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Location: Renewable Product Technology Research

2011 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.

3. Progress Report
This report documents accomplishments for the research project 3620-41000-135-00D, entitled Improving Biochemical Processes for the Production of Sustainable Fuels and Chemicals. 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 2011, Agricultural Research Service (ARS) scientists in Peoria, IL, made significant progress toward these objectives, as demonstrated by the following accomplishments. Native enzymes that are capable of releasing sugar from lignocellulosic biomass were purified and characterized from fungal species of Acremonium. Several genes encoding hydrolytic enzymes were identified in the genome of the fungus Fusarium, and recombinant forms of these enzymes were expressed in Escherichia coli. Strains of lactic acid bacteria that could tolerate growth in the presence of butanol were isolated. These butanol-tolerant strains are good candidates for genetic engineering to express the butanol production pathway. Bacterial contaminants from commercial dry-grind and wet-mill ethanol facilities have been isolated and identified. Strains that contaminate sugar processing facilities were characterized with respect to polysaccharide production and biofilm formation. A study to determine the fate of antibiotics used to control contamination during ethanol production was conducted at the pilot plant facilities available at the National Corn-to-Ethanol Research Center, Edwardsville, IL. Diverse strains of fungi were screened for production of the enzyme laccase, which has application in conversion of lignin into valuable bioproducts.

4. Accomplishments
1. Fate of antibiotics used during fuel ethanol production. Antibiotics are used to control bacterial contamination at commercial fuel ethanol facilities, but the fate of these drugs in dried distillers grains (DDG), a byproduct of ethanol production, is unknown. DDG is typically incorporated into animal feed, so determining if these drugs are degraded during the fermentation or drying process is important in monitoring antibiotic use in livestock. Scientists in the Renewable Product Technology Research Unit at the USDA-ARS-National Center for Agricultural Utilization Research, Peoria, IL, collaborated with researchers at the National Corn-to-Ethanol Research Center (NCERC), Edwardsville, IL, to measure the biological activity of the antibiotic virginiamycin throughout the ethanol production process. Pilot plant trials conducted at NCERC demonstrated that biologically active antibiotic persisted in DDG produced from fermentations that were treated with virginiamycin. This work provides new information for the ethanol and feed industries to facilitate development of improved management practices with DDG used in animal feed.

2. Production of novel enzymes for the biomass conversion industry. Agricultural wastes and energy crops represent a viable and abundant renewable resource for the production of biofuels and commodity chemicals, provided that the efficiency of breaking this biomass down to fermentable sugars is improved. Enzymes capable of digesting biomass are available, but the economic feasibility of utilizing this approach is often hampered by poor efficiencies of conversion. Scientists in the Renewable Product Technology Research Unit at the USDA-ARS-National Center for Agricultural Utilization Research, Peoria, IL, collaborated with researchers at the Federal University of Vicosa, Vicosa, Brazil, to examine strains of fungi for enzymes that degrade biomass with high efficiency. Two strains of a fungus, called Acremonium, were found to produce enzymes capable of rapidly degrading certain biomass components and could release the majority of the sugars in sugarcane bagasse, a fibrous waste-product of the sugar refining industry. These enzymes can supplement commercially available enzyme products for application to regional feedstocks, which will enable the economic conversion of biomass to sustainable fuels and chemicals.

3. Isolation of novel butanol-tolerant bacteria. Butanol is recognized as a promising alternative liquid transportation fuel because it can easily be stored, transported, and blended with gasoline, and it can be produced by microbial fermentation using agriculturally-derived sugars, including waste residues or energy crops. Efforts to improve the economic viability of microbial butanol production are hampered by growth inhibition that results from the accumulating butanol. Scientists in the Renewable Product Technology Research Unit at the USDA-ARS-National Center for Agricultural Utilization Research, Peoria, IL, identified several bacterial isolates from commercial ethanol facilities that were capable of growth in high levels of butanol. These bacterial strains provide the necessary foundation to utilize genetic engineering to develop robust butanol-producing strains for application in bio-based refinery platforms that convert renewable agricultural materials to biofuels.

4. Genome sequencing of an ethanol-tolerant bacterium. The ability to convert agricultural wastes or energy crops to biofuels requires microorganisms that are capable of fermenting the many types of sugars that are produced from biomass feedstocks. Yeasts typically used for biofuels production lack this trait, while bacteria that can utilize mixed sugars are often limited by growth inhibition by the ethanol. Scientists in the Renewable Product Technology Research Unit at the USDA-ARS-National Center for Agricultural Utilization Research, Peoria, IL, identified a bacterium, called Lactobacillus buchneri, that is tolerant to high concentrations of ethanol, and can ferment the sugars derived from biomass to produce ethanol and other chemicals. In collaboration with scientists at the University of California, Davis, CA, they sequenced the genome of this organism, which was released through the National Center for Biotechnology Information (available at Genome information from this strain will provide necessary information on the genetic pathways involved in ethanol production, and will be used to facilitate development of new strains for conversion of biomass to biofuels and bioproducts.

