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Location: Renewable Product Technology Research

2012 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:
In FY12, ARS scientists made significant progress toward the objectives of this research project, as demonstrated by the following activities: • Genes for acetylxylan and ferulate esterases, auxiliary enzymes used in deconstructing lingnocellulosic biomass, have been isolated from fungal and bacterial microorganisms, and tested for expression in recombinant hosts. • Microorganisms that produce ethanol and butanol were exposed to ultraviolet radiation to produce strains with improved biocatalytic properties. Properties targeted for improvement included the ethanol yield of the yeast Kluyveromyces marxianus and the butanol tolerance of the bacterium Clostridium. • A survey of bacteria that inhabit commercial ethanol plants continued, and strains collected from the plant were identified and characterized for their ability to form biofilms and inhibit ethanol fermentation. In addition, new antibacterial agents were tested against contaminating bacterial strains. • Reaction conditions were developed for an enzyme-based system to convert the chemical components of lignin into more valuable chemicals. Specifically, products from p-coumaryl alcohol and sinapyl alcohol were produced in sufficient quantity for identification and further characterization. • A low-calorie protein sweetener, brazzein peptide, was expressed in Kluyveromyces marxianus, an ethanol producing yeast capable of using all available sugars from lignocellulosic biomass. • Corn residues were identified as promising feedstocks for production of biobased polymers, which exhibited chemical and physical properties similar to those of commercially produced polymers. Progress achieved during FY12 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.

4. Accomplishments
1. Virginiamycin residues in distillers grains do not select for antibiotic resistant bacteria in cattle. Antibiotics are frequently used to control bacterial contamination at commercial fuel ethanol facilities, but there is concern that residues of these drugs could enter the food chain through the distillers grains coproducts that are incorporated into animal feed. Scientists in the Renewable Product Technology Research Unit at the USDA, ARS, National Center for Agricultural Utilization Research, Peoria, Illinois and the Food and Feed Safety Research Unit at the USDA, ARS, Southern Plains Agricultural Research Center, College Station, Texas, examined the antibiotic susceptibility of enteric bacteria of cattle fed distillers grains that contained antibiotic residues. No significant differences were observed in antibiotic resistance phenotypes and genotypes of enteric bacteria from cattle fed virginiamycin-containing distillers grains and controls. This information will be used by the ethanol industry, animal feed industry, and government regulatory agencies to facilitate development of policies and practices that will permit antibiotics to be used where necessary without compromising public health.

2. Novel antibacterial agent with specificity against gram-positive bacterial pathogens. Antibiotics are routinely used to control bacterial contamination of fuel ethanol fermentations, but drug resistance and regulatory constraints may limit the future use and effectiveness of these agents. The fermentation industry needs new low-cost, non-antibiotic intervention strategies to eliminate or reduce contaminating bacteria. Scientists in the Renewable Product Technology Research Unit at the USDA, ARS, National Center for Agricultural Utilization Research, Peoria, Illinois have characterized a small protein, called laparaxin, which is produced by a bacterial strain of Lactobacillus paracasei. Laparaxin was found to inhibit the growth of several Gram-positive bacteria, including bacteria like Lactobacillus that contaminate ethanol plants and bacteria like Staphylococcus aureus that cause disease in humans and animals. The peptide has potential application in clinical and veterinary medicine, as well as in controlling contamination in the food and feed industries.

3. Enzymatic production of chemicals from lignin. In order to improve the economic production of fuels and chemicals from lignocellulosic feedstocks, it is necessary to identify valuable coproducts from lignin – the primary residual by product. Scientists in the Renewable Product Technology Research Unit at the USDA, ARS, National Center for Agricultural Utilization Research, Peoria, Illinois identified two renewable chemicals that can be biochemically produced from lignin-related model compounds. This work provides a foundation for further development of enzyme-mediated conversion of lignin into renewable chemicals that can be evaluated in a wide variety of consumer, food, and industrial applications.

4. Novel ferulate esterase for breaking down lignocellulosic biomass. A variety of enzyme activities are required to degrade the complex structure of lignocellulosic biomass (e.g. agricultural and forestry residues) into fermentable sugars. Scientists in the Renewable Products Technology Research Unit at the USDA, ARS, National Center for Agricultural Utilization Research, Peoria, Illinois isolated and characterized an enzyme called ferulate esterase from a strain of the bacterium Lactobacillus. The enzyme breaks a specific cross-link between lignin and hemicellulose, which is important for full hydrolysis of lignocellulosic substrates. This enzyme may prove suitable for industrial application in the conversion of biomass to sugars for production of fuel ethanol or other valuable fermentation products, as well as in the feed, textile, and pulp and paper industries.

