Industrial Oil Crops

Industrial Oil Crops

Editors

Thomas A. McKeon

United States Department of Agriculture, Agricultural Research Service, Western Regional Research Center, Albany, CA, USA

Douglas G. Hayes

Department of Biosystems Engineering and Soil Science, University of Tennessee, Knoxville, TN, USA

David F. Hildebrand

Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, USA

Randall J. Weselake

Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton, AB, Canada

Table of Contents

Cover image

Title page

Copyright

List of Contributors

Editor Biographies

Preface

Chapter 1. Introduction to Industrial Oil Crops

Introduction

What Is an Industrial Oil Crop?

Why Do We Care About Industrial Seed Oils?

Where Do Industrial Oils Come From?

How Are Industrial Oil Crops Improved?

Genetic Engineering of Fatty Acid Biosynthesis

Genetic Engineering of Oil Biosynthesis

Technical and Social Issues Related to Plant Genetic Engineering

Conclusions

Chapter 2. Biodiesel and Its Properties

Introduction

Historical Background

Biodiesel Production

Biodiesel Analysis

Biodiesel Properties

Disclaimer

Chapter 3. Polymeric Products Derived From Industrial Oils for Paints, Coatings, and Other Applications

Introduction

Preparation of New Monomers From Unsaturated and Polyunsaturated Fatty Acids

Preparation of Polyols From Unsaturated Fatty Acids

Conjugated Oils as Coatings: Direct Free Radical Polymerization

Cationic Polymerization of Unsaturated Oils

Metathesis Polymerization of Unsaturated Oils

Vulcanization of Polyunsaturated Oils

Estolides and Polycondensation Products From Hydroxy Fatty Acids

Conclusions

Chapter 4. Castor (Ricinus communis L.)

Introduction

Agronomy

Castor Breeding

Ricin and RCA

Ricin Detection

Castor Seed Allergen

Processing

Castor Oil Biochemistry and Molecular Biology

Castor Oil Production and Applications

Miscellaneous Uses of the Castor Plant

Summary

Chapter 5. Brassica spp. Oils

Introduction

Characteristics of Brassica Oilseed Species and Industrial Uses of Their Oils

Production of Brassica Oilseed Species

Breeding of Brassica Oilseed Species

Brassica Seed Oil as a Feedstock for Biodiesel Production

Chemical Modification of Brassica Oils for Other Industrial Applications

Genetic Engineering of Brassica Oilseed Species to Produce Industrial Oils

Closing Comments

Chapter 6. Flax (Linum usitatissimum L.)

Introduction

Differentiation of Fiber Flax and Oilseed Flax

Flax Genetics and Breeding

Flaxseed Oil

Closing Comments

Chapter 7. Crambe (Crambe abyssinica)

Origin and History

Crambe Botany

Seed Composition

Crambe as an Industrial Oil Crop

Crambe Cultivation

Genetic Resources and Conventional Breeding

Crambe Improvement Using Gene Technology

Future Perspectives

Chapter 8. Camelina (Camelina sativa)

Introduction

Camelina Production as an Oil Seed Crop

Camelina Oil Composition and Stability

Camelina Meal Quality and Uses in Livestock

Antinutritive Compounds in Camelina Meal

Camelina Genome

Agrobacterium-Mediated Transformation of Camelina

Outcrossing Potential for Camelina

Genetically Engineering Camelina Fatty Acid Composition

Increasing the Agronomic Potential of Camelina

Conclusion

Chapter 9. Coconut (Cocos nucifera)

Introduction

Botany, Cultivation and Production

Yields and Production

Coconut Oil

Emerging Applications

Premium Grade Monolaurin and HIV/AIDS

Chapter 10. Tung (Vernicia fordii and Vernicia montana)

Introduction

The History of Tung in the United States: The Tung Belt

Research on Tung

Tung’s Demise in the United States

Industrial Utilization of Tung Oil

Tung Trees Meet the Molecular Age

Conclusions

Chapter 11. Emerging Industrial Oil Crops

Introduction

Chapter 11.1. Chia (Salvia hispanica L.)

Chapter 11.2. Cuphea (Cuphea spp.)

Chapter 11.3. Hemp (Cannabis sativa L.)

Chapter 11.4. Jatropha (Jatropha curcas L.)

Chapter 11.5. Jojoba (Simmondsia chinensis)

Chapter 11.6. Lesquerella (Physaria spp.)

Chapter 11.7. Meadowfoam (Limnanthes alba)

Chapter 11.8. Pennycress (Thlapsi spp.)

Chapter 11.9. Perilla (Perilla frutescens)

Chapter 11.10. Pili (Canarium ovatum)

Chapter 11.11. Epilogue

Chapter 12. Successful Commercialization of Industrial Oil Crops

Introduction

Grow

Make

Sell

Conclusion

Chapter 13. Applications of Doubled Haploidy for Improving Industrial Oilseeds

Introduction

DH Methodology

Brassica Species

Other Cruciferous Species

Apiaceae Species

Compositae Species

Other Species

Conclusion

Chapter 14. Genetic Transformation of Crops for Oil Production

Introduction

Multipurpose and Technical Oils

Oils—An Essential Dietary Requirement for Humans

Plant Oils as an Alternate to Petroleum—Biofuels

Oilseed Crop Platforms for Animal Feedstock

Conventional Methods of Oil Crop Improvement

Recombinant DNA Technology—Methods and Tools for Obtaining Transgenic Plants

Genetic Transformation of Oil Crops for Sustainable Development

Soybean

Oil Palm

Rapeseed

Castor

Sunflower

Cotton

Flax

Maize

Jatropha

Camelina

Transgenic Oil Crops—Means and Challenges

Development of Clean Marker-Free Transgenic Plants

Targeted Genome Engineering for Locus-Specific Modifications

The Debate on GM Crops

Oil Crops of the Future

Conclusion

Chapter 15. Engineering Oil Accumulation in Vegetative Tissue

Introduction

Overview of Acyl-Lipid Metabolism in Developing Seeds and Leaves

Transcription Factors Promote Increased Triacylglycerol Production in Vegetative Tissue

