Location: Bioenergy Research
2016 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
This project is focused on developing technologies that promote commercialization of lignocelluloses based biorefineries. Cultivating perennial grasses could produce up to 400 million dry tons of biomass per year. However, success requires high production and processing quality. ARS researchers in Lincoln, Nebraska, recently released a new switchgrass cultivar bred to be winter adapted and high biomass yields. We are evaluating this new cultivar for production of sugars and ethanol. We have characterized a replicated set of field samples grown by ARS researchers located in Madison, Wisconsin, for chemical composition and have begun to optimize conditions for their treatment using ARS’s moist ammonium pretreatment. This research relates to Objective 1 to the development of herbaceous bioenergy crops.
Once the biomass reaches the refinery’s gates, it is milled, pretreated to deconstruct the plant cell wall, and sugars are extracted from the hemicellulose and cellulose. Commercial xylanases are limited in their ability to extract sugars from hemicellulose. We hypothesize that yield losses occur because commercial enzyme mixtures are missing activities needed for removing side groups; hemicelluloses have a complex assortment of these. We have verified this hypothesis by testing pretreated biomass with commercial enzymes and identifying oligosaccharides products. These products contain unusual chemical linkages present in the side-branches. These resistant products can now be used for testing commercial enzyme mixtures for needed activities and determining the prevalence of recalcitrant linkages in other biomass sources. This research is fulfillment of Objective 2A, to promote development of better hemicellulases by identifying the structure of residual oligomer carbohydrates.
We are also seeking to improve preparations for hydrolyzing hemicelluloses by identifying relevant enzymes with exceptional kinetics using a rigorous structure function approach. We continued characterizing a new class of ß-xylosidases based upon their protein structure. The recently discovered family of enzymes requires divalent metal cofactor. Within the class, we have identified enzymes that display the best overall reaction kinetics and are relatively unaffected by glucose compared to others, making them highly suitable for the end stage of saccharification where xylooligosacharide concentration is low and glucose concentration is high. This research relates to Objective 2C, which is discovering a new accessory enzyme with exceptional kinetic profiles.
The final step is fermentation of the biomass hydrolysate (e.g. sugars) into biofuels. A primary consideration when fermenting biomass hydrolysate is the presence of side-products, produced during the pretreatment step, which are inhibitory to growth and microbial product formation. In the prior project, Saccharomyces yeasts were identified with exceptional robustness for growth in the presence of these inhibitors. This work is being expanded to enhance this native resistance to inhibitors by molecular engineering. The construction of six different transcription factors associated with stress response into three different plasmid expression vectors has been completed. The three plasmid expression vectors differ only in the expression level: simply high, medium, and low, based on the promoter/transcription terminator combination. The multi-level expression strategy was pursued as a way to thoroughly test the impact of these transcription factors and achieve the best possible outcome on inhibitor tolerance. The plasmids are termed low copy number, meaning there are only 2-3 copies of the plasmid construct in the cell, since expending energy to make additional plasmid DNA is viewed as detrimental to the overall goal of improving inhibitor tolerance. The expression constructs have been transformed into three different strains previously identified in an inhibitor screening project. High-throughput testing of the various strains and constructs on individual inhibitors is currently underway. This research is in partial fulfillment of Objective 3A, which is to engineer microorganisms with improved robustness for growth on hydrolysates generated from biomass.
Water recycling is important for lignocellulosic based processes; however, a potential hurdle to recycling process water is the presence of microbial inhibitors that are found in biomass sugars and that could build up in recycled water streams. A microbe that metabolizes inhibitors present in biomass sugars was investigated for use in a process to improve the fermentability of recycled process water at lab scale. We used recycled liquor for biomass pretreatment and determined that the microbe can improve the subsequent ethanol fermentation. Xylose-fermenting microbes (E. coli and two yeast strains) were compared to identify the best microbe for use in a laboratory model of process water recycling. Strains with known sensitivities to environmental conditions were beginning to be evaluated with the goal of discerning which aspects of recycled process water will be most relevant for recycling. This research relates to objective 3B, to use biological inhibitor abatement to increase the proportion of process water that can be recycled and to decrease overall use of process water in biomass conversion processes.
Accomplishments
1. Recycling process water in cellulosic ethanol production. A major obstacle to recycling process water is microbial inhibitors because organic inhibitors and salts can build up to the point that they could impede fermentation. When water is recycled all of these inhibitors will accumulate with each recycle stage and, therefore, it will be critical to control their concentrations and effects. ARS researchers in Peoria, Illinois, have shown that reuse (recycling) of process water can be improved by using a microbe to “clean up” the water used to produce cellulosic ethanol by microbial fermentation at lab scale. We have also shown that a strain, sensitive to osmolar stress, and is defective in growth on biomass sugars. These findings may point the way to improved strains or processes for recycling biomass water. This is important because locating a cellulosic ethanol plant can be limited by water availability and because excess process water usage is an added cost.
