Investigation of Solvent Toxicity in Bacterial Strains Involved in Butanol Production

Reduction in dependency on imported petroleum and the quest to identify renewable energy sources has led to a search for innovative biofuels derived from renewable biomass, which promise long-term reductions in greenhouse gas emissions. Butanol is one possible biofuel. It is an industrial fuel that can be produced from crops using acetone-butanol (AB) fermentation by butanolagenic microbes, such as Clostridium spp. However, high yields of butanol production by microbial AB fermentation strategies has been limited by butanol toxicity. To make AB fermentation economically tractable, two major shortcomings of this fermentation process that uses Clostridium spp, must be overcome: (1) butanol toxicity to the clostridial cells at low concentrations (e.g., < 2 percent in final fermentation broth); and (2) the inefficient regulation of electron flow and distribution in C. acetobutylicum that leads to loss of reducing equivalents and an incorrect redox balance. This phase I project aims to fully annotate, metabolically reconstruct, and compare the genomes of C. acetobutylicum and C. beijerinckii with specific emphasis on reconstructing metabolic pathways associated with improving solvent tolerance and bioenergetics. Furthermore, the genomes of the Clostridium spp. will be compared to other known microbial genomes capable of survival at high levels of organic solvents (e.g., Pseudomonas putida) to identify key differences and similarities in genes relevant to conferring solvent resistance. The proposed work will lay the foundation for creating a commercial microbial butanol producing system. The immediate goal is to use the information about the presence or absence of enzymes related to key aspects of metabolic sub-systems in strain engineering of known C. acetobutylicum strains to create more effective and productive microorganisms suitable for economical butanol biofuel production. The initial commercial target is to create a bioproduction system that is economically competitive with the chemical synthesis of butanol from petroleum sources. To do this, the current production levels of C. acetobutylicum simply need to be doubled—a readily achievable goal. The ultimate target of this project is to create a butanol bioproduction system that is competitive with the ethanol now being produced for fuel. The higher energy content, miscibility, octane-improving power, and low volatility of butanol are important advantages over ethanol for use in mixtures with gasoline.