Increasing the Availability of Building Blocks for Triacylglycerol Assembly by Modifying Enzyme Action

Increasing Triacylglycerol Assembly

Decreasing Triacylglycerol Turnover

Multigene Strategies for Increasing Triacylglycerol Content in Vegetative Tissue

Enriching Leaf Triacylglycerol in Industrially Useful Fatty Acids

Engineering C4 High-Biomass Plants to Produce Triacylglycerol in Vegetative Tissue

Conclusion

Index

Copyright

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List of Contributors

Maythem AL–Amery,     Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, United States

Helen Booker,     Crop Development Centre, Department of Plant Sciences, University of Saskatchewan, Saskatoon, SK, Canada

Karen L. Caswell,     National Research Council, Saskatoon, SK, Canada

Guanqun Chen,     Department of Biological Sciences, University of Manitoba, Winnipeg, Manitoba, Canada

Yicun Chen,     Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, Zhejiang, China

Jonathan Curtis,     Agricultural Lipid Biotechnology Program, Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton, AB, Canada

Marie-Josée Dumont,     Department of Bioresource Engineering, McGill University, Ste-Anne-de-Bellevue, QC, Canada

Alison M.R. Ferrie,     National Research Council, Saskatoon, SK, Canada

Hirotada Fukushige,     Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, United States

Linda M. Hall,     Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada

Douglas G. Hayes,     Department of Biosystems Engineering and Soil Science, University of Tennessee, Knoxville, TN, United States

Andrew Hebard,     Technology Crops International, Winston-Salem, NC, United States

David F. Hildebrand,     Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, United States

Amit J. Jhala

Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada

Department of Agronomy and Horticulture, University of Nebraska-Lincoln, Lincoln, NE, United States

Gerhard Knothe,     U.S. Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research, Peoria, IL, United States

Igor Kovalchuk,     University of Lethbridge, Lethbridge, AB, Canada

Hu Lisong,     Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, Zhejiang, China

Priti Maheshwari,     Lethbridge Research Station, Lethbridge, AB, Canada

Thomas A. McKeon,     United States Department of Agriculture, Agricultural Research Service, Western Regional Research Center, Albany, CA, United States

Peter B.E. McVetty,     Department of Plant Science, University of Manitoba, Winnipeg, MB, Canada

Elzbieta Mietkiewska,     Agricultural Lipid Biotechnology Program, Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton, AB, Canada

Eric J. Murphy

Department of Basic Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND, United States

Agragen, LLC, Cincinnati, OH, United States

Agragen OY, Helsinki, Finland

Tolibjon Omonov,     Agricultural Lipid Biotechnology Program, Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton, AB, Canada

Shreya Patel,     Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, United States

Laura J. Pham,     Oils and Fats Laboratory, National Institute of Molecular Biology and Biotechnology (BIOTECH), University of the Philippines at Los Baños, Laguna, Philippines

Tim Phillips,     Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, United States

Timothy Rinehart,     United States Department of Agriculture-Agricultural Research Service, Thad Cochran Southern Horticultural Laboratory, Poplarville, MS, United States

William Serson,     Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, United States

Jay Shockey,     United States Department of Agriculture-Agricultural Research Service, Southern Regional Research Center, New Orleans, LA, United States

Rodrigo M.P. Siloto,     Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada

David C. Taylor,     National Research Council of Canada, Saskatoon, SK, Canada

Randall J. Weselake,     Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton, AB, Canada

Wang Yangdong,     Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, Zhejiang, China

Zhan Zhiyong,     Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, Zhejiang, China

Li-Hua Zhu,     Department of Plant Breeding, Swedish University of Agricultural Sciences, Alnarp, Sweden

Editor Biographies

Thomas A. McKeon is a research chemist at the Western Regional Research Center of the U.S. Department of Agriculture, Agricultural Research Service (USDA-ARS WRRC) in Albany, CA. He received his bachelor’s of science in chemistry from Worcester Polytechnic Institute and PhD in biochemistry from the University of California at Berkeley. He was a postdoctoral associate in plant lipid enzymology in the Department of Biochemistry and Biophysics at UC Davis and visiting scientist in postharvest biochemistry and ethylene regulation in The Department of Vegetable Crops at UC Davis. He started his career at WRRC as a research chemist in 1981, initially in the area of postharvest biochemistry and starting research on castor oil biosynthesis and ricin detection in 1992. He has published 120 papers in peer-reviewed journals and book chapters. Tom has served as a division officer for the American Oil Chemists Society (AOCS) Biotechnology (BIO) Division from 2004 to 2010 and continues to serve the BIO Board on special assignment. He has served on the AOCS Books and Special Publications Committee and on the AOCS Board of Directors (2008–2010). He also served on the secretariat of the US-Japan Natural Resources (UJNR) Food and Agriculture Panel from 2006 to 2015. Tom is an editor for Biocatalysis and Agricultural Biotechnology and board member for the International Society for Biocatalysis and Agricultural Biotechnology (ISBAB). His current research interests include development of technologies to improve the processing of castor seed and elucidation of enzymology that regulates isoprenoid synthesis in plants.

