Glycerol is a highly available by-product generated in the biodiesel industry. It can be converted into higher value products such as hydrogen using biological processes. The aim of this study was to optimize a continuous dark fermenter producing hydrogen from glycerol, by using micro-aerobic conditions to promote facultative anaerobes. For that, hydrogen peroxide (H2O2) was continuously added at low but constant flow rate (0.252 mL/min) with three different inlet concentrations (0.2, 0.4, and 0.6% w/w). A mixture of aerobic and anaerobic sludge was used as inoculum. Results showed that micro-oxidative environment significantly enhanced the overall hydrogen production. The maximum H-2 yield (403.6 +/- 94.7 mmolH(2)/molGly(consumed)) was reached at a H2O2 concentration of 0.6% (w/w), through the formate, ethanol and butyrate metabolic pathways. The addition of H2O2 promoted the development of facultative anaerobic microorganisms such as Klebsiella, Escherichia-Shigella and Enterococcus sp., likely by consuming oxygen traces in the medium and also producing hydrogen. Despite the micro-oxidative environment, strict anaerobes (Clostridium sp.) were still dominant in the microbial community and were probably the main hydrogen producing species. In conclusion, such micro-oxidative environment can improve hydrogen production by selecting specific microbial community structures with efficient metabolic pathways.