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United States Department of Agriculture

Agricultural Research Service


Location: Bioenergy Research

2013 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:
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.

4. Accomplishments
1. 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.

2. 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.

3. 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.

Review 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.

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., 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.

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.

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.

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.

Last Modified: 08/21/2017
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