anteaters, and capybaras.
Any high-volume spill of vegetable oil can be harmful to aquatic wildlife.
Oils that saturate a bird’s feathers can render it flightless, putting it at greater risk
to predators. Otters and beavers with oil-impregnated fur suffer the risk of
hypothermia, and fish may find it impossible to breathe. The EPA classifies any
oil spill as harmful under terms of the Clean Water Act (Randall and Point
1999). When a biodegradable biodiesel breaks down, it provides nutrient
material to naturally occurring marine life. In contrast, it is more difficult to

accelerate the breakdown of spilled petroleum diesel fuels (Randall and Point
1999). Biodiesel contains soy esters that are flammable, and spills that occur in
streams, rivers, and lakes can lead to surface fires that kill plants and wildlife. The
soy esters can form solvents that are strong enough to dissolve engine seals and
gaskets, so their effect on delicate ecosystems may be devastating. If a biofuel
spill occurs in drinking water reservoirs, the water will not be potable until it is
thoroughly filtered and treated. The Convention on Biological Diversity (CBD)
has emphasized the need for the adoption of adequate policy frameworks to
ensure that the production of biodiesel is sustainable. Sustainable production
and use of biodiesel is promoted by most of the countries while taking into
account the full life cycle and acting in accordance with the precautionary
principle. However, current biodiesel targets appear to have been developed by
different countries with little consideration for environmental consequences of
biodiesel production (Campbell and Doswald 2009). Table 23-7 illustrates the
potential effects and mitigation for biodiversity.
Recent studies on biodiversity changes from biodiesel production have
highlighted some serious concerns. For example, the Cerrado, located in Brazil’s
central highlands, is a particular concern. The Cerrado is the world’s most wildlife-
rich savannah and listed as a biodiversity hotspot with a high level of endemism by
Conservation International. It contains 837 bird species, including the critically
endangered cone-billed tanager (Conothraupis mesoleuca) and nearly 200 species
of mammals, with threatened species such as the giant anteater, pampas cat and
maned wolf. Together with around 7,000 species of plants and hundreds of species
of reptiles and freshwater fish (Klink and Machado 2005), this area is of huge
importance for wildlife. By 2004, large-scale soya bean and other farming had
reduced the size of this unique habitat to 43% of its original size. Around 1% of the
remaining Cerrado is lost every year (Butler 2007), and only 2.2% of the Cerrado is
legally protected. Moreover, the Cerrado soil and vegetation have high levels of
stored carbon. If Cerrado is cleared to cultivate soya bean for biodiesel production,
it is estimated that it would take 37 years to repay the carbon debt created by the
land conversion (Fargione et al. 2008).
Another example of biodiesel consequence on biodiversity is of European
grassland. Because of the recent boom in biofuels, much land under the Common
Agricultural Policy set-aside scheme has been turned into maize and rapeseed
crops. This has caused further reduction in habitats available for many European
farmland birds, such as little bustard (Tetrax tetrax) and red kite (Milvus milvus).
Many studies have confirmed the beneficial role of fallow pockets like set-asides on
farmland birds (Bracken and Bolger 2006; Wretenberg et al. 2007) and mammals
(Macdonald et al. 2007). Such land is important for birds because it provides food
in winter and undisturbed nesting sites in spring. However, the European
Table 23-7. Potential Effects of Biodiesel Production on Wildlife and Habitat, Land Use, and Its Mitigation.
620
Characteristics and
Activity Effects On Potential Effects mitigation) Mitigation Effects References
Land conversion Land use, Destruction or Effects and impacts • Beneficial impacts on Medium to Webb and
to biodiesel wildlife and fragmentation with existing natural biodiversity were only high Coates
crops from habitat of habitat, vegetation and set expected from (2012)
undisturbed disturbed aside/marginal/ conversion of
natural natural abandoned land cropland or grassland
vegetation vegetation, • Nature: negative to grass feedstocks or

biodiversity • Magnitude: high/ woody feedstocks for
loss medium biofuels.
• Spatial: local • Neutral impacts were
• Timing: farming and recorded on set-aside,
operation marginal, and
• Duration: short term abandoned land for
• Reversibility: medium only grass or woody
• Likelihood: medium feedstocks
Excavation, Wildlife and Destruction or Effects and impacts • Use existing trails or Low AAFC
vegetation habitat fragmentation only exist if wildlife or roads where possible (2011)
clearing, and of habitat, and wildlife habitat is • Keep disturbance
dewatering noise impacts present within development
• Nature: negative area(s)
• Magnitude: medium • Limit activity during
• Spatial: local breeding windows as
• Timing: construction identified in wildlife
• Duration: short term analysis
• Reversibility: medium
Construction or Wildlife and Destruction or Effects and impacts • Use existing trails or Medium AAFC
modification habitat fragmentation only exist if wildlife or roads where possible (2011)
of linear of habitat, and wildlife habitat is • Limit the length of
features (e.g., noise impacts present fences/size of fenced
roads, rail • Nature: negative area(s)
lines, fences) • Magnitude: medium • Avoid known
• Spatial: local migration corridors
• Timing: construction and other high use
and operation areas as identified in
• Duration: continuous wildlife analysis
• Reversibility: medium • Recreate habitat in
accessible areas
(Continued)
621
Table 23-7. Potential Effects of Biodiesel Production on Wildlife and Habitat, Land Use, and Its Mitigation. (Continued)
622
Characteristics and
Activity Effects On Potential Effects mitigation) Mitigation Effects References
Construction Land use Increased Effects and impacts • Plan and design Low to AAFC
and operation vehicular traffic with existing or construction entrance medium (2011)
during planned land use to ensure traffic has
construction adjacent to facility/no the least impact on
and operation existing or planned surrounding land
land use adjacent to uses

facility • Schedule
• Nature: negative construction traffic to
• Magnitude: medium avoid interfering with
• Spatial: local other activities or
• Timing: construction uses in the area.
or operation
• Duration: intermittent
• Reversibility: high/
medium
Operation Land Use The potential for Effects and impacts • Consult with adjacent Low to AAFC
decreasing the with existing or property owners prior medium (2011)
value of planned land use to project initiation
adjacent adjacent to facility/no • Conduct independent
properties existing or planned assessment of the
land use adjacent to potential impact of
facility development on
• Nature: negative property values
• Magnitude: • Choose site that is
low/medium compatible with
• Spatial: local adjacent and nearby
• Timing: operation land uses
• Duration: continuous
• Reversibility:
high/medium
• Likelihood:
low/medium
623
Commission has completely abolished compulsory set-aside from 2008 as part of

the Health Check of the CAP, jeopardizing biodiversity conservation. The
justification put forward for this abolition has been the high price of cereals,
partly driven by the growth in biofuels.
Besides pollutions by runoff, the irrigation needed for biofuel crops would
impact the surrounding wildlife, particularly on wetland ecosystems. A current
example is a large-scale sugarcane plantation development project in the Tana
River Delta on the northeast coast of Kenya. Developers plan to establish a
20,000 ha (49,421 ac) sugarcane plantation 30 km (18.6 mi) upstream of the
delta, partly for bioethanol production. The project intends to extract 28 m3/s
of water (a third of the river water volume during the dry season) from the
Tana River to irrigate the sugarcane (Mireri et al. 2008). This huge irrigation will
cause severe competition for water resources between the sugar project,
other development projects, and downstream domestic, livestock, wildlife,
fisheries, and ecosystem needs, affecting not only wildlife, but local livelihoods
as well.
The Tana River Delta consists of a series of complex and seasonally flooded
habitats and is an important bird area (IBA: KE022) with more than 345 species of
birds including the threatened Basra reed warbler (Acrocephalus griseldis) and
Tana River cisticola (Cisticola restrictus). No less than 22 species with interna-
tionally important populations have been recorded there, making the delta one of
the key sites in Kenya for bird conservation. After it was made public in 2007,
strong advocacy and awareness campaigns against the project were raised and a
court injunction temporarily stopped it. However, in June 2009, Kenya’s high
court ruled in favor of the project, allowing the government to give tenure rights
and ownership to the developers for the growth of maize and rice.
23.6 GREENHOUSE GAS EMISSIONS
23.6.1 Reduction in Greenhouse Gas Emissions

Biodiesel is considered as a credible source of low-carbon energy that delivers
GHG savings compared with fossil fuels. Fossil fuels release carbon dioxide that
has been stored for millions of years beneath the earth’s surface, whereas biodiesel
produced from biomass has the potential to be carbon neutral over the life cycle
because combustion only returns to the atmosphere the carbon dioxide absorbed
from the air by feedstock crops through photosynthesis (Lin et al. 2011). The
consensus regarding biodiesel is that it has the capacity to lower GHG emissions
compared to those of fossil fuels. Biodiesel effects from land-use change have the
potential to cause even more emissions than that what would be caused by using
fossil fuel alone. Carbon dioxide is one of the major GHGs. Although the burning
of the biodiesel produces carbon dioxide emissions similar to those from ordinary
fossil fuels, the plant feedstock used in the production absorbs carbon dioxide
from the atmosphere when it grows. Plants absorb carbon dioxide through
photosynthesis, which allows them to store energy from sunlight in the form of
sugars and starches. After the biomass is converted into biodiesel and burned as
fuel, the energy and carbon is released again. Some of that energy can be used to
power an engine while the carbon dioxide is released back into the atmosphere.
Biodiesel from soy oil results in a 57% reduction in GHGs compared with fossil
diesel, and biodiesel produced from waste grease results in an 86% reduction (EPA
2010). According to a model developed by the Argonne National Laboratory
(ANL), neat (100%) biodiesel from soybeans can cut global warming pollution by
more than half relative to conventional petroleum-based diesel. The emissions
benefits are higher for canola oil. In the future, nonconventional sources like algae
may have the potential to provide dramatic (90%) reductions in global warming
pollution. However, significant technological hurdles remain before algae and
other advanced feedstocks can be processed into biodiesel for commercial
purposes.
The worldwide efforts have been put forward by different nations to tackle
GHG emissions. In that context, biodiesel is considered to be a key factor by most
of these nations (Lin et al. 2011). For example, biodiesel demand in Europe is likely
to increase in the near future because Europe has set targets to cut GHG emissions
by one-fifth by 2020, partly through demanding that one in 10 vehicles are fueled
by biofuels. In December 2008, European leaders agreed that 10% of all trans-
portation in the European Union should run on renewable energy by 2020
(Renssen 2011). This target is very beneficial and viable because more than half
of Europe’s cars run on diesel. Former US President Barack Obama announced in
2010 that the federal government will reduce its GHG pollution by 28% by 2020.
The Chinese government announced plans to control GHG emissions by 40% to
45% by 2020 from its 2005 level. The Chinese government also decided to help
African countries to develop their clean energy projects. The Japanese government
has a GHG emission reduction target of 60% to 80% by 2050 from its current level.
Moreover, India, Brazil, South Africa, and other countries have also set their GHG
reduction targets and development programs of substitute energy. Thus, biodiesel
industry is likely to have rapid and significant development in the near future.
23.6.2 Uncertainties about the GHG Emissions Savings

The impact of biodiesel on the environment is not necessarily reducing GHG
emissions compared with fossil fuels as often assumed. The development of the
biodiesel industry may directly or indirectly cause other negative effects on the
environment. The increased GHG emissions from converting rain forests, peat
lands, or grasslands to produce biofuels in the tropics and the United States
was 17 to 420 times larger than the GHG reductions these biodiesels could
provide by displacing fossil fuel use (Fargione et al. 2008). Biofuels were
considered to be environmentally friendly based on the theory that they only
emit as much as carbon as they absorbed during growth, but that has been
undermined by a new concept known as indirect land-use change (ILUC),
which scientists are still struggling to accurately quantify (Dunmore 2011).
According to this concept, it is estimated that switching the crops to biofuel
will increase food shortages. Moreover, most of the new farmland (80%) is
created by cutting down forests.
The European Environmental Agency’s (EEA) scientific committee said that
when energy crops take the place of forests, which would have stored more carbon,
they can actually lead to a net increase in carbon dioxide in the atmosphere
(Renssen 2011). According to ANL, biodiesel from soybeans can cut global
warming pollution; however, this model does not consider changes in land use.
When soybeans are used for fuel, they are taken out of the food market, which
increases prices and stimulates demand that farmers around the world respond to
by bringing more land into cultivation. Therefore, it is possible that the life cycle
global warming pollution of soybean biodiesel is even higher than petroleum diesel
once indirect land-use changes are considered. Therefore, biodiesel can be an
expensive option for reducing GHG emissions and improving energy security
because, to grow the biodiesel crops, additional land must be brought into
production. In June 2010, three Brussels-based nongovernmental organizations
(NGOs) issued a report that pointed out that harvesting trees for energy creates a
carbon debt. When forest is cut down and burned for energy, the carbon in the
trees is emitted right away, whereas new trees take years to grow back (Renssen
2011). Deforestation or clearing rain forests is required for biodiesel crop
plantation. These forests are one of the world’s largest carbon sinks. The clearing
of the rain forest and peat lands for biodiesel crops results in a sudden release of
large amounts of carbon dioxide. Clearing of rain forests can cause climate change
in some regions. Recent droughts observed in Southeast Asia have been directly
related to the clearance of many forests for energy crops by many Southeast Asian
countries. Rain forests can hold excess rainwater to release it in the dry season.
Because of large-scale deforestation, the water storage and conservation capability
of soil has been reduced. Haze caused by forest fires, where farmers in some
developing countries use fire to clear land for agriculture uses, is also a point of
concern. Overall, there is uncertainty about GHG savings if direct and indirect
land-use change is considered (Searchinger et al. 2008).
The European Commission denies that the EEA’s paper causes fresh pro-
blems for its biofuels policy. There is a complex ongoing debate over how to
account for the phenomenon called indirect land use change (ILUC) when judging
biofuel’s contribution to climate change (Renssen 2011). The effect of ILUC is
tricky to calculate. Scientists now think that ILUC wipes out any carbon-emissions
savings made by avoiding fossil fuels, as reports pointed out by the Commission’s
Joint Research Centre in Ispra, Italy, and by the International Food Policy
Research Institute (IFPRI) in Washington, DC (Noorden 2012). The ILUC debate
has occupied European policymakers for several years. ILUC refers to the
displacement of one use of land for another, as a result of energy crops being
grown somewhere else. In practice, ILUC is often the displacement of tropical
forest by food production because the former food croplands have been used to
grow energy crops. The result is the unintended release of GHG emissions as large
natural carbon sinks are destroyed. Figure 23-3 is a modified bar chart from
Renssen (2011) for biodiesel. The dashed and solid lines across the bars show the
120 Indirect land-use change emissions

