Location: Bioenergy Research
2015 Annual Report
Objectives
Objective 1. Develop platform yeast technology to enable commercial conversion of lignocellulose-derived xylose to chemicals such as triacetic acid lactone (4-hydroxy-6-methyl-2-pyrone). Sub-objective 1.A. Develop an expanded xylose inducible expression system with various expression levels for tunable control of gene expression in Saccharomyces yeasts. Sub-objective 1.B. Generate a xylose-specific transporter that is not significantly inhibited by glucose. Sub-objective 1.C. Engineer industrial Saccharomyces cerevisiae strains to produce triacetic acid lactone from xylose.
Objective 2. Develop technologies that enable the commercial production of itaconic acid (methylene succinic acid) from all the carbohydrates in lignocellulosic feedstocks. Sub-objective 2.A. Screen Aspergillus terreus strains for itaconic acid production from xylose and arabinose. Sub-objective 2.B. Adapt the best performing itaconic acid producing A. terreus strain to (i) dilute acid pretreated wheat straw hydrolyzate for inhibitor tolerance and (ii) high concentrations of itaconic acid for itaconic acid tolerance. Sub-objective 2.C. Optimize process parameters for batch production of itaconic acid from dilute acid pretreated wheat straw hydrolyzate by (i) separate hydrolysis and fermentation (SHF) and (ii) simultaneous saccharification and fermentation (SSF). Sub-objective 2.D. Demonstrate the batch itaconic acid production from wheat straw at a pilot scale (100 L).
Objective 3. Develop technologies that enable the commercial production of xylitol from lignocellulosic hydrolyzates. Sub-objective 3.A. Optimize xylitol production by Coniochaeta ligniaria C8100, a fungal strain that produces xylitol from xylose but does not grow on xylose. Sub-objective 3.B. Clone and express heterologous xylose reductase in C. ligniaria C8100.
Objective 4. Develop technologies that enable the commercial production of butanol from sweet sorghum bagasse. Sub-objective 4.A. Develop efficient pretreatment and enzymatic saccharification processes for generation of fermentable sugars from sweet sorghum bagasse. Sub-objective 4.B. Integrate enzymatic hydrolysis, fermentation and product recovery schemes for conversion of pretreated sweet sorghum bagasse to butanol. Sub-objective 4.C. Evaluate process economics of butanol production from sweet sorghum bagasse.
Approach
Hypothesis 1.A. Expressing xylose metabolism genes from tunable xylose-inducible expression modules will improve yield and productivity from both glucose and xylose. Hypothesis 1.B. Enhanced co-utilization of xylose and glucose will increase the xylose utilization rate. Goal 1.C. Integrate the genes required for triacetic acid lactone (TAL) production from xylose into an industrial S. cerevisiae strain and produce TAL from lignocellulosic feedstocks.
Goal 2.A. Through screening of Aspergillus terreus strains from varied sources, identify a strain that effectively produces itaconic acid from all sugars typically present in a lignocellulosic hydrolyzate. Goal 2.B. Determine if the mixed sugar utilizing and itaconic acid (IA) producing A. terreus strain will be able to tolerate the common fermentation inhibitors typically present in dilute acid hydrolyzates of lignocellulosic feedstock and high concentrations of IA through adaptive evolution. Goal 2.C. Develop efficient SHF or SSF process for itaconic acid production from pretreated lignocellulosic feedstocks. Goal 2.D. Scale up the itaconic acid production process from one L to 100 L.
Goal 3.A. Optimize xylitol production from hemicellulosic hydrolyzates by the inhibitor-tolerant fungus C. ligniaria C8100. Hypothesis 3.B. Increasing xylose reductase activity in C. ligniaria strain 8100 will enhance xylitol production from xylose by the recombinant fungal strain.
