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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

2012 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. Scientists working in the Agricultural Research Service (ARS) Bioenergy Research Unit (BER) at the National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, have made substantial progress during FY12 in each of these areas. One way to improve ethanol yields is to develop better quality bioenergy cultivars. To this end large data sets have been constructed for Napier and Reed Canary grass that consist of ethanol/sugar yields for genetically diverse plant samples. The data sets are presently being applied to develop near infrared scanning methods for rapid grading of cultivars specifically for use in biorefineries. Another cost-sensitive step is enzymatic saccharification of biomass into fermentable monosaccharides. Earlier we reported on a xylan degrading enzyme that is 10 times more efficient than any similar type of enzyme previously reported. We have recently discovered that its activity is enhanced 30 times by the presence of specific species of metal cations. This is a novel discovery and had not previously been shown for this “family” of enzymes. It represents an important step in further characterizing this commercially important enzyme and is relevant to the study of other related enzymes. The enzyme’s thermal stability has also been improved by +8°C and this is important for separate hydrolysis and fermentation schemes. Cellulose structure plays an important role in biomass recalcitrance. We have begun to evaluate protein factors from nature that interact directly with cellulose in such a way as to disrupt its structure and to enhance the action of cellulases. We have expressed five of these factors in recombinant yeast that will allow their full characterization. We have also broadened our work with the unique fungus Coniochaeta ligniaria, which was first isolated by BER. C. ligniaria is unique for its ability to remove inhibitory chemicals formed during pretreatment that if left to persist would block fermentation. It is widely known that class of compounds referred to as furans are potent fermentation inhibitors and we have demonstrated that C. ligniaria readily mineralizes these when present in hydrolysates. We have now determined that furan removal alone is not sufficient and acetate can also directly impede xylose fermentation by GMO yeast. This is an important discovery because acetate is naturally present in all grasses. We are presently expanding our biological abatement approach to also remove acetate. In a related development, in the course of altering xylose metabolism in C. ligniaria, we have fortuitously constructed a strain that was determined to be very efficient at converting xylose to the food ingredient xylitol. Xylitol production can be a valuable co-product further enhancing the economics of a biomass biorefinery.


4.Accomplishments
1. Rapid analysis of Switchgrass for composition and ethanol yield. Analyzing biomass samples for compositions and actual ethanol yields costs hundreds of dollars per sample and is too slow to assist crop breeding efforts. As part of team research, ARS scientists including those in the Bioenergy Research Unit at the National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, have released a near infrared spectroscopy (NIRS) method that allows for the non-destructive measurement of 20 compositional properties and ethanol yield. The method is inexpensive because it is reagent free and only takes minutes per sample. It should be feasible to improve ethanol yields per hectare by improving both biomass yield and conversion efficiency by using NIRS analyses to quantify differences among cultivars and management practices. The technique should also be invaluable for future ethanol producers for rating Switchgrass loads at the factory gate.

2. Discovered a GH43 ß-xylosidase that is activated 30-fold by divalent metal cations. Enzymes with higher specific activity for converting biomass to sugars are needed to lower operating costs for cellulosic biorefineries. Researchers within the Bioenergy Research Unit at the National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, have discovered that one such enzyme works better in the presence of divalent metals. Prior to this discovery, it was not known that adding a divalent metal cation was needed to activate this commercially important enzyme or others from this extensive family of related enzymes. This work is important for ensuring the efficient function of this xylosidase, which was discovered by unit scientists and is the most efficient ever described, and for further screening of enzymes from this family.

3. Use of a biological process to improve usability of rice hulls for making ethanol. Rice hulls, as a byproduct of rice production, are a potential feedstock for ethanol production. A technical barrier to fermenting the sugars obtained from rice hulls (termed rice hull hydrolysates) into ethanol or other products is the presence of inhibitory compounds formed during the necessary pretreatment. A process has been developed by Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, for the successful conversion of rice hulls to ethanol. The process entails biological conditioning of rice hull hydrolysates using a fungus, Coniochaeta ligniaria. Biological conditioning is advantageous because it does not require additional chemical inputs and also doesn’t generate additional chemical waste.