Direct emissions
(g CO₂ equivalent per MJ)

Greenhouse gas emissions
100
80
60 35%
50%
40 threshold
20
0
Palm oil Soybean Rapeseed Sunflower
Biodiesel from different sources
Figure 23-3. Greenhouse gas emissions from direct and indirect land-use change
for different energy crops.
Source: Modified from Renssen (2011).
threshold for a 50% and 35% emission saving, respectively, compared with fossil
fuels. Initially biodiesels will have to deliver a 35% saving under the EU law, but
this will rise to 50% in 2017. Indeed, when policymakers talk about raising the
threshold in the context of the ILUC debate, they are reportedly talking about
raising it to 50%.
Figure 23-3 shows that according to what is known about the scale of ILUC,
this policy approach would not solve the problem. The European Commission
will officially accept such assessments when it makes legislative proposals on
biofuels and ILUC in 2012. When the commission recently reviewed its fuel
directive, it proposed classifying oil from tar sands in Canada as producing 107 g
of CO2-equivalent gases per mega joule (CO2eq/MJ) of energy—worse than crude
oil’s 87.5 g CO2eq/MJ. Leaked figures suggest that biodiesel from palm oil,
soybean, and rapeseed will be classified close to tar sands oil, at 105, 103, and
95 g CO2eq/MJ, respectively. The Institute for European Environment Policy
(IEEP) calculates that when ILUC is considered, the plans of EU countries for
biofuel use to 2020 would actually lead to between 81% and 167% more GHG
emissions than if fossil fuels were used instead (Bowyer 2011). ILUC has cancelled
out most of biodiesel’s climate benefits and, furthermore, diesel from EU rapeseed,
Asian palm oil, and South American soybeans turns out to be worse for the climate
than fossil diesel (Dunmore 2011).
The estimates in the literature for GHG mitigation from biodiesel vary
depending on the country and pathways (Sims et al. 2010). Most analyses indicate
that biodiesel shows at least some net benefit in terms of GHG emissions reduction
and energy balances. However, because of the limited scope for cost reductions
and the increasing global demand for food and fiber, little improvement in these
mitigation potentials can be expected in the short term. Consequently, there is
need to find a way to address the GHG balance of using biodiesel for fuel. For
addressing GHG emissions the life cycle assessment (LCA) has been applied to
different biofuels with varying results (Andreoni et al. 2004). LCA of GHG balance
is complex; all factors can have a considerable influence on results, including
planting and harvesting of crops; fertilizer and pesticide use; irrigation technology;
soil treatment; processing the feedstock into biodiesel; transporting the feedstock
and final fuel; and storing, distributing, and retailing the biodiesel (Lin et al. 2011).
23.7 PUBLIC SAFETY AND HEALTH
Recent rapid expansion of the biodiesel industry and projected continued trends
in the near future have raised concerns about associated impacts on public safety
and health (Swanson et al. 2007, McKone et al. 2011, Ridley et al. 2012, Scovronick
and Wilkinson 2014). Biodiesel may impact human health through a range of
pathways such as occupational hazards; increased food prices affecting overall
nutrition; and water, soil, air pollution during the production as well as by using
the biodiesel. In general, biodiesel is expected to improve air quality up to a certain
level, especially in urban areas. However, it is important to evaluate all the health
impacts associated with biodiesel production and the use of biodiesel. Given the
uncertainties about health impacts and the multiple ways in which biodiesel
production might influence them, there is a need for further life cycle assessments
(Phalan 2009). Assessment of health impacts of biodiesel at all stages in its life
cycle can make a significant contribution to the development of biodiesel and/or
agricultural policies.
23.7.1 Occupational Health Hazards

Human health may be affected by occupational risks involved at various stages of
biodiesel production and its use, such as exposure to hazardous chemicals used in
the production process, exposure to water/air pollution caused at different stages of
biodiesel life cycle, exposure to carcinogens, high levels of noise, and the possibility
of injury. More than 800,000 deaths/year are reported recently from occupational
risks (Lim et al. 2012). In general, there is some degree of occupational health risk in
conventional fuel industries, and the agricultural sector usually has one of the
highest rates of injury and disease (Takala 1999, Sumner and Layde 2009). Common
causes for injuries in the agricultural sector where manual harvesting is practiced
include those resulting from strenuous and repetitive movements, exhaustion,
poisoning, burns, and accidents with machetes or heavy machinery. Some biodiesel
crops themselves are toxic, for example, jatropha fruits contain irritants, which can
affect laborers (Achten et al. 2008). Modernizing the agricultural sector can reduce
these health hazards in the agriculture sector. Although injury and death rates in the
agricultural sector are high, the same is true of fossil fuel extraction, and therefore
substituting biodiesel for fossil fuels may not lead to a meaningful change in
occupational injury burdens (Sumner and Layde 2009).
During the biodiesel processing many biochemical and chemicals are used.
Exposure to these biological and chemical reagents used in biodiesel is another
concern. Biodiesel production involves reacting lipids with methanol using a

catalyst such as caustic sodium or potassium hydroxide to produce biodiesel and
glycerol (Swanson et al. 2007, Law et al. 2011). These chemicals are associated with
a range of health issues (Swanson et al. 2007). Other noted risks to workers
involved in biodiesel production include those from air pollution, explosions and
fires, and falls (Gunderson 2008, Law et al. 2011). Release of H2S is very hazardous
for human health. Inhalation, dermal contact, or consumption can bring water
and soil pollutants into contact with humans. Infectious, pathogenic, or chemical
nature of pollutants along with exposure time determines the health impacts. Such
impacts range from skin irritation to a carcinogenic level (Scovronick and
Wilkinson 2014). There is need for health effect research and hygiene standards
to stay current considering that the technologies in the industry are evolving
rapidly with increased investment and as future-generation biodiesel becomes
viable (Gunderson 2008).
Outside of the biofuel industry, minimizing exposure to diesel exhaust has
been highlighted as a priority in certain industries, because occupational exposures
can be orders of magnitude higher than ambient concentrations (Cantrell and
Watts 1997). There has been sustained interest in the potential benefits of using
biodiesel at work sites to reduce exposure, although results are mixed. For
example, a study at a small recycling center in the United States found substantial
reductions in fine particulate matter (PM2.5) and some, but not all, air toxics when
using 20% biodiesel (B20) compared to petroleum diesel (Traviss et al. 2010). An
experiment on the impact to air pollution of switching to B50 or B100 (50% and
100% biodiesel, respectively) in mine shaft operations in general showed positive
results for PM concentrations, particularly for B100, although results were
strongly dependent on vehicle operating conditions (Bugarski et al. 2010). Some
of the benefits were offset by an increase in the fraction of particle-bound VOCs.
23.7.2 Impact of Soil Pollution on Health

Exposure to soil pollutants can occur from consumption, inhalation, or dermal
contact. Health impacts depend on the type of pollutant (pathogenic or chemical)
as well as the extent of exposure. Health concerns from exposure to soil pollution
range from skin irritation and poisoning to exposure to infectious agents and
carcinogens (Scovronick and Wilkinson 2014).
Growing a crop for biodiesel feedstock does not normally differ from growing
the same crop for food. In the United States, row crops like corn have some of the
highest rates of soil erosion and nutrient application (Dominguez-Faus et al.
2009). In terms of relative potential for soil pollution, a US analysis reported that
soy-based biodiesel releases only a fraction of the nitrogen, phosphorous, and
pesticides of corn ethanol per unit energy yield (Hill et al. 2006). A more recent
analysis similarly reported relatively lower nitrogen requirements for soybean
biodiesel compared to ethanol from either corn or switch grass (used for second-
generation ethanol), but confidence intervals were wide (Dominguez-Faus et al.
2009). Of the three, however, soybeans had the highest pesticide use and switch
grass the lowest. There is still some debate about fertilizer and pesticide use for
second-generation biofuel feedstock crops (e.g., switch grass) because there is little
precedent for managing them as cash crops (Dominguez-Faus et al. 2009).
The soil simulations on spills from different biodiesel blends also showed that
diesel contaminants produced cytotoxic effects, whereas components of the
biodiesel produced genotoxic and mutagenic effects (Leme et al. 2011, 2012).
There is a scarcity of information on epidemiological studies to directly link
biodiesel industry human health (Scovronick and Wilkinson 2014). Proximity to
land contaminated by petroleum fuels has been investigated and associated
with, for example, increased incidence of cancer and adverse birth outcomes
(San Sebastian et al. 2001, 2002). Any hazardous components of petroleum fuels
would also be present in biodiesel blends.
23.7.3 Impact of Water Pollution on Health

Similar to soil, exposure of water pollutants can occur from consumption,
inhalation, or dermal contact. Health impacts depend on the type of pollutant
(pathogenic or chemical) as well as the extent of exposure. Health concerns from
exposure to water pollution range from skin irritation and poisoning to exposure
to infectious reagents and carcinogens (Scovronick and Wilkinson 2014).
In general, growing a crop for biodiesel feedstock does not normally differ
from growing the same crop for food. The water quality is more likely to be
affected through the large-scale cultivation of biodiesel crops. However, bringing
new areas of land into production of biodiesel crops and intensifying production
on existing croplands are both likely to increase nitrate, phosphate, and
pesticide runoff into waterways. Grave environmental and human health effects
are linked with agricultural industries comprising water pollution from ferti-
lizers, use of various agrochemicals to protect the crop from pests, discarding
unsatisfactorily treated wastewater from the processing plants, soil erosion,
among others.
Comparatively low nitrogen input is required for soybean biodiesel matched
with ethanol from corn or switch grass (Dominguez-Faus et al. 2009). However,
soybeans had the highest pesticide use and switch grass the lowest. The inorganic
fertilizers and pesticides used on biodiesel crops enter surface or subsurface water
bodies and can pose a serious threat to human health. Risk factors mostly include
the carcinogenic characteristics of agrochemicals (McKelvey et al. 2004, Brody
et al. 2006). Some pesticides have been identified as endocrine disruptors.
Endocrine systems of mammals (birth defects and infertility), diabetes, cancer,
and even behavioral changes are caused by estrogenic activity of agrochemicals,
especially pesticides. Chlorine derivative pesticides can cause cancer (Vieira et al.
2008). Nitrates (soluble in water, a macronutrient of the plants, originated from
nitrogen, and provided by inorganic fertilizer and animal manure) are the primary
form of nitrogen and are present in the groundwater of agricultural lands because
they easily pass through soil to the groundwater table. Nitrates are very stable and
can persist in groundwater for decades, so they may accumulate to a high level and
promote risk of many types of cancer in human stomach and colon (Ward et al.
2005, Irigaray et al. 2007). Newborns, especially below 6 months of age, are more
vulnerable. Pesticide contamination has also been associated with breast cancer
(O’Leary et al. 2004, Brody et al. 2006, Mittal et al. 2013).
Biodiesel spills during storage and distribution is another source of contami-
nation. Attention has focused on potential adverse effects of exposure to con-
taminants in the biodiesel, but also on the possibility that the properties of the
blends could intensify exposure to hazardous compounds found in the petroleum-
based fuels, for instance, by acting as a dispersant, prolonging persistence in the
environment, or increasing solubility (Scovronick and Wilkinson 2014). Aquifer
keeps distinct water quality that is disturbed by the nitrates, potassium, chloride,
calcium, and magnesium that originated from the aforementioned pollutants.
Leme et al. (2012, 2011) conducted the simulations on spills from different
biodiesel blends and found that the addition of biodiesel reduced the amount of
polyaromatic hydrocarbons released into the water. Only B0 and B5, the blends
with the lowest biodiesel content, produced cytotoxic effects and the authors
therefore attributed toxicity to the diesel constituents. They did not find evidence
that biodiesel worsened the impact of the diesel contaminants. Proximity to water
contaminated by petroleum fuels has been associated with increased cancer and
adverse birth defects (San Sebastian et al. 2001, 2002). Any hazardous components
of petroleum fuels would also be present in biodiesel blends.
In addition to water pollution, biodiesel expansion may contribute to water
scarcity. However, the way potential biodiesel-induced water scarcity may impact
health has not been yet quantified (Scovronick and Wilkinson 2014).
23.7.4 Impact of Air Pollution on Health

