2012 Annual Report
1a.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.
1b.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.
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 without 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 National Center for Agricultural Utilization Research, Peoria, IL, have found that 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 of corn stover by the fungal strain under optimized conditions is an effective pretreatment option.
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 that 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.
Robust yeast strains for biofuels and bioproducts. Although a number of different pretreatment processes have been undertaken to reduce inhibitory 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 Agricultural 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 tolerant yeast strains will serve as a platform for development and afford the opportunity to improve productivity and cost-effectiveness of biofuels and bioproducts.
Developed a method to recover butanol biofuel from fermentation broth. Butanol can be produced from economically available agricultural residues such as corn stover, wheat straw, and barley straw by fermentation where a bacterial culture converts the sugars generated from agricultural residues 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 inhibits 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 enables more efficient production of butanol by fermentation.
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: http://dx.doi.org/10.1016/j.bcab.2011.08.008.
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.