A metabolic network model for Clostridium butyricum was developed using six different carbon sources (sucrose, fructose, galactose, mannose, trehalose and ribose) to study the fermentative H-2 production. The model was used for investigation of H-2 production and the ability of growth on different substrates to predict the maximum abilities for fermentative H-2 production that each substrate can support. NADH fluxes were calculated by the model as an important cofactor affecting on H-2 production. Butyrate and acetate production were used as model assumptions and biomass formation was chosen as the objective function for flux analysis calculations. Among the substrates selected, sucrose and trehalose supported the maximum growth and H-2 yields. The Cell Net Analyzer metabolic network model developed for H-2 estimation revealed good correlation with experimental data and could be further used to study the effect of environmental conditions and substrates concentration on H-2 yield. (C) 2014 Elsevier Ltd. All rights reserved.