In bioleaching processes using autotrophic bacteria, CO2 is the carbon source for the growth of the microorganisms and its availability is dependent on gas mass transfer. The objective of this study was to investigate the demand in CO2 in complex copper concentrate bioleaching operations and to optimize CO2 supply. Batch tests in 2L-stirred reactors at 10%w/v solid load were performed to study the need for CO2-supplementation and to determine the adequate CO2 partial pressure in the gas inlet. The results show that Fe oxidation (and thus microbial activity) is delayed when air is injected without CO2-supplementation. CO2-supplementation improves leaching kinetics since Cu dissolution rate increases from 84 mg/L/h with air solely to 120 mg/L/h when CO2 is added to air. The study proposes also a methodology to determine G/L transfer components and to asses CO2 limitations in the system. It shows that the microorganisms are not only sensitive to the transfer rate of CO2 from the gas to the liquid phase, but also to the availability of CO2 in solution.