Nowadays mixed cultures are considered as a serious alternative to pure cultures in biotechnological processes. Mixed cultures can use various organic substrates and be operated under unsterile and continuous conditions. Although most of studies dealing with fermentative hydrogen production have focused on dominant species, sub-dominant bacteria can also have a significant effect despite their low abundance. The determination of their exact ecological role is essential for better understanding microbial metabolic networks in mixed cultures. In this work, the contribution of sub-dominant bacteria to fermentative H-2 production was investigated using chemostats continuously fed with a glucose-based medium. Interestingly, Clostridium pasteurianum was dominant in six assays on seven at steady state, and only bacterial populations in low abundance differed. Acting as keystone species, these bacteria impacted substantially the microbial metabolic network of the overall ecosystem despite their low abundance. While Bacillus spp. and Lactobacillus spp. lowered the H-2 yields by diverting a part of the H-2 potential to lactate production, the presence of Escherichia coli increased the H-2 yield by redirecting the metabolic network to acetate and butyrate hydrogen-producing pathways.