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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
2006 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? Why does it matter?
U.S. fuel ethanol production in 2005 exceeded 4 billion gallons. Most of this ethanol was produced from over 1.5 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 by year the currently approved milestones (indicators of research progress)
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 Characterize furfural degradation pathway.

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.List the single most significant research accomplishment during FY 2006.
DEVELOPING A NEW EXPRESSION SYSTEM FOR PRODUCING HYDROLYTIC ENZYMES. This directly contributes to increasing process efficiencies for converting lignocellulosic biomass to ethanol as outlined in the ethanol component of National Program 307. Anaerobic fungi produce high active enzymes for lignocellulose degradation but they do not produce these enzymes in high yields. To capture the advantages of these enzymes, one will need to genetically engineer the genes coding for the enzymes into commonly used production hosts such as Trichoderma reesei and Aspergillus niger. We have successfully over-expressed the Orpinomyces xylanase A gene in T. reesei, and the heterologous xylanase is secreted into culture medium under cellulase production conditions. The engineered T. reesei strain produces high levels of xylanases in addition to its own cellulases, and the enzyme should enhance the lignocellulose conversion into fermentable sugars.


4b.List other significant research accomplishment(s), if any.
NOVEL ENZYME FOR RELEASING SUGARS FROM BIOMASS. This also directly contributes to increasing process efficiencies for converting lignocellulosic biomass to ethanol as outlined in the ethanol component of National Program 307. Xylose is the second most common sugar present in biomass after glucose. Sources of beta-xylosidase are needed for converting xylan to xylose for subsequent bioconversion to ethanol. We determined that the beta-xylosidase from Selenomonas ruminantium is the most catalytically efficient enzyme known (at least 15-fold better than those reported in the literature) for catalyzing the hydrolysis of xylooligosaccharides to xylose and has good properties of temperature and pH stability. Additionally, the enzyme can be efficiently produced in Escherichia coli (>4 g enzyme/liter). These properties place the enzyme at the forefront for development as a saccharification catalyst. Details of the enzyme mechanism and binding of substrates and inhibitors provide new insights that apply to other glycohydrolases.

IMPROVED ENZYMES FOR CONVERTING CORN FIBER TO FERMENTABLE SUGARS. This also directly contributes to increasing process efficiencies for converting lignocellulosic biomass to ethanol as outlined in the ethanol component of National Program 307. Corn fiber, a low-value co-product of corn wet milling, is a potential feedstock for ethanol production. Treating corn fiber with liquid hot-water is one proposed method for preparing the carbohydrates for fermentation. Liquid hot water (LHW) treatment breaks down most of the carbohydrates to oligomers, which are then converted to monosaccharides by treating with hydrolytic enzymes. Unfortunately, these oligomers are too chemically complex for commercial enzymes to completely digest. So, we developed custom enzyme preparations – prepared by growing fungi on LHW treated corn fiber - and demonstrated arabinose, glucose, and xylose yields of 80%, 100%, and 80%, respectively. This technology was developed as part of a current collaboration with Purdue University and Aventine Renewable Energy to demonstrate Purdue’s LHW technology at one of Aventine’s ethanol facilities.


4c.List significant activities that support special target populations.
None.


5.Describe the major accomplishments to date and their predicted or actual impact.
Research accomplishments are directed towards increasing the economic competitiveness for converting lignocellulose to biomass as outlined in the ethanol component of National Program 307. 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 the first two years of this 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. Information on this technology has been shared with the Pea and Lentil Association and has been expanded to investigate corn/pea mixtures. The technology has also been widely published in U.S. and Canadian trade journals and has led to numerous discussions with potential users. A new beta-xylosidase has been characterized that is significantly improved compared to currently available enzymes. Discussions are currently underway to have DuPont evaluate the enzyme as part of their ongoing commercial interests in biomass processing. Finally, a medium throughput ethanol assay for ranking herbaceous biomass in terms of yields has been developed. This has led to many requests within and outside of ARS to evaluate samples. This assay is expected to lead to the first effort to develop energy crop cultivars selected for increased ethanol yield.