2. Conversion of switchgrass and corn stover extracted sugars into single cell oil. ARS researchers in Peoria, Illinois, working with a private company demonstrated the feasibility of directly converting sugars extracted from plants into lipids, which are expected to be suitable for conversion into either biodiesel or jet fuel. The significance of this effort is that the sugars extracted without the aid of enzymes, as is typically done. This is advantageous because enzymes are expected to be a major cost for producing sugars from plant fibers. Therefore, this shows that a commercial process for producing sugars for plant fibrous feedstocks is suitable for producing a renewable intermediate for biodiesel production.
Review Publications
Mertens, J.A., Bowman, M.J. 2016. Kinetic properties of Rhizopus oryzae RPG1 endo-polygalacturonase hydrolyzing galacturonic acid oligomers. Biocatalysis and Agricultural Biotechnology. 5:11-16. doi: 10.1016/j.bcab.2015.12.005.
Kim, D., Ximenes, E.A., Nichols, N.N., Cao, G., Frazer, S.E., Ladisch, M.R. 2016. Maleic acid treatment of biologically detoxified corn stover liquor. Bioresource Technology. 216:437-445. doi: 10.1016/j.biortech.2016.05.086.
Jordan, D.B., Braker, J.D. 2015. Rate-limiting steps of stereochemistry retaining ß-d-xylosidase from Geobacillus stearothermophilus acting on four substrates. Archives of Biochemistry and Biophysics. 583:73-78. doi: 10.1016/j.abb.2015.08.004.
Jordan, D.B., Braker, J.D., Wagschal, K., Lee, C.C., Chan, V.J., Dubrovska, I., Anderson, S., Wawrzak, Z. 2015. X-ray crystal structure of divalent metal-activated ß-xyloisdase, RS223BX. Applied Biochemistry and Biotechnology. 177:637-648. doi: 10.1007/s12010-015-1767-z.
Price, N.P.J., Labeda, D.P., Naumann, T.A., Vermillion, K.E., Bowman, M.J., Berhow, M.A., Metcalf, W.W., Bischoff, K.M. 2016. Quinovosamycins: New tunicamycin-type antibiotics in which the alpha, beta-1", 11'-linked N-acetylglucosamine residue is replaced by N-acetylquinovosamine. Journal of Antibiotics. 69(8):637-646. doi: 10.1038/ja.2016.49.
Chen, M.H., Bowman, M.J., Cotta, M.A., Dien, B.S., Iten, L.B., Whitehead, T.R., Rausch, K.D., Tumbleson, M.E., Singh, V. 2016. Miscanthus x giganteus xylooligosaccharides: Purification and fermentation. Carbohydrate Polymers. 140:96-103. doi: 10.1016/j.carbpol.2015.12.052.
Xue, S., Uppugundla, N., Bowman, M.J., Cavalier, D., Da Cousta Sousa, L., Dale, B.E., Balan, V. 2015. Sugar loss and enzyme inhibition due to oligosaccharides accumulation during high solids-loading enzymatic hydrolysis. Biotechnology for Biofuels. 8:195. doi: 10.1186/s13068-015-0378-9.
Dien, B.S., Zhu, J.Y., Slininger, P.J., Kurtzman, C.P., Moser, B.R., O'Bryan, P.J., Gleisner, R., Cotta, M.A. 2016. Conversion of SPORL pretreated Douglas fir forest residues into microbial lipids with oleaginous yeasts. RSC Advances. 6(25):20695-20705. doi: 10.1039/c5ra24430g.
Dien, B.S., Slininger, P.J., Kurtzman, C.P., Moser, B.R., O'Bryan, P.J. 2016. Identification of superior lipid producing Lipomyces and Myxozyma yeasts. AIMS Environmental Science. 3(1):1-20. doi: 10.3934/environsci.2016.1.1.
Qureshi, N., Dien, B.S., Saha, B.C., Iten, L., Liu, S., Hughes, S.R. 2015. Genetically engineered Escherichia coli FBR5 to use cellulosic sugars: Production of ethanol from corn fiber hydrolyzate employing commercial nutrient medium. European Chemical Bulletin. 4(3):130-134. https://doi.org/10.17628/ecb.2015.4.130-134
Casler, M.D., Cherney, J., Brummer, E., Dien, B.S. 2015. Designing selection criteria for reed canarygrass as a bioenergy feedstock. Crop Science. 55:2130-2137.
Scully, E.D., Gries, T.L., Sarath, G., Palmer, N.A., Sattler, S.E., Baird, L., Serapiglia, M., Dien, B.S., Boateng, A.A., Funnell-Harris, D.L., Twigg, P., Clemente, T.E. 2016. Overexpression of SbMyb60 impacts phenylpropanoid biosynthesis and alters secondary cell wall composition in sorghum bicolor. Plant Journal. 85:378-395.