Air pollution is one of the most important exposures in terms of population health
and therefore is the most well-studied exposure with regard to biodiesel. Air
pollution occurs at all stages in the biodiesel life cycle (Scovronick and Wilkinson
2014). Ambient outdoor air pollution is associated with increased morbidity and
mortality from cardiovascular and respiratory diseases, certain cancers, and with
all-cause mortality. The strongest effects are normally associated with PM, which
is a heterogeneous mix of solid and/or liquid particles suspended in the air, but
other pollutants of public health concern include ground-level ozone, carbon
monoxide, SO2, oxides of nitrogen, and various air toxics such as benzene and
acetaldehyde. The mortality burden from outdoor air pollution was recently
estimated at more than three million deaths annually (Lim et al. 2012).
Burning land for biodiesel crop production can contribute to smoke pollu-
tion. Although legislation is phasing out the burning of land in most countries, it is
still common practice in many areas, enabling easier clearing of fields of
undesirable wildlife. The biomass burning is the predominant source of particulate
matter during the burning season and it increases a toxicological risk during the
burning season, particularly in younger children and asthmatics. It may also lead
to higher disruptions in mucociliary clearance in children and adolescents, living
near burning sites, compared with a control site with no burning (Sisenando et al.
2012). Mucociliary clearance is involved in removing inhaled particles and
microorganisms from the respiratory system. The increased cases of respiratory
issues and morbidity have been associated with burning biomass (Scovronick and
Wilkinson 2014). Important unresolved issues include understanding how such
pronounced exposure seasonality influences long-term health effects; why biomass
burning sometimes shows a comparatively strong respiratory but weaker cardio-
vascular effect when compared to urban emissions (Arbex et al. 2010); and
determining to what extent biomass burning induces new disease rather than
exacerbates existing conditions.
The effect on vehicle emissions of switching from fossil fuels to biodiesels or
blends is not straightforward. The effect of adopting biodiesel is influenced by the
vehicle studied (age and type), the operating conditions, the blend ratio, and
possibly to the biodiesel’s feedstock (Anderson 2012). Lower-proportion blends
tend to show emissions benefits with regard to PM10, hydrocarbons, and carbon
monoxide, but increases in oxides of nitrogen (Scovronick and Wilkinson 2014). An
important feature of lower-proportion biodiesel blends is the fuel’s high volatility,
leading to evaporative emissions that may contribute to increased ozone-forming
potential. Very high ambient concentrations of certain VOCs can be partially
attributed to the combustion and evaporation of biodiesel. A last factor to note is
that differences in health risks for PM do not depend entirely on the level of
emissions but also on their composition and size, which may differ for different fuel
blends. For example, Hemmingsen et al. (2011) recently found that particles emitted
from 20% biodiesel blends were larger and equally or less potent than those from B0.
Another recent study reported lower PM2.5 emissions from cooking oil-derived
B100 and B50 compared to B0 but suggested that the toxicity and oxidative stress for
the biodiesel blends was higher, at least for certain engine loads (Betha et al. 2012).
Morris et al. (2003) found reductions in premature mortality from air toxics of
around 2% and 5% assuming 50% and 100% penetration of B20, respectively, in the
heavy-duty diesel fleet in southern California. They also found that the changes in
ambient concentrations of PM and ozone were very small.
Air pollution occurs at all stages in the biofuel life cycle, not only from
biomass burning and vehicle tailpipe emissions. However, there is scarcity of
information on contribution to air pollution from other stages. Existing air quality
modelling outlines the importance of a life-cycle approach, showing that life cycle
emissions of some pollutants may be higher for biodiesels when compared to
conventional fuels, largely owing to the emissions associated with feedstock
production and biofuel processing (Scovronick and Wilkinson 2014). However,
these upstream stages mainly occur in rural locations, whereas any vehicle
emission benefits that accrue will be experienced in more population-dense areas,
suggesting spatial variation in health impact. Life cycle health impact modelling
has been identified as one of the challenging areas in biofuel research (McKone
et al. 2011).
Health impact studies can offer some valuable insights into possible health
burdens under specific biodiesel scenarios, but the results are difficult to generalize
because of the very small number of studies, different pollutants studied, and the
different health outcomes assessed. The studies also tend to emphasize air
quality modelling rather than health impact calculations. Therefore, a better
understanding of the likely health burdens associated with vehicle emissions

requires additional policy scenarios as well as more comprehensive health impact
methodologies, particularly when considering the complexities of biofuel-related
emissions identified in the literature: co-varying risk factors, influences on
multiple related health outcomes, and impacts on diseases with long induction
periods (e.g., cancer).
23.7.5 Impact of Food Security Issues on Health

Biodiesel production has the potential to influence food security mainly through
potential reduction of food supplies and potential increases in food price
(Scovronick and Wilkinson 2014). Recent estimates suggest that undernutrition
is responsible for 45% of all child deaths, and diet-related risk factors (including
physical inactivity) account for 10% of the total global burden of disease (Lim et al.
2012). Possibly the fiercest criticism of biodiesels revolves around claims
that biodiesel production will compromise food security and contribute to
undernutrition. However, biofuel feedstock crops currently occupy only around
2% of cropland globally (Fargione et al. 2008), and if it doubles or even triples, as
some have predicted (FAO 2008), proportional reductions in food supply
would still not create global shortages. Global per capita food supply is already
above recommended intake levels and is expected to increase in the coming years
(FAO 2006).
Undernutrition is much less a problem of food supply than of food distribu-
tion, and therefore the remaining question is whether and how biofuels affect the
latter. Complex linkages between the agricultural and energy markets make it
possible for biodiesel production to influence the price of food and hence, the food
distribution. Biodiesel demand can increase the price of the feedstock crops and
also the prices of other food commodities as markets adjust (Scovronick and
Wilkinson 2014). The adverse impacts of higher food prices could be offset to
some degree by increases in wages for those involved in the biofuel or agricultural
industries or by policies aimed at absorbing price shocks (Ewing and Msangi
2009). Higher food prices can adversely affect the intake of foods and key nutrients
in low-income populations (Rosegrant et al. 2008). The increased biofuel produc-
tion in line with current production targets would lead to between 80 and
140 million additional undernourished people by 2020 when compared to a
scenario of no extra biofuel growth (Fischer et al. 2009). However, at present there
is not enough quantitative and direct evidence linking changes to food security
and/or undernutrition (Scovronick and Wilkinson 2014). The eventual market
penetration of future-generation liquid biofuels will likely reduce pressure on food
prices and undernutrition, because these fuels should not need to compete for
cropland or food crops to the same degree as first-generation biofuels (Rosegrant
et al. 2006, Fischer et al. 2009). The issues around food production and food price
can also impact the health of wealthier populations. Food prices may affect food
purchasing decisions and consumption in even the richest countries. However,
there is lack of information on this aspect, although many health issues are linked
with dietary habits in wealthy countries or populations (Lim et al. 2012).
23.7.6 Impact of Indirect Pathways on Health

Indirect pathways to health associated with biodiesel production and use have
potentially high population burdens and could possibly outweigh those from some
exposures already discussed. The first relates to concerns about the impact of land-
use change on health. Biofuel-induced land-use change has been documented

empirically, and modelling studies suggest the likelihood of direct and indirect
future land-use impacts (Wicke et al. 2011). How land-use change influences health
is a relatively new research area in many respects, but studies have linked it to
infectious diseases in various habitats and to changes in air quality (Foley et al. 2005).
The development of a biodiesel industry alters employment patterns, local
wealth production, and in some circumstances may affect national-scale economic
performance (Ewing and Msangi 2009). These factors may have significant effect
on health. Increased incomes are often strongly associated with population health,
although the exact nature of the relationship is complex and dependent on factors
including the level of development, aspects of income inequality, and the choice of
the health metric (Deaton 2007, 2003).
Many biofuel programs and policies have been implemented to help mitigate
anthropogenic climate change, which is expected to have an adverse impact on
health overall (IPCC 2007). The mitigation potential of different biofuels is fiercely
debated in the literature. The direct impact on greenhouse gases of most biofuels
usually seems favorable when compared to fossil fuels, although indirect impacts
(e.g., those from land-use change) may compromise these benefits (FAO 2008,
Fargione et al. 2008). Second-generation biofuels are likely to fare better in this
regard (FAO 2008), but liquid biofuels appear unlikely to be a major contributor
to mitigation targets overall, at least when compared to many other potential
mitigation options (FAO 2008).
23.7.7 Safe Handling

Biodiesel manufacture is a potentially dangerous industrial process, and therefore
it has mandatory guidelines for safety. Although biodiesel is safer than conven-
tional diesel fuel, there are certain safety precautions to consider during the
production of biodiesel. Biodiesel is nontoxic and biodegradable (NREL 2009,
Leme et al. 2012). Thus, biodiesel creates lesser environmental or health issues in
the case of accidental release or spillage. Important biodiesel properties from safe
handling and a health point of view are discussed in this section. The properties of
biodiesel fuel produced vary with its production feedstock (Table 23-8).
Countries that have adopted biodiesel stipulate their own specifications for
biodiesel. Table 23-9 illustrates the biodiesel properties. The density of the
biodiesel is similar to that of diesel, so it can mix excellently with diesel and
does not require new refueling stations.
23.7.7.1 Biodiesel Toxicity

According to NREL, biodiesel contains no hazardous materials and is usually
regarded as safe. The acute oral lethal dose (LD50) of biodiesel is greater than
Table 23-8. Some Properties of Vegetable Oils Commonly Used in Biodiesel Production.
Vegetable oil Kinematic viscosity Density Cetane Flash point Cloud point Pour point
type (mm2/s, at 40 °C) (g/cm³, at 21 °C) number (°C) (°C) (°C)
Soybean 33.1 0.914 38.1 254 −3.9 −12.2

Rapeseed 37.3 0.912 37.5 246 −3.9 −31.7
Sunflower 34.4 0.916 36.7 274 7.2 −15.0
Corn 35.1 0.910 37.5 277 −1.1 −40.0
Safflower 31.6 0.914 36.7 246 −3.9 −31.7
Cottonseed 33.7 0.915 33.7 234 1.7 −15.0
Peanut 40.0 0.903 34.6 271 12.8 −6.7
Tallow 51.2 0.920 40.2 201 — —
635
Table 23-9. Biodiesel Specifications.

636
Country/biodiesel Petroleum
properties United States Europe Germany Canada Japan Australia Brazil China India diesel
Density at 15 °C — 0.86–0.9 0.875–0.9 0.86–0.9 0.86–0.9 0.86–0.90 0.82–0.90 0.86–0.90 0.8

(g/cm3)
Flash point (°C, min) 100 120 110 >40 °C 120 120 100 130 120 54
Water and sediments 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
(% vol. Max.)
Water content — — 300 500 500 500 —
(mg/kg, max)
Solid matter — — 20 10–16 24 24 None 24 —

(mg/kg, max)
Kinematic viscosity at 1.9–6.0 3.5–5.0 3.5–5.0 1.3–4.1 3.5–5.0 3.5–5.0 3.5–5.0 1.9–6.0 3.5–5.0 2.3
40 °C (mm2/sec)
Ash (% wt., max) — 0.03 0.01
Sulfated ash 0.02 0.02 — 0.02 0.02 0.02 0.02 0.02 —
(% mass, max)
Sulphur 0.0015 0.001 0.001 0.005 0.001 0.005 0.005 0.05–0.005 0.005 IB
(% mass, max)
Copper strip Class 3 Class 1 No. 1 Class 1 No 3 Class 1 1 Class 1 IB
corrosion, max.
Cetane, min 47 51 49 40 51 51 45 49 51 50
Free glycerin 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 —
(% mass, max)
Total glycerin (% 0.24 0.25 0.25 0.025 0.25 0.25 0.25 0.24 0.25 —
mass, max)
References Leung (2001), Goto and Shiotani (2007), BBFSEAWG (2010)
17.4 g/kg body weight. In comparison, table salt (NaCl) is nearly 10 times more
toxic (CLF 2013). Biodiesel does not smell as bad as regular diesel fuel when it
burns; sometimes biodiesel exhausts smells like French fries. A 24 h human patch
test indicated that undiluted biodiesel produced very mild irritation. The irritation
was less than the result produced by a 4% soap and water solution. A 96 h
lethal concentration for bluegill of biodiesel-grade methyl esters was greater than
1,000 mg/L. Lethal concentrations at these levels are usually deemed insignificant
according to National Institute for Occupational Safety and Health (NIOSH)
guidelines in its Registry of the Toxic Effects of Chemical Substances (RTECS
1997).
23.7.7.2 Biodiesel Biodegradability

Biodiesel has desirable degradation attributes, which is why it is considered a
nonpolluting fuel. Several studies found that biodiesel biodegrades much more
rapidly than conventional diesel (DeMello et al. 2007). Biodiesel degrades about
four times faster than petroleum diesel. Studies at the University of Idaho
compared the biodegradation of biodiesel in an aqueous solution to diesel fuel
and dextrose (sugar). Biodiesel samples degraded more rapidly than dextrose and
were 95% degraded at the end of 28 days. The diesel fuel was approximately 40%
degraded after 28 days. Blending biodiesel with diesel fuel accelerates its biode-
gradability. For example, blends of 20% biodiesel and 80% diesel fuel degrade
twice as fast as No. 2 diesel. Thus, biodiesel use has demonstrated biodegradability
benefits at levels lower than 100%. Simply stated, neat biodiesel degrades as fast as
sugar, and a B20 blend will degrade twice as fast as petroleum-based diesel fuel.
Users in environmentally sensitive areas such as wetlands, marine environments,
and national parks have taken advantage of this property by replacing toxic
petroleum diesel with biodiesel.
DeMello et al. (2007) reported that in the event of a biodiesel mixture spill,
FAMEs will be consumed by bacteria, and samples from a contaminated area may
be indistinguishable from a conventional fossil diesel spill after only a short period.
They also suggested that FAMEs will not affect the rate of evaporation of
petroleum hydrocarbons. In addition, FAMEs themselves will likely biodegrade
before evaporating from spilled biodiesel. The physical properties of FAMEs may
alter the environmental behavior of petroleum hydrocarbons. FAMEs will stabilize
biodiesel droplets in the water column, and this may influence the transport,
weathering rate, and ecological impact of spilled biodiesel. By stabilizing small oil
droplets in the water column, FAMEs might also enhance dissolution rates of
conventional hydrocarbons (DeMello et al. 2007).
23.7.7.3 Biodiesel Flash Point

The flash point of a fuel is defined as the lowest temperature at which the vapor
above a combustible liquid can be made to ignite in air. Biodiesel’s flash point is
greater than 260 °F (126 °C), well above petroleum-based diesel fuel’s flash point
of around 125 °F (51 °C) (NREL 2009). Testing has shown the flash point of
biodiesel blends increases as the percentage of biodiesel increases. Therefore,
biodiesel and blends of biodiesel with petroleum diesel are safer to store, handle,
and use than conventional diesel fuel.
Fire safety precautions must be taken as for the case of any fuel. No placards
or warning signs are required for the transport of pure biodiesel. However,
biodiesel blends with diesel and kerosene are required to be transported in

placarded trucks if the flash point of the blend is lower than 200 °F (93 °C),
according to federal department of transportation (DOT) regulations. Biodiesel
blends are of great concern because they may contain kerosene or petroleum diesel
(NREL 2009). The flash point of biodiesel is higher than 212 °F (100 °C), so it is
considerably less dangerous. However, biodiesel blends will have flash points
intermediate to the two liquids. Kerosene is highly flammable with a flash point of
100° to 162 °F (38 to 72 °C) and conventional petroleum diesel fuel is also
highly flammable with its flash point of 126° to 204 °F (52 to 96 °C). The US DOT
considers a blend flammable and the Resource Conservation and Recovery Act of
1976 (RCRA) considers it to be ignitable if the flash point is lower than 140 °F
(60 °C) or combustible if the flash point is 140° to 200 °F (60 to 93 °C) (per DOT).
Pure biodiesel can be extinguished with dry chemical, foam, halon, CO2, or
water spray, although the water stream may splash the burning liquid and spread
fire. Oil-soaked rags can cause spontaneous combustion if they are not handled
properly. Before disposal, oil-soaked rags must be washed with soap and water
and then left for dry in well-ventilated areas. Because biodiesel could burn if
ignited, it must be always kept away from oxidizing agents, excessive heat, and
ignition sources.
23.7.7.4 Biodiesel Safe Handling and Storage

