In this study, comparative enhancement effect of nanoparticle form and bulk form of Fe(SO4)OH(H2O)(2) on hydrogen production by mixed culture of Rhodobacter sphaeroides NMBL-02 and Escherichia coli NMBL-04 was investigated in batch process. The maximum cumulative hydrogen production and % substrate conversion efficiencies were observed at 300 mg/L Fe(SO4)OH(H2O)(2) concentration (Fe-concentration) with nano as well as bulk form. But the results obtained with nano form of Fe(SO4)OH(H2O)(2) were 1.2 fold higher in contrast to bulk form. Further optimization studies were also performed with two more factors namely pH and malate concentration. Response surface methodology (RSM) in accordance with Box-Behnken Design was employed to evaluate the interactive effects of these factors. From RSM the optimum values of pH and, malate concentration and Fe-concentration were obtained as 5.603, 3.948 g/L and 312.168 mg/L respectively. Response surface methodology analysis showed best fitting of experimental data with statistical model. At the optimal conditions, the cumulative hydrogen production was reported as 2046 mL H-2/L of medium which was in vicinity of predicted value (2029.405 mL H-2/L). Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.