Progress 11/16/09 to 09/30/14

Outputs
Progress Report 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 the recovery of biofuels, biorefinery co-products, and/or bioproducts from dilute fermentation broths. 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%. This is the final report for this project which expires in November of 2014. A new project, 5325-41000-054-00D, �Technologies for Improving Industrial Biorefineries that Produce Marketable Biobased Products�, was just certified by the Office of Scientific Quality Review after NP 213, Bioenergy, peer panel review, and will begin on October 1, 2014. For objective 1, ARS scientists cloned a new and novel thermally-stable protease from a thermophilic bacterium (Coprothermobacter proteolyticus) that was isolated from thermophilic anaerobic digester. The protease gene was cloned into E. coli and bacillus and the expressed enzyme showed activity up to ~63C (with a temperature optimum well above 45C (at alkaline pH). This enzyme has significant potential applications in food processing and detergents; an industrial detergent producer requested samples. This enzyme was developed in collaboration with the National Institute for Biotechnology and Genetic Engineering (NIBGE) in Faisalabad, Pakistan, in a project funded by United States Agency for International Development (USAID) and Pakistan�s Commission of Higher Education, Women in Science program. Under objective 2, in collaboration with multiple partners, including the National Aeronautics and Space Administration (NASA), ARS scientists developed multi-functional enzymes with significantly better biomass- degrading properties. The team tethered as many as 5 active enzymes to a nanoscale chaperonin complex, which can bind up to 18 different enzymes, and showed that this immobilized enzyme was more active than an enzyme cocktail free in solution. In another example that enzymes tethered to scaffolding proteins (self-assembling nanostructure chaperonin complex) can exhibit greater synergism than if the same enzymes are free in solution, several enzymes were tethered to together to convert glucose to glucaric acid, a commercially-viable molecule formed in multiple enzymatic steps. In collaboration with almond growers and processers, the Almond Hullers and Processors Association (AHPA), ARS researchers successfully completed a survey of almond production for the Industry with a wide-spread sampling and analysis program to determine sugars and other components (including aflatoxin) in almonds, hulls and shells based on their location, age, and harvesting/storage conditions. Recommendations were made to the industry for improving shelf-life by advising on improved storage conditions. Research will continue within the new project plan. For objective 3, ARS researchers discovered and characterized a bi- functional enzyme that hydrolyzes both mannans and mixed glucans, two distinct types of polysaccharides present in plant cell wall. Bi- functional enzyme enables two hydrolytic reactions to proceed more efficiently than corresponding individual reactions. Via �gene mining� ARS researchers discovered and then expressed a thermostable pectin methylesterase from Thermotoga sp. that is being tested on commercial biomass waste; specifically for converting citrus and beet food processing waste streams to value-added chemicals. We discovered and characterized 4 novel uronate dehydrogenase enzymes that are being tested on the same �real world� biomass wastes, specifically citrus and beet food processing waste to value-added chemicals. Under objective 4, ARS researchers were granted a patent (US Patent 8617395) for a novel method to fabricate thin-film composite membranes for separating organic solvents including ethanol from microbial broths. Membranes made by this method use porous supports with much higher porosity, resulting in both higher flux and higher separation factor. Work continues on finding a suitable technology transfer partner for this patented research. Accomplishments 01 Adding value to agricultural waste. To provide sufficient quantities of biomass sources between growing seasons, ARS researchers in Albany, California, developed a large pilot scale biorefinery located at the Salinas Crazy Horse Landfill that converts rural and urban solid waste into ethanol, biogas, compost, and/or value-added recyclables. They showed that food processing waste can be converted into ethanol, yielding 65 gallons per ton. Conversely, if the same biomass source is converted to liquefied natural (bio)gas it yields the equivalent of 108 gallons of transportation fuel, which has been utilized to power diesel turbines. Together, the USDA and the City of Salinas are creating an �energy park� that converts both agriculturally derived biomass, as well as curb-collected garbage, into bioenergy at the same biorefinery, thus exhibiting remarkable flexibility in handling different feedstock supplies.

