2010 Annual Report
1a.Objectives (from AD-416)
The broad goal of this project is to improve the biochemical processes for converting agricultural materials to fuels and chemicals, which will consequently enable cost reductions and increase profitability of biorefining. Research will focus on significant technical challenges that must be overcome to achieve cost-competitive conversion of agricultural feedstocks to biofuels.
Objective 1: Develop commercially-viable technologies based on novel deconstruction enzymes for hydrolyzing ligno-cellulose.
Objective 2: Develop, via genetic engineering, strains of pentose-utilizing gram-positive bacteria that enable commercially-viable processes for producing fuel-grade ethanol or butanol from ligno-cellulosic hydrolyzates.
Objective 3: Develop commercially-preferred methods for preventing, detecting, controlling, and/or correcting economically-harmful microbial contamination in ethanol production facilities.
Objective 4: Develop, via high-throughput methods, enzyme and/or microbial-based technologies that enable new commercially-viable coproducts from ethanol fermentations.
1b.Approach (from AD-416)
The growth and sustainability of bioenergy production in the United States (U.S.) are hindered by a number of technical and commercial barriers. Biochemical conversion of lignocellulosic biomass to fuels and chemicals is technically feasible, but inefficiencies in the process make it economically impractical. Accomplishing the objectives will help overcome significant technical challenges to producing sustainable fuels and chemicals from agricultural feedstocks.
This report documents accomplishments for the research project 3620-41000-135-00D, entitled Improving Biochemical Processes for the Production of Sustainable Fuels and Chemicals. Portions of this work are a continuation of research performed under project 3620-41000-135-00D (Microbial Catalysts to Produce Fuel Ethanol and Value Added Products), which was terminated in FY2009.
Research focuses on four objectives: 1. Develop novel deconstruction enzymes for hydrolyzing lignocellulose; 2. Develop Gram-positive bacteria for producing biofuels from lignocellulosic hydrolysates; 3. Develop methods for controlling bacterial contamination of fuel ethanol fermentations; 4. Develop technologies to enable new coproducts from biofuel production.
In FY 2010, ARS scientists made significant progress toward these objectives. Enzymes that are capable of degrading lignocellulosic biomass were characterized from the fungus Acremonium zeae. Strains of lactic acid bacteria were transformed to functionally express an enzyme involved in butanol production. New methods were developed to characterize bacterial biofilms, which are contributing factors in chronic and acute infections of fuel ethanol fermentations. An antibacterial peptide that has potential to inhibit the bacterial strains that contaminated ethanol plants was purified and characterized. Substantial progress has been made toward developing programs for the robotic workcell to generate transformed strains of the yeast Saccharomyces cerevisiae.
Progress achieved during FY 2010 has potential scientific impact for researchers in industry, government and academia, and will facilitate development and improvement of efficient processes that lower the production costs of fuels and chemicals from renewable agricultural materials.
ENGINEERING LACTIC ACID BACTERIA FOR PRODUCING BUTANOL. Lactic acid bacteria are well suited to industrial fermentation environments and have the potential to be developed into biocatalysts for the production of butanol from agricultural feedstocks. Renewable Product Technology Research Unit (RPT) scientists at the USDA-ARS-National Center for Agricultural Utilization Research (NCAUR), Peoria, Illinois, engineered strains of lactic acid bacteria to express a gene for the enzyme thiolase, which is the first step of the butanol production pathway in the butanol-producing bacterium Clostridium berjerinckii. Thiolase enzyme activity was detected in the engineered strains, indicating the enzyme is functionally active in lactic acid bacteria. This work has potential industrial application in bio-based refinery platforms that need new microbial biocatalysts to convert agricultural materials to biofuels.
PRODUCTION OF LIPASE ENZYME IN SACCHAROMYCES CEREVISIAE. Developing value-added coproducts from biofuel production is important to ensure stable, profitable, and sustainable operation of second generation biorefineries. Renewable Product Technology Research Unit (RPT) scientists at the USDA-ARS-National Center for Agricultural Utilization Research (NCAUR), Peoria, Illinois have modified a gene that encodes an enzyme called lipase, and expressed it in a yeast strain that makes ethanol. The modified enzyme catalyzed the formation of fatty acid ethyl esters (biodiesel) from ethanol and soybean oil. This work has potential to increase the profitability of an integrated biorefinery by combining bioethanol production with coproduction of a low-cost biocatalyst that converts corn oil to biodiesel.