Goal 4.A. Develop an optimized process of sweet sorghum bagasse pretreatment and enzymatic hydrolysis to release sugars that are efficiently fermented to butanol by Clostridium beijerinckii P260. Goal 4.B. Develop an integrated process for butanol production from pretreated sweet sorghum bagasse by combining enzymatic saccharification, fermentation, and product recovery. Goal 4.C. Perform economic analysis of conversion of sweet sorghum bagasse to butanol.
Progress Report
Under Sub-objective 1.A., significant progress was made toward improving the ability of industrial yeast strains to use biomass-derived sugars. Several variations of a xylose-inducible promoter were tested to improve the fold-induction of the promoter. A non-radioactive method to analyze xylose uptake into the cell was developed. This new method was used to identify new transporters capable of increasing xylose transport into the cell. Four potential xylose transporters were isolated from a newly identified xylose-utilizing yeast that originated from the Brazilian rain forest. These transporters were evaluated for the ability to improve growth and production of ethanol from xylose. Two yeasts isolated from Brazilian fuel ethanol plants were engineered to ferment xylose and strain development continued using a novel xylose isomerase. Using a strain that was evolutionarily adapted for improved growth on xylose, the complete genome was sequenced and analyzed to determine the genetic changes that result in increased ability to use xylose. Progress toward production of the platform chemical triacetic acid lactone (TAL) continued. It was discovered that high productivity of TAL resulted in modification and degradation of TAL. The modification was identified and several genes were evaluated for their contribution to TAL modification.
Under Sub-objective 2.A., one hundred Aspergillus terreus strains obtained from ARS Culture Collection, Peoria, Illinois, were screened for production of itaconic acid from glucose, xylose, and arabinose separately (80 g/L) in shake flasks. Itaconic acid is one of the 12 identified building block chemicals and a platform chemical that can be produced by fermentation. The goal is to identify an A. terreus strain that can effectively produce itaconic acid from all sugars typically present in a lignocellulosic hydrolyzate. A total of 15 strains were found to produce itaconic acid from glucose, xylose and arabinose as substrates. Among these, 10 strains were then subjected for secondary screen to evaluate itaconic acid production from a mixture (80 g/L) of glucose, xylose, and arabinose as the carbon source. The best performing strain with respect to productivity and yield was identified. This strain will be used for adaptation to dilute acid pretreated wheat straw hydrolyzate for inhibitor tolerance and high concentrations of itaconic acid for itaconic acid tolerance.
Under Sub-objective 3.A., research to develop a microbe that makes xylitol, a sugar substitute from biomass derived sugar xylose was continued. A micriobial strain was indentified that converts xylose to xylitol. The microbe also has intrinsic tolerance for and grows on the mixture of inhibitory compounds that commonly occur along with the sugars. The growth conditions including aeration, pH, temperature, and glycerol concentration were optimized in controlled small-scale fermentations. The microbe produced up to 0.44 g xylitol/g xylose in a rich medium containing 5% xylose. The activity of the enzyme responsible for xylitol production was also assayed under these conditions. This matters because biomass must be harshly pretreated to release sugars, there are typically inhibitory chemicals present in biomass sugars. Use of a microbe that withstands the inhibitory milieu avoids use of a separate step to remove inhibitors. When confronted with hydrolyzates, the inhibitor-tolerant fungus removed inhibitory chemicals while producing xylitol from biomass sugar xylose.
Under Sub-objective 4.A., liquid hot water pretreatment conditions with respect to temperature (160-200°C) and duration of pretreatment (0-15 min) were optimized for generation of fermentable sugars from sweet sorghum bagasse after enzymatic hydrolysis using a mixture of 3 commercial enzyme preparations (cellulase, ß-glucosidase, and xylanase). The enzymatic hydrolyzate of sweet sorghum bagasse was then subjected to fermentation to butanol (acetone-butanol-ethanol or ABE with butanol being the major product) by a butanol producing anaerobic bacterium. Butanol is a superior transportation biofuel compared to ethanol with an energy content close to gasoline. It also has several superior fuel properties such as burning clean (less shoot), and transportation in existing pipelines due to being less corrosive. For the fermentation purpose, three parameters were studied: i) inhibition effect due to generated sugar degradation products; ii) accumulation of butanol/aceton-butanol-ethanol (ABE) in the fermentation broth; and iii) rate of production of butanol/ABE (productivity). The hydrolyzate as such was found not to be inhibitory to the culture at all and the fermentation resulted in accumulation of significant amount of ABE (13.27-15.50 g/L). The butanol productivity was higher (0.43-0.65 g/L.h) than when using glucose or corn (0.35 g/L.h) as a feedstock.
