Progress 06/04/04 to 06/03/09

Outputs
Progress Report 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). 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. Significant Activities that Support Special Target Populations 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. Technology Transfer Number of Active CRADAS: 1 Number of New/Active MTAs(providing only): 2 Number of Invention Disclosures submitted: 1

Impacts
(N/A)

Publications

• Casler, M.D., Stendal, C., Kapich, L., Vogel, K.P. 2007. Genetic diversity, plant adaptation regions, and restoration gene pools for switchgrass. Crop Science. 47:2261-2273.
• Casler, M.D., Vogel, K.P., Taliaferro, C.M., Ehlke, N.J., Berdhal, J.D., Brummer, E.C., Kallenbach, R.I., West, C.P. 2007. Latitudinal and longitudinal adaptation of switchgrass populations. Crop Science. 47:2249- 2260.
• Vadas, P.A., Barnett, K.H., Undersander, D.J. 2008. Economics and energy of ethanol production from alfalfa, corn, and switchgrass in the Upper Midwest, USA. Bioenergy Research. 1:44-55.
• Boateng, A.A., Weimer, P.J., Jung, H.G., Lamb, J.F. 2008. Response of thermochemical and biochemical conversion processes to lignin concentration in alfalfa stems. Energy and Fuels. 22:2810-2815.
• 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.
• 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.

Progress 10/01/06 to 09/30/07

Outputs
Progress Report 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). 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. Accomplishments a. Biosynthesis of bio-based adhesive. An extracellular polysaccharide produced by several anaerobic bacteria appears to have some utility as a bio-based adhesive, but how the bacteria produce this adhesive is not known. We have identified seven genes likely to be involved in the biosynthesis of precursors for the bio- based adhesive of Clostridium thermocellum (a thermophilic ethanol- producing bacterium useful for consolidated bioprocessing of biomass) have been identified from whole-genome searching, and PCR primers have been designed and manufactured for study of gene expression by this organism under different growth conditions. Elucidation of the biosynthetic mechanism and its regulation should allow the material to be produced more cheaply and in higher yield. This accomplishment addresses NP 306 Component 2. (New Processes, New Uses, and Value-Added Foods and Biobased Products), Convert low value agricultural residues into higher value products; and NP307 Action Plan Component 1 (Ethanol), Coproduct development. b. On-farm pretreatment technologies for improving enzymatic degradability of cellulose and hemicelluloses in perennial grasses. Biomass needs to be pretreated prior to further processing because the carbohydrates are largely contained in complex cell wall structures that impede their enzymatic conversion into fermentable sugars. We demonstrated that sulfuric acid and lime serve as effective pretreatments under mild ambient conditions, allowing up to 80% conversion to ethanol (theoretical basis), albeit at high chemical loadings. Significantly increasing the degradability of the biomass while in storage is expected to add value by either allowing milder or possibly eliminating the need for pretreatment at the biorefinery, thereby, providing better return for farmers. This accomplishment addresses NP 306 Action Plan Component 2. (New Processes, New Uses, and Value-Added Foods and Biobased Products), Convert low value agricultural residues into higher value products; and NP307 Action Plan Component 1 (Ethanol), Coproduct development. c. Harvesting and storage of perennial grasses. Information on the harvesting and storage losses, and on adding value by field fractionation during harvesting is needed to determine practical yields, product value, and economics of biofuels production from perennial grasses. We measured dry matter losses under various storage methods to range from 4 to 10%, with least loss occurring for indoor or under-tarp storage, and most losses occurring in bales wrapped with twine and stored outdoors. We also prepared bales of switchgrass and reed canarygrass at different initial moisture levels, and prepared bales of these same forage species harvested with leaf-stripping machinery to produce fractions that differ in leaf and stem proportions, in order to determine their storage characteristics; analysis of these materials will be performed at the end of this storage period (autumn 2007). The research was carried out by cooperators at the University of Wisconsin- Madison (K.J. Shinners, see Progress Report 3655-41000-004-02S), with analytical support by the US Dairy Forage Research Center. This research will evaluate the technical and economic feasibility of harvesting and storage technologies. This accomplishment addresses NP 306 Action Plan Component 1 (Quality Characterization, Preservation, and Enhancement), Preservation and/or Enhancement of Quality and Marketability. d. Energy balances and economics of biomass production systems. Switchgrass and alfalfa have been identified as a promising feedstock for biofuels, but the energy requirements and economics of production have not been quantified, particularly as part of an integrated production system. We assessed the economics and energy of potential cellulosic ethanol production in the Upper Midwest by estimating production costs and energy balances for three crop systems: continuous corn, continuous switchgrass, and an alfalfa-corn rotation. Results show alfalfa-corn competes well with continuous corn or switchgrass on an energy balance basis, and may offer the greatest farm income. These studies lay the foundation for assessment of biofuel economics in the upper Midwestern U. S. This accomplishment addresses NP307 Action Plan Components 1 (Ethanol) and 4 (Energy Crops). e. Biomass screening. Screening large numbers of biomass samples for their bioconversion potential is limited by the difficulty of conventional SSF assays, which require operation under aseptic conditions to prevent bacterial contamination. We used a rapid in vitro ruminal gas production assay to screen several hundred biomass samples (primarily switchgrass, reed canarygrass, and corn stover) from 6 different collaborators (3 within ARS) for fermentability to identify germplasm, processing conditions, and harvesting/storage conditions that improve biodegradability. The data will allow collaborators to reduce the number of samples to test via SSF to identify the most useful germplasm and bioprocessing conditions. This research addresses NP 306 Action Plan Component 1 (Quality Characterization, Preservation, and Enhancement), Factors and Processes that Affect Quality. Significant Activities that Support Special Target Populations We have a collaborative research project K-1251 with the Institute of Industrial Biotechnology, Stepnogorsk, Republic of Kazakhstan, to develop enzyme technology for upgrading biomass materials through enzyme addition during ensiling. The project is administered through the International Science and Technology Council in Moscow, through funds from USDA-ARS Office of International Programs and U.S. Department of State�s Office of Cooperative Threat Reduction. The purpose of the program is to redirect former Soviet weapons scientists into agricultural research. Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings: 6 Number of Newspaper Articles,Presentations for NonScience Audiences: 9

