Hydrogen can be produced via dark and photo-fermentation using either single-stage or two-stage processes. The advantage of a two-stage system is that it is possible to separately optimize and control culture conditions for the dark and photo-fermentative bacteria. In the present study a mixture of starch and glucose was used as carbon source for the dark fermentation step. Response surface methodology (RSM) with a Box-Behnken design (BBD) was applied to the photo-fermentation stage for the optimization of key parameters: inoculum concentration (Rhodopseudomonas palustris), substrate concentration (dark fermentation effluent (DFE)) and pH. In this sequential two-stage system, the highest overall hydrogen yield (8.3 +/- 0.1 mmol H-2/gCOD), overall hydrogen production (1.62 mmol) and the photo fermentation yield (7.21 +/- 0.2 mmol H-2/gCOD) were achieved at inoculum of 9 mL (1.64 +/- 0.7 x 10(8) cells), a DFE dilution of 2.5x and a pH of 7.5, which were center points of the design. 97% COD removal was achieved at the highest dilution of DFE (lowest concentration of carbon source) at pH 6.5. (C) 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.