Oilseed crops for biodiesel and animal feed

Back in the early days of this blog, I posted some general information about the variety of crops and other plant species that were being used, or contemplated for use, in producing biofuels, and about some of the companies known at that time to be developing genetically modified plants for this purpose. Generally speaking, these plant species can be divided into two categories – plants that are primarily used as a source of carbohydrates like sugar or cellulose for the production of ethanol, butanol and other fuels; and plants that are used as a source of oils (fatty acids) which can be extracted and chemically converted into biodiesel via transesterification. Included in these two categories are plant species such as the following:

	Traditional Species	New Species
Sugar/Cellulose sources	corn, sugarcane, beets, forestry wastes, other cellulosic materials such as corn stover, bagasse, etc.	sorghum, switchgrass, Miscanthus, energy cane, Arundo donax, woody trees like Eucalyptus
Oilseed crops	soybean, canola (rapeseed), cotton, palm, vegetable oil	Camelina, Jatropha, castor beans

What’s notable from an economic standpoint is that, for just about all of these crop species, the potential exists to derive additional value from materials that are left over after the harvested crop material has served its purpose in biofuel production. Many of the plants used as carbohydrate sources can be used in animal feed, in the form of distillers grains (often combined with the spent biomass of the yeast or other microorganism used to ferment the sugars); and the protein-rich portion of oilseed crops can also be used in animal feed as oilseed meal after the fatty acids have been extracted for biodiesel production. These are both longstanding uses for such plant-derived materials, which may play a significant role in improving the economics of biofuel production processes by creating byproducts having tangible economic value.

The market for dried distillers grains (DDGs) is significant: U.S. production alone is reportedly about 35 million metric tons per year, with a value of somewhere around US $10 billion, depending on the somewhat volatile market price; with over 9 million metric tons of U.S. production exported in 2013. Many companies developing novel microorganisms to produce biofuels by fermentation of these feedstocks are hoping to have their microbial strains approved for inclusion in DDGs that are fed to animals. I hope to devote a future blog entry to discussing the DDG market in more detail, but for today I’d like to discuss the markets for the oilseed crops that can be used for biodiesel production and as a source of seed meal for animal feed.

The term biodiesel refers to an automotive fuel that can be used in place of petroleum-derived diesel, which is produced from any of several renewable sources of fats or oils. Although several methods are used, biodiesel is generally produced by chemical transesterification of the fatty acids to produce mono-alkyl esters. In addition to vegetable oils and animal fats, oils extracted from several oilseed crops are commonly used to produce diesel. Historically, these include soybean, canola (rapeseed), palm and sunflower, but other oil-rich seed crops are being developed or have begun being used for biodiesel production, particularly including Camelina and Jatropha. Under the U.S. EPA Renewable Fuel Standard (RFS), there are approved pathways to produce renewable diesel from most of these sources, including soybean, canola, and Camelina, while pathway petitions for use of Jatropha are pending; biodiesel produced according to any approved pathway (at a facility registered with EPA) would be qualified to issue Renewable Identification Numbers (RINs) to take advantage of economic benefits under the RFS. Under the EU Renewable Energy Directive (RED), biodiesel made from these plants would qualify as a “renewable fuel” if the production process met the specified greenhouse gas emission reduction (currently 35%) and satisfied the other sustainability criteria in Article 17 of the Directive.

According to a 2012 publication of the Food and Agriculture Organization, there were almost 18 million metric tons (5.34 billion gallons) of biodiesel produced in the world in 2010. This amount is expected to rise dramatically due to incentives and mandates around the world such as the U.S. RFS and the EU RED, and one estimate by the FAO and the Organization for Economic Cooperation and Development is that by 2022 world production will rise to 41 billion liters (approximately 10 billion gallons) per year. The 2012 FAO report (citing an estimate from F.O. Licht) says that in 2010, most of the biodiesel produced globally was derived from rapeseed oil (5.75 million metric tons), soybean oil (5.70 million MT), and palm oil (2.44 million MT), followed by animal fats (2.23 million MT) (See Figure below, from FAO 2012). Among other sources, 211,000 MT of sunflower oil was used to produce biodiesel, and from these figures it appears that about 12 million MT of oils derived from oilseed crops were used in 2010 to make biodiesel. Based on approximate figures for the percentage of oil in the oilseed mass this amount of oil would have been produced by approximately 68 million MT of oilseed crops.

Feedstock Usage for Bioideisel. Source: FAO 2012

However, this amount of oilseed used for biodiesel production represents only a small percentage of oilseed crops grown around the world. Recent estimates by the USDA Foreign Agriculture Service (downloadable from here) are that the world produces around 500 million metric tons of all oilseed crops combined, with soybean alone accounting for over half this amount with approximately 286 million MT, and rapeseed a distant second at around 70 million MT.

Oilseed crops are used for a variety of purposes, including the creation of oilseed meal, a protein-rich material that can be used in human foods and animal feed. The USDA FAS estimated at the end of 2013 that total worldwide oilseed meal production in that year was about 281 million MT, the great majority of which was attributed to soybean (190 million MT). Using these figures and the approximate market prices for oilseed meals at the end of 2013, I’ve estimated that the overall world market for oilseed meal (i.e. the economic value of the 281 MMT of production) was about US $123 billion.