Impacts
(N/A)

Publications

• Weimer, P.J., Springer, T.L. 2007. Fermentability of eastern gamagrass, big bluestem and sand bluestem grown across a wide variety of environments. Bioresource Technology. 98:1615-1621.
• Weimer, P.J., Price, N.P., Kroukamp, O., Joubert, L.M., Wolfaardt, G.M., Van Zyl, W.H. 2006. Studies of the extracellular glycocalyx of the anaerobic ruminal bacterium Ruminococcus albus 7. Applied and Environmental Microbiology. 72(12):7559-7566.
• Shinners, K.J., Binversie, B.N., Muck, R.E., Weimer, P.J. 2007. Comparison of wet and dry corn stover harvest and storage. Biomass and Bioenergy. 31:211-221.
• Shinners, K.J., Adsit, G.S., Binversie, B.N., Digman, M.F., Muck, R.E., Weimer, P.J. 2007. Single-pass, split-stream harvest of corn grain and stover. Transactions of the ASABE. 50:353-363.
• Lynd, L.R., Weimer, P.J., Wolfaardt, G.M., Zhang, Y.P. 2007. Cellulose hydrolysis by Clostridium thermocellum: A microbial perspective. In: Kataeva, I.A., editor. Cellulosome: Molecular Anatomy and Physiology of Proteinaceous Machines. Hauppage, NY:Nova Science Publishers. p. 95-117.

