<P><B>Abstract</B></P> <P>Hydrogen (H<SUB>2</SUB>) 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<SUB>2</SUB>-consuming species and/or enrich the microbial community with H<SUB>2</SUB>-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<SUB>2</SUB> production but aeration caused unstable H<SUB>2</SUB> production. In addition, biokinetic control was key in <I>Clostridium</I> selection as dominant species in all microbial communities. Lower and unstable H<SUB>2</SUB> production were associated with a higher relative abundance of <I>Enterobacteriaceae</I> family members. Our results allow a better understanding of H<SUB>2</SUB> production in continuous systems and how the microbial community is affected. This provides key information for efficient selection of operating conditions for future applications.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Microbial community in inocula has a great impact on pre-treatments efficiency. </LI> <LI> In aerobic sludge no pre-treatment is required to increase hydrogen yield. </LI> <LI> Biokinetic control has a strong influence on the <I>Clostridiaceae</I> family selection. </LI> <LI> Low/unstable hydrogen production is associated with the <I>Enterobacteriaceae</I> family. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>