| The expanding global population will need to look to alternative fuel sources in order to meet the increasing demand for transportation fuel, mitigate carbon emissions, and promote energy security. A promising alternative to traditional fossil fuels is the generation of biofuels through the conversion of highly abundant and renewable cellulosic biomass to liquid fuel products, such as ethanol. Clostridium thermocellum is a thermophilic anaerobic bacterium that rapidly solubilizes cellulose and produces ethanol as a byproduct of its metabolism. As such, C. thermocellum is a candidate for the industrial production of renewable bio-ethanol. The ability of C. thermocellum to produce ethanol is dependent on the metabolic activity of the organism. Therefore, it is important to understand factors that render C. thermocellum metabolically inactive and cause the development of non-growth states.;The aim of this work is to understand what factors trigger the formation of two non-growth states, spores and L-forms, in C. thermocellum. We determined that spore formation was induced by exposure to oxygen and changes in substrate composition, whereas L-form formation was induced by starvation conditions during exponential growth. In addition, we sought to identify genes involved in the onset of spore formation. Through the use of targeted gene deletions, gene overexpression, and selective gene expression, we were able to identify four histidine kinases involved in the onset of spore formation, and one kinase with an apparent role in L-form formation. In order to study the role of kinases via selective gene expression, a novel inducible expression system was created for C. thermocellum . This expression system was used to show that sporulation frequencies increase with the expression of clo1313_1492, a gene encoding a sporulation kinase identified in this study.;The ability to control and enhance growth in C. thermocellum cultures is important for optimal biomass deconstruction and ethanol production. In this work we have identified factors that contribute to the development of spores and L-forms in this organism. Detailed understanding of C. thermocellum non-growth states can be used to develop strategies for the effective cultivation of C. thermocellum in industrial biofuel applications, as well as provide insight into Clostridium biology. |
