2010 Annual Report 1a.Objectives (from AD-416) Objective 1: Develop enzyme-based technologies (based on cleaving specific covalent crosslinks which underlie plant cell wall recalcitrance) thereby enabling new commercially-viable* saccharification processes. Objective 2: Develop new enzyme-based technologies that enable the production of commercially-viable* coproducts such as specialty chemicals, polymer precursors, and nutritional additives/supplements from raw or pretreated lignocellulosic biomass. Objective 3: Develop pretreatment technologies that enable commercially-viable* biorefineries capable of utilizing diverse feedstocks such as rice straw, wheat straw, commingled wastes (including MSW), sorghum, switchgrass, algae, and food processing by-products. Objective 4: Develop new separation technologies that enable commercially-viable* and energy-efficient processes for biofuel and/or co-product recovery from dilute fermentation broths. 1b.Approach (from AD-416) Novel enzymes for pretreatment of lignocellulosic feedstocks will be developed and improved by (1) creation of new genomic DNA libraries from diverse environments that are known to contain microbes that digest plant biomass, (2) development of novel rapid screening assays for identifying enzymes that have a specific activity, and (3) optimization of different enzyme cocktails for different biomass sources via multivariant, combinatorial optimization protocols. Greener routes toward production of styrene, terephthalic acid, vanillin and ferulic acid derivatives will be developed by a combination of biochemical and chemical synthetic pathways. Enzymes will be applied to created these bioproduct feedstocks. Engineering process models, economic analysis, and process parameters for developing integrated biorefineries using biomass from MSW and other under-utilized biomass sources as feedstock will be developed to create a source of cellulose that is consistent, easily converted to bioenergy and available during all seasons. Develop novel separation methods to reduce energy use and costs for recovering and purifying biofuels/bioproducts from low concentration fermentation broths, especially those resulting from lignocellulosic feedstocks where product concentrations are typically below (sometimes far below) 6 wt%. Replacing 5325-41000-046-00D (11/09). 3.Progress Report This project was replaced by 5325-41000-046-00D This project meets the program requirements for National Program 213, Bioenergy and Energy Progress in enzyme production for biomass pretreatment resulted in overexpression, production, and characterization of glucuronoyl esterase from Schizophyllum commune for the investigation of lignin-carbohydrate bond cleavage in plant cell wall. This work was performed in collaboration with scientists from Slovac Academy of Science, Slovakia. In related research, BCE scientists isolated, cloned, and expressed exo- and endo-xyloglucanases from rumen microbes. This group of enzymes is known to act on crosslinks between cellulose, pectin, and xylan. Results were reported and published. BCE researchers successfully created a family-shuffled DNA library, using the ß-xylosidase gene. This library was screened for mutants with improved thermal stability, resulting in discovery of 5 mutants with significant improvements in thermal stability. In all five cases, the activity level for these more stable enzymes on its industrially relevant natural substrate xylobiose (X2) were not compromised. In collaboration with the USDA NCAUR, Peoria, we completely characterized a ß-xylosidase from Bacillus halodurans that has the second highest rate (kcat) known for hydrolysis of the industrially relevant substrate xylobiose. For vanillin biosynthesis from ferulic acid, we have designed a ferulic acid synthase (fcs) gene from Burkholdia sp. that is codon-optimized for shuffling with a fcs gene from Pseudomonas sp. Both genes were made and cloned into optimal expression plasmids, and were found to readily express soluble enzyme suitable for e development. We also designed an enoyl co-A hydratase/lyase enzyme (ech)from Thermus sp. that is codon-optimized to a ech gene from Pseudomonas sp. These constructs will be used to complete the pathway to biosynthesis of vanillin and will serve as starting blocks for additional enzyme engineering efforts to convert ferulic acid to vanillin. In separation engineering, ethanol was shown to be an effective separation aid. BCE researchers showed that ethanol can be used to effectively remove water from solids. Very significant reduction of process energy related to drying biorefinery solids are revealed. This strategy was shown to improve energy use in a variety of biomass refining technologies that also make ethanol. In related separation engineering research, BCE scientists discovered new solvents (carboxylic acids) that were shown to be more effective than industry standards in a solvent extraction process. This process was introduced as a lower-energy alternative to distillation for recovery of fuel ethanol from fermentation broths Studies on the toxicity of these solvents to yeast showed that these solvents would not slow yeast growth or ethanol production during fermentation. In membrane separation research, studies on the performance-enhancing components of membranes for recovery of fuel ethanol showed the benefits and limitations of zeolite mixed-membrane separation systems. 4.Accomplishments 1. Solvent Extraction in Fuel Ethanol Recovery Process. In fuel ethanol production, recovery of the ethanol from the fermentation broth by distillation is energy-intensive. The energy required climbs rapidly as the ethanol concentration of the broth drops, which happens when shifting feedstock from corn starch to biomass. ARS Researchers in Albany, California studied the performance of new solvents (selected carboxylic acids) for recovery of fuel ethanol by a solvent extraction process. Toxicity of the solvents to yeast was also determined. Structure-performance relationships were mapped. Compared to distillation, solvent extraction was shown to use less energy to accomplish the separation. Review Publications Offeman, R.D., Franquivillanueva, D.M., Cline, J.L., Robertson, G.H., Orts, W.J. 2010. Extraction of ethanol with higher carboxylic acid solvents and their toxicity to yeast. Separation and Purification Technology. 72, 180-185. Li, R., Kibblewhite, R.E., Orts, W.J., Lee, C.C. 2009. Molecular cloning and characterization of multidomain xylanase from manure library. World Journal of Microbiology and Biotechnology. doi:10:1007/s11274-009-011106 Wagschal, K.C., Heng, C., Lee, C.C., Robertson, G.H., Orts, W.J., Wong, D. 2008. Purification and characterization of a glycoside hydrolase family 43 Beta-xylosidase from Geobacillus thermoleovorans IT-08. Applied Biochemistry and Biotechnology. 155(1-3):1-10. Jordan, D.B., Wagschal, K.C. 2010. Properties and applications of microbial beta-D-xylosidases. Applied Microbiology and Biotechnology. 86(6):1647-1658 Fan, Z., Yuan, L., Jordan, D.B., Wagschal, K.C., Heng, C., Braker, J.D. 2010. Engineering lower inhibitor affinities in beta-D-xylosidase. Applied Microbiology and Biotechnology. 86(4):1099-1113. Li, R., Zhang, Y., Lee, C.C., Lu, R., Huang, Y. Development and validation of a hydrophilic interaction liquid chromatographic method for determination of aromatic amines in environmental water. Journal of Chromatography, 2010. A 1217(11), pp 1799-1805.