Enterobacter aerogenes have a known ability to convert glycerol (GL) in a fermentative process to yield hydrogen and ethanol as the main by-products. The concentration of some media constituents was optimized to maximize biohydrogen yield and rate of production. E. aerogenes were cultured in aerobic conditions, and then transferred into anaerobic conditions before being cultured in a minimum mineral synthetic media (MMSM) containing 15 g/L GL. The concentration of selected salts were optimized in the following ranges: 0-300 mg/L MgSO4, 0-14 g/L Na(2)EDTA, 0-10 mg/L CaCL2, 0-10 g/L Na2HPO4, and 0 9.7 g/L KH2PO4. The results of the full factorial design indicated that the production of biohydrogen required a minimal concentration of 3.5 mg/L EDTA, 200 mg/L MgSO4 center dot 7H(2)O and no CaCl2 center dot 2H(2)O. A significant interaction between EDTA and MgSO4 was also observed. Results from the phosphate salts optimization showed that Na2HPO4 gave better results than KH2PO4. The optimal conditions determined using pure glycerol (commercial grade glycerol), were successfully applied to the fermentation of crude glycerol from biodiesel production. The results indicated promising yields of 0.79 and 0.84 mol/mol of glycerol for bioethanol and biohydrogen, respectively, and this at a faster rate than reported previously for E. aerogenes. (C) 2012 Elsevier Ltd. All rights reserved.