2011 Annual Report 1a.Objectives (from AD-416) Develop pretreatment and fermentation processes to convert lignocellulosic materials such as barley straw, barley hulls, DDGS, and other crop biomass into fuel ethanol and value-added coproducts. This work complements present ARS work to develop advanced bioconversion process for production of cellulosic ethanol and value-added co-products that can be integrated into an existing fuel ethanol plant. The first step in a lignocellulosic biomass bioconversion process normally is pretreatment to open up the structure and subsequently allow increased enzymatic hydrolysis. Soaking in aqueous ammonia (SAA) has been proven to be effective as a pretreatment method of agricultural residues, such as corn stover, corn fiber, barley hull, switchgrass, etc. In this study, SAA will be employed for the pretreatment step and subsequent conversion of SAA pretreated biomass will be investigated. 1b.Approach (from AD-416) The composition of specific types of biomass studied in this project will be determined using standard procedures for biomass analysis. Then, pre-treatment processes based upon Soaking in Aqueous Ammonia (SAA) technology will be developed and optimized for each biomass source. SAA will open up the structure of the biomass and subsequently allow increased enzymatic hydrolysis. This pretreated biomass will be subject to saccharification using commercial cellulases with xylanase supplements. The conversion of biomass to free sugars will be monitored by HPLC and standard methods of analysis. Once saccharified, the sugars will be fermented by traditional brewers yeast. In other studies, pre-treated biomass will be simultaneously saccharified and fermented to directly produce ethanol from the pretreated biomass in one step. 3.Progress Report A simple pretreatment method using anhydrous ammonia was developed to minimize water and ammonia inputs for cellulosic ethanol production, termed the low moisture anhydrous ammonia (LMAA) pretreatment. In this method, corn stover with 30 - 70% moisture was contacted with anhydrous ammonia in a reactor under nearly ambient conditions. After the ammoniation step, biomass was subjected to a simple pretreatment step at moderate temperatures (40 – 120 degrees C) for 48 - 144 h. Pretreated biomass was saccharified and fermented without an additional washing step. With 3% glucan loading of LMAA-treated corn stover under the best treatment conditions found (0.1 g-ammonia + 1.0 g-water per g biomass, 80 degrees C, and 96 h), simultaneous saccharification and cofermentation using recombinant E. coli KO11 and enzymes (15 FPU cellulase + 30 CBU beta-glucosidase + 1,000 GXU xylanase per g-glucan) resulted in 24.2 g/l of ethanol in the final beer (89 % of theoretical ethanol yield based on glucan + xylan in corn stover). This pretreatment system has several benefits over traditional cellulosic ethanol pretreatments, including less requirements for acid and chemical resistant reactors, lower system pressures, and less formation of byproducts that inhibit the cellulosic enzymes and fermentative organisms. A poster on the technology was presented and a manuscript has been submitted to a peer reviewed journal. Invention reports on this technology have been filed at Iowa State University and in ARS. Progress on this project was monitored in a number of ways. In addition to numerous e-mails and telephone exchanges, both ARS scientists involved in the project visited Ames Iowa in October 2010 to coordinate research activities with the Iowa State PI and his graduate student and staff.