PROCESS TECHNOLOGIES FOR PRODUCING BIOFUELS AND COPRODUCTS FROM LIGNOCELLULOSIC FEEDSTOCKS

Goals / Objectives
Objective 1: Starting with industrial strains of yeast, develop new commercially-viable strains that have (1) improved inhibitor tolerance and (2) wide sugar-substrate specificity for fermenting lignocellulosic hydrolyzates to fuel ethanol. Objective 2: Develop (1) microbial based pretreatment and (2) simultaneous saccharification and fermentation (SSF) technologies that will enable commercially-viable processes for converting lignocellulosic feedstocks to fuel ethanol. Objective 3: Develop novel technologies that enable commercially-preferred processes for producing fuel-grade butanol from lignocellulosic feedstocks. Objective 4: Develop fermentative and enzymatic based technologies that will enable commercially-preferred processes for the production of xylitol from lignocellulose hydrolyzates.

Project Methods
The overall goal of this project is to develop commercially-targeted, integrated bioprocess technologies for production of biofuels and value-added coproducts from lignocellulosic feedstocks. The plan will emphasize microbiologically based approaches to overcome technical constraints that impede industrial applications. Our target is to use corn stover as a model lignocellulosic feedstock for ethanol, butanol, and xylitol production. This research will focus on screening for yeast (Saccharomyces) strains that can tolerate the fermentation inhibitors typically formed during certain pretreatments of lignocellulosic biomass and developing a recombinant S. cerevisiae strain that can efficiently ferment both glucose and xylose derived from lignocellulosic feedstocks. We will develop a microbial pretreatment at the laboratory scale and a simultaneous saccharification and fermentation (SSF) process for production of ethanol from a microbially pretreated feedstock using the recombinant S. cerevisiae strain developed in this project plan. We will identify and characterize the fermentation stimulating/enhancing chemicals present in dilute acid hydrolyzate of wheat straw and develop an integrated SSF with product recovery (SSFR) using ionic liquid or vacuum for efficient production of butanol which is very toxic to the fermentative bacterium. Finally, we will develop batch and fed-batch fermentation processes for production of xylitol from the hemicellulosic hydrolyzates of corn stover and a cell-free enzymatic method with cofactor regeneration for its production. This research project will greatly help to overcome the fermentation related challenges associated with the production of biofuels and coproducts from lignocellulosic feedstocks.

Progress 11/27/09 to 08/28/14

Outputs
Progress Report Objectives (from AD-416): Objective 1: Starting with industrial strains of yeast, develop new commercially-viable strains that have (1) improved inhibitor tolerance and (2) wide sugar-substrate specificity for fermenting lignocellulosic hydrolyzates to fuel ethanol. Objective 2: Develop (1) microbial based pretreatment and (2) simultaneous saccharification and fermentation (SSF) technologies that will enable commercially-viable processes for converting lignocellulosic feedstocks to fuel ethanol. Objective 3: Develop novel technologies that enable commercially- preferred processes for producing fuel-grade butanol from lignocellulosic feedstocks. Objective 4: Develop fermentative and enzymatic based technologies that will enable commercially-preferred processes for the production of xylitol from lignocellulose hydrolyzates. Approach (from AD-416): The overall goal of this project is to develop commercially-targeted, integrated bioprocess technologies for production of biofuels and value- added coproducts from lignocellulosic feedstocks. The plan will emphasize microbiologically based approaches to overcome technical constraints that impede industrial applications. Our target is to use corn stover as a model lignocellulosic feedstock for ethanol, butanol, and xylitol production. This research will focus on screening for yeast (Saccharomyces) strains that can tolerate the fermentation inhibitors typically formed during certain pretreatments of lignocellulosic biomass and developing a recombinant S. cerevisiae strain that can efficiently ferment both glucose and xylose derived from lignocellulosic feedstocks. We will develop a microbial pretreatment at the laboratory scale and a simultaneous saccharification and fermentation (SSF) process for production of ethanol from a microbially pretreated feedstock using the recombinant S. cerevisiae strain developed in this project plan. We will identify and characterize the fermentation stimulating/enhancing chemicals present in dilute acid hydrolyzate of wheat straw and develop an integrated SSF with product recovery (SSFR) using ionic liquid or vacuum for efficient production of butanol which is very toxic to the fermentative bacterium. Finally, we will develop batch and fed-batch fermentation processes for production of xylitol from the hemicellulosic hydrolyzates of corn stover and a cell-free enzymatic method with cofactor regeneration for its production. This research project will greatly help to overcome the fermentation related challenges associated with the production of biofuels and coproducts from lignocellulosic feedstocks. This report constitutes the final report of the project plan. Substantial progress was made in all objectives, which address research needs to develop commercially-targeted, integrated bioprocess technologies for production of biofuels and coproducts from biomass. These objectives emphasized microbiologically based approaches to overcome technical constraints that impede industrial applications. Under objective 1, over 150 yeast species were screened for tolerance to inhibitors resulting from biomass pretreatment. The six strains selected from a primary screen were equivalent and in some cases superior, depending on the inhibitor, to the benchmark strain, a commonly used industrial strain. Tolerant strains have been provided to support additional projects. A unique soil fungus metabolizes and broadly removes inhibitory chemicals found in biomass sugars. The strain was used to condition agricultural residues including rice hulls, and genetic tools were developed for the fungus. A behavioral response to furan-type inhibitors was also discovered in bacteria. These findings may enable biomass conversion. ARS made significant progress in developing industrial yeast strains with improved fermentation capacity using biomass hydrolyzate feedstock. A xylose inducible expression system for brewer�s yeast was developed for improved control of gene expression (patent application in preparation). A robust industrial yeast strain was engineered to express a new xylose isomerase from a rumen bacterium. This strain was adapted under aerobic conditions to generate a strain with improved ability to grow on xylose. A patent was awarded for this new enzyme. Production of triacetic acid lactone (a platform chemical) was improved by 350%. Under objective 2, we screened 27 white rot fungal strains for powerful delignification ability growing them under solid state cultivation (SSC) on corn stover. SSC was optimized using 3 selected strains for maximum sugar release after enzymatic hydrolysis of pretreated corn stover. Good yields of ethanol were achieved from fungal pretreated corn stover. Under objective 3, efficient bioreactors (14 fold productive) and an energy efficient product recovery technique (vacuum) were developed for butanol production from economical and sustainable feedstock corn stover which can be obtained for $24-60/ton as compared to corn at $170-275/ton. A novel cost- effective integrated bioprocess technology was developed for butanol production from corn stover by simultaneous hydrolysis of pretreated corn stover to sugars, fermentation of sugars to butanol, and product recovery using vacuum in one unit operation by simultaneous saccharification, fermentation and product recovery (SSFR). The butanol production cost from corn stover by this SSFR process was estimated to be $3.42/gal. Under objective 4, xylitol production from xylose and corn stover hemicellulosic hydrolyzate was optimized by one yeast strain that has the ability to produce xylitol from xylose but little or no arabitol from arabinose from a mixture of xylose and arabinose. A co-factor (NADH) regeneration system for enzymatic production of xylitol was investigated. Significant Activities that Support Special Target Populations: Developed and led two 75-min �Bioenergy for the Future� workshops for educators at the Science, Technology, Engineering, Architecture/Art, and Mathematics (STEAM) conference held at Northeastern Illinois University, a Hispanic serving Institution. Developed and led three 75-min �Microbiology & Molecular Biology� high school science sessions for Peoria ELITE Youth Outreach. This program makes science and science careers accessible to local minority and at- risk students. Accomplishments 01 Industrial yeast strains for producing triacetic acid lactone (TAL). TAL is a promising platform chemical if produced in sufficient quantity could be converted into a wide range of chemicals such as flavoring agents, and precursors for production of food preservatives and plastics. For large-scale TAL production, genetically-stable, robust, industry-friendly yeast strains are needed that are able to consume the majority of biomass-derived sugars. Agricultural Research Service scientists in the Bioenergy Research Unit at the National Center for Agricultural Utilization Research in Peoria, Illinois, engineered several industrial yeasts, selected from a screen for yeasts with increased tolerance to inhibitors present in hydrolyzate, to produce TAL. Using the best strain, culture conditions were developed to maximize production and the TAL concentration was further increased by 350%. Since many commodity chemicals are currently derived from petroleum-based feedstock, producing these from agricultural biomass will reduce our dependence on petroleum. These inhibitor tolerant, engineered, industrial yeast strains are essential for achieving high levels of TAL required for an economic process. 02 Developed cost-effective process technology for butanol production from corn stover. Butanol is an advanced biofuel that packs 30% more energy than ethanol on per gallon basis. It is produced via fermentation of sugars; however, butanol should be removed simultaneously (as it is produced) from the fermentation because butanol above a certain concentration inhibits its further production. Thus, the key to produce butanol economically from corn stover is to saccharify pretreated corn stover to sugars using enzymes, ferment the sugars to butanol, and recover butanol simultaneously. Agricultural Research Service scientists in the Bioenergy Research Unit at the National Center for Agricultural Utilization Research in Peoria, Illinois, have developed a novel SSFR process for production of butanol from dilute acid pretreated corn stover using vacuum distillation for recovery of butanol that allowed integration of these three process steps. The production cost for butanol from corn stover by this process was estimated to be at $3.42/gal whereas the production cost of butanol from corn was $4.39/gal. Thus the newly developed process enables more cost-effective production of butanol by fermentation. 03 Fermentation based process for making xylitol. Xylitol is a commercially valuable product that could be produced by fermentation of agricultural biomass derived sugars; especially hemicellulose hydrolyzates that are rich in xylose. However, these feedstocks typically contain a mixture of xylose and arabinose and xylitol producing yeasts generally convert arabinose to arabitol. It is very difficult to separate xylitol and arabitol adding significantly to the cost of product purification and loss of yield. Agricultural Research Service scientists in the Bioenergy Research Unit at the National Center for Agricultural Utilization Research in Peoria, Illinois, have identified a yeast strain after evaluating 105 selected yeast strains from ARS Culture Collection (Peoria, Illinois) which has the ability to produce xylitol from xylose but no or little arabitol from arabinose from a mixture of xylose and arabinose. The process of making xylitol from corn stover hydrolyzate by the yeast strain was optimized in batch and fed-batch fermentations. This yeast strain has potential to be used in commercial production of xylitol from various hemicellulosic hydrolyzates. Xylitol has established commercial use as an alternative low-calorie sweetener in mouth wash, tooth paste, chewing gum and food. It has been identified as one of the 12 value-added materials to be produced from biomass, thereby serving as key economic driver for biorefineries.

