2008 Annual Report 1a.Objectives (from AD-416) Develop cost-effective pretreatment, enzymatic saccharification, fermentation and downstream processing technologies, and their integration for production of biofuels from lignocellulosic biomass. 1b.Approach (from AD-416) The overall goal is to produce biofuel from waste lignocellulosic agricultural residues and processing byproducts at a price competitive with corn starch-based fermentation process. Specific approaches have the following components: 1. Developing an effective pretreatment strategy that will greatly increase the efficiency of enzymatic hydrolysis of lignocellulose. For this, various pretreatment options will be evaluated. Dilute acid pretreatment at high temperature generates furfural, hydroxymethyl furfural, levulinic acid, and unknown aromatic compounds which are inhibitory to fermentative microorganisms. Chemical and biological methods will be explored to detoxify these hydrolyzates. Mechanisms of such detoxification will be studied. 2. Developing high productivity fermentation system for production of biofuels (ethanol, butanol) from lignocellulosic hydrolyzates. For this, batch and continuous fermentations with cell recycle will be studied. Tolerance of the fermentative microorganism to high substrate, fermentation inhibitors, organic acids, and alcohols will be studied with an aim to identify robust organisms. 3. Pervaporation, gas stripping, and liquid-liquid extraction as well as conventional distillation methods will be investigated to economically recover biofuels from dilute fermentation broths. 4. In order to be economic, the steps involved in any bioconversion process need to be integrated. Research will be conducted to integrate enzymatic saccharification, fermentation, and downstream processing technologies for production of biofuels. The technologies for both ethanol and butanol production will be demonstrated at 100-L scale, and a preliminary cost analysis for each process will be performed. 3.Progress Report The conventional yeast strain cannot utilize xylose, limiting the amount of ethanol that can be made from biomass feedstocks. Yeast strains have been engineered to ferment xylose. However, they consume xylose at sub-optimal rates for industrial use. One limitation in these strains is the lack of a xylose-specific transport system for getting xylose inside the cell. Xylose transporters were expressed in the yeast cells engineered to utilize xylose. It was shown that the presence of a xylose transport system to allow better uptake of xylose into the cell improved xylose utilization. Up to 2-fold more xylose was co-consumed with glucose, and ethanol concentration and specific productivity were increased. Metabolic limitations to xylose fermentation were investigated using engineered yeast strains that utilize xylose by reduction/oxidation. A novel dehydrogenase was identified based on its induced expression during growth on xylose. Yeast cells lacking the gene that codes for the dehydrogenase were generated and shown to be more sensitive to oxidative damage, which requires nicotinamide adenine dinucleotide phosphate (NADPH) for maximum tolerance. The protein was purified and partially characterized. The gene was determined to be an alcohol dehydrogenase. The effect of microwave pretreatment on the enzymatic saccharification and fermentation of wheat straw by a mixed sugar utilizing ethanologenic bacterium was investigated. Continuous production of ethanol by the recombinant bacterium from alkaline peroxide pretreated enzymatically saccharified wheat straw hydrolyzate was studied for over 120 days. During this period, ethanol was produced continuously by the bacterium. The bacterium was able to maintain its plasmid, and cell viability did not decrease. The simultaneous saccharification and fermentation (SSF) of dilute acid pretreated wheat straw was studied using the recombinant bacterium. The effects of various mitigation techniques for removal of fermentation inhibitors on SSF were investigated. The fed-batch approach decreased the time of SSF and improved ethanol productivity. The production of ethanol from xylose in a membrane cell recycle reactor was studied using the recombinant bacterium. In this reactor, very high ethanol productivity was obtained which is about 60-fold increase over the batch fermentation. The butanol production from dilute acid pretreated corn stover, barley straw, and switchgrass was investigated. Several butanol recovery systems including adsorption, gas stripping, pervaporation, and liquid-liquid extraction were studied to recover butanol from fermentation broth simultaneously. It was found that a combination of two product recovery systems such as pervaporation and adsorption is more cost-effective and hence will be applied to recover butanol from fermentation broth. This research addresses NP 307, Component I. 4.Accomplishments 1. IMPROVED CONVERSION OF XYLOSE TO ETHANOL BY ENGINEERED YEAST. The conventional yeast cannot utilize xylose (sugar derived from hemicellulose) and ferment it to ethanol. Yeast have been engineered to consume xylose and make ethanol, but suffer from numerous problems that limit their ability to convert xylose to ethanol. One of these problems is a lack of proteins required for efficient transport of xylose into the cells. A xylose transporter from another organism was expressed in the engineered yeast which increased xylose transport into the cells resulting in increased xylose consumption and ethanol production. A yeast strain that efficiently ferments xylose to ethanol is needed to decrease the cost of ethanol production from agricultural residues, making ethanol production from these feedstocks economically feasible. The research addresses under NP 307, Component I, Problem Area "Ethanol cannot be produced from any agricultural feedstock today at a selling cost competitive with petroleum sources." 