2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? In the U.S., the production of corn grain based ethanol reached 4.5 billion gallons in 2005, a fraction of the 140 billion gallons of transportation fuel used annually. The goal is to displace 30% of the nation’s current gasoline use with ethanol by 2030, and this will require production levels equal to roughly 60 billion gallons a year. If all corn grain now grown in the U.S. is converted to ethanol, it can satisfy approximately 15% of the current transportation fuel needs. Thus, developing ethanol as fuel, beyond its current role as fuel oxygenate, will require developing lignocellulose as feedstock because of its abundance. In particular, various agricultural residues (corn stover, wheat straw, rice straw), agricultural processing by-products (corn fiber, rice hulls, sugar cane bagasse), and energy crops (switchgrass) can be used as low-cost attractive sources of sugars for biofuel production. Environmentally friendly methods for pretreatment, efficient and rapid enzymatic saccharification to fermentable sugars, high productivity fermentation of mixed sugar streams, and cost-effective recovery of dilute products need to be developed in order to economically use these materials as feedstocks for production of biofuel and other value-added commodity chemicals. The objective of this project is to develop cost-effective pretreatment, enzymatic saccharification, fermentation and downstream processing technologies, and their integration for production of biofuels (ethanol, butanol) from lignocellulosic biomass. It has four components: . 1)To develop environmentally friendly pretreatment and enzymatic saccharification methods to generate fermentable sugars from lignocellulosic biomass,. 2)to develop high productivity fermentation systems to convert lignocellulosic hydrolyzates to biofuels,. 3)to develop downstream processing technologies to recover biofuels from fermentation broth, and. 4)to perform process integration, economic evaluation, and pilot scale demonstration of lignocellulosic biomass conversion. Our initial target is to use a more complex lignocellulosic substrate, such as wheat straw, than corn fiber as a model biomass substrate. The U.S. produces 77.1 million metric tons of wheat straw annually, which has the potential to give 54 million metric tons of fermentable sugars. Any lignocellulosic biomass is resistant to enzymatic hydrolysis in native form. Attempts will be made to develop an effective pretreatment strategy that will greatly aid in the rapid enzymatic hydrolysis of cellulose, help to reduce the enzyme doses required for such conversion, and minimize the formation of fermentation inhibitors. We propose to develop simple methods to detoxify the inhibitory effects of these compounds on fermentative microorganisms. We will develop high productivity fermentation systems for production of biofuels from lignocellulosic hydrolyzates. For this, we will study batch and continuous fermentations with cell recycle. We will develop methods to recover butanol by using membrane based technologies. Finally, we will integrate the enzymatic saccharification and fermentation for production of ethanol, and enzymatic saccharification, fermentation, and downstream processing technologies for production of butanol in order to simplify the process options, demonstrate the technologies for both ethanol and butanol production at 100-L scale, and perform a preliminary cost analysis for each process. The research falls under National Program (NP) 307 - Bioenergy and Energy Alternatives (70%). Component 1. Ethanol. This research will contribute to new technologies that integrate feedstock pretreatment, biological conversion, and product recovery processes, as well as fundamental knowledge regarding lignocellulose decrystallization, generation and detoxification mechanisms of fermentation inhibitors, fermentation, and membrane separation. The information gained will result in the reduction of capital and processing costs associated with biofuel production. The research also falls under National Program (NP) 306 - Quality and Utilization of Agricultural Products (30%). Component 2. New Processes, New Uses, and Value-Added Foods and Biobased Products. Problem Area 2a-New Product Technology, Problem Area 2b-New Uses for Agricultural By-products, and Problem Area 2c-New and Improved Processes and Feedstocks will be addressed by the development of new products from unutilized and underutilized agricultural residues via fermentation and biocatalytic processes. The research aims to produce biofuels from waste and low-value