2005 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? What does it matter? In the U.S., the production of fuel alcohol from corn starch reached 3.4 billion gallons in 2004. 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 byproducts (corn fiber, rice hull, sugar cane bagasse), and energy crops (switchgrass) are particularly 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 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 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 wheat straw as a model biomass substrate. 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 (ethanol, butanol) 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. 2.List the milestones (indicators of progress) from your Project Plan. The milestones from the project plan are as follows: Component 1 1A. Dilute acid pretreatment and enzymatic saccharification. 1B. Alkaline peroxide pretreatment and enzymatic saccharification. 1C. Identification and characterization of fermentation inhibitors produced during pretreatments. 1D. Develop methods of detoxification and study mechanism of detoxification by lime. 1E. Choose a pretreatment option and optimize the sugar yield. Component 2 2A. Batch separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). 2B. Continuous fermentation (SHF, SSF). 2C. High cell density and cell recycle fermentation (SHF, SSF). 2D. Choose a fermentation method and optimize the conditions for rapid and efficient fermentation. Component 3 3A. Butanol recovery by pervaporation. 3B. Butanol recovery by gas stripping. 3C. Butanol recovery by liquid liquid extraction. 3D. Choose a product recovery method for butanol production and optimize conditions. Component 4 4A. Integration of SSF and butanol recovery with pervaporation. 4B. Integration of SSF and butanol recovery by gas stripping. 4C. Integrate SSF for ethanol production. 4D. Choose one technology option for each fermentation product (ethanol and butanol) and demonstrate the technology at 100L scale using the model biomass substrate; perform a preliminary cost analysis for each process. 4a.What was the single most significant accomplishment this past year? CONVERSION OF WHEAT STRAW TO FUEL ETHANOL. Wheat straw contains 70% complex carbohydrate that can serve as a low-cost feedstock for production of fuel ethanol. Batch dilute acid pretreatment, separate enzymatic hydrolysis and fermentation (SHF), and simultaneous enzymatic saccharification and fermentation (SSF) systems have been evaluated for production of ethanol from wheat straw. We have demonstrated that wheat straw pretreated with dilute acid at a moderate temperature can be easily saccharified enzymatically to fermentable sugars with a very good yield. The work will contribute to the development of an integrated bioprocess technology for fuel ethanol production from lignocellulose. 4b.List other significant accomplishments, if any. SIMULTANEOUS FERMENTATION AND PRODUCT RECOVERY FOR BUTANOL. A major problem associated with butanol fermentation was the low yield of butanol in the fermentation broth due to strong inhibition of the fermentative microorganism by butanol. A simultaneous recovery method for butanol during fermentation using pervaporation has been developed in cooperation with a university partner. This will solve the problem of product inhibition of the fermentative microorganism and thus reduce the production cost of butanol significantly as a result of integration of fermentation with recovery. 4c.List any significant activities that support special target populations. None. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. The current project, started in September 2004, is based on a new interim project that was initiated in 2002. Two SY positions are still vacant. 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. Simultaneous fermentation of glucose to butanol and butanol recovery by pervaporation 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 cell recycle fermentation method for production of mannitol has been developed under the Cooperative Research and Development Agreement (CRADA). The above accomplishments are linked to milestone 1A, 3A, and 4A. These are directly linked to the National Program 307 Component 1, National Program 306 Component 2, and ARS Strategic Plan Goals 1 and 2. 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 from corn derived glucose in collaboration with a university partner and a corn processing company 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 U.S. patent was issued for a method for making mannitol by fermentation. The CRADA partner received FDA approval of the mannitol production process. 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). Taking the next step toward growing our own fuel. College of Agriculture, Consumer, and Environmental Sciences, University of Illinois Press Release, Urbana, IL, October 6, 2004. Continuous butanol fermentation of starch. Industrial Bioprocessing, 27(1), p. 2, 2005. Butanol fermentation based on starch. Industrial Bioprocessing, 27(1), p. 4, 2005. Biological processes for manufacturing mannitol. Industrial Bioprocessing, 27(3), p. 2-3, 2005. FDA paves way for mannitol launch. Food Ingredient News, 12(12), p. 1-2, December 2004. FDA paves way for launch of mannitol sweetener made with new process. Soya and Oilseed Industry News. Innovative Food Solutions. January 7, 2005. zuChem launches first mannitol sweetener. Food Navigator.com. Breaking News on Food and Beverage Development. January 11, 2005. Bacteria-based production method patented. ARS New Service. March 30, 2005. Bacteria-based production method patented. http://www.Sciencedaily.com/releases/2005/04/050421235139.htm. New biobased