2013 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.
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. Agricultural Research Service (ARS), Bioenergy Research Unit (BER) scientists have made substantial progress during FY13 in each of these areas. One way to improve ethanol yields is to develop better quality bioenergy cultivars. Switchgrass has been screened for the affects of ecotype and harvest maturity on conversion yield. It was observed that if harvested at post-frost, the higher biomass switchgrass was superior in conversion quality compared to the low yielding ecotype. Another cost-sensitive step is enzymatic saccharification of biomass into fermentable monosaccharides. We are seeking to develop better enzymes for saccharification of xylan and pectin. We continued work on generating and characterizing genetically engineered thermostable ß-xylosidases. We corrected the literature on several instances of incorrect data and interpretations of ß-xylosidase mechanism of action. We followed up on divalent metal requirements of ß-xylosidases by adding more thorough biochemical studies. We prepared for further studies on xylan decomposition. The most active exo- and endo-polygalacturonase enzymes from Rhizopus oryzae, along with pectin methyl esterase and cellulase enzymes, were evaluated to determine the impact of polygalacturonase enzymes on the enzymatic hydrolysis of hot-water pretreated alfalfa biomass. Multiple experiments using different concentrations of polygalacturonase and cellulase enzymes demonstrated that the polygalacturonase enzymes had little to no impact on the rate or final concentrations of simple sugars obtained from pretreated alfalfa biomass. This finding contrasts with previous work using acid-hydrolyzed corn stover that suggested addition of a commercially available pectinase formulation improved enzymatic hydrolysis. Finally, we have continued our research on developing a biological abatement step for conditioning hydrolysates for fermentation. This method is based upon a unique fungus, which was first isolated by BER scientists. The fungus is unique for its ability to remove inhibitory chemicals formed during pretreatment that if left to persist would block fermentation. While the fungus removes inhibitors first, if incubated for prolonged periods, the fungus also metabolizes glucose. As a first step to develop a strain that is unable to metabolize glucose, we are isolating the genes involved in glucose metabolism. We have determined that multiple genes are involved and are now proceeding to sequence the genome to identify all of the relevant genes. The same inhibitors that impede fermentation have recently been discovered to inhibit cellulases. We have teamed up with researchers at Purdue University to demonstrate that biological removal of inhibitors increased cellulose conversion 1.2 -1.5 fold.
Better quality switchgrass. Switchgrass is a perennial grass native to the Midwest prairies that is being developed by ARS as a bioenergy crop because of its high biomass yields. Two ecotypes, high and lowland varieties, exist for switchgrass. Upland is preferred for forage applications because it is more digestible by ruminant livestock (e.g. cattle) but lowland gives higher biomass yields. Agricultural Research Service (ARS) scientists in the Bioenergy Research Unit at the National Center for Agricultural Utilization Research, Peoria, Illinois, determined that when harvested post-frost, lowland was as easy or easier to convert to sugars and ethanol than upland switchgrass. This result suggests that lowland varieties are the preferred ecotypes for bioenergy crop development based upon both plant yield and conversion quality, important determinants of overall process economics.
Biological abatement of cellulase inhibitors. Side-products produced when preparing cellulosic biomass for enzymatic hydrolysis inhibit the very enzymes used for production of sugars. Agricultural Research Service (ARS) scientists in the Bioenergy Research Unit at the National Center for Agricultural Utilization Research, Peoria, Illinois, previously developed biological abatement as a waste-free method to improve fermentation efficiency by removing chemicals that interfere with fermentation. Now, these ARS researchers also showed that bioabatement improves the activity of enzyme in biomass hydrolysates. In work with academic collaborators (Purdue University, West Lafayette, Indiana), biological conditioning improved conversion of cellulose by 20-50%. This should directly benefit facilities producing renewable fuels and chemicals by reducing the cost of enzyme needed in these processes.
Agler, M.T., Werner, J.J., Iten, L.B., Dekker, A., Cotta, M.A., Dien, B.S., Angenent, L.T. 2012. Shaping reactor microbiomes to produce the fuel precursor n-butyrate from pretreated cellulosic hydrolysates. Environmental Science and Technology. 46(18):10229-10238.
Bowman, M.J., Dien, B.S., O'Bryan, P.J., Sarath, G., Cotta, M.A. 2012. Comparative analysis of end point enzymatic digests of arabino-xylan isolated from switchgrass (Panicum virgatum L) of varying maturities using LC-MS(n). Metabolites. 2(4):959-982.
Khullar, E., Dien, B.S., Rausch, K.D., Tumbleson, M.E., Singh, V. 2013. Effect of particle size on enzymatic hydrolysis of pretreated Miscanthus. Industrial Crops and Products. 44:11-17.
Dunlap, C.A., Bowman, M.J., Schisler, D.A. 2013. Genomic analysis and secondary metabolite production in Bacillus amyloliquefaciens AS 43.3: A biocontrol antagonist of Fusarium head blight. Biocontrol. 64(1):166-175.
Jordan, D.B., Lee, C.C., Wagschal, K., Braker, J.D. 2013. Activation of a GH43 ß-xylosidase by divalent metal cations: Slow binding of divalent metal and high substrate specificity. Archives of Biochemistry and Biophysics. 533:79-87.
Jordan, D.B., Wagschal, K.C., Grigorescu, A.A., Braker, J.D. 2013. Highly active ß-xylosidases of glycoside hydrolase family 43 operating on natural and artificial substrates. Applied Microbiology and Biotechnology. 97:4415-4428.
Lan, T.Q., Gleisner, R., Zhu, J.Y., Dien, B.S., Hector, R.E. 2013. High titer ethanol production from SPORL-pretreated lodgepole pine by simultaneous enzymatic saccharification and combined fermentation. Bioresource Technology. 127:291-297.
Lee, C.C., Braker, J.D., Grigorescu, A.A., Wagschal, K.C., Jordan, D.B. 2013. Divalent metal activation of a GH43 ß-xylosidase. Enzyme and Microbial Technology. 52(2):84-90.