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United States Department of Agriculture

Agricultural Research Service

2010 Annual Report

1a.Objectives (from AD-416)
1) Develop new germplasm of perennial forage species that display increased yield and bioconversion potential..
2)Develop new commercially viable technologies for harvest, storage and/or on-farm pretreatment and biorefining of perennial bioenergy crops, and use modeling to assess the economic and environmental impacts of integrating these new technologies into sustainable farming systems..
3)Develop technologies based on mixed culture ruminal fermentation that enable commercially viable processes for producing hydrocarbon and alcohol fuels from lignocellulosic biomass via volatile fatty acid intermediates..
4)Develop technologies to enable commercially viable consolidated bioprocessing (CBP) of lignocellulosic biomass to fuel ethanol and adhesive co-products.

1b.Approach (from AD-416)
1) Use conventional breeding methods and molecular analytical tools to develop and characterize new varieties of switchgrass adapted to growth in the northern United States..
2)Develop equipment and technology for harvesting perennial grasses and alfalfas at reduced cost or producing fractions having higher value and different end uses (e.g., stem fraction as biofuels feedstock and leaf fraction as animal feed). Evaluate practicality and economics of on-farm biomass pretreatment with acid, lime, ozone, and/or other reagents. Evaluate economics and environmental impact of biofuels and biogas production systems and assess opportunities for integration into dairy farming systems..
3)Modify cultivation methods and use selective pressure to improve mixed culture fermentations for converting cellulosic biomass to volatile fatty acids (VFA) mixtures. Economically prepare fermentation broths for further processing. Demonstrate and improve electrolytic conversion of VFA to hydrocarbons in aqueous systems using Kolbe and Hofer-Moest reactions..
4)Identify secondary plant cell wall structural factors that limit plant cell wall biodegradation. Improve fermentation of plant cell wall materials to ethanol and adhesive-containing fermentation residue. Improve bacterial strains and culture media to increase yield of adhesive material, and improve adhesive properties through further chemical modification.

3.Progress Report
Progress was made toward the development of new biofuels feedstock germplasm. Harvesting was completed on existing field experiments of switchgrass, designed to evaluate new and novel germplasm as candidate cultivars. New field experiments were planted to initiate marker-selection protocols designed to improve the efficiency of selection. Feasibility was demonstrated for using mixed ruminal bacteria in a consolidated bioprocessing platform in which bacteria produce their own biomass-degrading enzymes and convert the resulting sugars to energy-dense products. The mixed cultures are able to convert a wide variety of biomass materials to mixtures of volatile fatty acids (VFA) for subsequent conversion to alkanes and alkenes by electrolysis. VFA yields were determined for the fermentation of a wide variety of polysaccharides, protein preparations and nucleic acid components. Successful electrolytic conversion of VFA-containing fermentation broths was demonstrated using low voltages and inexpensive graphite electrodes. A U.S. patent application was filed for the combined fermentation/electrochemical process and its components. To add value to the fermentation component of the process, additional experiments were conducted to develop the fermentation residues (remaining biomass plus microbial cells and their sticky extracellular products) as bio-based adhesives. Fermentation residues were produced at approximately 1-kg scale from alfalfa stems and from eastern gamagrass. Production of chemical modifiers for these fermentation residues was initiated. These proprietary modifiers are designed to simulate monolignols in structure and reactivity and, hence, encourage cross-linking to fermentation residues via either free radical and/or ionic initiators. A spreadsheet model was developed to estimate fuel use for farm-scale field operations, including tillage, planting, spraying, fertilizing, and harvest. The model will be combined with machinery and materials cost estimates to assess farm-scale economic and energy balances for next-generation bioenergy feedstocks. A field experiment was initiated to investigate the effect of nitrogen fertilizer rates and harvest times on potential use of switchgrass as a bioenergy feedstock. Field plots were established in Arlington and Marshfield, WI, and the first-year fertilizer treatments were applied. An analysis was conducted to estimate the potential for beef pasture intensification to increase land available for bioenergy feedstock production. The analysis showed that intensifying pastures so they produce the same yield as average reported hay yields could make 125 million ha of pastureland available.

