2009 Annual Report 1a.Objectives (from AD-416) 1. Develop harvesting, fractionation and storage processes for forages and bioenergy crops that are economical, and that retain product quality. 2. Identify specific varieties of energy crops that display maximum fermentability when grown at specific locations under defined environmental conditions. 3. Develop switchgrass germplasm having broad adaptation to the northern USA and improved fermentability for conversion to value-added products. 4. Develop and improve fermentations for direct bioconversion of cellulosic biomass to value-added products (viz., ethanol, chemical feedstocks and novel bioadhesive components). 1b.Approach (from AD-416) New harvesting strategies will be developed that economically separate forages and bioenergy crops into higher- and lower-value fractions. An in vitro ruminal fermentation assay will be used to rapidly screen large numbers of biomass samples from several bioenergy crop species, provided by ARS agronomists from throughout the U.S. The data will be correlated to ethanol bioconversion capability, and NIRS calibration equations will be developed for ruminal fermentability and ethanol production. Switchgrass germplasm improvement will be carried out by recurrent phenotypic selection for vigor, lodging, and disease resistance to extend adaptation and biomass yield in several eco-regions. Switchgrass hybrids will be selected for enhanced biomass yield and fermentability. Consolidated bioprocessing of bioenergy crops, using anaerobic bacteria that produce their own cellulolytic enzymes and ferment the products to ethanol and other valuable products, will be improved through optimization of strains and culture conditions. Value-added co-products, such as adhesives produced by the fermentative bacteria, will be identified and their utility will be determined. 3.Progress Report Field experiments established in 2008 to support Objectives 3a and 3b are being managed to generate biomass yield data and quality samples. These activities relate to the development of new germplasm. Studies investigating the efficacy of dilute acid and alkali for preservation and storage were conducted at the laboratory, and on pilot- and farm-scale. After acid pretreatment and anaerobic storage, conversion of cell wall glucose to ethanol ranged from 22 to 83% of total cellulose for reed canarygrass and from 16 to 46% for switchgrass depending on moisture, storage duration, and chemical loading. Glucose conversion after calcium hydroxide pretreatment and anaerobic storage ranged from 21 to 55% and 18 to 54% for reed canarygrass and switchgrass, respectively. These on-farm pretreatments were scaled up with minimal difficulty, although stronger precautions for worker safety became necessary when handling sulfuric acid at farm-scale. Chemical costs for biomass pretreatment applied at a medium level of 50 g (kg of dry matter [DM])-1 were estimated to be as low as $4.05 and $5.20 per Mg DM for calcium hydroxide and sulfuric acid, respectively. A quantitative evaluation of cellulosic biomass digestion in dairy cattle revealed that the process displays several advances in proposed systems for consolidated bioprocessing (CBP) of biomass to produce ethanol. Included among these are a novel and effective physical pretreatment, a stable microbial consortium, operation at high solids loading (15% by weight), and an ability to convert essentially all carbohydrate, protein, and nucleic acids in the biomass to a mixture of volatile fatty acids (VFA), methane, and carbon dioxide. Approximately 72% of the total energy content of the carbohydrate portion of the forage is retained as VFA, and 16% is retained as methane. This total energy content (88%) is similar to the theoretical maximum energy retained in ethanol in an engineered CBP system. The ruminal microbial fermentation was operated outside the rumen for 80 successive transfers without contamination control and consistently produced 0.15 M total VFA, a concentration that could be effectively subjected to chemical conversion to hydrocarbon fuels. The CBP bacterium was grown on cellulose and on the cellulose breakdown product, cellobiose, at different growth rates to examine gene expression. 