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.
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
Harvesting was completed on existing fields of switchgrass designed to evaluate new and novel germplasm as candidate cultivars. New fields were planted to initiate marker-selection protocols designed to improve the efficiency of selection. Several methods to extract proteins from cell walls of alfalfa cell cultures and alfalfa stems were developed and used to prepare protein samples for use in proteomics analyses. It is expected that comparative proteomics analyses (e.g., between younger, highly digestible stems and older less digestible stems) to be carried out in the near future will identify proteins involved in cell wall cross-linking that can be targeted for modification to improve the bioconversion potential of plant biomass. Progress was also made to improve sustainability and profitability of biomass production. A spreadsheet model was developed to estimate production costs and fuel use for harvesting corn stover, using conventional and experimental technology and storing stover on-farm with several storage options. In a field experiment investigating the effect of nitrogen fertilizer rates and harvest times on switchgrass yields, the first-year harvest cycle and soil sampling were completed for field plots in Arlington and Marshfield, WI; in the second year, fertilizer treatments were applied. Soil samples are now being analyzed for nitrogen. Progress was made toward the development of new bioconversion processes to produce hydrocarbon fuels and value-added co-products. A new strain of the bacterium, Clostridium kluyveri, was isolated from the cow rumen and characterized for conversion of mixed fermentation broths containing ethanol and acetic acid to butyric and caproic acids. We previously demonstrated that these acids can be electrolytically converted to fuel hydrocarbons. In collaboration with scientists from other institutions, the genomes of two important ruminal biomass-fermenting bacteria have been sequenced for enzyme discovery research. To add value to the fermentation component of the process, more experiments were conducted to develop the fermentation residues (remaining biomass plus microbial cells and their sticky extracellular products) as bio-based adhesives. Production of chemical modifiers for these fermentation residues was initiated, as an approach to improve the adhesive properties of the residues. These proprietary modifiers are designed to simulate monolignols in structure and reactivity and, hence, encourage cross-linking of fermentation residues to wood via either free radical and/or ionic initiators. The cross-linking approach involves generation of novel organic compounds capable of undergoing either free radical or ionic reactions with existing functionality present on the fermentation residues on the particle surface. The criteria of these cross-linking agents for improved bio-adhesive production include: 1) facile, low-cost synthesis; 2) reliable sourcing of synthetic precursors; 3) low volatility; and 4) nontoxicity of precursors or products. To date, three of these novel cross-linking agents have been synthesized and are awaiting further tests.
Suen, G., Weimer, P.J., Stevenson, D.M., Aylward, F.O., Boyum, J., Deneke, J., Drinkwater, C., Ivanova, N., Mikhailova, N., Chertkov, O., Goodwin, L.A., Currie, C.R., Mead, D., Brumm, P.J. 2011. The complete genome sequence of Fibrobacter succinogenes S85 reveals a cellulolytic and metabolic specialist. PLoS One. 6(4):e18814.