5. Improving economic value of waste products from biomass conversion. Lignin is a major structural component of agricultural wastes and energy crops that has limited commercial value. Developing new products from lignin could significantly improve the economics of converting biomass to fuels and chemicals. In collaboration with a visiting scientist from Rangsit University, Pathumtani, Thailand, scientists in the Renewable Product Technology Research Unit at the USDA-ARS-National Center for Agricultural Utilization Research, Peoria, IL, examined genetically diverse strains of the fungus Aureobasidium for the production of an enzyme, called laccase, that is involved in chemical modification of lignin and many other compounds. A specific genetic group of strains was identified that produces this enzyme. This work provides a foundation for further development of sustainable chemicals from lignin that will increase the value of biorefinery coproducts.

6. Development of a yeast strain for conversion of lignocellulosic sugars to ethanol. Economically producing fuels and chemicals from agricultural waste products and energy crops requires microorganisms capable of fully utilizing all available sugars resulting from the biomass conversion process. Scheffersomyces stipitis is a yeast that can use these sugars to make ethanol, but it requires specific oxygen levels that are difficult to achieve in large-scale fermentation facilities. Scientists in the Renewable Product Technology Research Unit at the USDA-ARS-National Center for Agricultural Utilization Research, Peoria, IL, isolated several strains of this yeast that were capable of growing in the absence of oxygen on two of the most common sugars obtained from biomass conversion, xylose and glucose. These strains serve as candidates for further improvement to produce ethanol from agricultural residues.

Review Publications
Bischoff, K.M., Callaway, T.R., Edrington, T.S., Genovese, K.J., Crippen, T.L., Nisbet, D.J. 2010. Antimicrobial use: Alternatives. In: Ullrey, D.E., Baer, C.K., Pond, W.G., editors. Encyclopedia of Animal Science, Second Edition. Boca Raton, Fl: CRC Press. 1(1):43-45.

Liu, S., Li, Y., Azaizeh, H., Cui, F., Tafesh, A., Bischoff, K.M. 2010. Production of value-added products by lactic acid bacteria. In: Hou, C.T., Shaw, J.-F., editors. Biocatalysis and Molecular Engineering. New York: John Wiley and Sons. p. 421-435.

Rich, J.O., Bischoff, K.M., Leathers, T.D., Cote, G.L., Liu, S. 2010. Lactic acid bacteria – Friend or Foe? Lactic acid bacteria in the production of Polysaccharides and fuel ethanol. KKU Research Journal. 15(5):424-435.

Liu, S., Bischoff, K.M., Qureshi, N., Hughes, S.R., Rich, J.O. 2010. Functional expression of the thiolase gene THl from Clostridium beijerinckii P260 in Lactococcus lactis and Lactobacillus buchneri. New Biotechnology. 27(4):283-288.

Rastogi, G., Bhalla, A., Adhikari, A., Bischoff, K.M., Hughes, S.R., Christopher, L.P., Sani, R.K. 2010. Characterization of thermostable cellulases produced by Bacillus and Geobacillus strains. Bioresource Technology. 101(22):8798-8806.

Rich, J.O., Leathers, T.D., Nunnally, M.S., Bischoff, K.M. 2011. Rapid evaluation of the antibiotic susceptibility of fuel ethanol contaminant biofilms. Bioresource Technology. 102(2):1124-1130.

Hughes, S.R., Moser, B.R., Harmsen, A.J., Bischoff, K.M., Jones, M.A., Pinkelman, R., Bang, S.S., Tasaki, K., Doll, K.M., Qureshi, N., Liu, S., Saha, B.C., Jackson Jr, J.S., Cotta, M.A., Rich, J.O., Caimi, P. 2010. Production of Candida antaractica Lipase B gene open reading frame using automated PCR gene assembly protocol on robotic workcell and expression in ethanologenic yeast for use as resin-bound biocatalyst in biodiesel production. Journal of the Association for Laboratory Automation. 16(1):17-37. DOI: 10.1016/j.jala.2010.04.002.

Songstad, D., Lakshmanan, P., Chen, J., Gibbons, W., Hughes, S.R., Nelson, R. 2011. Historical perspective of biofuels: Learning from the past to rediscover the future. In: Tomes, D., Lakshmanan, P., Songstad, D., editors. Biofuels. New York Dordrecht Heidelberg London: Springer. p. 1-8.

Gibbons, W., Hughes, S.R. 2011. Integrated biorefineries with engineered microbes and high-value co-products for profitable biofuels production. In: Tomes, D., Lakshmanan, P., Songstad, D., editors. Biofuels. New York Dordrecht Heidelberg London: Springer. p. 265-284.

Rich, J.O., Manitchotpisit, P., Peterson, S.W., Leathers, T.D. 2011. Laccase production by diverse phylogenetic clades of Aureobasidium pullulans. Rangsit Journal of Arts and Sciences. 1(1):41-47.

Walton, S.L., Bischoff, K.M., Van Heiningen, A.R., Van Walsum, G. 2010. Production of lactic acid from hemicellulose extracts by Bacillus coagulans MXL-9. Journal of Industrial Microbiology and Biotechnology. 37:823-830.

Liu, S., Leathers, T.D., Copeland, A., Chertkov, O., Goodwin, L., Mills, D.A. 2011. Complete genome sequence of Lactobacillus buchneri NRRL B-30929, a novel strain from a commercial ethanol plant. Journal of Bacteriology. 193(15):4019-4020. DOI: 10.1128/JB.05180-11.