Bioconversion of crude glycerol to hydrogen has gained importance as it addresses both sustainable energy production and waste disposal issues. Until recently, statistical optimizations of crude glycerol bioconversion to hydrogen have been greatly focused on pure strains. In this study, biohydrogen production from crude glycerol by an enriched microbial culture (predominated with Clostridium species) was improved by statistical optimization of media components. Plackett-Burman design identified MgCl2 center dot 6H(2)O and KCl with negative effect on hydrogen production and selected NH4Cl, K2HPO4 and KH2PO4 as significant variables. Box-Behnken design indicated the optimal region beyond design area and studies were continued by ridge analysis. Central composite face centered design envisaged a maximal hydrogen yield of 1.41 mol-H-2/mol-glycerol(consumed) at concentrations 4.40 g/L and 2.27 g/L for NH4Cl and KH2PO4 respectively. Confirmation experiment with the optimized media (NH4Cl, 4.40 g/L; K2HPO4, 1.6 g/L; KH2PO4, 2.27 g/L; MgCl2 center dot 6H(2)O,10 g/L; KCl, 1.0 g/L; Na-acetate center dot 3H(2)O, 1.0 g/L and tryptone, 2.0 g/L) revealed an excellent correlation between predicted and experimental hydrogen yield. Optimization of media components by design of experiments enhanced hydrogen yield by 29%. (C) 2014 Elsevier Ltd. All rights reserved.