BIOPROCESS AND METABOLIC ENGINEERING TECHNOLOGIES FOR BIOFUELS AND VALUE-ADDED COPRODUCTS

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
511	1510	1040	70%
511	1510	2020	30%

Progress 12/15/00 to 08/08/04

Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? U.S. fuel ethanol production in 2003 exceeded 2.8 billion gallons. Most of this ethanol was produced from over 1 billion bushels of corn. Expanding fuel ethanol production will require conversion of lower cost feedstocks. Such conversions are currently possible but are cost prohibitive (currently enzyme costs are approximately 50 cents/gallon of ethanol produced with a reasonable target being 5 cents/gallon) if using plant biomass other than starch. This is because agricultural material is made of many different polymers that must first be broken down into simple sugars that microorganisms can then use for the formation of products possessing higher value. Furthermore, a major technical hurdle to converting biomass to ethanol is developing an appropriate microorganism for the fermentation of mixed sugars. Our overall objective is to develop efficient global processes for converting crop cellulose and hemicellulose to ethanol and develop high-value co-products that will substitute for petrochemical derived industrial products. This project directly addresses the Ethanol Component of National Program 307. Technologies are needed to reduce the cost of producing ethanol from corn and biomass. The lack of cost effective enzyme preparations for saccharifying biomass and industrially robust microorganisms for their conversion to bioethanol have been identified as the two most significant technical restraints to developing a domestic lignocellulose ethanol industry. This project also addresses Quality and Utilization of Agricultural Products, National Program 306. Specifically, this project addresses Component 2, New Processes, New Uses, and Value- Added Foods and Biobased Products. Specific areas addressed are Problem Areas 2a (New Product Technology), 2b (New Uses for Agricultural By- Products), and 2c (New and Improved Processes and Feedstocks). These areas will be addressed by developing new products from unutilized and underutilized agricultural residues via fermentation and biocatalytic processes. The customer base for this renewable biofuel and coproduct research is international in scope and covers farmers, commodity groups, industry groups such as enzyme producers, grain processing companies, fermentation industry, etc., and scientists with other government agencies, universities, and private industry. 2. List the milestones (indicators of progress) from your Project Plan. The major milestones of this project were to develop new biocatalysts for production of ethanol and lactic acid from sugar mixtures consistent with those produced from saccharifying fibrous biomass feedstocks, to develop integrated pretreatment and fermentation processes for conversion of corn fiber to ethanol, to develop novel enzymes for xylan degradation, and finally to develop fermentation processes for production of sugar alcohols, a group of fine chemicals used as a non-caloric sweeteners. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004, and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. 1. Development of improved Gram positive strains for ethanol production. Progress: A synthetic operon was developed that should be suitable for re-direction fermentation products in lactic acid producing bacteria towards ethanol. To enhance production, a synthetic version of the critical gene, pyruvate decarboxylase, was developed that is preferably encoded for expression in Gram positive bacteria. 2. Development of integrated pretreatment and fermentation processes. Progress: A demonstration corn fiber pretreatment process has been successfully used as a feedstock for conversion to ethanol using ethanologenic strains developed in-house. 3. Scale-up of mannitol and xylitol fermentations. Progress: The highly productive scientist that directed the later research was switched to an alternate CRIS during FY 2003. B. List the milestones that you expect to address over the next 3 years (FY 2005, 2006, and 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? This is the final Report of Progress (AD-421) for this project. A new project plan was certified on 7/21/04 by the Office of Scientific Quality Review as having completed NP 307, Bioenergy and Energy Alternatives Panel Review. The replacement project, 3620-41000-118-00D, is entitled "Industrially robust enzymes and microorganisms for production of sugars and ethanol from agricultural biomass." Milestones for the next 3 years on the new project are: Objective 1: Integrated processes. FY 2005 Test effect of harvest maturity. Develop screening assay for ethanol yield. FY 2006 Evaluate pretreated corn fiber. FY 2007 Relate forage quality and ethanol yield. Objective 2: Novel hydrolytic enzymes. FY 2006 Screen enzymes for biomass hydrolysis. Isolate candidate novel enzyme genes. FY 2007 Develop hosts for enzyme production. Objective 3: New biocatalysts. FY 2005 Construct synthetic pdc gene w/Gram+ signals and vectors. Isolate Klebsiella oxytoca mutants. Evaluate Lactobacillus for xylose fermentation. FY 2006 Characterize xylose metabolism for Lactobacillus. Conduct assessment of for K. oxytoca and Lactobacillus; decision point for future development. FY 2007 Select alternate host organisms. Objective 4: Bioabatement. FY 2005 Apply bioabatement to Escherichia coli/yeast SSF. Verify cloned genes function in furoic acid growth. FY 2006 Evaluate other strains for inhibitor removal. Clone glucokinase gene and construct knockout. FY 2007 Develop LC/MS methods to detect pathway intermediates and measure these. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004: Integrated processes for corn fiber conversion to ethanol: Converting corn fiber to ethanol can increase the yield from a bushel of corn by 10%. Sugar mixtures generated from a pilot plant demonstration project (e.g., 43 gallons per minute of slurry) situated at a local ethanol producer's facility were fermented to ethanol using our recombinant Escherichia coli strains. The demonstration project is the outcome of a highly successful collaboration among an academic instruction (principal investigator (PI)), ethanol producer (co-PI), Agricultural Research Service (ARS), and Department of Energy. In particular, E. coli FBR16, which has been engineered to co-ferment arabinose, glucose, and xylose sugars, had ethanol yields that were 84-97% of the maximum possible based upon beginning sugar concentrations. These results demonstrate the ability of our strains to ferment hydrolysates generated by industrially sized hydrolysis processes. B. Other significant accomplishment(s), if any: New ethanol producing microorganisms: Inexpensive crop residues are potential feedstocks for production of ethanol for fuel. However, there are no naturally-occurring microbes that produce ethanol from the abundant pentose sugars in agricultural residues, and genetic engineering of a microbe that efficiently ferments pentose sugars is a major barrier to establishment of a biomass-to-ethanol industry. Toward engineering a new, industrially-hardened microorganism that efficiently produces ethanol from biomass sugars, we synthesized a pyruvate decarboxylase gene that has been designed to be efficiently produced in the lactic acid group of bacteria. The optimized gene has also been constructed with the appropriate genetic signals. A new microbe engineered to produce ethanol will be useful to the ethanol industry and to those seeking to make value- added products from biomass. Improved enzymes of biomass hydrolysis: New xylanase preparations are needed for converting biomass to fermentable sugars. Xylanases are categorized into different families. We have discovered that members of family 10 are much more efficient at hydrolyzing corn fiber than members of family 11. Therefore, enzyme preparations to be used on corn fiber should be enriched for family 10 xylanases. This work will be of direct benefit to the ethanol industry as producers seek to expand current feedstocks beyond grain starch. C. Significant activities that support special target populations: none. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Bioprocess and metabolic engineering technologies have been developed that expand biofuel feedstocks and add value to agricultural wastes. Development of new and more active biomass hydrolyzing enzymes, along with robust genetically engineered microbes capable of fermenting multiple sugars, are recognized as major technical breakthroughs for the economic conversion of biomass to fuel ethanol and chemical feedstocks that can be used in a variety of renewable products. Specific accomplishments have included: Development of novel ethanologenic Escherichia coli strains that selectively convert sugars to ethanol or lactic acid at near to theoretical yields, discovery of a fungal microorganism that is adapted for removing organic byproducts from biomass derived hydrolysates that retard fermentation, and the isolation and expression of a novel ferulic esterase enzyme that will enhance the action of cellulases for saccharification of biomass. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? We have discovered and developed genetically engineered fermentation strains on which an ARS patent has been granted, and the strains have been tested by international and corporate collaborators under Material Transfer Agreements. Specific aspects of the research (hydrolysis and fermentation) are being extended with an extramural funded industrial scale project between ARS, academia, and private industry. An additional patent has been filed regarding discovery of microorganisms suitable for the bio-abatement of sugar solutions prepared from biomass, and these microorganisms are also being evaluated by an international collaborator. We have produced and purified enzyme samples which we have made available to ARS and international collaborators. This has contributed to two U.S. patents (6,365,390 and 6,534,286). Members of this group have been invited to present their results at the annual meeting of the American Institute for Chemical Engineers, International Starch Conference, Society of Industrial Microbiology, and Mie Bioforurm 2003, Biotechnology of Lignocellulose Degradation and Biomass Utilization held at Mie University, Japan. Collaborators have been invited to share results at the Corn Utilization and Technology Conference and the 20th Annual International Fuel Ethanol Workshop and Trade Show. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Chem Manufacturers Curious about ARS's Ethanol Production. Federal Laboratory Coordinator Newslink. April 2004. Article: Phenolic acid esterase ease biomass breakdown. In Industrial Bioprocessing, 25:9, p. 7.

