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Monitoring the environmental factors during shake-flask culture of microorganisms can help to optimise the initial steps of bioprocess development. Herein, we developed a circulation direct monitoring and sampling system (CDMSS) that can monitor the behaviour of CO 2  and O 2  in the gas–liquid phases and obtain a sample without interrupting the shaking of the culture in Erlenmeyer flasks capped with breathable culture plugs. Shake-flask culturing of  Escherichia coli  using this set-up indicated that a high concentration of CO 2  accumulated not only in the headspace (maximum ~100 mg/L) but also in the culture broth (maximum ~85 mg/L) during the logarithmic phase (4.5–9.0 h). By packing a CO 2  absorbent in the gas circulation unit of CDMSS, a specialised shake-flask culture was developed to remove CO 2  from the headspace. It was posited that removing CO 2  from the headspace would suppress increases in the dissolved CO 2  concentration in the culture broth (maximum ~15 mg/L). Furthermore, the logarithmic growth phase (4.5–12.0 h) was extended, the U.O.D. 580  and pH value increased, and acetic acid concentration was reduced, compared with the control. To our knowledge, this is the first report of a method aimed at improving the growth of  E. coli  cells without changing the composition of the medium, temperature, and shaking conditions.

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Development of a circulation direct sampling and monitoring system for O2 and CO2 concentrations in the gas–liquid phases of shake-flask systems during microbial cell culture