Analysis and application of the influence of accumulating CO2 in
the headspace during shake-flask culture on microorganism andmicrobial community
Masato TAKAHASHI
Faculty of Life and Environmental Sciences, University of Tsukuba.
Report:Accumulation of CO2 in the gas phase in microbial culture was shown to affect theshake-flask culture. CO2 accumulation was found to be dependent not only on the shape ofthe flask, but also on the type of breathable culture-stopper. We also demonstrated that thenew culture method in which the CO2 concentration in the flask gas phase is controlled
constantly or intermittently, presented different microbial behavior from that of theconventional method, illustrating that this method may affect various physiologicalactivities.
Research aims
It was difficult to investigate and control culture factors in a shake-flask culture without theinterruption of shaking, because of high speed of revolutions 1). Therefore, it is difficult toelucidate the effects of various flask conditions (e.g.., type of culture-stopper, flask shape,and shaking conditions), and consequently, these conditions have not been studied. In orderto maximize the potential of shake-flask culture, some techniques are necessary formonitoring culture environments in flasks and selecting appropriate flask conditions. This
study focused on CO2 produced during microbial culture, using a developed Circulation
Direct Monitoring and Sampling System (CDMSS); wherein sampling and monitoring are
possible without interrupting shaking 2) (and references therein). CDMSS was used toevaluate the ventilation capacity of the entire incubator comprising of a conventional flaskwith a culture-stopper (quantification of the total gas transfer rate between the flask gas
phase and the outside air) and creation of a novel flask condition. In addition, this study also
analyzed the effects of novel flask conditions on microorganisms.
Methods
After filling the flask gas phase with CO2, CO2 concentration was monitored using CDMSS
using culture-stoppers with varying air permeability in combination with various flasks, andthe time at which the CO2 concentration in the flask was halved was calculated. In addition,
we developed a unique system which can control the CO2 concentration in the flask gas

phase by connecting the CDMSS and aerationdevice with a PID control device using the thus
obtained CO2 ventilation capacity. This system
was able to function using a conventional
culture-stopper and flask. This newly developed
CO2-controlled method for shake-flask culture
was compared with the conventional method.
Results
The ventilation capacity and oxygen supply
capacity of Erlenmeyer flasks, Sakaguchi flasks,and cylindrical flasks were evaluated for thepurpose of creating novel flask conditions 3).
Results of our study on shaking cultures ofEscherichia coli using various flask shapes
showed that not only the oxygen supply capacity,but also the ventilation capacity is important (Fig.

Fig. 1 Growth course of E. coli in shake culture
with equivalent oxygen supply capacity butdifferent ventilation capacity. ● , Erlenmeyer
flask; □, Cylindrical flask.

1) 3). The half-life of CO2 was used as an index toevaluate the ventilation performance of plug-type
and cap-type culture-stoppers made of silicone
foam, which are widely used as breathable
culture-stoppers. The CO2 half-life values
obtained for both stoppers were different (Fig. 2),
thus it was clear that this difference affects theFig. 2 Ventilation capacity of the breathable
culture-stopper. Black, plug type; ash, cap type.

growth of microorganisms 4). A significant
difference was observed in the culture-stopper
depending on the material. There was almost nochange in the half-life of CO2 due to shaking
conditions. In order to compare the newly
developed CO2-controlled shake flask culture
method with the conventional method, a pureFig. 3 Behavior of combustion gas containing
high concentration CO2 generated by flamesterilization operation in the sampling process.

culture of Pelomonas saccharophila showed
increased growth upon controlling the high concentration of CO2 in the flask gas phase 4). Asa factor that increases the CO2 concentration in the flask gas phase, we studied flamesterilization operation at the time of sampling in the next experiment. We showed that highconcentrations of CO2 accumulate in the flask gas phase by utilizing a measurement
imitating the actual operation of flame sterilization and simulation of combustion gas by

CFD (Fig. 3). Shaking culture was performed by mimicking only the intermittent increase inCO2 concentration seen during sampling over time, and as a result, Acetobacter pasteurianus,
P. saccharophila, cultures showed increased growth, however, E. coli and Saccharomyces
cerevisiae cultures did not show this change 5).
Conclusion
It is necessary to select breathable culture-stoppers based on to microbial culture conditions
including the medium used. In the selection of culture-stoppers, it was shown that theirshape (such as cap-type and plug-type) and material, and the foaming method used, allow
reproducible microbial culture. The flask environment of the gas phase during aerobic
conventional shake culture is an important culture factor along with the culture medium used.
The constant or intermittent control of the CO2 concentration in the headspace provides
novel flask conditions. It is suggested that shake-flask culture allowing CO2 accumulation
affects the growth and various physiological activities, and it is expected to contribute to
upstream bioprocess development wherein the shake-flask culture method is widely used. In
addition, as a model of the microbial flora, moromi was cultured under various gas phaseconditions. As a result, the culture microbial community structure obtained was different
from the conventional one, depending on the conditions of flask gas phase.
References
1） Takahashi, M., Aoyagi, H. (2018) Practices of shake-flask culture and advances inmonitoring CO2 and O2. Appl. Microbiol. Biotechnol. 102: 4279-4289.
2） Takahashi, M., Aoyagi, H. (2018) 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. 126: 96-101.
3） Takahashi, M., Aoyagi, H. (2020) Analysis and effect of conventional flasks in shaking
culture of Escherichia coli. AMB Express 10: 77.
4） Takahashi, M., Aoyagi, H. (2020) Analysis of porous breathable stopper and developmentof PID control for gas phase during shake-flask culture with microorganisms. Appl.
Microbiol. Biotechnol. 104: 8925-8936.
5） Takahashi, M., Honzawa, T., Tominaga, R., Aoyagi, H. (2020) Analysis of the influence offlame sterilization included in sampling operations on shake-flask cultures ofmicroorganisms. Sci. Rep. 10: 10385.