Safety should be the top priority in any biodiesel operation, above all other goals.
Accidents involving chemicals and or large volumes of vegetable oil, biodiesel, or
byproducts can cause injury, loss of life, property damage, or environmental
contamination. A comprehensive approach to safety begins with a whole-system
consideration of all potential areas for risk, followed by thorough plans for
accident prevention such as spontaneous combustion. All the problems associ-
ated with processing biodiesel can be handled with a properly engineered design
and standard practices for ventilation, methanol fumes, and make-up air,
washing fuel, prewashing fuel for soap reduction, drying fuel, and waterless
soap removal, and for safe handling of chemicals such as methanol, NaOH, and
KOH.
Biodiesel can be stored and handled by the same standard storage and
handling procedures used for petroleum diesel. Biodiesel should be stored in a
clean, dry, dark environment, and standard safety practices should be in place for
oil collection, chemical storage, and labeling stored fluids. Recommended materi-
als for storage tanks include aluminum, steel, polyethylene, polypropylene, and
Teflon, but not concrete-lined storage tanks. Storage tanks should not include any
copper, brass, lead, tin, zinc, or rubber fittings if possible (practically speaking,
brass ball valves are used by many with no major ill effect). Because biodiesel is an
organic liquid, the use of an algaecide or fungicide additive is recommended
whenever the biodiesel is stored during warm weather. Storage time for biodiesel
and petroleum diesel should be limited to 6 months for best performance (Steiman
et al. 2008).
23.8 CHALLENGES
The energy content of the biodiesel is approximately 11% lower than that of
petroleum diesel (Radich 2004). Biofuels have been accused by the United Nations
and others of pushing up world food prices and exacerbating the effect of the most
severe drought in the United States in half a century. It has been recently claimed
by German researchers that European-produced biodiesel does not meet the
sustainability targets (Macalister 2012). The researchers (Friedrich Schiller Uni-
versity in Jena), also observed that eight of the 10 tests on locally produced
rapeseed biodiesel failed to show the 35% GHG savings promised. In most cases it
was less than 30%. Pehnelt and Vietze (2012) also claim their work has been
undermined by a lack of cooperation from the European Union, which they
believe is on the defensive over championing local energy crops. Water resources,
soil pollution, emissions, greenhouse gases, and land use have been well-repre-
sented in the literature, whereas biodiversity and human health were not. There is
lack of information about these topics in some parts of the world such as the
Southern Hemisphere where the greatest potential socioeconomic benefits, as well
as environmental damages, may occur.
The production of biodiesel should be further perfected to promote biodiesel
properties and quality. The further development in additives to improve con-
sumption of biodiesel could favor power recovery, economy, and emissions,
especially for NOx emissions. Carbonyl compounds emissions have discordant
results for biodiesel, although it is widely accepted that biodiesel increases these
oxidants emissions because of higher oxygen content. Further studies on non-
regulated emissions of biodiesel should be carried out to determine a conclusive
trend, especially for the carbonyl compounds emissions. The methodology or the
instrumentation used for measurements needs to be improved to fulfill the
expected requirements.
23.9 SUMMARY
The biodiesel industry should pursue more interdisciplinary research to assess

complex global environmental and health concerns. Currently there is very
scattered information available about the sustainability of biodiesel production
in many parts of the word because of noncommercialization of the same in those
countries. It can be concluded from the limited literature that the use of biodiesel
favors reduced carbon deposit and wear of the key engine parts compared with
diesel. It is attributed to the lower soot formation, which is consistent to the
reduced PM emissions of biodiesel and the inherent lubricity of biodiesel. Most
studies have shown that PM emissions for biodiesel are significantly reduced
compared with diesel. The higher oxygen content and lower aromatic compounds
have been regarded as the main reasons. The vast majority of studies agree that
NOx emissions will increase when using biodiesel, mainly caused by higher oxygen
content for biodiesel. Moreover, the cetane number and different injection
characteristics also have an impact on NOx emissions for biodiesel. It is commonly
accepted that CO emissions reduce when using biodiesel because of the higher
oxygen content and the lower carbon-to-hydrogen ratio in biodiesel compared
with diesel. It is the predominant viewpoint that HC emissions reduce when
biodiesel is fueled instead of diesel. This reduction is mainly contributed to the
higher oxygen content of biodiesel, but the advance in injection and combustion of
biodiesel also favor lower total hydrocarbon emissions (THC) emissions.
Inconsistent conclusions exist on biodiesel-associated CO2 emission. Some
research indicated that the CO2 emission reduces for biodiesel as a result of the low
carbon-to-hydrocarbons ratio, but others showed that the CO2 emission increases
or remains similar because of more effective combustion. In any event, the CO2
emission of biodiesel reduces greatly from the view of the life cycle circulation of
CO2. Most research showed that aromatic and polyaromatic compound emissions
for biodiesel reduce with regard to diesel. It can be concluded that the blends of
biodiesel with small content by volume could replace diesel to help in controlling air
pollution and easing the pressure on scarce resources to a great extent without
significantly sacrificing engine power and economy.
Increased biodiesel crops require large amounts of water, and thus there will
be an increased pressure on natural water resources. Water quality issues are likely
to be increased from manufacturing biodiesel as well as the contamination of
water resources from the biodiesel crops. Increase in irrigation, use of fertilizers
and pesticides, and soil erosion from land activities are the future challenges for
maintaining the productivity and quality of the soil. Deforestation and conversion
of food agricultural lands into fuel crops may occur in biodiesel producing
countries. Therefore, there is a concern about the imbalances in biodiversity as
well as global food reserves. There is inconsistency in GHG emissions resulting
from biodiesel production. Indirect land use has been the most critical parameter
while judging the global warming contributions of biodiesel production. The
speculative nature of a reduction in health effects based on chemical composition
of biodiesel exhaust needs to be followed up with investigation in biologic systems.
Sincere thanks to the Killam Trusts for providing the Killam Postdoctoral
Fellowship to Tanaji More at the University of Calgary, Schulich School of
Engineering, Calgary, Alberta, Canada. We also thank the Natural Sciences and
Engineering Research Council of Canada (Grant A 4984, Canada Research Chair)
for their financial support. Views and opinions expressed in this chapter are those
of the authors.
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CHAPTER 24
Biodiesel: Socioeconomic
and Political Aspects

H. Panidepu
S. K. Ram
V. Cheernam
R. D. Tyagi
24.1 INTRODUCTION
Biodiesel has been promoted on the premise that they are replenishable; could
reduce carbon emissions, increase farmer income, improve energy security, and
create new jobs; and they have properties similar to oil (Deepak Rajagopal 2008).
However, this view is like the sociologist Max Weber’s concept of Ideal Type,
wherein a one-to-one mapping is done and other factors that contribute to this
development or what other implications this development entails are ignored. In
this chapter we shall look into some of the issues beyond technology in the realm
of economics, politics, and sociology; into the ideology of different sovereign
countries and their take on biodiesels; the international fora and their mulling over
proliferation of biodiesel technology; the oil sector and its humungous power; the
social and environmental developments occurring with biodiesel development;
and most important, the implications for a common man and to a community as
the consumer/producer/observer of the biodiesel production phenomenon.
24.2 POLICY IMPETUS TO BIODIESEL PRODUCTION IN VARIOUS

COUNTRIES
The European Union in 2005 released the Biomass Action Plan outlining 20 actions
to stimulate the development and diffusion of bioenergy in Europe. Increasing jobs,
curbing over-dependency on oil, and bringing oil prices down have been its major
objective. In 2008, the Renewable Transport Fuel Obligation (RTFO) became the
653
first policy instrument formulated to promote biofuels for transport to reduce

greenhouse gas emissions (GHGs). Similarly in 2015, the European Parliament
voted to approve a new legislation so that the member states can meet the target of
10% of their transport fuel requirements from renewable components by 2020.
Countries like Germany have been promoting biodiesel since 2004 with
policies of tax exemptions. Later in similar lines with the EU policies, in 2006,
Germany mandated the blending of biofuels with other fossil fuels. The blending
was benchmarked in terms of calorific value and not volume or any other
parameter. Minimum blending percentages for various fuels (petrol, diesel) have
been stipulated. Through the years until 2010, the blending quotas have been
increasing and reached a maximum of 6.25% of calorific value (the amount of heat
evolved per kilogram of substance). Not adhering to these blending quotas
attracted penalties, whereas sticking to those norms could exonerate the compa-
nies from paying heavy tariffs. Thus, Germany has been simultaneously incentiv-
izing and necessitating the use of biofuels. Currently, GHGs are the new
benchmark in Germany rather than calorific value (Sievers and Schaffer 2016).
Switzerland has the concept of preferential tax status, which confers tax
rebates only to a few selected biodiesel producers who have a better environmental
rating rather than everyone who produces biodiesel. In other words, those who
produce biofuels from waste products, waste oils, or woody biomass only shall be
encouraged by the policy of preferential tax status (Zhao 2015).
In Brazil, the National Programme for the Production and Use of Biodiesel
(PNPB) started in 2004 by the federal government to put the biodiesel production
into an organized framework. Apart from providing conduciveness by a policy in
place, it also mentions social protection to farmers in terms of sustainable income
to farmers, encouraging nonagricultural crops like oilseeds in fallow lands. Many
farmers are part of PNPB, thereby increasing their per capita income, and now
there is a diversified production of feedstocks for biofuel production like canola,
soybean, and castor beans, among others. A law was enacted in 2005, following
which it is mandatory to blend 5% (v/v) biodiesel with 95% petroleum diesel, the
mixture being called B5. A program called Social Fuel Seal was envisaged by the
Brazilian government whereby the producers of biodiesels and the producers of
raw materials like small farmers and households were closely linked to each other.
Under this program, the farmers get a better price for their products and have
more price negotiation capability because they are assisted by intermediaries like
family farmer cooperatives. Thus, Brazil has been supporting the growth of
biodiesel production with quality assurance being checked at each level of
production and also with an agenda to fend off social inequalities (Ubrabio 2007).
Since 2005 in Canada, there has been mandation to blend biofuels with
conventional oil. With the exception of British Columbia’s low carbon fuel standard
and Ontario’s green diesel requirements, all the renewable fuel standards of provinces
have been met. Scientific studies also indicate reduction in carbon emissions.
Qualitatively, renewable fuel standards and low-carbon fuel standards have reduced
annual carbon pollution in 2014 by 4.3 Mt CO2 eq (equivalent to taking one million
cars off the road) and have promoted biofuel use to 3.9 million m3, equivalent to
BIODIESEL: SOCIOECONOMIC AND POLITICAL ASPECTS 655
5% of all gasoline and diesel use in Canada. Canada is looking forward to significantly
increase its biofuel capacity by 2050 (Moorhouse 2016).
In China, research in biodiesel started in 1930. The renewable energy law of
the People’s Republic of China was a most important decree in the country, aiming
at utilization and promotion of renewable energy resources. The National

Development Reform Commission (NDRC) has set a goal that China should
meet the production target of 12 million tons of biofuels by 2020, accounting for
15% of transportation fuel needs. There is considerable emphasis on cellulose-
based biofuels because there is financial support from the government in terms of
reduction of tax and other procedural fees. Similarly, since 2011, there is waiver of
5% consumption tax for biodiesel producers who use cooking oils not fit for
human consumption or non-lipid oils from animal slaughterhouses (Zhao 2015).
In Japan, according to the Kyoto protocol, which called for reduction of its
GHGs by 6% of its 1990 level, the Japanese government has formulated a policy
called Global Warming Countermeasures Basic Bill, which plans to increase the
share of biofuels and renewables to 10% of its primary energy supply in 2020 amid
reduction of GHGs to 10% of its 1990 levels in the years between 2008 and 2012
and 60% of its 1990 levels by 2050.
In the United States, many acts and policies support biodiesel production.
The most important one of them all is the Renewable Fuel Standard (RFS), under
which the EPA sets targets of biofuel production over the coming years. This
comes from the Energy Policy Act (2005) and Energy Independence and Security
Act (2007). There is high support from Congress for biodiesel promulgation as can
be seen in their open letter in 2016 to EPA administrator Gina McCarthy to
increase the limits of renewable volume obligations (RVOs). The EPA’s rule of
2018 has capped the growth of biodiesel production to 378.5 million liters over
2017; however, the senators want it to be increased by 1,514 million liters and
thereby want the production to reach 9.5 billion liters, citing reasons of not being
able to take full advantage of the American biodiesel production capacity, ignoring
the fact that numerous jobs have been created in the biodiesel industry hitherto,
and the surplus of tax revenues the biofuel sector generates (Biodiesel.org 2016).
Thus, there is a massive support to biofuel technologies and production in the
United States, and surplus jobs have also been created there dating back to 2004
with the American Job Creation Act.
Despite policy support, sometimes owing to technological, organizational,
and coordination impediments, certain industries find many challenges. In the
United States, the first-generation traditional biofuel industry is dominated by
production of biodiesel from corn-based feedstock oil. There was also a presence
of well-developed value chain and associated industries for this established
production process. However, when the US industries had received all the policy
support and when they began deciding to move toward the second-generation
biofuel production from switchgrass, the switchgrass-based biofuel production
could not take off in an economically competitive way to replace the corn-
based fuel production, simply resulting from the lack of development of other
switchgrass-associated processing industries like the grain processing industry
present in the corn production value chain per se. Therefore, such situation calls
for the federal governments to provide policy support and aid in capital invest-
ments to the associated industries for promoting development of greener and
more efficient fuels (Kedron and Bagchi-Sen 2017).
24.3 THE OIL SECTOR: ITS POLITICAL POWER AND SENSITIVITY
24.3.1 Oil Price Change

