Progress 10/01/99 to 06/02/04

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 its 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. 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. 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 simultaneous saccharification and fermentation (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) 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. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004, and indicate, which ones were not fully or substantially met, briefly explain why not, and your plans to do so. Objective 1. Establish stands of alfalfa, switchgrass and reed canarygrass. Milestone met. Objective 2. Compare in vitro gas production and SSF methods. Milestone met. Screen gamagrass and bluestem samples. Milestone met. Objectove 3a. Completion of cycle 2 of switchgrass selection and production of maternal seed. Milestone met. Objective 3b. Transplant crossing blocks. Milestone met. Objective 4b. Large-scale production of fermentation residues for adhesives testing. Milestone met. FY2004 work supporting achievement of FY2005 milestones is on schedule. B. List the milestones that are scheduled to be addressed over the next 3 years (FY 2005, 2006, and 2007). 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. FY2005: Screen switchgrass and bermudagrass samples for in vitro fermentability. FY2006: Screen alfalfa samples for in vitro fermentability. FY 2006-2008: Develop expert system for predicting fermentability. Objective 3a. FY 2005-2006: Cycle 3 selection of switchgrass for vigor, lodging resistance and disease resistance. FY2007-2008: Field trials of CO, C1, C2, and C3 selections. Objective 3b. FY2005: Harvest hybrid seed (lowland x upland, upland x lowland) from crosses selected for yield and energy conversion. 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 2005-2006: Medium minimization and mass balance determination for production of ethanol and biological adhesive. FY2006- 2008: Optimization of yield and product concentration. Objective 4b. FY 2005: Structural characterization of glycocalyx. Optimization of adhesive formulations and applications. FY2006-2007: Technology transfer to manufacturers. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004: In collaboration with scientists at the USDA Forest Service, Forest Products Laboratory (FPL), we have extended our previous discovery that fermentation residues, left over from the bacterial fermentation of cellulosic biomass to ethanol, can serve as novel components of adhesive formulations for the production of plywood. In particular, we have extended the range of feedstocks to include alfalfa fiber, a potentially abundant waste product of a wet-fractionation process (developed in the previous CRIS project) used to produce a high-protein juice fraction. Moreover, we have extended the range of bioadhesivee-producing organisms to include Clostridium thermocellum, a thermophilic bacterium that produces considerably more ethanol co-product than do the ruminococci we have used previously. These residues have potential as adhesive or adhesive extenders in wood construction applications. B. Other significant accomplishment(s), if any: 1. A simple screening method has been developed for determining the fermentability of biomass materials. The method involves measurement of gas production in sealed vessels containing the biomass plus a microbial inoculum taken from the rumens of cattle. Gas production is easily measured with a digital pressure gauge. Collaborative work with ARS scientists at USDA-ARS-NCAUR (Peoria, IL) has revealed that the method can predict, with fair accuracy, the amount of ethanol that can be produced in a conventional enzyme/yeast bioconversion system. Because the rumen assay can be run in 24 hours without sterilizaton of the medium or use of aseptic technique, considerable savings in research costs (labor and materials) can be realized in a biomass screening program. 2. The second cycle of phenotypic selection of cultivar WS4U was completed in 2004 with the elimination of 90% of the nursery on the basis of poor vigor/low biomass, lodging, disease susceptibility, and low seed production. To develop varieties of switchgrass with optimum yield and biomass fermenatablility, hybrid crossing blocks (upland x lowland and lowland x upland) were successfully established at Prairie du Sac, WI. 3. Concentration of soluble leaf protein from alfalfa juice (prepared by wet-fractionation [R.G. Koegel]) could be increased to 8% (w/v) using a standard Romicon low-shear hollow fiber ultrafiltration device. Evaluation of an in-house-designed crossflow ultrafiltration system revealed that head pressure was the primary determinant of protein concentration. High rotor speed had a detrimental effect on permeate flux and increased protein damage, possibly due to higher shear rates. Lower shear rates (lower rotor speed) and lower temperatures are expected to improve recovery of protein. 4. In a specific cooperative agreement with the University of Wisconsin- Madison (CRIS project 3655-41000-004-01S), storage losses from corn stover have been quantified under a variety of conditions. Substantial benefits (reduced dry matter losses) have been demonstrated for storage under low moisture anaerobic conditions, or from storage of conventional bales wrapped with nylon netting (as opposed to plastic or sisal twine), particularly indoors. C. Significant activities that support special target populations: None. D. Progress Report: Residues remaining after fermentation of authentic cellulosic biomass materials by several strains of thermophilic anaerobic bacteria have been shown to serve as biologically derived adhesives that can replace a large fraction of the petroleum-derived phenol-formaldehyde resin used in plywood manufacture, without significant loss of adhesive properties. 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. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. The wood adhesive research was disseminated as the feature story in the July 20, 2004 edition of the News section of the ARS website: http://www.ars.usda.gov/is/pr/2004/040720.htm