Douglas G. Hayes is a professor of biosystems engineering at the University of Tennessee (UT). Doug received his bachelor’s of science and PhD in chemical engineering at Iowa State University (1986) and the University of Michigan (1991), respectively, and served as a postdoctoral research chemist at the USDA/ARS/NCAUR (Peoria, IL) from 1991 to 1994. Doug served as a faculty member in the Department of Chemical and Materials Engineering at the University of Alabama in Huntsville before joining UT in 2004. Doug also holds an adjunct professorship in the UT Department of Chemical and Biomolecular Engineering and guest professorships at Wuhan Polytechnic University and Jinan University in China and is a UT–Oak Ridge National Laboratory Joint Faculty. Doug is a senior associate editor (SAE) for the Journal of the American Oil Chemists’ Society and an AE for Journal of Surfactants and Detergents. Doug has served AOCS as an officer for the Biotechnology Division (Chair from 2014–2015), chair of the AOCS Professional Educators’ Common InterestGroup, and a member of the AOCS Books and Special Publications Committee. Doug has published over 65 articles in peer-reviewed publications, 15 book chapters, and one edited book. Doug’s research interests include applied biocatalysis, biobased surfactants and polymers, soft matter colloidal systems, and small-angle scattering methods.

David Floyd Hildebrand is a professor of plant & soil sciences at the University of Kentucky, Lexington, KY. David earned a BS in chemistry from the University of Maryland in 1977 and a second BS in agronomy, also from the University of Maryland. David earned an MS in plant breeding and genetics from the University of Illinois in 1980 and a PhD in plant breeding and genetics, also from the University of Illinois in 1982. David worked at the Kyoto University Research Institute for Food Science under a Japanese Society for the Promotion of Science Fellowship. David’s research focuses on oil biosynthesis, breeding, and metabolic engineering of plants for improved edible and industrial uses. He has authored or co-authored more than 150 publications and 10 patents. David serves on several editorial boards including the JAOCS and Biocatalysis and Agricultural Biotechnology and served as the chairperson of the AOCS biotechnology division. David’s recent and current teaching is a graduate-level plant biochemistry course, http://www.uky.edu/∼dhild/biochem/welcome.html, and an introductory course on Food, Agriculture, Energy and the Environment.

Randall J. Weselake is a professor and Tier I Canada Research Chair in Agricultural Lipid Biotechnology in the Department of Agricultural, Food and Nutritional Science at the University of Alberta (Edmonton, Alberta, Canada). Since 1987, when he was a research associate at the National Research Council of Canada in Saskatoon, Randall’s research has focused on the biochemistry and molecular biology of storage lipid metabolism in Brassica napus and other oil crops. Recently, he served as scientific director of the Alberta Innovates Phytola Center, which specializes in oilseed innovations, including research on the development of industrial oil crops. From 2007 to 2013, Randall was leader of the Bioactive Oils Program funded by AVAC Ltd., and from 2006 to 2011, he was co-leader of the Genome Canada/Genome Alberta project Designing Oilseeds for Tomorrow’s Markets. From 1989 to 2004, Randall was with the Department of Chemistry and Biochemistry at the University of Lethbridge (Alberta, Canada), serving as chair from 1996 to 1999. His doctoral research in plant biochemistry was conducted at the University of Manitoba and Grain Research Laboratory of the Canadian Grain Commission. Randall is joint editor-in-chief of the American Oil Chemists’ Society (AOCS) Lipid Library, associate editor for Lipids, and editor for Biocatalysis and Agricultural Biotechnology. Randall has published more than 160 papers in refereed journals and books and is editor of the book Teaching Innovations in Lipid Science, which was published by the Taylor & Francis Group of the CRC Press and the AOCS Press. He is a fellow of both the AOCS and the International Society of Biocatalysis and Agricultural Biotechnology.

Preface

While seed oils and other vegetable oils are mostly thought of as food ingredients, many of these oils are also used for nonfood industrial applications. This book describes plants that produce oils that are chiefly used for industrial purposes and provides relevant information about the seeds and vegetative parts that produce oils and their uses. The initial chapters focus on key applications of industrial oils which include biodiesel polymers, paints, and coatings. Subsequent chapters deal with long-established crops, and entire chapters are devoted to these. Over the years, many plants that produce oils well suited to industrial use have been identified. Often these were wild or decorative species, referred to as new crops, which are not well suited to large-scale production or cultivation without considerable agronomic improvement. We have identified and described the more prominent new crops in the subchapters collected in the chapter entitled Emerging Oil Crops. This chapter is followed by a case study on the economic and marketing strategies underlying successful introduction of a new crop, in this case, the Ahiflower™.

In addition to describing the properties and uses of industrial oil crops, many chapters include information about attempts to genetically engineer the crop or to transfer genes from the industrial crop to a more manageable transgenic crop with more favorable agronomic characteristics. Some of the novel technologies developed for oil crop modification are more closely examined and explained in chapters specifically dedicated to these topics. The application of these and related technologies are brought to fruition in the final chapter, which is devoted to the development of crops that produce oil throughout the vegetative portion of the plant.

This volume went through several evolutionary steps. We would like to thank our current management team, Janet Brown and Lori Weidert of AOCS Press as well as Caroline Johnson of Elsevier, who has shepherded the final version for AOCS in conjunction with Elsevier Press. We would also like to thank the AOCS Press and the AOCS Books and Special Publications Committee, especially Magdi Mossoba, former Committee Chair for continuing patience and support from the early stages of the book’s development. We hope this volume serves as a valued source of information in the area of industrial oil crops and believe that expanded cultivation of the crops described can play a significant role in replacing some petroleum-based products with products derived from these renewable resources.

