According to the results obtained from this study, it is concluded that using higher initial glucose concentration (36.9 g/L) could lead to a higher cumulative hydrogen production, but much more initial glucose concentration (46.11 g/L) would result in substrate inhibition for H2-producing microorganism (Lee et al., 2008). Another reason for the decrease of cumulative hydrogen production could be the increase in the viscosity of glucose solution, thereby affecting the mass transfer of substrate to microorganism.