Recent research has focused on finding new sources of renewable energy and the H2 could be considered as renewable if produced from biomass. In that purpose, H2 can be produced biologically by dark fermentation from a great variety of organic materials, including industrial waste such as glycerol. The aim of this study was to evaluate the effect of inoculum pretreatment on H2 yield in a CSTR. Aerobic enrichment as well as thermal shock pretreatment were investigated on two types of mixed inoculums: aerobic and anaerobic sludge. H2 yields showed no significant difference between the use of non pretreated aerobic sludge (control), and anaerobic or aerobic sludge pretreatment after heat shock or aerobic enrichment for 4 weeks. The best yield was around 0.55 molH2 mol−1glycerol. The operational conditions of the continuous system had a higher influence on H2 yields than inoculum pretreatments. It was observed a specific selection of hydrogen producers-microbial community and clearly distinct with non-producers- microbial community.