Design of Experiments (DoE) was applied to improve the ability of enriched activity sludge to efficiently convert crude glycerol from biodiesel industry into hydrogen and ethanol, using a very simple synthetic medium. Based on Plackett-Burman screening design, glycerol concentration, temperature and initial pH were identified as significant variables. Box-Behnken design and Response Surface Method (RSM) were then used for optimization. The maximum hydrogen yield of 0.96 mol H-2/mol glycerol was estimated at the temperature of 37.0 degrees C, initial pH of 7.9 and glycerol concentration of 15.0 g/L. Maximum hydrogen production rate of 2191 mL/L/d was estimated at the temperature of 37.3 degrees C, initial pH of 8.0 and glycerol concentration of 15.2 g/L. Finally maximum ethanol production of 7.92 g/L was estimated at an initial pH of 8.0 and glycerol concentration of 15.0 g/L (temperature had no significant effect). These results show that it is possible to obtain both, high yield and production of hydrogen and ethanol together, using a very simple synthetic medium, without trace element- and vitamin solution, tryptone or yeast extract. Copyright (c) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.