1a.Objectives (from AD-416)
1. Develop harvesting, fractionation and storage processes for forages and bioenergy crops that are economical, and that retain product quality. 2. Identify specific varieties of energy crops that display maximum fermentability when grown at specific locations under defined environmental conditions. 3. Develop switchgrass germplasm having broad adaptation to the northern USA and improved fermentability for conversion to value-added products. 4. Develop and improve fermentations for direct bioconversion of cellulosic biomass to value-added products (viz., ethanol, chemical feedstocks and novel bioadhesive components).

1b.Approach (from AD-416)
New harvesting strategies will be developed that economically separate forages and bioenergy crops into higher and lower-value fractions. An in vitro ruminal fermentation assay will be used to rapidly screen large numbers of biomass samples from several bioenergy crop species, provided by ARS agronomists from throughout the U.S. The data will be correlated to ethanol bioconversion capability, and NIRS calibration equations will be developed for ruminal fermentability and ethanol production. Switchgrass germplasm improvement will be carried out by recurrent phenotypic selection for vigor, lodging and disease resistance to extend adaptation and biomass yield in several eco-regions. Switchgrass hybrids will be selected for enhanced biomass yield and fermentability. Consolidated bioprocessing of bioenergy crops, using anaerobic bacteria that produce their own cellulolytic enzymes and ferment the products to ethanol and other valuable products, will be improved through optimization of strains and culture conditions. Value-added co-products, such as adhesives produced by the fermentative bacteria, will be identified and their utility will be determined.

4.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.

5.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.

6.Technology Transfer

Number of non-peer reviewed presentations and proceedings	6
Number of newspaper articles and other presentations for non-science audiences	9

Review 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.