Abstract:In this study, batch biohydrogen production by co-digestion of raw rice straw and activated sewage sludge was investigated with different inoculum heat treatment, pH, S/X ratio (based on VS) and substrate sizes under mesophilic condition. In order to achieve a high bio-hydrogen yield and methanogens activity inhibition, heat treatment of inoculum was optimized at different exposure times (30, 45 & 60 min) and temperature ranges (80, 90 and 100 °C) prior to dark fermentation process. Collected data was analysed using response surface methodology (RSM).The heat treatment of inoculum at 100 °C for 60 minutes produced the highest bio-hydrogen yield of 14.22 NmL H2/g VS at concentration of 70.97 % and Production of 0.073 NmLCH4/gVS at 0.17% concentration in total produced biogas. The raw rice straw was also co-digested with heat-treated inoculum at different ratios of volatile solids (2:1, 4:1 and 6:1) and initial pH (4, 4.75 and 5.5) as numerical variables and 4 categories of substrate size ( (250-500 µm], (500 µm-2mm], (2-20mm), [20-30mm]) . The highest bio-hydrogen yield of 14.70 NmL/g VS was recognized at the optimum initial pH of 5.01 and S/X ratio of 4.54:1 using 2-20 mm rice straw.