Pichia stipitis: Oxygen and carbon dioxide transfer rate
Pichia stipitis is a species of yeast distantly related to brewer’s yeast, Saccharomyces cerevisiae. Pichia stipitis has been extensively studied because of its natural ability to directly ferment xylose. Converting the hemicellulose to ethanol makes this yeast interesting for the production of renewable fuels. In contrast to brewer’s yeast, fermentation in the crabtree-negative Pichiais not induced by high concentrations of sugar, but repressed in presence of oxygen.
With the aid of the Respiration Activity Monitoring System (RAMOS), it is already possible to determine oxygen- (OTR) and carbon dioxide- (CTR) transfer rates online in the shake flask. The following example of Pichia cultivation (fig.1) illustrates, how data generated from RAMOS allow conclusions to be drawn on the actual consumed carbon source and the availability of oxygen for the cells.
Fig. 1: Oxygen (OTR) and carbon dioxide (CTR) transfer rate during cultivation of Pichia stipitis in RAMOS, concentrations of glucose and ethanol were determined offline in parallel entrained shake flasks with cotton plug; temperature 30 °C, liquid volume 10 mL, flask volume 250 mL, shaking speed 100 rpm, shaking diameter 50 mm, complex medium with 20 g/L glucose.
After an initial rise, the oxygen transfer rate (OTR) reaches a plateau after 5 hours and remains static on a level of 0.02 mol L-1 h-1 till 21 hours of cultivation. This curve is typical for an oxygen limited culture. The carbon dioxide transfer rate (CTR) increases even after 5 hours. Due to the limited availability of oxygen, the cells must have converted the sugar aerobically as well as anaerobically to CO2 and ethanol during this time. This is demonstrated in figure 1 by the offline determined EtOH concentration.
After 10 hours, primary carbon source glucose is exhausted, CTR drops. Pichia now begins to degrade the ethanol formed earlier. CTR declines below the level of OTR, resulting in a respiratory quotient (RQ) < 1, a typical value for carbon sources more reduced than glucose. End of fermentation is reached after 21 hours, with both OTR and CTR dropping.
Figure 1 illustrates, that online data from RAMOS and offline results complement one another and provide a comprehensive picture of the metabolic state of the culture. Gassing in RAMOS measuring flasks can be adapted to the respective type of closure of the parallel sampling flaks, so that ventilation in both types of bioreactors is comparable. This becomes apparent in terms of the volatile component ethanol: Depletion of the offline determined EtOH concentration corresponds with the decline of OTR and CTR in RAMOS flasks
Different fermentation stages (respiratory, fermentative) can be exactly detected with RAMOS, without need for additional sampling. Hence, expense of time and money for offline analytics can be diminished. RQ can be calculated with OTR and CTR and allows drawing conclusions to the kind of carbon source consumed by the cells.
Corynebacterium glutamicum: Recognition of limitations on different levels
RAMOS is applicable for media optimization, as shown by the following example with Corynebacterium glutamicum, a gram-positive soil bacterium.
Fig. 2a: Oxygen transfer rate of Corynebacterium glutamicum in RAMOS with different filling volumes of the flasks, addition of substrate A and/ or B after 24 h, supplement solutions do not contain any carbon source; mineral medium with glucose, shaking speed 300 rpm, shaking diameter 50 mm, flask volume 250 mL, temperature 30 °C, in cooperation with IBT2, Forschungszentrum Jülich.
The flasks in the experiment of figure 2a exhibit different filling volumes from 10 to 20 mL. However, course of oxygen transfer rates (OTR) in all flasks is similar: OTR mounts at the beginning, reaches its maximum after 9 hours to decline in the following. This curve is typical for a substrate limited culture. Though, the carbon source glucose is available in sufficient amounts at that time (data not shown). Hence, growth of C. glutamicum must be restricted by the availability of a different nutrient. To determine, which substrate constitutes the limiting factor at this cultivation, two mineral solutions (without carbon sources) A and B were added solely or in combination.
Fig. 2b: Oxygen transfer rate of Corynebacterium glutamicum in RAMOS with different filling volumes of the flasks, addition of substrate A and/ or B after 24 h, supplement solutions do not contain any carbon source; mineral medium with glucose, shaking speed 300 rpm, shaking diameter 50 mm, flask volume 250 mL, temperature 30 °, C in cooperation with IBT2, Forschungszentrum Jülich.