Chapter 1

Introduction to Industrial Oil Crops

Thomas A. McKeon     United States Department of Agriculture, Agricultural Research Service, Western Regional Research Center, Albany, CA, United States

Douglas G. Hayes     Department of Biosystems Engineering and Soil Science, University of Tennessee, Knoxville, TN, United States

David F. Hildebrand     Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, United States

Randall J. Weselake     Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton, AB, Canada

Abstract

Vegetable oils are derived from the seeds or fruit of certain crops and are most often used for food or animal feed. These oils are composed of triacylglycerols (TAG) and trace amounts of various organic compounds including sterols and antioxidants. For most of the commodity vegetable oils, the TAG are acylated with varying proportions of the same five fatty acids, the two saturates palmitate (16:0) and stearate (18:0), the monounsaturate oleate (18:1Δ⁹), and the polyunsaturates linoleate (18:2Δ⁹,¹²) and α-linolenate (18:3Δ⁹,¹²,¹⁵) (double bonds all cis) (Fig. 1.1). Even though they are primarily consumed as food, for thousands of years the oils containing these fatty acids have also served in certain nonfood applications. The oils, hydrolyzates of the oils, or alkyl esters prepared from transesterification of the oils have been used in producing fuel for lighting, lubricants, soaps, cosmetics, and lacquers. These end uses have been supplied by seed oils including what are now major commodity oils derived from palm (e.g., Elaeis guineensis), soybean (Glycine max), canola (Brassica napus), sunflower (Helianthus annuus), cottonseed (Gossypium hirsutum), olive (Olea europaea), corn (Zea mays), peanut (Arachis hypogaea), and other seeds, as well as animal fats.

Keywords

Fatty acids; Fatty acid biosynthesis; Genetic engineering; Industrial oil crops; Oil crops; Seed oils; Triacylglycerols; Vegetable oils

Introduction

Vegetable oils are derived from the seeds or fruit of certain crops and are most often used for food or animal feed. These oils are composed of triacylglycerols (TAG) and trace amounts of various organic compounds including sterols and antioxidants. For most of the commodity vegetable oils, the TAG are acylated with varying proportions of the same five fatty acids, the two saturates palmitate (16:0) and stearate (18:0), the monounsaturate oleate (18:1Δ⁹), and the polyunsaturates linoleate (18:2Δ⁹,¹²) and α-linolenate (18:3Δ⁹,¹²,¹⁵) (double bonds all cis) (Fig. 1.1). Even though they are primarily consumed as food, for thousands of years the oils containing these fatty acids have also served in certain nonfood applications. The oils, hydrolyzates of the oils, or alkyl esters prepared from transesterification of the oils have been used in producing fuel for lighting, lubricants, soaps, cosmetics, and lacquers. These end uses have been supplied by seed oils including what are now major commodity oils derived from palm (eg, Elaeis guineensis), soybean (Glycine max), canola (Brassica napus), sunflower (Helianthus annuus), cottonseed (Gossypium hirsutum), olive (Olea europaea), corn (Zea mays), peanut (Arachis hypogaea), and other seeds, as well as animal fats.

Figure 1.1  Fatty acids present in vegetable oils used primarily for food.

What Is an Industrial Oil Crop?

While any seed oil can fill certain nonfood applications, there are hundreds of seed oils containing a different complement of fatty acids that impart physical and chemical properties making the oil and associated fatty acids especially useful for industrial and other nonfood uses. These differences may lie in chain length, degree or nature of unsaturation, or the presence of unusual functional groups on the fatty acid chain. While some of these oils are also valued for food, such as coconut (Cocos nucifera) (see chapter: Coconut (Cocos nucifera)) and palm kernel oils with high laurate (12:0) content (Fig. 1.2), and linseed oil with high linolenate content (see chapter: Flax (Linum usitatissimum L.)), there are other oils with no nutritive value such as castor oil (see chapter: Castor (Ricinus communis L.)), which is composed of 90% ricinoleate (12-OH 18:1Δ⁹; Fig. 1.2), and tung oil (Aleurites fordii) (see chapter: Tung (Vernicia fordii and Vernicia montana)), which is composed of 80% eleostearate (18:3Δ⁹cis,11trans,13trans; Fig. 1.2). These oils are uniquely valuable for industrial applications due to their chemical structure. This book will provide descriptions and uses of the most important and widely used oils mentioned above and examine progress in expanding production of some industrial oil crops.

Figure 1.2  Some fatty acids present in seed oils used for industrial applications.

Table 1.1

World Oil Consumption, 2014a

a http://www.bp.com/en/global/corporate/about-bp/energy-economics/statistical-review-of-world-energy/review-by-energy-type/oil/oil-production.html.

Why Do We Care About Industrial Seed Oils?

More than 33 billion barrels of petroleum are used annually worldwide (Table 1.1). While the primary use of petroleum is in fuel and energy production, up to 30% is used for a range of products including polymers, lubricants, detergents, resins, paints, plasticizers and coatings, cosmetics, inks, solvents, and assorted other chemicals. Seed oils can provide products equal or superior to those derived from petroleum, as the conserved chemical structure of the different oils provides a more uniform starting material with less refining needed. A comparison of some products derived from petroleum with those derived from seed oils is provided in Table 1.2. A key difference of these products is the biodegradability of many of those derived from seed oils, even after chemical modifications for specific uses. This property is especially important in reducing environmental contamination of soil and water. Contamination of soil, aquifers, and waterways resulting from spills of petroleum-based products poses a long-term problem requiring great expense for cleanups or detrimental health effects for local populations. Use of renewable oils such as seed oils to replace petroleum products is a major goal of research in sustainable development, and products currently derived from these oils include a broad representation of the types currently provided by petroleum. Specific applications including biodiesel (see chapter: Biodiesel and Its Properties) and polymers and coatings (see chapter: Polymeric Products Derived From Industrial Oils for Paints, Coatings, and Other Applications) will be covered in separate chapters in this volume. Other products, applications, and sources will be covered throughout this volume in chapters describing individual crops.

Table 1.2

Comparison of Products Derived From Petroleum and Seed Oils

Where Do Industrial Oils Come From?