Impacts
(N/A)

Publications

Cotta, M.A., Nichols, N.N., Dien, B.S., Li, X. 2003. Development of microbial biocatalysts to produce fuels from agricultural biomass. Proceedings 32nd annual United States Japan Natural Resources Protein Panel. p. 387-393.
Dien, B.S., Cotta, M.A., Jeffries, T.W. 2003. Bacteria engineered for fuel ethanol production: current status. Applied Microbiology and Biotechnology. 63:258-266.
Dien, B.S., Bothast, R.J., Nichols, N.N., Cotta, M.A. 2003. Increasing the ethanol yield from a bushel of corn; prospects for biomass conversion [abstract]. International Starch Technology. Paper No. 2.
Dien, B.S., Nagle, N., Singh, V., Moreau, R.A., Nichols, N.N., Johnston, D. , Cotta, M.A., Hicks, K.B., Nguyen, Q., Bothast, R.J. 2004. Fermentation of "quick fiber" produced from a modified corn milling process into ethanol and recovery of corn fiber oil. Journal of Applied Biochemistry and Biotechnology. 115:937-950.
Dien, B.S., Nichols, N.N., Cotta, M.A. 2003. Fermentation of hexose and pentose sugar mixtures to lactic acid by recombinant bacteria [abstract]. American Institute Of Chemical Engineers. Paper No. 161D.
Dien, B.S., Iten, L.B., Nichols, N.N., Cotta, M.A. 2003. Conversion of lignocellulose to ethanol using a recombinant E. coli strain [abstract]. Society of Industrial Microbiology. p. 102.
Dien, B.S., Whitehead, T.R., Nichols, N.N., Cotta, M.A. 2004. Engineering klebsiella oxytoca for production of lactic acid from hexose and pentose [abstract]. Biotechnology for Fuels and Chemicals. Paper No. 2-08.
Mosier, N.S., Kim, Y., Zeng, M., Hendrickson, R., Dien, B.S., Welsh, G., Ladisch, M. 2003. Optimization of controlled pH liquid hot water pretreatment of corn fiber and stover [abstract]. American Institute Of Chemical Engineers. Paper No. 163d.
Lopez, M.J., Nichols, N.N., Dien, B.S., Moreno, J., Bothast, R.J. 2004. Isolation of microorganisms for biological detoxification of lignocellulosic hydrolysates. Applied Microbiology And Biotechnology. 64:125-131.
Li, X., Dien, B.S., Cotta, M.A. 2004. Growth of and enzyme production by Trichoderma reesei on corn fiber fractions [abstract]. Biotechnology For Fuels And Chemicals. p. 25.
Li, X., Ximenes, E.A., Chen, H., Cotta, M.A., Ljungdahl, L.G. 2003. Cloning and sequencing of two highly homologous cellulase genes, celh and celi from the anaerobic fungus orpinomyces strain pc-2 [abstract]. MIE Bioforum. p. 120.
Li, X., Ljungdahl, G. 2003. Protein production in Aureobasidium pullulans. U.S. Patent 6,534,286.
Li, X., Ljungdahl, L.G., Azain, M.J., Davies, E.T., Shah, A.K., Blum, D.L., Kataeva, I. 2003. Phenolic acid esterases, coding sequences, and methods. U.S. Patent 6,602,700.
Li, X., Ljungdahl, L.G., Ximenes, E.A., Chen, H., Felix, C.R., Cotta, M.A., Dien, B.S. 2004. Properties of a recombinant beta-glucosidase from the polycentric anaerobic fungus Orpinomyces PC-2 and its application for cellulose hydrolysis. Journal of Applied Biochemistry and Biotechnology. 113:233-250.
Nichols, N.N., Dien, B.S., Guisado, G.M., Lopez, M.J. 2004. Use of the ascomycete coniochaeta ligniaria to remove inhibitors from biomass-derived sugars [abstract]. Biotechnology For Fuels And Chemicals Symposium. Paper No. 2-10.
Mosier, N., Hendrickson, R., Dreschel, R., Dien, B.S., Bothast, R.J., Welch, G., Ladisch, M.R. 2003. Principles and economics of pretreating cellulose in water for ethanol production [abstract]. American Chemical Society. Paper No. 103.
Tumbleson, M., Lemuz, C., Dien, B.S., Singh, V., Belyea, R., Rausch, K. 2004. A standardized laboratory procedure for ethanol yield measurement [abstract]. International Fuel Ethanol Workshop. Paper No. 61.

