Shake-flask cultures of microorganisms involve flame sterilization during sampling, which produces combustion gas with high CO2 concentrations. The gaseous destination has not been deeply analyzed. Our aim was to investigate the effect of flame sterilization on the headspace of the flask and on the shake-flask culture. In this study, the headspace CO2 concentration was found to increase during flame sterilization ~0.5–2.0% over 5–20 s empirically using the Circulation Direct Monitoring and Sampling System. This CO2 accumulation was confirmed theoretically using Computational Fluid Dynamics; it was 9% topically. To evaluate the influence of CO2 accumulation without interference from other sampling factors, the flask gas phase formed by flame sterilization was reproduced by aseptically supplying 99.8% CO2 into the headspace, without sampling. We developed a unit that can be sampled in situ without interruption of shaking, movement to a clean bench, opening of the culture-plug, and flame sterilization. We observed that the growth behaviour of Escherichia coli, Pelomonas saccharophila, Acetobacter pasteurianus, and Saccharomyces cerevisiae was different depending on the CO2 aeration conditions. These results are expected to contribute to improving microbial cell culture systems.