In 2016, the crude oil price fell to USD 27 per barrel (Karl 2016). This is the
steepest fall in prices in recent years. One of the biggest challenges to innovation
and funding in biodiesel technologies and production in biofuels is the price of
crude oil. Pricing of oil or any commodity in a free market is regulated on the basic
premises of demand and supply. The entire world is primarily dependent on oil
extracted from oil rigs for its energy requirements so far. The prices were hovering
at around USD 100/barrel until recently. There was an influx of surplus money to
the oil exporting countries in terms of profits and likewise to the governments in
terms of higher tax duties. When money is surplus in the hands of companies, they
have a greater flexibility to allocate a higher amount of budget for research and
development, which could be either directed toward increasing efficiency of their
fuel combustion characteristics or alternatively promoting investment toward
development of clean energy projects given the contemporary harangue about
climate change and more precisely about GHG reduction.
Similarly, when the high oil price brings extra revenue to the federal govern-
ment in the form of tax levies, the governments too have more leg room to increase
their budget expenditure in terms of capital investments to support infrastructure
projects, to expend on social programs, and most important, to fund their own clean
energy projects. These projects unlike the projects sponsored by the private
corporations are not most likely and obviously profit-oriented, but rather have a
problem-solving mindset that stems from the tenets of the larger good. Thus,
increased federal government tax revenue is also most likely to fund research
laboratories and institutes working toward cleaner fuels like biodiesel production
per se. Unfortunately in 2016, the price of crude oil took a steep fall, and there is no
ubiquitous incentive to fund all projects directed toward the development of
biofuels; in a more general way, a temporary hiatus prevails. The most obvious
question is Why should the consumer be interested to pay any higher for an
alternative fuel that is in its developmental phase and trying its best to match the
standards of fossil fuels? This, however, is a short-sighted thought because it ignores
the cataclysmic effects of climate change and the limitation of fossil fuels.
24.3.2 Oil and Its Impact on Geopolitics

Apart from the decrease in investment in biodiesel and other clean energy
investments, the decrease in oil price has crippled economies whose prime source
of income is the export of oil. The 2016 oil price slump had a negative impact on
the economies of Saudi Arabia, Venezuela, and Russia, to name a few. One of the
reasons for this effect is that the largest purchaser of oil, the United States, has
started to invest heavily in shale gas/oil, thereby reducing their exclusive depen-
dence on oil for their energy needs. Naturally when demand falls, prices fall,
income reduces, and unrest may increase in the supply country (Arezki and
Blanchard 2015).
Allies and poles are created in the politics of fuels. For instance, Saudi Arabia’s
social and economic system so far was based on subsidy to the enterprises involved
in exploration, processing, and export of oil because this is a sector that brings
huge currency into the country’s economy. In case of the 2016 price slump, a
supplier country would wish to deliberately cut short the supply to create supply
shortage and maintain the artificial high price. However if it does so, other
suppliers in the world such as Iran, on whom the economic sanctions against them
were recently lifted owing to their nuclear program transparency, would compete
with Saudi Arabia. If that happens, Saudi is most likely to lose its market share of
oil, which would be more disastrous for them than exporting oil at a loss to other
countries. Given the Shia–Sunni political differences already existing between the
rival countries, they would always try to remain competent in the market even
though it comes at a heavy cost to its exchequer (Bush 2007, George 2012).
A matter of speculation that many worldwide believe to be a possibility is a
covert link between oil revenue earned by some of the countries in the Middle East
and a plausible indirect funding to radical groups like ISIS and Al-Qaeda. If this
assumption is true, logically the funding to terror groups decreases and so will
their terrorism-associated activities. Civilian per capita income shall also decrease,
and the most likely outcome is social unrest. This would be more disastrous
because hitherto much time and resources have been spent to curb the Arab
Spring, such as events in Middle Eastern countries since 2010. The protest that
started with Tunisia also spread like a fire to other countries of the world.
Whatever the basis for movements—rationally questioned or malignantly moti-
vated—it did try to change existing realities in the countries. The governments of
all such countries have therefore been very vigilant, and numerous measures have
been taken to preempt any uprising. All these causes and consequences are directly
proportional to the economy of a country. For countries solely based on
conventional oil exports, if a price change of oil could have these many
sociopolitical effects and myriad dimensions, what could be the effect on them
when the world starts moving their dependence to alternative renewable cleaner
energies? Certainly, this transition would not be smooth; however, these countries
should necessarily emphasize on building an alternative basis of the economy to
maintain their current economic status quo (Johnstone and Mazo 2011).
Another detrimental policy to deter biodiesel innovations would be the
subsidies given to petroleum products. Venezuela is a classic example to describe
the mayhem and disaster caused by surplus. The country has abundant oil
resources and is also part of the Organization of the Petroleum Exporting
Countries (OPEC). Because of the country’s socialistic foundations, it had
subsidized oil products by the philosophy that perhaps if we are in a position
to export oil to other countries, we certainly are supposed to avail the oil at
negligible cost. The country is reeling under hyperinflation and the currency is
exponentially loosing value. In fact, the cost of printing currency is 20% more than
the face value of the currency, and gunnysacks of Venezuelan currency are being
carried by the public to buy even basic consumer products from retail shops.
Certainly, such economies supported with bad economic policies are not a
conducive atmosphere to innovation in biodiesel technologies. The process of
subsidizing producers of biodiesel creates an ecosystem that survives primarily on
public intervention. Although this prima facie might seem beneficial to the
country that wants to promote biodiesel, such artificially protected environments
damage domestic markets and are not healthy for an economy. The detrimental
effect is more acute in a developing country when domestic markets are disrupted
and poverty rises. Rising poverty is the biggest enemy not only to people but also
to the environment, and one of the most obvious outcomes shall be environmental
degradation (Zhao 2015).
Biodiesel is and shall be also intricately linked to all these existing and
emergent political factors given that it is assumed to replace the conventional oils,
the process of which has already begun by the blending of oil and biodiesel. At a
political level, many questions are arising with the advent of biodiesel technology.
Which country will benefit more from the advent of biodiesel technologies?
Does that question the existing status quo? Is the new country building
capabilities to be worthy of that new position? Is such a country ready to find
the next and the right opportunity to take the leap toward green technologies
and find itself in a dominant position and circumventing the labyrinth of
uncertainties, possibilities, and realities? All these are some of the very real
political factors and complexities associated with the development and prolifer-
ation of technologies in the oil sector. To sum up, the way politics intersects with
economic processes and ecological conditions is something that shapes how a
biofuel intervention plays out.
New alliances and partnerships are already in the process of engagement. For
instance, in the past in the United States, Monsanto and Cargill collaborated at the
time to form Renessen LLC (Jakel and Ulrich 2004), whereby animal feed
and biofuels were integrated in such a way that a genetically modified crop
could also be used as a feed for biofuel production; in other words, animal feed
could also become a byproduct of biomass fuel production. These are highly
interwoven sophisticated production processes with implications reaching far
into the socioeconomic and environmental domain. Such high potential colla-
borations would make companies highly difficult to escape from industrial
farming, a highly sensitive topic that possibly could disrupt the occupation of
farmers. The Malaysian government is soon turning into a palm oil conglomer-
ate. Brazil is collaborating with India, China, Mozambique, and South Africa to
make an ethanol alliance. It is also hoping to be the biggest exporter of biodiesel
by exporting to the United States and European countries, a dream that can
come true if the United States relaxes its import tariffs. This dream is also
supported by collaborations between a prominent biodiesel company of Brazil,
Cosan, and the Royal Dutch Shell company. Now Brazil is hoping to double its
ethanol production and ship the alternative renewable fuel to the world (Borras
et al. 2010).
24.4 SOCIAL AND ENVIRONMENTAL AFTERMATH OF BIODIESEL

PRODUCTION
The emphasis on biodiesel has brought a myriad of other issues to the forefront.
Governments worldwide have been promoting biofuel feedstock production
hoping to fight climate change, to invigorate the rural economy by providing
employment, with a vision to nurture an alternative energy resource that is no
more limited by limitation of crude oil in the near future, either by natural or
political constraints. However, perpetuating this change has caused a domino
effect and brought about other changes too.
24.4.1 Land Use Changes

Following the rapid expansion of production of biofuel feedstocks, land usage has
also changed. The most evident outcome to this development is deforestation. In
principle, biofuel feedstock production is expected to minimize GHG production
by an inherent nature of less pollution from combustion in automobiles, but the
process of producing biodiesel itself by deforestation and other similar ecology
perturbing activities is not contributing to lessen GHG emissions. Therefore, the
net benefit or impact of this entire production process and promotion of
technology is something that should be mulled and contemplated.
To substantiate, in Indonesia, 80% of forest lands have been cut down for oil
palm plantations. The Indonesian Oil Palm Research Institute (IOPRI) in 2004
estimated that 3% of all oil palms in Indonesia had been established in primary
forest and 63% in secondary forest and bush. In Malaysia, the Malaysian Palm Oil
Association (MPOA) calculated that 66% of plantations had been converted from
other agricultural uses, and the remainder established from logged-over forest
land. These numbers of Indonesia and Malaysia speak for themselves and show
that the accentuation of biofuel feedstock production could come at a tremendous
cost of food security and climate change perpetuated by mass deforestation
(Upham et al. 2009). In the Amazon, many indigenous populations have been
evacuated for the same. Devil Orchards is the name used in the Amazon for the
sugar cane and palm oil plantations being massively cultivated in the forest areas
(Borras et al. 2010). In Asia, however, some literature suggests there has not been a
very significant land use change specifically associated with biodiesel production,
the reason for which is attributed to lesser demand for them (both natural and
policy induced) compared with other products that cause land use change like
food, timber, feed, and fiber. However, time will only tell of any plausible
implications with the advent of new policies (Phalan 2009).
The biodiesel market is an additional consumer of the products of agricultural

farmers. Thus, the technology development has a direct impact on the economic
development of farmers. For third world countries with primarily agrarian
economies, the impact would be substantial. However, statements like “biofuels
may mean filling the fuel tank at the cost of emptying the stomach of the poor”
(Runge and Senauer 2007) are heard. A case study done on Jatropha cultivation in
the wastelands of Tamil Nadu, India, reveals that it was neither beneficial nor pro-
poor. It rather just benefited the middle- to rich-class farmers who had access to
capital (Borras et al. 2010).
In recent years, a multitude of lands have exchanged hands. According to a
World Bank report, 45 million ha of land have been transacted between 2005 and
2009 (Deininger and Byerlee 2011). There are private–private leases and public–
private leases for biodiesel production. Many such speculative deals happen in
sub-Saharan Africa, and the political ramifications of such large capital invest-
ments and exchanges could be something as sudden as when the ruling govern-
ment was brought down as in Madagascar in 2009. Other talk making the rounds
is that investors are just hoarding more and more land and preparing for the next
global crisis.(Borras et al. 2011).
There has been a paradigm shift of biofuel feedstocks from sugarcane and
food crops (first generation) toward nonfood feedstocks (second generation)—
namely, jatropha and palm oil, toward raw material like algae (third generation).
Currently, the progress is toward fourth generation raw material sources—
microbial oils. This ostensibly will compensate for the world shortages of arable
land, uncertainty of climate, and increasing salinity in the soil, as mentioned in a
2014 Intergovernmental Panel on Climate Change (IPCC) report (Field et al.
2014).
Every country should grow a suitable crop, in which it has a relative
advantage, to meet its domestic fuel needs after due research and contemplation.
A study in Bangladesh finds the production of biofuels from rice bran oil has
higher prospects because of the country’s suitability for high rice production albeit
at the cost of food security, if overexploited (Wakil et al. 2016). Every country has a
unique inherent natural topography and climate, or perhaps surplus manpower
availability, a different form of domestic demand, and also plausible advantageous
export-orientated growth prospects. Therefore, it could be a double-whammy to
the country and its producers, when it finds an optimum technology and
production process tailored to its inherent framework and thereby aid in its
socioeconomic development. The way the energy flows, the way agrodiversity is
affected, and the way carbon is sequestered are hugely different in every country
just because their poverty and livelihoods are different. With the proliferation of
biodiesel production, chemical input to the soil shall likely be increased in terms of
specific required fertilizer to the crops to increase the yield. Increased chemical
input with more irrigation is likely to make the soil more saline and likely to cause
soil damage in the long run. Needless to say, the habitat of many species
indigenous to any forest or the flora might also get depleted owing to deforesta-
tion. Rainfall patterns may change along with a possible microclimate change.
24.4.2 Labor Rights

The utopian and propagated school of thought says the biodiesel industry could
supplement farm income. Today however, a decrease in farm income has become
a major ailment of the primary sector economy. Biodiesel production creates more
job opportunities because it has higher labor requirements compared with fossil
fuel production. The silver lining is increased employment in rural areas and the
hinterland, which is where most of the feedstock production is likely to take place,
which is likely to curb rural–urban migration in a country.
In Indonesia, in the run-up to enhance biodiesel production, the country has
witnessed displacement of local communities in the event of oil plantation
deforestation. In particular, labor rights have been compromised because the
production is large scale and land tenure is poorly defined. In India, widespread
cultivation of Jatropha has denied the villagers of their community lands, which
hitherto provided them with wood for their chulhas (Cotula et al. 2008).
Resettlement is not a viable option most of the time, because they lose their
land in this process and also plausibly witness social disarticulation—namely,
alienation from their tribe or community.
Rights and benefits are innate in any workforce and should be upheld in a
more sanctifying way in an economic activity. However, various stakeholders are
involved in an economic activity and like civilian groups, peasants, and industries,
they have interests relevant to them, but sometimes they are in conflict to others,
which makes policy framing all the more challenging. Various responses range
from unconditional embrace to outright opposition. In the case of peasant groups,
two major transnational farmer groups are relevant in the biodiesel policy
formulation scenario—namely, the International Federation of Agricultural Pro-
ducers (IFAP) and La Via Campesina. IFAP is dominated by farmers of developed
countries, and it primarily echoes the interests of the middle-class rich farmers
from developed countries. La Via Campesina typically comprises farmers of
developing countries, most of them poor and with small land holdings; the key
agrarian interests they raise emanate from the class interest of their mass bases
(Borras et al. 2010).
24.5 CONCLUSIONS
Overall, there should be responsible biodiesel production. But what constitutes

responsible? A subjective question! Open to debate and perhaps at the whims of
winners of electoral politics as is the case in the 21st century. The biggest problem is
sociological! Unlike an engineering laboratory experiment that quite frequently uses
the regression technique to fit a straight line through various data points, wherein
the data points and variance of the data points are just some points that are static in
a finite experiment that might yield a poor R2 value in a worst-case scenario,
sociologically, these variant points, when they exist, are individuals, entities or
organizations that are dynamic, free, and chaotic. A policy that is legislated for the
society is an experiment that runs infinitely and has infinite socioeconomic and
political implications on future generations. An eminent sociologist Karl Marx talks
of technological determinism, which basically means technology is responsible for
every change that has happened in the society historically. Evidently, the present
generation is booming with technology in every sphere—information and com-

munication technology, biotechnology, materials science and quantum science—the
change, as is obvious, makes it to the national headlines and that’s when some part
of the world knows about it. However, what if the change is subtle? What if the
change that has materialized is having unhealthy repercussions on society? Is it
possible to go back in time? When it becomes obvious to the masses that the
technology-induced change has indeed turned out malignant, would there be a
thought that in hindsight perhaps the policy makers should have had more
foresight? These issues are quite complicated. A logical mind might wish it was
possible to do something analogous to response surface methodology to bring in an
optimum policy to get the best result. However, the crux of the problem involves
thinking and talking across generations. Marx of the 21st century would perhaps say
the technology of the contemporary world is in the hands of scientists and
researchers. The technology could become a change when supported by think
tanks, nurtured in technology incubation centers, funded by financial institutions or
governments or enterprises, thereby facilitating its technology transfer.
Everyone has a crucial responsibility and plays a vital role toward this
foreseeable or unforeseeable change. Biodiesel as a commodity is constructed
through social, political, and economic relationships in ways that must be
understood as a whole and located within wider, often global processes (Sassen
2006). To be competitive with conventional diesel, biodiesel will require more than
the combined assistance of carbon tax credits and the cost savings resulting from
reduced diesel emissions to make it competitive with conventional diesel, unless
the price of crude oil is maintained at prices significantly higher than historical
averages. Biodiesel corroborated with new transit networks, electric vehicle use,
and efficient cities is going to be the new reality of the coming centuries.
Conclusively, all these social, economic, political, and environmental aspects
should be given a quick thought while developing and scaling up a technology,
and that would perhaps constitute responsible.
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Index
Page numbers followed by e, f, or t indicate equations, figures, or tables.
acetyl-CoA, 207, 211–214, 216–217, air quality: agrochemicals and, 594,