4.Accomplishments 1. On-farm biomass pretreatment for ethanol production: Storage and pretreatment of biomass at the biorefinery prior to conversion to ethanol are major costs for cellulosic ethanol production. We have found that pretreatment of biomass at the farm can effectively be accomplished by storing harvested biomass with sulfuric acid or lime for several months without heating in sealed plastic bags typically used for silage production. The pretreatments resulted in greater ethanol yields from an enzyme-yeast conversion system. The low cost ($4-5 chemical/dry ton) of on-farm pretreatment can reduce capital and storage costs for biorefining, and can add value to the biomass for the farmer, with resulting benefits to rural economies. 2. Increasing the efficiency of the process to convert cellulosic biomass to fuels: Proposed schemes for conversion of cellulosic biomass to fuel ethanol generally require a chemical pretreatment step, are subject to culture contamination, and do not use all of the carbohydrates of the biomass. We have analyzed the process of forage digestion by cattle and have identified several strategies that may be transferable to industrial biomass fermentations to produce fuels. These include a new way of grinding biomass to avoid chemical pretreatment and the use of adapted mixed rumen microbial cultures that do not require sterilization of the biomass or fermentation equipment. In addition, these methods can convert the protein and nucleic acid components of the biomass, as well as the cellulosic carbohydrates. Using a variation of existing chemical technology for the conversion of the organic acid fermentation products to organic fuel molecules (alkanes) could yield a practical, more economical system for converting biomass to fuels. 3. Regulation of genes involved in conversion of cellulose to ethanol and bio-based adhesives: Production of ethanol from cellulosic biomass is not yet economically feasible on an industrial scale due, in part, to the lack of co-products that can augment the value of ethanol. In collaboration with colleagues at the University of Wisconsin-Madison, we have examined the expression of over three thousand genes from Clostridium thermocellum, a leading candidate bacterium for cellulosic ethanol production, under twelve different growth conditions. The results provided genetic information necessary to optimize production of both ethanol and a unique bio-based adhesive compound produced by the bacterium during growth on cellulose. The bio-adhesive could effectively replace a large portion of the petroleum phenol-based adhesives used to produce wood laminates (i.e., plywood), resulting in more environmentally friendly wood products. 6.Technology Transfer Number of Active CRADAs 1 Number of the New/Active MTAs (providing only) 2 Number of Invention Disclosures Submitted 1 Review Publications Lorenz, A.J., Coors, J.G., De Leon, N., Wolfrum, E.J., Hames, B.R., Sluiter, A.D., Weimer, P.J. 2009. Characterization, Genetic Variation, and Combining Ability of Maize Traits Beneficial to the Production of Cellulosic Ethanol. Crop Science. 49:85-98. Shinners, K.J., Boettcher, G.C., Hoffman, D.S., Munk, J.T., Muck, R.E., Weimer, P.J. 2009. Single-Pass Harvest of Corn Grain and Stover: Performance of Three Harvester Configurations. Transactions of the ASABE. 52(1):51-60. Weimer, P.J., Russell, J.B., Muck, R.E. 2009. Lessons From the Cow: What the Ruminant Animal Can Teach Us About Consolidated Bioprocessing of Cellulosic Biomass. Bioresource Technology. 100:5323-5331. Weimer, P.J., Morris, J.B. 2009. Grasses and Legumes for Bio-Based Products. In: Wedin, W.F., Fales, S.L. editors. Grassland: Quietness and Strength for a New American Agriculture. Madison, WI: American Society for Agronomy/Crop Science Society of America/Soil Science Society of America. p. 221-233. Kiniry, J.R., Lynd, L., Greene, N., Johnson, M., Casler, M.D., Laser, M.S. 2008. Biofuels and water use: Comparison of maize and switchgrass and general perspectives. In: Wright, J.H., Evans, D.A., editors. New Research on Biofuels. Nova Science Publishers, Inc. p. 17-30. Casler, M.D., Heaton, E., Shinners, K.J., Jung, H.G., Weimer, P.J., Liebig, M.A., Mitchell, R., Digman, M.F. 2009. Grasses and Legumes for Cellulosic Bioenergy. In: Wedin, W.F. and Fales, S.L., editors. Grassland: Quietness and Strength for a New American Agriculture. Madison, Wisconsin: ASA-CSSS-SSSA. p. 205-219. Lorenz, A.A., Anex, R.P., Isci, A., Coors, J.G., deLeon, N., Weimer, P.J., Wolfrum, E.J. 2009. Forage Quality and Composition Measurements as Predictors of Ethanol Yield from Maize (Zea mays L.) Stover. Biotechnology for Biofuels. 2:5. El Nashaar, H.M., Banowetz, G.M., Griffith, S.M., Casler, M.D., Vogel, K.P. 2009. Genotypic Variability in Mineral Composition of Switchgrass. Bioresource Technology. 100:1809-1814.