ANTIBACTERIAL PEPTIDE PRODUCED BY LACTOBACILLUS PARACASEI. Bacterial contamination of commercial fermentation cultures is a common and costly problem to the fuel ethanol industry. Renewable Product Technology Research Unit (RPT) scientists at the USDA-ARS-National Center for Agricultural Utilization Research (NCAUR), Peoria, Illinois have purified an antibacterial peptide produced by a strain of Lactobacillus paracasei, and partially characterized its amino acid sequence. The antibacterial peptide has a broad spectrum of activity against bacteria that contaminate ethanol plants and against some disease-causing bacteria. The peptide has potential application in the biofuels industry to treat infections of ethanol fermentations, and in the food industry to control contamination by food-borne pathogens.
BIOFILM-FORMING CONTAMINANTS. New and improved methods are needed to rapidly identify microbial strains that produce problematic biofilms in fuel ethanol production. Renewable Product Technology Research Unit (RPT) scientists at the USDA-ARS-National Center for Agricultural Utilization Research (NCAUR), Peoria, Illinois, tested methods for high-throughput screening of biofilm-forming contaminants of fuel ethanol production. A rapid assay was developed to simultaneously compare multiple strains of bacteria for their ability to form biofilms in pure culture. This work has potential impact for research to develop new methods and approaches for control of fuel ethanol contamination.
PURIFICATION AND CHARACTERIZATION OF NEW ENZYMES THAT RELEASE SUGAR FROM BIOMASS. Improved enzymes are needed to degrade complex lignocellulosic biomass into fermentable sugars. Renewable Product Technology Research Unit (RPT) scientists at the USDA-ARS-National Center for Agricultural Utilization Research (NCAUR), Peoria, Illinois examined a fungus that infects corn for enzymes that release arabinose sugars from biomass. Two forms of the enzyme arabinofuranosidase were purified from the fungus Acremonium zeae, and they were shown to act synergistically to release sugar from corn fiber arabinoxylan. The enzymes from this organism may prove suitable for industrial application in the conversion of biomass to sugars for production of fuel ethanol or other valuable fermentation products.
Hughes, S.R., Rich, J.O., Bischoff, K.M., Hector, R.E., Qureshi, N., Saha, B.C., Dien, B.S., Liu, S., Jackson Jr, J.S., Sterner, D.E., Butt, T.R., Labaer, J., Cotta, M.A. 2009. Automated yeast transformation protocol to engineer S. cerevisiae strains for cellulosic ethanol production with open reading frames that express proteins binding to xylose isomerase identified using robotic two-hybrid screen. Journal of the Association for Laboratory Automation. 8:200-212.
Gibbons, W.R., Hughes, S.R. 2009. Integrated biorefineries with engineered microbes and high-value co-products for profitable biofuels production. In Vitro Cellular and Developmental Biology - Plants. 45:218-228.
Liu, S., Qureshi, N. 2009. How microbes tolerate ethanol and butanol. New Biotechnology. 26(3/4):117-121.
Songstad, D.D., Lakshmanan, P., Chen, J., Gibbons, W., Hughes, S.R., Nelson, R. 2009. Historical perspective of biofuels: Learning from the past to rediscover the future. In Vitro Cellular and Developmental Biology - Plants. 45:189-192.
Rastogi, G., Muppidi, G.L., Gurram, R.N., Adhikari, A., Bischoff, K.M., Hughes, S.R., Apel, W.A., Bang, S.S., Dixon, D.J., Sani, R.K. 2009. Isolation and characterization of cellulose-degrading bacteria from the deep subsurface of the Homestake Gold Mine, Lead, South Dakota, USA. Journal of Industrial Microbiology and Biotechnology. 36:585-598.
Bischoff, K.M., Liu, S., Hughes, S.R., Rich, J.O. 2010. Fermentation of corn fiber hydrolysate to lactic acid by the moderate thermophile Bacillus coagulans. Biotechnology Letters. 32:823-828.
Hughes, S.R., Hector, R.E., Rich, J.O., Qureshi, N., Bischoff, K.M., Dien, B.S., Saha, B.C., Liu, S., Jackson Jr, J.S., Sterner, D.E., Butt, T.R., Labaer, J., Cotta, M.A. 2009. Automated yeast mating protocol using open reading frames from Saccharomyces cerevisiae genome to improve yeast strains for cellulosic ethanol production. Journal of the Association for Laboratory Automation. 8:190-199.
Hughes, S.R., Qureshi, N. 2010. Biofuel demand realization. In: Vertes, A., Qureshi, N., Blascheck, H.P., Yukawa, H., editors. Biomass to Biofuels: Strategies to Global Industries. UK:John Wiley & Sons Limited. p. 55-69.