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United States Department of Agriculture

Agricultural Research Service

Research Project: INDUSTRIALLY ROBUST ENZYMES AND MICROORGANISMS FOR PRODUCTION OF SUGARS AND ETHANOL FROM AGRICULTURAL BIOMASS
2005 Annual Report


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 2004 exceeded 3.1 billion gallons. Most of this ethanol was produced from over 1 billion bushels of corn. Grain ethanol is expected to grow until it meets 4-5% of our automotive fuel needs. Expanding fuel ethanol production further to meet 10-15% or more of our fuel needs will require developing alternative fibrous feedstocks, such as agricultural residues and herbaceous energy crops. Such conversions are currently possible but are cost prohibitive. 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.
Major milestones are broken down by objective number and year of completion.

FY 2005 1.2 Test effect of harvest maturity. 1.2 Develop screening assay for ethanol yield. 3.1 Synthetic pdc gene w/Gram+ signals and vectors. 3.2 Isolate Klebsiella oxytoca mutants. 3.2 Evaluate Lactobacillus for xylose fermentation. 4.1 Bioabatement and Escherichia coli/yeast simultaneous saccharification and fermentation (SSF). 4.2 Verify cloned genes function in furoic acid growth.

FY 2006 1.1 Evaluate pretreated corn fiber. 2.1 Screen enzymes for biomass hydrolysis. 3.2 Characterize xylose metabolism for Lactobacillus. 3.2 Decision point for Klebsiella oxytoca and Lactobacillus. 4.1 Evaluate other strains for inhibitor removal. 4.1 Clone glucokinase gene and construct knockout. 4.2 LCMS to detect pathway intermediates.

FY 2007 1.2 Relate forage quality and ethanol yield. 2.3 Develop hosts for enzyme production. 3.1 Select alternate host organisms.

FY 2008 1.2 Develop new pretreatments. 1.2 Test hemicellulases. 2.2 Isolate candidate novel enzyme genes. 3.1 Add adh gene to Pdc-expressing organisms. 3.2 Introduce and stabilize further genetic changes. 4.1 Construct glucose non-metabolizing mutant. 4.2 Enzyme assays and synthesis of CoA compounds. 4.2 Express genes in Escherichia coli and Pseudomonas putida.

FY 2009 1.2 Integrate pretreatments, enzymes, and microbes. 2.3 Protein engineering of selected enzymes. 2.4 Evaluate engineered enzyme mixtures. 3.1 Measure Pdc activity and fermentation products. 3.1 Begin inactivating chromosomal metabolic genes. 4.1 Evaluate mutant function in bioabatement. 4.2 Structure-function studies for bioabatement.


4a.What was the single most significant accomplishment this past year?
DEVELOPING AN ALTERNATE CROP FOR FUEL ETHANOL PRODUCTION. Field pea production in northern U.S. is growing, and producers are looking towards expanding this market. Field peas are high in starch and, as such, represent a potential ethanol feedstock. We developed processes for dry fractionating field peas into enriched protein and starch streams and fermenting the pea starch to ethanol. Ethanol yields from fermenting peas were comparable to that of corn (on a starch basis), and the enriched protein stream was similar in protein content to high-protein soy meal, with a well balanced amino acid profile. Farmers and ethanol producers should directly benefit from this alternate feedstock, and we have been working with North Dakota Dry Pea and Lentil Association to inform ethanol producers situated in areas where field peas are cultivated.


4b.List other significant accomplishments, if any.
IMPROVED ENZYME PRODUCING FUNGI. The carbohydrates available for fermentation to ethanol that are typically found in biomass include cellulose and hemicellulose. Enzyme technology for converting hemicellulose (e.g., xylanase and others) to fermentable sugars has lagged behind that for converting cellulose (i.e., cellulase) to glucose. We have addressed this problem by expressing a highly active xylanase protein in Trichoderma reesei, an industrial enzyme producing fungus. The engineered strain produced elevated yields of xylanase. This strain will be of interest to biotechnology companies marketing or researching enzymes for application to the biomass conversion and animal feed markets.

IMPROVED ENERGY CROPS FOR ETHANOL PRODUCTION. Herbaceous biomass from grasses is a potential feedstock for ethanol production. Many of these potential grass crops have been long used as animal forages, and research in this area indicates that harvest maturity is an important factor in deciding upon conversion efficiency. No one has looked at the effect of maturity on herbaceous grasses for ethanol conversion. We tested three grasses at different maturities for their glucose yields, an indirect measure of ethanol yields. The results showed that maturity was an important factor and that high glucose recoveries were measured for younger and middle maturity harvested biomass compared to those from older plants. This important result will be of great interest to plant breeders working in this area and eventually farmers looking to develop energy crops.


4c.List any 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.
This research program is a continuation of a previous project (3620-41000-084-00D) and, as part of that project, process and metabolic engineering technologies were 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 by-products 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.

In this first year of the project, technology has been developed for converting field peas to fuel ethanol, production of novel xylanase enzymes, and increasing the efficiency of herbaceous energy crops to ethanol. The field pea work has potential as an alternative crop for ethanol production because it is grown in regions that have ethanol fermentation facilities, and yields of peas are increasing. A variety of enzymes useful for hydrolyzing biomass has been identified, characterized, and produced in transgenetic hosts. Technology from this project has already begun to be transferred to processing laboratories for inclusion into their research either directly or through our cooperation in the Midwest Consortium for Biobased Products and Bioenergy. Finally, the research on energy crops has been communicated to our collaborators, and we are in the process of extending this work with the goal of breeding superior yielding cultivars for ethanol production.


