In this study, a flux balance analysis (FBA) was adopted to estimate the activity of acetogenic H-2-consuming reaction. Experimental data at different substrate concentrations of 10, 20, and 30 g COD/L showing the lowest, medium, and highest H-2 yields, respectively, were used in the FBA to calculate the fluxes. It was interesting to note that the hydrogenase activity based on R12 (2Fd(+) + 2H(+) -> 2Fd(2+) + H-2, ferredoxin (Fd)) flux was most active at 10 g COD/L. The flux of RI7 (4H(2) + 2CO(2) -> CH3COOH), a mechanism for reutilizing produced H-2, increased in steps of 0.030, 0.119, and 0.467 as the substrate concentration decreased. Contradictory to our general understanding, acetate production found to have a negligible or even negative effect on the final H-2 yield in dark fermentation. (C) 2013 Elsevier Ltd. All rights reserved.