Progress 10/01/02 to 09/30/03

Outputs
1. What major problem or issue is being resolved and how are you resolving it? U.S. fuel ethanol production in 2002 exceeded 2.2 billion gallons. Most of this ethanol was produced from over 800 million bushels of corn. Expanding fuel ethanol production will require conversion of lower cost feedstocks. Such conversions are currently possible but are cost prohibitive (currently enzyme costs are approximately 50 cents/gallon of ethanol produced with a reasonable target being 5 cents/gallon) if using plant biomass other than starch. This is because agricultural material is made of many different polymers that must first be broken down into simple sugars that microorganisms can then use for the formation of products possessing higher value. Furthermore, a major technical hurdle to converting biomass to ethanol is developing an appropriate microorganism for the fermentation of mixed sugars. Our overall objective is to develop efficient global processes for converting crop cellulose and hemicellulose to ethanol and develop high-value co-products that will substitute for petrochemical derived industrial products. 2. How serious is the problem? Why does it matter? Recent estimates predict that world oil production will peak in the next decade and begin to rapidly decline. Since the U.S. consumes 26% of the world's production, declining oil production and increasing prices are expected to have profound effects on the U.S. economy. Even if U.S. consumption stopped increasing, dependence upon the global oil market (now at 60% and rising) would continue to grow because U.S. production has been decreasing since 1970 as domestic reserves have become depleted. Biofuels, such as ethanol, can substitute for oil as a transportation fuel. Ethanol has a long history of being used as an automotive fuel and is currently blended with about 15% of the gasoline sold in the U.S. Conversion of corn fiber produced in corn processing plants could increase ethanol production by 70 million gallons/year. Fermentation of corn fiber would also serve as a test bed for other plant feedstocks. Available biomass reserves in the U.S. are approximately 200 million dry tons a year and, unlike fossil fuels, are continually renewed. Substituting biofuels for oil will also help significantly lower national carbon dioxide emissions. As evidenced by the recent Kyoto conference on global warming, human associated carbon dioxide emissions have become an international concern. In the U.S., transportation needs account for 35% of domestic greenhouse gas emissions. Biofuels will lower our net carbon dioxide emissions because much of the released carbon dioxide is recycled as plant matter. Other benefits associated with biofuels include reducing the U.S. trade debt (exceeding 50 billion dollars worth of oil imported), increasing employment by an estimated 30,000 jobs, and creating value for agricultural wastes. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? Our research supports the missions of National Program 306, Quality and Utilization of Agricultural Products and National Program 307, Bioenergy and Energy Alternatives, by focusing on conversion of low-value agricultural residues to fuel ethanol and commodity products. NP306: Quality and Utilization of Agricultural Products (40%) Corn used for production of fuel ethanol provides a significant market for corn grown by American farmers and is projected to consume one fifth of the nation's corn crop by 2012. In addition, we are developing methods, enzymes, and biocatalysts for production of ethanol from other, lower-value agricultural materials such as crop residues. NP307: Bioenergy and Energy Alternatives (60%) Our research is developing new technologies for production of fuel ethanol in areas such as feedstock pretreatment, biological conversion, product recovery, and milling. Information gained from our work will result in reduction of capital and processing costs associated with production of biofuels, for example, via development of a process for conversion of low-value corn fiber to ethanol. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2003: Converting biomass to ethanol requires first breaking down the biomass into its constitutive sugars. Producing sugars is most efficiently done enzymatically; however, commercially available enzymes have low activity, meaning too much enzyme is required, and often result in lower than desirable sugar yields. We have isolated hydrolyzing enzymes with very high specific activities from novel sources (i.e., anaerobic fungus Orpinomyces PC-2) and have applied recombinant DNA technology to construct hosts capable of producing these enzymes in large quantities. Developing cost-effective enzymes for sugar production from biomass will allow current ethanol producers to greatly expand production from current levels by expanding their feedstock beyond starch containing grains. B. Other significant accomplishment(s), if any: Ethanol producers that rely on dry grinding corn, which accounts for 60% of U.S. ethanol production, depend upon selling the animal feed Distiller's Dry Grain with Soluble (DDGS) to remain profitable. However, DDGS is declining in value because of increased production. We have researched processes for converting DDGS and related fibers to ethanol using our advanced fermentation microorganisms. The results are of interest to farmer co-operatives for potentially adding profit to ethanol production. Preparing biomass for ethanol fermentation often involves pretreating with high temperature and chemicals, which generates undesirable chemicals that impede fermenting the biomass into ethanol. We have isolated novel microorganisms that grow on these chemicals, thereby, removing them. The best of these organisms (a fungus) is the subject of a submitted patent. Developing a bio-remediation strategy for removing inhibitors from pretreated biomass streams will be of interest to clients seeking to produce ethanol from non-traditional fibrous feedstocks. C. Significant activities that support special target populations: none. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Bioprocess and metabolic engineering technologies have been developed that expand biofuel feedstocks and add value to agricultural wastes. Development of new and more active biomass hydrolyzing enzymes along with robust genetically engineered microbes capable of fermenting multiple sugars are recognized as major technical breakthroughs for the economic conversion of biomass to fuel ethanol and chemical feedstocks that can be used in a variety of renewable products. 6. What do you expect to accomplish, year by year, over the next 3 years? Over the next 3 years, we plan to continue development of process components for more efficient conversion of lignocellulosic feedstocks to value-added fermentation products. FY 2004: New enzymatic activities and microorganisms will be characterized for the conversion of cellulose and xylan components of biomass into fermentable sugars for valuable, renewable chemicals. We will apply novel enzymatic and microbial based catalysts to lignocellulosic hydrolysates and validate novel models for corn to ethanol. FY 2005: Continue to evaluate enzymes and recombinant microorganisms, while creating improved or novel enzymes and microorganisms for fermentation and apply the corn ethanol model to a variety of corn hybrids to measure starch availability. FY2006: Evaluate microorganisms and enzymes for processing of a variety of lignocellulosic biomasses. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Discovery and development of genetically engineered fermentation strains on which an ARS patent has been granted and the strains tested by international and corporate collaborators under Material Transfer Agreements. Specific aspects of the research (hydrolysis and fermentation) are being extended with an extramural funded industrial scale project between ARS, academia, and private industry. An additional patent has been filed regarding discovery of microorganisms suitable for the bio-abatement of sugar solutions prepared from biomass, and these microorganisms are also being evaluated by an international collaborator. Dr. Nancy Nichols is hosting a visiting scientist from that laboratory this summer. Dr. Xin Liang Li has made purified enzyme samples available to ARS and international collaborators. Dr. Li's work has contributed to two U.S. patents (6,365,390 and 6,534,286). Collaborations have also begun with a university, and Dr. Bruce Dien is serving on the committee of a graduate student. Members of this group have been invited to present their results at the Annual Meeting of the Society of Industrial Microbiology, International Starch Conference, Annual Meeting of the Distiller's Dry Grains Technology Council, and the Annual Meeting of the Corn Dry Millers Association. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). Do ethanol yields differ in Bt, non-Bt corn varieties? The Land, August 30, 2002. Dry milling destroys Bt Protein. AgJournal, July 8, 2002. (www.agjournal. com). USDA agricultural bioenergy research. IEA Bioenergy Task 39 Newsletter No. 4, p. 16, August 2002. Thinking ahead: shaping ethanol's future. Ethanol Producer Magazine, p. 14-19, September 2002. Scientists monitor Bt protein in corn ethanol. ARS News Information. July 5, 2002. Where's the BT? Tracking the protein's fate during ethanol production. CropBiotech Net Knowledge Center. July 12, 2002. (www.anbio.org.br) Where's the BT? Tracking the protein's fate during ethanol production. Iowa Grain Quality Initiative, Iowa State University. July 5, 2002. (www. extension.iastate.edu)