Impacts
(N/A)

Publications

• Weimer, P.J., Conner, A.H., Lorenz, L.F. 2003. Fermentation residues from ruminococcus cellulose fermentations as components of wood adhesive formulations. Applied Microbiology and Biotechnology. 63:29-34.
• Weimer, P.J., Kroukamp, O., Joubert, L.M., Wolfaardt, G.M., Van Zyl, W.H. 2004. Characterization of the glycocalyx of cellulose-grown ruminococcus albus. [abstract]. 104th General Meeting of the American Society for Microbiology. B-428.
• Casler, M.D., Boe, A.R. 2003. Cultivar x environment interaction in switchgrass. Crop Science. 43(6):2226-2233.
• CASLER, M.D., VOGEL, K.P., TALIAFERRO, C.M., WYNIA, R.L. LATITUDINAL ADAPTATION OF SWITCHGRASS POPULATIONS. CROP SCIENCE. 2003. v. 44. pp 293- 303.
• Lamsal, B.P., Koegel, R.G., Boettcher, M.E. 2003. Separation of protein fractions in alfalfa juice: effects of some pretreatment methods. American Society of Agricultural Engineers. 46:715-720.

Progress 10/01/02 to 09/30/03

Outputs
1. What major problem or issue is being resolved and how are you resolving it? 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 its 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. How serious is the problem? Why does it matter? 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. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? National Program 306, Quality and Utilization of Agricultural Products (60%) and National Program 307, Bioenergy and Energy Alternatives (40%) This project addresses National Program 306 - Quality and Utilization of Agricultural Products component - New Processes, New Uses, and Value- Added Products, one objective of which is to "create technology leading to an expanded range of food and non-food products from commodities and undervalued byproducts of agriculture." This project also addresses National Program 307 - Bioenergy and Energy Alternatives component - Ethanol, two objectives of which are to develop processes for converting cellulosic biomass to ethanol, and to develop value-added co-products that enhance the economics of ethanol production. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2003: Biologically derived adhesive materials based on soy protein display poor stability when wetted and their use diverts soy from the protein market. In collaboration with scientists at the USDA Forest Service, Forest Products Laboratory (FPL), we examined the adhesive properties of the residues of biomass fermentations produced by bacteria that produce a sticky cell coating during fermentation. We showed that the fermentation residues produced by several Ruminococcus bacterial strains could partially replace phenol-formaldehyde resins (at up to 73% on a dry weight basis) as an adhesive for plywood construction; we also demonstrated adhesive properties of the fermentation residue of alfalfa fiber, a more industrially relevant substrate. These residues have potential as adhesive or adhesive extenders in wood construction applications. B. Other significant accomplishment(s), if any: 1.Our ability to predict the ability of a bacterium's extracellular coating to serve as a biologically derived adhesive is limited by a lack of knowledge of the chemical composition and structure of these coatings. We performed a partial chemical characterization of the residues from the fermentations of several bacteria whose cell coating can serve as an adhesive compound. These residues were shown to possess a novel polysaccharide composition and substantial amounts of protein that was remarkably similar across strains, suggesting that differences in adhesive properties are due to the yield of extracellular polymers produced by each strain. These results provide information necessary to unveil the means by which these compounds can serve as adhesive agents. 2.Prairie remnant populations of switchgrass from hardiness zones 3, 4, and 5 have not been characterized or evaluated for agronomic potential, limiting our understanding of the potential for using switchgrass as a biofuel crop in these hardiness zones. Fifty-seven populations were evaluated for several agronomic and biofuel traits. Phenotypic variability among prairie remnant populations is closely associated with both the USDA hardiness zone (defined by minimum cold temperatures) and ecoregion (defined by historic native vegetation) from which a population was collected. This is the first experimental and quantitative data to support the hypothesis that switchgrass populations have spatial structure at the landscape level, information that will be useful in identifying optimal germplasm for breeding, conservation, and production. 