Animal feed represents only a portion of the market for oilseed meal. One published estimate (apparently from 2009) was that about 22 MMT of oilseed meal was used in animal feed in Europe, about 68% of which was soybean meal. The European Union represents about 20% of the world market for all compound animal feed, and using this figure to extrapolate, world usage of oilseed meal in animal feed might be approximately 110 MMT. Using approximate percentages attributing this amount of production to the 3 or 4 major crop species, and applying the approximate 2013 commodity prices to the result, gives an estimate that the world market for oilseed meal used in animal feed might be as high as US $50 billion, with about $10 billion in the EU. These are very rough approximations, but they are consistent with other figures I’ve found – for example, from figures published by the U.S. Soybean Board, about 27 MMT of soybean meal was used in the 2011/12 growing season in animal feed in the U.S., giving an estimated U.S. market size for soybean meal in animal feed of US $14 billion, which is consistent with my calculated value for soybean meal used in the EU (15 MMT for a value of US $7.7 billion).

These calculations combine published estimates from different sources and different years, and embody a number of assumptions, and so the figure of a $50 billion world market for oilseed meal in animal feed should be considered as an approximation. The market is currently dominated by soybean and canola, and no doubt includes some amount of the oilseed meal derived from crops already used for biodiesel production. But this figure represents a significant market for developers of biodiesel crops – even gaining an 0.1% market share for these byproducts of biodiesel production would represent perhaps US $50 million in annual sales.

The animal feed market is segmented in several ways, notably by target animal. According to published data (Alltech’s survey of the amounts of compound feed produced globally in 2012), the overall market breaks down as 44% poultry, 26% ruminants (primarily cattle), 23% pigs, 4% aquaculture, 2% companion animals and 1% horses (Alltech’s more recent survey of 2013 production gives similar results, although poultry’s share has risen and ruminants’ has decreased). Other published estimates provide similar percentages. Different mixes of oilseed meals are preferred for different target animals, and so crop-by-crop percentages within the different sectors differ, but it is clear that the market is dominated by soybean meal, which tends to have a protein content, amino acid balance, and other features that make it well suited for most animal diets. However, each of the other oilseed meals used in animal feed supplement the nutritional value of soybean meal in certain ways, and each type of meal makes up a specific portion of the diet for each target animal species.

It is worth noting that most oilseed meal has achieved regulatory approval for use in animal feed. Traditional oilseed meals like soy, cotton, and palm have long been used in animal feed and are considered to be generally recognized as safe (GRAS) in the U.S. and elsewhere in the world. However, because naturally occurring rapeseed contains potentially toxic levels of erucic acid, GRAS status for canola and other artificially-bred low-erucic acid varieties of rapeseed had to be achieved through appropriate testing, with approvals around the world coming in the 1980s. More recently, the oilseed crop Camelina has achieved approval for use in certain animal feeds, in the U.S. and elsewhere, but the potential use of Jatropha meal in feed likely faces the same challenge as did rapeseed, since Jatropha seeds contain high levels of toxic materials such as phorbol esters. Methods to detoxify Jatropha or to breed nontoxic varieties are under development but have yet to gain regulatory approval for use in feed.

While the focus of oilseed crop strain development is clearly on improving oil yield or other traits important for diesel production, developers should keep in mind the potential value of the oilseed meal as an animal feed ingredient. For most oilseed meals, markets and marketing channels exist and regulatory approvals to enable market access; while for some of the newer crops some work remains to be done to establish regulatory approvals and gain market entry. However, the animal feed market remains an important opportunity for biofuel feedstock developers to improve the economics of their products.

D. Glass Associates, Inc. is a consulting company specializing in government and regulatory affairs support for renewable fuels and industrial biotechnology. David Glass, Ph.D. is a veteran of over thirty years in the biotechnology industry, with expertise in industrial biotechnology regulatory affairs, U.S. and international renewable fuels regulation, patents, technology licensing, and market and technology assessments. More information on D. Glass Associates’ regulatory affairs consulting capabilities, and copies of some of Dr. Glass’s prior presentations on biofuels and biotechnology regulation, are available at www.slideshare.net/djglass99 and at www.dglassassociates.com. The views expressed in this blog are those of Dr. Glass and D. Glass Associates and do not represent the views of any other organization with which Dr. Glass is affiliated. Please visit our other blog, Biofuel Policy Watch.

DOE Support for Development of Engineered High Energy Crops

Some of my early (2010-11) posts on this blog discussed strategies being pursued to use advanced biotechnology to create improved varieties of crops, trees, and other plants for use in biofuel production, as well as the companies who have been active in this field (see links below). Although the timeframes for such research projects are inherently somewhat long, due to the need to conform to the life cycles (often annual) of the plants being developed and the need for several years of field testing at escalating acreage, research and commercial activities in this field have continued and progressed. One important driver for this activity has been the funding of the U.S. Department of Energy (DOE), including the Advanced Research Projects Agency-Energy (ARPA-E) division of DOE.