309, 311, 336, 430, 437, 442 601, 601f; biodiesel and, 12–13;
acid value, 478, 498, 499t, 500t; combustion emissions and,
catalyst selection and, 96, 100, 103, 584–594, 586t, 595–597t, 598–600t;
164; of lard products, 128, 130; particulate emissions, 23–24;
oxidation and, 3, 11; of waste physicochemical characteristics of
cooking oils, 155–156, 156e, biodiesel and, 18; public health and,
166–168 631–633
acid washing: biodiesel purification alcohol-to-substrate molar ratio,
and, 459–460; techno-economic enzyme-catalyzed
evaluation of, 554, 554f, 555t, 556, transesterification and, 62–63, 63t
556t algae: genetic engineering and,
acid-based catalysis, 56–57, 80, 100, 437–440, 438f. See also microalgae,
130, 134, 158, 283–284, 549, biodiesel from
564–565, 570, 572 algal biomass, residue from
acidolysis, waste cooking oils and, agricultural biomass and, 505
161, 162f algal oil: techno-economic evaluation,
activated carbon, biodiesel and single cell oil compared, 539,
purification and, 463–464 540–541t, 542, 543t, 544; techno-
adenosine monophosphate (AMP), economic evaluation of, 535–536,
212–214, 309 537t, 538
adsorbents/adsorption: physical alkali-based catalysis, 29, 56, 80, 100,
technologies, 68, 71–72, 405; 133–134, 134e, 157–158, 160t, 162,
purification comparative studies, 164–165, 170t, 354
494–496, 495t, 497t, 498, 498t, 499t, alkali-based transesterification, 56;
500t; research on improvements, waste cooking oils and, 157–161,
498, 500–501, 500e, 501e 158f, 159f, 160t, 162, 164–165
agricultural biomass, residue and Amberlite, 71, 354, 463, 486, 490t,
coproducts of, 504–506, 507t, 508 494, 495t, 496, 498, 499t, 500t, 552
agricultural wastes, biodiesel anaerobic digestion, and biogass
production from oleaginous production, 509
microorganisms, 230, 233–234, animal fat biodiesel, 123–143;
235t, 236–238t advantages of, 124–125, 143;
agrochemicals: air quality and, 594, comparative studies of free fatty
601, 601f. See also pesticides acids, 124e, 133–137, 136t, 137f;
665
666 INDEX
economic impacts of, 140–142, 21t, 22; responsible production

141t; effects of metals on, 137–139, and, 661–662; sociological and
138f; genetically modified crops political aspects of, 653–662;
and, 658; health hazards of animal standards for, 5, 6–7t, 17; storage,
fats, 130–131; physical and stability, and transportation of,

chemical properties of, 142; 10–11, 477–478, 479; technical
production statistics, 124, 140–141; definition of, 16; toxicity and, 634,
pros and cons of, 142–143; sources 637; variations, properties,
of animal fats, 124–133, 140–141 comparison with petro-diesel,
animal feed, coproduct management 15–24. See also properties, of
and, 511–512 biodiesel
animal oils, plant oils contrasted, 202 Biodiesel Diesel Analyzer 1.1, 18
aromatic compounds, 89, 269, 272, biodiversity, biodiesel’s effect on,
284, 516–517, 591–592, 640 618–619, 620–623t, 624
ATP-binding cassette (ABC) bioethanol. See agricultural wastes
proteins, lipid milking and, biogas. See agricultural wastes
391–392, 392f biomass harvesting: microalgae and,
ATP-citrate lyase (ACL) enzyme, 212, 191–192; techno-economic
213, 217, 221, 309–311, 313, 335 evaluation, 544, 545t, 546
Australia, 5, 91, 141, 219, 528, 530 biorefinery concept, 517
automobiles, ethanol and, 527–528 blending, of biodiesel, 9–10, 16, 23
autotrophic microalgae, 176, 177, brake thermal efficiency (BTE),
182–186, 188 biodiesel compared to other
biorefinery products, 9
Bangladesh, 660 Brazil, 654, 658–659
bead milling, lipid extraction and, butyl-methyl imidazolium hydrogen
360, 370, 374 sulfate, 155
bentonite, 495–496, 497t
biocatalysts. See enzyme-catalyzed Canada: biodiesel economy in, 577t;
transesterification biodiesel production in, 141,
biocompatible organic solvents, lipid 229, 530, 576, 627, 654–655;
milking and, 387, 389, 390f feedstocks and, 54, 55, 89–90,
biodiesel: advantages of, 12, 583; 152, 239, 264
basics of, 1–13; biodegradability carbon assimilation, genetic
and, 637; blending of, 9–10, 16, 23; engineering of algae, 440
chemical composition of, 2; carbon dioxide: animal fat biodiesel
contaminants in, 22–23; and, 139–140; carbon dioxide
development history, 1–2, 201, 422, emissions, 587; microalgae
527–528; emissions and cultivation and, 176
environmental hazards and, 12–13; carbon monoxide emissions, 586
genetic engineering and, 440–442; carbon source: lignocellulosic
meaning of term, 1; other biomass and, 349–350; microalgae
biorefinery products compared, 5, cultivation and, 185–186
8t, 9; physicochemical carbon to nitrogen (C-to-N) molar
characteristics of, 17–18, 19t, 20t, ratio: biodiesel production from
INDEX 667
oleaginous microorganisms, closed photobioreactor systems,

249–250; crude glycerol conversion microalgae cultivation and,
study, 319t, 320t, 321t, 322–323, 190–191
322t; single cell oil and, 208–209, cloud point (CP), 5, 17, 18
213, 221, 318–319; wastewater clustered regularly interspaced short

sludge extraction and, 295 palindromic repeats (CRISPRs),
carbon-limited conditions, single cell 426
oil and, 210–212, 211f coalescing, as biodiesel separation
carbonyl compounds, air quality and, technique, 457–458
592–594 cold filter plugging point (CFPP), 5,
cash flow analysis, economic 17–18, 22, 169
feasibility evaluation and, 566, cold flow properties, 4–5
566e, 568f cold soak filtration test (CSFT), 5
cell disruption, as lipid extraction compression ignition engine (CIE),
technology, 360, 361–362t starting and, 4–5
cellulosic polymers. See consolidated bioprocessing (CBP),
lignocellulosic biomass 233
centrifugation: as biodiesel separation continuous fermentation,
technique, 456–458, 485, 487t; lignocellulosic biomass and, 349
microalgae harvesting and, 192 coproducts, of biodiesel production,
ceramic membranes, biodiesel 503–518; adding value to, 518; from
purification and, 466–467 agricultural biomass, 504–506,
cetane number: components of, 22; 507t, 508; biorefinery concept, 517;
efficiency and, 16; ignition delay management and uses of, 508–517,
time and, 2–3; particulate emissions 508t
and, 588–589; tallow products and, corn fiber hydrolysate, lipid
127–128 accumulation and, 349–350
chain length of alcohol, enzyme- cost analysis. See economic impacts;
catalyzed transesterification and, techno-economic evaluation
61–62, 62f cost of unit production, economic
charcoal, animal fat biodiesel and, 140 feasibility evaluation and, 570,
chemical gene transfer methods, 570e, 573t, 574f
424–425 covalent bonding technique, for
chemical technologies, of lipid lipase transesterification, 72, 405
separation, 359, 360, 377; organic cross-linking technique, for lipase
solvent extraction, 365–367, transesterification, 72–73, 405
368–369t, 371–373t, 374; crude glycerol, conversion to lipids
supercritical fluid extraction, and biodiesel, 305–336, 306f, 306t;
374–375, 376t analysis of studies reported in
chicken fat. See poultry fat literature, 325, 326–330t;
China, 613, 658; biodiesel production characteristics and composition of,
in, 655; feedstocks, 55, 91, 133, 152 307–308, 307t; C/N ratio, media
climate change. See environmental components, fermentation
and public health impacts, of parameters study, 318–319, 319t,
biodiesel 320t, 321t, 322–323, 322t, 335;
668 INDEX
coculture system study, 324; economic impacts: animal fat

cultivation mode study, 334–335; biodiesel, 140–142, 141t; single cell
fermentation cultivation mode oil biodiesel, 218–220, 219f; waste
study, 314–318, 315t, 316t, 317t, cooking oils, 169–170, 169t, 170t;
318f; genetic and metabolic wastewater sludge extraction

engineering and, 336; laboratory techniques, 279, 281–283, 281t,
scale studies, 313–325; metabolism 282t. See also economic feasibility
for glycerol uptake, 309–313, 310f, evaluations; techno-economic
311f, 312t; pretreatment study, evaluation
323–324, 323t, 324t; rationale for, edible and inedible tallow, animal fat
308–309, 309f; strain selection biodiesel and, 123, 125–128
study, 313–314, 314e edible oils, as feedstocks, 89, 90–91
electrophoresis, 192, 428
de novo pathway, single cell oil electroporation, 389, 391, 423–424,
production and, 203, 204, 208, 210, 439
213, 217, 221 emissions. See air quality
density, of biodiesel, 4, 126–127, 134 entrapment/encapsulation technique,
Diesel, Rudolf, 1, 24, 527 for lipase transesterification, 73–74,
dissolved oxygen (DO), biodiesel 405
production from oleaginous environmental and public health
microorganisms, 247, 247f, impacts, of biodiesel, 12–13,
248–249, 248e 583–640; on air quality, 584–601,
DNA. See genetic engineering 631–633; of biodiesel feedstocks,
downstream processing: genetic 263; on biodiversity, 618–624;
engineering and, 442; techno- challenges of, 639; food-versus-
economic evaluation of, 544, 545t, food controversy, 54, 89–91, 114,
546–547, 547t 152, 166–167, 201, 220, 393, 531,
dry washing: biodiesel purification 535, 626, 633; greenhouse gas
and, 461–464, 462f, 479, 488, 492, emissions, 624–628; industrial
500–501, 552, 559–563, 565; as waste and, 239; land use changes,
biodiesel separation technique, 485, 659–660; occupational health
487t; techno-economic evaluation, hazards, 628–629; safe handling
551–553, 559–562, 559f, 560t, 561f, concerns, 634–639; on soil,
561t, 562t; waste cooking oils 612–618, 629–630; waste cooking
and, 165 oil and, 152; wastewater sludge
disposal, 265; on water resources,
E85 fuel, 528 601–611, 630–631
economic feasibility evaluations: cash enzymatic alcoholysis, waste cooking
flow analysis, 566, 566e, 568f; cost oils and, 161–162, 162f, 163t
of unit production, 570, 570e, 573t, enzyme immobilization,
574f; investments, profits, revenues, nanotechnology and
and credits, 568, 569t, 570, 571t; transesterification: methods,
payback period, 572, 572e, 575t; 404–405, 405t, 406t; nano-
return on investment, 565–566, biocatalysis-assisted biodiesel
565e, 567t, 568f production, 408–409, 409f, 410t,
INDEX 669
411, 412t, 413–416, 413f, 414f; fermentation: biodiesel production

nanoparticles as support matrix, from oleaginous microorganisms,
406–408, 407f, 407t, 408t, 409f, 409t 250–251; crude glycerol conversion
enzyme lysis–assisted solvent studies, 314–319, 315t, 316t, 317t,
extraction, 374 318f, 319t, 320t, 321t, 322–323,

enzyme-catalyzed transesterification, 322t, 325, 331, 331–332f, 333,
30, 30f, 32–34t, 53–81, 658–659; 335–336; findings from previous
advantages of, 53, 57; chemical studies and future perspectives,
reaction of, 54f; cost analysis of, 80; 333–336, 333f; history of, 527;
extracellular lipases, 61; lignocellulosic biomass and,
immobilization of lipases, 67–75, 345–346, 347t, 348–352, 353t
68f, 69t, 70t; intracellular lipases, fertilizers, coproduct management
61; lipase enzyme production, and, 517
59–60; lipase sources, 58–59; filtration, microalgae harvesting and,
parameters affecting, 61–67, 62t, 192
64t; of plant oils, 106, 108, fire safety precautions, 638
109–110t; reaction mechanism of, fish waste: animal fat biodiesel and,
60, 60f; reactors for immobilized, 133–134, 135; effects of metals on,
75–79, 76f 137–139, 138f
enzymes, coproduct management flash point of biodiesel, public health
and, 514 and, 637–638
ethanol: automobiles and, 527–528; flotation, microalgae harvesting and,
blending of biodiesel and, 10; 192
coproduct management and, fluidized-bed reactor (FBR), for
509–510 immobilization lipase
European Union: biodiesel economy transesterification, 75, 76f, 79
in, 574, 576; biodiesel policies, 5, foil photobioreactor systems,
138, 142–143, 201, 529, 575–576, microalgae cultivation and,
625, 653–654 190–191
ex-novo pathway, single cell oil food-versus-fuel controversy, 54,
production and, 208, 213, 221 89–91, 114, 152, 166–167, 201,
expeller pressing, lipid separation 220, 300, 393, 531, 535, 626,
and, 363, 364t 633
extracellular lipases, 61 fractionation, of lignocellulosic
biomass, 344–345
fatty acid content, of biodiesel, 17–18 free fatty acids: comparative studies
fatty acid synthesis (FAS): genetic of, 133–137, 134e, 136t, 137f;
engineering and, 435–436, 437; nanotechnology and lipid
single cell oil production in yeast extraction, 400–401
and, 213–215, 213e, 214e, 214f fuel viscosity. See viscosity
fed-batch fermentation,
lignocellulosic biomass and, 349 gas chromatography (GC), 18, 473,
feedstocks, generally: plant oils as, 89, 476
90–91, 114–115; for gene gun (ballistic) transfer
transesterification, 54–55, 80 mechanism, 423–424
670 INDEX
genetic engineering, 421–446; heterotrophic microalgae, 176–177,

advances in tools, 425–426; in 182–183, 185, 186, 230, 308, 314,
algae, 437–440, 438f; basic gene 325; nanotechnology and, 398–399,
transfer methods, 422–425; 416
constructs and synergy of operons, high-pressure homogenization, lipid

