Proteomic Analysis of Ethanol Sensitivity and Tolerance of Thermophilic Anaerobic Bacteria

There is renewed interest in the development of technologies for producing chemicals, materials, and energy using biologically-based processes. The Biobased Products and Bioenergy Vision calls for a 3-fold increase in the use of bioproducts and bioenergy by 2010 and a lO-fold increase by 2020. Much of this has been prompted by recognition that continued increases in fossil fuel use will have negative impacts on the environment, economy, and national security. The production of bio-ethanol offers considerable promise for decreasing fuel oil consumption and increasing domestic markets for agricultural and forestry commodities. Bioconversion of fibrous organic material by thermophilic anaerobic bacteria circumvents some of the problems associated with currently used yeast fermentations. Perhaps the greatest advantage of anaerobic bacteria is their ability to degrade and metabolize structural plant carbohydrates. This latter advantage is particularly important since fibrous biomass is relatively inexpensive and is expected to become the predominant feedstock for liquid fuels within the next 20 years. However, one of the technological barriers to efficient biomass conversion by bacteria is the relatively low ethanol tolerance that nearly all bacteria have when compared to yeast. The effects of ethanol on some thermophilic bacteria have been examined and putative modes of inhibition have been proposed, but there is still relatively little detailed information on the mechanisms causing cellularalterations. State-of-the-art proteomic approaches are now available to efficiently characterize protein expression changes. Our basic hypothesis is that exposure of thermophilic anaerobic bacteria to ethanol causes alterations in protein expression patterns. The overall goal of this project is to identify and define these changes using proteomic approaches. Several organisms will be used to model the effects that ethanol has on the proteomes of thermophilic anaerobes. Specifically, Clostridium thermocellum and Thermoanaerobacter ethanolicus produce ethanol, degrade polymeric carbohydrates, and have been proposed for use in biomass conversion systems. The specific objectives are to: (i) characterize alterations in the proteomic profile of C. thermocellum and T. ethanolicus in response to ethanol challenge; (ii) determine the proteomic profile of ethanol resistant strains; (iii) examine whether proteomic changes elicited by ethanol are similar to those caused by other environmental stresses including altered temperature, pH, and organic solvents; and (iv) evaluate alternative approaches to identify and quantify changes in proteomes of thermophilic bacteria. The recent completion of the C. thermocellum genome sequence will greatly aid in the identification and putative function of isolated proteins. The proposed work directly addresses Agricultural Issue #6 (Natural Resources and Environmental Quality) in that basic and specific knowledge will be developed on organisms that can be used to develop more sustainable energy technologies. These experiments will provide basic information needed to overcome present process limitations and to optimize microbial conversion of biomass to alcohol, solvents and other useful products.