agricultural residues and by-products at a selling cost-competitive price with imported petroleum. It will improve basic scientific information on the structure, biodegradation, and biotransformation of lignocellulosic biomass. The research will help to expand the use of biofuel, thereby, reducing the nation's dependence on foreign oil and create new and expanded markets for various unutilized and underutilized renewable agricultural residues and energy crops. This will help to create jobs and economic activity in rural America. In addition, it will result in the reduction of environmental pollution. This research will also help to create a lignocellulosic sugar platform biorefinery that can be used in making other value-added fermentation products. 2.List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2005) 1A. Dilute acid pretreatment and enzymatic saccharification. 2A. Batch separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). 3A. Butanol recovery by pervaporation. Year 2 (FY 2006) 1A. Continue dilute acid pretreatment and enzymatic saccharification. 1B. Alkaline peroxide pretreatment and enzymatic saccharification. 2A. Continue batch separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). 2B. Continuous fermentation (SHF, SSF). 3B. Butanol recovery by gas stripping. 4A. Integrate SSF and butanol recovery with pervaporation. Year 3 (FY 2007) 1B. Continue alkaline peroxide pretreatment and enzymatic saccharification. 1C. Identification and characterization of fermentation inhibitors produced during pretreatments. 2B. Continue continuous fermentation (SHF, SSF). 3C. Butanol recovery by liquid liquid extraction. 4B. Integrate SSF and butanol recovery by gas stripping. Year 4 (FY 2008) 1C. Continue identification and characterization of fermentation inhibitors produced during pretreatments. 1D. Develop methods of detoxification of lignocellulosic hydrolyzates and study mechanism of detoxification by lime. 2C. High cell density and cell recycle fermentation (SHF, SSF). 3D. Choose a product recovery method for butanol and optimize conditions. 4C. Integrate SSF for ethanol production. Year 5 (FY 2009) 1E. Choose a pretreatment option and optimize the sugar yield. 2D. Choose a fermentation method and optimize the conditions for rapid and efficient fermentation. 3D. Choose a product recovery method for butanol and optimize conditions. 4D. Choose one technology option for each fermentation (ethanol, butanol), demonstrate the technology at 100-L scale using the model biomass substrate; perform a preliminary cost analysis for each process. 4a.List the single most significant research accomplishment during FY 2006. COMPLETE SACCHARIFICATION AND FERMENTATION OF WHEAT STRAW TO FUEL ETHANOL. This research contributes to resolving “Ethanol cannot be produced from any agricultural feedstock today at a selling cost competitive with petroleum sources” outlined in the “Component 1 Ethanol” of National Program 307. Wheat straw contains 70% complex carbohydrate that can serve as a low-cost feedstock for production of fuel ethanol. Batch alkaline peroxide pretreatment, separate enzymatic hydrolysis and fermentation (SHF), and simultaneous enzymatic saccharification and fermentation (SSF) systems have been developed for production of ethanol from alkaline peroxide pretreated wheat straw. We have demonstrated that wheat straw pretreated with alkaline peroxide can be enzymatically saccharified to fermentable sugars completely. No common fermentation inhibitors were produced. Both SHF and SSF approaches worked equally well for production of ethanol from the alkaline peroxide pretreated wheat straw by an ethanologenic recombinant bacterium capable of utilizing multiple sugars (glucose, xylose, arabinose). The work will greatly contribute to the development of an integrated bioprocess technology for fuel ethanol production from lignocellulose. 4b.List other significant research accomplishment(s), if any. PRODUCTION OF BUTANOL FROM WHEAT STRAW HYDROLYZATE BY FERMENTATION AND SIMULTANEOUS PRODUCT RECOVERY. This research aligns with “National Program 307 Component 1 Ethanol” to the problem “Ethanol cannot be produced from any agricultural feedstock today at a selling cost competitive with petroleum sources.” Butanol can serve not only as fuel but also as a chemical. Using dilute acid pretreated, enzymatically saccharified wheat straw, we have been successful in producing butanol with a high yield without using any detoxification step (lime treatment) typically required for dilute acid pretreated substrate. The results demonstrate that the fermentative bacterium can efficiently utilize multiple sugars, and lignocellulosic hydrolyzates can be used for production of butanol. In order to reduce butanol (product) inhibition and utilize all the sugars present in the bioreactor, butanol was recovered simultaneously by gas stripping during fermentation. This solves the problem of notoriously well-known product inhibition of the fermentative bacterium and will thus help to reduce the production cost of butanol significantly as a result of integration of fermentation with recovery. It is anticipated that the developed process would be economical to produce butanol from wheat straw. 