Location: Bioenergy Research
2018 Annual Report
Objectives
Objective 1: In collaboration with ARS plant production laboratories, identify agronomic practices that maximize the value to biorefiners of lignocellulosic feedstocks.
Objective 2: Develop commercially-viable technologies to improve the commercial production of fermentable sugars from arabinoxylan in lignocellulosic biomass. Sub-objective 2.A. Identification and subsequent characterization of glycosidic bonds that occur singly or in patterns that are unrecognized by arabinoxylan carbohydrases. Sub-objective 2.B. Characterize kinetic variation of soluble xylan products released from hydrothermal and acid catalyzed pretreatments. Sub-objective 2.C. Identify key and highly active enzymes for the hydrolysis of natural substrates that compose ß-xylan: a-L-arabinofuranosidases acting on arabinoxylan, a-glucuronidases acting on uronoxylan, and ß-xylosidases acting on oligoxylans.
Objective 3: Develop technologies to manage hydrolyzate inhibitors in commercial-scale second generation biorefineries, including biorefineries utilizing significant water recycling. Sub-objective 3.A. Investigate multiple paths to engineering furan aldehyde tolerance in Saccharomyces cerevisiae for purpose of developing a platform biocatalyst. Sub-objective 3.B. Use biological inhibitor abatement to facilitate water recycling in conversion of biomass lignocellulose hydrolyzates.
Approach
Goal 1. Demonstrate that agronomic decisions directly affect biomass conversion yields to sugars and biofuels by changes in cell wall structure and composition.
Goal 2.A. Show that plant cell wall xylan contains conserved glycosidic linkages across candidate biomass sources that are not hydrolyzed by commercially available enzymes.
Goal 2.B. Establish that bimodal kinetic hydrolysis of xylan release, frequently observed for acidic and hydrothermal treatments is a result of compositional variation within the xylan structure.
Goal 2.C. Discover key and highly active accessory enzymes for hydrolysis of heteroxylans by using activity on native substrates as a guide.
Hypothesis 3.A. Application of multiple molecular methods, including increased expression of transcriptional regulators and engineering of a catabolic pathway, will yield yeast strains with increased tolerance to furan inhibitors.
Goal 3.B. Determine the ability of an inhibitor-tolerant fungal strain that metabolizes fermentation inhibitors to facilitate reuse of process water streams.
Progress Report
Progress has been made on all three Objectives, all of which fall under National Program 306, Component 3, Biorefining.
Objective 1:
• Liberty switchgrass field samples were analyzed for composition and a low-moisture ammonium pretreatment process was successfully used for pretreatment. The pretreated switchgrass samples were hydrolyzed with a commercial enzyme mixture and the extracted sugars fermented to ethanol.
• Bioenergy grasses were pelletized and converted to ethanol. The pelletized grasses, which showed no decrease in conversion to ethanol compared to the non-pellets controls, significantly improved material handling problems associated with transportation of bulk grasses in their un-pelletized form.
Objective 2A:
• To produce fuels and chemicals from biomass, the polymeric carbohydrate components in biomass must first be converted to monomeric sugars. Yield losses occur at this step because commercial enzyme mixtures are missing activities needed to remove the complex side groups present on carbohydrates derived from biomass. Complex saccharides were characterized from barley straw, wheat straw, rice straw, rice hull, corn stover, sorghum, and miscanthus that are resistant to digestion with commercial enzyme mixtures.
• The structures of complex polymeric sugar products were determined to aid in identification of enzyme activities needed to increase the conversion of biomass to usable sugars.
• A panel of enzymes was evaluated to identify new candidate enzymes capable of cleaving the complex sugar products to generate fermentable sugars.
Objective 2B:
• Optimal enzymatic cleavage of cellulose to usable sugars requires removing 80% of xylan, the non-cellulose component of biomass, during hydrothermal pretreatment. However, part of the xylan fraction is resistant to removal. Xylan was isolated and analyzed from pretreated switchgrass throughout a hydrothermal pretreatment process to identify the chemical basis for its resistance to cleavage and removal.
Objective 3A:
• Genes for enzymes important for inhibitor detoxification were expressed in two different environmental Saccharomyces cerevisiae (Brewer’s yeast) strains. The impact of expressing the enzymes toward increasing inhibitor tolerance and thereby increasing cell growth and productivity was determined.
• Two enzymes important for inhibitor detoxification were expressed in the yeast Pichia pastoris for the purposes of fully characterizing the activity of the enzymes.
Objective 3B:
• Water recycling is a necessary strategy for reducing the ecological impact associated with fermentation processes. However, when using a water recycle strategy, inhibitors in the process streams are concentrated and inhibitory effects are amplified in the recycled water streams. “Unfermentable” recycled process water was generated in a model system, followed by bioabatement to detoxify of the process water.
• Due to high particulate concentrations in process streams containing biomass-derived sugars, it is very difficult to measure cell growth using conventional methods. To address this problem, a fluorescent yeast strain was constructed to enable convenient monitoring of microbial growth under these challenging conditions. This strain is being used to identify the inhibitors (e.g., metals and phenolics) responsible for the reduced fermentation of recycled biomass hydrolysate.