Impacts
(N/A)

Publications

• Singh, S.K., Heng, C., Braker, J.D., Chan, V.J., Lee, C.C., Jordan, D.B., Yuan, L., Wagschal, K.C. 2013. Directed evolution of GH43 �-xylosidase XylBH43 thermal stability and L186 saturation. Journal of Industrial Microbiology and Biotechnology. 41(3):489-498. DOI: 10.1007/s10295-013- 1377-0.
• Offeman, R.D., Holtman, K.M., Covello, K.M., Orts, W.J. 2014. Almond hulls as a biofuels feedstock: Variations in carbohydrates by variety and location in California. Biomass and Bioenergy. 54:109-114.
• Offeman, R.D., Ludvik, C.N. 2011. A novel method to fabricate high permeance, high selectivity thin-film composite membranes. Journal Membrane Science. 380:163-170.
• Wagschal, K.C., Lee, C.C. 2012. Microplate-based active/inactive 1 screen for biomass degrading enzyme library purification and gene discovery. Analytical Biochemistry. 89: 83-85.
• Mao, J., Holtman, K.M., Franquivillanueva, D.M. 2010. Chemical structures of corn stover and its residue after dilute acid prehydrolysis and enzymatic hydrolysis: Insight into factors limiting enzymatic hydrolysis. Journal of Agricultural and Food Chemistry. 58(22):11680-11687.
• Mcclendon, S.D., Mao, Z., Shin, H., Wagschal, K.C., Chen, R.R. 2012. Designer xylanosomes: protein nanostructures for enhanced xylan hydrolysis. Biomacromolecules. 167:385-411.
• Holtman, K.M., Bozzi, D.V., Franquivillanueva, D.M., Orts, W.J. 2010. Biofuels and bioenergy production from municipal solid waste commingled with agriculturally-derived biomass. In: Braun, R, Karlen, D, Johnson, D., editors. Sustainable alternative fuel feedstock opportunities, challenges and roadmaps for six U.S. regions. Washington, D.C.:Soil and Water Conservation Society. p. 237-247.
• Jha, A.K., Tsang, S., Ozcam, A., Offeman, R.D., Balsara, N.P. 2012. Master curve captures the effect of domain morphology on ethanol pervaporation through block copolymer membranes. Journal Membrane Science. 401-402:125- 131.
• Gong, L., Xu, Q., Lee, C.C., Zhang, H. 2012. Selenium speciation analysis of Misgurnus anguillicaudatus selenoprotein by HPLC-ICP-MS and HPLC-ESI-MS/ MS. European Food Research and Technology. 235(1):169-176.
• Santos, C., Polo, C., Costa, M., Nascimento, A., Mezal, A.N., Cota, J., Hoffmam, Z.B., Honorato, R., Oliveira, P.S., Goldman, G.H., Prade, R.A., Ruller, R., Squina, F.M., Wong, D., Murakami, M. 2014. Mechanistic strategies for catalysis adopted by evolutionary distinct family 43 arabinanases. Journal of Biological Chemistry. 289(11):7362-7373.
• Majeed, T., Tabassum, R., Orts, W.J., Lee, C.C. 2013. Expression and characterization of Coprothermobacter proteolyticus alkaline serine protease. The Scientific World. DOI: 10.1155/2013/396156.
• Olivera, J.A., Moraes, E.A., Marconini, J.M., Mattoso, L.H., Glenn, G.M., Medeiros, E.S. 2013. Miscibility of poly(lactic acid) and poly(ethylene oxide) solvent polymer blends and nanofibers made by solution blow spinning. Polymer Journal. 129(6):3672-3681.

