COST-EFFECTIVE BIOPROCESS TECHNOLOGIES FOR PRODUCTION OF BIOFUELS FROM LIGNOCELLULOSIC BIOMASS

Goals / Objectives
Develop cost-effective pretreatment, enzymatic saccharification, fermentation and downstream processing technologies, and their integration for production of biofuels from lignocellulosic biomass.

Project Methods
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. BSL-1 and Risk Group RG1 recertified April 17, 2008.

Progress 09/01/04 to 07/19/09

Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations The overall objective of this research program is to develop cost- effective pretreatment, enzymatic saccharification, fermentation and downstream processing technologies, and their integration for production of biofuels from lignocellulosic biomass. Following evaluation of pretreatment options for wheat straw conversion and saccharification, dilute acid was selected as the best pretreatment option and simultaneous saccharification and fermentation (SSF) using a recombinant ethanologen was identified as the best strategy for fermentation. Inhibitors produced during dilute acid pretreatment were mitigated by growing an inhibitor metabolizing fungus on the hydrolyzate prior to ethanol fermentation. These processes were optimized, combined, and demonstrated in an integrated process at the 10 L scale. Demonstration at 100 L scale and preliminary cost analysis are underway and will help evaluate the commercial potential for this integrated process. In similar research using barley straw as the feedstock, four pretreatment (liquid hot water, dilute acid, lime, and alkaline peroxide) methods coupled with enzymatic saccharification were evaluated as conversion options. Ethanol production by a recombinant bacterium from dilute acid, lime, and alkaline peroxide pretreated and enzymatically saccharified barley straw was investigated further. In order to better understand the physical, chemical, and biochemical processes being applied to convert plant cell wall polysaccharides to fermentable sugars, analytical methods (robust and medium throughput labeling, liquid chromatography/mass spectrometry/MS2) were developed for the study of oligosaccharide components of lignocellulose that influence the severity of pretreatment and enzymatic saccharification. Tandem mass spectrometry provided a powerful tool for analyzing oligosaccharides as it combined high sensitivity with the ability to determine structural information via the fragmentation patterns of molecules. Because the conventional fermentation yeast (Saccharomyces cerevisiae) cannot utilize xylose for ethanol production, we have engineered strains to make ethanol from xylose. More robust industrial recombinant yeast strains were generated that showed improved xylose fermentation. Xylose fermentation by the yeast also results in metabolic insufficiencies that increase the cell�s sensitivities to numerous stresses. A recombinant xylose fermenting yeast strain with enhanced resistance to redox stress was developed by over-expressing two identified proteins. Based on the results of our previous work on the simultaneous recovery of butanol from fermentations, it was decided to use a combination of gas stripping and pervaporation for a bench scale (10 L) demonstration using wheat straw hydrolyzate (dilute acid) as the model substrate in a separate enzymatic hydrolysis and fermentation (SHF) process. Inhibitor detoxification was not required and fermentation proceeded without additional conditioning of the hydrolyzate. Demonstration of the technology at 100 L scale and preliminary cost analysis of the process are planned and will help move this technology toward commercial application. Technology Transfer Number of New CRADAS: 1 Number of New/Active MTAs(providing only): 2

Impacts
(N/A)

