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United States Department of Agriculture

Agricultural Research Service

Research Project: ADDING VALUE TO BIOFUELS PRODUCTION SYSTEMS BASED ON PERENNIAL FORAGES
2011 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.


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.


4.Accomplishments
1. Plant biomass can be converted to hydrocarbon fuels by combining biological and electrochemical processes. Consolidated bioprocessing (CBP) of biomass to ethanol using anaerobic bacteria has several advantages over conventional processing using enzymes and yeast. Both processes require pretreatment of feedstock and methods to control contamination. Neither process can convert non-carbohydrate components of biomass to fuels. ARS researchers in Madison, Wisconsin have previously shown that fermentation of biomass by bacteria from the cow rumen can convert almost all of the components of biomass to organic acids that can then be converted to hydrocarbon fuels by electrolysis. To extend the length of carbon chains in the fuel molecules, we isolated and characterized a novel strain of Clostridium kluyveri from the rumen that converts mixtures of ethanol and acetic acid (easily produced by both ruminal and non-ruminal CBP bacteria) to butyric and caproic acid, which can be electrochemically converted to hydrocarbon fuels. This technology can increase conversion of biomass feedstocks to longer-chain “drop-in” hydrocarbon fuels. Such technology would greatly increase the flexibility of biofuels for wider use.

2. Genomic sequencing of ruminal cellulolytic bacteria helps fine-tune biomass-hydrolyzing capabilities. Fibrobacter succinogenes and Ruminococcus albus are two of the most important fiber-degrading bacteria in the rumen and in rumen-derived CBP cultures for biomass conversion to biofuels. ARS researchers in Madison, Wisconsin, in collaboration with several cooperating institutions, determined and annotated the complete genome sequences of these two species. Parallel physiological experiments have allowed interpretation of the sequence in terms of the known biomass-hydrolyzing capabilities of these species. This research has identified new enzymes that can break down biomass, with the potential to increase the efficiency of biofuels production.

3. Corn stover harvest and storage systems can have economical payoffs. Corn stover has potential as a bioenergy feedstock in North America. ARS scientists in Madison, Wisconsin compared production costs for stover harvest and storage systems. The cheapest system was single-pass, whole-plant harvest with outdoor-wrapped bales. The next cheapest systems were:.
1)two-pass bale harvest and outdoor-wrapped bale storage,.
2)two-pass chop harvest and bag storage, and.
3)three-pass bale harvest and outdoor-wrapped bale storage. Three-pass chop harvest with silage bag storage, and single-pass ear-snap harvest with silage bag storage were the most expensive. The analysis suggests all harvest and storage systems have tradeoffs, and several systems can be economically viable. The results provide guidance to farmers seeking to maximize profits from corn stover production for biofuels use.


Review Publications
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.

Suen, G., Scott, J.J., Aylward, F.O., Adams, S.M., Tringe, S.G., Pinto-Tomas, A.A., Foster, C.E., Pauly, M., Weimer, P.J., Barry, K.W., Goodwin, L.A., Bouffard, P., Osterberger, J., Harkins, T.T., Slater, S.C., Donohue, T.J., Currie, C.R. 2010. An insect herbivore microbiome with high plant biomass-degrading capacity. PLoS Genetics. 6(9):e1001129.

Vogel, K.P., Dien, B.S., Jung, H.G., Casler, M.D., Masterson, S.D., Mitchell, R. 2011. Quantifying actual and theoretical ethanol yields for switchgrass strains using NIRS analyses. BioEnergy Research. 4(2):96-110. DOI: 10.1007/s12155-010-9104-4.

Weimer, P.J. 2011. End product yields from the extraruminal fermentation of various polysaccharide, protein and nucleic acid components of biofuels feedstocks. Bioresource Technology. 102:3254-3259.

Jakubowski, A., Casler, M.D., Jackson, R. 2010. Landscape composition and configuration predict the abundance of Phalaris arundinacea L. in Wisconsin wetlands. Wetlands. 30:685-692.

Jakubowksi, A., Casler, M.D., Jackson, R. 2010. The benefits of harvesting wetland invaders for cellulosic biofuel: an ecosystem services perspective. Restoration Ecology. 18:789-795.

Tahir, M., Casler, M.D., Moore, K.J., Brummer, E. 2010. Biomass yield and quality of reed canarygrass under five harvest management systems for bioenergy production. BioEnergy Research. 4:111-119.

Zalapa, J., Price, D., Kaeppler, S., Tobias, C.M., Okada, M., Casler, M.D. 2010. Hierarchical classification of switchgrass using SSR and chloroplast sequences: ecotypes, ploidies, gene pools, and cultivars. Theoretical and Applied Genetics. 122:805-817.

Costich, D., Friebe, B., Sheehan, M.J., Casler, M.D., Buckler IV, E.S. 2010. Genome-size variation in switchgrass (Panicum virgatum): flow cytometry and cytology reveal rampant aneuploidy. The Plant Genome. 3:130-141.

Last Modified: 8/27/2014
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