3. In a specific cooperative agreement with the University of Wisconsin- Madison (project 3655-41000-003-07S), harvesting of corn stover at higher moisture level (40% moisture) and subsequent storage in plastic film under non-ensiling conditions were shown to: economize field operations, increase the rate and efficiency of harvesting, and to reduce storage losses of dry matter to under 5%. C. Significant activities that support special target populations: none. D. Progress Report: Residues remaining after fermentation of cellulose by several strains of Ruminococcus bacteria have been shown to serve as biologically derived adhesives that can replace most of the petroleum-derived phenol- formaldehyde resin used in plywood manufacture, without significant loss of adhesive properties. 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 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 73% fermentation residue plus 27% 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 do you expect to accomplish, year by year, over the next 3 years? Year One: a) Screen large numbers of biomass materials from different growth environments for fermentability (collaboration with several ARS scientists). b) Isolate and characterize bacterial strains from nature that display enhanced production of ethanol and/or bioadhesive materials. c) Improve fermentations to enhance production of ethanol and bioadhesives from alfalfa fiber. d) Determine levels of Random Amplified Polymorphic DNA (RAPD) marker variability among and within prairie remnant collections and cultivars. e) Evaluate ultrafiltration for concentration of soluble protein from clarified plant juice. f) Determine effects of harvesting methods on yield and quality of corn stover, an abundant agricultural residue (via specific cooperative agreement with the University of Wisconsin-Madison). Year Two: a) Establish quantitative relationships between the fermentability of biomass materials and growth environment of different varieties of several biomass crop species. b) Determine optimum process conditions (e.g., time, temperature, pressure) for incorporation of fermentation residues into adhesive mixtures. c) Complete the compositional and structural analysis of the bioadhesive materials produced by fiber-digesting bacteria. d) Complete cycle 2 of selection for high biomass and disease resistance in switchgrass cultivar WS4U.e) Determine the effect of cutting height and maturity at harvest on the yield, quality, and persistence of perennial grasses (via specific cooperative agreement with the University of Wisconsin-Madison). Year Three: a) Integrate fermentability data and biomass production data into expert systems for predicting quality of biomass feedstocks (in collaboration with ARS scientists in research project 3655-31000-018-00D). b) Transfer bioadhesive technology to industry. c) Complete cycle 3 of selection for earliness and winterhardiness in switchgrass cultivar Kanlow. 7. 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 provisional U.S. patent application has been filed covering the use of cellulosic fermentation residues as bioadhesive materials.

Impacts
(N/A)

Publications

• Lynd, L.R., Weimer, P.J., Van Zyl, W.H., Pretorius, I.S. Microbial cellulose utilization: fundamentals and biotechnology. Microbiology and Molecular Biology Reviews. 2002. v. 66. p. 506-577.
• Fukushima, R.S., Weimer, P.J., Kunz, D.A. Photocatalytic interaction of resazurin N-oxide with cysteine optimizes preparation of anaerobic culture media. Anaerobe. 2002. v. 8. p. 29-34.
• Fukushima, R.S., Weimer, P.J., Kunz, D.A. Use of photocatalytic reduction to hasten preparation of culture media for saccharolytic Clostridium species. Brazilian Journal of Microbiology. 2002. v. 33. p. 1-5.
• Fukushima, R.S., Weimer, P.J., Kunz, D.A. Growth of Clostridium species in anaerobic media reduced by photocatalytic interaction of cysteine and RNO. American Society for Microbiology. 2002. Abstract p. 260-261.
• Lynd, L.R., Zhang, Y., Fan, Z., Desai, S., Tyurin, M., La Grange, D., Gundilapalli, S., Otero, R.C., Van Zyl, W., Pretorius, I.S., Weimer, P.J. The microbial cellulose utilization paradigm: fundamentals and implications for consolidated bioprocessing. Proceedings of 24th Symposium on Biotechnology of Fuels and Chemicals. 2002. Abstract p. 11.
• Stevenson, D.M., Weimer, P.J. Isolation and characterization of a Trichoderma strain capable of converting cellulose to ethanol. American Society for Microbiology. 2002. Abstract p. 350.