Progress 10/01/12 to 09/30/13

Outputs
Progress Report 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 the recovery of biofuels, biorefinery co-products, and/or bioproducts from dilute fermentation broths. 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). Scientists within the project discovered and characterized a bi- functional enzyme that hydrolyzes both mannans and mixed glucans, two distinct types of polysaccharides present in plant cell wall. Bi- functional enzymes enable two hydrolytic reactions to proceed more efficiently than corresponding individual reactions. In another aspect of the project, Western Regional Research Center(WRRC) and National Center for Agricultural Utilization Research (NCAUR) scientists collaborated to improve the thermal stability of �-xylosidase. Both the WRRC and NCAUR high-activity �-xylosidases have relatively low thermal and pH stability compared to the parent enzymes. Site-directed mutagenesis and directed evolution were used to improve the thermal stability of the WRRC �- xylosidase. The enzyme was stable and highly active at temperatures 8�C higher than normal. This �-xylosidase exhibited 1/3 lower end-product inhibition but had about 2/3 the activity of the very-highly active �- xylosidase developed at NCAUR. Another aspect of the project focused on developing biobased caprolactone. Caprolactone is used in the manufacture of polyurethane and although it is biodegradable, it is petroleum-based. Biobased replacements for caprolactone were developed from derivatized glycerols and derivatized di-lactide. This work was performed in collaboration with an industrial partner and an invention disclosure was filed. Scientists within the unit received a 3-year, $479K NIFA grant to develop multi-functional enzymes with significantly better biomass- degrading properties. Multi-functional enzymes have multiple active sites. Work has shown that enzymes tethered to scaffolding proteins (self- assembling nanostructure chaperonin complex) can exhibit greater synergism than the same enzymes free in solution. The work is being initiated with cellulases and hemicellulases. This research is being performed in collaboration with multiple partners from both industry and a government agency (NASA). Accomplishments 01 Adding value to almond byproducts. In California, the world�s leader in almond production with 85% of the international market, roughly ~3.3 billion lbs of almond waste (mostly hulls) and 1.3 billion lbs of almond shells are produced annually and generally sold as cattle feed. Researchers in Albany, California, working with the Almond Hullers & Processors Association and a commercial partner, created value-added uses for the sugars derived from these almond wastes. Extraction analysis showed that nonpareil almond hulls (which make up 75-80% of production) contain more than 30% simple sugars (glucose, fructose, sucrose) that can readily be obtained by hot-water extraction. This team, in collaboration with growers and processers throughout California, initiated a wide-spread sampling and analysis program to determine sugars and other components (including aflatoxin) in almond hulls based on their variety, locality, age, and harvesting/storage conditions. They then showed the industry that simple sugars derived from almond hulls are viable as natural food ingredients, such as a natural sugar in �nutrient bars� or could be converted to ethanol in commercially viable processes, potentially adding hundreds of millions of dollars to the value of these waste products. 02 Enzymatic breakdown of covalent linkages in plant cell wall biopolymers. Utilization of biomass requires a cascade of enzymes, and a key process involves cleavage of the covalent bonds crosslinking individual polymers in plant cell wall. Agricultural Research Service scientists at Albany, California, applied cloning techniques to produce and test mportant new enzymes to break down crosslinks in various natural biopolymers. The research has helped industrial partners to improve processing of forage crops into biofuels and feed ingredients. These processes add value to underutilized agricultural wastes and forage crops. 03 Biomass degradation by enzyme nanoassemblies. The breakdown of lignocellulosic biomass into simple sugars requires many different enzymes which add to the overall cost of conversion. ARS scientists at Albany, California, assembled different enzyme combinations onto nanoscale ring scaffolds. They demonstrated that enzymes mounted onto the scaffolds had higher specific activities relative to enzymes free in solution. This research will lead to decreased enzyme usage and, hence, improved process economics. The ultimate goals is to create environmentally chemicals using enzymes and not harsh chemicals. 04 Selection and characterization of novel Uronate Dehydrogenase enzymes. Conversion of sugars and sugar acids to value-added sugar dicarboxylic acids, a Department of Energy top-ten target for biobased building blocks, can provide a bio-based chemical platform for polymers. The use of enzymes for this conversion is environmentally relatively benign and potentially can be more cost effective than harsh chemical procedures such as nitric acid oxidation. Researchers at Albany, Californa, identified five uronate dehydrogenase genes from three different bacterial genera that converted sugar uronic acids to sugar dicarboxylic acid building blocks. The kinetic and biophysical properties of these enzymes were extensively characterized, showing that this process may be commercially viable because it results in chemicals that can be used to make environmentally friendly plastics. 05 Engineering a GH43 xylosidase enzyme for improved thermal stability. GH43 enzymes are used to convert woody biomass including rice and wheat straw into simple sugars and value added products. Researchers at the Western Regional Research Center in Albany, California, in collaboration with researchers at the Peoria labs, used the enzyme engineering technique of directed evolution to improve the thermal stability of a highly active xylosidase by up to 8 degrees celcius. Improved thermostability facilitates commercial viability by (1) lowering enzyme cost, and (2) allowing the process to take place at elevated temperatures where reaction rates are higher and the risk of bacterial contamination lessened. Several commercial partners have indicated interest in these enzymes for improving their biorefinery strategies.

