This Small Business Technology Transfer Research Phase I project aims to enhance fermentation yield of four carbon (C4) chemicals by instating mixotrophic fermentation. In order to realize the most cost effective fermentation for commodity chemical and biofuel production, the process should achieve maximum conversion of feedstock. In carbohydrate fermentations, CO2 and H2 are commonly evolved, which negatively impacts yield of desired products. We hypothesize that mixotrophic fermentation can recapture that yield loss. We define mixotrophic fermentation as the simultaneous consumption of organic and inorganic substrates. Improvements in yield from mixotrophic fermentation can be very significant. Moreover, certain clostridial organisms in theory can perform such fermentation, but relatively little is known about this. Moreover, the genetic tools to manipulate these microorganisms are underdeveloped. Consequently, this Phase I STTR will develop a genetic toolbox for these microorganisms, interrogate their ability for simultaneous substrate utilization of both carbohydrate and gas, and demonstrate the potential to produce C4 chemicals from mixotrophic fermentation.<br/><br/>The broader impact/commercial potential of this project is to develop renewable and domestic chemical production and transportation fuel technologies that are cheaper, greener and more sustainable. Project outcomes, have the potential to increase product yield 10 ? 50%, which greatly reduces production-operating expense. The potential to utilize CO2 in the fermentation, minimizes the carbon footprint of the process. Lastly, process sustainability is enhanced since a greater diversity of feedstocks can be concurrently used such as complex carbohydrates, five and six carbon sugar monomers, biodiesel waste, hydrolyzed biomass, syngas, waste gas, and activated methane molecules. Overall, the project has the commercial potential to improve the triple bottom line of many chemical companies. Furthermore, this project could significantly enhance scientific and technological understanding of microbial physiology and metabolism during gas and carbohydrate fermentation.