Biohydrogen produced by fermentation depends of the operating conditions. The objective of this experimental work was to establish a reaction pathway based on measured values of compound concentrations and to estimate the relative effect of environmental conditions like pH, stirring velocity and hydraulic retention time (HRT). The experiments were carried out at 37 °C using a continuous stirred tank reactor fed with molasses. A principal component analysis was used to determine the main reactions implied in the degradation of sucrose into acetate, propionate, butyrate, hydrogen, inorganic carbon and biomass. Two reactions explain 89% of the experimental variance. The estimation of the pseudo-stoichiometric coefficients, using SQP optimization, shows that the first reaction mainly produced butyrate, hydrogen and biomass whereas the second reaction mainly produced acetate and biomass. HRT ranged from 6 to 14 h, pH from 5.5 to 6 and the stirring velocity from 150 to 300 rpm. The maximum hydrogen production of 5.4 L d-1 L-1 was obtained for a pH of 5.5, a HRT of 6 h and a stirring velocity of 300 rpm, at a loading rate of 37.12 gCOD d-1 L-1, which corresponds to 2.5 mol H2/mol sucrose. Only linear effects of pH, HRT and stirring velocity were significant and not their interactions or quadratic effects.