Impacts
(N/A)

Publications

• Wong, D., Tenkanen, M., Vrsanaka, M., Sika-Aho, M., Puchart, V., Penttila, M., Salohelmo, M., Biely, P. 2012. Xylanase XYN IV from Trichoderma reesei showing exo- and endo-xylanase activity. FEBS Journal. 280: 295-301.
• Orts, W.J. 2008. Self-assembled films of cellulose nanofibers and poly (o- ethoxyaniline). Colloid and Polymer Science. 286(11):1265-1272.
• Wong, D., Chan, V.J., Liao, H., Zidwick, M. 2013. Cloning of a novel feruloyl esterase gene from rumen microbial metagenome and enzyme characterization in synergism with endoxylanases. Journal of Industrial Microbiology. 40:287-295.
• Holtman, K.M., Kodama, A.B., Klamczynski, A., Flynn, A., Bozzi, D.V., Torres, L., Franquivillanueva, D.M., Mao, J., Glenn, G.M., Orts, W.J. 2012. Thermal properties of Poly(ethylene terephthalate) recovered from municipal solid waste by steam autoclaving. Journal of Applied Polymer Science. 126(5):1698-1708.
• Bilbao-Sainz, C., Chiou, B., Glenn, G.M., Gregorski, K.S., Williams, T.G., Wood, D.F., Klamczynski, A., Orts, W.J. 2013. Solid lipid particles in lipid films to control diffusive release of 2-heptanone. Pest Management Science. 69(8):975-982. DOI:10.1002/ps.34162.
• Lee, C.C., Kibblewhite, R.E., Wagschal, K.C., Li, R., Orts, W.J. 2012. Isolation of alpha-glucuronidase enzyme from a rumen metagenomic library. Protein Journal. 31(3):206-211.
• Jordan, D.B., Lee, C.C., Wagschal, K., Braker, J.D. 2013. Activation of a GH43 �-xylosidase by divalent metal cations: Slow binding of divalent metal and high substrate specificity. Archives of Biochemistry and Biophysics. 533:79-87.
• Lee, C.C., Wagschal, K.C., Ruiping, L., Robertson, G.H., Kibblewhite, R.E., Orts, W.J. 2012. Isolation and characterization of a novel GH67 a- glucuronidase from a mixed culture. Journal of Industrial Microbiology and Biotechnology. 39:1245-1251. DOI:10.1007/s10295-012-1128-7.

