Research Project: Technologies for Improving Process Efficiencies in Biomass Refineries

Location: Bioenergy Research

2019 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: Three cultivars of bioenergy switchgrass were compared for production of sugars and ethanol. The cultivars were established in northern Wisconsin and harvested from replicated field plots following a killer frost. Samples were pretreated with low-moisture ammonium hydroxide and converted to sugars and ethanol. Major conclusions were that Liberty out-yielded Summer and that Kanlow did not overwinter. The maximum ethanol yield was 4.96 Ml/ha, which compares to 5.3 Ml for an average corn field. Objective 2A: To produce fuels and chemicals from biomass, the polymeric carbohydrate components in biomass must first be converted to monomeric sugars. Glucose yield losses occur at this step due to inhibition of the cellulolytic enzymes by complex biomass xylan-derived oligosaccharides that are resistant to digestion. The inhibitory effect of the recalcitrant oligosaccharides was assessed on purified enzyme components using small glucooligomers. 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 time course of hydrothermal pretreatment to identify the fine structure of the oligomers responsible for resistance to cleavage and removal. Objective 3A: The genes for three aldehyde/alcohol dehydrogenase/oxidase enzymes from three different organisms were expressed in two different Saccharomyces cerevisiae strains. The engineered strains did not convert 5-hydroxymethylfurfural (HMF), a fermentation inhibitor present in lignocellulosic hydrolysates, to the product expected as a result of over-expressing the enzymes. Consistent with the enzymes not detoxifying hydroxymethylfurfural, no reduction in the lag growth phase relative to the wild-type strains was observed. The enzymes mentioned above were tagged with a green fluorescent protein (GFP) and visualized by microscopy, confirming that the enzymes were expressed. This result suggests that while the bacterial enzymes could be expressed weakly, they were not able to shift equilibrium toward the expected product. As a result, lag phase of growth was not improved. Since engineering the first step in the pathway into yeast did not convert HMF to the expected product (furan dicarboxylic acid), or reduce the growth lag, original plans to engineer the rest of the pathway to furoic acid were halted and the contingency plan was activated. ADH7 alcohol dehydrogenase was expressed in two S. cerevisiae strains, resulting in a decreased lag phase of roughly 1 hr. Additional dehydrogenase enzymes such as aryl-alcohol dehydrogenase enzymes are also being over-expressed in these strains with characterization of their effect on inhibitory tolerance ongoing. Objective 3B: Fermentability of recycled process water was evaluated using an ethanol-producing E. coli strain as well as a recombinant, xylose-fermenting yeast. Utilization of xylose was improved under certain conditions. Saccharomyces with heightened sensitivity to fermentation inhibitors served as a reporter for the inhibitory effect of recycled process water. Genetic markers and a transformation method were developed for a new fungal strain that grows in a microbial consortium on biomass. A fluorescent version of the microbe was constructed, a feature that will help understand how the microbe “makes a living” by breaking down biomass polymers.

Accomplishments
1. Biological abatement for recycling process water at a biorefinery. Biofuel manufacturers recycle process water to minimize their water footprint and to save operating costs. Cellulosic based biofuel processes are chemically more complex than grain-based process steams and contain inhibitory chemicals released during biomass pretreatment. ARS researchers in Peoria, Illinois, developed a method for removing many of these inhibitory chemicals based upon a novel fungus isolated from an industrial site. This microorganism can be beneficial for reusing process water by minimizing losses in productivity and yield of biorefinery processes. Minimizing net water usage is important for zoning advanced biofuel plants and reducing negative impacts of water use.

Review Publications
Singh, V., Dien, B.S., Cheng, M.H., Huang, H. 2019. The costs of sugar production from different feedstocks and processing technologies. Biofuels, Bioproducts, & Biorefining (Biofpr). 13(3):723-739. https://doi.org/10.1002/bbb.1976.
Saunders, L.P., Bischoff, K., Bowman, M.J., Leathers, T.D. 2018. Inhibition of Lactobacillus biofilm growth in fuel ethanol fermentations by Bacillus. Bioresource Technology. 272:156-161. https://doi.org/10.1016/j.biortech.2018.10.016.
Morrow, E.A., Terban, M.W., Thomas, L.C., Gray, D.L., Bowman, M.J., Billinge, S.L., Schmidt, S.J. 2018. Effect of amorphization method on the physicochemical properties of amorphous sucrose. Journal of Food Engineering. 243:125-141. https://doi.org/10.1016/j.jfoodeng.2018.08.036.
Anderson, W.F., Dien, B.S., Masterson, S.D., Mitchell, R. 2018. Development of near infrared reflectance spectroscopy (NIRS) calibrations for traits related to ethanol conversion from genetically variable napiergrass (Pennisetum purpureum Schum.). BioEnergy Research. 12(1):34-42. https://doi.org/10.1007/s12155-018-9946-8.
Knight, C., Bowman, M.J., Frederick, L., Day, A., Lee, C., Dunlap, C.A. 2018. The first report of antifungal lipopeptide production by a Bacillus subtilis subsp inaquosorum strain. Microbiological Research. 216:40-46. https://doi.org/10.1016/j.micres.2018.08.001.
Nichols, N.N., Hector, R.E., Frazer, S.E. 2019. Genetic transformation of Coniochaeta sp. 2T2.1, key fungal member of a lignocellulose-degrading microbial consortium. Biology Methods and Protocols. 4:1-5. https://doi.org/10.1093/biomethods/bpz001.
Nichols, N.N., Hector, R.E., Frazer, S.E. 2019. Factors affecting production of xylitol by the furfural-metabolizing fungus Coniochaeta ligniaria. Current Trends in Microbiology. 12: 109-119.
Mertens, J.A., Kelly, A., Hector, R.E. 2018. Screening for inhibitor tolerant Saccharomyces cerevisiae strains from diverse environments for use as platform strains for production of fuels and chemicals from biomass. Bioresource Technology. 3:154-161. https://doi.org/10.1016/j.biteb.2018.07.006.
Quarterman, J.C., Slininger, P.J., Hector, R.E., Dien, B.S. 2018. Engineering Candida phangngensis – an oleaginous yeast from the Yarrowia clade – for enhanced detoxification of lignocellulose-derived inhibitors and lipid overproduction. Federation Of European Microbiological Societies Yeast Research. 18(8):foy102. https://doi.org/10.1093/femsyr/foy102.
Dien, B.S., Mitchell, R.B., Bowman, M.J., Jin, V.L., Quarterman, J.C., Schmer, M.R., Singh, V., Slininger, P.J. 2018. Bioconversion of pelletized big bluestem, switchgrass, and low-diversity grass mixtures into sugars and bioethanol. Frontiers in Energy Research. 6:129. https://doi.org/10.3389/fenrg.2018.00129.
Kim, S.M, Lee, D., Thapa, S., Dien, B.S., Tumbleson, M.E., Rausch, K.D., Singh, V. 2018. Cellulosic ethanol potential of feedstocks grown on marginal land. American Society of Agricultural and Biological Engineers. 61(6):1775-1782. https://doi.org/10.13031/trans.12945.