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. An industrial partner has indicated interest in evaluating our beta-xylosidase as part of their ongoing program in biomass conversion. 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 two federal and three 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).
Plant biofuels. Dec. 16, 2005. MicrobeWorld Radio Show. American Society for Microbiology and Fingerlakes Production Radio. Peas pose option for ethanol. April 13, 2006. Sean Pratt. Western Producer. p. 21. Pea starch a source of ethanol? April 25, 2006. Geoff Dale, Farmcentre.com. Fuel in the fridge. July/Sept. 2006. Agricultural Innovation. 15(3):9. Ethanol from pea starch. 2006. Industrial Bioprocessing. 28(5):7. NDDPLA collaborates with ARS in studying peas in ethanol. March 2006. Anonymous. NDDPLA Newsletter. Development of new biocatalysts for the conversion of lignocellulose to ethanol. M. Cotta. Presented at the Workshop on Sustainable Bioenergy: Focus on the Future of Biofuels and Chemicals. April 13-14, 2006. University of Illinois at Urbana-Champaign. Imagine-fuel alcohol from pea starch! March 28, 2006. Jan Suszkiw. ARS Magazine. This article by Jan Suszkiw was also reprinted or adapted by: Agriculture Industry Today, Alternate Energy Resource Network, Agnet (Food Safety Network, Univ. of Guelph), Agra-net.com, Agri-View, Alternate Energy Resource Network, American Vegetable Grower, Axcess News, Biofuels Journal, CommunityDispatch.com, Doane Agricultural Services, DomesticFuel.com, Environmental Observatory, FarmAssist.ca, Farmers National Company, Farms.com, FoodIngredients.com, High Plains/Midwest Ag Journal, John Deere Ag News, MidAmerica CropLife Association, Monsanto.com, Ohio Farm Bureau Federation, ScienceBlog.com, Statpub.com, The Bay Net (Maryland) News, The Soy Daily, TradingCharts.com.


Review Publications
Nichols, N.N., Dien, B.S., Wu, Y., Cotta, M.A. 2005. Ethanol fermentation of starch from field peas. Cereal Chemistry. 82(5):554-558.

Hughes, S.R., Riedmuller, S., Mertens, J.A., Li, X., Bischoff, K.M., Cotta, M.A., Farrelly, P. 2005. Development of a liquid handler component for a plasmid-based functional proteomic robotic workcell. Journal of the Association for Laboratory Automation. 10(5):287-300.

Liu, S., Dien, B.S. 2005. Metabolic engineering of Lactobacillus brevis for ethanol production [abstract]. The World Congress on Industrial Biotechnology and Bioprocessing. p. 23.

Liu, S., Nichols, N.N., Dien, B.S., Cotta, M.A. 2006. Metabolic engineering of a Lactobacillus plantarum double LDH knockout strain for enhanced ethanol production. Journal of Industrial Microbiology and Biotechnology. 33(1):1-7.

Nichols, N.N., Dien, B.S., Bothast, R.J., Cotta, M.A. 2006. The corn ethanol industry. In: Minteer, S., editor. Alcoholic Fuels. Boca Raton, FL: CRS Press. p. 59-78.

Liu, S., Dien, B.S., Cotta, M.A., Bischoff, K.M., Hughes, S.R. 2005. Lactobacillus brevis: a potential biocatalyst for lignocellulosic biomass to ethanol [abstract]. Society of Industrial Microbiology. Paper #P05.

Mertens, J.A., Skory, C.D. 2005. Development of plasmids for expression of heterologous proteins in Rhizopus oryzae [abstract]. Society of Industrial Microbiology. p. 100.

Nichols, N.N., Dien, B.S., Cotta, M.A. 2005. Ethanol fermentation of sugars in corn stover dilute acid hydrolysates [abstract]. Society of Industrial Microbiology. p. 85.