Progress 10/01/01 to 09/30/02

Outputs
1. What major problem or issue is being resolved and how are you resolving it? 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 its 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. How serious is the problem? Why does it matter? 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. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? This project addresses National Program 306 - Quality and Utilization of Agricultural Products, component - New Processes, New Uses, and Value- Added Products, one objective of which is to "create technology leading to an expanded range of food and non-food products from commodities and undervalued byproducts of agriculture. This project also addresses National Program 307 - Bioenergy and Energy Alternatives, component - Ethanol, two objectives of whjch are to develop processes for converting celluosic biomass to ethanol, and to develop value-added co-products that enhance the economics of ethanol production. 4. What was your most significant accomplishment this past year? A. Single Most Significant Accomplishment During FY2002: Wet fractionation technology was improved to permit separation of alfalfa herbage into a high-protein juice fraction and a low-value fiber fraction. Protein recovery by ultrafiltration or pH adjustment was accomplished, and the fiber fraction was successfully fermented to a residue having bioadhesive properties. B. Other Significant Accomplishment(s), if any: Alfalfa processing. A novel juice heater was developed that functions by direct passage of electrical current through the juice. Biological adhesives. In collaboration with Dr. Tony Conner, U.S. Forest Products Laboratory, we have shown that the residues from the fermentation of cellulose by the ruminal bacteria Ruminococcus albus and R. flavefaciens can serve as effective bioadhesives in aspen plywood in the presence of variable amounts of phenol-formaldehyde as a co-adhesive. Novel cellulolytic fungi. A collaboration with ARS postdoctoral research associate Dr. David Stevenson has resulted in the isolation of a novel strain of the fungus Trichoderma harzianum, that ferments cellulose to ethanol under non-growing conditions in the absence of oxygen and in a completely minimal medium. Moreover, mutant complementation studies reveal the presence of parasexuality in this strain, which should permit the facile development of a gene transformation system. Improved anaerobic culture methods. A collaboration with Drs. R.S. Fukushima and D.A. Kunz (both recent visiting scientists to the USDFRC) has resulted in the discovery of a novel means of accelerating the preparation of culture media for strictly anaerobic bacteria. The method exploits the photocatalytic reaction of the dye resazurin-N-oxide with cysteine to render pregassed culture media anaerobic media within minutes of cysteine addition under high light intensities, as opposed to hours at normal light intensities. C. Significant Accomplishments that Support Special Target Populations: none. D. Progress Report: An improved means of establishing strictly anaerobic conditions for cultivation of our fermentative cultures has been developed, and 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 collaboration with the U.S. Forest Products Laboratory has shown that 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 73% fermentation residue plus 27% phenol-formaldehyde resin. 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. 5. Describe your 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 have been shown to be fermentable to ethanol by Ruminococcus albus, and the fermentation residues (containing indigestible fiber, bacterial cells, and bacterial glycocalyx) have been shown to have adhesive properties whose improvement may permit their use as a plywood adhesive. 6. What do you expect to accomplish, year by year, over the next 3 years? Year 1. A) Complete larger scale fermentations of alfalfa fiber, determine mass balance of substrates and products, and recover sufficient material (several hundred grams) for testing of bioadhesive properties. B) Supply fermentation residues to collaborators at the U.S. Forest Products Laboratory (USDA-Forest Service) for testing of adhesive properties. C) Improve culture medium to minimize need for added nutrients. D) Evaluate ultrafiltration for concentration of soluble protein from clarified plant juice. Year 2. A) Improve ethanol yield and final product concentration using new isolates or adapted strains of anaerobic cellulolytic bacteria. B) Develop predictive equations to determine the optimum combination of processing conditions (time, temperature, pressure, residue composition) for using fermentation residues as bioadhesives in plywood panel construction. Year 3. A) Continue improvement of ethanol yield and product concentration. B) Expand range of biomass substrates to include other biomass crop residues, paper mill sludge, etc. C) Transfer technology for bioadhesive production to partners. 7. What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? Because at least one engineering firm has expressed interest in working with us in technology transfer, we anticipate that such transfer may occur soon after demonstration of utility and cost-effectiveness. Subsidies and credits for ethanol production from biomass are likely to hasten the entrance of successful technologies into the fuel marketplace. As is common for the entrance of any "green technology" into the marketplace, it is likely that producers of competitive petroleum-based products (e.g., adhesives) would aggressively protect their market.

