Biohydrogen from microalgal biomass has shown particular advantage due to its high growth rate and high bioenergy production. As a representative of microalgae, Chlorella vulgaris was chosen as substrate along with digested sludge (DS) as inoculum in this research. In order to improve the hydrolysis of algal biomass and enhance biohydrogen production, pretreatment methods like acid and thermal pretreatment were employed. Thermal pretreatment showed better results than acid pretreatment of microalgal biomass. 100 degrees C for 60 min was identified as the optimum condition for the thermal pretreatment of C vulgaris by response surface methodology (RSM) analysis. Experiments were also carried out to identify the optimum substrate to inoculum ratio (SIR) for the process. SIR of 8 generated the highest hydrogen yield of 190.90 mL H-2/g-VS. Moreover, the overall energy balance of the process was evaluated and the results showed a positive energy balance of 1790.13 kJ/kg. The results indicated that optimization of pretreatment methods and substrate to inoculum ratio was effective in enhancing biohydrogen production from microalgal biomass and digested sludge. (C) 2018 Elsevier Ltd. All rights reserved.