Applied Microbiology and Biotechnology

, Volume 102, Issue 15, pp 6637–6645 | Cite as

Monitoring of CO2 and O2 concentrations in the headspace of Sakaguchi flasks during liquid culture of microorganism

  • Masato Takahashi
  • Hideki AoyagiEmail author
Applied microbial and cell physiology


CO2 and O2 in the Sakaguchi flask headspace during culture was monitored via circulation direct monitoring and sampling system (CDMSS), a device with circulation bypass system. In static culture with Saccharomyces cerevisiae (circulation rate, 50 mL/min), a vertical CO2 concentration gradient (maximum gap ~ 2% (v/v) [height from the bottom of flask 45 mm, 7%; 155 mm, 5%]) in the Sakaguchi flask headspace was observed; no concentration O2 gradient was observed. However, shake flask culture showed vertical gradient concentrations for both CO2 and O2 (maximum gap of CO2 and O2 concentrations: 2 and 4% [heights from the bottom of flask 115 mm, 6.0 and 9.5%; 175 mm, 4.0 and 13.5%], respectively). When the CDMSS circulation rate in the Sakaguchi flask headspace was 300 or 400 mL/min, the gaseous environment was uniformly distributed so that no vertical gradient concentration was observed. In shaking culture with Escherichia coli under these conditions, CO2 was accumulated at high concentrations in the headspace and culture broth (maximum values 8%, in the headspace; 120 mg/L, in the culture broth). Most of the accumulated CO2 in the headspace could be removed by inserting a column packed with CO2 adsorbent at the bypass port of the CDMSS gaseous circulation. Thus, dissolved CO2 was maintained at a lower concentration, and the final UOD (unit optical density) value of culture was increased compared with that of the control. This study is the first to demonstrate that vertical gradients of CO2 and O2 concentrations exist in the headspace of Sakaguchi flask during culture.


Batch culture Carbon dioxide Monitoring device Oxygen Sampling operation Shake-flask culture 



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

Funding information

This work was supported by a grant-in-aid from the Japan Society for the Promotion of Science (JSPS) (fellowship 16J00800). This work was a funded by a JSPS KAKENHI (grant number 15H04569), JSPS KAKENHI Challenging Research [Exploratory] (grant number 17K19218), and The Sumitomo Electric Industries Group Corporate Social Responsibility Foundation.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Authors’ information

Masato Takahashi (Postdoctoral researcher; Research fellow of Japan Society for the Promotion of Science). Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305–8572, Japan.

Hideki Aoyagi (Professor and Principal investigator). Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305–8572, Japan.

MT and HA designed the research. HA supervised the research. MT created the CDMSS. MT performed all culture experiments. MT and HA analysed the data. MT and HA wrote the manuscript. All authors read and approved the final manuscript.