Among the more prominent DOE efforts is the funding program initiated by ARPA-E in 2011 known as PETRO — “Plants Engineered To Replace Oil”. According to the PETRO website, the goals of this program have been to use advanced biotechnology methods to develop non-food crops that directly produce transportation fuel, at prices that are cost-competitive with petroleum. DOE says that at the present time, biofuel production is limited by both the inefficient capture of solar energy by plants and the inefficient processes they use to convert CO2 from the atmosphere into usable fuels.The PETRO program aims to redirect the processes for energy and CO2 capture in plants toward fuel production, to create dedicated energy crops that serve as a domestic alternative to petroleum-based fuels and deliver more energy per acre with less processing prior to the pump. More specifically, the program was set up to fund research efforts to genetically engineer new classes of crops that produce fuels which can be extracted directly from the plants themselves. The funded projects feature research on various plants–including pine trees, tobacco, sugarcane, and sorghum–to create molecules already found in petroleum-based fuels that can be dropped directly into the tanks of existing vehicles.

The following are the programs funded by PETRO beginning in 2011, with links to project descriptions as found on the ARPA-E website.

The PETRO program has been back in the news in recent months, because of DOE’s announcement that it intends to implement programs to support the field testing of “engineered high energy crops” (EHECs), including those developed with PETRO funding, and that in order to comply with the National Environmental Policy Act (NEPA) in doing so, DOE intends to prepare a Programmatic Environmental Impact Statement. Environmental Assessments and/or Environmental Impact Statements are required under NEPA for major federal government actions, and any DOE program to support field testing of genetically modified plants would likely be considered to be subject to this requirement. DOE first issued a public Request for Information in April 2013, and this was followed up with a June 21, 2013 Federal Register announcement formally stating its intent to prepare the Programmatic Environmental Impact Statement (PEIS) and inviting public comments on the proposed scope of the PEIS. There is a substantial amount of information about this effort available on the PEIS website, and although the public comment period is expiring soon (July 22), I thought it would be worth mentioning this effort in the blog.

DOE anticipates that its program will include providing financial assistance for the field trials that are needed to evaluate the performance of EHECs. Such trials could range in size from small, development-scale (5 acres or less), to pilot-scale (up to 250 acres), and possibly also demonstration-scale, which could entail field plots of up to 15,000 acres. As DOE notes on the website, these field activities would likely require permits from the U.S. Department of Agriculture under that department’s biotechnology regulations administered by the Animal and Plant Health Inspection Service (APHIS) and would need to be conducted under conditions of “confinement” that are common in field tests of GMO plants, to limit their potential environmental impact. DOE currently plans to limit its funding activities to field trials that would take place in states in the southeastern U.S., including Alabama, Georgia, Kentucky, Mississippi, North and South Carolina, Tennessee, Virginia, and parts of Florida.

During the course of the public comment period, DOE has held public “scoping meetings” in several states in the region, and also hosted a webinar on July 17. Public comments will be accepted until July 22 (with instructions for commenting found on here on the website), but comments submitted after that date will be considered to the extent possible.

As I’ve previously discussed in the blog, efforts to develop improved plant varieties to use as biofuel feedstocks will likely prove very important for the ability of countries around the world to efficiently produce significant amounts of biofuels from non-food crops. Aside from Syngenta’s Enogen corn (expressing alpha-amylase and phosphomannose isomerase), there are no other genetically modified plant species yet being used in commercial fuel production in the U.S. or any other major market, although there are several examples of plant species improved through classical breeding that are being used or investigated for this purpose. In view of the history of controversies surrounding agricultural biotechnology, and in particular the history of USDA’s biotechnology regulations and the long record of NEPA litigation leveled against it, it is inevitable that biotechnology critics will pounce on any large-scale DOE program intended to support or promote field trials of engineered plants, and express concerns over alleged or hypothetical environmental risks. The route of proactively preparing a PEIS is probably the right one for DOE, and it is certainly wise to begin the effort at an early stage, to be sure that the needed EIS can pass muster under NEPA and be completed in time to enable the planned funding to go forward. It will be interesting to see what the range might be of the public comments DOE receives either during this scoping period, or when a draft EIS is eventually published for public comment.

Earlier blog posts on use of transgenic plants as biofuel feedstocks:

Strategies to Engineer Plants for use as Biofuel Feedstocks, January 25, 2010.
Companies Developing Modified Plant Varieties as Biofuel Feedstocks. Part 1, March 25, 2010
Companies Developing Modified Plant Varieties as Biofuel Feedstocks. Part 2, March 28, 2010
Companies Developing Modified Plant Varieties as Biofuel Feedstocks. Part 3, March 29, 2010
USDA Regulations Affecting Use of Genetically Modified Plants as Biofuel Feedstocks, May 18, 2010.
Impact of USDA Regulations on the Use of Genetically Modified Plants as Biofuel Feedstocks, Part 1, June 1, 2010
Impact of USDA Regulations on the Use of Genetically Modified Plants as Biofuel Feedstocks, Part 2, June 4, 2010

D. Glass Associates, Inc. is a consulting company specializing in government and regulatory affairs support for renewable fuels and industrial biotechnology. David Glass, Ph.D. is a veteran of over thirty years in the biotechnology industry, with expertise in industrial biotechnology regulatory affairs, U.S. and international renewable fuels regulation, patents, technology licensing, and market and technology assessments. Dr. Glass also serves as director of regulatory affairs for Joule Unlimited Technologies, Inc. More information on D. Glass Associates’ regulatory affairs consulting capabilities, and copies of some of Dr. Glass’s prior presentations on biofuels and biotechnology regulation, are available at www.slideshare.net/djglass99 and at www.dglassassociates.com. The views expressed in this blog are those of Dr. Glass and D. Glass Associates and do not represent the views of Joule Unlimited Technologies, Inc. or any other organization with which Dr. Glass is affiliated. Please visit our other blog, Biofuel Policy Watch.

