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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Bioenergy Research » Research » Research Project #427438

Research Project: Biochemical Technologies to Enable the Commercial Production of Biofuels from Lignocellulosic Biomass

Location: Bioenergy Research

2016 Annual Report

Objective 1. Develop technologies that enable the commercial production of marketable lipid-based advanced biofuels from lignocellulosic biomass hydrolyzates. Objective 2. In collaboration with industrial biorefiners, develop technologies that enable widespread commercial production of cellulosic ethanol from lignocellulosic biomass.

Goal 1. Develop oleaginous yeast and associated processes for converting hydrolyzates of lignocellulosic biomass to lipids for biodiesel and valuable co-products for other uses. Goal 2. Apply novel patented stress-tolerant yeast strains under commercial conditions to convert hydrolyzates of lignocellulosic biomass to ethanol.

Progress Report
The utility of an elite few oleaginous yeasts identified among many in the ARS Culture Collection were demonstrated for production of single cell oil using various types of lignocellulosic hydrolyzates prepared from corn stover, switchgrass, and Douglas fir forest residues. This is the first time that unconditioned hydrolysate from woody biomass has been used for microbial lipid production. Our maximum lipid titer (18 g/L) is the highest reported for use of woody biomass. Applying an advanced two-stage process to manage sugar and nitrogen supplies in enzyme saccharified dilute acid-pretreated switchgrass, top strains were able to use acetic acid and nearly all of the diverse sugars available to rapidly accumulate 50-65 % of cell biomass as lipid, which corresponded to 30 g/L, exceeding all other literature reports to date for any undetoxified hydrolyzate. One conclusion of this study was that the benefit of amino acid source increased in importance as hydrolyzate concentration was increased to support production of high lipid concentrations. Research in our laboratory has also suggested that appropriate nitrogen (N) delivery to ethanologenic yeast strains may increase yield and productivity in hydrolyzates by at least four fold. In order to deliver amino N more economically to fortify yeast fermentations, various protein sources were screened to determine which had the amino acid profiles needed to support the fermentation of post frost switchgrass hydrolyzates which are low in available amino nitrogen. Low cost agricultural materials (at < $0.25/lb) containing high protein contents (45-60%) such as soyflours, cottonseed flour, and corn steep powder were examined along with processes to support primary amino acid release. Amino acid release kinetics and yield were examined based on standard methods, including enzymatic primary amino nitrogen content (PAN). Additionally, a high performance liquid chromatograhphy (HPLC) method and associated equipment for the analysis of the individual essential amino acids were acquired and applied in our laboratory. The yeasts used in this testing were the inhibitor-tolerant evolved S. cerevisiae strain Y-50049 developed in our laboratory best xylose-engineered strain derived from the best hydrolyzate tolerant evolved native xylose fermenting strain S. stipitis. N source utilization was monitored, including overall PAN, urea, and ammonia, and individual amino acid uptake studies to indicate key amino acids needed by the yeast during growth and bioconversion of hydrolyzates to ethanol or lipids. Most useful commercial amino acid sources were thus identified and are being studied with respect to identifying the hydrolysis methods supporting most effective amino acid release. Determining the suitability of microbial lipids for biodiesel production requires knowledge of fatty acid composition. A gas chromatography based method was successfully adapted from the literature to measure relative fatty acid percentages and will be further developed for quantitative determination of fatty acids using in situ esterification as required for GC analysis. Additionally, carotenoid production was observed in our processes, and further studies are planned as followup since carotenoids, as higher value co-products, are expected to greatly enhance the economic feasibility of lipid production for conversion to biodiesel fuel. Both HPLC and UV-visible spectrophotometry methods for quantitation of individual carotenoids and total carotenoids, respectively, are being developed. These will support efficient next stage advancement of oleaginous biocatalysts, through screening of isolates from nature and targeted evolution of strains showing promise for economical commercial application. Two staining protocols were developed that allow for identification of yeast producing lipids microscopically – nile red and sudan black. Currently enrichment protocols are being developed to support work to concentrate and isolate oleaginous yeast from natural environments. Yeast isolations are planned to begin the end of the FY. New insights into industrial yeast tolerance were obtained at the genome level for ARS patented yeast strain Saccharomyces cerevisiae NRRL Y-50049. ARS scientists at Peoria, Illinois, in collaboration with scientists from Chinese Academy of Sciences conducted comparative genome sequence analysis and revealed DNA sequence mutations from the tolerant yeast strain involved in mitogen-activated protein kinases (MAPK) signaling pathway. During collaboration with scientists from New Mexico State University at least 44 downstream pathways were rewired from the tolerant yeast in response to challenges of furfural and 5-hydroxymethylfurfural. These findings confirm earlier discoveries of the impact of glycolysis and pentose phosphate pathways on yeast tolerance and extend the tolerant interactions to the global level. Three forms of new beta-glucosidase enzymes from ARS patented yeast strain Clavispora NRRL Y-50464 were discovered to enable the yeast to produce sufficient beta-glucosidase enzymes for cellobiose hydrolysis resulting in lower cost ethanol production from cellulosic materials. The enzymes demonstrate high resistance to glucose product inhibition, tolerance to ethanol, and resistance to inhibitors furfural and HMF. In collaboration with scientists from India, cellulosic ethanol production of 36.7 g/L from green solvent-pretreated rice straw was achieved in 36 h, further indicating commercial promise of Y-50049.