  1. Ali M, Ismaini, Depamede SN, Setyono BDH, Mukhlis A, Amin M, Ashari M (2015) Stirred bioreactor for the robustness production of recombinant GST.VP28 in fed-batch cultivation of Escherichia coli. Sci Study Res Chem Chem Eng Biotechnol Food Ind 16:245–252Google Scholar
  2. Amoabediny G, Buchs J (2010) Determination of CO2 sensitivity of micro-organisms in shaken bioreactors. I. Novel method based on the resistance of sterile closure. Biotechnol Appl Biochem 57:157–166CrossRefPubMedGoogle Scholar
  3. Anderlei T, Buchs J (2001) Device for sterile online measurement of the oxygen transfer rate in shaking flasks. Biochem Eng J 7:157–162CrossRefPubMedGoogle Scholar
  4. Anderlei T, Zang W, Papaspyrou M, Buchs J (2004) Online respiration activity measurement (OTR, CTR, RQ) in shake flasks. Biochem Eng J 17:187–194CrossRefGoogle Scholar
  5. Diederichs S, Korona A, Staaden A, Kroutil W, Honda K, Ohtake H, Buchs J (2014) Phenotyping the quality of complex medium components by simple online-monitored shake flask experiments. Microb Cell Factories 13:149CrossRefGoogle Scholar
  6. Dixon NM, Kell DB (1989) The inhibition by CO2 of the growth and metabolism of microorganisms. J Appl Bacteriol 67:109–136CrossRefPubMedGoogle Scholar
  7. Flitsch D, Ladner T, Lukacs M, Buchs J (2016) Easy to use and reliable technique for online dissolved oxygen tension measurement in shake flasks using infrared fluorescent oxygen-sensitive nanoparticles. Microb Cell Factories 15:45CrossRefGoogle Scholar
  8. Ikeno Y, Ozaki A (1968) Factors affecting oxygen transfer into shaken flask. Agric Biol Chem 32:912–915CrossRefGoogle Scholar
  9. Jones RP, Greenfield PE (1982) Effect of carbon-dioxide on yeast growth and fermentation. Enzym Microb Technol 4:210–222CrossRefGoogle Scholar
  10. Kato I, Tanaka H (1998) Influence of CO2 ventilation on microbial cultivation in shake-flasks. Biotechnol Tech 12:325–328Google Scholar
  11. Kluyver A, Perquin L (1933) Zur methodik der schimmel-stoffwechseluntersuchung. Biochem Z 266:68–81Google Scholar
  12. Losen M, Frolich B, Pohl M, Buchs J (2004) Effect of oxygen limitation and medium composition on Escherichia coli fermentation in shake-flask cultures. Biotechnol Prog 20:1062–1068CrossRefPubMedGoogle Scholar
  13. Matsuda F, Kinoshita S, Nishino S, Tomita A, Shimizu H (2017) Targeted proteome analysis of single-gene deletion strains of Saccharomyces cerevisiae lacking enzymes in the central carbon metabolism. PLoS One 12:e0172742CrossRefPubMedPubMedCentralGoogle Scholar
  14. Okazaki F, Aoki J, Tabuchi S, Tanaka T, Ogino C, Kondo A (2012) Efficient heterologous expression and secretion in Aspergillus oryzae of a llama variable heavy-chain antibody fragment V-HH against EGFR. Appl Microbiol Biotechnol 96:81–88CrossRefPubMedGoogle Scholar
  15. Omura S, Iwai Y, Hirano A, Nakagawa A, Awaya J, Tsuchya H, Takahashi Y, Masuma R (1977) A new alkaloid AM-2282 OF Streptomyces origin taxonomy, fermentation, isolation and preliminary characterization. J Antibiot 30:275–282CrossRefPubMedGoogle Scholar
  16. Scheidle M, Klinger J, Buchs J (2007) Combination of on-line pH and oxygen transfer rate measurement in shake flasks by fiber optical technique and respiration activity monitoring system (RAMOS). Sensors 7:3472–3480CrossRefPubMedGoogle Scholar
  17. Schneider K, Schuetz V, John GT, Heinzle E (2010) Optical device for parallel online measurement of dissolved oxygen and pH in shake flask cultures. Bioprocess Biosyst Eng 33:541–547CrossRefPubMedGoogle Scholar
  18. Shiota H, Sakaguchi K (1950) Studies on the production of mold protease in the submerged culture. Part I. J Agric Chem Soc Japan 23:426–429Google Scholar
  19. Takahashi M, Sawada Y, Aoyagi H (2017) 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. AMB Express 7:163CrossRefPubMedPubMedCentralGoogle Scholar
  20. Takahashi M, Aoyagi H (2018a) Effect of intermittent opening of breathable culture plugs and aeration of headspace on the structure of microbial communities in shake-flask culture. J Biosci Bioeng.
  21. Takahashi M, Aoyagi H (2018b) Practices of shake-flask culture and advances in monitoring CO2 and O2. Appl Microbiol Biotechnol 102:4279–4289. CrossRefPubMedGoogle Scholar
  22. Takahashi Y, Iwai Y, Omura S (1983) Relationship between cell morphology and the types of diaminopimelic acid in Kitasatosporia setalba. J Gen Appl Microbiol 29:459–465CrossRefGoogle Scholar
  23. Van Gool MP, Vancso I, Schols HA, Toth K, Szakacs G, Gruppen H (2011) Screening for distinct xylan degrading enzymes in complex shake flask fermentation supernatants. Bioresour Technol 102:6039–6047CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Faculty of Life and Environmental SciencesUniversity of TsukubaTsukubaJapan

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