Biofuel Technologies Available for Licensing

From time to time, I’ll be posting descriptions of inventions or other research discoveries arising in academic or other non-profit laboratories that may enable improved techniques for development or production of renewable fuels, which are available for licensing for commercial development. For each invention, I’ll include a link where you can find further information, as well as contact information for the technology transfer office handling each invention. I welcome questions or comments on these listings, and I would also be happy to hear from tech transfer offices about other inventions for inclusion in later posts.

Microorganisms and Enzymes for Improved Biofuel Production

Metabolic Engineering of Caldicellulosiruptor bescii for the Production of Biofuels and Bioproducts. Caldicellulosiruptor bescii, a thermophilic bacterium with native cellulolytic activity, has a high affinity for decomposing lignocellulosic biomass including agricultural residues such as rice straw, switchgrass, as well as hard- and softwoods. These characteristics make this organism especially appealing for use in biomass processing, but in order to economically produce compounds of economic interest, the organism must be modified to enable its use in industrial processes for the production of biofuels and other commodity chemicals. The inventors have developed methods for genetic manipulation of Caldicellulosiruptor species, for example by introducing genes from Clostridium thermocellum, which is the best-known thermophilic ethanol producer, enabling the production of ethanol and hydrogen from biomass. Other genes from C. thermocellum can similarly be introduced allowing the bacterium to convert acetate to acetaldehyde. These modified strains of C. bescii can be used to economically produce fuels and chemicals from lignoceullolosic feedstocks. Contact: Gennaro J. Gama, Ph.D., Senior Technology Manager, University of Georgia Research Foundation, Inc., Phone: 706-583-8088, Email: GJG@uga.edu. Reference: Case Number 1936.

Chimeric Enzymes with Improved Cellulase Activities. Production of cellulosic biofuels continues to face the challenge of finding cost-effective ways to release the sugars from the complex polymers found in these feedstocks for conversion to ethanol or other fuels. The use of cellulolytic enzymes to catalyze and improve extraction yields of sugars from cellulosic biomass could help to overcome this challenge. The inventors have developed a novel and modified version of cellobiohydrolase from Clostridium thermocellum (CbhA) that exhibits enhanced catalytic activity in the saccharification of cellulose. Clostridium thermocellum has the potential to be used in the production of ethanol, because of its ability to directly convert cellulose to ethanol, which is catalyzed through a cellular surface organelle called a cellusome, which possesses a complex of many different catalytic subunits. This invention provides recombinant methods and molecules for producing chimeric CbhA polypeptides, incorporating polypeptides from other organisms, that exhibit enhanced cellulolytic activity. The chimeric CbhA exhibits cellulolytic activities that are at least two-fold greater than the wild-type CbhA for degrading cellulosic biomass. Contact: Eric Payne, Senior Licensing Executive, National Renewable Energy Laboratory, Phone: 303-275-3166, E-mail: eric.payne@nrel.gov. Reference: Case Number: NREL ROI 12-33.

Genetically-engineered Microorganism for the Conversion of Feedstock into Fatty Acids. Rutgers University researchers have developed a process for genetically engineering microorganisms for the efficient production of fatty acids. This process makes use of genome-scale flux-balance analysis (FBA) modeling for the in silico design of engineered strains of microbes that overproduce diverse targets, and allows E. coli and other bacteria to be engineered for high-efficiency production of fatty acids, for conversion into biofuel. Although other methods exist for engineering bacteria to produce fatty acids, the advantage of this process is to enhance the efficiency of bacterial production, thus increasing fatty acid yield and reducing process costs. Contact: Rick Smith, MSEE, MBA, Assistant Director, Physical Sciences and Engineering Licensing, Office of Technology Commercialization, Rutgers, The State University of New Jersey, Phone: 732-932-0115 x3010, Email: rismith@otc.rutgers.edu. Reference: Technology Number 2011-049.

Algal and Biodiesel Technologies

Enhanced Lipid Production in Algae. Algae are considered among the best potential candidates for production of advanced biofuels in view of growing golabl energy concerns. They are an attractive option over terrestrial crops due to their ability to grow fast, produce large quantities of lipids, carbohydrate and proteins, thrive in poor quality waters, sequester and recycle carbon dioxide and remove pollutants from industrial, agricultural and urban wastewaters. This invention features a method to enhance algal biomass productivity (i.e. production of lipids) per unit of area. The method comprises inducing a specific stress into microalgae, which in turn results in increased production of lipids. Lipid productivity increased 70% – 336% over control, which is expected to translate to a corresponding increase in the production of bio-oils per unit of area of growth pond. Contact: Gennaro J. Gama, Ph.D., Senior Technology Manager, University of Georgia Research Foundation, Inc., Phone: 706-583-8088, Email: GJG@uga.edu. Reference: Case Number 1549.