While some plants produce seeds with a high starch content to provide energy for germination, some seeds store and utilize TAG for the germinating seed. These oilseeds utilize oxidation of fatty acids derived from the oil to produce energy for germination and early growth. Oils that carry uncommon fatty acids such as those described earlier are a result of an evolutionary process that may provide the seed with a selective advantage resulting from a protective effect against disease or predation. While the time scale of evolution may be very long, the process has provided a rich collection of resources containing potentially useful fatty acids. In many cases, though, the plants that produce these fatty acids are not suitable as crops. The adoption of these plants as crops is stymied by the need for growers to have a stable market and for the industrial user to have a stable supply of the crop. Moreover, additional research is usually needed to develop plants that are better adapted to cultivation, harvesting, and high yield. Ultimately, a commercial sponsor is required to enable the adoption of such crops. The challenges and economics of introducing these emerging oilseed crops are presented in Chapters Emerging Industrial Oil Crops and Successful Commercialization of Industrial Oil Crops, respectively.

How Are Industrial Oil Crops Improved?

Breeding

Traditional breeding techniques have been used to alter levels of fatty acids present in seed oil. An important example is the development of canola from rapeseed (B. napus), which resulted in a reduction of erucic acid (22:1Δ¹³) content from ∼50% to <2% in the seed oil. The success of this breeding resulted from the availability of suitable germplasm that could provide a low-erucate background for incorporation into highly productive rapeseed varieties (Stefansson et al., 1961; Downey, 1964) (see chapter: Brassica spp. Oils). Breeding can also be used to enhance the content of specific fatty acids. The development of high-oleic safflower oil (Carthamus tinctorius) is an early example (Knowles, 1985), with success based on identification of a single locus associated with oleate content. Such enrichment is especially valued for industrially significant fatty acids as it reduces the cost of purifying the desired component. Traditional methods of self-crossing can require 5–10  years to develop homozygous strains to incorporate in a breeding program. However, there is technology for producing haploid strains of crops in tissue culture and then doubling their chromosomes to provide homozygous strains within 1  year, greatly accelerating the ability to breed and screen for crop improvement (see chapter: Applications of Doubled Haploidy for Improving Industrial Oilseeds).

While traditional breeding relies on screening of physical and chemical traits, the introduction of marker-based breeding expanded the ability of breeding programs to screen crosses. Molecular markers such as restriction and amplified fragment length polymorphisms are used to generate quantitative trait loci tied to desired traits. This molecular marker approach in breeding has proven especially useful evaluating crosses in tree crops such as palm that take several years to produce seed (Singh et al., 2009). The availability of economical genome sequencing introduced the application of single nucleotide polymorphisms and high-throughput sequencing in breeding programs (Wilson, 2012). However, even with such advances, breeding ultimately relies on the availability of suitable germplasm, and traditional breeding cannot introduce a new fatty acid or any other component not already present in the genetic background of the plants used in crosses.

Mutagenesis

Mutagenesis using chemical agents or radiation has also been effective in modifying fatty acid composition through alteration of the genome followed by screening and breeding (Knowles, 1985). Mutagenesis is especially useful in eliminating genes or reducing or blocking gene expression, and has been successfully used to produce high-linoleate linseed (Linum usitatissimum) oil (Green, 1986) (see chapter: Flax (Linum usitatissimum L.)). The biosynthetic map of plant fatty acid and lipid biosynthesis was developed through screening of mutagenized Arabidopsis thaliana (Browse and Somerville, 1991), and led to the identification and cloning of genes involved in fatty acid and oil biosynthesis. The identification of genes encoding enzymes involved in fatty acid biosynthesis provided the foundation for the application of Targeting Induced Local Lesions IN Genomes (TILLING) to modify fatty acid composition. TILLING relies on mutagenesis followed by high-throughput DNA sequence screening of the M2 generation seeds to identify those seeds that carry stop codons in the sequence of the gene to be silenced (Henikoff and Comai, 2003). Plant selections carrying these mutated genes can then be screened directly for desired characteristics. The TILLING process thus moves most of the screening effort into the laboratory, considerably reducing the population that would otherwise have to be grown in the field for phenotypic screening.

Although not specifically a form of mutagenesis, gene suppression has also proven to be an effective means for altering fatty acid composition. Gene suppression was initially discovered as an occasional byproduct of gene overexpression, and was useful in altering fatty acid composition of Brassica seed oil (Knutzon et al., 1992). Usually, the correct or sense strand of DNA is transcribed to provide the mRNA that will be translated into the desired protein, but in such gene-suppressed plants, the wrong or antisense strand of DNA is transcribed, limiting protein synthesis from the sense mRNA. Direct use of antisense technology led to the discovery that expression of small pieces of RNA can suppress gene expression (Smith et al., 2000). This RNA silencing technology has been used to develop crops ranging from tomatoes with delayed softening during ripening to high-oleic acid soybeans (Frizzi and Huang, 2010) (see chapter: Genetic Transformation of Crops for Oil Production).

Genetic Engineering of Fatty Acid Biosynthesis

Years of biochemical characterization of fatty acid and oil biosynthesis provided the foundation for rapid advances in identification and cloning of genes involved in these processes. In order to modify the fatty acid composition of seed oil through genetic engineering, it is necessary to understand the processes involved in oil biosynthesis. A combination of biochemical characterization of enzymes involved in fatty acid and oil biosynthesis plus generation of mutants in lipid biosynthesis provided the means to identify genes essential for directing the synthesis of the desired fatty acids and their incorporation in oil (Browse and Somerville, 1991). The widespread availability of information on genes that direct biosynthesis of certain fatty acids are key steps in oil biosynthesis providing the means to introduce novel activities and components into oilseeds (see chapter: Genetic Transformation of Crops for Oil Production).

