2007 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. 4.Accomplishments SCREENING SWITCHGRASS CULTIVARS FOR ETHANOL YIELDS. Switchgrass has been viewed as one of the most promising candidates for an energy crop, but more efficient cultivars are needed for conversion to ethanol. Over 100 samples of switchgrass were pretreated and converted to ethanol by simultaneous saccharification and fermentation. Samples varied widely in environmental growth conditions and genetic traits. The results demonstrated a wide-spectrum in conversion yields for the different samples and validate the need for further plant breeding of energy crops. This data is currently being applied to develop a quick scanning near-infrared (NIR) model that will be used for breeding switchgrass cultivars for superior ethanol yield. This work applies directly to National Program 307, Bioenergy and Energy Alternatives, Component I and IV, Ethanol and Energy Crops, because it relates to conversion of biomass into ethanol and processing of energy crops. DISCOVERY AND ISOLATION OF A GENE ENCODING FOR A NOVEL ENZYME ACTIVITY FOR BIOMASS DEGRADATION FROM TRICHODERMA REESEI. New enzymes are needed for breaking down plant cell walls for conversion to ethanol because current enzyme blends are not always effective. The fungus T. reesei is the most common source for commercial cellulases, and novel genes related to biomass conversion are extremely important for further improvement of industrial cellulases. The enzyme discovered is a glucuronic acid esterase; glucuronic acids are present in a wide variety of biomasses and an ability to remove them is expected to directly lower yields by interfering with the release of neutral sugars. This work applies directly to National Program 307, Bioenergy and Energy Alternatives, Component I, Ethanol, because it relates to conversion of biomass into ethanol. THE BIFUNCTIONAL BETA-D-XYLOSIDASE/ALPHA-L-ARABINOFURANOSIDASE FROM SELENOMONAS RUMINANTIUM IS THE BEST CATALYST KNOWN (KCAT, KCAT/KM) FOR PROMOTING HYDROLYSIS OF 1,4-BETA-D-XYLOOLIGOSACCHARIDES, AND IT HAS POTENTIAL FOR USE IN SACCHARIFICATION PROCESSES. Highly active biomass conversion enzymes are required for economic and efficient saccharification of agricultural biomass. Active-site amino acid residues that are involved in substrate distortion and increasing enzyme activity have been identified. As well, active-site amino acid residues that are involved in preferring xylose glycosides over arabinose glycosides have been identified. Enzyme-inhibitor complexes that could be formed in saccharification processes have been demonstrated. This work applies directly to National Program 307, Bioenergy and Energy Alternatives, Component I, Ethanol, because it relates to conversion of biomass into ethanol. IMPROVING FERMENTABILITY OF BIOMASS SUGARS. Inhibitors arising during conversion of biomass to sugars are impediments to fermentative production of ethanol. Over three dozen chemical by-products were detected in hydrolyzed corn and evaluated for removal by a biological treatment system. The fermentability of hydrolysates was improved by biological abatement, due to removal of a number of inhibitory compounds present in acid-pretreated corn stover. This work is being expanded to include an alkaline-pretreated feedstock, which has a different array of inhibitory compounds. The inhibitor abatement methods remove side-products of pretreatment and can greatly enhance the yield and rate of production for biofuels. This work applies directly to National Program 307, Bioenergy and Energy Alternatives, Component I, Ethanol, because it relates to conversion of biomass into ethanol. ISOLATION OF HYDROLYTIC ENZYMES FROM RHIZOPUS ORYZAE SPECIES. The filamentous fungus Rhizopus oryzae contains a large and diverse complement of biomass degrading enzymes that are of interest for improving the efficacy and efficiency of biomass degradation. Three unique previously unidentified glucoamylase genes (glucoamylase aids in the breakdown of starch) from two different Rhizopus strains were isolated, expressed, and initial biochemical characterization performed. Two of the enzymes were highly active, while the third enzyme is inactive, even though it is a truncated version of a previously isolated enzyme. Indepth biochemical characterization of the active enzymes is in progress with an eye toward the potential use of these enzymes as processing aids in biomass degradation. This work applies directly to National Program 307, Bioenergy and Energy Alternatives, Component I, Ethanol, because it relates to conversion of biomass into ethanol. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of new CRADAs and MTAs 7 Number of active CRADAs and MTAs 10 Number of patent granted 1 Number of non-peer reviewed presentations and proceedings 18 Number of newspaper articles and other presentations for non-science audiences 12 Review Publications Dien, B.S., Jung, H.G., Vogel, K.P., Casler, M.D., Lamb, J.F., Iten, L.B., Mitchell, R., Sarath, G. 2006. Chemical composition and response to dilute-acid pretreatment and enzymatic saccharification of alfalfa, reed canarygrass, and switchgrass. Biomass and Bioenergy. 30:880-891. Dien, B.S., Li, X., Iten, L.B., Jordan, D.B., Nichols, N.N., O Bryan, P.J., Cotta, M.A. 2006. Enzymatic saccharification of hot-water pretreated corn fiber for production of monosaccharides. Enzyme and Microbial Technology. 39:1137-1144. Mertens, J.A., Skory, C.D. 2007. Isolation and characterization of a second glucoamylase gene without a starch binding domain from Rhizopus oryzae. Enzyme and Microbial Technology. 40:874-880. Bischoff, K.M., Rooney, A.P., Li, X., Liu, S., Hughes, S.R. 2006. Purification and characterization of a family 5 endoglucanase from a moderately thermophilic strain of Bacillus licheniformis. Biotechnology Letters. 28:1761-1765. Jordan, D.B., Li, X.-L., Dunlap, C.A., Whitehead, T.R., Cotta, M.A. 2007. Structure-function relationships of a catalytically efficient beta-D-xylosidase. Applied Biochemistry and Biotechnology. 141:51-76. Jordan, D.B., Li, X., Dunlap, C.A., Whitehead, T.R., Cotta, M.A. 2007. Beta-D-xylosidase from Selenomonas ruminantium of glycoside hydrolase family 43. Applied Biochemistry and Biotechnology. 136-140:93-104. Lopez, M.J., Vargas-Garcia, M., Suarez-Estrella, F., Nichols, N.N., Dien, B.S., Moreno, J. 2007. Lignocellulose-degrading enzymes produced by the ascomycete Coniochaeta ligniaria and related species: application for a lignocellulosic substrate treatment. Enzyme and Microbial Technology. 40:794-800. Li, X., Skory, C.D., Ximenes, E.A., Jordan, D.B., Dien, B.S., Hughes, S.R., Cotta, M.A. 2007. Expression of an AT-rich xylanase gene from the anaerobic fungus Orpinomyces sp. strain PC-2 in and secretion of the heterologous enzyme by Hypocrea jecorina. Applied Microbiology and Biotechnology. 74:1264-1275. Ximenes, E., Dien, B.S., Ladisch, M.R., Mosier, N., Cotta, M.A., Li, X. 2007. Enzyme production by industrially relevant fungi cultured on coproduct from corn dry grind ethanol plants. Applied Biochemistry and Biotechnology. 136-140:171-183. Mertens, J.A., Skory, C.D. 2007. Isolation and characterization of two genes that encode active glucoamylase without a starch binding domain from Rhizopus oryzae. Current Microbiology. 54:462-466. Chen, H., Hopper, S.L., Li, X., Ljungdahl, L.G., Cerniglia, C.E. 2006. Isolation of extremely AT-rich genomic DNA and analysis of genes encoding carbohydrate-degrading enzymes from Orpinomyces sp. strain PC-2. Current Microbiology. 53:396-400. Chen, H., Li, X., Xu, H., Ljungdahl, L.G., Cerniglia, C.E. 2006. High level expression and characterization of the cyclophilin B gene from the anaerobic fungus Orpinomyces sp. strain PC-2. Protein and Peptide Letters. 13:727-732. Hughes, S.R., Dowd, P.F., Hector, R.E., Riedmuller, S.B., Bartolett, S., Mertens, J.A., Qureshi, N., Liu, S., Bischoff, K.M., Li, X., Jackson Jr, J.S., Sterner, D., Panavas, T., Cotta, M.A., Farrelly, P.J., Butt, T. 2007. High-throughput fully automated construction of a multiplex library of mutagenized open reading frames for an insecticidal peptide using a plasmid-based functional proteomic robotic workcell with improved vacuum system. Journal of Laboratory Automation. 12(4):202-212.