Impacts
(N/A)

Publications

Avci, A., Saha, B.C., Kennedy, G.J., Cotta, M.A. 2013. High temperature dilute phosphoric acid pretreatment of corn stover for furfural and ethanol production. Industrial Crops and Products. 50:478-484.
Saha, B.C., Cotta, M.A. 2014. Alkaline peroxide pretreatment of corn stover for enzymatic saccharification and ethanol production. Industrial Biotechnology. 10(1):34-41.
Nichols, N.N., Hector, R.E., Saha, B.C., Frazer, S.E., Kennedy, G.J. 2014. Biological abatement of inhibitors in rice hull hydrolyzate and fermentation to ethanol using conventional and engineered microbes. Biomass and Bioenergy. 67:79-88.
Qureshi, N., Singh, V., Liu, S., Ezeji, T.C., Saha, B.C., Cotta, M.A. 2014. Process integration for simultaneous saccharification, fermentation, and recovery (SSFR): Production of butanol from corn stover using Clostridium beijerinckii P260. Bioresource Technology. 154:222-228.
Qureshi, N., Cotta, M.A., Saha, B.C. 2014. Bioconversion of barley straw and corn stover to butanol (a biofuel) in integrated fermentation and simultaneous product recovery bioreactors. Food and Bioproducts Processing. 92:298-308.
Hughes, S.R., Bang, S.S., Cox, E.J., Schoepke, A., Ochwat, K., Pinkelman, R., Nelson, D., Qureshi, N., Gibbons, W.R., Kurtzman, C.P., Bischoff, K.M., Liu, S., Cote, G.L., Rich, J.O., Jones, M.A., Cedeno, D., Doran-Peterson, J., Riano, N.M. 2013. Automated UV-C mutagenesis of Kluyveromyces marxianus NRRL Y-1109 and selection for microaerophilic growth and ethanol production at elevated temperature on biomass sugars. Journal of Laboratory Automation. 18(4):276-290.
Liu, S., Bischoff, K.M., Leathers, T.D., Qureshi, N., Rich, J.O., Hughes, S.R. 2013. Butyric acid from anaerobic fermentation of lignocellulosic biomass hydrolysates by Clostridium tyrobutyricum strain RPT-4213. Bioresource Technology. 143:322-329.