442–443; crude glycerol and, 336; extraction and, 360, 370
future prospects, 429f, 445–446; homogeneously catalyzed
improved biodiesel production and, transesterification process, 29–30,
440–442; lipid production 29e ,94, 96, 97–98t, 99–100, 99f,
strategies, 429f, 430, 431–433t, 101–102t, 103, 103f, 115
435–437; metabolic pathway design hybrid cultivation, of microalgae, 191
challenges, 443–445; planning hydrocarbon emissions, 587–588
knowledge needed, 426–430, 427f, hydrochloric acid, as catalyst, 57, 155
429f; single cell oil production and, hydrodynamic cavitation, oil/fat
217–218, 221–222 transesterification to biodiesel
geopolitics, impact of biodiesel on, and, 38
656–659 hydrogen, coproduct management
Germany, 265, 528; biodiesel policies, and, 510–511
654; biodiesel production in, 1, hydrolytic reactions, effects on waste
218–219, 576, 639 cooking oils, 153–156
glutaraldehyde (GLA), 72–75, 414 hydrophobic solvents, 65, 387
glycerol, washing with, techno- hydrophobic substrates, single cell oil
economic evaluation, 555f, production in yeast and, 216–217
556–559, 556f, 557t. See also crude
glycerol, conversion to lipids and ignition time, cetane number and,
biodiesel 2–3
glycerol backbone synthesis, single immobilization technique, for lipase
cell oil production in yeast and, transesterification, 67; cost analysis
215, 215f of, 80; covalent bonding technique,
gravity sedimentation: as biodiesel 72; cross-linking technique, 72–73;
separation technique, 456; entrapment/encapsulation
microalgae harvesting and, 192 technique, 73–74; nanotechnology
greenhouse gases (GHGs), 12, 135, and, 74–75; oil/fat
584, 640, 659; reduction of, transesterification to biodiesel,
624–628, 627f 30–31; physical adsorption
technique, 68, 71–72; of plant oils,
heat. See temperature and heat 115; reaction temperature and, 66;
hemicellulose moieties. See reactors for immobilized, 75–79,
lignocellulosic biomass 76f; techniques, 67–68, 68f, 69t, 70t
hemicellulous polymer, 345 impurities: animal fat and, 125, 126,
heterogeneous catalytic 135, 140, 143; crude glycerol, 306,
transesterification, 31, 36–37t, 47, 315, 317, 334; effects of, 485, 486t,
103–104, 105t, 106, 107t 550; wastewater sludge, 263,
heterologous protein expression, 298–299. See also purification
genetic engineering and, 426 entries
INDEX 671
in situ biodiesel production, genetic light: genetic engineering of algae,

engineering and, 441–442 439; microalgae cultivation and,
in situ transesterification, 28, 39–40, 176, 183–184
40f, 44, 283–284 light harvesting complexes (LHC), 439
incineration, of wastewater sludge, lignocellulosic biomass (LCB),

265 341–355; advantages of, 341–342,
India, 5, 54, 91, 505, 625, 658, 355; challenges of, 342; common
660–661 sources of, 342; fermentation and,
Indonesia, 54, 91, 576, 659, 661 345–346, 347t, 348–352, 353t; lipid
industrial farming, 658 production from, 342–345, 343t;
industrial wastes, biodiesel pretreatment inhibitors, 352,
production from oleaginous 354–355; subunits of, 342
microorganisms, 239, 240–241t, lipase: as biocatalyst, 57–58; catalytic
242 transesterisfication, 30, 30f; three-
intracellular lipases, 61 dimensional structure of, 57, 58f
investments, profits, revenues, and lipid extraction techniques, 359–377;
credits, economic feasibility cell disruption and, 360, 361–362t;
evaluation and, 568, 569t, 570, 571t chemical technologies, 359, 360,
iodine value (IV), 5, 22, 124, 129–130 365–367, 368–369t, 370, 371–373t,
ion-exchange resins: biodiesel 374–375t, 376t, 377; coproduct
purification and, 463, 501; case management and, 514, other
study on use in biodiesel technologies, 375, 377; physical
purification, 488, 491–492, 491f, technologies, 359, 360, 363, 364t,
491t; purification comparative 365, 377; techno-economic
studies, 494–496, 495t, 497t, 498, evaluation, 546–547, 547t
498t, 499t, 500t; research on lipids, milking from oleaginous
improvements, 498, 500–501, 500e, microorganisms, 383–416;
501e; techno-economic evaluation, advantages of, 385, 385f;
552, 559–560, 559f, 560t, 565 commercial processes, 383–384,
ionic liquid solvent systems, 65, 464 384f; future prospects, 392–393;
Iran, 528, 657 methods, 387, 388t, 389–392, 390f,
isocitrate dehydrogenase (ICDH), 390t, 392f; production potentials,
209, 212–213, 309 385, 386t, 387t
lipoplexes, 424–425
Japan, 5, 152, 265, 625, 655 liquid homogenization, lipid
extraction and, 360
kinematic viscosity, 18, 23, 135, 168 low-temperature flow test (LTFT), 5
Krebs cycle, single cell oil and, 211 lubricity, biodiesel compared to other
biorefinery products, 9
labor rights, effects of biodiesel on,
661 Magnesol: dry washing and, 461–462,
land use changes, 613, 617, 634 474t, 487–488, 489t, 490t, 492, 493t,
lard products: animal fat biodiesel 496, 497t, 498t, 501, 551; techno-
and, 125, 128–130; effects of metals economic evaluation, 560–562,
on, 137–139, 138f 561f, 561t, 562t, 565
672 INDEX
magnetofection, 423–424 microbe whole-cell utilization,

Malaysia, 54, 91, 113, 658, 659 oil/fat transesterification to
malic enzyme (ME) activity, 209, 215, biodiesel, 30
310–311, 436–437 microbial biomass, residue from
malonyl-CoA, 214, 309, 311, 336, 430, agricultural biomass and, 505–506
436–437 microemulsions, biodiesel production
mechanical-chemical extraction, and, 27, 56, 89, 94, 151
from wastewater sludge, 273–277, microreactor, transesterification in,
273f, 275f, 275t, 276f, 277, 403–404, 403f
284; economics and, 281–283, microwave-assisted processes, 39,
282t 370, 371, 374
media components, crude glycerol mixotrophic microalgae, 176–177,
conversion study, 318–319, 319t, 182, 536
320t, 321t, 322–323, 322t molecular distillation, waste cooking
membrane reactors: biodiesel oils and, 166, 167f
purification and, 467, 469, 469f, municipal solid waste, biodiesel
470t, 471–472, 472f; problems production from oleaginous
associated with, 485, 487t microorganisms, 242
membrane technology: biodiesel
purification and, 464–467, 465f, nanotechnology, 397–416;
468t, 469, 469f, 470t, 471–472, advantages of, 397–398, 416;
472f, 479; as biodiesel separation defined, 397; lipase
technique, 485, 487t; techno- transesterification and, 74–75; lipid
economic evaluation, 562–563, extraction and, 377, 399–400; lipid
563f, 563t, 564t, 565; waste cooking milking and, 416; lipid production
oils and, 166 and, 398–399, 399t; process
membrane transport proteins, lipid flowchart, 398f; purification of
milking and, 391–392, 392f hydrocarbons from oil, 400–402,
metals, effects of animal fat 402t; transesterification reaction
feedstocks, 137–139, 138f and, 402–416; wastewater sludge
microalgae, biodiesel from, 41, 43f, and, 284
175–193; advantages of, 175, 193; nicotinamide adenine dinucleotide
biomass harvesting, 191–192; phosphate (NADPH), 213–215,
cultivation conditions, 183–188; 310–311, 436–437
cultivation systems, 188–191, 189t; noncatalytic transesterification,
industrial waste and, 239, 242; 28, 29
microalgae diversity and nonedible oils, as feedstocks, 89, 91
classifications, 176–177, 178t; Novozyme 435, 61, 63, 65, 66, 71, 75,
microalgae oil’s composition, 177, 78, 79, 161, 408
179t, 180; microalgae oil’s content nutrient sources, microalgae
and productivities, 180; nutrient cultivation and, 182–183, 186–187
sources, 182–183, 186–187;
production steps, 175–176; occupational health hazards, 628–629
prospects for, 183; strains of octane number, 16
microalgae, 180, 181t, 182 octyltriethoxysilane (OTES), 72
INDEX 673
oil cakes, residue from agricultural parameters, 243–251, 245t, 246,

biomass and, 504–505, 509, 512, 246e, 246f, 247f, 248e; residential
514, 517–518 wastes, 242–243
oil-bearing substances, Organization of the Petroleum
transesterification and, 39–40, 40f Exporting Countries (OPEC),

oil/fat, transesterification to biodiesel 251–252, 657
and, 28–39, 29e, 30f, 32–34t, oxidative reaction, effects on waste
35–36t; alcohol type effect on, 31, cooking oils, 153, 155f
37; alcohol-to-oil ratio and, 29e, 38; oxidative stability, of biodiesel, 3, 10,
assisted transesterification, 39–40; 11, 22–23
catalyst impact, 29–31, 29e; oil type oxides of nitrogen emissions,
effect on, 37–38 589–590
oleaginous microorganisms: lipid
accumulation of, in wastewater packed-bed reactor (PBR), for
sludge, 296, 298; single cell oil immobilization lipase
biodiesel, 203, 204, 205–206t. See transesterification, 75, 76f, 77–79
also lignocellulosic biomass (LCB); particulate matter (PM) emissions,
lipids, milking from oleaginous 23–24, 588–589, 639–640
microorganisms; organic waste, as payback period, economic feasibility
raw materials evaluation and, 572, 572e, 575t
omega-3 polyunsaturated fatty acids, pervaporation, biodiesel purification
133, 512–515, 517 and, 472, 472f, 479
1,3-propanediol, 513 pesticides, impacts of, 594, 601,
open pond systems: microalgae 605–606, 618, 629–631, 640
cultivation and, 176, 182, 186, petroleum diesel: biodiesel compared,
189–190, 191; techno-economic 5, 8t, 9; blended with animal fat
evaluation, 536 diesel, 123
operons, 216, 442–443 petroleum sector, reactions to
organic acids, coproduct biodiesel, 656–659
management and, 513–514 pH, biodiesel production from
organic membranes, biodiesel oleaginous microorganisms, 244,
purification and, 465–466 246, 246e, 246f
organic solvents: biodiesel photobioreactors (PBRs), 182–184,
purification and, 460; enzyme- 188, 190–191, 536, 542
catalyzed transesterification and, photoheterotrophic microalgae, 176,
63, 65; lipid separation and, 177, 182
365–367, 368–369t, 371–373t, 374; photo-inhibition, genetic engineering
techno-economic evaluation, 551 of algae, 439
organic wastes, as raw materials, physical adsorption technique, for
229–253, 231t, 232t; agricultural lipase transesterification, 68, 71–72,
wastes, 230, 233–234, 235t, 405
236–238t; case studies, 251–252; physical gene transfer mechanisms,
economics of, 230, 252–253; 423–424
industrial wastes, 239, 240–241t, physical technologies, of lipid
242; lipid accumulation separation, 359, 360, 377; expeller
674 INDEX
pressing, 363, 364t; thermal proteomics, genetic engineering’s

extraction, 363, 365. See also future and, 444
ultrasonification/ultrasonic public health. See environmental and
harvesting public health impacts, of biodiesel
physicochemical characteristics, of pulsed electric fields (PEFs), lipid

biodiesel, 17–18, 19t, 20t, 21t, 22 milking and, 389–391, 390t
ping-pong Bi-Bi mechanism, 60, 60f purification, techno-economic
plant oils, 89–115; animal oils evaluation of, 550; dry washing,
contrasted, 202; biodiesel 551–553, 559–562, 559f, 560t, 561f,
production methods from, 89–90; 561t, 562t, 565; membrane
challenges of, 113–114; feedstocks technology, 562–563, 563f, 563t,
for, 90–91, 114–115; future work 564t, 565; methods reported in
and prospects, 114; physical and literature, 553t; wet washing, 551,
chemical properties of, 92, 92t, 93, 554, 554f, 555t, 556–559, 556f, 556t,
93t, 95t. See also plant oil, 557t, 558f, 558t, 559t, 563, 565
transesterification processes purification, using resins and
plant oils, transesterification adsorbents, 485–501; adsorbent,
processes, 94, 96, 96f; enzyme- ion-exchange resins, and wet-
catalyzed transesterification, 106, washing methods comparative
108, 109–110t; heterogeneously studies, 494–496, 495t, 497t, 498,
catalyzed transesterification, 498t, 499t, 500t; effects of
103–104, 105t, 106, 107t; impurities on performance, 485,
homogeneously catalyzed 486t; ion-exchange and adsorbents
transesterification, 94, 96, 97–98t, research on improvements, 498,
99–100, 99f, 101–102t, 103, 103f; 500–501, 500e, 501e; ion-exchange
supercritical transesterification, case study, 488, 491–492, 491f,
108, 111–112t, 113 491t; problems associated with
plasmid vectors, gene transfer and, commons methods of, 485, 487;
423 resins used on, 485–487; silica as
poly(hydroxyalkanoates) (PHA), 513 adsorbent case study, 487–488,
polyunsaturated free fatty acids, 489t, 490t; wastes as adsorbent case
nanotechnology and lipid study, 492, 493t, 494
extraction, 401–402, 402t purification and recovery, of
poultry fat: animal fat biodiesel and, biodiesel, 453–480; advantages
125, 130–131, 133–134; effects of and disadvantages of techniques,
metals on, 137–139, 138f 174–175t, 473, 485, 487t; biodiesel
pour point, cold flow properties, 5 separation techniques, 455–458;
prions, animal fats and, 124, 126, 143 conventional purification
properties, of biodiesel: cetane techniques, 458–464, 459f, 462f;
number, 2–3; chemical future prospects, 478–479;
composition, 2; cold flow membrane technology, 464–467,
properties, 4–5; density, 4; heat of 465f, 468t, 469, 469f, 470t, 471–472,
combustion (heating value), 3–4; 472f, 479; quality testing and, 473,
iodine value (IV), 5; oxidative 476, 479; steps in, 453, 454f;
stability, 3; viscosity, 4 storage, stability, and
INDEX 675
transportation, 477–478, 479; waste 208–210; genetic engineering and,

