Location: Bioenergy Research
2017 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 was made on all three Objectives and their Subobjectives of this project, as part of National Program 213: Biorefining; Component 1: Biochemical Conversion.
Objective 1: 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 give high biomass yields. ARS researchers in Peoria, Illinois, evaluated the new cultivar for enzymatic sugar and fermentative ethanol production. This is the first time that sugar and ethanol yields are reported on a hectare basis for an advanced USDA switchgrass cultivar.
Objective 2: For Biochemical conversion of biomass, the polymeric carbohydrate components must be converted to monomeric sugars. Commercial enzymes are limited in their ability to hydrolyze the hemicellulose component of the biomass (typically 20-30% of the biomass content). Yield losses occur because commercial enzyme mixtures are missing activities needed to remove the complex side groups present in hemicelluloses. We have previously characterized oligosaccharide products from digestion of switchgrass xylan. Screening for enzyme supplementation candidates capable of digest these components is ongoing. The switchgrass-derived resistant products were used to determine the prevalence of the same recalcitrant products in other biomass sources. These same products were identified in xylan digestions from barley straw, wheat straw, rice straw, rice hull, corn stover, sorghum, and miscanthus. These additional sources contain unidentified recalcitrant oligosaccharides of different composition than the previously characterized oligosaccharides; therefore, in-depth characterization is required to determine their structures.
Objective 3: A primary consideration when fermenting biomass hydrolysate to biofuels is the presence of pretreatment side-products that inhibit 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 the native resistance to inhibitors by molecular engineering. The construction of transcription factors into three different plasmid expression vectors has been completed. The expression constructs have been transformed into three different strains. High-throughput testing of the various strains and constructs on individual inhibitors has been completed for furfural and 5-hydroxymethylfurfural. The individual factors tested have little to no impact on reducing the lag phase of growth, and in some cases were slightly detrimental to growth. Overexpression of an enzyme has been shown to improve lag times in Saccharomyces and does so in the selected strains we are working with. The improvement is minimal, but consistent and contingency work in an attempt to gain greater reduction in lag time. An additional issue of fermentation inhibitors in biomass hydrolysates is the effect on process water. Water recycling is important for lignocellulosic based processes; however, a potential hurdle to recycling process water is the accumulation of fermentation inhibitors in recycled water streams. In a model system, we succeeded in generating “unfermentable” recycled process water. A bioabatement process, improved the fermentability of the process water. Biomass hydrolysate provided by an industrial partner was evaluated; however, neither bioabatement nor chemical mitigation resulted in efficient fermentation of that recycled stream. Additionally, a yeast bioassay was established to report environmental conditions that are detrimental to fermenting microbes by comparing performance of a robust strain with mutants having specific defects in their ability to grow under inhibitory conditions.
Accomplishments
1. Robust yeast strains for production of biofuels and value-added products. Compounds produced during biomass pretreatment processes reduce productivity of fermentation processes due to inhibition of the microbial production hosts. ARS scientists in Peoria, Illinois tested the over-expression of factors in an attempt to further improve tolerance toward lignocellulosic inhibitors in yeast strains with greater natural tolerance to these inhibitors. The approach is appropriate for improving tolerance in less robust yeast strains. As such, the technology will be of value to researchers wishing to industrially harden laboratory yeast strains.
2. Conversion of switchgrass to bio-ethanol. An established field of Liberty switchgrass located in central Wisconsin was harvested in 2014 and 2015 and processed for production of ethanol. The field yields were 3,510 – 4,960 liters/hectare. By way of context, corn grown on good land with a harvest of 200 bu/acre would yield 5,300 liters/hectare. Liberty is the newest ARS released cultivar bred for biomass production. The yields far exceeded those of other popular switchgrass cultivars (summer and kanlow). This is a rare example of field to fermentation integrated study and the first using Liberty. Results from the study help to establish switchgrass as a viable industrial crop and are critical for farmers and ethanol processors considering production of advanced biofuels in the northern U.S.
Review Publications
de Souza, A.R., de Araujo, G.C., Zanphorlin, L.M., Ruller, R., Franco, F.C., Torres, F.A.G., Mertens, J.A., Bowman, M.J., Gomes, E., Da Silva, R. 2016. Engineering increased thermostability in the GH-10 endo-1,4-ß-xylanase from Thermoascus aurantiacus CBMAI 756. International Journal of Biological Macromolecules. 93:20-26. doi: 10.1016/j.ijbiomac.2016.08.056.
