Abstract The kinetic study was performed using a modified “initial rate-method” and the dynamic ones by the relaxation time methodology. The approach was tested on glucose as sole carbon source while the hydrogen forming bacteria HFB were obtained by acid treatment of anaerobic sludge. A large spectrum of substrate concentration from 5 g/l to 90 g/l was experimentally tested. During the test biogas evolution, gas composition, glucose concentration as well as pH and Red-Ox Potential (ROP) were monitored. At the end of the tests ethanol and VFA were measured to evaluate a reference molar H2 yield (Y*). The biogas composition ranged in (40–60%) for H2 and rest CO2, no CH4 was observed. A first order kinetic equation for glucose with a kinetic constant of 0.0041 h−1 and an inhibited kinetic equation for biogas evolution with a maximum production rate of 100 ml/l h were set-up. The dynamic study evidences the strong role of the pH in the regulation of activity of the Ferrodoxin and Hydrogenase pools. Lastly a test with a bioreactor of 2 l with pH adjustments validated the dynamics of the system showing an increase of 2.8 times of efficiency of glucose conversion into H2 compared with tests without pH adjustments and agitation.