Novel Plants as Biofuel Feedstocks

Higher Yielding Biomass Developed Using Newly Discovered Cell Wall Structures and Proteins. In spite of the promise of biomass as a renewable resource, the cost of biomass-based fuels historically has not been competitive relative to oil or other energy resources. A major barrier in converting biomass into fuels is that the complex of cellulose, hemicellulosic and pectic polysaccharides that make up a plant’s cell wall is naturally resistant to degradation by microbes and enzymes, making it hard to free up sugar resources. UGA researchers have created a transgenic plant that has much decreased recalcitrance and increased plant stem and root growth, which will directly translate into higher volumes of convertible sugar. The invention is based on the identification of novel plant cell wall structures in which cell wall pectin and hemicellulose glycans are linked to structural proteins, enabling a substantial reduction in recalcitrance while enhancing plant growth. This new research suggests the existence of a complex proteoglycan network in plant cell walls, which enables a potential new pathway for the development of transgenic plants having more degradable plant biomass available for biofuel production. Contact: Gennaro J. Gama, Ph.D., Senior Technology Manager, University of Georgia Research Foundation, Inc., Phone: 706-583-8088, Email: GJG@uga.edu. Reference: Case Number 1664.

Improved Biochemical Fermentation Utilizing Modified Transgenic Rice and Switchgrass. Biomass is a renewable resource that has shown promise to replace petroleum based fuels. A major barrier in converting lignocellulosic biomass into fuels is that the complex of cellulose, hemicellulosic and pectic polysaccharides that make up a plant’s cell wall is naturally resistant to degradation by microbes and enzymes, making it hard to free up sugar resources. Researchers at The University of Georgia have successfully modified rice and switchgrass, both of which are fast growing and rich in carbohydrates, to create transgenic varieties having improved growth, sugar yield and ethanol yield from fermentation. The invention involves the modification of plant pectins, the complex carbohydrates found in cell walls and other structures, through overexpression of an esterase gene in these plant species. These novel transgenic plants resulted in a larger biomass yield compared to study controls and wild rice. Furthermore, fermentation of sugars extracted from the novel plants led to improved ethanol yield that was18% to 56% greater than that seen from wild-type plants. Contact: Gennaro J. Gama, Ph.D., Senior Technology Manager, University of Georgia Research Foundation, Inc., Phone: 706-583-8088, Email: GJG@uga.edu. Reference: Case Number 1843.

D. Glass Associates, Inc. is a consulting company specializing in government and regulatory affairs support for renewable fuels and industrial biotechnology. David Glass, Ph.D. is a veteran of over thirty years in the biotechnology industry, with expertise in industrial biotechnology regulatory affairs, U.S. and international renewable fuels regulation, patents, technology licensing, and market and technology assessments. Dr. Glass also serves as director of regulatory affairs for Joule Unlimited Technologies, Inc. More information on D. Glass Associates’ regulatory affairs consulting capabilities, and copies of some of Dr. Glass’s prior presentations on biofuels and biotechnology regulation, are available at www.slideshare.net/djglass99 and at www.dglassassociates.com. The views expressed in this blog are those of Dr. Glass and D. Glass Associates and do not represent the views of Joule Unlimited Technologies, Inc. or any other organization with which Dr. Glass is affiliated. Please visit our other blog, Biofuel Policy Watch.

Strategies to Engineer Plants for Use as Biofuel Feedstocks

There has recently been increasing interest in using genetic engineering or other advanced biological technologies to improve the plants that are used as feedstocks for production of ethanol or other fuels. Some efforts are being directed at food crops like corn: even though most observers today consider corn ethanol to be a transitional fuel, the techniques for genetic engineering of corn are well known and easily practiced, and there are a number of companies that consider corn to be somewhat of a “model crop” and only a short-term product opportunity. Efforts are also ongoing to engineer the plant species that might be alternative biofuel feedstocks , including trees, switchgrass, oil-rich crops like canola or Jatropha and others. The following is an overview of some of the approaches being taken to engineer plants for biofuel use: see Sticklen 2006; Torney et al. 2007; Sticklen 2008; and Weng et al. 2008 for more comprehensive scientific reviews of research in this field.

Techniques for genetic engineering of plants have been well-established for over two decades, with the earliest methods for transforming dicotyledonous plants being developed in the mid-to-late 1980s and methods for transforming monocots (including most of the world’s important cereal crops) arriving several years later. In many ways, the methodology resembles the techniques used to engineer microorganisms, but with the added challenge of successfully transporting the exogenous DNA through the plants’ cell wall (a feature unique to plants) en route to the cell nucleus. Among the methodologies to accomplish this are the use of Agrobacterium, a microorganism naturally having the ability to inject its DNA into plant cells; electroporation, in which the DNA is transported into plant cells using electric current; and the “gene gun”, where DNA is adhered to extremely small nanospheres which are shot at high velocity into the cells.

Some of the possible technological approaches to using plant genetic engineering to improve biofuel production are summarized in below. The most common strategies for creating transgenic plants for use as biofuel feedstocks have as their goal the introduction of genetic changes into certain plant species to make them more useful for the production of ethanol and other fuels. For example, one might introduce genes encoding key biodegradative enzymes such as cellulases or ligninases into the plants, to enhance the conversion of cellulose into fermentable sugars, thereby improving the pretreatment of cellulosic feedstocks. Other companies and research groups are focusing on engineering oilseed crops to have altered or enhanced lipid content, to make them more suitable for production of biodiesel (analogous to strategies used for altering algal strains).

Genetic Engineering Strategies: Plants

Overexpress cell-wall hydrolysis enzymes.
- Cellulases
- Hemicellulases
- Ligninases
Increase plant biomass, cellulosic biomass.
- Cellulose biosynthetic enzymes.
Lignin modification to reduce need for pretreatment.
- Down-regulate lignin biosynthesis.
Regulated gene expression, so traits are active only when needed.