1. Microbial oil production from woody biomass. ARS researchers in Peoria, Illinois, successfully converted hydrolysate prepared from Douglas fir forest waste that was prepared by the Forest Product Laboratory in Madison, Wisconsin, to microbial oil using oleaginous yeasts. This demonstration is promising for commercial interest because the upfront process for treating the wood uses unit operations common to a pulp and paper plant, the sugar syrup did not need to be conditioned prior to microbial processing to lipids, and the lipid titer (18.0 g/l) is by far the highest ever reported for wood based hydrolysates. This technology will be of interest to operators of pulp and paper mills as a method to valorize forest waste through conversion to drop in fuels. DOE has estimated that a total of 1.3 billion tons of biomass are available for conversion to biofuels, including forest residues. If all were converted to biodiesel fuel, over 60% of diesel from petroleum could potentially be replaced by biodiesel from plant biomass.

2. Novel cellobiose-fermenting yeast for lower cost cellulosic ethanol production. Cellulolytic enzymes, including beta-glucosidase as a key member, are a major cost factor in the conversion of lignocellulose to ethanol. ARS scientists in Peoria, Illinois, developed a novel yeast strain Clavispora NRRL Y-50464 able to produce sufficient innate beta-glucosidase to convert cellulose to ethanol, potentially eliminating a portion of the enzyme cost. Scientists discovered that the success of the yeast lies in its unique three-member beta-glucosidase gene family. These enzymes not only showed a broad range of substrate specificity, but also demonstrated strong resistance to glucose product inhibition, high ethanol tolerance, and a superior resistance to furan aldehydes commonly present in hydrolyzates. Confirmation of its dual functions of cellulolytic and cellobiose-fermenting capability by ARS scientists made Clavispora NRRL Y-50464 a candidate for lower-cost cellulosic ethanol production using economic simultaneous saccharification and fermentation. In collaboration with scientists in India, a cellulosic ethanol production of 36.7 g/L in 36 h from green solvent-pretreated rice straw with a conversion efficiency of 90.1% was observed. This new technology would allow enzyme cost reduction and consolidated process efficiencies providing an estimated savings of ~$0.35/gal in the selling price of ethanol. This technology supports the rural economy and is expected to reduce risks and increase profitability in existing industrial biorefineries which produce ethanol and other products.