Progress 10/01/11 to 09/30/12

Outputs
Progress Report 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 the recovery of biofuels, biorefinery co-products, and/or bioproducts from dilute fermentation broths. 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%. Multifunctional enzymes for biomass-to-bioenergy conversion. Converting crop residue such as straw or stover into biofuels is a complex process that can require multiple enzymes working synegistically to release fermentable sugars. ARS researchers in Albany, California, in collaboration with multiple partners (including National Aeronautics and Space Administration and a corporate partner), developed multi-functional enzymes with significantly better biomass-degrading properties. These multi-functional enzymes are built into a protein structure called a chaperon structure that has multiple active sites and can bind up to eighteen different enzymes. Preliminary data showed that enzymes tethered to these chaperonin scaffolds proteins (self-assembling nanostructure chaperonin complex) can exhibit greater synergies than if the same enzymes are free in solution. ARS researchers in Albany, California, received a three year, $479K National Institute of Food and Agriculture grant to continue this research. Bi-functional enzyme that hydrolyzes both mannans and mixed glucans. Several polysaccharides present in the plant cell wall are often difficult to degrade into fermentable sugars for biofuels production, especially those containing mannan. ARS researchers in Albany, California, developed a bi-functional enzyme that enables two hydrolytic reactions to proceed more efficiently than the corresponding individual reactions that releases mannan-derived sugars (mannose) from complex cell wall components. In related work this team employed site-directed mutagenesis and directed evolution to increase the thermal stability of a highly-active beta-xylosidase by eight degrees Celsius, a significant increase in temperature stability with no loss of activity. This beta- xylosidase, which exhibits one third lower end-product inhibition, has about three fourths the activity of the highest beta-xylosidase reported in the literature but has a much broader pH and temperature range, making it potentially interesting in commercial applications. Biorefinery strategies to create polymers and polymer building blocks (i. e. green monomers). In order to optimize biorefinery strategies for the Western States, special effort must be made to ensure that multiple product outputs are developed and commercialized, ensuring that no fraction of the biomass is wasted. In two related projects, ARS researchers in Albany, California, have worked with researchers partners to (1) convert waste methane (biogas) to a biodegradable polymer, poly(hydroxy alkanoate), PHA. (2) convert algae- and kelp-derived sugars using specific enzymes to value added chemicals. The monomers produced will be a range of intersting aldaric acids that have been used in nylon production and in anti-freeze formulations. An ARS partnership with a research partner will continue to test the commercial viability of producing aldaric acids as substrates for polymer production at larger scales. Accomplishments 01 Producing biofuels and value-added products from almond hulls. Almond hulls are rich in "fermentable" sugar that could be used in biofuel production, but only if the sugars can be extracted in a commercially viable method. Working with the Almond Hullers & Processors Association and a commercial partner, ARS researchers in Albany, California, have developed novel way to extract free sugars from almond hulls on a continuous basis and are then creating value-added uses for these extrac Analysis of different almond species have shown that nonpareil almond hulls, which make up 75-80% of domestic production, contain ~33% simple sugars (glucose, fructose, and sucrose) which are extractable by hot-wat and can be converted to ethanol or other biofuels. The remaining ingredients are rich in various other valuable compounds that are being explored for use in food ingredients, nutraceuticals, or as additives in make-up, and are also being monitored for the presence of aflotoxins, which would potentially limit their applications. Approximately three billion pounds of almond waste (mostly hulls) are produced annually in t U.S., with 90% of that production in California. Adding value to this coproduct stream, which is presently sold as cattle feed, would improve market conditions for the almond producers. 02 Creating bioenergy from wood and agricultural waste. Crop residues are potential feedstocks for production of biofuels and bioenergy, but are often not used because of the difficulty in transporting unprocessed, lo density biomass to a biorefinery processing plant. Working with a resear partner, ARS researchers in ALbany, California, are developing a portabl system for converting biomass to "biocoal", so that biomass can be used a powerplant similar to coal. This heating/compression process, called torrefaction, adds density to agricultural wastes, thus improving the efficiency of transportating biomass from a remote source to a larger, central power plant. Preliminary pilot-scale results on wood chips, grap pomace, olive pomace, apple pomace, tomato pomace, almond shells, and walnut shells provide data that a portable unit may prove cost-effective in converting biomass to bioenergy. The group of ARS researchers in Albany, California, are presently creating a full-scale unit that will provide commercial-scale substitutes for coal and biochar that is to be used as a polymer filler.