Another approach is simply to engineer or breed biofuel crop species to grow faster and/or create more biomass. It has also been proposed that one might engineer plants, particularly trees, to have reduced lignin content, which might greatly facilitate the pretreatment steps now needed to process cellulosic feedstock into fermentable sugars. One intriguing step towards this goal was recently achieved by a group from Brookhaven National Laboratory, who used protein engineering to alter a plant enzyme that catalyzes the synthesis of certain lignin precursors, so that the enzyme instead produced molecules unable to serve as the building blocks for lignin (this engineered enzyme has not yet been tested in plants).

A number of companies and research groups are taking these genetic engineering approaches one step farther, with the goal of using gene expression promoters capable of being regulated so that key degradative enzymes would not be expressed in the plant tissue until the specific time they are needed. In this way, transgene expression would remain silent during the normal growth of the plant, but expression would be turned on during pretreatment or processing or at a similar stage when expression of the degradative enzyme is most needed. Companies pursuing this strategy include Agrivida and Farmacule Bioindustries.

Finally, there are also several companies that are developing genetically engineered plants as “factories” for the production of enzymes for use in fuel fermentation. Similarly to companies using microorganisms to produce cellulytic or other degradative enzymes, some of these companies (like Medicago) are ones that are already in the business of creating transgenic plants for the commercial production of enzymes or other industrial products, or pharmaceutical products. One such approach is being taken by the seed company Syngenta, which has received approval in several countries (but not the U.S.) to commercially sell a strain of corn that has been engineered to overexpress an amylase enzyme, for use in biofuel production.

Later entries in this blog will discuss the programs and strategies of commercial biotech companies that are developing modified plant varieties for biofuel use. At this writing, I’m aware of at least 15 companies in the U.S. and elsewhere in the world that are actively pursuing such goals.

D. Glass Associates, Inc. is a consulting company specializing in several fields of biotechnology. David Glass, Ph.D. is a veteran of nearly thirty years in the biotech industry, with expertise in patents, technology licensing, industrial biotechnology regulatory affairs, and market and technology assessments. This blog provides back-up and expanded content to complement a presentation Dr. Glass made at the EUEC 2010 conference on February 2, 2010 entitled “Prospects for the Use of Genetic Engineering in Biofuel Production.” The slides from that presentation are available at www.slideshare.net/djglass99.

Strategies to Engineer Algae for Biofuel Production

Researchers have investigated the possible use of algae for biofuel production for many years, dating back at least to efforts by the U.S. Department of Energy in the 1970s. These early efforts failed to result in production technologies that were economically competitive with petroleum-derived fuels, and so the field stagnated. However, recent years have seen an increased interest in the use of algae to produce biodiesel and other fuels. Within the past couple of years, most of this renaissance was fueled by news of several big business deals involving algal biofuel firms, particularly the research partnership between ExxonMobil and Synthetic Genomics for development of improved algal strains using synthetic biology. With this renewed interest has come increased activity in the application of biotechnology to improve the strains of algae that might be used in biofuel production, thus providing process improvements that could make such methods economically competitive. I’ll briefly summarize some of the biotechnology strategies that are being used or that have been proposed, again with the caveat that this is cannot be a comprehensive scientific review. Among published reviews are Rosenberg et al. 2008, Li et al. 2008; Angermayr et al. 2009 and Mayfield (undated).

Of the three classes of organism that are amenable to improvement for biofuel purposes, algae are the laggards with regard to the development of technologies to enable reliable, stable genetic transformation. A number of algal strains can be genetically engineered, and in fact, engineered algae have long been proposed for use in industrial production of pharmaceuticals and other high value products. But according to Rosenberg et al, “routine transformation has only been achieved in a few algal species” in spite of significant and ongoing progress in extending such techniques to other species. Techniques exist, or are under development, for targeting genetic changes either to nuclear DNA or chloroplast DNA of microalgae species.

The biofuel strategy most often contemplated for algae is the large-scale production of biodiesel, jet fuel, and other petroleum-derived fuels. Biodiesel is usually composed of a mixture of fatty acid methyl esters, which can be produced from any biologically-derived source of fatty acids, with the final product created by a transesterification reaction. Mixtures of these esters can mimic the composition of petroleum-derived fuels such as diesel, jet fuel and others, which are complex mixtures of linear and branched hydrocarbons and cyclic alkanes. So, most genetic engineering strategies contemplated for algae involve enhancing or altering natural lipid biosynthetic pathways, to create a mixture of fatty acids suitable for conversion to biodiesel or other hydrocarbon-based fuels. One example (described in Rosenberg et al) is to transform an algal strain to express one or more enzymes involved in lipid biosynthesis – the example given in Rosenberg et al. is the enzyme acetyl-CoA carboxylase, which in early experiments was engineered into an algal strain, albeit with little impact on lipid biosynthesis. It has been reported that Aurora Biofuels is pursuing a similar approach to the enhancement of lipid synthesis in algae.

