2011 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 United States 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 United States 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. Scientists working in the Agricultural Research Service (ARS) Bioenergy Research Unit at the National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, have made substantial progress during 2011 in each of these areas. In particular, a novel process has been developed for conversion of reed canarygrass into ethanol that relies on dilute ammonia, which can be recycled and that requires minimum preparation for fermentation. The pretreated biomass was successfully converted to ethanol using a xylose-fermenting yeast strain, developed by NCAUR scientists, at very good yields (e.g., 81-84%). We have also applied our novel technology for conditioning pretreated biomass for fermentation to rice hulls; a significant agricultural residue. Rice hulls are particularly difficult to ferment following pretreatment because the fermentation microbes die when introduced into the hydrolysates. We have successfully treated rice hulls with dilute-acid and fermented the sugars into ethanol, again using our xylose-fermenting yeast. The strategy relied on using our patented biological abatement approach to remove microbial inhibitors following pretreatment and prior to fermentation. Finally, we have made important strides in our enzyme work. Xylan represents 30-40% of the plant cell wall carbohydrates, and enzymes will be needed to convert xylan to simple sugars so that they can be fermented by yeasts. Enzymatic hydrolysis of this xylan fraction in biomass, even following extensive pretreatment, has given us poor yields (e.g. 28-40%). We have embarked on a rational approach to overcome this barrier. We have collected residual xylan following a yeast fermentation of pretreated switchgrass, which included commercial enzymes for conversion of the carbohydrates into fermentable sugars. There is currently no appropriate method for determining the structure of the recalcitrant xylan. We have developed a novel method for chemically modifying the side-branches of the xylan and thereby allowing determination of specific structure information. This information in turn will be used to improve yields by either altering pretreatment conditions to avoid these xylan structures or supplementing with enzymes capable of specifically hydrolyzing these groups.
Method development to facilitate the analysis of hemicellulose structure by mass spectrometry (MS). Hemicellulose, e.g., xylan, represents approximately one-third the mass of the plant cell wall. Switchgrass has been identified as a promising energy crop for the production of fuel ethanol or other bio/renewable products. However, there are several aspects of the plant structure that need to be delineated for the most efficient conversion to simple sugars. The characteristics and role of xylan, the second most abundant carbohydrate, in conversion is not fully understood. Therefore, a mild chemical treatement method was developed by Agricultural Research Service (ARS) Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, that provides detailed structural analysis. This method will allow for increased understanding of the structurally important sequences of xylan that may be responsible for impeding pretreatment and/or enzymatic hydrolysis, and the underlying method should be broadly applicable to studying the structure of warm season grasses.
Conversion of bean starch to ethanol. Recognition of the health benefits of beans has led to focused interest in micronutrient content and functional properties of beans. However, processing beans for those high-value components means that uses will be needed for the other parts of beans, such as starch, which makes up about 40% of beans by weight. Using a lab-scaled fermentation process, Agricultural Research Service (ARS) Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, showed that starch from several varieties of dry beans could be converted to ethanol. The average ethanol yield for the 8 bean types examined was 92% of the theoretical maximum and would yield 273 L/ton using this technology. This work showed that ethanol could be produced as a co-product to a higher-value use for another bean component such as phytochemicals, using the same process and enzymes used industrially for corn. This research was supported in part by a commodity organization.
Effects of Stenocarpella ear rot on corn ethanol production. Stenocarpella ear rot is a major corn disease occurring within the corn belt. While Stenocarpella ear rotted corn is not considered a risk for feeding livestocks, the affect it may have on corn ethanol production and Distiller's Dried Grains with Solubles (DDGS) quality is unknown. Agricultural Research Service (ARS) Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, demonstrated conclusively that inclusion of Stenocarpella ear rotted kernels did not negatively influence ethanol yield or productivity. It did, however, drastically lower test weights (e.g., lb/bu) and decreased oil contents. The decrease oil content led to reduced DDGS quality. This work is of broad interest to corn growers, seed companies, and corn ethanol producers as it directly details what is and is not an issue in regard to Stenocarpella ear rot. This work is important for understanding the affect of ear rotted corn on a major United States industry.
