Analysis of porous breathable stopper and development of PID control for gas phase during shake-flask culture with microorganisms

Applied Microbiology and Biotechnology volume 104, pages8925–8936(2020)Cite this article

Abstract

We evaluated the ventilation ability of two types (plug-type and cap-type) of culture-stoppers having standard air permeability. The culture-stoppers were evaluated using the circulation direct monitoring and sampling system with CO2 concentration in the gas phase of a shake-flask culture as an index. The half-lives of CO2 in the headspace of the shake flask with the plug-type and cap-type stoppers were about 51.5 min and about 30.3 min, respectively. Based on these half-lives, we formulated a model equation to simulate the behaviour of CO2 with different culture-stoppers. After validating the model equation by shake-flask culture with Saccharomyces cerevisiae, we investigated the effect of different ventilation abilities of the culture-stoppers on the growth of Pelomonas saccharophila and Escherichia coli: the sensitivity of the culture-stopper to the ventilation ability was dependent on the microorganism species. In the case of P. saccharophila, when the plug-type culture-stopper was combined with controlled CO2 concentration (6%) in the flask, the maximum yield increased by twofold compared to that of the control. This study shows the importance of ventilation in headspace and conventional culture-stoppers during the shake-flask culture of microorganisms. The problems that may occur between the conventional shake-flask culture approach using a breathable culture-stopper and the next-generation shake-flask culture without a conventional culture-stopper were clarified from the evaluation of gas-permeable culture-stoppers. The importance of controlled gaseous phase in the headspace during shake-flask culture of the microorganisms was also elucidated.

Key points

• Ventilation capacity of culture-stoppers was evaluated using the CO2 half-life concentration.

• Behaviour of microorganisms varies with the type of culture-stopper.

• Developed a PID system for control of CO2 in flask gas phase to enhance the shake-flask culture.

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Acknowledgements

The manuscript has been edited carefully by a native English-speaking professional editor from Editage, a division of Cactus Communications.

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Funding

This work was supported by NISR Young Investigator Research Grant and Japan Society for the Promotion of Science (JSPS) KAKENHI Grants-in-Aid for Early-Career Scientists (Grant Numbers JP20K15097) (grants to Masato Takahashi). This work was partly supported by JSPS KAKENHI Grant-in-Aid for Scientific Research B (Grant Number JP19H03086), Takahashi Industrial and Economic Research Foundation (Grant Number 10-003-183), and Sumitomo Electric Industries Group Corporate Social Responsibility Foundation, Japan (grants to Hideki Aoyagi).

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  1. Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8572, Japan

    Masato Takahashi & Hideki Aoyagi

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  1. Masato Takahashi
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  2. Hideki Aoyagi
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MT and HA designed the research. HA supervised the research. MT created the experimental design and performed all experiments. MT and HA analysed the data. MT and HA wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Hideki Aoyagi.

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Takahashi, M., Aoyagi, H. Analysis of porous breathable stopper and development of PID control for gas phase during shake-flask culture with microorganisms. Appl Microbiol Biotechnol 104, 8925–8936 (2020). https://doi.org/10.1007/s00253-020-10847-x

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Keywords

  • Breathable culture-stopper
  • Carbon dioxide
  • Monitoring device
  • PID control
  • Shake-flask culture