Fermentation of de-oiled Jatropha waste (DJW) hydrolyzate was carried out at 55 degrees C and pH 7 to evaluate the efficacy of the DJW pretreatment methods for enhancing the hydrogen production rate. The enzyme (7.5%) treated hydrolyzate showed a peak hydrogen production rate (HPR) and hydrogen yield (HY) of 5146 mL H-2/L-d and 98 mL H-2/g VSadded, respectively. The HCl (2.5%), H2SO4 (0.5%), ultrasonication (45 min), and heat treatment (100 degrees C, 45 min) treatments gave maximal HY of 43, 24, 23, and 34 mL H-2/g VSadded, respectively. Further enzyme (5%) treatment of the acid (2%) unhydrolyzed biomass showed HPR of 2947 mL H-2/L-d and HY of 106 mL H-2/g VSadded. Besides, it was also observed that higher DJW concentrations during pretreatment, and higher butyrate levels in the fermentation process, led to increased release of reducing sugar and increased HPR, respectively. The total bioenergy production analysis revealed nearly 10-fold increase in the energy values with peak HPR and HY of 55 kJ/L-d and 1.0 kJ/g VSadded, respectively. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.