Improved microbial conversion of de-oiled Jatropha waste into biohydrogen via inoculum pretreatment: process optimization by experimental design approach 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 o C, 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 o C, 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. Kumar et al. / Biofuel Research Journal 5 (2015) 209-214 Please cite this article as: Kumar G., Bakonyi P., Sivagurunathan P., Nemestóthy N., Bélafi-Bakó K., Lin C.Y. Improved microbial conversion of de-oiled Jatropha waste into biohydrogen via inoculum pretreatment and process optimization by experimental design approach. Biofuel Research Journal 5 (2015) 209-214.