Impacts
(N/A)

Publications

• Teixeira, E., Curvelo, A., Correa, A.C., Marconcini, J.M., Glenn, G.M., Mattoso, L.H. 2012. Properties of thermoplastic starch from cassave bagasse and cassava starch and their blends with poly (lactic acid). Industrial Crops and Products. 37: 61-68.
• Aouada, F.A., De Moura, M.R., Orts, W.J., Mattoso, L.H. 2011. Preparation and characterization of a novel micro- and nanocomposite hydrogels containing cellulosic fibrils. Journal of Agricultural and Food Chemistry. (17): 9433-9442.
• Chiou, B., Robertson, G.H., Rooff, L.E., Cao, T., Jafri, H.H., Gregorski, K.S., Imam, S.H., Glenn, G.M. 2010. Water absorbance and thermal properties of sulfated wheat gluten films. Journal of Applied Polymer Science. 116: 2638-2644.
• Yu, J., Kohel, R.J., Fang, D.D., Cho, J., Van Deynze, A., Ulloa, M., Hoffman, S.M., Pepper, A.E., Stelly, D.M., Jenkins, J.N., Saha, S., Kumpatla, S.P., Shah, M.R., Hugie, W.V., Percy, R.G. 2012. A high-density simple sequence repeat and single nucleotide polymorphism genetic map of the tetraploid cotton genome. Genes, Genomes, Genetics. 2:43-58.
• Robertson, G.H., Cao, T., Orts, W.J. 2008. Effect on dough functioned properties of partial fractionation, redistribution and in-site deposition of wheat flour gluten proteins exposed to ethanol and aqueous ethanol. Cereal Chemistry. 85(5): 599-606.
• Robertson, G.H., Cao, T., Orts, W.J. 2007. Wheat proteins extracted from flour and butter with aqueous ethanol at subambient temperatures. Cereal Chemistry. 84(5): 497-501.
• Barghin, A., Ivanova, V.I., Imam, S.H., Chielliniam, E. 2010. Poly- (epsilon-caprolactone)(PCL) and poly(hydroxy-butyrate)(PHB) blends containing seaweed fibers: morphology and thermal-mechanical properties. Journal of Polymer Science. 48: 5282-5288.
• Imam, S.H., Gordon, S.H., Mohamed, A., Harry O Kuru, R.E., Chiou, B., Glenn, G.M., Orts, W.J. 2006. Enzyme catalysis of insoluble cornstarch granules: impact on surface morphology, property and biogradability. Polymer Degradation and Stability. 91(12): 2894-2900.
• Orts, W.J., Shey, J., Imam, S.H., Glenn, G.M., Guttman, M.E. 2005. Application of cellulose microfibrils in polymer nanocomposites. Polymers and the Environment. 13: 4.
• Ogawa, Y., Orts, W.J., Glennm, G.M., Wood, D.F. 2003. A simple method for studying whole sections of rice grain. Biotechnic & Histochemistry. 78(5) :237-242.
• Jordan, D.B., Bowman, M.J., Braker, J.D., Dien, B.S., Hector, R.E., Lee, C. C., Mertens, J.A., Wagschal, K.C. 2012. Plant cell walls to ethanol. Biochemical Journal. 442:247-252.
• Bilbao-Sainz, C., Bras, J., Williams, T.G., Senchal, T., Orts, W.J. 2011. HPMC reinforced with different cellulose nanoparticles. Carbohydrate Polymers. 86(4): 1549-1557.
• Glenn, G.M., Imam, S.H., Orts, W.J., Holtman, K.M. 2012. Starch as a feedstock for bioproducts and packaging. Book Chapter. p. 255-269.
• Ogawa, Y., Glenn, G.M., Orts, W.J., Wood, D.F. 2003. Historical structures of cooked rice grain. Journal of Agricultural and Food Chemistry. 51:7019- 7023.

