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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 Interest
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
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.
TABLE 1.2 Comparison of Products Derived From Petroleum and Seed Oils
Petroleum Products
Fuels—gasoline, diesel
Paints and coatings
Lubricants and greases
Nylon 6,6; Nylon 12,12
Detergents
Polyurethanes
Polystyrene
Printing inks
Hydraulic fluids
Seed Oil–Derived Products
Fuel—biodiesel
Paints and coatings with low VOC
Biodegradable lubricants and greases
Nylon 11, Nylon 10,10
Soaps and detergents
Partially biodegradable polyurethanes
Polyesters
Biodegradable, nonsmudging inks
Biodegradable hydraulic fluids
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.
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 higholeic 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
Acetyl-ACP
Malonyl-CoA
Palmitoyl-CoA
Stearoyl-CoA
Oleoyl-CoA
Malonyl-ACP CO2
Acetyl-CoA
ACP ACP
Butyryl-ACP
Malonyl-ACP x 6
Palmitate
Stearate
Oleate
Palmitoyl-ACP
Malonyl-ACP
Stearoyl-ACP
Oleoyl-ACP
∆9-Desaturase
FIGURE 1.3 Pathway for fatty acid biosynthesis in plants.
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.
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
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
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 monoalkyl 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.
TABLE 2.1 Major Standards (ASTM and EN) Related to Biodiesel
D975 Diesel Fuel
D6584 Total monoglycerides, total diglycerides, total triglycerides, and free and total glycerin in B-100 by Gas Chromatography
D6751 Biodiesel blend stock (B100) for middle distillate fuels
D7321 Particulate contamination of B100 and biodiesel blends by laboratory filtration
D7371 Biodiesel (FAME) content in diesel fuel by mid infrared spectroscopy
D7398 Boiling range distribution of FAME in the range from 100 to 615°C by GC
D7462 Oxidation stability of B100 and blends of biodiesel with diesel fuel
D7467 B6–B20 blends
D7501 Determination of fuel filter blocking potential of B100 by cold soak filtration test (CSFT)
D7591 Free and total glycerin in biodiesel blends by anion exchange chromatography
D7797 FAME content in jet fuel using flow analysis by FTIR
D7806 FAME content of a blend of biodiesel and petroleum-based diesel Fuel oil using mid-IR
EN 590
EN 141078
Diesel Fuel
FAME in diesel fuel by IR
EN 14103
EN 14104
Ester and linolenic acid ester content
Acid value
EN 14105
EN 14106
EN 14107
EN 14108
EN 14109
EN 14110
EN 14111
EN 14112
Free and total glycerol, acylglycerols by GC
Free glycerol in the range 0.005–0.07% (m/m)
Phosphorus by ICP
Sodium content by AAS
Potassium content by AAS
Methanol content by GC
Iodine value
Oxidation stability
TABLE 2.1 Major Standards (ASTM and EN) Related to Biodiesel—cont’d
EN 14213
EN 14331
EN 14538
EN 15751
EN 15779
FAME as heating fuels
FAME analysis by LC/GC
Ca and Mg by ICP-OES
Modified oxidation stability
Determination of polyunsaturated (≥4 double bonds) in FAME by GC
two phases at the end, methyl esters and glycerol, although in case of higher esters there exists an increased tendency toward the formation of emulsions. The methyl ester and glycerol can generally be easily separated after the reaction. The methyl ester phase can be washed with water (temperature of the wash water may be slightly elevated) to remove remaining glycerol and catalyst. Minor components remaining in the biodiesel after completion of all steps include starting material (triacylglycerols), intermediates (di- and monoacylglycerols), glycerol, catalyst, and extraneous materials. These minor components can significantly influence biodiesel fuel properties, probably the most prominent examples being cold flow and oxidative stability.
The development of other catalysts or catalytic systems as well as variations of the transesterification reaction has received considerable attention by the research community in recent years. Countless other catalysts, the enumeration of which transcends this article, are to address issues with the conventional transesterification reaction such as increasing tolerance for water, reducing problems associated with the use of other alcohols or easy removal of the catalyst. Most notably, heterogeneous catalysts or catalyst systems as well as enzymatic catalysis have been researched extensively. Some review articles that deal with conventional transesterification or alternative catalytic systems are Ma and Hanna (1999), Lotero et al. (2005), Di Serio et al. (2008), Fjerbaek et al. (2009), Andrade et al. (2011), Abbaszaadeh et al. (2012), He and Van Gerpen (2012b), Kouzu and Hidaka (2012), Motasemi and Ani (2012), Veljkovic et al. (2012), Davison et al. (2013), Hama and Kondo (2013), Kralisch et al. (2013), Narwal and Gupta (2013), and Ramachandran et al. (2013).
TABLE 2.2 Fuel Property Specifications
Property Unit
in Biodiesel Standards
on grade of petrodiesel fuel to be blended with.
Other approaches to modifying the transesterification reaction include the use of a solvent to achieve a one-phase system or in situ transesterification in which the oil is not removed from the original feedstock, rather the feedstock containing the oil directly subjected to the transesterification reaction. Such procedures entail changes to the reaction such as a higher stoichiometric ratio
