2005 Annual Report
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.
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.
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.
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.
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.
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.