Control of carbon dioxide concentration in headspace of multiple flasks using both non-electric bellows pump and...

Current methods of controlling gas in the headspace involve constant speed aeration and proportional-integral-differential (PID) controlled aeration using improved monitoring devices or gas cylinders. However, these approaches are restricted and inconvenient to use. In this study, we propose a method to control the CO₂ concentration in the headspace while maintaining the convenience of shake-flask culture. A combination of a non-electric bellows pump for shake-flask (NeBP-sf) and a CO₂ incubator was used to control the flask gas phase by shaking without additional external power. The CO₂ half-life, as an indicator of the ventilation ability of the system, was measured using a circulation direct monitoring and sampling system, and the NeBP-sf was optimised. The ventilation capacity varied depending on the shaking speed, and under optimal conditions, was 10 min compared with 45 min when only a breathable culture plug was used. In conventional microbial shaking culture, the CO₂ concentration in the flask gas phase remained higher than the 5% set-value with a maximum of 9%, resulting in a large concentration difference with the set point. Therefore, the ventilation capacity of the conventional shake-flask culture was insufficient for aerobic culture. Cultivation of Escherichia coli and Lactiplantibacillus plantarum using the system showed no significant difference between the set point and real point values. Thus, the system combined an NeBP-sf and a gas incubator built-in shaking table to achieve the reproducibility of gas control while maintaining a high level of convenience.