Mixed culture fermentation has to be considered as a serious alternative to pure culture fermentation in environmental biotechnology for the production of hydrogen (H2) and other molecules of interest such as organic acids or alcohols. Indeed, a mixed culture can be operated continuously under unsterile conditions, at low cost and from various organic substrates. So far, most studies have focused on dominant species, such as Clostridium for H2 production, but sub-dominant bacteria can also have a significant effect on the ecosystem despite their low abundance. Thus the determination of the ecological function of these species in their environment is essential for better understanding the microbial processes in mixed cultures. In this work, the contribution of sub-dominant bacteria to H2 production by dark fermentation was investigated using continuous-flow stirred tank-reactors (CSTR) at steady state. Three complex ecosystems were used as initial inoculum: an anaerobic digested sludge, a pre-fermented manioc and freshly collected caecotrophs (i.e., soft faeces of rabbits). Moreover, a heat pre-treatment of each inoculum as well as a mixture of these 3 inocula were also evaluated. The operating conditions were the same for all the experiments and were considered as optimal for H2 production (i.e., 37°C; pH of 5.5; HRT of 6 h). Glucose was used as model substrate. For each assay, the diversity and dynamics of microbial communities were investigated by Capillary Electrophoresis Single-Strand Conformation Polymorphism (CE-SSCP). Along with microbiological analysis, the performance of H2 production and the distribution of the metabolic products were assessed. At steady state, seven different H2 producing ecosystems were obtained. The H2 yield varied between 1.3 and 2.6 mol H2 per mol glucose consumed. The H2 yield increased with a decrease of the diversity of the ecosystems. The results showed that the same dominant bacterial species was found six times on seven assays (i.e., C. pasteurianum). Thus, only the nature, the number and the relative abundance of the minority bacterial population differed from one ecosystem to another. The phylogenetic affiliation of the minority species seemed to be also important: E. coli influenced positively the H2 yield by redirecting the metabolic network to acetate and butyrate production, while Bacillus sp. and Lactobacillus sp. impacted negatively the H2 yields by diverting significantly a part of the H2 potential to lactate production. To conclude, this study showed that H2 production by dark fermentation is clearly influenced by the structure and the diversity of the bacterial community. The metabolic network of the ecosystem seems to be driven by minority bacteria. These sub-dominant bacteria are good candidates for acting as keystone species of such microbial ecosystems. The implementation of these species in an ecosystem should allow to control the microbial bioprocess for functioning optimally.