Progress 10/01/10 to 09/30/11

Outputs
Progress Report 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 the recovery of biofuels, biorefinery co-products, and/or bioproducts from dilute fermentation broths. 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 create 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). Xylosidase activity has recently been recognized as being a critical rate- limiting component of commercial cellulosic biomass saccharification enzyme cocktails, and as such these commercial cocktails are now in many cases spiked with additional xylosidase activity. We discovered that xylosidase XylBH43, developed in Albany, has the second largest kcat reported for xylobiose hydrolyis, implying its high commercial potential. Conceived and developed an E. coli in-vivo whole-cell active/inactive (digital) 1st-tier high-throughput screening assay utilizing fluorophore- tagged substrates, for (1) gene discovery and (2) random-mutagenesis derived library purification for enzyme engineering applications. Demonstrated its use for the following enzyme classes that are critical for enzymatic hydrolysis of biomass: (1) xylosidases; (2) arabinofuranosidases, which remove arabinose residues from xylan chains; (3) xylanases, which hydrolyze the hemicellulose backbone; (4) cellulases, which hydrolyze the internal beta-1,4-bonds of cellulose; and (5) ferulic acid esterases, which hydrolyze ferulic acid groups that link lignin with the the hemicellulose backbone. A novel fabrication method was developed to make thin-film membranes for recovering alcohols from fermentation broths. The membranes show better performance than commercial membranes. A patent application was filed. CRADA to study mixed matrix membranes for recovery of ethanol from fermentation broths was completed and closed. A terminal report was issued. CRADA partner has initiated manufacturing of its first commercial product, which is used to dry water-soaked books and documents. The product contains a superabsorbent originally invented by USDA. New classes of adsorbents for use in controlling humidity in closed environments were studied. A collaboration with Reliant Energy was started, with the aim of developing a process to ferment almond hulls and other agricultural wastes to ethanol. Studies were initiated to characterize and analyze feedstocks. Accomplishments 01 Improved ethanol separation from biomass fermentation using polymer membranes. In production of fuel ethanol from agriculturally-derived biomass, the recovery of ethanol from fermentation broths via distillati is energy-intensive with separation costs representing a high percentage of total cost. ARS researchers in Albany, CA, have invented a new proces to make alcohol-selective membranes with performance significantly bette than that of commercial membranes. Application of these new membranes reduces energy costs from cellulose-ethanol fermentation, especially for broths with low ethanol concentration. The improved performance of the alcohol-selective membranes makes them more cost-competitive for fuel ethanol operations, an advantage that makes them suitable for commercial application in the cellulose-ethanol industry. 02 Biorefineries that work at township- or farm-scale to convert agriculturally-derived biomass to bioenergy. Recent investment into biomass-to-ethanol biorefineries have been hampered by the large capital investment required to build very large facilities. ARS researchers in Albany, CA, have developed flexible technology that converts ag-derived biomass into bioenergy and chemical feedstocks by applying anaerobic digestion in concert with ethanol fermentation depending on feedstock ty and scale. Specifically, improvements in anaerobic digestion to produce biogas and/or commercial acids, have resulted in process flexibility whe the biorefinery can be sustainable at a small or large scale depending o feedstock availability. This flexibility allows scaling of the biorefine for optimization at farm- or township-scale operation, with revenue generated by anaerobic digestion complementing cellulose-to-ethanol production. 03 Improved enzyme �cocktails� for breaking down ag-derived biomass. Industrial enzymes to break down agriculturally-derived biomass have proven costly because of the presence of complex, hard-to-degrade chemic crosslinks within straw and woody materials. ARS scientists in Albany an Peoria, along with researchers at U. Kentucky have combined efforts to improve an enzyme, xylosidase, that specifically breaks-down one of thes complex �minor� linkages that are so critical in degrading biomass. This work not only resulted in one of the most active xylosidase enzymes ever reported in the literature but also development of high-throughput screening assays for this enzyme class, emonstration that this enzyme ca be selectivity improved for reduced product inhibition, and clear eviden that these class of enzymes can work synergistically with other similar enzymes to degrade cellulose-rich biomass. This information is importan for the cellulose-ethanol industry and can be used by enzyme companies t improve their enzyme biomass-degrading �cocktails."

