The biohydrogen production potential of E. coli XL1 blue was investigated with pure substrate (glucose), complex natural feedstock (Acid (HCl) +microwave assisted and enzymatic (Viscozyme mediated) hydrolysate) of Deoiled jatropha waste (DJW) in order to check the efficiency of the strain towards fermentative hydrogen production. The results show that the strain has a conversion efficiency of sugar degradation of more than 75% in all cases. The hydrogen formation from DJW hydrolysate revealed that the strain could work efficiently in complex substrates also. Applied immobilization technique effectively enhanced the production rates. The peak hydrogen production rate (HPR), specific hydrogen production rate (SHPR) and hydrogen yield (HY) were obtained as 3.18 ± 0.2 L H2/L-d, 7.95 ± 0.5 L H2/g DCW-d and 153.3 ± 3.3 mL H2/ g substrate added when substrate (glucose) concentration 5 g/L, incubation temperature 37 0C and biomass concentration was set to 0.4 g DCW/L (immobilized cells). These values indicated that process efficiency is almost doubled compared with free cells. The energy evaluation report states that the bioenergy produced from this process calculated as peak energy generation rate (EGR) and energy yield are 37.3 ±0.2 kJ/L-d and 1.8 ±0.6 kJ/g Substrate added.