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United States Department of Agriculture

Agricultural Research Service

Research Project: ADDING VALUE TO BIOFUELS PRODUCTION SYSTEMS BASED ON PERENNIAL FORAGES
2012 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:
Biomass yield, associated field traits, and biomass quality traits were measured on a total of nine existing field experiments of switchgrass. These experiments were designed to evaluate new and novel germplasm. Biomass quality data were generated on field experiments designed to evaluate numerous switchgrass cultivars. Proteomic analysis was carried out on less mature, more digestible apical alfalfa stems and more mature, less digestible basal alfalfa stems. Although differences between the two tissue types were not as great as expected, this cataloging of cell wall proteins should allow potential targets to be selected for modification in alfalfa to evaluate the impact on cell wall digestibility. Field experiments investigating the effect of nitrogen fertilizer rates and harvest times on switchgrass yields and soil nitrogen availability were evaluated over multiple years in Arlington and Marshfield, Wisconsin. Soil samples are now being processed for nitrogen analysis. Maximizing the capacity and subsequent efficiency of the forage harvester necessitates consolidation (raking or merging) of alfalfa cuttings. In this study, theoretical field capacity, headland time, leaf loss, and the efficacy of a tine pickup merger to incorporate ash (soil) into windrows was investigated by on-farm observation and through a controlled experiment. These data will serve as critical inputs to the economic analysis and models used to understand biomass production. Progress was made toward the development of bioconversion processes to produce hydrocarbon fuels and value-added co-products. This was based on a biomass fermentation to organic acids by mixed rumen bacteria in bioreactors, followed by electrolysis to produce alkanes and alkenes. Screening of enzymes provided by an industrial cooperator resulted in the identification of an enzyme preparation that augments biomass degradation and organic acid production by mixed cow rumen bacteria. One particular rumen bacterium proved to be a promising agent for conversion of lactic acid (an easily made product of other biomass fermentations) to organic acid precursors, particularly propionic and valeric acids. Pure cultures of ruminal bacteria grown in continuous culture at similar growth rates on cellulose or cellobiose revealed modest differences in expression of genes related to known proteins of the cellulosome organelle.


4.Accomplishments
1. Secrets of switchgrass evolution revealed. The choice of switchgrass as a national herbaceous model species for bioenergy feedstock development has led to an increase in the number of U.S. breeding programs (from two in 1995 to twelve in 2012). Because little was previously known about switchgrass evolution and diversity - knowledge that would greatly improve the effectiveness of switchgrass breeding programs - collaborative research between ARS researchers in Madison, Wisconsin and non-ARS colleagues set out to characterize genetic diversity in switchgrass across its native range in the U.S. These scientists demonstrated the existence of at least ten distinct types of switchgrass, ranging from highly drought-tolerant ecotypes of the Great Plains to highly heat-tolerant types of the Atlantic seaboard. This result should lead to improved biomass yield from switchgrass because it allows for the use of a wide range of genetic and geographically diverse switchgrasses in breeding programs, and it points to the distinct possibility of combining important biomass production traits such as late flowering, early nutrient recycling, and winter hardiness into these breeding programs, resulting in germplasm with increased biomass production efficiencies.

2. Natural hybrids reveal pathway to increased biomass yield and adaptation of switchgrass. While cellulosic biomass crops are receiving considerable research attention, economic and life-cycle analyses have uniformly indicated that low biomass yield is a major factor which limits adoption and deployment of new switchgrass cultivars. Collaborative research between ARS researchers in Madison, Wisconsin and non-ARS colleagues led to the discovery of the first documented natural hybrids between the two dominant ecotypes of switchgrass: upland and lowland ecotypes. Researchers showed that natural hybrids were created during the Ice Ages when upland and lowland ecotypes shared the same habitats. The scientists demonstrated that these hybrids are stable, able to survive under a wide range of conditions, and capable of sexual reproduction and seed production. This research forms the basis for broadening the breeding programs to utilize natural hybrids and/or to create new high-yielding and broadly adaptive hybrids between diverse ecotypes of switchgrass, thereby increasing biomass yield and adaptation across many geographical and climatic regions in the U.S.