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.
Sakakibara, Y., Saha, B.C., Taylor, P. 2009. Microbial Production of Xylitol from L-arabinose by Metabolically Engineered Escherichia coli. Journal of Bioscience and Bioengineering. 107(5):506-511.
Hector, R.E., Qureshi, N., Hughes, S.R., Cotta, M.A. 2008. Expression of a Heterologous Xylose Transporter in a Saccharomyces cerevisiae Strain Engineered to Utilize Xylose Improves Aerobic Xylose Consumption. Applied Microbiology and Biotechnology. 80(4):675-684.
Qureshi, N. 2009. Solvent Production. In: Schaechter, M., editor. Encyclopedia of Microbiology. Oxford: Elsevier. p. 512-528.
Saha, B.C., Bothast, R.J., Jordan, D.B. 2009. Enzymes, Industrial (Overview). In: Schaechter, M., editor. Encyclopedia of Microbiology. Oxford: Elsevier. p. 281-294.
Qureshi, N., Saha, B.C., Hector, R.E., Cotta, M.A. 2008. Removal of Fermentation Inhibitors from Alkaline Peroxide Pretreated and Enzymatically Hydrolyzed Wheat Straw: Production of Butanol from Hydrolysate Using Clostridium beijerinckii in Batch Reactors. Biomass and Bioenergy. 32(12):1353-1358.
Sakakibara, Y., Saha, B.C. 2008. Isolation of an Operon Involved in Xylitol Metabolism from a Xylitol-utilizing Pantoea ananatis Mutant. Journal of Bioscience and Bioengineering. 106(4):337-344.
Saha, B.C., Biswas, A., Cotta, M.A. 2008. Microwave Pretreatment, Enzymatic Saccharification, and Fermentation of Wheat Straw to Ethanol. Journal of Biobased Materials and Bioenergy. 2(3):210-217.
Canakci, S., Kacagan, M., Inan, K., Belduz, A.O., Saha, B.C. 2008. Cloning, Purification, and Characterization of a Thermostable Alpha-L- arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari. Applied Microbiology and Biotechnology. 81(1):61-68.
Liu, S., Bischoff, K.M., Hughes, S.R., Leathers, T.D., Price, N.P., Qureshi, N., Rich, J.O. 2009. Conversion of Biomass Hydrolysates and Other Substrates to Ethanol and Other Chemicals by Lactobacillus buchneri. Letters of Applied Microbiology. 48(3):337-342.
Hughes, S.R., Sterner, D.E., Bischoff, K.M., Hector, R.E., Dowd, P.F., Qureshi, N., Bang, S.S., Grynavyski, N., Chakrabarty, T., Johnson, E.T., Dien, B.S., Mertens, J.A., Caughey, R.J., Liu, S., Butt, T.R., Labaer, J., Cotta, M.A., Rich, J.O. 2008. Engineered Saccharomyces cerevisiae Strain for Improved Xylose Utilization with a Three-plasmid SUMO Yeast Expression System. Plasmid Journal. 61:22-38.

Progress 10/01/06 to 09/30/07

Outputs
Progress Report 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. Evaluate a variety of chemical and physical pretreatments of barely straw before saccharification and fermentation to ethanol. 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. Accomplishments NEAR COMPLETE SACCHARIFICATION AND FERMENTATION OF RICE HULLS TO FUEL ETHANOL. Rice hulls are a complex lignocellulosic feedstock with high lignin and ash content. Batch alkaline peroxide pretreatment, separate enzymatic hydrolysis and fermentation (SHF), and simultaneous enzymatic saccharification and fermentation (SSF) processes have been developed for conversion of this tenacious feedstock to ethanol. We have demonstrated that alkaline peroxide pretreated rice hulls can be enzymatically saccharified to fermentable sugars almost completely. Moreover, silica, a value-added coproduct, was easily recovered from the alkaline peroxide pretreated rice hull hydrolyzate. No common fermentation inhibitors were produced. Both SHF and SSF processes performed well for production of ethanol from the alkaline peroxide pretreated rice hulls by an ethanologenic recombinant bacterium capable of utilizing multiple sugars. The research will greatly contribute to the development of an integrated bioprocess technology for cost-effective fuel ethanol production from lignocellulosic feedstock. The research falls under National Program 307 - Bioenergy and Energy Alternatives, Component 1, Ethanol, and addresses the Problem Statement "Ethanol cannot be produced from any agricultural feedstock today at a selling cost competitive with petroleum sources." AN INTEGRATED PROCESS FOR BIOCONVERSION OF WHEAT STRAW TO BUTANOL. An integrated energy efficient process for conversion of wheat straw to butanol was developed. Dilute acid pretreated wheat straw was simultaneously saccharified enzymatically and fermented to butanol efficiently by an anaerobic bacterium without using the expensive detoxification step (overliming) typically required for dilute acid pretreated feedstock, thus offering significant cost savings. Since the accumulation of butanol is toxic to the fermentative organism, it was successfully removed simultaneously by employing gas stripping, thus solving the problem of strong product inhibition. The newly developed integrated process (simultaneous saccharification, fermentation, and recovery; SSFR) reduces the production cost of butanol significantly and thus will make the commercial production of butanol from lignocellulosic feedstock much more economical. The research aligns under National Program 307 - Bioenergy and Energy Alternatives, Component 1, Ethanol, and addresses the Problem Statement "Ethanol cannot be produced from any agricultural feedstock today at a selling cost competitive with petroleum sources." Technology Transfer Number of Active CRADAS and MTAS: 3 Number of Invention Disclosures submitted: 1 Number of Non-Peer Reviewed Presentations and Proceedings: 17 Number of Newspaper Articles,Presentations for NonScience Audiences: 4