cooking oils and, 165–166 217–218; oleaginous
Purolite, 463, 485, 490t, 494, 495t, microorganisms, 203, 204,
496, 498, 498t, 499t, 552 205–206t; origins of, 204; prospects
pyrolysis (thermochemical) and challenges, 220–221, 222;

extraction: biodiesel production substrates used for production, 204,
and, 27–28, 27e, 56, 89, 91, 205–206t, 207–208; techno-
139–140, 233; defined, 266, 516; economic evaluation of, 538–539,
from wastewater sludge, 266, 540–541t, 542, 543t, 544. See also
268–269, 269f, 270–271t, 272–273, genetic engineering
277, 279, 281, 281t, 284 site-specific genome editing, 426
sociological and political aspects, of
quality testing, of biodiesel, 473, biodiesel, 653–662; country
476, 479 policies, 653–656; labor rights, 661;
land use changes, 659–660; oil
rendering, of fats, 125–129 sector’s political power and,
return on investment, economic 656–659
feasibility evaluation and, 565–566, soil, biodiesel’s effect on: fuel spillage,
565e, 567t, 568f 617–618; public health and,
629–630; soil erosion, 613, 617;
saccharification: of cellulose, 345; soil productivity, 612–613,
lignocellulosic biomass and, 614–616t
350–351 sol–gel immobilization, 73, 76, 161
safe handling concerns, in biodiesel solid-state fermentation (SSF),
production, 634, 635t, 636t, lignocellulosic biomass and, 348
637–639 solvents: extraction from wastewater
salinity, microalgae cultivation and, sludge, 274–277, 275f, 275t, 276f,
185 277, 282, 284–285; organic, and
saponification value, 22, 473 enzyme-catalyzed
Saudi Arabia, 528, 657 transesterification, 63, 65
scum sludge, 263, 265–266, 274, South Africa, 5, 528, 625, 658
277–278, 283, 284 spontaneous oozing, lipid milking
silica: biodiesel purification and, and, 391
461–463, 462f, 501; case study on stirred tank reactor (STR), for
use as purification adsorbent, immobilization lipase
487–488, 489t, 490t transesterification, 75–77, 76f
single cell oil (SCO) biodiesel, submerged fermentation (SMF),
201–222; advantages of, 204; lignocellulosic biomass and,
biochemistry of yeast production 348, 351
of, 212–217, 213f, 214e, 214f, 215f, sulfonic acid, 57, 155
216f; carbon-limited conditions sulfuric acid, as catalyst, 57–58, 96,
and degradation of, 210–212, 211f; 115, 128, 131–134, 138, 155, 157,
characteristics of single cell oil, 276, 283–284, 463, 471, 534
202–204, 203f; economics and, supercritical fluid extraction (SFE),
218–220, 219f; factors affecting, lipid extraction and, 374–375, 376t
676 INDEX
supercritical transesterification thermal hydrolysis, agricultural

process, of plant oils, 108, wastes and, 233
111–112t, 113 thermal stability, transport and
Switzerland, 654 storage of biodiesel and, 11
synthesis reactions, waste cooking oils thermolytic reaction, effects on waste

and, 161, 162f cooking oils, 153
3-Glycidoxypropyl trimethoxysilane
tallow products: animal fat biodiesel (GPTMS), 72
and, 125–128, 135; effects of metals trace elements, biodiesel production
on, 137–139, 138f from oleaginous microorganisms,
tax regulations, animal fat biodiesel 250
and, 142–143 transcription activator-like effector
technological determinism, of Marx, nucleases (TALENs), 426
662 transesterification, nanotechnology
techno-economic evaluation, and, 402–403, 403f; by enzyme
527–578; of algal oil use, 535–536, immobilization, 404–416, 405f,
537t, 538; biodiesel development 405t, 406t, 407f, 407t, 408t, 409f,
history, 527–528; of downstream 410t, 412t, 413f, 414f; in
processing, 544, 545t, 546–547, microreactor, 403–404, 403f
547t; global diesel in future, 574, transesterification, production by,
576, 577–578, 577f, 577t; 27–47; catalytic, 28, 29, 47; defined,
importance of, 529–531; membrane 56; feedstocks and, 54–55; history
technology, 562t; of single cell oil of, 527; method overview, 55–58;
use, 538–539, 540–541t, 542, 543t, noncatalytic, 28, 29; oil-bearing
544; of transesterification process, substances to, 39–40, 40f; oil-fat to
547–550, 548t; of vegetable oil use, biodiesel, 28–39, 29e, 30f, 32–34t,
531, 532t, 533, 533f; of waste 35–36t; in situ, 28, 39–40, 40f, 44;
cooking oil use, 534–535, 534t. See synthetization methods, 41, 42–44f,
also economic feasibility 45–46t; techno-economic
evaluations; purification, techno- evaluation of, 547–550, 548t. See
economic evaluation of also enzyme-catalyzed
temperature and heat: biodiesel transesterification; plant oil,
production from oleaginous transesterification processes
microorganisms, 246–248, 247f; transesterification, waste cooking
effects on waste cooking oils, oils and, 151, 156, 156f; alkali-based
152–156, 154t, 155f, 156e; heat of transesterification, 157–161,
combustion (heating value), 3–4; 158f, 159f, 160t, 162, 164–165;
microalgae cultivation and, enzymatic alcoholysis, 161–162,
184–185; pyrolysis of wastewater 162f, 163t
sludge and, 269, 270–271t, 272; transformation techniques, genetic
reaction temperature and enzyme- engineering and, 425, 439
catalyzed transesterification and, triacylglycerol synthesis: single cell oil
65–66; single cell oil and, 209 production in yeast and, 217f;
thermal extraction, lipid separation single cell oil production in yeast
and, 363, 365 and, 215–216
INDEX 677
tricarboxylic acid (TCA) cycle, 209, and, 155–156, 156e, 166–168;

211, 212–213, 430 advantages of, 171; economic
impacts, 151–152; factors affecting,
ultra-low sulfur diesel (ULSD), 137, 152–156, 154t, 155f, 156e; physical
586, 590, 591 and chemical properties of,

ultrasonification/ultrasonic 152–153, 154t; process of, 41, 42f;
harvesting: lipid extraction and sources of, 152; techno-economic
separation, 360, 365, 370; evaluation of, 534–535, 534t;
microalgae and, 188, 192; oil/fat transesterification and, 156–165,
transesterification to biodiesel and, 156f, 158f, 159f, 162f, 163t
38; from wastewater sludge, waste stream effluents, single cell oil
273–274, 273f and, 207, 210
United States: biodiesel policies, wastes, case study on use in biodiesel
655–656; biodiesel economy in, purification, 492, 493t, 494
574, 576; biodiesel production in, 1, wastewater: as byproduct or biodiesel
16, 123, 140–142, 152, 219, production, 506, 508; lipase enzyme
657–658; economic and, 528–529, production and, 59–60
574–575; Energy Policy Act of wastewater sludge, 263–285; biodiesel
2005, 2, 123, 528, 655; environment from fermented lipids, 289–300;
and, 603, 605–606, 625, 629; biodiesel production from
ethanol and, 528; feedstocks, 54–55, oleaginous microorganisms,
89, 91, 127, 564–565; iodine value 242–243; challenges of and future
(IV), 5 perspective, 283–284, 293–299,
universal soil loss equation (USLE), 300; characteristics of, 266, 267t,
613, 617 290, 291t; disposal methods,
264–266, 264t, 265t; extraction
vectors, gene transfer via, 422–423 processes for sludge-derived lipids,
vegetable oils and fats: biodiesel 291–293, 292f, 293f; extraction
production from, 42f; techno- strategies for, 263–264, 264f; factors
economic evaluation of, 531, 532t, affecting lipid production, 293–296,
533, 533f. See also waste cooking oil 294t, 295e, 296e, 297t; lipid
(WCO), biodiesel from accumulation in oleaginous
Venezuela, 657–658 microorganisms, 293, 298;
viral vectors, gene transfer and, 423 mechanical-chemical extraction,
virgin oil: techno-economic 273–277, 273f, 275f, 275t, 276f;
evaluation, 535; waste cooking oils products from, 266; pros and cons
compared, 154t, 166–169, 168t of extraction methods, 277, 278t;
viscosity, 4; cold flow properties, 5; pyrolysis (thermochemical)
free fatty acids and, 134; kinematic, extraction, 266, 268–269, 268t,
18, 23, 135, 168; tallow products 269f, 270–271t, 272–273; sludge-
and, 126–127; vegetable oil and based oils compared to other oils,
diesel fuel, 56 277, 279, 279t, 280t, 281t; techno-
economic evaluation of extraction
waste cooking oil (WCO), biodiesel methods, 279, 281–283, 281t, 282t,
from, 55, 151–171, 242; acid value 538; types of, 263, 290
678 INDEX
water: biodiesel purification and, water washing: techno-economic

459, 459f; as contaminant, evaluation, 551, 558–559, 558f, 558t,
22–23; enzyme-catalyzed 559t; waste cooking oils and, 165
transesterification and, 66; fuel wet washing: biodiesel purification
degradation and, 3; oil/fat and, 458–460, 459f; purification

transesterification to biodiesel and, comparative studies, 494–496, 495t,
37–38 497t, 498, 498t, 499t, 500t; techno-
water resources, biodiesel’s effect on, economic evaluation, 551, 554,
601, 640; public health and, 554f, 555t, 556–557, 556f, 556t,
630–631; water availability, 557f, 557t, 558f, 558t, 559t
602–605, 602t, 603f; water
pollution, 605–606, 607–611t zinc finger nucleases (ZFNs), 426

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Biodiesel Production: Technologies, Challenges,

R. D. Tyagi, Rao Y. Surampalli,

Published by the American Society of Civil Engineers

of the Technical Committee on Hazardous, Toxic, and Radioactive Waste Engineering

Chapter 1 Biodiesel Basics ....................................................................................................1

Chapter 2 Biodiesel: Variations, Properties, and Comparison with

Chapter 3 Biodiesel Production by Transesteriﬁcation ...................................27

Chapter 4 Enzyme-Catalyzed Transesteriﬁcation for Biodiesel

4.4 Cost Analysis of Immobilized Lipase-Based Transesteriﬁcation .......... 80

Chapter 5 Plant Oil to Biodiesel.....................................................................................89

Chapter 6 Animal Fat Biodiesel ................................................................................... 123

Chapter 7 Biodiesel from Waste Cooking Oil ..................................................... 151

Chapter 8 Microalgae Oil Biodiesel ........................................................................... 175

Chapter 9 Single Cell Oil Biodiesel............................................................................ 201

9.4 Substrates Utilized for Single Cell Oil Production....................................204

Chapter 10 Biodiesel Production from Oleaginous Microorganisms

Chapter 11 Oil Extraction Using Wastewater Sludge .................................... 263

Chapter 12 Biodiesel Production Using Fermented Wastewater

12.5 Lipid Accumulation in Oleaginous Microorganisms Fed with

Chapter 13 Conversion of Crude Glycerol to Lipid and Biodiesel ........ 305

Chapter 14 Lignocellulosic Biomass: The Future Renewable

Chapter 15 Lipid Extraction Technologies............................................................ 359

Chapter 16 Milking of Lipids from Oleaginous Microorganisms ........... 383

Chapter 17 Application of Nanotechnology in Biodiesel

17.3 Lipid Extraction Using Nanotechnology .....................................................399

Chapter 18 Genetic/Metabolic Engineering and Synthetic Biology

Chapter 19 Recovery and Puriﬁcation Technologies of Biodiesel......... 453

Chapter 20 Puriﬁcation of Biodiesel Using Resins and Adsorbents .... 485

Chapter 21 Management of Coproducts from Biodiesel Production ..... 503

21.3 Coproduct Management.....................................................................................508

Chapter 22 Economic Evaluation of Biodiesel Production

Chapter 23 Biodiesel Impact on Environment and Health ........................ 583

Chapter 24 Biodiesel: Socioeconomic and Political Aspects ..................... 653

environment and human well-being, and Chapter 24 presents socioeconomic and

contributed to this book.

G. S. Anisha, Government College, Chittur, Palakkad, India

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R. D. Tyagi, Ph.D., is an internationally recognized professor at Institut National

Energy, Environment and Water Policy. He is a Distinguished Engineering

Member of the Russian Academy of Engineering, an elected Fellow of the Water

Journal of Environmental Engineering in 2012 and holds membership in Canadian

INRS University of Québec in 2013. She is currently working at the Harbin

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1.1 BIODIESEL HISTORY

environmental pollution, net carbon emissions, and dependence on fossil fuels,

similar phenomenon of a rapid increase in biodiesel production was observed

1.2 PROPERTIES OF BIODIESEL

1.2.1 Chemical Composition

1.2.2 Cetane Number

1.2.3 Oxidative Stability

1.2.4 Heat of Combustion

1.2.5 Density of Biodiesel

1.2.6 Viscosity of Biodiesel

1.2.7 Cold Flow Properties

1.2.8 Iodine Value

1.3 BIODIESEL STANDARDS

The produced biodiesel is tested to determine fuel characteristics based on ASTM

1.4 BIODIESEL VERSUS OTHER BIOREFINERY PRODUCTS

Biodiesel and other bioreﬁnery products, such as bioethanol, have similar

Flash point (°C) >101 — ISO CD 3679e 130 — D 93

Monoglyceride content (% by — 0.80 Pr EN 14105m — — —

1.5 BIODIESEL BLENDING, STORAGE, AND TRANSPORT