Abstract
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.
Highlights
Shake-flask cultures of microorganisms involve flame sterilization during sampling, which produces combustion gas with high CO2 concentrations
At both flame sterilizations of the angle of inclination of 25 ° and the routine manual work, as time increased by 1 s, the CO2 concentration increased by ~0.1% (v/v) (Figs. 2 and 3)
We demonstrated for the first time, to the best of our knowledge, that flame sterilization with a Bunsen burner during standard sampling of shake-flask cultures generates CO2 due to combustion and that high concentrations of CO2 accumulate in the headspace of the flasks
Summary
Shake-flask cultures of microorganisms involve flame sterilization during sampling, which produces combustion gas with high CO2 concentrations. We developed a new bypass-type device that can monitor O2 and CO2 concentrations by circulating the medium and gas in shake-flask cultures[5]. This differs from conventional monitoring devices[6,7,8,9,10,11]. Flask shaking is often interrupted in order to investigate changes in various culture parameters with time and the sampling procedure has no established restrictions, except for the prevention of contamination This process is dependent on the skills of the experimenter. Among the various operations in standard sampling, it is presumed that those causing changes to the gas environment of the flask (e.g., flame sterilization or opening the culture-plug) have significant effects on the microorganisms in the culture broth. The aim was to analyze the effect of flame sterilization on the headspace of the flask and on the shake-flask culture
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