Accomplishments
1. New industrial yeast strains for producing bio-ethanol. Brewer’s yeasts are the preferred organism for industrial ethanol production, but not all yeasts are tolerant to industrial processes. Two yeasts originally isolated from Brazilian fuel ethanol production facilities have been shown to perform well under harsh industrial conditions. These yeasts are extremely efficient at converting glucose to ethanol but they are not able to use xylose, the second most abundant sugar in lignocellulosic biomass. Agricultural Research Service scientists in Peoria, Illinois, have engineered these strains to express all of the proteins required for conversion of xylose to ethanol. One of these two strains was capable of rapid utilization of xylose when it was the only sugar available. The engineered strains consumed all of the xylose and made 70% more ethanol compared to other engineered industrial strains. Complete utilization of all biomass-derived sugars from the feedstock is important to achieve the highest productivity. This new technology is expected to promote the economics of cellulosic ethanol, furthering rural development.
2. Developed a novel pretreatment process for conversion of sweet sorghum bagasse to butanol. Sweet sorghum is potentially a high energy and biomass crop that is regarded as one of the most promising crops for biofuel production. Agricultural Research Service scientists in Peoria, Illinois, have developed a novel process to pretreat sweet sorghum bagasse with liquid hot water to generate fermentable sugars after enzymatic hydrolysis. The pretreated sweet sorghum bagasse after enzymatic hydrolysis was easily fermented to butanol (acetone-butanol-ethanol/ABE with butanol as major product) by ABE producing anaerobic bacterium. Additionally, butanol/ABE productivity was found to be 23-86% higher than when using glucose or corn as a feedstock. Butanol is an advanced biofuel that packs 30% more energy than ethanol on a per gallon basis. The developed process would benefit the biofuel companies and the United States transportation industry and reduce dependency on imported oil.
3. A microbe that produces xylitol. Xylitol, a natural sugar substitute used in foods and pharmaceuticals, can be produced from biomass sugars. However, due to necessarily harsh pretreatment used to break down biomass fibers, the biomass sugars typically contain inhibitors that are detrimental to the process. Agricultural Research Service scientists in Peoria, Illinois, developed a microbe that produces xylitol while also withstanding the mixture of inhibitory compounds that commonly occur along with the sugars. Culture conditions were optimized for maximum xylitol production. A microbe that withstands inhibitors would avoid use of a separate processing step to remove inhibitors and allow more economical production of xylitol. This approach will elevate lower-value biomass to a higher-value sugar substitute.
4. Itaconic acid production from lignocellulosic biomass. Itaconic acid is one of the 12 identified building block chemicals and a platform chemical that can be produced by fermentation. Agricultural Research Service scientists in Peoria, Illinois, that can produce itaconic acid from lignocellulosic biomass derived sugars such as glucose, xylose and arabinose with good yields. This strain has potential to be used for itaconic acid production from lignocellulosic hydrolyzates by fermentation. Itaconic acid is a promising product that can be manufactured from cellulosic biomass that will promote rural development.
Review Publications
Hughes, S.R., Cox, E.J., Bang, S.S., Pinkelman, R.J., Lopez-Nunez, J.C., Saha, B.C., Qureshi, N., Gibbons, W.R., Fry, M.R., Moser, B.R., Bischoff, K.M., Liu, S., Sterner, D.E., Butt, T.R., Reidmuller, S.B., Jones, M.A., Riano-Herrera, N.M. 2015. Process for assembly and transformation into Saccharomyces cerevisiae of a synthetic yeast artificial chromosome containing a multigene cassette to express enzymes that enhance xylose utilization designed for an automated platform. Journal of Laboratory Automation. 20(6):621-635. doi: 10.1177/2211068215573188.
