Present study investigated fermentative hydrogen production of two novel isolates of Enterobacter aerogenes HGN-2 and HT 34 isolated from oil water mixtures. The two isolates were identified as novel strains of E. aerogenes based on 16S rRNA gene. The batch fermentations of two strains from glucose and xylose were carried out using economical culture medium under various conditions such as temperature, initial pH, NaCl, Ni(+)/Fe(++), substrate concentrations for enhanced fermentation process. Both the strains favoured wide range of pH (6.5-8.0) at 37 degrees C for optimum production (2.20-2.23 mol H(2)/mol-glucose), which occurred through acetate/butyrate pathway. At 55 degrees C, both strains favoured 6.0-6.5 and acetate type fermentation was predominant in HT 34. Hydrogen production by HT 34 from xylose was highly pH dependant and optimum production was at pH 6.5 (circa 1.98 mol-H(2)/mol-xylose) through acetate pathway. The efficiency of the strain HGN-2 at pH 6.5 was 1.92-1.94 mol-H(2)/mol-xylose, and displayed both acetate and butyrate pathways. At 55 degrees C, very low hydrogen production was detected (less than 0.5 m mol/mol-xylose). (C) 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.