Biodiesel production from agricultural crops generates a considerable amount of crude glycerol as a by-product each year. The purification costs for this crude glycerol are high and market demand for refined glycerine is low, resulting in a large fraction of the crude glycerol being incinerated, adding to climate emissions and production costs. World Energy intends to commercialize the production of bio-butanol, a superior biofuel and chemical commodity, from this waste glycerol. A process has previously been developed for the conversion of glycerol to butanol using Clostridium pasteurianum, however the genetic changes that have occurred and the stability of the new strains are not well understood. The aim of this project is to improve the continuous fermentation process by gaining a better understanding of the genetic changes that have occurred in the engineered bacterial strains as well as enhance bio-butanol production and fatty acid tolerance through additional genetic modifications. Process scale up is also planned. World Energy generates approximately 66,000 MT of glycerol annually that can be used for bio-butanol production. With initial goals of 30% of the carbon from waste glycerol being converted to bio-butanol, up to $3.7 million additional revenue could be incurred per facility per year.