4c.List significant activities that support special target populations. None. 5.Describe the major accomplishments to date and their predicted or actual impact. The current project, started in September 2004, is based on a new interim project that was initiated in 2002. Efficient dilute acid pretreatment and highly effective enzymatic saccharification methods of wheat straw have been developed for its conversion to fermentable sugars without forming or minimizing the formation of major fermentation inhibitors such as furfural and hydroxymethyl furfural. A cost-effective method for generating fermentable sugars from wheat straw will greatly aid in commercialization of a wheat straw to ethanol process. Fermentation of dilute acid pretreated, enzymatically saccharified wheat straw hydrolyzates to butanol and simultaneous butanol recovery by gas stripping has been developed. The method developed has solved the problem of strong product inhibition of the fermentative microorganism and thus made the fermentative production of butanol much more economical. A continuous fermentation method for production of mannitol has been developed under a Cooperative Research and Development Agreement (CRADA) with a small company. A joint U.S. patent application has been filed (October 2005). The above first two accomplishments are directly linked to the “National Program 307 Component 1 Ethanol” to the problem “Ethanol cannot be produced from any agricultural feedstock today at a selling cost competitive with petroleum sources.” The third accomplishment addresses “National Program 306 Component 2. New Processes, New Uses, and Value-Added Foods and Biobased Products. Problem Area 2a-New Product Technology, Problem Area 2b-New Uses for Agricultural By-products, and Problem Area 2c-New and Improved Processes and Feedstocks.” 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Efforts on commercialization of butanol fermentation in collaboration with a university partner are in progress. The research and development work to develop and commercialize the mannitol bioprocess under the Cooperative Research and Development Agreement (CRADA) with a company is continuing. A major U.S. enzyme company has shown keen interest in our enzymatic saccharification work. We are helping a small biomass conversion technology company engaged in developing an effective pretreatment strategy for lignocellulose. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Massey, E. 2005. Corn-husk use studied for ethanol. Chicago Tribune. September 5. Anonymous. 2005. Sweet, versatile mannitol from microbial fermentation. Agricultural Research. October. p. 23. Anonymous. 2006. Cheaper raw material for mannitol fermentation. Industrial Bioprocessing. 28(3):2. Carpenter, D. 2006. Quest for energy alternatives heats up. Associated Press. May 28. Anonymous. 2006. Manufacturing butanol from corn fiber. Industrial Bioprocessing. 28(5):2-3. Presentations: Saha, B.C. Fuel ethanol production from lignocellulosic biomass: current state and prospects. ARS-Mexico 8th International Agriculture and Biotechnology Workshop, Monterrey, Mexico, April 24-28, 2006. Saha, B.C. Enzymes in biotechnology. ARS-Mexico 8th International Agriculture and Biotechnology Workshop, Monterrey, Mexico, April 24-28, 2006. Saha, B.C. Assay, purification, and characterization of cellulase. ARS-Mexico 8th International Agriculture and Biotechnology Workshop, Monterrey, Mexico, April 24-28, 2006. Qureshi, N. Downstream processing in acetone butanol (AB) fermentation and process economics. An invited presentation to visit Dow Chemicals Group at the University of Illinois, Urbana-Champaign, May 5, 2006. Saha, B.C. Bioprocess technologies for production of fuel ethanol from lignocellulose. VTT Technical Research Center of Finland, Espoo, Finland, June 22, 2006. Three poster presentations have been made at the Peoria NEXT Scientific Innovation Meeting, Peoria, Illinois, April 27, 2006. Review Publications Karcher, P., Ezeji, T.C., Qureshi, N., Blaschek, H.P. 2005. Butanol extraction from fermentation broth: mathematical equations. Biotechnology for Fuels and Chemicals Symposium. Paper No. 6-57. Qureshi, N., Dien, B.S., Nichols, N.N., Saha, B.C., Cotta, M.A. 2006. Genetically engineered Escherichia coli for ethanol production from xylose: substrate and product inhibition and kinetic parameters. Institution of Chemical Engineers Transactions. 