Progress 10/01/12 to 09/30/13

Outputs
Progress Report Objectives (from AD-416): Objective 1: Starting with industrial strains of yeast, develop new commercially-viable strains that have (1) improved inhibitor tolerance and (2) wide sugar-substrate specificity for fermenting lignocellulosic hydrolyzates to fuel ethanol. Objective 2: Develop (1) microbial based pretreatment and (2) simultaneous saccharification and fermentation (SSF) technologies that will enable commercially-viable processes for converting lignocellulosic feedstocks to fuel ethanol. Objective 3: Develop novel technologies that enable commercially- preferred processes for producing fuel-grade butanol from lignocellulosic feedstocks. Objective 4: Develop fermentative and enzymatic based technologies that will enable commercially-preferred processes for the production of xylitol from lignocellulose hydrolyzates. Approach (from AD-416): The overall goal of this project is to develop commercially-targeted, integrated bioprocess technologies for production of biofuels and value- added coproducts from lignocellulosic feedstocks. The plan will emphasize microbiologically based approaches to overcome technical constraints that impede industrial applications. Our target is to use corn stover as a model lignocellulosic feedstock for ethanol, butanol, and xylitol production. This research will focus on screening for yeast (Saccharomyces) strains that can tolerate the fermentation inhibitors typically formed during certain pretreatments of lignocellulosic biomass and developing a recombinant S. cerevisiae strain that can efficiently ferment both glucose and xylose derived from lignocellulosic feedstocks. We will develop a microbial pretreatment at the laboratory scale and a simultaneous saccharification and fermentation (SSF) process for production of ethanol from a microbially pretreated feedstock using the recombinant S. cerevisiae strain developed in this project plan. We will identify and characterize the fermentation stimulating/enhancing chemicals present in dilute acid hydrolyzate of wheat straw and develop an integrated SSF with product recovery (SSFR) using ionic liquid or vacuum for efficient production of butanol which is very toxic to the fermentative bacterium. Finally, we will develop batch and fed-batch fermentation processes for production of xylitol from the hemicellulosic hydrolyzates of corn stover and a cell-free enzymatic method with cofactor regeneration for its production. This research project will greatly help to overcome the fermentation related challenges associated with the production of biofuels and coproducts from lignocellulosic feedstocks. Substantial progress was made in all FY13 sub-objectives, which address research needs to develop commercially-targeted, integrated bioprocess technologies for production of biofuels and coproducts from biomass. The sub-objectives emphasize microbiologically based approaches to overcome technical constraints that impede industrial applications. The following are specific examples of significant research developments. Six yeast species were selected from a primary screen for tolerance against inhibitors resulting from biomass pretreatment processes. The selected strains were tested for both ethanol tolerance and productivity relative to a commercial strain and demonstrated ethanol productivity rates and ethanol tolerance that were equal to, and in some cases exceeded, the commercial strain. A microbe, Coniochaeta ligniaria, removes inhibitory compounds from biomass sugars by metabolizing them. To improve removal of acetate, a key inhibitor, acetyl-CoA synthase genes from two yeast strains were isolated. The work was aimed at placing the gene in an appropriate vector for C. ligniaria. Work to sequence the C. ligniaria genome and transcriptome was undertaken which will give insight into inhibitor tolerance. Strains expressing a new enzyme (xylose isomerase, XI) from a rumen bacterium were adapted under aerobic and fermentative growth conditions to generate a strain with improved ability to convert xylose to ethanol. A patent application was filed. The gene for XI was inserted into the genome of an industrial yeast and the strain was adapted for increased ability to produce ethanol. For increased production of a platform chemical triacetic acid lactone (TAL), several industrial yeasts, selected from a screen for yeasts with increased tolerance to inhibitors present in biomass hydrolyzate, were tested for its production. Each strain expressed the 2-pyrone synthase gene. Two different promoters and multiple sugars were evaluated. Using simple batch culture, 1.47 g TAL/L was produced. An integrated process for butanol production efficiently by simultaneous saccharification, fermentation, and recovery (SSFR) from dilute acid pretreated and detoxified (by overliming) corn stover was developed. A vacuum technique was used for recovery. The cost of butanol production by SSFR process from corn stover ($30-60/ton) was estimated by using commercially available software. One hundred five yeast strains were evaluated for their ability to produce xylitol from xylose but no arabitol from arabinose from a mixture of xylose and arabinose as substrate. Ten strains showed ability to produce xylitol from xylose but no arabitol from arabinose. The best performing strain was used to produce xylitol from corn stover hemicellulosic hydrolyzate in batch and fed-batch fermentations. The process of making corn stover hemicellulosic hydrolyzate by dilute sulfuric acid pretreatment was optimized with respect to generation of maximum sugars and minimum formation of fermentation inhibitors. A novel single stage dilute phosphoric acid pretreatment process for efficient production of furfural from corn stover was developed at the laboratory scale. Accomplishments 01 Developed a synthetic promoter for xylose-regulated gene expression in Brewer�s yeast. Brewer�s yeast is the preferred organism for industrial ethanol production. While this organism is extremely efficient at converting glucose to ethanol, it does not naturally use xylose, the second most abundant sugar in lignocellulosic biomass. Enzymes for conversion of xylose to ethanol have been engineered into the organism, but methods for expressing the enzymes only when needed were not available. A xylose-regulated expression system is required to fine-tune gene expression to enable the most efficient use of both sugars. Agricultural Research Service, Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research, Peoria, Illinois, have developed a promoter that allows for the first time in this organism the ability to control gene expression in response to the availability of xylose in the cell. This will improve the efficiency of growth, substrate utilization and produce yield in yeast. This new technology is applicable to any process using biomass- derived sugars. 02 Developed a novel process for making furfural and fuel ethanol from corn stover. Corn stover contains 68% carbohydrates that can be used for production of fuel ethanol and other value-added chemicals. Pretreatment is crucial because any biomass in its native state is resistant to enzymatic hydrolysis. Value-added coproduct or by-product development is necessary in order to reduce the cost of ethanol production from biomass. Agricultural Research Service, Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research, Peoria, Illinois, demonstrated that a single stage dilute phosphoric acid pretreatment of corn stover at high temperature generates furfural with a very good yield and the solid residues containing cellulose after enzymatic hydrolysis can be efficiently fermented to ethanol by using conventional baker�s yeast. Furfural is a useful chemical solvent with multiple industrial uses. These findings are important for development of a commercially viable biomass to furfural and ethanol production processes in a biorefinery approach. 03 Dilute acid pretreatment process of corn stover for ethanol production without removing fermentation inhibitors. Three steps are involved for conversion of corn stover to ethanol: pretreatment, enzymatic hydrolysis and fermentation. During dilute acid pretreatment of corn stover, unwanted compounds are produced that are inhibitory to fermentation. The removal of these inhibitory compounds (detoxification) involves an additional process step which results in 5-10% loss of sugars and adds cost and process complexity. Agricultural Research Service, Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research, Peoria, Illinois, developed a strategy for dilute sulfuric acid pretreatment of corn stover that reduces the formation of inhibitory compounds. As a result, a detoxification step is not required prior to fermentation while also maximizing the sugar yield. This process technology can play an important role in the development of a commercially viable biomass to ethanol conversion technology by reducing processing costs and improving yield.

Impacts
(N/A)