Progress 10/01/05 to 09/30/06

Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Both scientists and policymakers view as inevitable the transition of industrial economies from those based on petroleum to those based on renewable materials. Biomass is the only sustainable source of fuels and chemicals available to humanity, but biomass production for the purpose of conversion to fuels and industrial chemicals in the U.S. and other industrialized nations is based almost exclusively on grains, particularly corn. This represents a diversion of these grains from the food supply, and the high inputs and ecological consequences of grain- based fermentations at industrial scale have led many to question their long-term sustainability. Forages have many advantages in agricultural production systems, including high biomass yields, ability to be cultivated on marginal lands, low input requirements, and ecological sustainability. However, forage acreage is declining in the U.S., largely because the low digestibility of forage cell walls limits their use in livestock feeding, and because alternative end uses for forage fiber are not available. Use of forages as feedstocks for fuel and chemical production will require: i) increasing the geographic range of the more promising candidates to insure their productivity under the environmental constraints of particular geographic regions, and ii) development of harvesting and storage technologies that maximize yield and quality of the forage feedstock. A major objective of this project is to identify and develop novel value- added products from alfalfa and other forages. Two basic process configurations are being explored. The first uses dry fractionation to separate alfalfa herbage into a high-value, leaf fraction and a low-value stem fraction. The second process configuration uses wet fractionation to produce a high-value juice fraction rich in protein and other value- added biochemicals (e.g., phytase and other recombinant enzymes), and a low-value fiber fraction. Our intent is to use the fiber directly as a biofiltering agent (e.g., in heavy metal removal from water) or to upgrade the fiber by microbial fermentation to produce ethanol (a liquid fuel) or lactic acid (a food acidulent). During these fermentations a substantial portion of the fiber is lignified and resists fermentation, resulting in a fermentation residue that also contains bacterial cells and a sticky "glycocalyx" required by the bacteria to attach to the fiber. We intend to use the residue as a biological adhesive competitive with soy protein. The U.S. is faced with ballooning trade deficits that undermine all aspects of our economy. Petroleum, our major source of transportation fuels and industrial organic chemicals, is our single largest import item, and the fraction of our petroleum needs that are met with imported oil increases yearly as domestic production cannot keep up with demand. Agriculture provides a positive trade balance to the U.S. economy and is the underpinning of rural economies nationwide, but these contributions are imperiled by the marginal return that farmers receive for their products. Moreover, the sustainability of agriculture is threatened by cropping practices that do not adequately control soil erosion and nutrient losses. Development of value-added co-products from the forages offers the opportunity to farm sensitive lands in a more sustainable fashion; to yield products that increase farmers' return, thus improving rural economies; and to reduce dependency on unstable foreign sources of petroleum. This project supports National Program 306 Action Plan Components 1 (Quality Characterization, Preservation and Enhancement) and 2 (New Processes, New Uses and Value-Added Food and Bio-Based Products), and National Program 307 Action Plan Components 1 (Ethanol) and 4 (Energy Crops). 2. List by year the currently approved milestones (indicators of research progress) Objective 1. FY2006-2007: Determine effects of cutting height and winter harvest. FY2006-2008: Develop field-scale fractionation technology. Evaluate low-moisture storage technology. Objective 2. FY2006: Screen alfalfa samples for in vitro fermentability. FY 2006-2008: Develop expert system for predicting fermentability. Objective 3a. FY 2006: Complete cycle 3 selection of switchgrass for vigor, lodging resistance and disease resistance. FY2007-2008: Field trials of C0, C1, C2, and C3 selections. Objective 3b. FY2006-2008: Field trials of parents and hybrids. FY2007- 2008: Lab analysis and in vitro fermentability tests of materials from field trials. Objective 4a. FY 2006: Medium minimization and mass balance determination for production of ethanol and biological adhesive. FY2006- 2008: Optimization of yield and product concentration. Objective 4b. FY2006-2007: Technology transfer to manufacturers. 