The optimum concentration of volatile fatty acids that maximized the hydrogen production by photobacteria was determined using Box-Behnken and Central Composite designs. A photofermentative hydrogen-producing consortium, obtained from a bio-electrochemical system, was used as inoculum. Rhodopseudomonas palustris was identified in the consortium. Experiments were conducted in anaerobic batch reactors at 30-35 degrees C and illuminated by a constant radiation of 5000 lux. Hydrogen production decreased when propionic acid is higher than 715 mg L-1. Ammonia formation was responsible for the pH increase. A maximum hydrogen production rate of 6.1 mL d(-1) L-culture(-1) h(-1) and a maximal amount of hydrogen produced of 108.8 mmol L-culture(-1) were obtained. Optimal concentrations of acetic, propionic and butyric acids were 1200 mg L-1, 715 mg L-1 and 1571 mg L-1, respectively. Copyright (c) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.