1. New bioenergy crop germplasm. In collaboration with the University of Georgia, the Great Lakes Bioenergy Research Center, the Bioenergy Sciences Center, and China Agricultural University, ARS scientists identified the following five distinct lineages of switchgrass: Gulf Coast lowland, Southern Plains lowland, Southern Plains upland, Central Plains upland, and Eastern upland. All switchgrass populations are derived from three glacial refugia that served as tallgrass prairie reserves during the Pleistocene period: a dryland refuge in the southwestern U.S. and Mexico, a western Gulf Coast refuge of the Coastal Plain, and a lowland plains refuge along the northern and eastern Gulf Coast. The two eastern refugia served as sources of lowland plants that are adapted to warmer climates and wetter soils. The dryland refuge is the only source of upland plants that have adapted to northern climates and dryer soils. The two Gulf Coast refugia are also sources of a low frequency of remnant hybrids between upland and lowland ecotypes, probably originating hundreds of generations ago, before flowering time of upland and lowland ecotypes had significantly diverged. Repeated continental glaciation of North America, leading to repeated north-south migrations of the tallgrass prairie and savanna ecosystems, has resulted in the storage of massive amounts of genetic variability within localized switchgrass populations, leading to a wide array of morphologies and stress tolerances. These results will be useful to anyone who conducts breeding, agronomic, genetics, and ecological research on switchgrass, as well as to people involved in restoration and conservation of the tallgrass prairie and oak-savanna or pine-barrens ecosystems.

2. Biological/Electrochemical conversion of biomass to hydrocarbons fuels. Conversion of cellulosic biomass to ethanol in a single reactor (consolidated bioprocessing [CBP]) has numerous advantages over simultaneous saccharification and fermentation (SSF), but both processes share requirements for substrate pretreatment, contamination control, and an inability to convert noncarbohydrate components. We have combined the in vitro fermentation of biomass materials to volatile fatty acids (VFA) by mixed ruminal bacteria with a subsequent electrolysis of the VFA to produce mixtures of hydrocarbons useful as fuels, along with hydrogen gas. The fermentation can be performed on ground biomass without additional pretreatment and without sterilization of the biomass or the culture medium. The electrolysis can be conducted at low voltages with inexpensive graphite electrodes. A U.S. patent application has been filed, and commercial partners are being sought to assist with scale-up and development. Successful development of this technology will produce hydrocarbon fuels and valuable co-products from mixed biomass feedstocks with minimal preprocessing.

3. High throughput screening of bio-based adhesives. A prototype high-throughput (HTP) screening apparatus for rapid testing of adhesive properties of chemically modified fermentation residues has been designed and constructed. This apparatus requires only a fraction of adhesive material (less than 1 g) compared to normal plywood testing protocols which usually require approximately 10 g of material. This apparatus will allow for rapid screening of adhesive properties of trial formulations in-house. The reduced quantity of formulated material required using this apparatus enables us to focus on production of a larger selection of trial formulations due to reduced requirements of adhesive additives that must be synthesized. The HTP device will expand our capabilities to test our proprietary bio-based adhesives in a wider variety of practical end uses.

Review Publications
Pinto-Tomas, A.A., Anderson, M.A., Suen, G., Stevenson, D.M., Chu, F.S., Cleland, W.W., Weimer, P.J., Currie, C.R. 2009. Symbiotic Nitrogen Fixation in the Fungus Gardens of Leaf-Cutter Ants. Science. 326:1120-1123.

Casler, M.D., Cherney, J.H., Brummer, C.E. 2009. Biomass Yield of Naturalized Populations and Cultivars of Reed Canarygrass. BioEnergy Research. 2:165-173.

Shuai, L., Yang, Q., Zhu, J.Y., Lu, F.C., Weimer, P.J., Ralph, J., Pan, X.J. 2010. Comparative Study of SPORL and Dilute Acid Pretreatments of Spruce for Cellulosic Ethanol Production. Bioresource Technology. 101:3106-3114.

Casler, M.D. 2010. Changes in Mean and Genetic Variance During Two Cycles of Within-family Selection in Switchgrass. BioEnergy Research. 3:47-54.

Shinners, K.J., Boettcher, G.C., Muck, R.E., Weimer, P.J., Casler, M.D. 2010. Harvesting and Storing of Two Perennial Grasses as Biomass Feedstocks. Transactions of the ASABE. 53(2):359-370.

Last Modified: 4/23/2014
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