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United States Department of Agriculture

Agricultural Research Service

Research Project: INDUSTRIALLY ROBUST ENZYMES AND MICROORGANISMS FOR PRODUCTION OF SUGARS AND ETHANOL FROM AGRICULTURAL BIOMASS
2009 Annual Report


1a.Objectives (from AD-416)
Develop improved processes for converting herbaceous biomass to ethanol by incorporating new enzyme and biocatalyst technologies with modern pretreatment strategies. Evaluate potential for converting biomass derived sugars to hydrogen via fermentation.


1b.Approach (from AD-416)
Herbaceous biomass feedstocks will be converted to fermentable sugar mixtures using low-waste pretreatments and novel enzymatic preparations. The generated sugar mixtures will be fermented using recombinant microorganisms specifically engineered for producing ethanol from biomass sugars. Specific steps in this approach include: (1) working with plant breeders to develop cultivars especially suited for low-chemical usage, mild pretreatments, (2) generating new enzyme mixtures using genes recovered and over-expressed from highly active anaerobic fungi, (3) developing bioabatement methods for removing organic chemical that interfere with fermentation and, thereby, increasing the fermentability of the recovered biomass sugars, (4) engineering gram positive bacteria to selectively produce ethanol; members of this group have a long history of use in industrial fermentations, and (5) screen and evaluate hydrogen producing bacteria for capability to co-produce hydrogen from biomass feedstocks.


3.Progress Report
The overall goals of this project are to develop technologies for lowering the cost and increasing the efficiency with which biomass can be converted to ethanol and chemicals. As part of this effort, we are working in collaboration with Agricultural Research Service (ARS) plant scientists to develop superior quality bioenergy crops. We have extended our screen to include alfalfa cultivars and expect to complete the screening of a collection of plant samples (e.g., 120 cultivars) by early fall. We have also continued progress on development of an advance pretreatment strategy for preparing biomass for enzymatic conversion to sugars, resulting in increased yields for both glucose and pentose sugars.

Fermentation Biotechnology researchers reported the discovery of a beta-xylosidase that is 10 times more active than others previously reported. The bond this enzyme breaks is the second most abundant in lignocellulosic biomass and, therefore, its use is expected to lower the cost of enzymes for biomass conversions. We have continued to extend our kinetic analysis of the enzyme. Using a structure-function approach, it was determined that the enzyme contains two sub-sites. Further experiments, applying an elegant approach that made use of aminoalcohols, differentiated the properties of each site and led to a strategy to further improve the enzymes properties for treatment of biomass at higher-solids.

Pretreating biomass for enzymatic hydrolysis promotes the formation of chemicals that are undesirable and impede fermentation. Our group is working on a novel approach that uses biological abatement as a strategy for removing these undesirable chemicals. Earlier we had shown that the method is suitable for preparing corn stover hydrolysate for fermentation. We have now expanded this strategy to additional herbaceous energy crops, including alfalfa stems, reed canary grass, and switchgrass.

Finally, this year, group members prepared a new 5-year research plan and submitted it for ARS Office of Scientific Quality Review.


4.Accomplishments
1. ADVANCE SCREENING TECHNIQUES FOR ENGINEERING SUPERIOR BETA-XYLOSIDASE FOR IMPROVED PERFORMANCE. The Fermentation Biotechnology (FBT) Research Unit has discovered and extensively characterized a beta-xylosidase that is 10 times more active (specific activity) than any previously reported. This enzyme is important because it catalyzes the second most common bond present in biomass carbohydrates into fermentable sugars. Therefore, it is expected that by increasing its activity, enzyme costs can be reduced for producing cellulosic ethanol. There exists an opportunity to improve this enzyme's performance by engineering it to be less susceptible to end-product inhibition. In collaboration (United States Department of Agriculture-Agricultural Research Service, Albany, California; University of Kentucky), we demonstrated that candidates with improved end-product inhibition properties could be selected by high throughput screening in combination with kinetic characterization. The successful isolation of enzyme mutants with enhanced tolerance to monosaccharide inhibitors is expected to have superior performance in industrial applications for processes using high-solid process streams.

2. SCREENING FOR IMPROVED ALFALFA CULTIVARS FOR ETHANOL PRODUCTION. Alfalfa has been identified as a promising bioenergy crop whereby the leaves would be used as a valuable animal feed product and the stems converted into ethanol. A wet-chemistry method, originally developed by the Fermentation Biotechnology Research Unit for screening of warm season grasses, was adapted and validated for use on this legume. The refined method is now being used to screen a library of over 100 stem samples for preferred bioenergy candidates. This should result in the identification of alfalfa cultivars with superior quality traits for biochemical conversion into ethanol and other biofuels.


6.Technology Transfer

Number of the New/Active MTAs (providing only)1
Number of Invention Disclosures Submitted2
Number of Other Technology Transfer3

Review Publications
Arora, A., Dien, B.S., Belyea, R.L., Wang, P., Singh, V., Tumbleson, M.E., Rausch, K.D. 2009. Thin Stillage Fractionation Using Ultrafiltration: Resistance in Series Model. Bioprocess and Biosystems Engineering. 32(2):225-233.

Jordan, D.B., Braker, J.D. 2009. Beta-D-xylosidase from Selenomonas ruminantium: Thermodynamics of Enzyme-catalyzed and Noncatalyzed Reactions. Applied Biochemistry and Biotechnology. 155(1-3):330-346.

Li, X., Skory, C.D., Cotta, M.A., Puchart, V., Biely, P. 2008. Novel family of carbohydrate esterases, based on identification of the Hypocrea jecorina Acetyl Esterase Gene. Applied and Environmental Microbiology. 74(24):7482-7489.

Fornero, J.J., Rosenbaum, M., Cotta, M.A., Angenent, L.T. 2008. Microbial Fuel Cell Performance with a Pressurized Cathode Chamber. Environmental Science and Technology. 42(22):8578-8584.

Mertens, J.A., Burdick, R.C., Rooney, A.P. 2008. Identification, Biochemical Characterization, and Evolution of the Rhizopus oryzae 99-880 Polygalacturonase Gene Family. Fungal Genetics and Biology. 45(12):1616-1624.

Lemuz, C.R., Dien, B.S., Singh, V., Mckinney, J., Tumbleson, M.E., Rausch, K.D. 2009. Development of an Ethanol Yield Procedure for Dry-grind Corn Processing. Cereal Chemistry. 86(3):355-360.

Jordan, D.B., Mertens, J.A., Braker, J.D. 2009. Aminoalcohols as Probes of the Two-subsite Active Site of Beta-D-xylosidase from Selenomonas ruminantium. Biochimica et Biophysica Acta. 1794(1):144-158.

Skory, C.D., Mertens, J.A., Rich, J.O. 2009. Inhibition of Rhizopus lactate dehydrogenase by fructose 1,6-bisphosphate. Enzyme and Microbial Technology. 44(4):242-247.

Last Modified: 4/16/2014
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