Fatty acid biosynthesis in plants starts with acetyl-CoA carboxylation to malonyl-CoA through the catalytic action of the acetyl-CoA carboxylase (ACCase) followed by a series of condensation reactions that result in production of long chain saturated fatty acids. Malonyl-CoA is converted to the corresponding acyl carrier protein (ACP) derivative, malonyl-ACP, and the first condensation is initiated by the keto-acyl synthase (KAS) III using acetyl-CoA to initiate the condensation with malonyl-ACP. This condensation cycle is followed by six additional condensations with malonyl-ACP catalyzed by KAS I to yield palmitoyl-ACP. This may be followed by an additional condensation catalyzed by KAS II to yield stearoyl-ACP, as depicted in Fig. 1.3. A fatty acid thioesterase can then release free palmitate or stearate which are converted to acyl-CoA derivatives and incorporated into TAG and other glycerolipids. The stearoyl-ACP can also be desaturated to oleoyl-ACP, the oleate released by thioesterase action and converted to oleoyl-CoA. The oleoyl-CoA is then incorporated into glycerolipids for incorporation into TAG or further modification. These reactions leading to palmitate, stearate, and oleate occur in the plastids, separate from reactions leading to oil biosynthesis. Given the dependence of fatty acid production on malonyl-CoA production (to provide malonyl-ACP), ACCase is thought to play a regulatory role in fatty acid production and oil biosynthesis (Weselake et al., 2009).

The first commercial plant engineered to produce an industrial useful fatty acid was high-laurate B. napus (Voelker et al., 1992). Plants that make oils containing medium chain fatty acids such as California bay laurel (Umbellularia californica) and coconut have a fatty acid thioesterase that catalyzes the release of fatty acyl chains from the fatty acid synthesis complex after four or five condensation steps carried out by the catalytic action of KAS I. When the gene for such a thioesterase was inserted and expressed in B. napus, the result was a canola seed with oil biosynthesis rerouted to produce oil containing over 50% laurate.

Figure 1.3  Pathway for fatty acid biosynthesis in plants.

Genetic Engineering of Oil Biosynthesis

The introduction of genes that modify the fatty acid composition of oil have been essential to identifying specific steps in the oil biosynthetic pathway (TAG assembly) that control the amount and the composition of the oil produced by the seed. The engineering required to develop the high-laurate B. napus (Voelker et al., 1996) made it clear that simple insertion of a gene to alter fatty acid biosynthesis or modify a fatty acid (Broun and Somerville, 1997) is not sufficient to significantly alter the fatty acid composition of the oil. For example, in B. napus, insertion of the coconut lyso-phosphatidate acylCoA acyltransferase (LPAAT) gene was required for acylation of the sn-2 position of TAG, thereby increasing laurate content (Voelker et al., 1996) because expression of the California bay laurel thioesterase alone in B. napus resulted in acylation of only the sn-1 and sn-3 positions of TAG.

The pathway for TAG assembly from plastid-generated acyl-CoAs (Fig. 1.3) is presented in Fig. 1.4. The Kennedy pathway for TAG assembly was driven by thermodynamically favored reactions, namely acylation using acyl-CoA or ATP to form thioesters.

Considerable research on oil biosynthesis has demonstrated additional layers of complexity in TAG assembly (Bates et al., 2013). The acyl-CoA can be elongated on the ER or incorporated into glycerolipids or phospholipids. TAG can be synthesized from DAG via the acyl-CoA-dependent DGAT. The LPCAT incorporates an acyl group from acyl-CoA into the sn-2 position to form PtdC, an important component of membranes and a substrate for desaturation or fatty acid modification of the sn-2 fatty acyl. The sn-2 acyl-group on PC can be released by PLA2 and the free fatty acid converted to acyl-CoA or the sn-2 acyl-group can be enzymatically transferred by PDAT to DAG, forming TAG. In each case, the other product is LPC which is then available for re-acylation by LPCAT. The glycerol backbone is derived from sn-glycerol-3-phosphate which is subject to sequential acylations to produce phosphatidic acid. Phosphatidic acid phosphatase catalyzes the dephosphorylation of phosphatidic acid to produce sn-1, 2-DAG (DAG). DAG can also be converted to phosphatidylcholine (PtdC) via the action of DAG:CPT or (PDCT). PtdC can also be converted to DAG by PDCT. The LPCAT can catalyze acyl-exchange between the sn-2 position of PUFA-enriched PtdC and the acyl-CoA pool, as facilitated by the ACBP. Both PLA2 and PDAT have been implicated in removal of unusual FAs from the PtdC for incorporation into TAG. The DGAT activity is unique to TAG biosynthesis, and has long been thought to be a gatekeeper for oil biosynthesis as it diverts the products leading to phospholipid production for membrane biosynthesis into TAG (Weselake et al., 2009). Increased expression of DGAT activities has been closely tied to increased oil production in some oilseeds (Liu et al., 2012). Additional detail is available at http://lipidlibrary.aocs.org/content.cfm?ItemNumber=40314.

Figure 1.4  Generalized scheme for triacylglycerol (TAG) biosynthesis in developing seeds of oleaginous plants. MUFAs , monounsaturated fatty acids; SFA , saturated fatty acids; CoAs , acyl-Coenzyme As; FAE , fatty acid elongase; ER , endoplasmic reticulum; G3P , sn -glycerol-3-phosphate; PAP , phosphatidic acid phosphatase; PA , phosphatidic acid; DAG , sn -1, 2-diacylglycerol; DGAT , acyl-CoA:diacylglycerol acyltransferase; PtdC , phosphatidylcholine; CPT , sn -1,2-diacylglycerol:cholinephosphotransferase; PDCT , phosphatidylcholine:diacylglycerol cholinephosphotransferase; FAD1 and FAD2 , fatty acid desaturases 1 and 2; PUFA , polyunsaturated fatty acid; PLA2 , phospholipase A2; ACS , acyl-CoA synthetase; LPCAT , acyl-CoA: lyso -phosphatidylcholine acyltransferase; ACBP , low molecular mass soluble acyl-CoA-binding protein; PDAT , phospholipid:diacylglycerol acyltransferase; LPC , lyso-phosphatidylcholine; GPAT , acyl-CoA: sn -glycerol-3-phosphate acyltransferase; LPA , lyso -phosphatidic acid; LPAAT , acyl-CoA: lyso -phosphatidic acid acyltransferase; PC , phosphocholine.

