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United States Department of Agriculture

Agricultural Research Service

Research Project: Advanced Conversion Technologies for Sugars and Biofuels: Superior Feedstocks, Pretreatments, Inhibitor Removal, and Enzymes

Location: Bioenergy Research Unit

2010 Annual Report

1a.Objectives (from AD-416)
The long-term goal of this project is to develop lignocellulosic materials as a feedstock for producing sugars and biofuels. The research will focus more specifically on the following objectives: Objective 1: Develop commercially-viable analytical tools that producers and biorefiners of lignocellulosic feedstocks can use to evaluate the quality of harvested biomass for enzymatic and fermentative conversion to ethanol and butanol and plant breeders can use to select superior cultivars for biorefining. Objective 2: Develop new, commercially-viable enzyme and/or protein systems that increase the efficiency of lignocellulosic saccharification. Objective 3: Identify components in lignocellulosic hydrolysates which reduce saccharification or fermentation efficiencies and develop commercially-viable mitigation strategies. Objective 4: Develop new technologies that enable commercially-viable pretreatment processes for lignocellulosic biomass, inhibitor abatement strategies, and enzyme preparations which are optimized for saccharifying particular lignocellulosic feedstocks.

1b.Approach (from AD-416)
Renewable biofuels have the potential to reduce U.S. dependence on imported oil, lower greenhouse gas emissions, and to further develop the rural economy. Renewable fuels produced from lignocelluloses should be able to replace up to 30% of the U.S. oil consumption. While technologically proven, commercializing lignocellulosic biofuels is stymied by prohibitively high processing and capital costs. There are opportunities to reduce expenses by developing higher-quality feedstocks, more active enzymes, better managing side-products, and using faster/higher yielding biocatalysts. Our research targets improvements in each of these areas. Crop quality for bioprocessing will be improved by collaborating with plant scientists to breed bioenergy cultivars, including reduced lignin mutants that are more amenable to processing, without sacrificing production yield. The enzymes used are too expensive because their specific activities are too low. Activities can be improved by creating balanced mixtures from blending individual enzymes selected for superior kinetics and by discovery of auxiliary proteins that aid cellulase binding to cellulose. Processing of biomass prior to fermentation releases a wide range of biologically active byproducts that can retard or stall fermentation and frustrate water recycling schemes. Biological abatement has the promise to selectively remove complex organic compounds without generating further waste. It is expected that when combined with an advanced pretreatment and engineered microbes, the final result will be a significantly improved process for converting lignocelluloses into sugars and renewable fuels.

3.Progress Report
The overall goals of this project are to develop technologies for lowering the cost and increasing the efficiency for converting biomass to biofuels and chemicals. The specific areas targeted are developing higher-quality feedstocks, more active enzymes, and better managing undesirable side-products present in processing streams. We have made substantial progress in each of these areas. Switchgrass is being bred to increase yields of biofuels. It has been demonstrated that conversion quality differs for the plant stems and leaves and stems are harder to convert. Furthermore, high stem internodes (e.g., occurring further from the ground) are easier to convert than lower stem portions. This research will allow for better quality switchgrass cultivars to be bred quicker by focusing on stem, rather than whole plant. It also recommends a new harvest strategy that favors collection of the leaves and upper stem portions. We are working to develop enhanced enzyme mixtures for breaking down the xylan portion of the carbohydrates; xylan is 30-40% of the available material for conversion into biofuels. Past efforts to develop better enzyme mixtures have been slowed by the lack of knowledge regarding xylan structure or specifically the side-chain units. A novel method was developed to determine the structure of these side-chains. This method should allow for direct analysis of xylan structure from switchgrass and this knowledge should directly lead to better enzyme formulations. We are also working to improve the efficiencies of the individual enzymes within this mixture. One of the most significant enzymes within this mixture is beta-xylosidase. Earlier, a beta-xylosidase was discovered that is 10x more active than any other reported example of this enzyme. However, this enzyme loses activity in more concentrated sugar solutions. We have improved the enzyme by lowering the delirious effect of concentrated sugar concentrations on enzyme activity. We have also begun to develop a novel suite of enzymes for degrading pectin, the third most common plant carbohydrate. We have selected several enzymes as targets for further development based upon their commercially favorable kinetic properties. The eventual impact of all these efforts will be to lower the cost of producing biofuels from biomass by making the enzymes less expensive. In a separate effort, we are also seeking to improve the quality of pretreated biomass hydrolysates by selectively removing chemicals, often side-products of pretreatment, which inhibit conversion of the hydrolysate into biofuels. We have recently patented a novel biocatalyst that removes these chemicals, but it also removes some of the sugars. This biocatalyst has been improved by greatly slowing its use of xylose. This improved biocatalyst allows for the conversion of biomass hydrolysates to ethanol at higher yields than the original biocatalyst.

1. Engineering alfalfa plants for more efficient conversion of sugars and fuels. Alfalfa is being actively researched as a perennial bioenergy crop whereby the leaves would be used as a valuable animal feed product and the stems converted into ethanol. A collaboration of Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research in Peoria, IL, and St. Paul, MN, commodity groups, and seed companies, found that lignin modified mutants of alfalfa currently in commercial development are superior for biochemical conversion into sugars and ethanol. This demonstrates for the first time in any plant that lignin composition can be molecularly engineered for increased biomass conversion quality without impacting production yield in a commercial farm setting.