Impacts
(N/A)

Publications

GENG, X., LI, K., KATAEVA, I.A., LI, X., LJUNGDAHL, L.G. EFFECTS OF TWO CELLOBIOHYDROLASES, CBHA AND CELK, FROM CLOSTRIDIUM THERMOCELLUM ON DEINKING OF RECYCLED MIXED OFFICE PAPER. PROGRESS IN PAPER RECYCLING. 2003. V. 2. P. 29-32.
LI, X.-L. DEGRADATION OF LIGNOCELLULOSIC BIOMASS BY ENZYMES FROM ANAEROBIC FUNGI. 225TH AMERICAN CHEMICAL SOCIETY NATIONAL MEETING. 2003. PAPER NO. 44.
FREER, S.N., DIEN, B.S., MATSUDA, S. PRODUCTION OF ACETIC ACID BY DEKKERA/BRETTANOMYCES YEASTS UNDER CONDITIONS OF CONSTANT PH. WORLD JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY. 2003. V. 19. P. 101-105.
LI, X., LJUNGDAHL, L.G., CHEN, H., XIMENES, E.A., FELIX, C.R. PROPERTIES OF A RECOMBINANT BETA-GLUCOSIDASE FROM THE POLYCENTRIC ANAEROBIC FUNGUS ORPINOMYCES PC-2 AND ITS APPLICATION FOR CELLULOSE HYDROLYSIS. SYMPOSIUM ON BIOTECHNOLOGY FOR FUELS AND CHEMICALS. 2003. PAPER NO. 1B-13.
NAGLE, N., TUCKER, M.P., DIEN, B.S., COTTA, M.A., HICKS, K.B., MOREAU, R.A. , SINGH, V., NGUYEN, Q. POTENTIAL TO IMPROVE DRY MILL ECONOMICS BY INCREASING ETHANOL YIELD FROM CORN FIBER RESIDUE. 25TH SYMPOSIUM ON BIOTECHNOLOGY FOR FUELS AND CHEMICALS SYMPOSIUM. 2003. ABSTRACT P. 20.
SINGH, V., JOHNSTON, D.B., MOREAU, R.A., HICKS, K.B., DIEN, B.S., BOTHAST, R.J. PRETREATMENT OF WET-MILLED CORN FIBER TO IMPROVE RECOVERY OF CORN FIBER OIL AND PHYTOSTEROLS. CEREAL CHEMISTRY. 2003. V. 80. P. 118-122.
WEIL, J.R., DIEN, B.S., BOTHAST, R.J., HENDRICKSON, R., MOSIER, N.S., LADISCH, M.R. REMOVAL OF FERMENTATION INHIBITORS FORMED DURING PRETREATMENT OF BIOMASS BY POLYMERIC ADSORBENTS. INDUSTRIAL AND ENGINEERING CHEMISTRY RESEARCH. 2002. V. 41. P. 6132-6138.
NICHOLS, N.N., DIEN, B.S., BOTHAST, R.J. ENGINEERING LACTIC ACID BACTERIA WITH PYRUVATE DECARBOXYLASE AND ALCOHOL DEHYDROGENASE GENES FOR ETHANOL PRODUCTION FROM ZYMOMONAS MOBILIS. JOURNAL OF INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY. 2003. V. 30. P. 315-321.
DIEN, B.S., NICHOLS, N.N., LI, X., COTTA, M.A., BOTHAST, R.J. CONVERSION OF CORN FIBROUS MATERIAL INTO ETHANOL. 25TH SYMPOSIUM ON BIOTECHNOLOGY FOR FUELS AND CHEMICALS. 2003. ABSTRACT P. 68.
CHEN, H., LI, X., BLUM, D.L., XIMENES, E.A., LJUNGDAHL, L.G. CELF OF ORPINOMYCES PC-2 HAS AN INTRON AND ENCODES A CELLULASE (CELF) CONTAINING A CARBOHYDRATE-BINDING MODULE. JOURNAL OF APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY. 2003. V. 105-108. P. 775-785.
DIEN, B.S., NICHOLS, N.N., BOTHAST, R.J. FERMENTATION OF SUGAR MIXTURES USING ESCHERICHIA COLI CATABOLITE REPRESSION MUTANTS ENGINEERED FOR PRODUCTION OF L-LACTIC ACID. JOURNAL OF INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY. 2002. V. 29. P. 221-227.
DIEN, B.S., BOTHAST, R.J., ITEN, L.B., BARRIOS, L., ECKHOFF, S.R. FATE OF BT PROTEIN AND INFLUENCE OF CORN HYBRID ON ETHANOL PRODUCTION. CEREAL CHEMISTRY. 2002. V. 79. P. 582-585.