2. CONTINUOUS PRODUCTION OF ETHANOL FROM WHEAT STRAW HYDROLYZATE BY A RECOMBINANT BACTERIUM. Long-term plasmid stability and cell viability are some of the problems when using a recombinant organism in industrial fermentations. To assess these issues, continuous production of ethanol from alkaline peroxide pretreated enzymatically sacchraified wheat straw hydrolyzate was performed using a mixed sugar utilizing recombinant ethanologenic bacterium developed at our research unit. The bacterium produced ethanol continuously over 120 days without losing any productivity and with no contamination problem. During this period, the bacterium was able to maintain its plasmid stability and cell viability. Additionally, the ethanol productivity was increased 2-fold in comparison to batch fermentation. This research shows that the recombinant ethanologenic bacterium has the potential to be used in industrial production of ethanol from lignocellulosic hydrolyzates. The research addresses NP 307, Component I, Problem Area "Ethanol cannot be produced from any agricultural feedstock today at a selling cost competitive with petroleum sources." 3. PRODUCTION OF ETHANOL FROM XYLOSE IN MEMBRANE CELL RECYCLE BIOREACTOR. For lignocellullose based ethanol production to compete with corn starch based ethanol production, cost reductions need to be made at all stages of the production process including fermentation. A membrane cell recycle reactor was characterized for ethanol production from xylose by a mixed sugar utilizing recombinant bacterium. In the membrane cell recycle reactor, the ethanol productivity from xylose was improved by a factor of 60 in comparison to a batch reactor. This increase in productivity would reduce reactor size and process streams thus reducing capital cost of the plant. It is anticipated that such an increase in productivity would reduce ethanol production cost from lignocellulosic feedstock significantly. The research addresses NP 307, Component I, Problem Area "Ethanol cannot be produced from any agricultural feedstock today at a selling cost competitive with petroleum sources." 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of Active CRADAs 1 Number of the New MTA (providing only) 1 Number of New Commercial Licenses Executed 3 Number of Web Sites Managed 3 Number of Other Technology Transfer 1 Review Publications Qureshi, N., Ezeji, T.C., Ebener, J., Dien, B.S., Cotta, M.A., Blaschek, H.P. 2008. Butanol production by Clostridium beijerinckii. Part I. Use of acid and enzyme hydrolysed corn fiber. Bioresource Technology. 99:5915-5922. Ezeji, T.C., Qureshi, N., Blaschek, H.P. 2007. Production of acetone butanol (AB) from liquefied corn starch, a commercial substrate, using Clostridium beijerinckii coupled with product recovery by gas stripping. Journal of Industrial Microbiology and Biotechnology. 34:771-777. Racine, F.M., Saha, B.C. 2007. Production of mannitol by Lactobacillus intermedius NRRL B-3693 in fed-batch and continuous cell-recycle fermentations. Process Biochemistry. 42:1609-1613. Qureshi, N., Saha, B.C., Hector, R.E., Hughes, S.R., Cotta, M.A. 2008. Butanol production from wheat straw by simultaneous saccharification and fermentation using Clostridium beijerinckii: Part I - batch fermentation. Biomass and Bioenergy. 32:168-175. Saha, B.C., Cotta, M.A. 2007. Enzymatic hydrolysis and fermentation of lime pretreated wheat straw to ethanol. Journal of Chemical Technology and Biotechnology. 82:913-919. Qureshi, N., Saha, B.C., Cotta, M.A. 2008. Butanol production from wheat straw by simultaneous saccharification and fermentation using Clostridium beijerinckii: Part II - fed-batch fermentation. Biomass and Bioenergy. 32:176-183. Qureshi, N., Saha, B.C., Cotta, M.A. 2007. Butanol production from wheat straw hydrolysate using Clostridium beijerinckii. Bioprocess and Biosystems Engineering. 30:419-427. Liu, Z., Saha, B.C., Slininger, P.J. 2008. Lignocellulosic biomass conversion to ethanol by Saccharomyces. In: Wall, J., Harwood, C., Demain, A., editors. Bioenergy. Chapter 4. Washington, DC: ASM Press. p. 17-36. Woodyer, R.D., Wymer, N.J., Racine, F.M., Khan, S.N., Saha, B.C. 2008. Efficient production of L-ribose with a recombinant Escherichia coli biocatalyst. Applied and Environmental Microbiology. 74(10):2967-2975. Qureshi, N., Ezeji, T.C. 2008. Butanol (a superior biofuel) production from agricultural residues (renewable biomass): Recent progress in technology. Biofuels, Bioproducts, and Biorefining. 2:319-330. Saha, B.C., Racine, F.M. 2008. Production of mannitol by lactic acid bacteria: A review. In: Hou, C.T., Shaw, J.-R., editors. Biocatalysis and Bioenergy. Hoboken, NJ: John Wiley and Sons, Inc. p. 391-404.