5. Biobased polymers from agricultural biomass. New technologies are needed to convert agricultural materials into higher-value bioproducts, to create new and expanded markets for agricultural commodities and reduce our dependence on imported petroleum. Under a Reimbursable Agreement between ARS and the Agricultural and Food Research Initiative Competitive Grants Program (AFRI), Department of Agriculture, scientists in the Renewable Product Technology Research Unit at the USDA, ARS, National Center for Agricultural Utilization Research, Peoria, Illinois, tested a variety of renewable cellulosic materials as feedstocks for production of the biobased polymer, schizophyllan (a compound used for pharmaceutical applictions). Corn residues were identified as promising for schizophyllan production, and biomass-derived polymers were found to exhibit chemical and physical properties similar to those of commercially produced schizophyllan. Schizophyllan is a potential value-added bioproduct consistent with the biorefinery concept, and may have applications as a replacement for certain petroleum-based polymers.

6. Novel microbial enzyme for biotransformation of agricultural lipids. New sources of microbial enzymes are needed to enhance bioconversions of agricultural materials to high-value bioproducts. Scientists in the Renewable Product Technology Research Unit at the USDA, ARS, National Center for Agricultural Utilization Research, Peoria, Illinois, examined genetically diverse strains of a fungus for the production of an enzyme, lipase, useful for conversion of agricultural lipids to biofuels. A specific genetic group of strains was identified that produces this enzyme. This work has potential impact for research to develop value-added products from agricultural byproducts.

7. Noval antibacterial agents with activity against lactic acid bacteria. Lactic acid bacteria frequently contaminate commercial fuel ethanol fermentations, reducing yields and decreasing profitability of biofuel production. Scientists in the Renewable Product Technology Research Unit at the USDA, ARS, National Center for Agricultural Utilization Research, Peoria, Illinois collaborated with scientists at Rangsit University, Thailand, to examine species of Bacillus for production of antibacterial compounds. Four strains were identified that produce a small molecular weight compound with inhibitory activity against several species of Lactobacillus. The antibacterial agent produced by these strains has application in the fuel ethanol industry as an alternative to antibiotics for prevention and control of bacterial contamination.

8. Yeast strain for converting pectin in agricultural residues to ethanol. Economically producing fuels and chemicals from agricultural residues requires a microorganism to fully utilize all available sugars derived from biomass. Kluyveromyces marxianus is a yeast that can use a variety of sugars to make ethanol. Scientists in the Renewable Product Technology Research Unit at the USDA, ARS, National Center for Agricultural Utilization Research, Peoria, Illinois subjected K. marxianus to a technique called ultraviolet mutagenesis to produce strains with improved biocatalytic properties. Two improved strains were obtained that grew on glucose and pectin, and produced more ethanol than the original K. marxianus strain. These strains will serve as candidates for further improvement to produce ethanol from agricultural residues including fruit and coffee waste streams that are rich in pectin.

Review Publications
Liu, S., Bischoff, K.M., Leathers, T.D., Qureshi, N., Rich, J.O., Hughes, S.R. 2012. Adaptation of lactic acid bacteria to butanol. Biocatalysis and Agricultural Biotechnology. 1(1):57-61. DOI:

Hughes, S.R., Moser, B.R., Robinson, S., Cox, E.J., Harmsen, A.J., Friesen, J.A., Bischoff, K.M., Jones, M.A., Pinkleman, 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. 2012. Synthetic resin-bound truncated Candida antarctica lipase B for production of fatty acid alkyl esters by transesterification of corn and soybean oils with ethanol or butanol. Journal of Biotechnology. 159:69-77. DOI: 10.1016/j.jbiotec.2012.01.025.

Hughes, S.R., Bischoff, K.M., Gibbons, W.R., Bang, S.S., Pinkelman, R., Slininger, P.J., Qureshi, N., Liu, S., Saha, B.C., Jackson, J.S., Cotta, M.A., Rich, J.O., Javers, J. 2012. Random UV-C mutagenesis of Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 to improve anaerobic growth on lignocellulosic sugars. Journal of Industrial Microbiology and Biotechnology. 39(1):163–173.

Hector, R.E., Mertens, J.A., Bowman, M.J., Nichols, N.N., Cotta, M.A., Hughes, S.R. 2011. Saccharomyces cerevisiae engineered for xylose metabolism requires gluconeogenesis and the oxidative branch of the pentose phosphate pathway for aerobic xylose assimilation. Yeast. 28:645-660.

Bischoff, K.M., De Rezende, S.T., Larson, T.M., Liu, S., Hughes, S.R., Rich, J.O. 2011. Purification and characterization of arabinofuranosidase from the corn endophyte Acremonium zeae. Biotechnology Letters. 33(10):2013-2018. DOI: 10.1007/s10529-011-0658-9.

De Almeida, M.N., Guimaraes, V.M., Bischoff, K.M., Falkoski, D.L., Pereira, O.L., Goncalves, D., De Rezende, S.T. 2011. Cellulases and hemicellulases from endophytic Acremonium species and its application on sugarcane bagasse hydrolysis. Applied Biochemistry and Biotechnology. 165:594-610. DOI: 10.1007/s12010-011-9278-z.

Hughes, S.R., Butt, T.R., Bartolett, S., Riedmuller, S.B., Sterner, D., Farrelly, P. 2011. Design and construction of a first-generation high-throughput integrated robotic molecular biology platform for bioenergy applications. Journal of the Association for Laboratory Automation. p. 292-307. DOI: 10.1016/j.jala.2011.04.004.