2006 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 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.
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.
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.
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.
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.