This study investigated the effect of controlling pH (5.5) and heat pre-treatment of seed inoculum in dark fermentative hydrogen production. The results showed that only inoculum source plays an important role rather than pH and heat treatment. Seed source is vital factor despite, heat treatment and pH controlled at 5.5. Mesophilic fermentation resulted in CH4 generation, however, thermophilic fermentation while using thermophilic inoculum is opted for H-2 generation. In contrast promoted mesophilic inoculum (mesophilic to thermophilic) still documented for CH4 generation. Peak hydrogen production rate (HPR) and methane production rate (MPR) were noted as 90 and 117 mL/L-d, during the conditions of thermo inoculum (thermophilic, pH 5.5) and pH no control (mesophilic) experiments, respectively. Peak, total solids (TS) and chemical oxygen demand (COD) removal were achieved as 56 and 42% at mesophilic condition. Volatile fatty acid (VFA) profiling revealed the background of the process performances. Microbial community analysis via fluorescent in-situ hybridization (FISH) narrated that bacteria and archaea communities were enriched during thermophilic and mesophilic experiments, respectively. Besides, the presence of methanogens revealed that heat treatment and controlling moderately acidic pH (5.5) could not completely eliminate them and resulted in CH4 generation, rather than H-2 production. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.