4a List the single most significant research accomplishment during FY 2006. Fermentation technology has been improved for production of biomass fermentation residues for use as wood adhesives. The culture medium has been simplified to reduce costs and to prolong the fermentation to produce a greater yield and concentration of product, and improvements in downstream processing have increased product recovery. USDA-ARS-USDFRC scientists improved the medium formulation and performed mass culture fermentations with collaborators at the USDA-Forest Service-Forest Products Laboratory. The improved cultivation conditions have improved the economics of producing the bio-based adhesive material. This accomplishment addresses NP 306 Component 2. (New Processes, New Uses, and Value-Added Foods and Biobased Products), Convert low value agricultural residues into higher value products; and NP307 Component 1 (Ethanol), Coproduct development. 4b List other significant research accomplishment(s), if any. 1. Harvesting and storage of biomass materials. A whole plant head harvester adapted to a grain combine allowed single-pass harvesting of corn with separation and recovery of 90% of the stover fraction, although the ground speed of the harvester was reduced considerably compared to conventional grain harvest. The research was carried out by cooperators at the University of Wisconsin-Madison, with analytical support by the US Dairy Forage Research Center. This research has shown the technical feasability of single-pass harvesting and identified harvest rate as an impediment to adoption of this technology. This accomplishment addresses NP 306 Component 1 (Quality Characterization, Preservation, and Enhancement), Preservation and/or Enhancement of Quality and Marketability. Low-moisture storage in plastic silage bags over an 8-month period was shown to provide excellent protection of the quality of corn stover collected by a single-pass harvester. Dry matter losses during conventional storage represent a substantial income loss that would reduce the economic viability of stover-based biorefineries. The research was carried out by cooperators at the University of Wisconsin-Madison, with analytical support by the US Dairy Forage Research Center. This accomplishment addresses NP 306 Component 1 (Quality Characterization, Preservation, and Enhancement), Preservation and/or Enhancement of Quality and Marketability. Perennial grasses were successfully harvested and stored in plastic silage bags with very little deterioration (dry matter loss) over a 9- to 11-month storage period. Role: The research was carried out by cooperators at the University of Wisconsin-Madison, with analytical support by the U.S. Dairy Forage Research Center. This research has shown the technical feasibility of ensiling in plastic bags without field wilting, as a means of preserving feedstock mass and quality, for use in biorefinery applications. This accomplishment addresses NP 306 Component 1 (Quality Characterization, Preservation, and Enhancement), Preservation and/or Enhancement of Quality and Marketability. 2. Biomass screening. While alfalfa fermentability studies have been delayed by lack of samples from collaborators, screening for fermentability has been conducted on hundreds of samples from four other collaborators, including switchgrass, reed canarygrass, stover from corn hybrids, and wood pulps. This research has identified materials that display enhanced fermentabiility, and has advanced the research programs of several collaborating ARS scientists. This accomplishment addresses NP 306 Component 1 (Quality Characterization, Preservation, and Enhancement), Factors and Processes that Affect Quality. 3. Improved switchgrass. There are no switchgrass cultivars specifically adapted for biomass production in the north central and northeastern USA. We developed two populations of switchgrass for this region, based on collections made in native prairie remnants of the region and from plants selected for superior biomass production traits. These two populations represent the best adapted switchgrass germplasm for biomass production in this region and numerous seed requests have been received. Selection for high biomass yield of switchgrass is time consuming, resource intensive, and often inefficient. We identified two potential selection criteria that may improve the efficiency of breeding for higher biomass yield of switchgrass: yield per tiller or yield per phytomer. Future experiments will validate these traits as indirect selection criteria, determining their potential impact on breeding switchgrass for higher biomass yield. This accomplishment addresses NP 307 Component 4 (Energy Crops), Switchgrass germplasm with improved yields and biomass fuel characteristics. 