Publications

Qureshi, N., Saha, B.C., Cotta, M.A., Singh, V. 2013. An economic evaluation of biological conversion of wheat straw to butanol: A biofuel. Energy Conversion and Management. 65:456-462.
Qureshi, N., Dien, B.S., Liu, S., Saha, B.C., Hector, R.E., Cotta, M.A., Hughes, S.R. 2012. Genetically engineered Escherichia coli FBR5: Part I. Comparison of high cell density bioreactors for enhanced ethanol production from xylose. Biotechnology Progress. 28(5):1167-1178.
Qureshi, N., Dien, B.S., Liu, S., Saha, B.C., Cotta, M.A., Hughes, S.R., Hector, R.E. 2012. Genetically engineered Escherichia coli FBR5: Part II. Ethanol production from xylose and simultaneous product recovery. Biotechnology Progress. 28(5):1179-1185.
Saha, B.C., Yoshida, T., Cotta, M.A., Sonomoto, K. 2013. Hydrothermal pretreatment and enzymatic saccharification of corn stover for efficient ethanol production. Industrial Crops and Products. 44:367-372.
Avci, A., Saha, B.C., Dien, B.S., Kennedy, G.J., Cotta, M.A. 2013. Response surface optimization of corn stover pretreatment using dilute phosphoric acid for enzymatic hydrolysis and ethanol production. Bioresource Technology. 130:603-612.
Ezeji, T.C., Qureshi, N., Blaschek, H.P. 2013. Microbial production of a biofuel (acetone-butanol-ethanol) in a continuous bioreactor: Impact of bleed and simultaneous product removal. Bioprocess and Biosystems Engineering. 36(1):109-116.
Qureshi, N., Liu, S., Ezeji, T.C. 2012. Cellulosic butanol production from agricultural biomass and residues: Recent advances in technology. In: Lee, J.W., editor. Advanced Biofuels and Bioproducts. New York, NY: Springer Science and Business Media. p. 247-265.
Saha, B.C., Cotta, M.A. 2012. Ethanol production from lignocellulosic biomass by recombinant Escherichia coli strain FBR5. Bioengineered. 3(4) :197-202.
Saha, B.C., Nichols, N.N., Cotta, M.A. 2013. Comparison of separate hydrolysis and fermentation versus simultaneous saccharification and fermentation of pretreated wheat straw to ethanol by Saccharomyces cerevisiae. Journal of Biobased Materials and Bioenergy. 7(3):409-414.
Nichols, N.N., Lunde, T.A., Graden, K.C., Hallock, K.A., Kowalchyk, C.K., Southern, R.M., Soskin, E.J., Ditty, J.L. 2012. Chemotaxis to furan compounds by furan-degrading Pseudomonas strains. Applied and Environmental Microbiology. 78:6365-6368.
Avci, A., Saha, B.C., Kennedy, G.J., Cotta, M.A. 2013. Dilute sulfuric acid pretreatment of corn stover for enzymatic hydrolysis and efficient ethanol production by recombinant Escherichia coli FBR5 without detoxification. Bioresource Technology. 142:312-319.
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.
Biswas, A., Berfield, J.L., Saha, B.C., Cheng, H.N. 2013. Conversion of agricultural by-products to methyl cellulose. Industrial Crops and Products. 46:297-300.

Progress 10/01/11 to 09/30/12

Outputs
Progress Report Objectives (from AD-416): Objective 1: Starting with industrial strains of yeast, develop new commercially-viable strains that have (1) improved inhibitor tolerance and (2) wide sugar-substrate specificity for fermenting lignocellulosic hydrolyzates to fuel ethanol. Objective 2: Develop (1) microbial based pretreatment and (2) simultaneous saccharification and fermentation (SSF) technologies that will enable commercially-viable processes for converting lignocellulosic feedstocks to fuel ethanol. Objective 3: Develop novel technologies that enable commercially- preferred processes for producing fuel-grade butanol from lignocellulosic feedstocks. Objective 4: Develop fermentative and enzymatic based technologies that will enable commercially-preferred processes for the production of xylitol from lignocellulose hydrolyzates. Approach (from AD-416): The overall goal of this project is to develop commercially-targeted, integrated bioprocess technologies for production of biofuels and value- added coproducts from lignocellulosic feedstocks. The plan will emphasize microbiologically based approaches to overcome technical constraints that impede industrial applications. Our target is to use corn stover as a model lignocellulosic feedstock for ethanol, butanol, and xylitol production. This research will focus on screening for yeast (Saccharomyces) strains that can tolerate the fermentation inhibitors typically formed during certain pretreatments of lignocellulosic biomass and developing a recombinant S. cerevisiae strain that can efficiently ferment both glucose and xylose derived from lignocellulosic feedstocks. We will develop a microbial pretreatment at the laboratory scale and a simultaneous saccharification and fermentation (SSF) process for production of ethanol from a microbially pretreated feedstock using the recombinant S. cerevisiae strain developed in this project plan. We will identify and characterize the fermentation stimulating/enhancing chemicals present in dilute acid hydrolyzate of wheat straw and develop an integrated SSF with product recovery (SSFR) using ionic liquid or vacuum for efficient production of butanol which is very toxic to the fermentative bacterium. Finally, we will develop batch and fed-batch fermentation processes for production of xylitol from the hemicellulosic hydrolyzates of corn stover and a cell-free enzymatic method with cofactor regeneration for its production. This research project will greatly help to overcome the fermentation related challenges associated with the production of biofuels and coproducts from lignocellulosic feedstocks. Substantial progress was made in all FY12 sub-objectives of this project, which address research needs to develop commercially-targeted, integrated bioprocess technologies for production of biofuels from biomass. The sub- objectives emphasize microbiologically based approaches to overcome technical constraints that impede industrial applications. The following are specific examples of significant research developments. Over 150 yeast strains, of mixed genetic background and from a variety of habitats, were screened for tolerance against inhibitors resulting from biomass pretreatment processes. A number of strains showed promise with tolerance to inhibitors that exceed existing production and experimental strains. Ability of a bioabatement microbe to remove acetate from biomass hydrolyzate was determined. Using conventional yeast and strains engineered to ferment xylose, the effect of acetate on conversion of rice hull hydrolyzate to ethanol was examined. Acetate had a negative effect on xylose consumption by the engineered yeast strain. We discovered that natural xylose utilizing yeasts are able to produce several proteins required for xylose use, while brewers� yeast could not. Initial attempts to induce these genes led to growth inhibition due to inappropriate expression when the proteins were not needed. To fix this problem, we developed a new method to express the proteins only when needed. This method allows for control of protein production in response to xylose availability. Several enzymes for xylose use were identified from intestinal bacteria. The best enzyme was expressed in yeast which was then adapted for improved growth on xylose. This adapted yeast grows four- fold better on xylose and is being adapted further for better ethanol production. To develop microbial based pretreatment of biomass, we optimized the conditions for pretreatment of corn stover by three white rot fungal strains. Simultaneous saccharification and fermentations of corn stover pretreated under optimized conditions using one fungal strain were investigated using a laboratory yeast strain, a recombinant xylose utilizing yeast, and a mixed sugar utilizing ethanologenic recombinant bacterium. Good yields of fuel ethanol were achieved from corn stover pretreated by the fungus in all cases which demonstrate that pretreatment of corn stover by the selected fungus is an effective pretreatment option. Separation of butanol from fermentation broth using ionic liquids was investigated. The selected ionic liquid was found to have a high rate of butanol removal capacity from the fermentation. However, recycling of the ionic liquid encountered a number of problems such as high density, high viscosity, and difficulty in separating suspended particles. For this reason, a new product separation technique called vacuum fermentation was investigated. Using vacuum fermentation, simultaneous production, and recovery of butanol were efficiently achieved which successfully relieved the product toxicity. Accomplishments 01 Developed a method to recover butanol biofuel from fermentation broth. Butanol can be produced from economically available agricultural residue such as corn stover, wheat straw, and barley straw by fermentation where bacterial culture converts the sugars generated from agricultural residu into butanol. However, to make butanol production economically viable, butanol should be removed simultaneously (as it is produced) from the fermentation vessel because butanol above a certain concentration inhibi its further production by the bacterium. Agricultural Research Service (ARS), Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research, Peoria, IL, developed a novel product recovery technique that allows simultaneous recovery of butanol from the fermentation medium. In this process, vacuum was applied to the fermentation vessel where butanol was produced. As a result of vacuum, butanol was recovered simultaneously. The newly developed process enable more efficient production of butanol by fermentation. 02 Ethanol production from microbially pretreated corn stover. Typically, harsh chemical methods using acid and high temperature are used to pretreat corn stover before its breakdown to sugars by enzymes. This costly and energy-intensive pretreatment step is necessary because witho it, the corn stover is very resistant to breakdown by enzymes. However, the harsh pretreatment also generates compounds that inhibit the process for producing ethanol from sugars derived from corn stover. Agricultural Research Service (ARS), Bioenergy Research Unit scientists at the Nation Center for Agricultural Utilization Research, Peoria, IL, have found tha corn stover pretreated with a white rot fungus could be converted to fermentable sugars without production of inhibitors. The pretreated corn stover was converted to ethanol in good yield by simultaneous saccharification and fermentation. This demonstrates that pretreatment o corn stover by the fungal strain under optimized conditions is an effective pretreatment option. 03 Discovered chemotaxis to furans by furan-metabolizing bacteria. Furan molecules occur naturally in the environment and are also important because they interfere with green processes to convert biomass to fuels and chemicals. Agricultural Research Service (ARS), Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research, Peoria, IL, working with a scientist at the University of St. Thomas, St. Paul, MN, showed that some bacteria exhibit chemotaxis to furans. Chemotaxis is a behavior shown by bacteria, in which they detect and migrate to food sources in their environment. This finding showed th some bacteria have a behavioral response to an entirely new class of compounds that had not previously been examined. This framework for understanding furan metabolism contributes to efforts both to degrade furans in situations where they are undesirable (such as in biofuel production) and to make useful chemical building blocks from furans. 04 Robust yeast strains for biofuels and bioproducts. Although a number of different pretreatment processes have been undertaken to reduce inhibito substances in biomass feedstocks, challenges remain as the inhibitory compounds reduce productivity and cost-effectiveness. To overcome the problem associated with inhibitors, Agricultural Research Service (ARS), Bioenergy Research Unit scientists at the National Center for Agricultur Utilization Research, Peoria, IL, have collected and screened for yeast strains which are inhibitor-resistant with a number of strains demonstrating inhibitor tolerance that is equal to or greater than existing production and/or experimental strains. These inhibitor toleran yeast strains will serve as a platform for development and afford the opportunity to improve productivity and cost-effectiveness of biofuels a bioproducts.