Progress 10/01/01 to 09/30/02

Outputs
1. What major problem or issue is being resolved and how are you resolving it? U.S. fuel ethanol production in 2001 exceeded 1.7 billion gallons. Most of this ethanol was produced from over 650 million bushels of corn. Expanding fuel ethanol production will require conversion of lower cost feedstocks. Such conversions are currently possible but are cost prohibitive (currently enzyme costs are approximately 50 cents/gallon of ethanol produced with a reasonable target being 5 cents/gallon) if using plant biomass other than starch. This is because agricultural material is made of many different polymers that must first be broken down into simple sugars that microorganisms can then use for the formation of products possessing higher value. Furthermore, a major technical hurdle to converting biomass to ethanol is developing an appropriate microorganism for the fermentation of mixed sugars. Our overall objective is to develop efficient global processes for converting crop cellulose and hemicellulose to ethanol and develop high-value co-products that will substitute for petrochemical derived industrial products. 2. How serious is the problem? Why does it matter? Recent estimates predict that world oil production will peak in the next decade and begin to rapidly decline. Since the U.S. consumes 26% of the world's production, declining oil production and increasing prices are expected to have profound effects on the U.S. economy. Even if U.S. consumption stopped increasing, dependence upon the global oil market (now at 60% and rising) would continue to grow because U.S. production has been decreasing since 1970 as domestic reserves have become depleted. Biofuels, such as ethanol, can substitute for oil as a transportation fuel. Ethanol has a long history of being used as an automotive fuel and is currently blended with about 15% of the gasoline sold in the U.S. Conversion of corn fiber produced in corn processing plants could increase ethanol production by 70 million gallons/year. Fermentation of corn fiber would also serve as a test bed for other plant feedstocks. Available biomass reserves in the U.S. are approximately 200 million dry tons a year and, unlike fossil fuels, are continually renewed. Substituting biofuels for oil will also help significantly lower national carbon dioxide emissions. As evidenced by the recent Kyoto conference on global warming, human associated carbon dioxide emissions have become an international concern. In the U.S., transportation needs account for 35% of domestic green house gas emissions. Biofuels will lower our net carbon dioxide emissions because much of the released carbon dioxide is recycled as plant matter. Other benefits associated with biofuels include reducing the U.S. trade debt (exceeding 50 billion dollars worth of oil imported), increasing employment by an estimated 30,000 jobs, and creating value for agricultural wastes. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? Our research supports the missions of National Program 306, Quality and Utilization of Agricultural Products and National Program 307, Bioenergy and Energy Alternatives, by focusing on conversion of low-value agricultural residues to fuel ethanol and commodity products. NP306: Quality and Utilization of Agricultural Products (40%) Corn used for production of fuel ethanol provides a significant market for corn grown by American farmers and is projected to consume one fifth of the nation's corn crop by 2012. In addition, we are developing methods, enzymes, and biocatalysts for production of ethanol from other, lower-value agricultural materials such as crops residues. NP307: Bioenergy and Energy Alternatives (60%) Our research is developing new technologies for production of fuel ethanol in areas such as feedstock pretreatment, biological conversion, product recovery, and milling. Information gained from our work will result in reduction of capital and processing costs associated with production of biofuels, for example, via development of a process for conversion of low-value corn fiber to ethanol. 4. What was your most significant accomplishment this past year? A. Single most significant accomplishment during FY 2002: Advances in functional genomics, genetic engineering, and biochemical pathway analysis, collectively referred to as metabolic engineering, make it possible to manipulate the biosynthetic pathways of microorganisms. We have developed a series of biocatalysts for improved conversion of biomass sugars to fuel ethanol and polylactate plastics. Specifically, we improved our patented (allowed November 3, 2000) bacterial strains for increased ethanol production beyond current starch-based production and improved bacterial strains engineered to efficiently produce optically pure L-lactic acid for polylactate plastics. These strains co-utilize the sugars present in biomass, which is advantageous when working with complex materials such as agricultural biomass. B. Other significant accomplishment(s), if any: In order to improve the yield of ethanol from a bushel of corn, production of grains with tailored traits, e.g., corn with higher starch content and enhanced fermentability, are desirable attributes. In traditional alcohol fermentations, we found that the availability of starch was more important than the actual starch content. A new dry grind liquefaction model has been constructed that includes a jet cooker that will allow for a more realistic representation of the ethanol process. This is the only such scaled down liquefaction model incorporating a dry grind that we are familiar with to date. There is an urgent need for methods to efficiently convert biomass to fermentable sugars. We purified and characterized novel enzymes to convert fibrous biomass to sugars. We isolated xylanases from two fungal strains that are capable of breaking down complex corn fiber heteroxylan and refined a fermentation system developed last year for the production of the sugar alcohol, manitol, from corn syrups. Availability of effective enzymes for conversion of biomass to sugars is essential for cost effective production of green chemicals from renewable resources. C. Significant accomplishments/activities that support special target populations: none. 5. Describe your major accomplishments over the life of the project, including their predicted or actual impact? Bioprocess and metabolic engineering technologies have been developed that expand biofuel feedstocks and add value to agricultural wastes. Development of new and more active biomass hydrolyzing enzymes along with robust genetically engineered microbes capable of fermenting multiple sugars are recognized as major technical breakthroughs for the economic conversion of biomass to fuel ethanol and chemical feedstocks that can be used in a variety of renewable products. 6. What do you expect to accomplish, year by year, over the next 3 years? Over the next 3 years, we plan to continue development of process components for more efficient conversion of lignocellulosic feedstocks to value-added fermentation products. FY 2003: Characterize new enzymatic activities and microorganisms for the conversion of cellulose and xylan components of biomass into fermentable sugars for valuable, renewable chemicals and develop a novel model for industrial corn dry grinding ethanol fermentation. FY 2004: Apply novel enzymatic and microbial based catalysts to lignocellulosic hydrolysates and validate novel models for corn to ethanol. FY 2005: Continue to evaluate enzymes and recombinant microorganisms, while creating improved or novel enzymes and microorganisms for fermentation and apply the corn ethanol model to a variety of corn hybrids to measure starch availability. 8. List your most important publications and presentations, and articles written about your work (NOTE: this does not replace your review publications which are listed below) Suszkiw, J. Where's the Bt? Tracking the protein's fate during ethanol production. Agricultural Research Magazine, July 2002, v. 50(7). p. 14-16. Comis, D. Bioenergy Today. Agricultural Research Magazine, April 2002, v. 50(4). p. 4-8. Adams, P. The Politics Behind Ethanol. Peoria Journal Star, May 28, 2002.