Impacts
(N/A)

Publications

• Fukushima, R.S., Weimer, P.J., Kunz, D.A. Photocatalytic interaction of resazurin-N-oxide with cysteine optimizes preparation of anaerobic culture media. Anaerobe. 2002. v. 8. p. 29-34.
• Fukushima, R.S., Weimer, P.J., Kunz, D.A. Growth of Clostridium species in anaerobic media reduced by photocatalytic interaction of cysteine with resazurin-N-oxide. American Society for Microbiology. 2002. Abstract p. 260-261.
• Lynd, L.R., Zhang, Y., Fan, Z., Desai, S., Tyurin, M., La Grange, D., Gundilapalli, S., Otero, R.C., van Zyl, W., Pretorius, I.S., Weimer, P.J. The microbial cellulose utilization paradigm: fundamentals and implications for consolidated bioprocessing. 24th Symposium Fuels Chemicals. 2002. Abstract p. 11.
• Sreenath, H.K., Moldes, A.B., Koegel, R.G., Straub, R.J. Lactic acid production by simultaneous saccharification and fermentation of alfalfa fiber. Journal of Bioscience and Bioengineering. 2001. v. 92. p. 518-523.
• Stevenson, D.M., Weimer, P.J. Isolation and characterization of a Trichoderma strain capable of converting cellulose to ethanol. American Society for Microbiology. 2002. Abstract p. 350.