Qureshi, N. 2014. Consolidated processes for product recovery. In: Qureshi, N., Hodge, D., Vertes, A., editors. Biorefineries: Integrated Biochemical Processes for Liquid Biofuels. Amsterdam, The Netherlands: Elsevier. p. 141-160.
Qureshi, N. 2014. Integrated bioprocessing and simultaneous product recovery for butanol production. In: Qureshi, N., Hodge, D., Vertes, A., editors. Biorefineries: Integrated Biochemical Processes for Liquid Biofuels. Amsterdam, The Netherlands: Elsevier. p. 205-224.
Ezeji, T.C., Liu, S., Qureshi, N. 2014. Mixed sugar fermentation by Clostridia and metabolic engineering for butanol production. In: Qureshi, N., Hodge, D., Vertes, A., editors. Biorefineries: Integrated Biochemical Processes for Liquid Biofuels. Amsterdam, The Netherlands: Elsevier. p. 191-204.
Hector, R.E., Dien, B.S., Cotta, M.A., Mertens, J.A. 2013. Growth and fermentation of D-xylose by Saccharomyces cerevisiae expressing a novel D-xylose isomerase originating from the bacterium Prevotella ruminicola TC2-24. Biotechnology for Biofuels. 6:84.
Avci, A., Nichols, N.N., Saha, B.C., Frazer, S.E., Cotta, M.A., Donmez, S. 2015. A thermostable cyclodextrin glycosyltransferase from Thermoanaerobacter sp. 5K. Current Biotechnology. 3(4):305-312.
Qureshi, N., Singh, V. 2014. Process economics of renewable biorefineries: butanol and ethanol production in integrated bioprocesses from lignocellulosics and other industrial by-products. In: Qureshi, N., Hodge, D., Vertes, A., editors. Biorefineries: Integration Biochemical Processes for Liquid Biofuels. Amsterdam, The Netherlands: Elsevier. p. 237-252.
Hughes, S.R., Qureshi, N. 2014. Biomass for biorefining: Resources, allocation, utilization, and policies. In: Qureshi, N., Hodge, D., Vertes, A., editors. Biorefineries: Integrated Biochemical Processes for Liquid Biofuels. Amsterdam, The Netherlands: Elsevier. p. 37-58.
Qureshi, N., Friedl, A., Maddox, I.S. 2014. Butanol production from concentrated lactose/whey permeate: Use of pervaporation membrane to recover and concentrate product. Applied Microbiology and Biotechnology. 98:9859-9867.
Azam, M.M., Ezeji, T.C., Qureshi, N. 2014. Novel technologies for enhanced production of ethanol: impact of high productivity on process economics. European Chemical Bulletin. 3(9):904-910.
Saha, B.C., Nichols, N.N., Qureshi, N., Kennedy, G.J., Iten, L.B., Cotta, M.A. 2015. Pilot scale conversion of wheat straw to ethanol via simultaneous saccharification and fermentation. Bioresource Technology. 175:17-22.
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.
Huang, H., Qureshi, N., Chen, M., Liu, W., Singh, V. 2015. Ethanol production from food waste at high solid contents with vacuum recovery technology. Journal of Agricultural and Food Chemistry. 63:2760-2766.
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.
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
Saha, B.C., Qureshi, N., Kennedy, G.J., Cotta, M.A. 2015. Enhancement of xylose utilization from corn stover by a recombinant Escherichia coli strain for ethanol production. Bioresource Technology. 190:182-188.
Hohenschuh, W., Hector, R.E., Murthy, G.S. 2015. A dynamic flux balance model and bottleneck identification of glucose, xylose, xylulose co-fermentation in Saccharomyces cerevisiae. Bioresource Technology. 188:153-160.
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.