Impacts
(N/A)

Publications

Bothast, R.J., Iten, L.B., Dien, B.S. Strategies for enhanced ethanol yield from corn. 24th Symposium on Biotechnology for Fuels and Chemicals. 2002. Paper No. 1-01.
Cotta, M.A., Dien, B.S., Bothast, R.J., Nichols, N.N. Biotechnology: expanding the biofuel industry. Proceedings of the 2002 Illinois Crop Protection Technology Conference. 2002. p. 100-105.
Dien, B.S., Nichols, N.N., Bothast, R.J. Recombinant Escherichia coli engineered for production of L-lactic acid from hexose and pentose sugars. Journal of Industrial Microbiology and Biotechnology. 2001. v. 27. p. 259- 264.
Dien, B.S., Bothast, R.J., Nichols, N.N., Skory, C.D. Improved biocatalyst for production of bioethanol and lactic acid. National Meeting of the American Chemical Society. 2002. Paper No. 322.
Dien, B.S, Bothast, R.J., Nichols, N.N., Cotta, M.A. The U.S. corn ethanol industry: an overview of current technology and future prospects. International Sugar Journal. 2002. v. 104. p. 204-211.
Freer, S.N., Greene, R.V., Bothast, R.J. Gene structure of a bifunctional cellulase gene (celA) isolated from Teredinobacter turnerae. Himmel, M.E., Baker, J.B., Saddler, J.N., editors. ACS Symposium Series. Glycosyl Hydrolases from Biomass Conversion. 2001. p. 769.
Freer, S.N. Acetic acid production by Dekkera/Brettanomyces yeasts. World Journal of Microbiology and Biotechnology. 2002. v. 18. p. 271-275.
Keating, J.D., Robinson, J., Bothast, R.J., Saddler, J.N., Mansfiled, S.D. Characterization of a novel ethanologenic yeast in the fermentation of softwood lignocellulosic sugars. 24th Symposium on Biotechnology for Fuels and Chemicals. 2002. Paper No. 2-09.
Lopez, M.J., Nichols, N.N., Dien, B.S., Moreno, J., Bothast, R.J. Screening and discovery of microorganisms for inhibitor abatement in toxic fermentation substrates. Society for Industrial Microbiology Annual Meeting. 2002. Abstract p. 70.
Lopez, M.J., Nichols, N.N., Dien, B.S., Moreno, J., Bothast, R.J. Screening for microorganisms useful in detoxification of lignocellulosic hydrolysates. American Society for Microbiology. 2002. Paper No. 0-45.
Nichols, N.N., Dien, B.S. Identification of Pseudomonas putida genes involved in degradation of furfural and 2-furoic acid. 102nd General Meeting of the American Society for Microbiology. 2002. Abstract p. 354.
Saha, B.C. Production of fuels and chemicals from corn fiber. Japan Value Enhanced Grains Conference. 2002. Abstract p. 2.
Saha, B.C. Production of mannitol and lactic acid by Lactobacillus intermedius. Xth International Congress of Bacteriology and Applied Microbiology. 2002. Abstract p. 139.
Saha, B.C. Debittering of protein hydrolyzates. Biotechnology Advances. 2001. v. 19. p. 355-370.
Saha, B.C. Production, purification, and properties of xylanase from a newly isolated Fusarium proliferatum. Process Biochemistry. 2002. v. 37. p. 1279-1284.
Saha, B.C. Purification and characterization of an extracellular beta- xylosidase from newly isolated Fusarium verticillioides. Journal of Industrial Microbiology and Biotechnology. 2001. v. 27. p. 241-245.
Saha, B.C. Enzymatic saccharification of complex heteroxylan in corn fiber. Proceedings of the US-UJNR Protein Resources Panel 30th Annual Meeting. 2001. p. 184-190.
Saha, B.C. Enzymes in corn fiber saccharification. Japan Value Enhanced Grains Conference. 2002. Abstract p. 1.
Saha, B.C. Hemicellulose bioconversion. Society for Industrial Microbiology Annual Meeting. 2002. Abstract p. 82.
Saha, B.C. Production of mannitol by fermentation. American Chemical Society National Meeting. 2002. Paper No. 121.
Saha, B.C. Bio-based method for making mannitol. 2002. U.S. Patent Application S/N 10/146,616.
Weil, J.R., Dien, B.B., Bothast, R.J., Hendrickson, R., Mosier, N.S., Ladisch, M.R. Removal of furfural formed during biomass pretreatment by polymeric adsorbents. 24th Symposium on Biotechnology for Fuels and Chemicals. 2002. Paper No. 3-19.
Welch, G., Ladisch, M.R., Bothast, R.J., Hendrickson, R., Mosier, N.S., Brewer, M. Pilot-scale pretreatment of corn fiber using snake coil reactor system. 24th Symposium on Biotechnology for Fuels and Chemicals. 2002. Paper No. 5-08.
Bothast, R.J., Nichols, N.N., Dien, B.S., Cotta, M.A. Biocatalysts for production of bioethanol. Society for Industrial Microbiology Annual Meeting. 2002. Abstract p. 74.

