Enterobacter aerogenes has a known ability to convert glycerol during a fermentative process to yield hydrogen and ethanol as the main products. A Box-Behnken design and response surface methodology were used to determine the optimal concentration of some media constituents and oxygen to maximize the yield of biohydrogen. Results indicated that the concentration of the salts studied: NH(4)NO(3), FeSO(4), and Na(2)HPO(4) and; the presence of oxygen in the pre-culture significantly influence the production of biohydrogen. Optimal conditions were determined to be 7.5% O(2) in the inoculum transfer step, ratio of inocula 18%, 8 g/L of Na(2)HPO(4), 0.00625 g/L of FeSO(4) and 1.5 g/L of NH(4)NO(3). These optimal conditions resulted in a measured yield of 0.85 mol H(2)/mol glycerol at a substrate concentration of 15 g/L and a maximum predicted yield of 0.95 mol H(2)/mol glycerol at a substrate concentration of 21 g/L. These results were obtained using lower concentrations of salts than in previous studies, corresponding to a 76% cost savings. These experimental results also demonstrated the importance of optimizing the amount of oxygen present in the biological system rather than maintaining complete anaerobic conditions. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.