This study applies metabolic flux network analysis (MFA) to evaluate the metabolic flux of fermentative hydrogen production (FHP) with the use of Clostridium tyrobutyricum fed with either glucose or lactate/acetate as substrates. The MFA results suggest that hydraulic retention time (HRT) presents significant impact on hydrogen production from glucose. At HRT between 4 and 18 h, increase of HRT increased hydrogen production but decreased lactate production, while at HRT below 4 h decrease of HRT increased hydrogen production but decreased lactate production. The flux for lactate, butyrate and acetate seemed to affect H-2 production, due presumably to their impacts on the balance of NADH, ferredoxin and ATP. It is suggested that the MFA can be a useful tool to provide valuable information for optimization and design of the fermentative hydrogen production process. (c) 2013 Elsevier Ltd. All rights reserved.