4d Progress report. Structural and anatomical characteristics of the Ruminococcus albus glycocalyx, the active agent in biomass fermentation residue that displays wood adhesive properties, have been determined. Improved cellulosics fermentations and adhesive production have been obtained with Clostridium thermocellum, a related bacterium that grows more rapidly, produces more ethanol as a co-product, and has a more tractable genetic system. A modified culture medium with reduced levels of most nutrients has been developed. Elimination of most of the calcium from the medium inhibits undesirable sporulation, extends the growth phase of the organism, and results in a more extensive conversion of the biomass. Improved recovery of the adhesive residue was obtained by substituting continuous flow centrifugation in place of the previously-used filtration methods. Adhesive functionality of the fermentation residues varied markedly among different biomass feedstocks, and appears to be inversely related to the viscosity of the formulations during application to the wood panels. Improved switchgrass cultivars more adapted to climate and soil conditions of the upper midwestern U.S. have been developed, and potential selection criteria have been identified that may facilitate screening for enhanced biomass yield. A novel biomass fermentability assay based on in vitro ruminal gas production has been used for screening hundreds of biomass samples from collaborators across the U.S., as a first-stage screen for identifying the most promising materials for further bioconversion research. 5. Describe the major accomplishments to date and their predicted or actual impact. This project is aligned with NP306, Quality and Utilization of Agricultural Products and NP307, Bioenergy & Energy Alternatives. Strategies for processing alfalfa have been developed that include wet fractionation to produce high-protein juice and a fibrous solids material. The juice fraction can be treated by ultrafiltration or by pH adjustment to recover the protein, and has been shown to have high nutritional value as a protein supplement in foods that may be particularly useful in third- world countries whose populations eat protein-deficient diets. The solids remaining from wet fractionation of alfalfa have been shown to be fermentable to ethanol by the anaerobic bacteria, Clostridium thermocellum or Ruminococcus albus, although yields are currently low. A collaboration with the USDA Forest Service, Forest Products Laboratory has demonstrated that the fermentation residues (containing undegraded fiber, bacterial cells, and extracellular polymers produced by the bacteria) have adhesive properties and can partially replace petroleum- derived phenol-formaldehyde resins used in plywood manufacture. Acceptable shear strength and wood failure values were not obtained with rehydrated pure fermentation residues, but were obtained if the adhesive mixture was combined in a proportion of 30% fermentation residue plus 70% phenol-formaldehyde resin (dry weight basis). A novel screen for biomass fermentability, based on in vitro ruminal gas production, has been developed and validated for use with several species of perennial forages. Using classical plant breeding methods, switchgrass cultivars have been selected for improved performance in more northerly U.S. locations, and are under field evaluation. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Technologies for wet fractionation of herbage were transferred under a Cooperative Research and Development Agreement (CRADA) to a major plant biotechnology company. Parents of protein-deficient children were taught to make food supplements from green plants via fractionation. Technology for intensive conditioning of forage crops to accelerate drying and to increase digestibility was transferred under a CRADA to a major farm equipment manufacturer. A U.S. patent application was filed 06/15/2006 covering the use of cellulosic fermentation residues as bioadhesive materials. We have already been contacted by one company regarding the licensing of this technology, but are currently limited in our ability to scale the process to produce adhesives in quantities sufficient for commercial testing. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Behling, A. Beyond the Hay Shed, Hay and Forage Grower, March 2006, p.4. , describes use of alfalfa as a feedstock for production of bio-based adhesives. Weimer, P.J., Value-Added Products from Forages and Biomass Energy Crops, presentation to technology transfer representatives from Montana State University, at USDA-ARS-NCAUR, Peoria, IL, January 2006. Weimer, P.J., All Guts and No Glory: Cellulose Degradation by Ruminal Bacteria and its Potential Application to Biomass Conversion Systems, Invited seminar, Northern Illinois University, DeKalb, IL. April 2006. Weimer, P.J., Basic and Applied Aspects of Adherence to Cellulose by Anaerobic Cellulolytic Bacteria, Invited Seminar, University of Guelph, Guelph, Ontario, Canada, June 2006.

