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United States Department of Agriculture

Agricultural Research Service

Research Project: Improving Biochemical Processes for the Production of Sustainable Fuels and Chemicals

Location: Renewable Product Technology Research Unit

2013 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:
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 FY13, ARS Renewable Product Technology Research Unit scientists at the National Center for Agricultural Utilization Research, Peoria, Illinois, made significant progress toward the objectives of this research project, as demonstrated by the following activities: • A bacterial ferulate esterase, an auxiliary enzyme used in deconstructing lignocellulosic biomass, was isolated and its gene (faeA) was cloned and functionally expressed in Escherichia coli. • Novel sources of the enzyme laccase were identified. • Proteins that help confer tolerance to high concentrations of ethanol were identified from the bacterium Lactobacillus buchneri NRRL B-30929. • Bacterial contaminants from commercial fuel ethanol fermentations were screened for impact on yeast utilization of glucose and production of ethanol. • Novel inhibitory agents were tested for their ability to prevent biofilm formation of lactic acid bacteria that contaminate commercial fuel ethanol facilities. • Genes encoding antibacterial enzymes with activity against lactic acid bacteria were expressed in yeast during ethanol fermentation. • Conditions were optimized for the scale-up of schizophyllan production from agricultural biomass in controlled bioreactors. • Strains of Kluyveromyces marxianus were used to produce ethanol from lignocellulosic byproducts of coffee production. • Produced an artificial yeast minichromosome for stable expression of xylose isomerase and xylulokinase genes in fuel ethanol yeast.

Progress achieved during FY13 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. Antibacterial enzymes 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 the USDA-ARS-Animal Biosciences and Biotechnology Lab, Beltsville, Maryland, to develop antibacterial enzymes as inhibitors of bacteria during ethanol fermentation. Four enzymes, called phage endolysins, were found to inhibit the growth of lactic acid bacteria, and genes for two of the enzymes were expressed in an ethanol-producing yeast. This work has potential to improve the economics of cellulosic biofuel production by:.
1)increasing ethanol yield through elimination of competing bacteria;.
2)reducing production costs by avoiding large scale antibiotic use; and.
3)decreasing environmental impact by replacing broad range antibiotics with biodegradable, enzyme-based antibacterials.

2. 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 reducing our dependence on imported petroleum. Under a Reimbursable Agreement between ARS and the Agricultural and Food Research Initiative Competitive Grants Program, Department of Agriculture (AFRI), scientists in the Renewable Product Technology Research Unit at the USDA-ARS-National Center for Agricultural Utilization Research, Peoria, Illinois, optimized conditions for scale-up of schizophyllan production from agricultural biomass in controlled bioreactors. Bioreactor-produced polymers exhibited chemical and physical properties similar to those of commercially produced polymers. Schizophyllan is a potential value-added bioproduct consistent with the biorefinery concept, and may have a variety of commercial and industrial applications.

3. Novel microbial enzyme for transformation of agricultural substrates. 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, collaborated with scientists at Rangsit University, Thailand, to examine genetically diverse strains of a fungus for the production of an enzyme, laccase, useful for biotransformations of lignin and related materials. Specific genetic groups of strains were identified that produce this enzyme. This work has potential impact for research to develop value-added products from low-value agricultural materials.

4. Production of butyric acid from biomass. New valuable products are needed to improve the economics of biorefining agricultural residues. Scientists in the Renewable Product Technology Research Unit at the USDA-ARS-National Center for Agricultural Utilization Research, Peoria, Illinois, evaluated bacteria isolated from commercial ethanol facilities for the ability to produce valuable fermentation products. A strain of Clostridium tyrobutyricum was found to produce butyric acid, a valuable commodity chemical, from wheat straw, corn stover, corn fiber, rice hull, and switchgrass. Biological production of butyric acid may be preferable to consumers in food and cosmetic applications and potentially expands the availability of biobased products in the marketplace.

5. Production of ethanol from byproducts of coffee processing. 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, evaluated the ability of mutant strains of K. marxianus to make ethanol from waste products. Optimal conditions of temperature, agitation, and pH for large scale ethanol production were determined. These conditions will be used to improve the process to produce ethanol from agricultural residues.


