LAUSR

Abstract 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 × 10 8  cells), a DFE dilution of 2.5× 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.