Impacts
(N/A)

Publications

• Casler, M.D., Vogel, K.P., Beal, A.C. 2006. Registration of WS4U and WS8U switchgrass germplasms. Crop Science. 46:998-999.
• Muck, R.E., Shinners, K.J. 2006. Effect of inoculants on the ensiling of corn stover. Proceedings of ASABE Annual International Meeting. Paper No. 061013.

Progress 10/01/04 to 09/30/05

Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Both scientists and policymakers view as inevitable the transition of industrial economies from those based on petroleum to those based on renewable materials. Biomass is the only sustainable source of fuels and chemicals available to humanity, but biomass production for the purpose of conversion to fuels and industrial chemicals in the U.S. and other industrialized nations is based almost exclusively on grains, particularly corn. This represents a diversion of these grains from the food supply, and the high inputs and ecological consequences of grain- based fermentations at industrial scale have led many to question their long-term sustainability. Forages have many advantages in agricultural production systems, including high biomass yields, ability to be cultivated on marginal lands, low input requirements, and ecological sustainability. However, forage acreage is declining in the U.S., largely because the low digestibility of forage cell walls limits their use in livestock feeding, and because alternative end uses for forage fiber are not available. The objective of this project is to identify and develop novel value- added products from alfalfa and other forages. Two basic process configurations are being explored. The first uses dry fractionation to separate alfalfa herbage into a high-value, leaf fraction and a low-value stem fraction. The second process configuration uses wet fractionation to produce a high-value juice fraction rich in protein and other value- added biochemicals (e.g., phytase and other recombinant enzymes), and a low-value fiber fraction. Our intent is to use the fiber directly as a biofiltering agent (e.g., in heavy metal removal from water) or to upgrade the fiber by microbial fermentation to produce ethanol (a liquid fuel) or lactic acid (a food acidulent). During these fermentations a substantial portion of the fiber is lignified resists fermentation, resulting in a fermentation residue that also contains bacterial cells and a sticky "glycocalyx" required by the bacteria to attach to the fiber. We intend to use the residue as a biological adhesive competitive with soy protein. 2. List the milestones (indicators of progress) from your Project Plan. Objective 1. Harvesting, processing and storage technologies. i) Establish stands of alfalfa, switchgrass and reed canarygrass for harvesting research. ii) Determine effects of cutting height and winter harvest on yield, fermentability, and persistence of alfalfa, switchgrass and reed canarygrass. iii) Develop field-scale fractionation technology. iv) Evaluate low-moisture storage technology. Objective 2: Biomass screening. i) Compare in vitro gas production and SSF methods. ii) Screen gama grass and bluestem samples. iii) Screen switchgrass and bermuda grass samples. iv) Screen alfalfa samples. v) Develop expert system for predicting fermentability. Objective 3a. Selection of switchgrass for vigor, lodging resistance, and disease resistance. i) Complete cycle 2 of selection, produce maternal seed. ii) Complete cycle 3 of selection. iii) Field trials for C0, C1, C2 and C3 evaluation. Objective 3b. Switchgrass hybrids for yield and energy conversion. i) Transplant crossing blocks. ii) Harvest hybrid seed (upland x lowland and lowland x upland). iii) Field trials of hybrids and parents. iv) Lab work on samples from field trials and screening for fermentability. Objective 4a. Consolidated bioprocessing. i) Isolate and characterize microbial strains. ii) Medium minimization. iii) Mass balance determinations. iv) Optimization of yield and product concentration. Objective 4b: Bio-based adhesives. i) Structural characterization of glycocalyces. ii) Large-scale production of fermentation residues; iii) Optimization of adhesive formulations and applications. iv) Technology transfer to manufacturers. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Objective 1. Establish stands of alfalfa, switchgrass, and reed canarygrass. First-phase evaluation of storage technologies. Milestone Fully Met 2. Objective 2. Screen switchgrass and bermuda grass samples for fermentability. Milestone Fully Met 3. Objective 3a. FY 2005-2006: Cycle 3 selection of switchgrass for vigor, lodging resistance, and disease resistance. Milestone Substantially Met 4. Objective 3b. Harvest hybrid seed (lowland x upland, upland x lowland) from crosses selected for yield and energy conversion. Milestone Substantially Met 5. Objective 4a. Isolate new microbial strains. Milestone Not Met Redirection of Research focus due to change in priorities 6. Objective 4b. Structural characterization of glycocalyces. Optimization of adhesive formulations. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Objective 1. FY2006-2007: Determine effects of cutting height and winter harvest. FY2006-2008: Develop field-scale fractionation technology. Evaluate low-moisture storage technology. Objective 2. FY2006: Screen alfalfa samples for in vitro fermentability. FY 2006-2008: Develop expert system for predicting fermentability. Objective 3a. FY 2006: Complete cycle 3 selection of switchgrass for vigor, lodging resistance, and disease resistance. FY2007-2008: Field trials of C0, C1, C2, and C3 selections. Objective 3b. FY2006-2008: Field trials of parents and hybrids. FY2007- 2008: Lab analysis and in vitro fermentability tests of materials from field trials. Objective 4a. FY 2006: Medium minimization and mass balance determination for production of ethanol and biological adhesive. FY2006- 2008: Optimization of yield and product concentration. Objective 4b. FY2006-2007: Technology transfer to manufacturers. 4a What was the single most significant accomplishment this past year? Gene expression in ethanol-producing bacteria. Four genes involved in cellulose degradation by Clostridium thermocellum, a bacterium that grows at high temperature in the absence of oxygen, were shown to be regulated in a manner that varied in response to the conditions under which the bacterium is grown. The process of cellulose degradation (a key process in producing ethanol from cellulosic biomass) is poorly understood at the level of gene expression. This research -- the most complete study to date in any organism of the relationship between cell growth rate and gene expression -- provides new information on the regulation of cellulose degradation by an organism that shows particular promise for converting cellulosic biomass to both ethanol and a fermentation residue having desirable properties as a wood adhesive. The results provide new strategies for improving cellulose degradation and ethanol production by this bacterium. 4b List other significant accomplishments, if any. 1. Biomass screening. Fermentability data have been determined for Eastern gamagrass, bluestem, and switchgrass varieties grown at a variety of locations east of the 100th meridian, and NIR prediction equations have been developed for in vitro fermentability of these forages. 2. Improved switchgrass. Using conventional plant breeding methods, new experimental varieties crosses of switchgrass have been selected for improved performance in northern latitudes, for subsequent additional experimental evaluation in the coming year. 4d Progress report. The effect of growth conditions on gene expression in the cellulose- fermenting, ethanol-producing bacterium Clostridium. thermocellum ATCC 27405 was studied, using cells grown in continuous culture under cellobiose or cellulose limitation over approximately a tenfold range of growth rates. Fermentation product distribution displayed similar patterns in cellobiose- or cellulose-grown cultures, including substantial shifts in the proportion of ethanol and acetic acid with changes in growth rate. Expression of seventeen genes involved or potentially involved in cellulose degradation, intracellular phosphorylation, catabolite repression, and fermentation end-product formation was quantified by real-time PCR, with normalization to two calibrator genes (recA and 16S rDNA) to determine relative expression. Thirteen genes displayed modest (fivefold or less) differences in expression with growth rate or substrate type: sdbA (cellulosomal scaffoldin-dockerin binding protein), cdp (cellodextrin phosphorylase), cbp (cellobiose phosphorylase), hydA (hydrogenase), ldh (lactate dehydrogenase), ack (acetate kinase), one putative type IV alcohol dehydrogenase, two putative cAMP