Slininger, P.J., Dien, B.S., Gorsich, S.W., Liu, Z. 2005. Mineral and nitrogen source optimization enhance d-xylose conversion to ethanol by the yeast Pichia stipitis [abstract]. Society of Industrial Microbiology Annual Meeting. Paper No. P07.

Hughes, S.R., Riedmuller, S.B., Mertens, J.A., Li, X., Bischoff, K.M., Qureshi, N., Cotta, M.A., Farrelly, P.J. 2006. High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell. Proteome Science. 4:10.

Hughes, S.R., Riedmuller, S., Mertens, J.A., Li, X., Qureshi, N., Farrelly, P., Cotta, M.A. 2006. Automated strategy using a functional proteomic assay to identify and isolate cellulase F mutants with improved activity from multiplexed sets of plasmid [abstract]. PepTalk 2006. p. 10.

Hughes, S.R., Mertens, J.A., Li, X., Bischoff, K.M. 2005. Plasmid-based functional proteomic robotic workcell process for high-throughput screening of multiplexed libraries of mutagenized clones [abstract]. Laboratory Robotics Information Group. p. 1.

Nichols, N.N., Mertens, J.A., Dien, B.S. 2006. Identification and transcriptional profiling of furoic acid metabolism genes in Pseudomonas putida [abstract]. American Society for Microbiology. Paper No. Q-449.

Hughes, S.R., Riedmuller, S.B., Bischoff, K.M., Mertens, J.A., Li, X., Cotta, M.A., Farrelly, P.J. 2005. Development of a liquid handler component for a functional plasmid-based proteomic workcell that generates multiplex samples expresses in yeast [abstract]. Association for Laboratory Automation, LabAutomation 2005. Poster WP128.

Hughes, S.R., Riedmuller, S., Mertens, J.A., Li, X., Qureshi, N., Bischoff, K.M., Jordan, D.B., Cotta, M.A., Farrelly, P. 2005. Plasmid-based functional proteomic robotic workcell process for high-throughput screening of multiplexed libraries of mutagenized clones [abstract]. Optimization high-throughput Cultures for Bioprocessing 2005. p. 3.

Mertens, J.A., Skory, C.D., Ibrahim, A.S. 2006. Plasmids for expression of heterologous proteins in Rhizopus oryzae. Archives of Microbiology. 186:41-50.

Nichols, N.N., Lopez, M.J., Dien, B.S., Bothast, R.J. 2006. Culture containing biomass acid hydrolysate and Coniochaeta ligniaria fungus. U.S. Patent 7,067,303.

Hughes, S.R., Riedmuller, S.B., Mertens, J.A., Jordan, D.B., Li, X., Qureshi, N., Cotta, M.A., Farrelly, P.J., Bischoff, K.M. 2005. Functional proteomic workcell for high volume plasmid preparations for repeated in vitro protein expression and high throughput screening to identify mutant enyzmes for use at low pH [abstract]. Optimization High-throughput Cultures for Bioprocessing 2005. 13:3.

Hughes, S.R., Riedmuller, S.B., Mertens, J.A., Li, X., Bischoff, K.M., Liu, S., Qureshi, N., Cotta, M.A., Skory, C.D., Gorsich, S.W., Farrelly, P.J. 2006. Functional proteomic plasmid-based integrated workcell for high-throughput transformation of BL21 DE3 E. coli for expression in vivo with piromyces strain xylose isomerase [abstract]. Midwest Laboratory Robotics Information Group. p. 2.

Cotta, M.A., Dien, B.S., Jung, H.G., Vogel, K.P., Casler, M.D., Lamb, J.F., Weimer, P.J., Iten, L.B., Mitchell, R., Sarath, G. 2006. Development of forage crops as feedstocks for production of fuel ethanol [abstract]. International Conference on Bioenergy. p. I-5.

Dien, B.S., Nichols, N.N., Li, X., Cotta, M.A. 2006. An overview of recent advancements in lignocellulose to ethanol conversion technology [abstract]. International Conference on Bioenergy. p. 8.

Last Modified: 7/31/2014
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