Progress 10/01/00 to 09/30/01

Outputs
1. What major problem or issue is being resolved and how are you resolving it? United States fuel ethanol production in 2000 exceeded 1.6 billion gallons. Most of this ethanol was produced from over 600 million bushels of corn. Expanding fuel ethanol production will require conversion of lower cost feedstocks. Such conversions are currently possible but are cost prohibitive (currently enzyme costs are approximately 50 cents/gallon of ethanol produced with a reasonable target being 5 cents/gallon) if using plant biomass other than starch. This is because agricultural material is made of many different polymers that must first be broken down into simple sugars that microorganisms can then use for the formation of products possessing higher value. Furthermore, a major technical hurdle to converting biomass to ethanol is developing an appropriate microorganism for the fermentation of mixed sugars. Our overall objective is to develop efficient global processes for converting crop cellulose and hemicellulose to ethanol and develop high-value co-products that will substitute for petrochemical derived industrial products. 2. How serious is the problem? Why does it matter? Recent estimates predict that world oil production will peak in the next decade and begin to rapidly decline. Since the U.S. consumes 26% of the world's production, declining oil production and increasing prices are expected to have profound effects on the U.S. economy. Even if U.S. consumption stopped increasing, dependence upon the global oil market (now at 60% and rising) would continue to grow because U.S. production has been decreasing since 1970 as domestic reserves have become depleted. Biofuels, such as ethanol, can substitute for oil as a transportation fuel. Ethanol has a long history of being used as an automotive fuel and is currently blended with about 15% of the gasoline sold in the U.S. Conversion of corn fiber produced in corn processing plants could increase ethanol production by 70 million gallons/year. Fermentation of corn fiber would also serve as a test bed for other plant feedstocks. Available biomass reserves in the U.S. are approximately 200 million dry tons a year and, unlike fossil fuels, are continually renewed. Substituting biofuels for oil will also help significantly lower national carbon dioxide emissions. As evidenced by the recent Kyoto conference on global warming, human associated carbon dioxide emissions have become an international concern. In the U.S., transportation needs account for 35% of domestic green house gas emissions. Biofuels will lower our net carbon dioxide emissions because much of the released carbon dioxide is recycled as plant matter. Other benefits associated with biofuels include reducing the U.S. trade debt (exceeding 50 billion dollars worth of oil imported), increasing employment by an estimated 30,000 jobs, and creating value for agricultural wastes. 3. How does it relate to the National Program(s) and National Component(s)? National Program 306, Quality and Utilization of Agricultural Products (40%); National Program 307, Bioenergy and Energy Alternatives (60%). This research is focused on conversion of low-value agricultural residues to ethanol and value-added commodity fermentation products, thus developing new uses for agricultural residues. Fuel ethanol is produced by fermentation as an important source of bioenergy. It is an excellent energy alternative as well as an oxygenate for compliance with the Clean Air Act. Our research program coincides with the following goals of the national program: Microbes (i.e., ethanol producing Escherichia coli and Gram positive bacteria) capable of fermenting the multiple sugars found in biomass which provide ethanol yields suitable for industrial recovery. Construction of the necessary traits in selected microbes will be accomplished by genetic modification. Improved processes for enzymatic breakdown of corn fiber into component sugars (i.e., identifying and characterizing novel enzymes for biomass enzymatic hydrolysis), a necessary preliminary step for conversion of biomass to ethanol and other fermentative products. Developing new technologies in feedstock pretreatment, biological conversion, and product recovery processes, as well as fundamental knowledge regarding fermentation, milling, and membrane separations. The information gained will result in a reduction in capital and processing costs associated with biofuel production (i.e., development of process for conversion of corn fiber to ethanol). 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment During FY 2001 Year: Advances in functional genomics, genetic engineering, and biochemical pathway analysis, collectively referred to as metabolic engineering, make it possible to manipulate the biosynthetic pathways of microorganisms. We have developed a series of biocatalysts for improved conversion of biomass sugars to fuel ethanol and polylactate plastics. Specifically, we developed and patented (allowed November 3, 2000) a new bacterial strain for increased ethanol production beyond current starch-based production. We also developed several bacterial strains that efficiently produce optically pure L-lactic acid for polylactate plastics from biomass sugars. B. Other Significant Accomplishment(s), if any: In order to improve the yield of ethanol from a bushel of corn, production of grains with tailored traits, e.g., corn with higher starch content and enhanced fermentability, are desirable attributes. In traditional alcohol fermentations, we found that the availability of starch was more important than the actual starch content and that the insecticidal Bt protein was not detectable after fermentation of GMO corn hybrids. We also purified and characterized two novel enzymes from a recently isolated fungal strain that are capable of breaking down complex corn fiber heteroxylan and developed a fermentation system for the production of the sugar alcohol, manitol, from corn syrups. Current research activity follows from previous project 3620-41000-074-00D. C. Significant Accomplishments/Activities that Support Special Target Populations: Nothing to report. 5. Describe the major accomplishments over the life of the project including their predicted or actual impact. Bioprocess and metabolic engineering technologies have been developed that expand biofuel feedstocks and add value to agricultural wastes. Development of new and more active biomass hydrolyzing enzymes along with robust genetically engineered microbes capable of fermenting multiple sugars are recognized as major technical breakthroughs for the economic conversion of biomass to fuel ethanol and chemical feedstocks that can be used in a variety of renewable products. 6. What do you expect to accomplish, year by year, over the next 3 years? Over the next three years, we plan to evaluate alternate pretreatment processes to determine the factors critical to rapid and efficient enzyme function. We propose to discover and characterize new enzymatic activities for the conversion of cellulose and xylan components of biomass into fermentable sugars for valuable, renewable chemicals. Building on a novel strategy for creating ethanol producing microorganisms with enhanced genetic stability and high ethanol yields, we plan to construct new microorganisms, with a variety of genetic backgrounds, that selectively produce ethanol or lactic acid and screen these microorganisms for genetic stability in cultures that mimic the type of concentrations currently favored by the fermentation industry. These microbes will be engineered to redirect sugar catabolism to ethanol or lactic acid at the expense of other fermentation products. We also plan to develop fermentation processes for the production of value-added polyols such as xylitol and mannitol. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end user (industry, farmer, other scientists)? What are the constraints if known, to the adoption & durability of the technology product? Discovery and development of two novel bioprocess enzymes which are patented and licensed. Discovery and development of genetically engineered fermentation strains on which an ARS patent has been allowed November 3, 2000, and the strains tested by international collaborators under Material Transfer Agreements. Specific aspects of the research (hydrolysis and fermentation) are being extended with an extramural funded industrial scale project between ARS, academia, and private industry. Collaborative research is underway with the Department of Wood Science, University of British Columbia, on improved pretreatment processes for biomass conversion and discovery of unique metabolic pathways in yeast. Scientists assigned to this project are instrumental in the planning and participation of the Annual International Fuel Ethanol Workshop. 8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below) Anonymous. Enzyme system saccharifies corn fiber heteroxylan. Industrial Bioprocessing. June 2001. v. 23(6). p. 7-8.