binding proteins, three putative Hpr- like proteins, and a putative Hpr serine kinase. By contrast, four genes displayed > tenfold reduced levels of expression when grown on cellobiose at dilution rates > 0.05 h-1: cipA (cellulosomal scaffolding protein), celS (exoglucanase), manA (mannanase) and a second type IV alcohol dehydrogenase. The data suggest that at least some cellulosomal components are transcriptionally regulated, but that differences in expression with growth rate or among substrates do not directly account for observed changes in fermentation end-product distribution. These results suggest targets for genetic manipulation of this organism for enhanced cellulose degradation and ethanol production. In addition, this work represents the most complete study to date of the effect of growth rate on gene expression in any bacterial species. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Strategies for processing alfalfa have been developed that include wet fractionation to produce high-protein juice and a fibrous solids material. The juice fraction can be treated by ultrafiltration or by pH adjustment to recover the protein, and has been shown to have high nutritional value as a protein supplement in foods that may be particularly useful in third- world countries whose populations eat protein-deficient diets. The solids remaining from wet fractionation of alfalfa have been shown to be fermentable to ethanol by Ruminococcus albus, although yields are currently low. A collaboration with the USDA Forest Service, Forest Products Laboratory has demonstrated that the fermentation residues (containing undegraded fiber, bacterial cells, and extracellular polymers produced by the bacteria) have adhesive properties and can partially replace petroleum-derived phenol-formaldehyde resins used in plywood manufacture. Acceptable shear strength and wood failure values were not obtained with rehydrated pure fermentation residues, but were obtained if the adhesive mixture was combined in a proportion of 30% fermentation residue plus 70% phenol-formaldehyde resin (dry weight basis). An improved means of establishing strictly anaerobic conditions for cultivation of our fermentative cultures has been developed that is broadly applicable to other anaerobic microorganisms. A continuous flow, packed-bed reactor system has been constructed for cultivating the anaerobic bacterium Ruminococcus albus in a manner that produces more fermentation residue for bioadhesive testing. A novel strain of fungus has been isolated that can produce ethanol from cellulose under anaerobic, non-growing conditions in a completely mineral medium, and progress was made toward developing a genetic system for this strain. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Technologies for wet fractionation of herbage were transferred under a Cooperative Research and Development Agreement (CRADA) to a major plant biotechnology company. Parents of protein-deficient children were taught to make food supplements from green plants via fractionation. Technology for intensive conditioning of forage crops to accelerate drying and to increase digestibility was transferred under a CRADA to a major farm equipment manufacturer. A U.S. patent application was filed 05/05/2004 covering the use of cellulosic fermentation residues as bioadhesive materials. We have already been contacted by one company regarding the licensing of this technology.

Impacts
(N/A)

Publications

• Weimer, P.J., Dien, B.S., Springer, T.L., Vogel, K.P. 2005. In vitro gas production as a surrogate measurement of the fermentability of cellulosic biomass. Applied Microbiology Biotechnology. 67:52-58.
• Casler, M.D. 2005. Ecotypic variation among switchgrass population from the northern USA. Crop Science. 45:388-398.
• Contreras-Govea, F.E., Muck, R.E., Filya, I., Mertens, D.R., Weimer, P.J. 2005. In vitro gas production and bacterial biomass estimation for lucerne silage inoculated with one of three lactic acid bacterial inoculants. In: Park, R.S., Stronge, M.D., editors. Silage production and utilisation, XIVth International Silage Conference, July 3-6, 2005, Belfast, Northern Ireland. p. 207.
• Martin, N.P., Mertens, D.R., Weimer, P.J. 2004. Alfalfa: hay, haylage, baleage and other novel products. In: Proceedings of the Idaho Alfalfa and Forage Conference, February 24-25, 2004, Twin Falls, Idaho. p. 9-18.