Location: Bioenergy Research
2015 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 is the initial report for the project plan "Technologies for Improving Process Efficiencies in Biomass Refineries." 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. However, there is a lack of knowledge relating the effect of agronomic practices on conversion. Napier grass is one of the most productive crops for growth in the Southeastern U.S. We determined that harvesting grasses twice versus a single fall harvest increased carbohydrate yields by 8.1 to 15.1%. Also notable, two cuts was also found to be optimal for biomass production. These results are being confirmed using samples from a 2nd year harvest. In a separate project, we are measuring the effect of pelletizing switchgrass, big bluestem, and a low diversity mixture of prairie grasses on enzymatic sugar and microbial ethanol yields. Compressing biomass is essential to create a regional market for biomass because of the low bulk density of chopped grasses. 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 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. Biomass conversion enzymes are grouped into families. For one group of highly active enzymes (Family 52), we corrected the literature on several instances of incorrect data and interpretations ß-xylosidase mechanism of action. We conducted biochemical studies on two additional divalent metal activated ß-xylosidases. The newly identified subfamily of GH43 has high values of kcat/Km (e.g. high activity), which gives it practical importance. This research relates to Objective 2C, which is discovering 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 set of yeast was tested for their ability to sporulate and be transformed with plasmid DNA constructs, traits necessary to molecularly engineer them. Assays were also completed to determine baseline transcript levels of transcription factors that will be over-expressed in the next stage of the project to improve tolerance to major inhibitors. Finally, the genomes of the isolated strains were sequenced in an effort to understand what aspects of the genome potentially lead to these yeast isolates improved tolerance to inhibitors found in lignocellulosic hydrolysates. 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 used for corn ethanol facilities but has not been evaluated for lignocellulosic based processes. Water is expensive and the water footprint constrains facility site selection. We initiated research to determine whether a suitable microbe can be used to facilitate re-use of process water in conversion of biomass to fuels or chemicals. The microbe used for this work removes inhibitors commonly found in biomass sugars by metabolizing the inhibitory compounds. We showed that biomass fermentation process water, recycled up to four times in a laboratory model, has a modest negative effect on fermentation of glucose by conventional yeast. This result prompted us to examine the effect of recycled process water on engineered yeast and on fermentation of xylose, which is more sensitive to the presence of fermentation inhibitors than glucose. We have also begun to measure the effects on yeast of inhibitors in recycled process water. The amount of process water that can be recycled will depend upon the inhibitor load. 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. Enzymes for augmenting commercial biomass hydrolyzing enzymes. Xylan accounts for 30-40% of the carbohydrate present in biomass, but commercial enzymes are unable to completely digest biomass samples, even following extensive pretreatment. Agricultural Research Service scientists in Peoria, Illinois, identified several chemical linkages related to side-branches that are recalcitrant to commercial enzymes and limit the breakdown of xylan. With this information, scientists were able to identify enzymes that actively cleave these linkages improving the digestion of xylan. This research demonstrates that xylose yield loss is directly related to deficiencies in commercial xylanases and identifies a pathway to filling in gaps in activities needed for complete xylan hydrolysis. This is important because it will improve biofuel yield per ton and possibly lower enzyme costs, both of which are expected to lower the cost of biofuels production.
2. Napier grass for bioenergy production. Napiergrass (Pennisetum purpureum (L) Schum) is being developed as a bioenergy crop for production in the Southeastern U.S. In this study, Agricultural Research Service scientists in Peoria, Illinois, in collaboration with Agricultural Research Service researchers in Tifton, Georgia, demonstrated the conversion of Napiergrass to ethanol. The estimated ethanol yield, based upon laboratory scaled experiments, was up to 10,300 liters per hectare(l/ha). By way of comparison, we estimate a corn field yielding 180 bu/acre would produce 4,640 l/ha. This study promotes the feasibility of producing bioenergy crops in the southeastern U.S as a feedstock for production of liquid biofuels.
3. Xylooligosaccharides (XOS) production from Miscanthus x giganteus (MxG). XOS are marketed as an ingredient in health-promoting foods. Agricultural Research Service scientists in Peoria, Illinois, developed a new process for producing and purifying XOS from a bioenergy crop grown in the Midwest U.S. (e.g. MxG). This product has been assayed and found to be comparable in quality to the existing commercial product. It is expected, XOS will be a valuable co-product and for manufacturers of advanced biofuels.
Review Publications
Saunders, L.P., Bowman, M.J., Mertens, J.A., Da Silva, N.A., Hector, R.E. 2015. Triacetic acid lactone production in industrial Saccharomyces yeast strains. Journal of Industrial Microbiology and Biotechnology. 42:711-721.
