Hydrogen supersaturation in extreme-thermophilic (70 degrees C) mixed culture fermentation (MCF) was demonstrated for the first time by membrane inlet mass spectrometry. It was found that hydrogen supersaturation ratio (R-H2) increased dramatically (from 1.0 to 20.6) when H-2 partial pressure (P-H2) was reduced by N-2 flushing or sparging. The distribution change of metabolites was insignificant under low P-H2 (<0.30 atm) due to the high value of R-H2, which indicated that it was more relevant to the concentration of dissolved H-2 (H-2aq) rather than P-H2. To explain the cause of hydrogen supersaturation, the overall volumetric mass transfer coefficients (K(L)a) for H-2 were calculated. K(L)a changed slightly (similar to 7.0/h) with N-2 flushing, while it increased from 7.4 to 10.2/h when N-2 sparging rate increased from 0.3 to 17.9 mL/min/L. However, the required Kip values were orders of magnitude higher than the experimental ones when maintaining low R-H2 by gas sparging, which indicated that hydrogen supersaturation was likely inevitable in MCF. Moreover, to improve the hydrogen yield of MCF, the gas sparging rate was suggested as 2-10 times of the hydrogen production rate. (C) 2013 Elsevier Ltd. All rights reserved.