Impacts
(N/A)

Publications

Saha, B.C., Nichols, N.N., Qureshi, N., Cotta, M.A. 2011. Comparison of separate hydrolysis and fermentation and simultaneous saccharification and fermentation processes for ethanol production from wheat straw by recombinant Escherichia coli strain FBR5. Applied Microbiology and Biotechnology. 92:865-874.
Richter, H., Qureshi, N., Heger, S., Dien, B.S., Cotta, M.A., Angenent, L. T. 2012. Prolonged conversion of n-butyrate to n-butanol with Clostridium saccharoperbutylacetonicum in a two-stage continuous culture with in-situ product removal. Biotechnology and Bioengineering. 109:913-921.
Mariano, A.P., Qureshi, N., Filho, R.M., Ezeji, T.C. 2012. Assessment of in situ butanol recovery by vacuum during acetone butanol ethanol (ABE) fermentation. Journal of Chemical Technology and Biotechnology. 87:334-340.
Hector, R.E., Dien, B.S., Cotta, M.A., Qureshi, N. 2011. Engineering industrial Saccharomyces cerevisiae strains for xylose fermentation and comparison for switchgrass conversion. Journal of Industrial Microbiology and Biotechnology. 38(9):1193-1202.
Xu, J., Mohamed, A., Qureshi, N. 2011. The viscoelastic properties of the protein-rich materials from the fermented hard wheat, soft wheat and barley flours. International Journal of Agricultural Research. 6(4):347- 357.
Mariano, A.P., Qureshi, N., Filho, R.M., Ezeji, T.C. 2011. Bioproduction of butanol in bioreactors: new insights from simultaneous in situ butanol recovery to eliminate product toxicity. Biotechnology and Bioengineering. 108(8):1757-1765.
Hector, R.E., Mertens, J.A., Bowman, M.J., Nichols, N.N., Cotta, M.A., Hughes, S.R. 2011. Saccharomyces cerevisiae engineered for xylose metabolism requires gluconeogenesis and the oxidative branch of the pentose phosphate pathway for aerobic xylose assimilation. Yeast. 28:645- 660.
Qureshi, N., Bowman, M.J., Saha, B.C., Hector, R.E., Berhow, M.A., Cotta, M.A. 2012. Effect of cellulosic sugar degradation products (furfural and hydroxymethylfurfural) on acetone-butanol-ethanol (ABE) fermentation using Clostridium beijerinckii P260. Journal of Food and Bioproducts Processing. 90:533-540.
Liu, S., Bischoff, K.M., Leathers, T.D., Qureshi, N., Rich, J.O., Hughes, S.R. 2012. Adaptation of lactic acid bacteria to butanol. Biocatalysis and Agricultural Biotechnology. 1(1):57-61. DOI:
Saha, B.C., Nichols, N.N., Cotta, M.A. 2011. Ethanol production from wheat straw by recombinant Escherichia coli strain FBR5 at high solid loading. Bioresource Technology. 102(23):10892-10897.
Hughes, S.R., Moser, B.R., Robinson, S., Cox, E.J., Harmsen, A.J., Friesen, J.A., Bischoff, K.M., Jones, M.A., Pinkleman, R., Bang, S.S., Tasaki, K., Doll, K.M., Qureshi, N., Liu, S., Saha, B.C., Jackson, Jr., J.S., Cotta, M. A., Rich, J.O., Caimi, P. 2012. Synthetic resin-bound truncated Candida antarctica lipase B for production of fatty acid alkyl esters by transesterification of corn and soybean oils with ethanol or butanol. Journal of Biotechnology. 159:69-77. DOI: 10.1016/j.jbiotec.2012.01.025.