Impacts
(N/A)

Publications

Saha, B.C. Microbial production of xylitol from hemicellulosic biomass. Proceedings of the UJNR Protein Resources Panel 29th Annual Meeting. 2001. p. DD1-7.
Saha, B.C. Xylanase from a newly isolated Fusarium verticillioides capable of utilizing corn fiber xylan. Applied Microbiology and Biotechnology. 2001. DOI 10.1007/s002530100716.
Saha, B.C. Microbial production of xylitol from hemicellulosic biomass. 29th Annual Meeting of the U.S. Japan Natural Resources Protein Panel. 2000. Abstract No. 29. p. 26.
Dien, B.S., Nichols, N.N., Bothast, R.J. Production of L-lactic acid using recombinant Escherichia coli. Annual Meeting of the Society for Industrial Microbiology. 2001. Abstract p. 94.
Dien, B.S., Bothast, R.J., Barrios, L. Fate of the BT protein in dry/wet milled corn for ethanol production. International Starch Technology Conference. 2001. Abstract p. 126.
Saha, B.C. Enzymes in corn fiber saccharification. American Chemical Society Spring National Meeting. Cell. 2001. Paper No. 120.
Saha, B.C. Production, purification and characterization of xylanase and beta-xylosidase from a newly isolated Fusarium proliferatum. Annual Meeting of the Society for Industrial Microbiology. 2001. Abstract p. 91.
Cowles, C.E., Nichols, N.N., Harwood, C.S. BenR, a XylS homologue, regulates three different pathways of aromatic acid degradation in Pseudomonas putida. Journal of Bacteriology. 2000. v. 182. p. 6339-6346.
Nichols, N.N., Dien, B.S., Bothast, R.J. Use of catabolite repression mutants for fermentation of sugar mixtures to ethanol. Applied Microbiology and Biotechnology. 2001. v. 56. p. 120-125.
Cotta, M.A., Dien, B.S., Nichols, N.N. Metabolic engineering of Escherichia coli for improved production of ethanol and lactic acid. 23rd Symposium on Biotechnology for Fuels and Chemicals. 2001. Paper No. 2-22.
Bothast, R.J., Dien, B.S., Iten, L.B. Fermentation of high starch and GMO corn. Annual Meeting of the Society for Industrial Microbiology. 2001. Abstract p. 92.
Nichols, N.N., Dien, B.S., Bothast, R.J. Construction of an operon for production of ethanol in Lactococcus lactis. Annual Meeting of the Society for Industrial Microbiology. 2001. Abstract p. 92.
Keating, J.D., Robinson, J., Boussaid, A., Bothast, R.J., Saddler, J.N., Mansfield, S.D. Screening and characterization of ethanologenic yeast in the fermentation of lignocellulosic sugars. 23rd Symposium on Biotechnology for Fuels and Chemicals. 2001. Paper No. 2-36.
Bura, R., Boussaid, A., Mansfield, S.D., Saddler, J.N., Bothast, R.J. SO2-catalysed steam explosion of corn fiber for ethanol production. 23rd Symposium on Biotechnology for Fuels and Chemicals. 2001. Paper No. 1-16.
Peterson, J.D., Slakovic, D., Bond, K., Jamison, A., Burke, J., Peterson, S.W., Bothast, R.J. Chemical pretreatment and enzymatic hydrolysis of sugar beet pulp for production of fuel ethanol using recombinant ethanologenic E. coli. 23rd Symposium on Biotechnology for Fuels and Chemicals. 2001. Paper No. 3-128.
Bothast, R.J., Dien, B.S., Nichols, N.N., Skory, C.D. Metabolic engineering technologies for improved production of ethanol and lactic acid. International Chemical Congress of Pacific Basin Societies. Agrochemistry. 2000. Paper No. 0042.