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United States Department of Agriculture

Agricultural Research Service

Research Project: Process Technologies for Producing Biofuels and Coproducts from Lignocellulosic Feedstocks

Location: Bioenergy Research Unit

2010 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.

3.Progress Report
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.

1. Development of industrial xylose-fermenting yeast strains. To economically convert lignocellulosic materials to ethanol and other bio-based products 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.

2. Identification of stimulators of butanol (a biofuel) fermentation. Butanol 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, have discovered that dilute acid pretreated and enzymatically saccharified wheat straw hydrolyzate contains stimulators of butanol fermentation that 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 levels for enhancement of butanol fermentation. An increase in fermentation rate of this magnitude would bring production of butanol closer to commercialization by reducing butanol production costs.

3. 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, have 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 the detoxified hydrolyzate using an anaerobic bacterium. These results are important for developing process technologies for butanol production from corn stover.

4. 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 cost feedstock for conversion to fuel ethanol. Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research in Peoria, IL, have developed a small pilot scale (100 L) process for converting wheat straw to ethanol. The process consists of pretreating the straw with dilute acid, detoxifying the pretreated hydrolyzate with a novel fungal strain able to utilize the generated fermentation inhibitors prior to sugar consumption. A good yield of ethanol was obtained from the 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 for sterilization of the pretreated feedstock to solve contamination problem was easily met by using a very low level of an antibiotic commonly used in corn to ethanol fermentation. The developed process will serve as a prototype for developing a wheat straw to ethanol conversion technology commercially.

Review Publications
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.

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.

Last Modified: 4/15/2014
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