Progress 10/01/10 to 09/30/11

Outputs
Progress Report Objectives (from AD-416) Objective 1: Starting with industrial strains of yeast, develop new commercially-viable strains that have (1) improved inhibitor tolerance and (2) wide sugar-substrate specificity for fermenting lignocellulosic hydrolyzates to fuel ethanol. Objective 2: Develop (1) microbial based pretreatment and (2) simultaneous saccharification and fermentation (SSF) technologies that will enable commercially-viable processes for converting lignocellulosic feedstocks to fuel ethanol. Objective 3: Develop novel technologies that enable commercially- preferred processes for producing fuel-grade butanol from lignocellulosic feedstocks. Objective 4: Develop fermentative and enzymatic based technologies that will enable commercially-preferred processes for the production of xylitol from lignocellulose hydrolyzates. Approach (from AD-416) The overall goal of this project is to develop commercially-targeted, integrated bioprocess technologies for production of biofuels and value- added coproducts from lignocellulosic feedstocks. The plan will emphasize microbiologically based approaches to overcome technical constraints that impede industrial applications. Our target is to use corn stover as a model lignocellulosic feedstock for ethanol, butanol, and xylitol production. This research will focus on screening for yeast (Saccharomyces) strains that can tolerate the fermentation inhibitors typically formed during certain pretreatments of lignocellulosic biomass and developing a recombinant S. cerevisiae strain that can efficiently ferment both glucose and xylose derived from lignocellulosic feedstocks. We will develop a microbial pretreatment at the laboratory scale and a simultaneous saccharification and fermentation (SSF) process for production of ethanol from a microbially pretreated feedstock using the recombinant S. cerevisiae strain developed in this project plan. We will identify and characterize the fermentation stimulating/enhancing chemicals present in dilute acid hydrolyzate of wheat straw and develop an integrated SSF with product recovery (SSFR) using ionic liquid or vacuum for efficient production of butanol which is very toxic to the fermentative bacterium. Finally, we will develop batch and fed-batch fermentation processes for production of xylitol from the hemicellulosic hydrolyzates of corn stover and a cell-free enzymatic method with cofactor regeneration for its production. This research project will greatly help to overcome the fermentation related challenges associated with the production of biofuels and coproducts from lignocellulosic feedstocks. To develop new yeast strains with improved inhibitor tolerance, over 100 yeast strains were collected from various sources to uncover strains that are more tolerant of inhibitors resulting from biomass pretreatment processes. Initial stage of the primary screen has been completed. A number of strains show promise for continuation in the primary screen and movement to the secondary screen. To identify genes for metabolism of fermentation inhibitors, the inhibitor abating fungal strain was subjected to ultraviolet (UV) mutagenesis and screened for the ability to grow on furoic acid. One mutant was identified that could not utilize furoic acid for growth, but grew on rich nutrients. We also isolated a second mutant that cannot grow on xylose and discovered that it also cannot grow on furfural, a fermentation inhibitor. To engineer yeast for xylose fermentation, xylose transporters were expressed in a yeast strain and assayed for xylose uptake and metabolism. For two of the transporters, cell growth in xylose was improved. Additionally, we identified a major barrier to xylose metabolism. Brewer�s yeast, grown on xylose medium, was not able to regulate key genes involved in metabolism that are found, and elevated, in yeasts with the natural ability to use xylose. Failure to induce these enzymes on xylose medium correlated with increased sensitivity to fermentation inhibitors. These results highlight an important area for improving brewer�s yeast. Separately, an improved enzyme for xylose utilization was identified from a rumen bacterium. Yeast expressing this enzyme showed increased xylose use and growth. To develop microbial based pretreatments, we screened 27 white rot fungal strains in 2010 for powerful delignification ability, growing them under solid state cultivation (SSC) using corn stover (CS) as feedstock. This year, the best three performing strains with respect to maximum lignin and minimum holocellulose degradation, maximum ligninolytic enzymes and minimum cellulase and xylanase activities, and maximum sugar release after enzymatic hydrolysis were used for optimizing SSC on corn stover. First, the effect of cultivation time on lignin and holocellulose degradation in SSC of CS over a period of 42 days by the 3 fungal strains was determined. Then the effects of moisture content and inoculums size on each fungal pretreatment were determined using response surface methodology. To develop novel processes for butanol production from biomass by fermentation, in 2010 we identified two fermentation enhancing compounds, furfural and hydroxymethyl furfural (HMF), in the dilute acid pretreated and enzymatically saccharified wheat straw hydrolyzate. This enhanced the butanol productivity from glucose by an anaerobic bacterium by at least two fold. This year, we found that these two fermentation enhancers could not stimulate the production of butanol by the bacterium from dilute acid pretreated and enzymatically saccharified corn stover hydrolyzate (CSH) detoxified by overliming. We were then able to enhance the butanol productivity by the anaerobic bacterium from the detoxified CSH by 14 fold using cell recycle technique. Accomplishments 01 Efficient production of ethanol by a newly modified yeast from a major sugar derived from agricultural residues and energy crops. To economically convert agricultural residues and energy crops (often referred to as biomass) to ethanol, microorganisms capable of efficientl using three to five kinds of sugars, typically generated from any biomas are required. The yeast, commonly used for producing ethanol from corn commercially, cannot use a major sugar present in the generated sugar mixture. Agricultural Research Service (ARS) Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, have discovered that the yeast does not make certai proteins at the levels needed for efficient utilization of this major sugar for ethanol production from it. They were able to genetically modi the yeast which overcomes this limitation and produces ethanol with increased ability to use the major sugar. The newly modified yeast produces more ethanol than the original yeast from biomass derived sugar which reduces the production cost of ethanol from agricultural residues and energy crops. A patent application is in process and the yeast strai has been supplied to a number of researchers world-wide. 02 Developed a genetic system for a useful fungus for making valuable chemicals. The fungus, isolated from soil, is useful for cleaning up th sugars obtained from agricultural residues and energy crops, which are often referred to as biomass. The sugars, once cleaned up so that they a useable, can be converted to fuel ethanol in a second step. Agricultural Research Service (ARS) Biorenergy Research Unit scientists at the Nation Center for Agricultural Utilization Research (NCAUR), Peoria, IL, have made the fungus even more useful, beyond its native ability to remove th toxic compounds from the biomass derived sugars. They have developed genetic tools that can be used to modify the fungus to directly convert the biomass derived sugars to fuel ethanol while removing the toxic compounds, rather than requiring the second step. These genetic tools wi be used to develop the fungus as a platform for producing valuable chemical building blocks, such as lactic acid (a biodegradable plastic component) from biomass derived sugars. 03 Enhancement of breakdown of agricultural residues to sugars by fungal pretreatment. Typically, harsh chemical methods using acid and high temperature are used to pretreat agricultural residues before their breakdown to sugars by enzymes. This costly and energy-intensive pretreatment step is necessary because the agricultural residues are ver resistant to breakdown by enzymes without it. But the harsh pretreatment process also generates compounds that inhibit the process for producing ethanol from sugars derived from agricultural residues. Agricultural Research Service (ARS) Bioenergy Research Unit scientists at the Nationa Center for Agricultural Utilization Research, Peoria, IL, have found a fungal strain with powerful ability to remove lignin (a component of agricultural residues that acts as a barrier of generating sugars from them) from agricultural residues. There was significant enhancement of breakdown of corn stover to sugars by enzymes after this fungal pretreatment which was mild, performed at room temperature, and did not generate any inhibitory compounds. This research demonstrates that pretreatment with a lignin degrading fungus can be used for pretreatment of agricultural residues prior to their breakdown to sugars by enzymes. 04 Enhancing butanol biofuel production rate from corn stover. Butanol is next generation advanced biofuel that can be produced from agricultural residues such as corn stover. Agricultural Research Service (ARS) Bioenergy Research Unit scientists at the National Center for Agricultur Utilization Research (NCAUR), Peoria, IL, have developed a highly efficient process (cell recycle) for producing butanol by an anaerobic (not needing oxygen for growth) bacterium from corn stover derived sugar Butanol production rate was enhanced 14 fold. The sugars were generated from corn stover after breakdown of dilute acid pretreated corn stover b enzymes. The newly developed process enables more efficient and cost- effective production of butanol biofuel from corn stover.

