Hydrogen is produced during fermentative metabolism of glycerol via conversion of pyruvate to formate, a reaction catalyzed by the enzyme pyruvate formate lyase (PFL). The formate is converted to carbon dioxide and hydrogen by the action of formate hydrogen lyase (FHL). This is probably essential for maintaining both the CO2 supply needed for cell growth (Dharmadi et al. 2006) and the production of a proton motive force (PMF) that is essential for cell viability (Bagramyan and Trchounian 2003; Hakobyan et al. 2005). Under certain environmental conditions, engineered E. coli was found to produce hydrogen at the rate of 4.64mmol/L/h with a specific yield of 0.96 mol H-/mol glycerol (Yazdani and Gonzalez 2008) . Biohydrogen production from glycerol containing waste generated from a biodiesel plant was studied using a natural isolate E. aerogenes. The rate of hydrogen production was determined to be 63 mmol/L/h with a specific yield of 0.85 mol H—mol glycerol (Ito et al. 2005; Nishio and Nakashimada 2007- . Production of biohydrogen by E. aerogenes using crude glycerol as substrate affords biodiesel industries the opportunity to add value to crude glycerol and potentially increase their revenue base.