84(2):114-122. Ezeji, T., Qureshi, N., Blaschek, H.P. 2005. Butanol production from agricultural residues: impact of degradation products on Clostridium beijerinckii growth and butanol fermentation [abstract]. World Congress on Industrial Biotechnology and Bioprocessing. p. 163. Ezeji, T.C., Qureshi, N., Karcher, P., Blaschek, H.P. 2006. Production of butanol from corn. In: Minteer, S., editor. Alcoholic Fuels. Boca Raton, FL: Taylor & Francis Group. p. 99-122. Racine, M., Saha, B.C. 2005. Optimization of mannitol production by Lactobacillus intermedius [abstract]. Society of Industrial Microbiology. p. 78. Saha, B.C., Racine, M. 2005. Fermentation process development for mannitol production by a heterofermentative lactic acid bacterium [abstract]. Symposium on Lactic Acid Bacteria Genetics Metabolism and Applications. Paper No. A036. Qureshi, N., Dien, B.S., Nichols, N.N., Liu, S., Iten, L.B., Saha, B.C., Cotta, M.A. 2005. Continuous production of ethanol in high productivity bioreactors using Escherichia coli FBR5: membrane and fixed cell reactors [abstract]. American Institute of Chemical Engineers. Paper No. 589g. Biswas, A., Saha, B.C., Lawton Jr, J.W., Shogren, R.L., Willett, J.L. 2006. Process for obtaining cellulose acetate from agricultural by-products. Carbohydrate Polymers. 64:134-137. Qureshi, N., Annous, B.A., Ezeji, T.C., Karcher, P., Maddox, I.S. 2005. Biofilm reactors for industrial bioconversion processes: employing potential of enhanced reactions rates. Microbial Cell Factories. 4:24. Available: http://www.microbialcellfactories.com/content/4/1/24. Saha, B.C., Cotta, M.A. 2005. Alkaline peroxide pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol. In: Proceedings of the 34th Meeting of the United States-Japan Cooperative Program in Natural Resources (UJNR) Food and Agriculture Panel, October 23-29, 2005, Susoni, Shizuoka, Japan. p. 168-172. Saha, B.C., Cotta, M.A. 2006. Ethanol production from alkaline peroxide pretreated enzymatically saccharified wheat straw. Biotechnology Progress. 22:449-453. Qureshi, N., Dien, B.S., Nichols, N.N., Liu, S., Hughes, S.R., Iten, L.B., Saha, B.C., Cotta, M.A. 2005. Continuous production of ethanol in high productivity bioreactors using genetically engineered Escherichia coli FBR5: membrane and fixed cell reactors [extended abstract]. American Institute of Chemical Engineers. Paper No. 589g. Qureshi, N., Li, X., Hughes, S.R., Saha, B.C., Cotta, M.A. 2006. Butanol production from corn fiber xylan using Clostridium acetobutylicum. Biotechnology Progress. 22:673-680. Hughes, S.R., Riedmuller, S., Li, X., Qureshi, N., Liu, S., Bischoff, K.M., Cotta, M.A., Farrelly, P. 2006. Mass transformation of plasmid libraries of cDNA or mutagenized clone sets into yeast or bacteria using a functional proteomic robotic workcell [abstract]. PepTalk 2006. p. 10. Qureshi, N., Ezeji, T.C., Blaschek, H.P. 2006. Application of alternative product recovery techniques to acetone butanol (AB) fermentation: improving fermentation parameters. Proceedings of the Ninth International Workshop on the Regulation of Metabolism, Genetics, and Development of the Solvent and Acid Forming Clostridia. p. 23. Saha, B.C., Cotta, M.A. 2005. Fuel ethanol production from lignocellulose [abstract]. Society of Industrial Microbiology. p. 74. Qureshi, N., Saha, B.C., Hughes, S.R., Cotta, M.A. 2006. Production of acetone butanol (AB) from agricultural residues using Clostridium acetobutylicum in batch reactors coupled with product recovery [abstract]. Proceedings of the Ninth International Workshop on the Regulation of Metabolism, Genetics, and Development of the Solvent and Acid Forming Clostridia. p. 29. Sakakibara, Y., Saha, B.C., Taylor, P., Wymer, N. 2006. Microbial production of xylitol from L-arabinose [abstract]. American Chemical Society. Paper No. AGFD 79. Saha, B.C., Sakakibara, Y., Cotta, M.A. 2006. Towards the development of a process technology for making xylitol from glucose: optimization of D-arabitol production from glucose by a newly isolated Zygosaccharomyces rouxii [abstract]. International Specialised Symposium on Yeasts. p. 145. Taylor, P., Wymer, N., Saha, B.C., Racine, M., Sakakibara, Y. 2006. A new approach to xylitol biosynthesis [abstract]. BIO 2006. Paper No. C32.