Impacts
(N/A)

Publications

• Offeman, R.D., Ludvik, C.N. 2010. Poisoning of mixed matrix membranes by fermentation components in pervaporation of ethanol. Journal Membrane Science. 367:288-295.
• Jha, A.K., Chen, L., Offeman, R.D., Balsara, N.P. 2011. Effect of nanoscale morphology on selective ethanol transport through block copolymer membranes. Journal Membrane Science. 373:112-120.
• Robertson, G.H., Offeman, R.D., Orts, W.J. 2010. Ethanol in the refining of agricultural materials: energy and material implications. Biofuels, Bioproducts, & Biorefining (Biofpr). 5:37-53.
• Sasagawa, T., Moriya, S., Lee, C.C., Kitamoto, K., Arioka, M. 2011. A high- throughput protein expression system in Pichia pastoris using a newly developed episomal vector. Plasmid Journal. 65(1):65-69.
• Wagschal, K.C., Jordan, D.B., Braker, J.D. 2011. Expression, characterization, and site-directed mutagenesis of �-D-xylosidase XylBH43 from Bacillus halodurans C-125. Process Biochemistry. doi:10.1016/j. procbio.2011.07.009.
• Wong, D., Chan, V.J., Batt Throne, S.B., Sarath, G., Liao, H. 2011. Engineering Saccharomyces cerevisiae to produce feruloyl esterase for the release of ferulic acid from switchgrass. Journal of Industrial Microbiology and Biotechnology. Epub ahead of print. DOI: 10.1007/s10295- 011-0985-9.
• Lee, C.C. 2010. Screening assays for biomass-degrading enzymes. Biofuels. 1(4):575-588.
• Wong, D., Chan, V.J., Mccormack, A.A. 2009. Functional cloning of an endo- alpha-1,5-L-arabinanase gene from a metagenomic library. Protein and Peptide Letters. 16:1411-1435.
• Wong, D., Batt Throne, S.B., Robertson, G.H., Lee, C.C., Wagschal, K.C. 2010. Chromosomal integration of recombinant alpha-amylase and glucoamylase genes in saccharomyces cerevisiae for starch conversion. Industrial Biotechnology. 6:112-118.
• Zhanmin, F., Wagschal, K.C., Wei, C., Montross, M., Lee, C.C., Yuan, L. 2009. Multimeric hemicellulases facilitate biomass conversion. Proceedings of the National Academy of Sciences. 75(6):1754-1757.
• Wong, D., Chan, V.J., Mccormack, A.A., Batt Throne, S.B. 2010. A novel xyloglucan-specific endo-beta-1,4-glucanase: biochemical properties and inhibition studies. Applied Microbiology and Biotechnology. 86:1463-1471.
• Wong, D. 2010. Applications of metagenomics for industrial bioproducts. Wong, D. Applications of metagenomics for industrial bioproducts. In: Marco, D., editor. Metagenomics: Theory, methods and applications. Norfolk, UK. Caister Academic Press.p. 141-158.
• Wong, D., Chan, V.J., Mccormack, A.A., Batt Throne, S.B. 2010. Cloning and characterization of an exo-xylglucanase from rumenal microbial metagenome. Protein and Peptide Letters. 17:803-808.
• Orts, W.J., Holtman, K.M., Seiber, J.N. 2008. Agricultural chemistry and bioenergy. Journal of Agricultural and Food Chemistry. 56:3892-3899.
• Lee, C.C., Kibblewhite, R.E., Wagschal, K.C., Robertson, G.H., Orts, W.J. 2009. Cloning and characterization of an alpha-glucuronidase from a mixed microbial population. Enzyme and Microbial Technology. 155(1-3):314-320.
• Li, R., Zhang, Y., Lee, C.C., Liu, L., Huang, Y. 2011. HILIC separation mechanisms of tetracyclines on amino bonded silica column. Journal of Separation Science. 34(13):1508-1516.

Progress 10/01/09 to 09/30/10

Outputs
Progress Report 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. 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). 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 �-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 �-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. Accomplishments 01 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 shiftin feedstock from corn starch to biomass. ARS Researchers in Albany, California studied the performa nce 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.

Impacts
(N/A)

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. Properties and applications of microbial beta- D-xylosidases. Applied Microbiology and Biotechnology. 2010. 86:1647- 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, 1099-113.
• 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.