2. Use of a fungus to remediate fermentation inhibitors from cellulosic sugar streams. The sugars that make up biomass can be converted to fuels or other products by microbes. One of the technical hurdles to biofuels production is the fact that the sugar mixtures obtained from biomass also contain compounds that are inhibitory to microbes. Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research in Peoria, IL, have developed a biological abatement method, using a novel fungus strain, and demonstrated its efficiency for conversion of corn stover into ethanol. We have further demonstrated that this method is broadly effective for preparing pretreated herbaceous biomass for ethanol fermentation, including alfalfa, reed canary grass, and switchgrass and is more effective than the traditional method. It is envisioned that this new treatment will lead to greater process efficiencies and reduce technical constraints on the biochemical conversion of biomass into biofuels.

3. Better enzymes are needed to lower the cost of processing biomass into sugars. The enzyme beta-D-xylosidase originating from the bacterium Selenomonas ruminantium is the most proficient for forming xylose from biomass; xylose can be used for fermentation to bioethanol and other bioproducts. High sugar concentrations impair the performance of the enzymes. Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research in Peoria, IL, developed mutated versions of this enzyme that were less affected by high sugar concentrations. The most promising of these demonstrated 3-fold more tolerance to sugars, thereby, allowing their use for producing more concentrated sugar solutions from biomass. The expected impact is to lower enzyme costs and thereby improving the profitability of producing biofuels.

Review Publications
Rosenbaum, M., Cotta, M.A., Angenent, L.T. 2010. Aerated Shewanella oneidensis in Continuously-fed Bioelectrochemical Systems for Power and Hydrogen Production. Biotechnology and Bioengineering. 105(5):880-888.

Fornero, J.J., Rosenbaum, M., Cotta, M.A., Angenent, L.T. 2010. Carbon Dioxide Addition to Microbial Fuel Cell Cathodes Maintains Sustainable Catholyte pH and Improves Anolyte pH, Alkalinity, and Conductivity. Environmental Science and Technology. 44(7):2728-2734.

Arora, A., Dien, B.S., Belyea, R.L., Singh, V., Tumbleson, M.E., Rausch, K.D. 2010. Nutrient Recovery from the Dry Grind Process Using Sequential Micro and Ultrafiltration of Thin Stillage. Bioresource Technology. 101(11):3859-3863.

Nichols, N.N., Dien, B.S., Cotta, M.A. 2010. Fermentation of Bioenergy Crops Into Ethanol Using Biological Abatement for Removal of Inhibitors. Bioresource Technology. 101(19):7545-7550.

Dien, B.S., Sarath, G., Pedersen, J.F., Sattler, S.E., Chen, H., Funnell-Harris, D.L., Nichols, N.N., Cotta, M.A. 2009. Improved Sugar Conversion and Ethanol Yield for Forage Sorghum (Sorghum bicolor L. Moench) Lines with Reduced Lignin Contents. BioEnergy Research. 2(3):153-164.

Hughes, S.R., Sterner, D.E., Bischoff, K.M., Hector, R.E., Dowd, P.F., Qureshi, N., Bang, S.S., Grynavyski, N., Chakrabarty, T., Johnson, E.T., Dien, B.S., Mertens, J.A., Caughey, R.J., Liu, S., Butt, T.R., Labaer, J., Cotta, M.A., Rich, J.O. 2009. Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system. Plasmid Journal. 61(1):22-38.

Jordan, D.B., Braker, J.D. 2010. Beta-D-xylosidase from Selenomonas ruminantium: Role of Glutamate 186 in Catalysis Revealed by Site-directed Mutagenesis, Alternate Substrates, and Active-site Inhibitor. Applied Biochemistry and Biotechnology. 161(1-8):395-410.

Zhu, J.Y., Zhu, W., O Bryan, P.J., Dien, B.S., Tian, S., Gleisner, R., Pan, X.J. 2010. Ethanol Production from SPORL-Pretreated Lodgepole Pine: Preliminary Evaluation of Mass Balance and Process Energy Efficiency. Applied Microbiology and Biotechnology. 86(5):1355-1365.

Nichols, N.N., Dien, B.S., Lopez, M.J., Moreno, J. 2010. Use of Coniochaeta ligniaria to Detoxify Fermentation Inhibitors Present in Cellulosic Sugar Streams. In: Hou, C.T., Shaw, J.-F., editors. Biocatalysis and Molecular Engineering. New York: John Wiley and Sons. p. 253-263.

Bowman, M.J., Jordan, D.B., Vermillion, K., Braker, J.D., Moon, J., Liu, Z. 2010. Stereochemistry of Furfural Reduction by a Saccharomyces cerevisiae Aldehyde Reductase That Contributes to In Situ Furfural Detoxification. Applied and Environmental Microbiology. 76(15):4926-4932.

Last Modified: 4/20/2014
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