Impacts
(N/A)

Publications

Saha, B.C., Cotta, M.A. 2007. Enzymatic saccharification and fermentation of alkaline peroxide pretreated rice hulls to ethanol. Enzyme and Microbial Technology. 41:528-532.
Ezeji, T.C., Qureshi, N., Blaschek, H.P. 2007. Production of acetone- butanol-ethanol (ABE) in a continuous flow bioreactor using degermed corn and Clostridium beijerinckii. Process Biochemistry. 42:34-39.
Saha, B.C., Sakakibara, Y., Cotta, M.A. 2007. Production of D-arabitol by a newly isolated Zygosaccharomyces rouxii. Journal of Industrial Microbiology and Biotechnology. 34:519-523.
Ezeji, T., Qureshi, N., Blaschek, H.P. 2007. Butanol production from agricultural residues: impact of degradation products on Clostridium beijerinckii growth and butanol fermentation. Biotechnology and Bioengineering. 97:1460-1469.
Canakci, S., Belduz, A.O., Saha, B.C., Yasar, A., Ayaz, F.A., Yayli, N. 2007. Purification and characterization of a highly thermostable alpha-L- arabinofuranosidase from Geobacillus caldoxylolyticus TK4. Applied Microbiology and Biotechnology. 75:813-820.
Ezeji, T.C., Qureshi, N., Blaschek, H.P. 2007. Bioproduction of butanol from biomass: from genes to bioreactors. Current Opinion in Biotechnology. 18:220-227.
Saha, B.C. 2006. A low-cost medium for mannitol production by Lactobacillus intermedius NRRL B-3693. Applied Microbiology and Biotechnology. 72:676-680.
Saha, B.C. 2006. Effect of salt nutrients on mannitol production by Lactobacillus intermedius NRRL B-3693. Journal of Industrial Microbiology and Biotechnology. 33(10):887-890.
Saha, B.C. 2006. Production of mannitol from inulin by simultaneous enzymatic saccharification and fermentation with Lactobacillus intermedius NRRL B-3693. Enzyme and Microbial Technology. 39:991-995.

Progress 10/01/05 to 09/30/06

Outputs
Progress 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 nations 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. 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.

Impacts
(N/A)

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.
Taylor, P., Wymer, N., Saha, B.C., Racine, M., Sakakibara, Y. 2006. A new approach to xylitol biosynthesis [abstract]. BIO 2006. Paper No. C32.
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.

Progress 10/01/04 to 09/30/05

Outputs
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. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. 1A. Dilute acid pretreatment and enzymatic saccharification. Milestone Substantially Met 2. 2A. Batch separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). Milestone Substantially Met 3. 3A. Butanol recovery by pervaporation. Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? 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. Dilute acid pretreatment option for wheat straw will be completed, and highly efficient enzymes needed for complete saccharification of dilute acid pretreated wheat straw to fermentable sugars will be identified. Comparative performances of both batch and continuous SHF and SSF for fuel ethanol production from wheat straw hydrolyzates will be evaluated. Method for butanol recovery by gas stripping will be developed, and integration of SSF with butanol recovery by pervaporation will be completed. 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. Alkaline peroxide pretreatment option for wheat straw will be completed, and highly efficient enzymes needed for complete saccharification of alkaline peroxide pretreated wheat straw to fermentable sugars will be identified. Identification and characterization of the key fermentation inhibitors produced during dilute acid pretreatment will be performed. Continuous fermentation of wheat straw hydrolyzates to ethanol will be studied. Method for butanol recovery by liquid liquid extraction will be developed, and integration of SSF with butanol recovery by gas stripping will be completed. 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. We will attempt to develop a simple, inexpensive method for detoxification of lignocellulosic hydrolyzates, and high cell density and cell recycle fermentation systems for production of ethanol and butanol, and integrate SSF for ethanol production. 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. 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.

Impacts
(N/A)

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.