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United States Department of Agriculture

Agricultural Research Service


Location: Bioenergy Research

2011 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
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.

4. Accomplishments

Review 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.

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.

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.

Saha, B.C., Racine, F.M. 2011. Biotechnological production of mannitol and its application. Applied Microbiology and Biotechnology. 89(4):879-891.

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.

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.

Last Modified: 10/17/2017
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