To maximise the yield from fermentative H2 production, H2 consumption must be minimised. This work demonstrated for the first time that H2 consumption exists in an established continuous-flow biohydrogen system. The rate of H2 consumption was found to be related to the concentration of CO2, with H2 consumption inhibited at both low and high CO2. N2 sparging of the continuous reactor at 31 mL/min/L-liquid increased the H2 yield from 1.31 to 1.87 mol H2/mol glucose, but did not significantly change the in-situ rate of H2 consumption (0.07–0.09 mM/h). Assuming sparging completely inhibited H2 consumption, it could only account for 2–11% of the H2 yield increase during sparging, based on H2 consumption rates measured in the reactor and in vials. Therefore, H2 consumption may be of minor concern for continuous biohydrogen systems.

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