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ARS Home » Midwest Area » Madison, Wisconsin » U.S. Dairy Forage Research Center » Cell Wall Biology and Utilization Research » Research » Research Project #418601


Location: Cell Wall Biology and Utilization Research

2013 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:
This report summarizes progress for a four-year period (2010-2013). Biomass yield, associated field traits, and biomass quality traits were measured in nine existing field experiments of switchgrass, designed to evaluate new and novel germplasm as candidate cultivars. Proteomic analysis was conducted on over 200 cell wall proteins from less mature (more digestible) apical alfalfa stems and more mature (less digestible) basal alfalfa stems, but no candidates for down-regulation to improve digestibility were identified. Farm-scale pretreatment of corn stover was successfully demonstrated using both sulfuric acid and lime, although both agents required special handling that reduced their practicality. A spreadsheet model was developed and used to estimate the costs of production and fuel use for harvesting corn stover using conventional and experimental technology and storing stover on-farm with several storage options. The concept of “extraruminal” fermentation (i.e., fermentation of biomass materials by mixed ruminal bacteria in bioreactors, without sterilization of vessels or feedstock) was introduced and evaluated. Extraruminal fermentations were maintained through over 100 sequential transfers in the laboratory and were easily scaled to 300 liters (our limit of available reactor size). End-product yields from these extraruminal fermentations of various carbohydrate, protein, and nucleic acid components of biomass were determined. Prototype machinery was built that successfully separated alfalfa at harvest into leaf and stem fractions. Alfalfa leaves were readily fractionated by squeezing under pressure through a cone press to produce a solid fibrous residue that is readily utilized in extraruminal fermentations, and a green juice fraction that is rich in proteins and sugars. The sugars in the green juice were readily fermented over several days by lactic acid bacteria residing on the leaves or inoculated during harvest, but the fermentation time could be cut to 8 hours by inoculation with the ruminal bacterium, Streptococcus bovis, and incubating at 39 degrees C. The fermentations produced a brown juice that contained up to 46 grams of lactic acid/liter and an acidic pH that precipitated the leaf proteins for recovery as animal feed. Upon adjustment to pH 5.2 and inoculation with the bacterium Megasphaera elsdenii, lactic acid was converted to a mix of volatile fatty acids that can be converted to fuels and industrial chemicals. In collaboration with non-ARS colleagues, the first complete genome sequences were obtained for two of the most actively fiber-digesting ruminal bacteria. These sequences revealed fundamental differences in fiber-digesting strategies of these bacteria, and identified the genes for numerous novel enzymes for fiber digestion. In collaborative work with non-ARS scientists, analysis of current land use patterns and production inputs for animal agriculture revealed that large amounts of land could become available if Americans substituted chicken for beef in dietary scenarios aimed at improving human health.

4. Accomplishments
1. More protein is extractable from macerated leaves. Maceration of alfalfa leaves improves protein capture in a wet fractionation process designed to find higher-value uses for the crop. When working with grains such as soybeans, corn, or cereal crops, the farmer separates the value streams (e.g., grain and straw) for different uses. If alfalfa leaves were separated from the plant’s stem in a similar way, flexible uses for the crop could result, from customized leaf and stem blends in livestock feed rations to the opening of new markets for alfalfa's use in bioproducts and biofuels. ARS researchers at Madison, Wisconsin demonstrated a new three-step process to accomplish this: 1) mechanical leaf separation during harvest, 2) dewatering the leaves, and 3) fermentation of the resulting juice. To quantify the effectiveness of different methods of leaf dewatering, they studied five dewatering treatments: maceration and four different levels of screw-press back pressures. The scientists found that maceration did not affect the amount of water extracted, but did increase the protein concentration of the liquid press filtrate, or “green juice.” All moisture levels of recovered solids (press cake) were successfully ensiled for use as livestock feed. These results suggested that leaf stripping, combined with wet fractionation, can potentially provide an alternative method of alfalfa harvest and provide alternative uses for the valuable protein found in alfalfa leaves.