Other approaches to engineering algae are described in the table below. Such ideas include finding ways to enhance the efficiency of photosynthesis in algal production strains (e.g. by engineering the strains to synthesize large amounts of photoreceptor molecules), or to enable algae to utilize alternative food sources, particularly the ability to metabolize and ferment sugars. Solazyme has conducted research in these fields, and has pending patent applications relating to engineering light utilization and to alternate feedstocks for industrial algae strains. One unique approach that has been taken by Algenol Biofuels is to engineer cyanobacteria to express the enzymes pyruvate decarboxylase and alcohol dehydrogenase, allowing the algae to convert the common Krebs cycle molecule pyruvate first to acetaldehyde and then to ethanol, a strategy which the company is beginning to commercialize. Finally, it is also possible that genetic strategies might be used to attack what is a significant problem facing the use of algae to produce biodiesel: the need to separate the lipid products of the organisms from the aqueous medium in which the algae are grown. This often requires costly, energy-intensive physical processes, so that finding a biological method for secretion and sequestration of lipids from microalgae could be a very important development for the biofuels industry. Synthetic Genomics is reportedly trying to accomplish this goal using its synthetic biology expertise.

Genetic Engineering Strategies for Algae.

Enhance algal growth rate.
Enhance or alter lipid biosynthesis.
Enhance photosynthesis.
Enable use of alternate food sources.
Enable secretion of lipids to aid oil/water separation.

The renewed interest in algal biofuels over the past several years has led to a lot of activity in the field, with many companies operating, building, or announcing plans to build pilot or demonstration plants, many of which might use algae modified using some of these approaches. I’ll discuss the companies in this sector of the industry, their technologies and commercial plans in later installments of this blog.

Strategies to Engineer Microorganisms for Biofuel Production

Much of today’s commercial activity using advanced biotechnology for biofuel production focuses on the creation, selection or improvement of strains of desired microorganisms having enhanced properties for functions important for biofuel production. Longstanding methods for producing ethanol or other fuels, generally rely on the use of one or more selected microbial strains to drive the fermentation process. Traditionally, these methods have made use of naturally-occurring or classically selected microorganisms, but in recent years the power of the new biotechnologies to develop enhanced strains is being investigated or used by numerous companies. The following is a brief overview of some of the strategies that are being pursued. This is not meant to be a comprehensive summary of such strategies, and it is well beyond the scope of this blog to try to provide a thorough review of the multitude of published scientific papers describing genetic engineering approaches to improving biofuels organisms. For those seeking more scientific detail, there have been numerous review articles published in recent years, including Dien et al. 2003; Jarboe et al. 2007; Stephanopoulos 2007; Atsumi and Liao 2008; Fortman et al. 2008; Lee et al. 2008; Rude and Schirmer 2009 and Connor and Liao 2009.

Typical strategies include:

Overexpress desired enzymes within host fermentation organisms (e.g. to improve ability to process or degrade cellulosic feedstocks).
- Alter the timing or amount of expression of native enzymes
- Express heterologous enzymes from other species
- Express synthetic or engineered enzymes
Engineer microorganisms to manufacture novel or improved industrial enzymes for stand-alone use in biofuel production.
Create novel or synthetic microorganisms to enable production of renewable fuels.

Perhaps the most common strategy employed to date has been the use of recombinant DNA techniques to engineer microorganisms to overexpress desired enzymes, including heterologous enzymes derived from other species which are not naturally found in the host species. This is the strategy most often used by companies developing new microbial strains for cellulosic ethanol production: it would be advantageous from a process perspective to have a single microbial strain that can express the cellulases needed to break down the complex components of cellulosic feedstocks while also possessing the pathways to ferment the resulting sugars into ethanol, but there are few, if any, naturally occurring microorganisms that combine these features. One approach is therefore to introduce into host organisms that are already optimized for ethanol fermentation genes from other species encoding the desired cellulytic enzymatic activities. As another example, genetic engineering could be used to give ethanol fermenting microbes the metabolic pathways needed to utilize sugar sources such as the 5-carbon xyloses and other pentoses that are released from hydrolysis of woody biomass. The alternative approach would be to splice genes encoding the enzymes making up the ethanol fermentation pathway into an organism lacking that trait but having the ability to digest the complex cellulosic components. Strategies like these are being pursued by companies like Mascoma Corporation, Verenium Corporation and others. It is also possible to use genetic engineering simply to enhance the efficiency of naturally-occurring ethanol-producing strains that in some cases already have sufficient activity to be used commercially: an example of a company pursuing this strategy is Qteros, which is moving towards commercial use of a natural microbial isolate while beginning in-house research to improve its activity using biotechnology.

A variation of this strategy is to create and use engineered microbes to manufacture novel or improved industrial enzymes, which can then be used as a catalyst in fuel fermentations to enhance or accelerate biofuel production processes. Enzymes like cellulases, amylases, and other degradative enzymes can be used to pretreat cellulosic feedstocks, or can be added to an ethanol production process at any other suitable time. Companies pursuing this strategy are generally companies already manufacturing and selling other industrial enzymes, including several companies having decades of experience in this sector of the industry, such as Novozymes, the Genencor division of Danisco, and others. The field has also attracted some newer players, such as Iogen Corporation and Dyadic International, some of which are devoting a significant portion of their effort to the manufacture and sale of biofuel enzymes.