Progress 10/01/00 to 09/30/01

Outputs
1. What major problem or issue is being resolved and how are you resolving it? We are addressing the problems of nonrenewable resource depletion, air and water quality, and global competitiveness. Our approach is to develop new methods for processing alfalfa and other perennial legumes. Perennial legumes are a highly sustainable crop, having outstanding soil and water conservation characteristics, yet require low energy and fertilizer inputs and yield numerous useful products. Industrially valuable enzymes, specialt chemicals, and commodity chemicals are being created by genetic engineering by fractionation of plant herbage, and by fermentation of plant fiber. The enzyme phytase, produced in transgenic alfalfa, can save poultry and swine producers money while reducing phosphorus loading to the environment. Production of fuels by fermentation of plant-derived materials can reduce net greenhouse gas emissions and reduce our dependence on foreign petroleum 2. How serious is the problem? Why does it matter? Resource depletion, environmental quality, and global competitiveness are among the nation's most important problems. Maintaining our high standard of living and high quality of life requires finding satisfactory solutions to these problems. 3. How does it relate to the National Program(s) and National Component(s)? This work involves new uses, products, and materials including biofuels. It creates new uses and value-added products from agricultural commodities and crop residues via fractionation, and subsequent processing, of herbage into nontraditional products. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2001: Research was continued to create and evaluate a number of coproducts resulting from the fractionation of forage crops. These included food-grade protein concentrates, fiber board, biofilters, and adhesives (the latter two in collaboration with US Forest Products Laboratory). Alfalfa juice was clarified by centrifugation and the soluble protein in it concentrated by ultrafiltration. This could result in valuable food-grade protein being produced directly from the herbage of forage crops. B. Other Significant Accomplishment(s), if any: Three strains of Ruminococcus bacteria were shown to produce a glycocalyx compound potentially useful as a bioadhesive. The organisms were grown on cellulose and the resulting glycocalyx material was made available to Anthony Conner of the US Forest Products Laboratory for evaluation. The material was shown to be an effective adhesive for plywood. This bioadhesive could become a substitute or partial substitute for petroleum-based adhesives in engineered wood products.A novel continuous forage juice press with capabilities exceeding those of conventional presses was further developed and evaluated (see MS thesis listed under Scientific Publications). C. Significant Accomplishments/Activities that Support Special Target Populations: Nothing to report. 5. Describe the major accomplishments over the life of the project including their predicted or actual impact. The accomplishments are the same as those listed above. 6. What do you expect to accomplish, year by year, over the next 3 years? Year One: a) Improve equipment and technology for wet fractionation and for clarification of plant juice by removal of particulates). b) Develop analytical methods for characterizing bioadhesive materials produced by fiber-digesting bacteria. Year Two: a) Develop and evaluate methods for production of fiber-digesting enzymes on solid substrates. b) Evaluate ultrafiltration for concentration of soluble protein from clarified plant juice. c) Determine structure and properties of bioadhesives. d) Develop fermentation process for ethanol production from cellulose using Ruminococcus bacteria. Year Three: a) Determine functional properties of soluble protein from plant juice. b) Quantify production of lactic acid using enzymes produced on solid substrates. c) Quantify ethanol production from alfalfa fiber using Ruminococcus bacteria. 7. 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 & durability of the technology product? Technologies for wet fractionation of herbage were transferred under a 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. 8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)

Impacts
(N/A)

Publications

• Sreenath, H.K., Rosin, B.D., Koegel, R.G., Aktar, M. Solid substrate cultivation of Aspergillus japonicus NRRL 360 for xylanase production. Proceedings of 23rd Symposium on Biotechnology for Fuels and Chemicals. 2001. Section 3. p. 129-135.
• Sreenath,H.K., Koegel, R.G., Moldes, A.B., Jeffries, T.W., Straub, R.J. Lactic acid production from agricultural residues. Proceedings of Biotechnology 2000, 11th International Biotechnology Symposium and Exhibition. 2000. Section V.1. p. 139-144.
• Austin-Phillips, S., Koegel, R.G., Straub, R.J., Cook, M.E. Animal feed compositions containing phytase derived from transgenic alfalfa and methods of use thereof. 2001. U.S. Patent 6,248,938B1.
• Evans, E.J. A comparison and evaluation of three different forage material presses with a full-scale proposal of a field-going continuous press. M.S. Thesis. 2001. University of Wisconsin-Madison. 103 p.
• Sreenath, H.K., Koegel, R.G., Moldes, A.B., Jeffries, T.W., Straub, R.J. Ethanol production from alfalfa fiber fractions by saccharification and fermentation. Process Biochemistry. 2001. v. 36. p. 1199-1204.
• Sreenath, H.K., Koegel, R.G., Moldes, A.B., Straub, R.J. Lactic acid production from agriculture residues. Biotechnology Letters. 2001. v. 23(3). p. 179-184.