2. New fermentation process for alfalfa juice reduces time and inputs needed to obtain useful end products. “Green juice” produced by squeezing alfalfa leaves (part of a three-step process designed to find higher-value uses for alfalfa) is rich in proteins (useful as animal feed) and sugars (useful in biofuel production). However, it is difficult to recover either from the juice economically. One way to do this is to ferment the juice over a period of 3 to 21 days with bacteria already present in it; this produces a “brown juice” in which the sugars are converted to lactic acid. This acid fermentation precipitates the proteins from solution, facilitating their recovery. ARS researchers at Madison, Wisconsin have shown that the fermentation time can be shortened to as low as eight hours by inoculating the juice with Streptococcus bovis, a bacterium from the cow rumen. In addition, a secondary fermentation of the brown juice can be initiated by adjusting the pH above 5.2 and inoculating it with another ruminal bacterium, Megaspheara elsdenii. This converts the lactic acid to a mixture of volatile fatty acids (acetic, propionic, butyric, and valeric) at concentrations substantially higher than amounts previously obtained with an alternative method (carboxylate platform for biomass conversion using “stuck” anaerobic digestion). These volatile fatty acids can be recovered and converted to hydrocarbon and alcohol fuels using known chemical and electrochemical technologies. A major advantage of the fermentation is that it can be conducted without sterilization of the feedstock and without the addition of any other nutrients to the juice, providing a cost-effective way of producing value-added products from alfalfa.

3. Bacteria improves conversion of carbohydrates to fuel precursors. Megasphaera elsdenii is a bacterium from the cow rumen that is one of the few organisms known to convert carbohydrates and related compounds to five- and six-carbon volatile fatty acids which have potential value as fuel precursors. ARS researchers at Madison, Wisconsin demonstrated that M. elsdenii can ferment up to 19 grams of lactic acid per liter to produce 9 grams of mixed volatile fatty acids per liter. However, they also found that the bacterium ferments glucose poorly due to its ability to store up to 60% of the added sugars as glycogen. Alternative methods of fermentation of glucose can enhance the production of two volatile fatty acids (butyric and caproic), which are easily extracted and can be converted to hydrocarbons and alcohol fuels.

4. Dietary changes impact the amount of land available for growing biomass for biofuel production. Fuels produced from biomass can potentially supply a substantial portion of current U.S. motor fuels, but there are concerns about having enough farmland to grow biomass, or the impact on the food supply when land is used to grow biomass for biofuels. ARS researchers at Madison, Wisconsin participated in a collaboration led by non-ARS scientists at Dartmouth College to examine existing and potential patterns of human food and animal feed consumption to estimate land required to grow biomass for biofuel production. If food consumption followed recommended USDA nutrition guidelines, current land could produce enough biofuels to replace half the U.S. gasoline consumption without affecting food supply. If human protein consumption shifted from beef to poultry, which requires less land, about 60% of current gasoline use could be replaced by biofuels. This research provides consumers and policymakers with options for balancing food and fuel needs for U.S. citizens.

Review Publications
Verdonk, J.C., Hatfield, R.D., Sullivan, M.L. 2012. Proteomic analysis of cell walls of two developmental stages of alfalfa stems. Frontiers in Plant Science. DOI: 10.3389/fpls.2012.00279.

Weimer, P.J., Moen, G.N. 2013. Quantitative analysis of growth and volatile fatty acid production by the anaerobic ruminal bacterium Megasphaera elsdenii T81. Applied Microbiology and Biotechnology. 97(9):4075-4081.

Digman, M.F., Runge, T.M., Shinners, K.J., Hatfield, R.D. 2013. Wet fractionation for improved utilization of alfalfa leaves. Biological Engineering (ASABE). 6(1):29-42.

Digman, M.F., Shinners, K.J., Boettcher, M.E. 2013. Crop mergers: Management of soil contamination and leaf loss in alfalfa. Applied Engineering in Agriculture. 29(2):179-185.

Vadas, P.A., Digman, M.F. 2013. Production costs of potential corn stover harvest and storage systems. Biomass and Bioenergy. 54:133-139.

Weimer, P.J., Digman, M.F. 2013. Fermentation of alfalfa wet-fractionation liquids to volatile fatty acids by Streptococcus bovis and Megasphaera elsdenii. Bioresource Technology. 142:88-94.