Impacts
(N/A)

Publications

Qureshi, N. 2010. Agricultural residues and energy crops as potentially economical and novel substrates for microbial production of butanol (a biofuel). Commonwealth Agricultural Bureaux International. 5(59):1-8.
Saha, B.C., Cotta, M.A. 2011. Continuous ethanol production from wheat straw hydrolysate by recombinant ethanologenic Escherichia coli strain FBR5. Applied Microbiology and Biotechnology. 90(2):477-487.
Saha, B.C., Racine, F.M. 2011. Biotechnological production of mannitol and its application. Applied Microbiology and Biotechnology. 89(4):879-891.
Qureshi, N., Hughes, S.R., Ezeji, T. 2010. Production of liquid biofuels from biomass: Emerging technologies. In: Blaschek, H.P., Ezeji, T.C., Scheffran, J., editors. Biofuels from Agricultural Wastes and Byproducts. Ames, IA: Wiley-Blackwell. p. 11-18.
Dunlap, C.A., Jackson, M.A., Saha, B.C. 2010. Compatible solutes of sclerotia of Mycoleptodiscus terrestris under different culture and drying conditions. Biocontrol Science and Technology. 21(1):113-123.
Liu, S., Bischoff, K.M., Qureshi, N., Hughes, S.R., Rich, J.O. 2010. Functional expression of the thiolase gene THl from Clostridium beijerinckii P260 in Lactococcus lactis and Lactobacillus buchneri. New Biotechnology. 27(4):283-288.
Nichols, N.N., Szynkarek, M., Skory, C.D., Gorsich, S.W., Lopez, M.J., Guisado, G.M., Nichols, W.A. 2011. Transformation and electrophoretic karyotyping of Coniochaeta ligniaria NRRL30616. Current Genetics. 57(3) :169-175.
Hughes, S.R., Moser, B.R., Harmsen, A.J., Bischoff, K.M., Jones, M.A., Pinkelman, R., Bang, S.S., Tasaki, K., Doll, K.M., Qureshi, N., Liu, S., Saha, B.C., Jackson Jr, J.S., Cotta, M.A., Rich, J.O., Caimi, P. 2010. Production of Candida antaractica Lipase B gene open reading frame using automated PCR gene assembly protocol on robotic workcell and expression in ethanologenic yeast for use as resin-bound biocatalyst in biodiesel production. Journal of the Association for Laboratory Automation. 16(1):17- 37. DOI: 10.1016/j.jala.2010.04.002.

Progress 10/01/09 to 09/30/10

Outputs
Progress Report Objectives (from AD-416) Objective 1: Starting with industrial strains of yeast, develop new commercially-viable strains that have (1) improved inhibitor tolerance and (2) wide sugar-substrate specificity for fermenting lignocellulosic hydrolyzates to fuel ethanol. Objective 2: Develop (1) microbial based pretreatment and (2) simultaneous saccharification and fermentation (SSF) technologies that will enable commercially-viable processes for converting lignocellulosic feedstocks to fuel ethanol. Objective 3: Develop novel technologies that enable commercially- preferred processes for producing fuel-grade butanol from lignocellulosic feedstocks. Objective 4: Develop fermentative and enzymatic based technologies that will enable commercially-preferred processes for the production of xylitol from lignocellulose hydrolyzates. Approach (from AD-416) The overall goal of this project is to develop commercially-targeted, integrated bioprocess technologies for production of biofuels and value- added coproducts from lignocellulosic feedstocks. The plan will emphasize microbiologically based approaches to overcome technical constraints that impede industrial applications. Our target is to use corn stover as a model lignocellulosic feedstock for ethanol, butanol, and xylitol production. This research will focus on screening for yeast (Saccharomyces) strains that can tolerate the fermentation inhibitors typically formed during certain pretreatments of lignocellulosic biomass and developing a recombinant S. cerevisiae strain that can efficiently ferment both glucose and xylose derived from lignocellulosic feedstocks. We will develop a microbial pretreatment at the laboratory scale and a simultaneous saccharification and fermentation (SSF) process for production of ethanol from a microbially pretreated feedstock using the recombinant S. cerevisiae strain developed in this project plan. We will identify and characterize the fermentation stimulating/enhancing chemicals present in dilute acid hydrolyzate of wheat straw and develop an integrated SSF with product recovery (SSFR) using ionic liquid or vacuum for efficient production of butanol which is very toxic to the fermentative bacterium. Finally, we will develop batch and fed-batch fermentation processes for production of xylitol from the hemicellulosic hydrolyzates of corn stover and a cell-free enzymatic method with cofactor regeneration for its production. This research project will greatly help to overcome the fermentation related challenges associated with the production of biofuels and coproducts from lignocellulosic feedstocks. The harsh methods used to generate usable sugars from biomass result in sugar mixtures that are hard to work with, because inhibitors are formed along with the sugars. Inhibitors, especially the type known as furans, block efficient conversion of the sugars to products because they are toxic to microbes used to carry out conversion. Some microbes, however, can actually grow on furans. Toward understanding furan metabolism, transposon promoter-probe mutagenesis was carried out to identify bacterial genes that are activated in the presence of furoic acid. The disrupted genes were sequenced and compared to a database of known genes to determine the nature of the mutations. From nine mutants, five distinct gene sequences were identified, three of which had not previously been associated with furan metabolism. This may help us design a way to clean up the furans in biomass sugars or engineer fermenting microbes to tolerate the inhibitors. We have made significant progress in developing industrial yeast strains with improved fermentation capacity using biomass hydrolyzate feedstocks. Several industrial strains have been engineered by integrating xylose utilizing genes from another yeast strain to ferment xylose to ethanol. The ethanol production by these engineered strains from switchgrass hydrolyzate was increased by 15% compared to parent strains. Additionally, combining the first two enzymatic steps of the xylose metabolism in yeast into a single reaction could lead to further improvements. To address this, an improved enzyme (xylose isomerase) for xylose utilization was identified from a rumen bacterium. This novel enzyme was expressed in a haploid laboratory yeast strain for screening and analysis. We have screened 27 carefully selected basidomycete (white rot fungus) strains for powerful delignification ability growing them under solid state cultivation using corn stover as feedstock. There was a wide variability of delignification ability among the strains studied. Five strains showed promise as an option for microbial pretreatment of lignocellulosic feedstock for enhanced enzymatic saccharification. Fermentation of dilute acid pretreated and enzymatically saccharified wheat straw hydrolyzate demonstrated two fold improvement in acetone butanol ethanol (ABE) productivity by an anaerobic bacterium in comparison to glucose fermentations. It was speculated that this enhancement in productivity was due to the presence of one or more fermentation stimulators present in the hydrolyzate. Detailed analysis of the organic solvent extracted material of the wheat straw hydrolyzate confirmed that hydroxymethyl furfural and furfural at certain concentrations were responsible for enhancement of ABE productivity. Such an increase in productivity would greatly help to reduce the production cost of butanol. Corn stover is an important feedstock that can be converted to butanol. It was successfully fermented to butanol (ABE) in very good yield after pretreatment with dilute acid, enzymatic saccharification, overliming to remove the fermentation inhibitors, and fermentation of the overlimed hydrolyzate using an anaerobic bacterium. Accomplishments 01 Development of industrial xylose-fermenting yeast strains. To economical convert lignocellulosic materials to ethanol and other bio-based product at industrial scale, biocatalysts (i.e., microorganisms) capable of fermenting both hexose and pentose sugars from biomass feedstocks are required. Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research in Peoria, IL, have engineered several industrial Saccharomyces strains to ferment xylose. These yeasts were evaluated for their xylose fermentation capability using lignocellulosic hydrolyzate as feedstock. Several industrial yeasts were identified with superior performance compared to laboratory strains and other industrial yeasts included in the analysis. These strains, and the materials to engineer other industrial yeasts, have been made available to other university researchers and are advancing biofuel research nationally and internationally. 02 Identification of stimulators of butanol (a biofuel) fermentation. Butan is a next generation biofuel that can be produced by fermentation from lignocellulosic hydrolyzates. Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research in Peoria, IL, hav discovered that dilute acid pretreated and enzymatically saccharified wheat straw hydrolyzate contains stimulators of butanol fermentation tha enhance the rate of butanol production by an anaerobic bacterium by a factor of two or more. They were able to identify two fermentation stimulating compounds in the wheat straw hydrolyzate and their dose leve for enhancement of butanol fermentation. An increase in fermentation rat of this magnitude would bring production of butanol closer to commercialization by reducing butanol production costs. 03 Conversion of corn stover to butanol. Corn stover is an important lignocellulosic feedstock that is economically available in the Midwest for production of butanol. Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research in Peoria, IL, hav successfully converted corn stover to butanol (ABE) in very good yield after pretreatment with dilute acid, enzymatic saccharification, overliming to remove the fermentation inhibitors, and fermentation of th detoxified hydrolyzate using an anaerobic bacterium. These results are important for developing process technologies for butanol production fro corn stover. 04 Fuel ethanol production from wheat straw: demonstration of technology at the 100 liter scale. Wheat straw, a globally abundant byproduct of wheat production, contains about 70% carbohydrates that can serve as a low cos feedstock for conversion to fuel ethanol. Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research Peoria, IL, have developed a small pilot scale (100 L) process for converting wheat straw to ethanol. The process consists of pretreating t straw with dilute acid, detoxifying the pretreated hydrolyzate with a novel fungal strain able to utilize the generated fermentation inhibitor prior to sugar consumption. A good yield of ethanol was obtained from th bioabated wheat straw hydrolyzate by simultaneous saccharification using commercial cellulase enzymes and fermentation using a recombinant bacterium capable of producing ethanol from multiple sugars. The need fo sterilization of the pretreated feedstock to solve contamination problem was easily met by using a very low level of an antibiotic commonly used corn to ethanol fermentation. The developed process will serve as a prototype for developing a wheat straw to ethanol conversion technology commercially.

