Understanding how bioleaching systems respond to the availability of CO2 is essential to developing operating conditions that select for optimum microbial performance. Therefore, the effect of inlet gas and associated dissolved CO2 concentration on the growth, iron oxidation and CO2-fixation rates of pure cultures of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum was investigated in a batch stirred tank system. The minimum inlet CO2 concentrations required to promote the growth of At. ferrooxidans and L. ferriphilum were 25 and 70?ppm, respectively, and corresponded to dissolved CO2 concentrations of 0.71 and 1.57 mu M (at 30 degrees C and 37 degrees C, respectively). An actively growing culture of L. ferriphilum was able to maintain growth at inlet CO2 concentrations less than 30?ppm (0.310.45 mu M in solution). The highest total new cell production and maximum specific growth rates from the stationary phase inocula were observed with CO2 inlet concentrations less than that of air. In contrast, the amount of CO2 fixed per new cell produced increased with increasing inlet CO2 concentrations above 100?ppm. Where inlet gas CO2 concentrations were increased above that of air the additional CO2 was consumed by the organisms but did not lead to increased cell production or significantly increase performance in terms of iron oxidation. It is proposed that At. ferrooxidans has two CO2 uptake mechanisms, a high affinity system operating at low available CO2 concentrations, which is subject to substrate inhibition and a low affinity system operating at higher available CO2 concentrations. L. ferriphilum has a single uptake system characterised by a moderate CO2 affinity. At. ferrooxidans performed better than L. ferriphilum at lower CO2 availabilities, and was less affected by CO2 starvation. Finally, the results demonstrate the limitations of using CO2 uptake or ferrous iron oxidation data as indirect measures of cell growth and performance across varying physiological conditions. Biotechnol. Bioeng. 2012; 109:16931703. (C) 2012 Wiley Periodicals, Inc.