Review Publications
Rastogi, G., Gurram, R.N., Bhalla, A., Gonzalez, R., Bischoff, K.M., Hughes, S.R., Kumar, S., Sani, R.K. 2013. Presence of glucose, xylose, and glycerol fermenting bacteria in the deep biosphere of the former Homestake gold mine, South Dakota. Frontiers in Microbiology. 4(2):1-8.

Hughes, S.R., Bang, S.S., Cox, E.J., Schoepke, A., Ochwat, K., Pinkelman, R., Nelson, D., Qureshi, N., Gibbons, W.R., Kurtzman, C.P., Bischoff, K.M., Liu, S., Cote, G.L., Rich, J.O., Jones, M.A., Cedeno, D., Doran-Peterson, J., Riano, N.M. 2013. Automated UV-C mutagenesis of Kluyveromyces marxianus NRRL Y-1109 and selection for microaerophilic growth and ethanol production at elevated temperature on biomass sugars. Journal of Laboratory Automation. 18(4):276-290.

Liu, S., Wilkinson, B.J., Bischoff, K.M., Hughes, S.R., Rich, J.O., Cotta, M.A. 2012. Novel antibacterial polypeptide laparaxin produced by Lactobacillus paracasei strain NRRL B-50314 via fermentation. J Pet Environ Biotechnol. 3:121. DOI: 10.4172/2157-7463.

Khullar, E., Kent, A.D., Leathers, T.D., Bischoff, K.M., Rausch, K.D., Tumbleson, M.E., Singh, V. 2013. Contamination issues in a continuous ethanol production corn wet milling facility. World Journal of Microbiology and Biotechnology. 29(5):891-898.

Manitchotpisit, P., Bischoff, K.M., Price, N.P., Leathers, T.D. 2013. Bacillus spp. produce antibacterial activities against lactic acid bacteria that contaminate fuel ethanol plants. Current Microbiology. 66(5):443-449.

Rich, J.O., Leathers, T.D., Anderson, A.M., Bischoff, K.M., Manitchotpisit, P. 2013. Laccases from Aureobasidium pullulans. Enzyme and Microbial Technology. 53(1):33-37.

Qureshi, N., Dien, B.S., Liu, S., Saha, B.C., Hector, R.E., Cotta, M.A., Hughes, S.R. 2012. Genetically engineered Escherichia coli FBR5: Part I. Comparison of high cell density bioreactors for enhanced ethanol production from xylose. Biotechnology Progress. 28(5):1167-1178.

Qureshi, N., Dien, B.S., Liu, S., Saha, B.C., Cotta, M.A., Hughes, S.R., Hector, R.E. 2012. Genetically engineered Escherichia coli FBR5: Part II. Ethanol production from xylose and simultaneous product recovery. Biotechnology Progress. 28(5):1179-1185.

Liu, S., Bischoff, K.M., Leathers, T.D., Qureshi, N., Rich, J.O., Hughes, S.R. 2013. Butyric acid from anaerobic fermentation of lignocellulosic biomass hydrolysates by Clostridium tyrobutyricum strain RPT-4213. Bioresource Technology. 143:322-329.

Sutivisedsak, N., Leathers, T.D., Nunnally, M.S., Price, N.P., Biresaw, G. 2013. Utilization of agricultural biomass in the production of the biopolymer schizophyllan. Journal of Indusrial Microbiology and Biotechnology. 40(1):105-112. DOI: 10.1007/s10295-012-1208-8.

Sutivisedsak, N., Leathers, T.D., Bischoff, K.M., Nunnally, M.S., Peterson, S.W. 2013. Novel sources of ß-glucanase for the enzymatic degradation of schizophyllan. Enzyme and Microbial Technology. 52(3):203-210.

Larson, T.M., Anderson, A.M., Rich, J.O. 2012. Combinatorial evaluation of laccase-mediator system in the oxidation of veratryl alcohol. Biotechnology Letters. 35(2):225-231. DOI: 10.1007/s10529-012-1078-1.

Bhalla, A., Bansal, N., Kumar, S., Bischoff, K.M., Sani, R. 2013. Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes. Bioresource Technology. 128:751-759.

Roach, D.R., Khatibi, P.A., Bischoff, K.M., Hughes, S.R., Donovan, D.M. 2013. Bacteriophage-encoded lytic enzymes control growth of contaminating Lactobacillus found in fuel ethanol fermentations. Biotechnology for Biofuels. 6(1):20.

Last Modified: 10/1/2014
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