Chapters Castor (Ricinus communis L.), Brassica spp. Oils, Flax (Linum usitatissimum L.), Crambe (Crambe abyssinica), Camelina (Camelina sativa), Tung (Vernicia fordii and Vernicia montana), Emerging Industrial Oil Crops, and Engineering Oil Accumulation in Vegetative Tissue provide additional descriptions of oil biosynthesis including TAG incorporating uncommon fatty acids and production of oil by vegetative tissue. Recent developments in plant fatty acid and oil biosynthesis can be found in the Lipid Library (http://lipidlibrary.aocs.org/Biochemistry/content.cfm?ItemNumber=40300&navItemNumber=19219) maintained by AOCS.

Technical and Social Issues Related to Plant Genetic Engineering

As mentioned, the earliest commercialized oil crop genetically engineered (GE) primarily to produce an industrial oil product was high-laurate B. napus (Voelker et al., 1992). The oil was developed for use by the surfactant and detergent industry, and the crop represented the first source of laurate available from a temperate crop that had suitable agronomic characteristics. Because the seed meal remaining after oil extraction was to be used as animal feed, the crop required regulatory approval for use in food. Although the crop was not a commercial success, it provided a model for the regulatory process in developing industrial crops and also an example of the difficulty in achieving commercial success.

Two incidents involving GE industrial crops pointed to some inherent weaknesses in the regulatory system and resulted in increased oversight of industrial GE crop introduction (McKeon, 2003; Lemaux, 2008). The first incident involved StarLink corn, a variety engineered to produce the insect resistance protein CrY9c, because this protein had some properties consistent with allergens, the corn was approved only for animal feed or industrial use. Since animal feed is cheaper than human food, some unscrupulous or unknowing producers and users of this corn sold it as food for humans. This problem resulted in a 7% decline in corn prices for at least a year and reduced acceptance of U.S. corn exports in a number of countries (Carter and Smith, 2007). Moreover, this incident altered U.S. regulation of GE crop introductions, requiring that all such crops, even those solely for industrial use, be evaluated for risk to humans if the product were consumed (McKeon, 2003). The second incident involved soybeans that were planted in a field used in the previous year to grow ProdiGene’s GE corn that had been engineered to produce an industrial protease. Residual corn seed that germinated the following season in the soybean field was harvested with the soybeans, thereby contaminating the soy crop with the GE corn. The end result was that, upon discovery of the contamination, the harvested soybeans co-mingled with the contaminated soybeans were destroyed, resulting in a multi–million dollar loss and additional calls for increased regulation of GE crops (Fox, 2003).

These two incidents served as a warning to the agencies that regulate the plant biotechnology industry, leading to increased oversight in the granting of applications for introducing transgenic crops. It is now assumed that oilseeds engineered to produce industrial oil will produce a seed meal that may be used as food or feed, thus entering the food supply even if it is not intended to be used as food or. As a result of this possibility, even these byproducts of transgenic oilseeds must pass the substantial equivalence test for regulatory approval of the crop (Stewart and McLean, 2004).

There are currently 27 countries that produce GE food crops and 36 countries have approved GE crops for environmental release or use as food or feed (James, 2014). The major oilseed GE crops planted include soy, corn, canola (low–erucic acid rapeseed), and cotton. Although controversies regarding GE crops remain, eg, labeling of products derived from these crops, the regulations governing their production and use in food and feed have presumably been met in the countries that have approved their use. In most cases, an extensive regulatory oversight has been developed to assure safety of introduced GE crops. For example, the United States has three regulatory agencies that oversee approval of GE crops. The US Department of Agriculture Animal and Plant Health Inspection Service (USDA-APHIS) ensures that the plant is not invasive. The Environmental Protection Agency (EPA) addresses concerns that any plants carrying biopesticides or requiring application of pesticides or herbicides will not cause environmental problems. The Food and Drug Administration (FDA) ensures the crop is substantially equivalent to the unmodified crop or, if there are changes resulting in a significant difference, eg, different fatty acid composition, that the alteration does not pose a hazard for human or animal consumption (McKeon, 2003). The requirement that such crops are substantially equivalent to the non-GE crops is especially important when considering the crop for food and feed, to ensure that introduced genes do not reduce the safety or nutritive value of the crop. A number of specific concerns regarding GE crops are listed in Table 1.3.

The common practice of including antibiotic resistance as a marker for selection led to concerns that wholesale antibiotic resistance would be acquired by gut bacteria leading to disastrous untreatable infections. Although it is unlikely that these genes would survive the digestive process, the introduction of recombinase systems that can specifically excise these selection genes obviates the issue (Srivastava and Ow, 2015). Also, nonantibiotic selection systems are available, and antibiotic selection genes can be introduced at unlinked loci that segregate out from the traits of interest.

In most cases, GE plants will include genes that express proteins or enzymes; therefore, the expressed protein can potentially introduce a new allergen. As a result, the proteins and products resulting from introduced genes must be demonstrated to be readily digestible and comply with food safety regulations. The required testing for susceptibility to pepsin digestion serves as one indicator that the GE plant will not introduce a new allergen, but additional criteria including evaluation of the protein structure and allergenicity of the source of the protein are also used (Ladics, 2008).