4. Identification of xylo-oligomers resistant to saccharification. For biochemical processing of biomass into biofuels, enzymes are used to convert carbohydrates into fermentable sugars. The major carbohydrates present in grasses are cellulose and xylan. Xylan polymers are decorated with various sugars and organic acids and, therefore, require a complex mixture of enzymes to achieve complete digestion. There is no method currently available for uncovering deficiencies in the enzyme mixture. Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, have developed a method for predicting which enzymes are missing. When Switchgrass was pretreated, digested with enzymes, and fermented with yeast, it was discovered that the unused portion was composed of short chain carbohydrates with multiple arabinose sugar side groups and will help direct the development of better enzyme cocktails for the conversion of Switchgrass.


Review Publications
Jordan, D.B., Braker, J.D. 2011. Opposing influences by subsite -1 and subsite +1 residues on relative xylopyranosidase/arabinofuranosidase activities of bifunctional beta-D-xylosidase/alpha-L-arabinofuranosidase. Biochimica et Biophysica Acta. 1814:1648-1657.

Jordan, D.B., Braker, J.D., Bowman, M.J., Vermillion, K., Moon, J., Liu, Z. 2011. Kinetic mechanism of an aldehyde reductase of Saccharomyces cerevisiae that relieves toxicity of furfural and 5-hydroxymethylfurfural. Biochimica et Biophysica Acta. 1814:1686-1694.

da Cruz, S.H., Dien, B.S., Nichols, N.N., Saha, B.C., Cotta, M.A. 2012. Hydrothermal pretreatment of sugarcane bagasse using response surface methodology improves digestibility and ethanol production by SSF. Journal of Industrial Microbiology and Biotechnology. 39:439-447.

Dien, B.S., Wicklow, D.T., Singh, V., Moreau, R.A., Moser, J.K., Cotta, M.A. 2012. Influence of Stenocarpella maydis infected corn on the composition of corn kernel and its conversion into ethanol. Cereal Chemistry. 89:15-23.

Barr, C.J., Mertens, J.A., Schall, C.A. 2012. Critical cellulase and hemicellulase activities for hydrolysis of ionic liquid pretreated biomass. Bioresource Technology. 104:480-485.

Bowman, M.J., Dien, B.S., Hector, R.E., Sarath, G., Cotta, M.A. 2012. Liquid chromatography-mass spectrometry investigation of enzyme-resistant xylooligosaccharide structures of switchgrass associated with ammonia pretreatment, enzymatic saccharification, and fermentation. Bioresource Technology. 110:437-447.

Mertens, J.A., Hector, R.E., Bowman, M.J. 2012. Subsite binding energies of an exo-polygalacturonase using isothermal titration calorimetry. Thermochimica Acta. 527:219-222.

Rosenbaum, M.A., Bar, H.Y., Beg, Q., Segre, D., Booth, J., Cotta, M.A., Angenent, L.T. 2012. Transcriptional analysis of Shewanella oneidensis MR-1 with an electrode compared to Fe(III)citrate or oxygen as terminal electron acceptor. PLoS ONE. 7(2):e30827. DOI: 10.1371/journal.pone.0030827.

Dien, B.S., Casler, M.D., Hector, R.E., Iten, L.B., Nichols, N.N., Mertens, J.A., Cotta, M.A. 2012. Biochemical processing of reed canarygrass into fuel ethanol. International Journal of Low-Carbon Technologies. 7:338-347.

Sarath, G., Dien, B.S., Saathoff, A.J., Vogel, K.P., Mitchell, R., Chen, H. 2011. Ethanol yields and cell wall properties in divergently bred switchgrass genotypes. Bioresource Technology. 102:9579-9585.

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.

Easson, M.W., Condon, B.D., Dien, B.S., Iten, L.B., Slopek, R.P., Yoshioka-Tarver, M., Lambert, A.H., Smith, J.N. 2011. The application of ultrasound in the enzymatic hydrolysis of switchgrass. Applied Biochemistry and Biotechnology. 165(5):1322-1331.

Schmer, M.R., Vogel, K.P., Mitchell, R., Dien, B.S., Jung, H.G., Casler, M.D. 2012. Temporal and spatial variation in switchgrass biomass composition and theoretical ethanol yield. Agronomy Journal. 104:54-64.

Jordan, D.B., Bowman, M.J., Braker, J.D., Dien, B.S., Hector, R.E., Lee, C.C., Mertens, J.A., Wagschal, K.C. 2012. Plant cell walls to ethanol. Biochemical Journal. 442:247-252.

Last Modified: 10/20/2014
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