Bowman, M.J., Dien, B.S., Vermillion, K.E., Mertens, J.A. 2015. Isolation and characterization of unhydrolyzed oligosaccharides from switchgrass (Panicum virgatum, L.) xylan after exhaustive enzymatic treatment with commercial enzyme preparations. Carbohydrate Research. 407:42-50.
Slininger, P.J., Shea-Andersh, M.A., Thompson, S.R., Dien, B.S., Kurtzman, C.P., Balan, V., da Costa Sousa, L., Uppugundla, N., Dale, B.E., Cotta, M.A. 2015. Evolved strains of Scheffersomyces stipitis achieving high ethanol productivity on acid- and base-pretreated biomass hydrolyzate at high solids loading. Biotechnology for Biofuels. 8:60.
Dunlap, C.A., Bowman, M.J. 2014. The use of genomics and chemistry to screen for secondary metabolites in bacillus spp. biocontrol organisms. In: Gross, A.D., Coats, J.R., Duke, S.O., Seiber, J.N., editors. Biopesticides: State of the Art and Future Opportunities. Washington, D.C.: American Chemical Society. p. 95-112.
Dunlap, C.A., Schisler, D.A., Bowman, M.J., Rooney, A.P. 2015. Genomic analysis of Bacillus subtilis OH 131.1 and coculturing with Cryptococcus flavescens for control of fusarium head blight. Plant Gene. 2:1-9.
Wagschal, K.C., Jordan, D.B., Lee, C.C., Younger, A.R., Braker, J.D., Chan, V.J. 2014. Biochemical characterization of uronate dehydrogenases from three Pseudomonads, Chromohalobacter salixigens, and Polaromonas naphthalenivorans. Enzyme and Microbial Technology. 69:62-68.
Xue, Y.-P., Jin, M., Orjuela, A., Slininger, P.J., Dien, B.S., Dale, B.E., Balan, V. 2015. Microbial lipid production from AFEXTM pretreated corn stover. The Royal Society of Chemistry Advances. 5:28725-28734.
Chen, M., Dien, B.S., Vincent, M.L., Below, F.E., Singh, V. 2014. Effect of harvest maturity on carbohydrates for ethanol production from sugar enhanced temperate x tropical maize hybrid. Industrial Crops and Products. 60:266-272.
Bowman, M.J., Dien, B.S., Vermillion, K., Mertens, J.A. 2014. Structural characterization of (1-2)-ß-xylose-(1-3)-alpha-arabinose-containing oligosaccharide products of extracted switchgrass (Panicum virgatum, L.) xylan after exhaustive enzymatic treatment with alpha-arabinofuranosidase and ß-endo-xylanase. Carbohydrate Research. 398:63-71.
Nichols, V.A., Miguez, F.E., Jarchow, M.E., Liebman, M.Z., Dien, B.S. 2014. Comparison of cellulosic ethanol yields from midwestern maize and reconstructed tallgrass prairie systems managed for bioenergy. BioEnergy Research. 7:1550-1560.
Boateng, A.A., Serapiglia, M., Mullen, C.A., Dien, B.S., Fawzy, H.M., Dadson, R.B. 2014. Bioenergy crops grown for hyperaccumulation of phosphorus in the delmarva peninsula and their biofuels potential. Environmental Management. 150:39-47.
Jin, M., Slininger, P.J., Dien, B.S., Waghmode, S.B., Moser, B.R., Orjuela, A., Da Costa Sousa, L., Balan, V. 2015. Microbial lipid based lignocellulosic biorefinery: feasibility and challenges. Trends in Biotechnology. 33(1):43-54.
Ramchandran, D., Wang, P., Dien, B.S., Liu, W., Cotta, M.A., Singh, V. 2015. Improvement of dry fractionation ethanol fermentation by partial germ supplementation. Cereal Chemistry. 92:218-223.
Cao, G., Ximenes, E., Nichols, N.N., Frazer, S.E., Kim, D., Cotta, M.A., Ladisch, M. 2015. Bioabatement with hemicellulase supplementation to reduce enzymatic hydrolysis inhibitors. Bioresource Technology. 190:412-415.
Zhou, H., Lan, T., Dien, B.S., Hector, R.E., Zhu, J.Y. 2014. Comparisons of five Saccharomyces cerevisiae strains for ethanol production from SPORL pretreated lodgepole pine. Biotechnology Progress. 30(5):1076-1083.
