The conversion of carbon monoxide (CO) into hydrogen (H-2) by a Carboxydothermus hydrogenoformans pure culture was investigated and optimized in a 35 L gas-lift reactor. The reactor was operated with a continuous supply of gas for 3 months. Reactor performance was evaluated under various operational conditions, such as gas recirculation rates (0.3 and 1.5 L min(-1)), CO feeding rates (from 0.05 to 0.46 mol L-reactor(-1) day(-1)) and bacto-peptone addition to the medium. Overall, the H-2 yields were constant at 95 +/- 1% and 82 +/- 1% (molH(2).mol(CO)(-1)) with growth supported by peptone and unsupported respectively, regardless of the operational conditions tested. At the highest biomass density, a maximum CO conversion activity of 0.17 mol(CO) L-reactor(-1) day(-1) or 3.79 L-CO L-reactor(-1) day(-1) was achieved. The ratio of gas recirculation over CO feed flow rates (Q(R):Q(in)) was the major parameter that impacted both biological activity and volumetric gas-liquid mass transfer. The CO conversion performance of the gas-lift reactor was kinetically limited over a Q(R):Q(in) ratio of 40, and mass transfer limited below that ratio, resulting in a maximum conversion efficiency of 90.4 +/- 0.3% and a biological activity of 2.7 +/- 0.4 mol(CO) g(VSS)(-1) day(-1). Overall, the CO conversion performance in the gas-lift reactor was limited by a low cell density, typical of C. hydrogenoformans planktonic growth. This limitation was found to be the most restrictive factor for higher CO loading rates. (C) 2013, Crown Copyright and Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.