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?
Results from our research have been transferred to academic and ARS researchers as well as industrial groups. Results from the field pea experiments have been communicated to an appropriate commodity group and commercial ethanol producers. Results from work on producing hydrolytic enzymes have been used by academic and government laboratories to further their own research into fiber utilization and its conversion to chemicals. The enzymes are also being used to develop processes for converting Distillers' Dry Grains with Solubles (DDGS) to ethanol by a multi-partner collaborative research group consisting of four universities and three federal laboratories. Members of this group were recently the recipient of a Federal Research Biomass Directed Grant from the Department of Energy. A commercial enzyme producer has also shown interest in possibly marketing an esterase developed by this group. Research on crop maturity and ethanol production has been used by several federal laboratories with expertise in plant breeding to aid in planning future plant breeding experiments. The work has also resulted in a set of biomass calibration standards that has generated considerable interest in those researching energy crops and has already been requested by one federal and two university research groups.

This research program is a follow up of a previous project (3620-41000-084-00D). Recombinant strains developed for ethanol and lactic acid fermentations from that effort continue to be requested by research groups. Groups that have requested these strains include Federal, industrial, and university laboratories.


7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
Anonymous. 2005. Enzymes generate sugars from corn fiber. Industrial Bioprocessing: Technical Insights. Frost and Sullivan. 27:7, p. 3.


Review Publications
Lemuz, C.R., Dien, B.S., Tumbleson, M.E., Singh, V., Rausch, K.D. 2004. Ethanol yield determination for dry grind corn processing. Proceedings Corn Utilization and Technology Conference. p. 61.

Gorsich, S.W., Slininger, P.J., Liu, Z., Nichols, N.N., Dien, B.S. 2004. Identification of Saccharomyces cerevisiae genes involved in furfural tolerance during fermentation [abstract]. Proceedings of the Yeast Genetics and Molecular Biology. Abstract No. 166A, p. 104.

Li, X., Dien, B.S., Cotta, M.A., Wu, Y., Saha, B.C. 2005. Profile of enzyme production of Trichoderma reesei grown on corn fiber fractions. Applied Biochemistry and Biotechnology. 121-124:321-334.

Nichols, N.N., Dien, B.S., Guisado, G.M., Lopez, M.J. 2005. Bioabatement to remove inhibitors from biomass-derived sugar hydrolysates. Applied Biochemistry and Biotechnology. 121-124:379-390.

Dien, B.S., Iten, L.B., Skory, C.D. 2005. Converting herbaceous energy crops to bioethanol: a review with emphasis on pretreatment processes. In: Hou, C.T., editor. Handbook of Industrial Biocatalysis. Chapter 23. Boca Raton, FL: Taylor & Francis Group. p. 1-11.

Gorsich, S.W., Dien, B.S., Nichols, N.N., Slininger, P.J., Liu, Z. 2004. The Saccharomyces cerevisiae pentose phosphate pathway gene, rpe1, functions in furfural tolerance during fermentation [abstract]. Proceedings of the 11th International Congress on Yeasts in Science and Biotechnology. Paper No. PM24.

Weimer, P.J., Dien, B.S., Springer, T.L., Vogel, K.P. 2005. In vitro gas production as a surrogate measurement of the fermentability of cellulosic biomass. Applied Microbiology Biotechnology. 67:52-58.

Nichols, N.N., Dien, B.S., Lopez, M.J. 2004. A biological approach to removing inhibitory compounds from biomass sugars to be used for fermentation [abstract]. Great Lakes Regional American Chemical Society Symposium. Paper No. 36.

Wu, Y.V., Nichols, N.N. 2005. Fine grinding and air classification of field pea. Cereal Chemistry. 82(3):341-344.

Dien, B.S., Nagle, N., Singh, V., Moreau, R.A., Tucker, M.P., Nichols, N.N., Johnston, D., Cotta, M.A., Hicks, K.B., Nguyen, Q., Bothast, R.J. 2005. Review of process for producing corn fiber oil and ethanol from "Quick Fiber." International Sugar Journal. 107(1275):187-191.

Nichols, N.N., Dien, B.S., Wu, V., Cotta, M.A. 2005. Use of field pea starch as a feedstock for ethanol fermentation [abstract]. International Starch Technology. p. 96.

Dien, B.S., Li, X., Jordan, D.B., Nichols, N.N., Iten, L.B., Cotta, M.A. 2005. Enzymatic saccharification of pretreated corn fiber for production of sugars [abstract]. International Starch Technology. p. 90.

Calabrese, J.C., Jordan, D.B., Boodhoo, A., Sariaslani, S., Vannelli, T. 2004. Crystal structure of phenylalanine ammonia lyase: multiple helix dipoles implicated in catalysis. Journal of Biochemistry. 43:11403-11416.

Jordan, D.B., Calabrese, J.C. 2005. Active-site models of riboflavin synthase [abstract]. International Symposium on Flavins and Flavoproteins. p. 104.

Dien, B.S., Li, X., Cotta, M.A. 2005. Enzymatic saccharification of pretreated corn fiber for production of sugars [abstract]. Biotechnology for Fuels and Chemicals. Paper No. B-36.

Dien, B.S., Whitehead, T.R., Nichols, N.N., Skory, C.D., Cotta, M.A. 2004. Recombinant biocatalysts for converting sugar mixtures to lactic acid [abstract]. Great Lakes Regional American Chemical Society Symposium. Paper No. 40.

Singh, V., Dien, B.S., Johnston, D., Hicks, K.B., Cotta, M.A. 2004. A comparison between conversion of pericarp and endosperm fiber from corn into ethanol. Proceedings of ASAE Annual International Meeting. p. 1-15.

Last Modified: 4/18/2014
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