The fermentative metabolism of glycerol in the Enterobacteriaceae has long been considered a property of species that synthesize 1,3-propanediol (1,3-PDO). However, we have discovered that E. coli, an organism lacking the 1,3-PDO pathway, can fermentatively metabolize glycerol. We provide genetic and physiological evidence supporting: (i) the fermentative nature of this process, (ii) the role of fermentative pathways, and (iii) the incorporation of glycerol into cell mass. A novel trunk pathway responsible for the conversion of glycerol into glycolytic intermediates was identified. In addition, the formate hydrogen-lyase and F0F1-ATPase systems are required for this metabolic process. We propose a new paradigm for the 1.3-PDO-independent fermentation of glycerol in enteric bacteria in which trunk and auxiliary pathways work in partnership to attain redox balance. We demonstrated that glycerol fermentation in E. coli was not previously observed due to the use of culture conditions that impair the aforementioned pathways. Finally, we illustrated the significance of our findings for the development of microbial platforms that will convert low-priced glycerol (an inevitable byproduct of biodiesel fuel production) to higher value fuels and chemicals. Our current efforts focus on the identification of genetic and environmental determinants of glycerol fermentation by using system-level techniques, including global gene and protein expression analysis in combination with in silico and in vivo flux analysis. We will discuss at the meeting the implications of our findings for: (1) the elucidation of glycerol fermentation in enteric bacteria and (2) the metabolic engineering of E. coli to convert glycerol into reduced chemicals.