mannitol technology patented. IFT Weekly E-mail Newsletter. March 30, 2005. Bacteria based production method patented by ARS. Discovery based on ARS mission to create new, value-added markets for corn and soybeans. The Soy Daily. March 30, 2005. Biobased mannitol method receives patent. Food Ingredient News, 13(40), p. 7,10, April 2005. Agricultural Research Service patents bacteria-based method of producing sweetener. NewsTarget.com. July 19, 2005. Mannitol method to slash price for low cal sugar replacer? Food Navigator.com. Breaking News on Food Development in the U.S. March 31, 2005. Presentations: (invited) Qureshi, N. Downstream processing in acetone butanol (AB) fermentation. Institute for Genomic Biology, University of Illinois, Urbana, IL, April 9, 2005. Saha, B.C. Fuels and chemicals from biomass: challenges and opportunities, American Chemical Society University of Missouri Local Section Meeting, Columbia, MO, April 25, 2005. Saha, B.C. Enzymes in biotechnology: green chemistry challenges, American Chemical Society St. Louis Local Section Meeting, St. Louis, MO, April 26, 2005. Saha, B.C. Fuels and chemicals from biomass: challenges and opportunities, American Chemical Society Southern Illinois Local Section, Carbondale, IL, April 27, 2005. Saha, B.C. Fuels and chemicals from biomass: challenges and opportunities, American Chemical Society Ozark Local Section Meeting, Springfield, MO, April 28, 2005. Qureshi, N. Strategies to produce glutamic acid by fermentation. North Carolina State University, Raleigh, NC, June 13, 2005. Review Publications Saha, B.C. 2004. Xylitol production by yeasts: current status and future prospects [abstract]. International Congress on Yeasts. p. 52. Saha, B.C. 2005. Enzymes as biocatalysts for conversion of lignocellulosic biomass to fermentable sugars. In: Hou, C.T., editor. Handbook of Industrial Biocatalysis. Boca Raton, FL: CRC Press Taylor and Francis Group. p. 24-1-24-12. Ezeji, T.C., Qureshi, N., Blaschek, H.P. 2005. Continuous butanol fermentation and feed starch retrogradation: butanol fermentation sustainability using Clostridium beijerinckii BA101. Journal of Biotechnology. 115:179-187. Liu, S., Saha, B.C., Cotta, M.A. 2005. Cloning, expression, purification, and analysis of mannitol dehydrogenase gene mtlK from Lactobacillus brevis. Applied Biochemistry and Biotechnology. 121-124:391-402. Karcher, P.M., Ezeji, T.C., Qureshi, N., Blaschek, H.P. 2004. Acetone butanol ethanol (ABE) fermentation by Clostridium beijerinckii BA101: effect of bubble size on the performance of a gas stripping-based recovery system [abstract]. Biotechnology for Fuels and Chemicals. Paper No. 3-08. Qureshi, N., Ezeji, T.C., Blaschek, H.P., Cotta, M.A. 2004. A novel biological process to convert renewable biomass to acetone and butanol (AB) [abstract]. American Institute of Chemical Engineers. Paper No. 29d. Qureshi, N., Maddox, I.S. 2005. Reduction in butanol inhibition by perstraction: utilization of concentrated lactose/whey permeate by Clostridium acetobutylicum to enhance butanol fermentation economics. Transactions of the Institution of Chemical Engineers. 83(C1):43-52. Saha, B.C., Iten, L.B., Cotta, M.A., Wu, Y. 2004. Fuel ethanol production from wheat straw: current status and technical prospects. In: Van Swaaij, W.P.M., Fjallstrom, T., Helm, P., Grassi, A., editors. Proceedings of the 2nd World Conference on Biomass for Energy, Industry, and Climate Protection, May 10-14, 2004, Rome, Italy, p. 1481-1483. Ezeji, T.C., Qureshi, N., Blaschek, H.P. 2004. Butanol fermentation research: upstream and downstream manipulations. The Chemical Record. 4:305-314. Racine, M., Terentieva, E., Saha, B.C., Kennedy, G.J. 2004. Production of mannitol by fermentation [abstract]. Great Lakes Regional American Chemical Society Symposium. Paper No. 166. Qureshi, N., Brining, H.R., Iten, L.B., Dien, B.S., Nichols, N.N., Saha, B.C., Cotta, M.A. 2004. Adsorbed cell dynamic biofilm reactor for ethanol production from xylose and corn fiber hydrolysate [abstract]. Great Lakes Regional American Chemical Society Symposium. p. 179. Ezeji, T.C., Karcher, P.M., Qureshi, N., Blaschek, H.P. 2005. Improving performance of a gas stripping-based recovery system to remove butanol from Clostridium beijerinckii fermentation. Bioprocess and Biosystems Engineering. 27:207-214. Saha, B.C., Iten, L.B., Cotta, M.A., Wu, Y. 2004. Rice hull as substrate for production of fuel ethanol. In: Cherry, J.P., Pavlath, A.E., editors. Proceedings of the 33rd Annual Meeting of the United States-Japan Cooperative Program in Natural Resources (UJNR), December 11-18, 2004, Honolulu, Hawaii. p. 181-185. Qureshi, N., Li, X., Saha, B.C., Cotta, M.A. 2005. Production of acetone butanol from corn fiber xylan using Clostridium beijerinckii P260 [abstract]. Biotechnology for Fuels and Chemicals Symposium. p. 87. Saha, B.C., Iten, L.B., Cotta, M.A., Wu, Y. 2005. Dilute acid pretreatment, enzymatic saccharification, and fermentation of rice hulls to fuel ethanol. Biotechnology Progress. 21:816-822. Saha, B.C. 2005. The present and future of biorefinery research at USDA [abstract]. International Workshop on Biorefinery. p. 19-20. Karcher, P.M., Ezeji, T.C., Qureshi, N., Blaschek, H.P. 2005. Microbial production of butanol: product recovery by extraction. In: Satyanarayann, T., Johri, B.N., editors. Microbial Diversity: Current Prospectives and Potential Applications. New Delhi:I.K. International. p. 865-880. Saha, B.C. 2005. Status of biorefinery research and development at USDA-ARS [abstract]. International Biomass Forum: The Leading Edge of Biomass Research. Paper No. 4. Saha, B.C. 2005. Method for making mannitol with Lactobacillus intermedius. U.S. Patent 6,855,526.