Hydrogen (H2) 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 H2-consuming species and/or enrich the microbial community with H2-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 H2 production but aeration caused unstable H2 production. In addition, biokinetic control was key in Clostridium selection as dominant species in all microbial communities. Lower and unstable H2 production were associated with a higher relative abundance of Enterobacteriaceae family members. Our results allow a better understanding of H2 production in continuous systems and how the microbial community is affected. This provides key information for efficient selection of operating conditions for future applications.