Table 1.3

Issues Related to Genetically Engineered Crops

Introduction of antibiotic resistance

Random insertion of gene

Undesirably or unpredictably altered composition

Harmful effects on beneficial insects

Outcrossing

Introduction of allergen

Crops for both food and industrial applications

End uses of residues

Identity preservation

The outcrossing of GE crop traits to native germplasm has occurred in some cases and introduction of herbicide resistance to closely related species that are invasive weeds is a major concern (Warwick et al., 2009). Engineered crops can be planted with a buffer zone of nonmodified crops for containment of any outcrossing to native germplasm or related weeds and to reduce the development of insect resistance to biopesticides introduced in the GE crop. Buffer zones are also used for non-GE crop plants such as canola and rapeseed, where outcrossing can occur over some distance resulting in seed with undesirable composition.

The economics of producing GE oilseeds is beyond the scope of this text. One key issue related to economics, however, is identity preservation. The investment in research and development of a transgenic oilseed is often considerable. It is generally recognized that only crops to be grown on a large scale or are of very high value will be profitable for the seed developer. Furthermore, the transgenic crop products with the industrial oil trait(s) must be kept separate from other related seed in order to maintain its premium value. There are protocols developed for the process of identity preservation that are currently applied to crops such as organic and/or non-GE soybeans (Bullock and Desquilbet, 2002).

Conclusions

There is considerable recognized value for development of oilseed crops that provide biomaterials and feedstocks to the chemical industry. Oleochemicals already make an important contribution to biomaterial production in the form of surfactants, lubricants, paints, coatings, polymers, and biofuels. Successful development of approaches to genetically engineer crops to produce oils containing industrially useful fatty acids will aid the introduction of additional oil-derived biomaterials and provide renewable resources that can reduce the need for petrochemical production.

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Chapter 2

Biodiesel and Its Properties

Gerhard Knothe     U.S. Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research, Peoria, IL, United States

Abstract

Biodiesel, defined as the mono-alkyl esters of vegetable oils or animal fats, is an alternative diesel fuel that is becoming accepted in a steadily growing number of countries around the world. Since the source of biodiesel varies with the location and other sources such as recycled oils are continuously gaining interest, it is important to possess data on how the various fatty acid profiles of the different sources can influence biodiesel fuel properties. The properties of the various individual fatty esters that comprise biodiesel determine the overall fuel properties of the biodiesel fuel. In turn, the properties of the various fatty esters are determined by the structural features of the fatty acid and the alcohol moieties that comprise a fatty ester. Structural features that influence the physical and fuel properties of a fatty ester molecule are chain length, degree of unsaturation, and branching of the chain. Important fuel properties of biodiesel that are influenced by the fatty acid profile and, in turn, by the structural features of the various fatty esters are cetane number and, ultimately, exhaust emissions, cold flow, oxidative stability, viscosity, and lubricity.

Keywords

Biodiesel; Fatty acid methyl esters; Fuel properties; Transesterification

Introduction

Biodiesel (Knothe et al., 2010; Mittelbach and Remschmidt, 2004) is a biogenic alternative to conventional diesel fuel (DF) obtained from vegetable oils, animal fats, or other materials consisting largely of triacylglycerols (triglycerides). Reacting an oil or fat with an alcohol, usually methanol, in the presence of a catalyst, commonly sodium methoxide, affords the corresponding mono-alkyl esters. These mono-alkyl esters are defined as biodiesel. Glycerol is obtained as a co-product. Fig. 2.1 depicts the principle of the transesterification reaction.

While the suitability of any material as fuel, including biodiesel, can be influenced by contaminants arising from production or other sources, the nature of the major fuel components ultimately determines the fuel properties. Some of the properties included in standards can be traced to the structure of the fatty esters comprising biodiesel. Since biodiesel consists of fatty acid esters, not only the structure of the fatty acids but also that of the ester moiety derived from the alcohol can influence the fuel properties of biodiesel. Furthermore, the aforementioned mono-alkyl esters that comprise biodiesel are a mixture corresponding in its fatty acid profile to that of the parent oil or fat from which it is produced with each ester component contributing to the properties of the fuel.

The properties of a biodiesel fuel that are determined by the structure of its component fatty esters include ignition quality, cold flow, oxidative stability, viscosity, and lubricity. The present work discusses the influence of the structure of fatty esters on these properties. Not all of these properties have been included in biodiesel standards, although all of them are essential to the proper functioning of the fuel. This article begins, however, with brief summaries on the historical background, production, and analysis of biodiesel.

Figure 2.1 CH 3 ).

Historical Background

The use of vegetable oils or their derivatives in diesel engines is nearly as old as the diesel engine itself. While the engine was developed in the 1890s by Rudolf Diesel (1858–1913), the apparently first recorded run of a diesel engine using a vegetable oil as fuel, peanut oil, occurred at the World Exposition in Paris in 1900 at the request of the French government. The French government at that time was interested in developing alternative fuels for its European colonies that could be obtained or produced locally. Diesel himself made remarks supporting the concept of using vegetable oils as fuel in later writings as well as carrying out some experiments himself and describing the first test in 1900 (Diesel, 1912). Through the 1940s, numerous references can be found that describe the use of vegetable oils as diesel fuel. Probably under current aspects of using biodiesel, the most remarkable document in this connection is a Belgian patent granted in 1937 to C.G. Chavanne of the University of Brussels in which the synthesis of ethyl esters of vegetable oils for fuel use is discussed (Chavanne, 1937). A related publication details the production and use of ethyl esters in the Belgian Congo and in Belgium, including the probably first use of biodiesel in commercial buses (van den Abeele, 1942). Interest in alternative fuels waned after approximately 1945 and this lack of interest continued into the 1970s. In the United States, a few reports from The Ohio State University and Georgia Institute of Technology, after approximately the end of World War II, through the 1970s, apparently little interest existed in vegetable oil–based fuels, or alternative fuels in general.

The energy crises of the 1970s then spawned renewed interest in alternative fuels, among them vegetable oil–based fuels. In this connection, the alkyl esters of vegetable oils were