Progress 10/01/99 to 09/30/00

Outputs
1. What major problem or issue is being resolved and how are you resolving it? We are addressing the problems of nonrenewable resource depletion, air and water quality, and global competitiveness. Our approach is to develop new methods for processing alfalfa and other perennial legumes. Perennial legumes are a highly sustainable crop, having outstanding soil and water conservation characteristics, yet require low energy and fertilizer inputs and yield numerous useful products. Industrially valuable enzymes, special chemicals, and commodity chemicals are being created by genetic engineering by fractionation of plant herbage, and by fermentation of plant fiber. The enzyme phytase, produced in transgenic alfalfa, can save poultry and swine producers money while reducing phosphorus loading to the environment. Production of fuels by fermentation of plant-derived materials can reduce net greenhouse gas emissions and reduce our dependence on foreign petroleum. 2. How serious is the problem? Why does it matter? Resource depletion, environmental quality, and global competitiveness are among the nation's most important problems. Maintaining our high standard of living and high quality of life requires finding satisfactory solutions to these problems. 3. How does it relate to the National Program(s) and National Component(s)? This work involves new uses, products, and materials including biofuels. It creates new uses and value-added products from agricultural commodities and crop residues via fractionation, and subsequent processing, of herbage into nontraditional products. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2000 year: In order to carry out wet fractionation (juice expression) on-farm, less massive and more energy-efficient equipment is needed to eliminate the inconvenience and expense of hauling the harvested alfalfa to a separate site for fractionation. A one-quarter scale continuous rotary press with a converging chamber bounded by six slender conical rollers was constructed and evaluated. This press expressed more juice from the herbage and require less energy than conventional presses. This more efficient press could improve the profitability of on-farm wet fractionation. B. Other Significant Accomplishment(s), if any: Other technologies were developed to increase the efficiency or product value of wet fractionation. A rotating membrane filter was developed in the microfiltration range to clarify juice of plant chloroplasts, or in the ultrafiltration range to concentrate proteins from clarified juice. Plant fiber derived from wet fractionation, or recovered from cow manure, was evaluated as a construction material, as a bio-filter medium, and as a substrate for hydrolysis and fermentation to industrial chemicals (ethanol and lactic acid) or specialty chemicals (novel bioadhesives). Dairy production is enhanced under conditions that promote growth of the rumen bacterium Ruminococccus albus. A novel antimicrobial agent from cultures of this bacterial species was purified and characterized. The agent may be useful in controlling less desirable competitors in the cow's digestive tract, and during ethanol fermentation of waste agricultural materials. C. Significant Accomplishments/Activities that Support Special Target Populations: Nothing to report this fiscal year. D. Progress Report: Nothing to report this fiscal year. 5. Describe the major accomplishments over the life of the project including their predicted or actual impact. As this is the first year of the project, the accomplishments are the same as those listed above. 6. What do you expect to accomplish, year by year, over the next 3 years? Year One: a) Improve equipment and technology for wet fractionation and for clarification of plant juice by removal of particulates). b) Develop analytical methods for characterizing bioadhesive materials produced by fiber-digesting bacteria. Year Two: a) Develop and evaluate methods for production of fiber-digesting enzymes on solid substrates. b) Evaluate ultrafiltration for concentration of soluble protein from clarified plant juice. c) Determine structure and properties of bioadhesives. d) Develop fermentation process for ethanol production from cellulose using Ruminococcus bacteria. Year Three: a) Determine functional properties of soluble protein from plant juice. b) Quantify production of lactic acid using enzymes produced on solid substrates. c) Quantify ethanol production from alfalfa fiber using Ruminococcus bacteria. 7. 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 & durability of the technology product? Technologies for wet fractionation of herbage were transferred under a 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. 8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below) Anonymous. Alfalfa may produce bio-degradable plastic. Stockman Grass Farmer. January 2000.

Impacts
(N/A)

Publications

• Koegel, R. G., Straub, R.J., Boettcher, M. E. In-field wet fractionation of transgenic leguminous herbage. Paper 001040. Proceedings of the 93rd International ASAE Meeting. Milwaukee, WI. July 9-12, 2000.
• Chase, K. B., Koegel, R. G., Straub, R. J. Design and evaluation of a continuous press with a multiple conical roll chamber. Paper 001039. Proceedings of the 93rd International ASAE Meeting. Milwaukee, WI. July 9-12, 2000.
• Converse, J. C., Koegel, R. G., Straub, R. J. Nutrient separation of dairy manure. Paper 001039. Proceedings of the Eighth International Symposium on Animal Agricultural, and Food Processing Wastes. Des Moines, IA. Oct. 9-12, 2000.
• Chen, J., Weimer, P. J., Hatfield, R. D. Identification and purification of a bacteriocin from the ruminal cellulolytic bacterium Ruminococcus albus 7. Proceedings of the Annual Meeting of the Society of Industrial Microbiologists. San Diego, CA. July 23-27, 2000. p. 108.