Both dark and photo-fermentation can be used for biological hydrogen production; either performed separately, in two-stage systems, or in co-culture. A single stage process is less laborious and costly; however, the two types of microorganisms have different nutritional requirements requiring optimization of culture conditions. Here a response surface methodology (RSM) with a Box-Behnken design was used to optimize microorganism ratio and substrate and buffer concentrations, and to evaluate their interactive effects for maximization of hydrogen yield. Clostridium butyricum and Rhodopseudomonas palustris were grown on a potato starch/glucose base medium at 30 degrees C under continuous illumination (40 W m(-2) light intensity). The highest hydrogen yield, 6.4 +/- 1.3 mol H-2/mol glucose, was obtained with a substrate concentration of 15 g/L, buffer concentration of 50 mM, and microorganism ratio of 3. The observed strong interaction between buffer and substrate concentration is most likely due to the need to optimize the pH for co-cultures. (C) 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.