Impacts
(N/A)

Publications

Qureshi, N., Saha, B.C., Dien, B., Hector, R.E., Cotta, M.A. 2010. Production of Butanol (a Biofuel) from Agricultural Residues: Part I - Use of Barley Straw Hydrolysate. Biomass and Bioenergy. 34(4):559-565.
Qureshi, N., Saha, B.C., Hector, R.E., Dien, B., Hughes, S., Liu, S., Iten, L., Bowman, M.J., Sarath, G., Cotta, M.A. 2010. Production of butanol (a Biofuel) from agricultural residues: Part II - Use of corn stover and switchgrass hydrolysates. Biomass and Bioenergy. 34(4):566-571.
Saha, B.C., Cotta, M.A. 2010. Comparison of Pretreatment Strategies for Enzymatic Saccharification and Fermentation of Barley Straw to Ethanol. New Biotechnology. 27(1):10-16.
Hughes, S.R., Hector, R.E., Rich, J.O., Qureshi, N., Bischoff, K.M., Dien, B.S., Saha, B.C., Liu, S., Jackson Jr, J.S., Sterner, D.E., Butt, T.R., Labaer, J., Cotta, M.A. 2009. Automated yeast mating protocol using open reading frames from Saccharomyces cerevisiae genome to improve yeast strains for cellulosic ethanol production. Journal of the Association for Laboratory Automation. 8:190-199.
Hughes, S.R., Rich, J.O., Bischoff, K.M., Hector, R.E., Qureshi, N., Saha, B.C., Dien, B.S., Liu, S., Jackson Jr, J.S., Sterner, D.E., Butt, T.R., Labaer, J., Cotta, M.A. 2009. Automated yeast transformation protocol to engineer S. cerevisiae strains for cellulosic ethanol production with open reading frames that express proteins binding to xylose isomerase identified using robotic two-hybrid screen. Journal of the Association for Laboratory Automation. 8:200-212.
Saha, B.C., Racine, F.M. 2010. Effects of pH and Corn Steep Liquor Variability on Mannitol Production by Lactobacillus intermedius NRRL B- 3693. Applied Microbiology and Biotechnology. 87(2):553-560.
Hughes, S.R., Qureshi, N. 2010. Biofuel demand realization. In: Vertes, A., Qureshi, N., Blascheck, H.P., Yukawa, H., editors. Biomass to Biofuels: Strategies to Global Industries. UK:John Wiley & Sons Limited. p. 55-69.
Skory, C.D., Hector, R.E., Gorsich, S., Rich, J.O. 2010. Analysis of a functional lactate permease in the fungus Rhizopus. Enzyme and Microbial Technology. 46(1):43-50.
Qureshi, N., Blaschek, H.P. 2010. Clostridia and Process Engineering for Energy Generation. In: Vertes, A.A., Qureshi, N., Blaschek, H.F., Yukawa, H., editors. Biomass to Biofuels. Strategies for Global Industries. United Kingdom: Wiley and Sons. p. 347-358.
Ditty, J.L., Nichols, N.N., Parales, R.E. 2010. Measurement of Hydrocarbon Transport in Bacteria. In: Timmis, K.N., editor. Handbook of Hydrocarbon and Lipid Microbiology. Berlin Heidelberg: Springer-Verlag. p. 4213-4222.
Qureshi, N. 2010. Beneficial Biofilms: Wastewater and Other Industrial Applications. In: Fratamico, P.M., Annous, B.A., Gunther IV, N.W., editors. Biofilms in Food and Beverage Industries. Oxford: Woodhead Publishing Limited. p. 474-498.
Liu, S., Qureshi, N. 2009. How microbes tolerate ethanol and butanol. New Biotechnology. 26(3/4):117-121.
Hector, R.E., Bowman, M.J., Skory, C.D., Cotta, M.A. 2009. The Saccharomyces cerevisiae YMR315W gene encodes an NADP(H)-specific Oxidoreductase regulated by the transcription factor Stb5p in response to NADPH limitation. New Biotechnology. 26(3/4):171-180.