Accomplishments
1. Microbe for consumption of inhibitors. Microbial growth in biomass process streams can be difficult to measure in real time using conventional methods because the streams are discolored, contain suspended solids, and because some strains grow as filaments rather than single cells. ARS scientists in Peoria, Illinois, constructed a yeast that produces a fluorescent protein, which enables convenient monitoring of microbial growth in process streams. Use of a microbe to consume fermentation inhibitors is a promising method to improve the conversion of biomass sugars to fuels and chemicals. The fluorescent strain allows more efficient and accurate experimentation, which is necessary to measure the impact of various inhibitors on cell growth under industrial process conditions.
2. Understanding biomass recalcitrance. Almost all of the carbohydrates available for fermentation in plant fibers are in the cellulose and hemicellulose fractions. Fermentation of them requires their extraction as simple sugars. ARS scientists in Peoria, Illinois, examined xylan, a complex polymeric carbohydrate that is a fundamental component of biomass, from eight different biomass sources. Specific chemical structures were identified that resist conversion to simple sugars when treated with commercially relevant enzyme mixtures. Characterization and isolation of the complex sugar structures resistant to fermentation is the first step toward identifying enzymes that target these points of resistance. These new enzymes are required to improve sugar yields and profitability when using biomass feedstocks.
3. Biomass pellets. Ensuring a reliable supply of biomass for cellulosic ethanol producers is problematic because its low bulk density, even when baled, makes it expensive to store and transport long distances. ARS scientists in Peoria, Illinois, working with a commercial feed mill, pelletized three bioenergy grass crops at an industrial 20-ton scale. The pelletizing process was beneficial for processing biomass to ethanol and did not affect product yield. Manufactured biomass pellets are four times denser and in a small cylindrical form that is ideal for storage and shipping. Trucks are able to hold 50% more pellets compared to bales, which significantly decreases transportation costs for any process using biomass-derived sugars.
Review Publications
Wang, Z., Sharma, V., Dien, B.S., Singh, V. 2018. High-conversion hydrolysates and corn sweetener production in dry-grind corn process. Cereal Chemistry. 95:302-311. https://doi.org/10.1002/cche.10030.
Jordan, D.B., Stoller, J.R., Lee, C.C., Chan, V.J., Wagschal, K.C. 2016. Biochemical characterization of a GH43 ß-xylosidase from Bacteroides ovatus. Applied Biochemistry and Biotechnology. 182:250-260. doi: 10.1007/s12010-016-2324-0.
Vogel, K.P., Casler, M.D., Dien, B.S. 2017. Switchgrass biomass composition traits and their effects on its digestion by ruminants and bioconversion to ethanol. Crop Science. 57(1):275-281. https://doi.org/10.2135/cropsci2016.07.0625.
Wagschal, K.C., Stoller, J.R., Chan, V.J., Jordan, D.B. 2017. Expression and characterization of hyperthermostable exo-polygalacturonase RmGH28 from Rhodothermus marinus. Applied Biochemistry and Biotechnology. 183(4):1503-1515. https://doi.org/10.1007/s12010-017-2518-0.
Dias-Lopes, D., Rosa, C.A, Hector, R.E., Dien, B.S., Mertens, J.A., Ayub, M.A.Z. 2017. Influence of genetic background of engineered xylose-fermenting industrial Saccharomyces cerevisiae strains for ethanol production from lignocellulosic hydrolysates. Journal of Industrial Microbiology and Biotechnology. 44(11):1575-1588. doi: 10.1007/s10295-017-1979-z.
Nichols, N.N., Quarterman, J.C., Frazer, S.E. 2018. Use of green fluorescent protein to monitor fungal growth in biomass hydrolysate. Biology Methods and Protocols. 3(1)bpx012. doi: 10.1093/biomethods/bpx012.
Wang, Z., Dien, B.S., Rausch, K.D,. Tumbleson, M.E., Singh, V. 2018. Fermentation of undetoxified sugarcane bagasse hydrolyzates using a two stage hydrothermal and mechanical refining pretreatment. Bioresource Technology. 261:313-321. https://doi.org/10.1016/j.biortech.2018.04.018.
Seshadri, R., Leahy, S.C., Attwood, G.T., Hoong Teh, K., Lambie, S.C., Eloe-Fadrosh, E.A., Pavlopoulos, G.A, Hadjithomas, M., Varghese, N.J., Paez-Espino, D., Hungate1000 Project Collaborators*: Palevich, N., Janssen,P.H.,Ronimus, R.S., Noel, S., Soni, P., Reilly, K., Atherly, T., Ziemer, C., Wright, A.D., Ishaq, S., Cotta, M., Thompson, S.R., Crosley, K., Mckain, N., Wallace, R.J., Flint, H.J., Martin, J.C., Forster, R.J., Gruninger, R.J., McAllister, T., Gilbert, R., OuwerkerK, D., Klieve, A., Jassim, R.A., Denman, S., McSweeney, C., Rosewarne, C., Koike, S., Kobayashi, Y., Mitsumori, M., Shinkai, T., Cravero, S., Ceron Cucchi, M.*, Perry, R., Henderson, G., Creevey, C.J., Tarrapon, N., Lapebie, P., Drula, E., Lombard, V., Rubin, E., Kyrpides, N.C., Henrissat, B., Woyke, T., Ivanova, N.N, Kelly, W.J. 2018. Cultivation and sequencing of rumen microbiome members from the Hungate1000 Collection. Nature Biotechnology. 36:359-367. doi: 10.1038/nbt.4110.
