Improving Metabolic Engineering and Characterization of <i>Clostridium thermocellum</i> for Improved Cellulosic Ethanol Production

Biofules are an important option for humanity to move away from its dependence on fossil fuels. Transitioning from food crops to lignocellulosic alternatives for the production of biofuels is equally important. Most commonly, biofuels are produced using a crop such as corn or soybeans to feed sugars to the yeast, Saccharomyces cerevisiae for the fermentation of ethanol. Lignocellulosic biofuel production would eliminate the need for food crops and transition to biomass such as switchgrass, poplar, or corn stover. Currently, lignocellulosic biofuel production is limited primarily because of the cost of converting the biomass to fermentable sugars than can then be metabolized by yeast. To overcome this barrier, a process must be employed that can convert lignocellulosic biomass directly to fuels and chemicals quickly and affordably. Clostridium thermocellum is one of the most promising candidates for the production of advanced biofuels because of its potential ability to convert cellulose directly to ethanol without the expensive addition of enzymes. Challenges to implementing C. thermocellum on an industrial scale still exist including side product formation, slow growth, limited titers, inhibition on high solids loadings, and a limited ability to perform genetic engineering. This thesis considers all of these concerns with C. thermocellum and attempts to systematically improve each characteristic to produce an industrially relevant strain of C. thermocellum for advanced biofuel production. Metabolic engineering is applied for the elimination of undesirable fermentation products. Laboratory evolution and medium supplementation are used to improve and understand the mechanisms that influence growth rate, and systematic approaches are used to improve transformation for more efficient genetic engineering of C. thermocellum in the future.