This project will develop a very low-cost process for production of ethanol from cellulosic biomass, using a genetically engineered bacterium that grows at high temperatures. The process should expand fuel production from indigenous, renewable resources, and establish a process concept for renewable production of industrial chemicals The conversion of cellulosic biomass to commodity products via biological processing could benefit society by improving energy security, reducing greenhouse gas emissions, and promoting rural economies. However, commercial application is impeded by the current absence of low-cost technology to overcome the recalcitrance of cellulosic biomass. This project will develop a strain of Clostridium thermocellum, a rapid cellulose-fermenting microorganism that produces ethanol as well as organic acids, for producing industrial ethanol from pretreated biomass in a consolidated bioprocessing (CBP) configuration. Phase I established the technical capability to metabolically engineer C. thermocellum. A high efficiency gene transfer system for this organism, using custom apparatus and an optimized protocol, was developed. Site-specific gene integration into genes associated with acetic acid formation was demonstrated. Fermentation studies showed a dramatic decrease in acetic acid production compared to the wild-type C thermocellum. Phase II will develop engineered strains of C. thermocellum that produce ethanol at high yield, along with commercially recoverable concentrations from pretreated corn stover in the absence of added saccharolytic enzymes. Particular objectives include: (1) demonstrating the efficacy and removal of additional genetic markers; (2) developing C. thermocellum strains that produce ethanol at high yield; (3) developing ethanol-tolerant C. thermocellum strains; (4) demonstrating the effectiveness of AFEX pretreatment for C. thermocellum fermentation; and (5) analyzing process economics and business opportunities. Commercial Applications and Other Benefits as described by awardee: The technology should expand fuel production from indigenous, renewable resources, and also establish a process concept that can be applied to the production of industrial chemicals
