In the context of a global energy transition, sustainable ways to produce hydrogen need to be intensively investigated. Among these ways, the degradation of organic matter through dark fermentation processes is considered as a solution to avoid the use of hydrogen derived from fossil fuels. Several anaerobic bacterial species belonging to the genus Clostridium are known to perform such a fermentation, since they are able to convert hexoses into hydrogen, organic acids and solvents during their growth. The aim of this work was first to evaluate the operational parameters (pH, temperature, concentration of substrate), as well as the culture medium, to perform batch cultures in a bioreactor. Then, four strains of Clostridium (C. butyricum DSM 5431, C. pasteurianum ATCC 6013-W5, C. acetobutylicum ATCC 824 and C. tyrobutyricum CNRZ 510) were cultured under identical conditions to make comparative analyses in term of gas production (specific production rate and H2/CO2 ratio) using gas chromatography. The kinetics of metabolites production were also evaluated (mainly acids and solvents) and were used to estimate theoretical H2 productions, based on well-known metabolic pathways. Acetate and butyrate productions were particularly regarded because each mole of glucose converted into these metabolites is accompanied by the formation of 4 and 2 moles of H2 respectively. From this point of view, C. pasteurianum showed the most interesting performances in association with the best growth kinetic parameters. C.pasteurianum was then selected for further investigations during continuous culture processes in order to optimize the hydrogen production using a design of experiment with the pH, the dilution rate and the limiting glucose concentration as the input variables. Our preliminary results indicated that the dilution rate and the pH are the most important parameters, favoring the acetate production pathway, with a strong impact on H2 productivity.