In this study various pretreatment methods of sewage sludge inoculum and the statistical process optimization of de-oiled jatropha waste have been reported. Peak hydrogen production rate (HPR) and hydrogen yield (HY) of 0.36 L H2/L-d and 20 mL H2/g Volatile Solid (VS) were obtained when heat shock pretreatment (95 oC, 30 min) was employed. Afterwards, an experimental design was applied to find the optimal conditions for H2 production using heat-pretreated seed culture. The optimal substrate concentration, pH and temperature were determined by using response surface methodology as 205 g/L, 6.53 and 55.1 oC, respectively. Under these circumstances, the highest HPR of 1.36 L H2/L-d was predicted. Verification tests proved the reliability of the statistical approach. As a result of the heat pretreatment and fermentation optimization, a significant (~ 4 folds) increase in HPR was achieved. PCR-DGGE results revealed that Clostridium sp. were majorly present under the optimal conditions.