Hector, R.E., Mertens, J.A. 2017. A synthetic hybrid promoter for xylose-regulated control of gene expression in Saccharomyces yeasts. Molecular Biotechnology. 59(1):24-33. doi: 10.1007/s12033-016-9991-5.
Jimenez, D.J., Hector, R.E., Riley, R., Lipzen, A., Kuo, R.C., Amirebrahimi, M., Barry, K.W., Grigoriev, I.V., Dirk van Elsas, J., Nichols, N.N. 2017. Draft genome sequence of Coniochaeta ligniaria NRRL 30616, a lignocellulolytic fungus for bioabatement of inhibitors in plant biomass hydrolysates. Genome Announcements. 5(4):e01476-16. doi: 10.1128/genomeA.01476-16.
Dunlap, C.A., Bowman, M.J., Schisler, D.A., Rooney, A.P. 2016. Genome analysis shows Bacillus axarquiensis is not a later heterotypic synonym of Bacillus mojavensis; Reclassification of Bacillus malacitensis and Brevibacterium halotolerans as heterotypic synonyms of Bacillus axarquiensis. International Journal of Systematic and Evolutionary Microbiology. 66:2438-2443. doi: 10.1099/ijsem.0.001048.
Palazzini, J.M., Dunlap, C.A., Bowman, M.J., Chulze, S.N. 2016. Bacillus velezensis RC 218 as a biocontrol agent to reduce Fusarium head blight and deoxynivalenol accumulation: Genome sequencing and secondary metabolite cluster profiles. Microbiological Research. 192:30-36. https://doi.org/10.1016/j.micres.2016.06.002.
Serapiglia, M., Dien, B.S., Boateng, A.A., Casler, M.D. 2017. Impact of harvest time and switchgrass cultivar on sugar release through enzymatic hydrolysis. BioEnergy Research. 10:377-387.
Saha, B.C., Kennedy, G.J., Qureshi, N., Bowman, M.J. 2017. Production of itaconic acid from pentose sugars by Aspergillus terreus. Biotechnology Progress. 33(4): 1059-1067. doi: 10.1002/btpr.2485.
Slininger, P.J., Shea-Andersh, M.A., Thompson, S.R., Dien, B.S., Kurtzman, C.P., Sousa, L.D., Balan, V. 2016. Techniques for the evolution of robust pentose-fermenting yeast for bioconversion of lignocellulose to ethanol. Journal of Visualized Experiments. 116:1-15. doi: 10.3791/54227.
Qureshi, N., Liu, S., Hughes, S., Palmquist, D., Dien, B., Saha, B. 2016. Cellulosic butanol (ABE) biofuel production from sweet sorghum bagasse (SSB): Impact of hot water pretreatment and solid loadings on fermentation employing Clostridium beijerinckii P260. BioEnergy Research. 9(4):1167-1179. doi: 10.1007/s12155-016-9761-z.
Serapiglia, M., Mullen, C.A., Boateng, A.A., Dien, B.S., Casler, M.D. 2017. Impact of harvest time and cultivar on conversion of switchgrass to bio-oils via fast pyrolysis. BioEnergy Research. 10:388-399.
Jordan, D.B., Braker, J.D., Wagschal, K.C., Stoller, J.R., Lee, C.C. 2015. Isolation and divalent-metal activation of a ß-xylosidase, RUM630-BX. Enzyme and Microbial Technology. 82:158-163. doi: 10.1016/j.enzmictec.2015.10.001.
Wagschal, K.C., Stoller, J.R., Chan, V.J., Lee, C.C., Grigorescu, A.A., Jordan, D.B. 2016. Expression and characterization of hyperthermostable exo-polygalacturonase TtGH28 from Thermotoga thermophilus. Molecular Biotechnology. 58(7):509-519. doi: 10.1007/s12033-016-9948-8.
Quarterman, J., Slininger, P.J., Kurtzman, C.P., Thompson, S.R., Dien, B.S. 2017. A survey of yeast from the Yarrowia clade for lipid production in dilute-acid pretreated lignocellulosic biomass hydrolysate. Applied Microbiology and Biotechnology. 101(8):3319-3334. doi: 10.1007/s00253-016-8062-y.