3. Prototype model biocatalyst detoxifies two major classes of biomass hydrolyzate inhibitors. Development of the next-generation biocatalyst able to overcome the inhibitor stress of biomass hydrolyzates is one of the most significant challenges for sustainable biofuels production. ARS scientists in Peoria, Illinois, developed a tolerant industrial yeast that is able to reduce two major classes of inhibitors found in hydrolyzates (phenols and aldehydes) into less or non-toxic compounds while producing ethanol, as demonstrated during collaborative research with China. The genetic basis of this inhibitor tolerance was elucidated in collaborative research with New Mexico State University and the Chinese Academy of Sciences, allowing this prototype yeast to serve as a resilient base strain for engineering the next more advanced generations of biocatalysts. This novel yeast should be of interest because it provides a scaffolding that can be built upon to significantly drop the minimum ethanol selling price well below $2 per gallon, as is needed to be competitive with the price of gasoline. This technology furthers our progress towards national priorities of energy independence, a stronger rural economy, and sustainable environment.

4. Method for targeted evolution of superior hydrolyzate resistant yeast. Traditional industrial yeasts do not ferment pentose sugars, which comprise about one-third of available sugars from biomass, nor are they able to colonize, survive, or ferment the toxic concentrated hydrolyzates needed to produce economically viable ethanol concentrations > 40 g/L. Adaptive evolution, isolation, and relative performance index evaluation techniques were designed and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified, undetoxified hydrolyzates and to accumulate over 40 g/L ethanol. Repeated culturing of the native pentose-fermenting yeast NRRL Y-7124 in concentrated hydrolyzates, combined with controlled ethanol exposure, was used to force targeted evolution. The new strains will be useful to the biofuels industry and to other scientists studying yeast genetics, metabolism, physiology, and designing improved processes and technologies for the production of ethanol biofuel. Use of this method to improve strains allows a $0.31/gal ethanol savings in selling price compared to the parent, and as a result, this methodology furthers our progress towards national priorities of energy independence, a stronger rural economy, and sustainable environment.


Review Publications
Slininger, P.J., Dien, B.S., Kurtzman, C.P., Moser, B.R., Bakota, E.L., Thompson, S.R., O'Bryan, P.J., Cotta, M.A., Balan, V., Jin, M., da Costa Sousa, L., Dale, B.E. 2016. Comparative lipid production by oleaginous yeasts in hydrolyzates of lignocellulosic biomass and process strategy for high titers. Biotechnology and Bioengineering. 113(8):1676-1690. doi: 10.1002/bit.25928.
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.
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.
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.
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.
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.
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.
Zhang, Y., Liu, Z.L., Song, M. 2015. ChiNet uncovers rewired transcription subnetworks in tolerant yeast for advanced biofuels conversion. Nucleic Acids Research. 43(9):4393-4407. doi: 10.1093/nar/gkv358.
Yi, X., Gu, H., Gao, Q., Liu, Z.L., Bao, J. 2015. Transcriptome analysis of Zymomonas mobilis ZM4 reveals mechanisms of tolerance and detoxification of phenolic aldehyde inhibitors from lignocellulose pretreatment. Biotechnology for Biofuels. 8(1):153. doi: 10.1186/s13068-015-0333-9.
Chapla, D., Parikh, B.S., Liu, L.Z., Cotta, M.A., Kumar, A.K. 2015. Enhanced cellulosic ethanol production from mild-alkali pretreated rice straw in SSF using Clavispora NRRL Y-50464. Journal of Biobased Materials and Bioenergy. 9(4):381-388(8).
Wang, X., Liu, Z.L., Weber, S.A., Zhang, X. 2016. Two new native ß-glucosidases from Clavispora NRRL Y-50464 confer its dual function as cellobiose fermenting ethanologenic yeast. PLoS One. 11(3):e0151293. doi: 10.1371/journal.pone.0151293.
Kumar, A.K., Parikh, B.S., Shah, E., Liu, L.Z., Cotta, M.A. 2016. Cellulosic ethanol production from green solvent-pretreated rice straw. Biocatalysis and Agricultural Biotechnology. 7:14-23. doi: 10.1016/j.bcab.2016.04.008.
Wang, X., Ma, M., Liu, Z.L., Xiang, Q., Li, X., Liu, N., Zhang, X. 2016. GRE2 from Scheffersomyces stipitis as an aldehyde reductase contributes tolerance to aldehyde inhibitors derived from lignocellulosic biomass. Applied Microbiology and Biotechnology. 100(15):6671-6682. doi: 10.1007/s00253-016-7445-4.