Saccharomyces cerevisiae: Crabtree-effect and consumption of ethanol
Due to its overflow metabolism (Crabtree effect), baker’s yeast Saccharomyces cerevisiae has the ability to produce ethanol from glucose even in the presence of oxygen. After total consumption of the sugar, accumulated ethanol can be oxidized as an energy source by baker’s yeast under aerobic conditions.
With the help of the BPM system, the impact of baffles on the oxygen supply to yeast cells was investigated. The above mentioned diauxic growth of S. cerevisiae with glucose and ethanol is illustrated with data generated by the BPM.
Fig.1: Dissolved oxygen concentration DO [%] and pH-value during cultivation of Saccharomyces cerevisiae in shake flasks (Corning, 250ml) with and without baffles; YEP-medium with 20g/L glucose, 200rpm shaking frequency, 50 mm shaking diameter, 25mL filling volume, temperature 37°C
Following an initial decline, the dissolved oxygen concentration in the medium rises after 7 hours of cultivation, indicating that glucose is totally exhausted. During this initial growth phase, biomass and CO2 are formed, with ethanol as additional by-product of the aerobic fermentation.
It can also be seen that shortly after the yeasts have adapted their metabolism to ethanol consumption, the DO decreases until alcohol is completely oxidized. In the baffled flask this point was reached after 23 hours of cultivation, noticeable as the soaring concentration of dissolved oxygen. DO does not drop below 50% saturation during the entire cultivation, indicating a sufficient supply of oxygen to the cells.
Ethanol consumption takes longer in the ordinary flask, with the dissolved oxygen concentration dropping below 10 % saturation after 16 hours, leading to oxygen limitation of the yeast. Both carbon sources were only exhausted after 27 hours and the breathability of the yeast cells declined significantly.
In both types of flask a rise in pH can be observed after complete consumption of the substrates (after 23 resp. 27 h), potentially caused by autolysis of the cells.
This demonstrates that the BPM system can easily detect online the different stages of fermentation in shake flasks without laborious sampling and subsequent analysis.
Escherichia coli: Overflow metabolism and acetate consumption
Because of an imbalance between the glucose uptake and the capacity of the central metabolism, the bacterium Escherichia coli in the presence of high glucose concentrations, forms the fermentation product acetate, even under aerobic conditions (overflow metabolism). After complete consumption of the sugar, E. coli is able to utilize acetate as a carbon and energy source in a type of diauxic behaviour.
Using data collected by the BPM system these phenomena can be observed online in a shake flask culture of E. coli (Figure 2).
Fig.2: Dissolved oxygen concentration DO [%] and pH during cultivation of Escherichia coli in shake flask (Corning, 250ml); mineral medium with 20g/L glucose, 250rpm shaking frequency, 50 mm shaking diameter, 10mL filling volume, temperature 37°C
At the beginning, the glucose is oxidized as the bacteria consumes oxygen and acetate is formed as a by-product. This is seen as a decrease in pH. After 9 h of cultivation the oxygen saturation drops below 10 % indicating an oxygen limitation of the cells.
Glucose is consumed completely after 11 hours. The oxygen concentration in the medium then rises until the cells shift their metabolism to acetate exploitation. Within 15 hours cultivation time, degradation of the acetate formed earlier begins and pH rises concomitantly. All carbon and energy sources are exhausted after 25 hours. Breathability of the cells declines following this, while the DO accumulates.
It was shown, that using the BPM-system the point of glucose exhaustion or cessation of cellular breathability can be determined easily, online and without intervention.
CHO - cell culture
CHO cell culture: Difference between baffled and ordinary shake flasks
The BPM system was used to investigate whether baffles in shake flasks affect the cultivation of mammalian CHO-cells.
Fig. 3: Dissolved oxygen concentration DO [%] during cultivation of CHO cells in shake flasks with and without baffles (Corning, 250 mL); Pro-CHO medium, shaking frequency 170 rpm, shaking diameter 50 mm, filling volume 100 mL, temperature 37°C, incubated with 5 % CO2; kindly supported by ExcellGene SA (Lausanne, CH).
In the baffled flask, dissolved oxygen concentration remains above 90 % saturation during the entire cultivation. In the ordinary flask, DO drops below 80 % after 3 days but later rises again. As expected, the oxygen supply to the cells is better in the baffled flask, but in both types of shaken bioreactors, the cultures are adequately accommodated without oxygen limitation.
Furthermore, baffles suggest additional mechanical stress for the cells and may also lead to foaming (Fig. 4). The impact of baffles on the growth behavior and viability of CHO-cells was therefore examined with the BPM system.
Fig. 4: CHO-cells in shake flasks (Corning, 250 mL), right with baffles, left without
Fig. 5: Viability [%] and cell density [cells/mL] of CHO-cells in shake flasks with and without baffles (Corning, 250 mL); Pro-CHO medium, shaking frequency 170 rpm, shaking diameter 50 mm, filling volume 100 mL, temperature 37°C, incubated with 5 % CO2; kindly supported by ExcellGene SA (Lausanne, CH).
Contrary to expected results, this CHO-cell line did not show significant differences between baffled and non-baffled flasks, neither in cell viability nor in growth behavior.
Despite the low breathing activity of mammalian cells, the BPM system can track the course of cultivation as consistently as offline data.