Research Article: 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

Date Published: August 23, 2017

Publisher: Springer Berlin Heidelberg

Author(s): Masato Takahashi, Yoshisuke Sawada, Hideki Aoyagi.


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 CO2 and O2 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 CO2 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 CO2 absorbent in the gas circulation unit of CDMSS, a specialised shake-flask culture was developed to remove CO2 from the headspace. It was posited that removing CO2 from the headspace would suppress increases in the dissolved CO2 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.

Partial Text

Shake-flask cultivation in Erlenmeyer flasks with breathable culture plugs was first developed in 1932 for the submerged culture of Aspergillus niger (Kluyver and Perquin 1933). Shake-flask culture is frequently used to screen for secondary metabolites and optimise culture conditions for microorganisms in the initial steps of bioprocess development, because a lot of samples can be aerobically batch cultured in parallel at a low cost (Van Gool et al. 2011; Diederichs et al. 2014). In evaluating cultured samples, it is important to understand the culture environment (e.g., dissolved O2 and CO2, pH, biomass, nutrition source, products, oxidation–reduction potential, etc.) during the shake-flask culture of microorganisms. However, since Erlenmeyer flasks (with culture plugs) containing culture broth are rotated at a high speed, monitoring multiple culture factors in both the headspace and culture broth simultaneously is difficult.

The results of the monitoring of O2 and CO2 concentration in the gas–liquid phases of the flask and the measurement of U.O.D.580, pH, and acetic acid concentration are shown in Figs. 2 and 3, respectively.

The monitoring system developed in this study has the following two advantages: (1) it allows the direct monitoring of CO2 and O2 in the gas–liquid phases during shake-flask culture using culture plug-capped Erlenmeyer flasks, and (2) it allows sampling without interruption of shaking. To our knowledge, there are no reports of real-time monitoring of CO2 and O2 in both the gas phase and the liquid phase (culture broth) of breathable plug-capped Erlenmeyer flasks during standard shake-flask culture. Further, sampling without interruption of shaking has not been reported in conjunction with the conventional shake-flask cultivation method. By using a filter (pore size 0.22 µm), the gas phase of CDMSS was circulated through a packed CO2 absorbent, and CO2 generated in the headspace of the flask during shaking culture was removed.




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