3. Identification of cell wall proteins in an effort to modify plants for improved digestibility. Cell walls are important for the growth and development of all plants. They are also valuable resources for feed and fiber, and more recently as a potential feedstock for bioenergy production. Chemical interactions of cell wall components provide structure and rigidity to the wall, but restrict digestibility of the complex carbohydrates inside the cells, thus, limiting available energy in both animal and bioenergy production systems. One approach to improve cell wall digestibility might be manipulation of proteins present in the cell wall (known as the cell wall proteome). ARS researchers in Madison, Wisconsin conducted an analysis of the cell wall proteins present in two maturities of alfalfa stems: apical stems (less mature, more digestible) and basal stems (more mature, less digestible), using improved methods for protein extraction and identification. A total of 224 proteins were identified in the alfalfa stem cell wall proteome, about half of which had not previously been identified in cell wall proteomic analyses. The resulting data will be useful for scientists who study plant cell wall biology and should provide targets for the modification of plant cell walls to improve their digestibility and, thus, enhance their value in animal and bioenergy production systems.

4. On-farm pretreatment of biomass with sulfuric acid increases cellulosic conversion to ethanol. In the cellulosic ethanol production process, plant biomass needs to be pretreated prior to processing to decrease the complexity of cell wall structures that impede enzymatic conversion of cellulose into fermentable sugars. The advantages of this pretreatment process are:.
1)prevents excess mass losses during storage through inhibition of microbial activity, and.
2)helps to prepare the biomass for biochemical processing to biofuels. ARS research scientists in Madison, Wisconsin employed laboratory-scale experiments to assess:.
1)the optimal amounts of dilute acid (sulfuric acid) that could be used in the treatment process,.
2)rates of moisture that affect the treatment, and.
3)how time affects that process. These findings were then used to test two farm-scale methods to apply sulfuric acid to reed canarygrass and switchgrass at harvest. Acid-pretreated grasses produced 19 and 7 percentage units more ethanol after fermentation for reed canarygrass and switchgrass, respectively, than did the untreated grasses. Based on ethanol yields obtained with sulfuric acid treatment at the laboratory, pilot and farm scale, chemical costs for sulfuric acid applied at a rate of 50 grams per kilogram of dry matter were estimated to be as low as $4.72 per ton of dry matter. These findings are important to cellulosic biorefiners who are considering on-farm storage for preservation, pretreatment, and delivery of biomass. Acid pretreatment can be a cost-effective method of increasing ethanol production from cellulosic biomass.


Review Publications
Digman, M.F., Dien, B.S., Hatfield, R.D. 2012. On-farm acidification and anaerobic storage for preservation and improved conversion of switchgrass into ethanol. Biological Engineering (ASABE). 5(1):47-58.

Weimer, P.J., Stevenson, D.M. 2012. Isolation, characterization and quantification of Clostridium kluyveri from the bovine rumen. Applied Microbiology and Biotechnology. 94:461-466.

Shinners, T.J., Digman, M.F., Panuska, J.C. 2012. Overlap loss of manually and automatically guided mowers. Applied Engineering in Agriculture. 28(1):5-8.

Zhang, Y., Zalapa, J.E., Jakubowski, A.R., Price, D.L., Acharya, A., Wei, Y., Brummer, E.C., Kaeppler, S.M., Casler, M.D. 2011. Natural hybrids and gene flow between upland and lowland switchgrass. Crop Science. 51:2626-2641.

Zhang, Y., Zalapa, J.E., Jakubowski, A.R., Price, D.L., Acharya, A., Wei, Y., Brummer, E.C., Kaeppler, S.M., Casler, M.D. 2011. Post-glacial evolution of Panicum virgatum: centers of diversity and gene pools revealed by SSR markers and cpDNA sequences. Genetica. 139:933-948.

Suen, G., Stevenson, D.M., Bruce, D., Chertkov, O., Copeland, A., Cheng, J., Detter, C., Detter, J.C., Goodwin, L.A., Han, C.S., Hauser, L.J., Ivanova, N.N., Kyrpides, N.C., Land, M.L., Lapidus, A., Lucas, S., Ovchinnikova, G., Pitluck, S., Tapia, R., Woyke, T., Boyum, J., Mead, D., Weimer, P.J. 2011. Complete genome of the cellulolytic ruminal bacterium Ruminococcus albus 7. Journal of Bacteriology. 193:5574-5575.

Price, D., Salon, P., Casler, M.D. 2012. Big bluestem gene pools in the Central and Northeastern United States. Crop Science. 52:189-200.

Jakubowski, A., Casler, M.D., Johnson, R.C., Hu, J., Jackson, R. 2011. Genetic diversity and population structure of Eurasian populations of reed canarygrass: cytotypes, cultivars, and interspecific hybrids. Crop and Pasture Science. 62:982-991.

Casler, M.D., Tobias, C.M., Kaeppler, S.M., Buell, R., Wang, Z., Cao, P., Ronald, P. 2011. The switchgrass genome: tools and strategies. The Plant Genome. 4:273-282.

Last Modified: 8/27/2014
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