Everything Under Control: Gas Monitoring in Shake Flask Culture

Results in baffled flasks

A baffled flask containing autoclaved YPD medium was inoculated with S. cerevisiae and placed on the SFR vario inside a shaker at 30 °C and 300 rpm. Monitoring of pCO₂, DO and biomass was then started and recorded for 24 h. In parallel, pCO₂ and OD 600 nm (optical density) off­line measurements were carried out for comparison.

The observed carbon dioxide production and the oxygen consumption profile are typical for the aerobic diauxic growth of the yeast culture. First a large fraction of glucose is catabolized to ethanol and carbon dioxide, while a smaller fraction is used to form biomass (1 — 4 h, see Fig. 2). By converting glucose to ethanol yeasts obtain a growth benefit in the natural environment. While glucose can be used as carbon source by the majority of microorganisms, ethanol is toxic for the competing organisms and cannot be used as nutrient. When almost all glucose is metabolized to ethanol, the yeast starts using ethanol as carbon source. This metabolic switch can be seen as a drop of pCO₂ from 12 to 1% and a slight drop in DO after 5 h. DO is consumed as long as ethanol is metabolized and used for biomass production (16 h). After cell death the DO increases again because the presence of baffles allows oxygenation.

In the pCO₂ profile after 5 h we can observe in the online measurement, as well as in the corresponding off­line measurement a peak corresponding to the ethanol production, decreasing during the metabolic switch followed by a slow increase during ethanol consumption. After 16 h, when cell death occurs, pCO₂ drops to zero.

Biomass increased during culturing and stagnates at the same time the DO increased and the pCO₂ decreased when cell division stops. The data for biomass recorded by the SFR vario were comparable to the discontinuous offline OD 600 nm sampling.

As Fig. 2 shows, the stopping of the shaker in order to take samples for the offline measurements causes disturbances in the oxygen content that must be eliminated afterwards in a cumbersome process. The measurement with the SFR vario overcomes this drawback, as can be seen in Fig. 3, where the same measurement was performed but without taking offline measurements, and thus guarantees a continuous oxygen and nutrient supply for the yeast cells.

Results in non-baffled flasks

In a second experiment, the inoculation was performed in a non-baffled flask under the same experimental conditions. PCO₂ offline measurements were once again gathered as an external reference for our measurement system.

The consumption of oxygen exceeds the oxygenation of the medium by shaking, because aeration is limited in the non-baffled flasks. DO was consumed approximately during the first 10 h and then remained at 0%, because it is a flask with flat bottom (see Fig. 4). However, the S. cerevisiae continued growing beyond these 10 h since it is a facultative anaerobic microorganism. During this period of time we can observe a similar profile in the pCO₂ to the one observed in the baffled flasks, although in this case the glucose and ethanol consumption are not as clearly differentiated as before. PCO₂ offline measurements are also in accordance with this tendency. Biomass increases continuously from the beginning of the experiment, which confirms the cell division.

Conclusion

The SFR vario device together with the Presens Flask Studio Software form a professional and scientific system allowing the continuous gathering of accurate data of several analyses important for monitoring cell growth.

The accuracy of the SFR vario was confirmed by validation with the performed offline measurements. The innovative device possesses many advantages, such as reduced risk of contamination of the culture and a higher number of experimental points since the complete curves can be recorded and not only scattered measurement points. Thus, the SFR vario enables the real time observation of nutrient or oxygen depletion as well as switches in the metabolism.

Besides the main parameters described before, the SFR vario also records temperature, agitation speed (rpm) and pressure, which could be convenient to future references during the analysis of experimental data.

* Dr. Gernot John et. al. Presens GmbH 93053 Regensburg/Germany

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