Finally, a more ambitious strategy is to use synthetic biology, metabolic engineering, or other advanced techniques to create novel or synthetic microorganisms possessing enzymatic capabilities not found in the original host organism. This strategy might involve designing an “optimal” organism using combinations of enzymes from other sources, or even completely new enzymes designed and created using protein engineering to have maximal catalytic activity. Strategies like these are most often being pursued to create microorganisms or algae optimized to produce “designer” mixtures of hydrocarbons mimicking the composition of diesel, jet fuels or other petroleum fuels, including efforts by companies like LS9, Amyris Biotechnologies, Joule Biotechnologies, and (in algae) by Synthetic Genomics. However, this approach is also being used to enable more efficient production of other fuels: for example, Joule Biotechnologies is using synthetic biology to create novel organisms that use photosynthesis to produce ethanol and other transportation fuels from sunlight and carbon dioxide without the need for a biomass feedstock.

I’ll have more to say about these and other companies in later installments of this blog, when I focus on specific sectors of the industry and the technology strategies being pursued in each.

Applications of Biotechnology to Renewable Fuel Production (Overview)

In recent years, as the need to develop alternative, non-petroleum-based transportation fuels has become more pressing, there has been a growing interest in using advanced biotechnologies to improve biofuel production. Specifically, numerous strategies have evolved by which biotechnology is being used to create improved biofuels products or processes, involving the creation of engineered or synthetic microorganisms for use in production of ethanol, biodiesel or other fuels, or genetically engineered (“transgenic”) plants as improved fuel feedstocks. These approaches can generally be summarized as follows.

Potential Applications of Biotechnology to Improve Renewable Fuel Production.

Enhanced or engineered microorganisms for fermentation of ethanol, butanol, other fuels.
Engineered microorganisms or plants to manufacture enzymes used in fuel production.
Improved algal strains for biofuel production.
Selected or engineered plant species with favorable traits for use as improved biofuel feedstocks.

There are a number of ways in which microorganisms, algae or plants can be modified for improved industrial performance. Some of the companies and technologies I’ll discuss make use of selected, often proprietary strains of production organisms, that have been derived from naturally occurring organisms using traditional techniques of mutation and selection, or in the case of plants by traditional crop breeding. These methods have been practiced in industry and in agriculture for decades, and in many cases their use can lead to significant process improvements, for example in the efficiency of ethanol fermentation.

However, in most cases, the technology strategies I’ll discuss in this blog will be ones that utilize genetic engineering methods based on recombinant DNA. Recombinant DNA methods enable the insertion of genes from any source in nature into a chosen “host” organism, thereby conferring on the host organism a genetic trait or a biochemical capability not naturally found in that organism. Genes function in nature by encoding the synthesis of specific protein molecules, most of which are enzymes whose role it is to catalyze specific biochemical reactions inside living cells; and so by transplanting a gene into a new host organism, under conditions in which the gene can actively direct the synthesis of its corresponding enzyme, one can impart on the host organism new or improved biochemical powers. In the years since recombinant DNA techniques were first developed in the mid-1970s, techniques have been worked out for the genetic engineering of almost any species of organism having medical, industrial or agricultural value, including most important plant species, almost any microorganism, and many algal species. These techniques are now being used to improve natural processes for synthesis of ethanol and other fuels.

There are also a number of companies and academic research laboratories using more advanced technologies for improvement of microbial or plant performance. Many of these methodologies utilize recombinant DNA, but in ways specifically designed to facilitate the creation of organisms improved for a specific desired function. These newer techniques, along with more traditional methods of organism improvement, are summarized as follows, with the understanding that there can be some overlap in the way these terms are defined. Some of the technologies, like directed evolution or DNA shuffling, use a combination of genetic tricks and enhanced selective pressure to greatly enhance the activity of a targeted enzyme or pathway, while other techniques like synthetic biology allow creation of novel or enhanced metabolic pathways in organisms never before possessing such traits.

Biotechnologies Applicable to Biofuels.

Classical mutation and selection or plant breeding: this encompasses a variety of well-known, decades-old techniques for selectively breeding or otherwise selecting naturally-occurring (or mutationally induced) variants of a starting strain or plant variety.
Recombinant DNA: insertion and expression of heterologous genes into a desired host organism (microorganism, algae or plant) to improve a desired trait or biochemical function in the host organism.
Directed evolution: this technique generally involves growing a desired microbial strain under certain limiting conditions that impose selective pressure under which those mutant overperforming strains that arise can eventually outcompete the starting strain.
DNA shuffling: this is a form of directed evolution where the gene encoding the enzyme targeted for improvement is mutated in millions of permutations using recombinant techniques, followed by selection and isolation of superior performers, often carried out in multiple iterations of selection.
Metabolic engineering: this term refers to the use of recombinant DNA technologies to create new metabolic or biosynthetic pathways in host organisms, or to enhance existing pathways, through the engineered, coordinated expression of several heterologous or enhanced enzymes in the desired pathway.
Synthetic biology: in this technique biochemical pathways or even entire microorganisms are created “from scratch”, to create pathways or organisms not previously found in nature. It can be viewed as a more ambitious approach to metabolic engineering, as it often involves creation of biochemical pathways never before existing in the host organism of choice.

The entries that will follow over the next several days will provide overviews of the approaches that are being taken to modify microorganisms, algae and plants to improve biofuel production. Following that, I’ll begin to review the companies that are active in using advanced biotechnology in the different sectors of the biofuel industry. Please contact me with any questions you may have.

Bio-Based Chemicals and Fuels: Business, Policy and Regulation

Discussing how advanced biotechnology is being used to improve the production of bio-based chemicals, renewable fuels and other industrial biotech products

Category Archives: Technologies