Investigation of Solvent Toxicity in Bacterial Strains Involved in Butanol Production

Reduction in dependency of imported petroleum and the quest to identify renewable energy sources has lead to a search for innovative biofuels derived from renewable biomass, that 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 Clositridium 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 Clositridium spp, must be overcome: first, butanol toxicity to the clostridial cells at low concentrations (e.g. d 2% in final fermentation broth). Second, the inefficient regulation of Electron flow and distribution in Clostridium acetobutylicum that leads to loss of reducing equivalents and the incorrect redox balance. This Phase I proposal aims to fully annotate, metabolically reconstruct and compare the genomes of Clostridium acetobutylicum and C. beijerinckii with specific emphasis on reconstructing metabolic pathways associated with improving solvent tolerance and bioenergetics. Furthermore the genomes of the Clositridium spp. will be compared to other known microbial genomes capable of survival at high levels of organic solvents (e.g. Pseudomonas putide) 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 know C. acetobutylicum strains to create more effective and productive microorganisms suitable for economical butanol biofuel production. The initial commercial target is to create a bio-production system that is economically competitive with the chemical synthesis of butanol from petroleum sources. In order to do this, the current production levels of C. acetobutylicum simply need to be doubled; a readily achievable goal. The ultimate target of this work is to create a butanol bio-production 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.