Quick and inexpensive method for measuring switchgrass composition and ethanol yield. Near-infrared reflectance (NIR) spectroscopy is a convenient method used throughout the agricultural industry to measure compositional and quality properties of grains, animal feeds, and biomass. However, using NIR spectroscopy depends upon developing an accurate calibration set of biomass samples. Agricultural Research Service (ARS) scientists, including those associated with the Bioenergy Research Unit at the National Center for Agricultural Research Service (NCAUR), Peoria, IL, have released a calibration set for measuring the chemical composition of switchgrass. The technique also incorporates fermentation data and estimates the expected ethanol yield. This method is of interest to those seeking to breed switchgrass cultivars for bioenergy uses and in the future for commercial cellulosic ethanol producers planning to use switchgrass. The NIR calibration is under evaluation by a commercial research partner.
Rosenbaum, M., Bar, H.Y., Beg, Q., Segre, D., Booth, J., Cotta, M.A., Angenent, L.T. 2011. Shewanella oneidensis in a lactate-fed pure-culture and a glucose-fed co-culture with Lactococcus lactis with an electrode as electron acceptor. Bioresource Technology. 102(3):2623-2628.
Bowman, M.J., Dien, B.S., O Bryan, P.J., Sarath, G., Cotta, M.A. 2011. Selective chemical oxidation and depolymerization of switchgrass (Panicum virgatum L.) xylan with oligosaccharide product analysis by mass spectrometry. Rapid Communications in Mass Spectrometry. 25(8):941-950.
Nichols, N.N., Sutivisedsak, N., Dien, B.S., Biswas, A., Lesch, W.C., Cotta, M.A. 2011. Conversion of starch from dry common beans (Phaseolus vulgaris L.) to ethanol. Industrial Crops and Products. 33(3):644-647.
Dien, B.S., Miller, D.J., Hector, R.E., Dixon, R.A., Chen, F., McCaslin, M., Risen, P., Sarath, G., Cotta, M.A. 2011. Enhancing alfalfa conversion efficiencies for sugar recovery and ethanol production by altering lignin composition. Bioresource Technology. 102(11):6479-6486.
Mertens, J.A., Bowman, M.J. 2011. Expression and characterization of fifteen Rhizopus oryzae 99-880 polygalacturonase enzymes in Pichia pastoris. Current Microbiology. 62(4):1173-1178.
Ximenes, E., Kim, Y., Mosier, N., Dien, B.S., Ladisch, M. 2011. Deactivation of cellulases by phenols. Enzyme and Microbial Technology. 48(1):54-60.
Arora, A., Seth, A., Dien, B.S., Belyea, R.L., Singh, V., Tumbleson, M.E., Rausch, K.D. 2011. Microfiltration of thin stillage: Process simulation and economic analyses. Biomass and Bioenergy. 35(1):113-120.
Fan, Z., Yuan, L., Jordan, D.B., Wagschal, K.C., Heng, C., Braker, J.D. 2010. Engineering lower inhibitor affinities in beta-D-xylosidase. Applied Microbiology and Biotechnology. 86(4):1099-1113.
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.
Saathoff, A.J., Sarath, G., Chow, E.K., Dien, B.S., Tobias, C.M. 2011. Downregulation of cinnamyl-alcohol dehydrogenase in switchgrass by RNA silencing results in enhanced glucose release after cellulase treatment. PLoS One. 6(1):e16416. DOI: 10.1371/journal.pone.0016416.
Kim, Y., Hendrickson, R., Mosier, N.S., Ladisch, M.R., Bals, B., Balan, V., Dale, B.E., Dien, B.S., Cotta, M.A. 2010. Effect of compositional variability of Distillers' Grains on cellulosic ethanol production. Bioresource Technology. 101(14):5385-5393.
Jordan, D.B., Wagschal, K.C. 2010. Properties and applications of microbial beta-D-xylosidases. Applied Microbiology and Biotechnology. 86(6):1647-1658