RAMOS instantly detects differences in the OTR curves after addition of the substrate solutions: If substrate A is added, OTR triplicates. In contrast, addition of substrate B causes only a slight increase of cellular breathability. A and B supplemented together lead to a 1.5 fold elevation of OTR. From these results can be deduced, that solution A contains the limiting factor. After compensation of the limitation, breathability of C. glutamicum increases significantly.
Potentially, solution B may have contained a limiting factor, too. But this flask has also the highest filling volume. Therefore, the positive effect of solution B on the cells breathability could have been disguised by an additional oxygen limitation. Though, this assumption is disproved by the results shown in figure 2c.
Here the oxygen transfer rate of C. glutamicum during cultivation in a medium optimized by the above results is depicted: With the optimized medium, the cells reach an OTR of 0.035 mol L-1 h-1 at 20 mL filling volume. That is twice as much OTR as was reached in figure 2b at the same filling volume.
Fig. 2c: Oxygen transfer rate of Corynebacterium glutamicum in RAMOS with different filling volumes of the flasks, optimized mineral medium with glucose; shaking speed 300 rpm, shaking diameter 50 mm, flask volume 250 mL, temperature 30 °C, in cooperation with IBT2, Forschungszentrum Jülich.
OTR in the optimized medium is more than doubled with 0.06 mol L-1 h-1 at 10 mL filling volume. The OTR-curve is increasing with rising number of cells, till glucose is consumed completely after 15 hours and breathability ceases slowly.
On the basis of RAMOS generated data, an additional oxygen limitation is detectable in the flasks with higher filling volume than 10 mL. OTR increases firstly here, but reaches a plateau of 0.045 mol L-1 h-1 (at 15mL filling volume) resp. 0.035 mol L-1 h-1 with 20 mL of liquid in the flask. Additionally, the oxygen limited cultures need more time to consume their carbon source completely.
It could be shown, that RAMOS is a practical tool for rapid and efficient media optimization. With one or more trials, the respective medium can be matched to the requirements of the cells without laborious sampling and analytics. The results are meaningful and reproducible, further limitations (e.g. due to oxygen availability) are not longer hidden and do not falsify following experiments.
Hybridoma cells: Identification of distinctions between two media
Hybridoma cells are hybrid cell lines, formed by fusion of an antibody producing B cell and a myeloma cell. Hybridoma cells are able to grow in tissue culture and produce the desired monoclonal antibody.
Cell cultures reach only a hundredth of the common microbial breathability, as shown in figure 3. RAMOS can determine even this very low oxygen transfer rates and provide information about the conditions of the animal or human cells, online and without sampling. In the following experiment, impact of two different media on hybridoma cells is investigated with RAMOS, in two parallel flasks each.
Fig. 3: Oxygen transfer rates of hybridoma cells cultivated in RAMOS with two different media 1 and 2; filling volume 100 mL, flask size 250 mL, shaking speed 100 rpm, shaking diameter 50 mm, temperature 37 °C, in cooperation with Celonic GmbH, Jülich.
Medium 1 contains no supplementary fatty acids, but in medium 2 fatty acids were added. All four cultures start with the same very low oxygen transfer level of 0.00005 mol L-1 h-1 and rise linearly until hour 35. For comparison: Microbial oxygen transfer rates remain in a range of around 0.05 mol L-1 h-1 (see Fig. 1 and 2). The oxygen transfer rate of the cultures with medium 1 keeps on increasing linearly, while the OTR of the cultures with medium 2 starts to rise exponentially. After hour 55, the cell cultures reach their maximum oxygen transfer rates. Afterwards, the OTR of all hybridoma cultures decrease very slowly until hour 138, when the cells turn off their breathability.
This experiment shows that the oxygen transfer measurement of cell cultures in shake flasks is applicable, even when the respiration activity is hundred times lower than that of microbial cultures. Furthermore, respiration activity differences are detected for the slightly different media. As this signal differences are significant and reproducible, the RAMOS device has proven to be suitable for efficient cell culture process development in small scale.