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.
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.
Developed a genetic system for a useful fungus for making valuable chemicals. The fungus, isolated from soil, is useful for cleaning up the sugars obtained from agricultural residues and energy crops, which are often referred to as biomass. The sugars, once cleaned up so that they are useable, can be converted to fuel ethanol in a second step. Agricultural Research Service (ARS) Biorenergy Research Unit scientists at the National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, have made the fungus even more useful, beyond its native ability to remove the 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 will 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.
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 very 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 National 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.
Enhancing butanol biofuel production rate from corn stover. Butanol is a 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 Agricultural 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 sugars. Butanol production rate was enhanced 14 fold. The sugars were generated from corn stover after breakdown of dilute acid pretreated corn stover by enzymes. The newly developed process enables more efficient and cost-effective production of butanol biofuel from corn stover.
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 efficiently using three to five kinds of sugars, typically generated from any biomass, 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 certain proteins at the levels needed for efficient utilization of this major sugar for ethanol production from it. They were able to genetically modify 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 sugars which reduces the production cost of ethanol from agricultural residues and energy crops. A patent application is in process and the yeast strain has been supplied to a number of researchers world-wide.
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.