Hydrogen is a strong inhibitor of dark fermentation. We aimed at directly correlating the hydrogen production by Thermotoga neapolitana with the supersaturation of hydrogen in the liquid phase (H-2aq), which is often disregarded. Different agitation speeds, biogas recirculation and bubble induction by AnoxK(TM) K1 carrier were tested to prevent the supersaturation of H-2aq. At 100 rpm agitation, the H-2aq was 29.7 (+/-1.4) mL/L, which is 3-times higher than 9.7 mL/L, i.e. the equilibrium concentration given by Henry's law. Increasing the agitation speed up to 600 rpm reduced the H-2aq until 8.5 (+/-0.1) mL/L., in 2 h and increased the hydrogen production rate (HPR) from 39 (+/-2) mL/L/h at 0 rpm to 198 (+/-4) mL/L/h at 600 rpm. Similar to 600 rpm, biogas recirculation and the presence of K1 carrier at 200 rpm maintained the H-2aq below the equilibrium concentration. This study demonstrates the reciprocal influence of HPR and H-2aq and revealed an inverse nonlinear correlation between the two parameters. Therefore, we conclude that an adequate gas-liquid mass transfer, efficiently provided by biogas recirculation or the presence of solid materials (e.g. a biomass carrier), is essential to remove H-2 from the liquid phase and prevent H-2 supersaturation. (C) 2019 Published by Elsevier Ltd.