Hydrogen (H-2) production by dark fermentation can be performed from a wide variety of microbial inoculum sources, which are generally pre-treated to eliminate the activity of H-2 consuming species and/or enrich the microbial community with H-2-producing bacteria. This paper aims to study the impact of the microbial inoculum source on pre-treatment behavior, with a special focus on microbial community changes. Two inocula (aerobic and anaerobic sludge) and two pre-treatments (aeration and heat shock) were investigated using glycerol as substrate during a continuous operation. Our results show that the inoculum source significantly affected the pre-treatment efficiency. In aerobic sludge no pre-treatment is necessary, while in anaerobic sludge the heat pre-treatment increased H-2 production but aeration caused unstable H-2 production. In addition, biokinetic control was key in Clostridium selection as dominant species in all microbial communities. Lower and unstable H-2 production were associated with a higher relative abundance of Enterobacteriaceae family members. Our results allow a better understanding of H-2 production in continuous systems and how the microbial-community is affected. This provides key information for efficient selection of operating conditions for future applications. (C) 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.