MV
M.A. Vieira Lara
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Livestock production forms a threat to the environment and human health, while meat consumption increases due to global population and income growth. Commercial scale cultivated meat production provides a credible alternative due to its resemblance to the taste, texture and looks of conventional meat. In order to commercialize cultivated meat, major technological improvements are needed to scale-up its production from laboratory to commercial scale. This requires an animal cell line that can provide a food-grade product. Quail suspension cell line CCX.E10 is not genetically modified, is spontaneously immortalized and cultivated in animal component free medium. To research the growth kinetics of the cell line for commercial scale production investigation at a laboratory scale is required. Through this research a seed-train protocol to scale-up CCX.E10 cell bank to a scale-down model, namely a DASbox mini bioreactor system, was improved. Additionally, the growth kinetics of CCX.E10 were characterized during batch cultivation, while utilizing this system. A Design-of-Experiments approach was developed to understand what variables have the largest impact on growth kinetics of CCX.E10 to improve operational conditions. Input variables of the approach include the aeration rate, agitation rate and dissolved oxygen concentration of the bioreactor. This research found an exponential specific growth rate of 0.016 h-1 and viable cell density of 2.1x10^6 cells/mL for batch cultivation of cell line CCX.E10 in a DASbox bioreactor. A cell-specific lactate production rate 30% higher than the cell-specific glucose consumption rate was observed during the lag phase of cell cultivation. The growth kinetics derived during cell proliferation suggest that cell line CCX.E10 exhibits the Warburg effect. The findings indicate that the operational conditions for cultivated meat production based on CCX.E10 require further improvement to design a commercial scale bioprocess.
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Livestock production forms a threat to the environment and human health, while meat consumption increases due to global population and income growth. Commercial scale cultivated meat production provides a credible alternative due to its resemblance to the taste, texture and looks of conventional meat. In order to commercialize cultivated meat, major technological improvements are needed to scale-up its production from laboratory to commercial scale. This requires an animal cell line that can provide a food-grade product. Quail suspension cell line CCX.E10 is not genetically modified, is spontaneously immortalized and cultivated in animal component free medium. To research the growth kinetics of the cell line for commercial scale production investigation at a laboratory scale is required. Through this research a seed-train protocol to scale-up CCX.E10 cell bank to a scale-down model, namely a DASbox mini bioreactor system, was improved. Additionally, the growth kinetics of CCX.E10 were characterized during batch cultivation, while utilizing this system. A Design-of-Experiments approach was developed to understand what variables have the largest impact on growth kinetics of CCX.E10 to improve operational conditions. Input variables of the approach include the aeration rate, agitation rate and dissolved oxygen concentration of the bioreactor. This research found an exponential specific growth rate of 0.016 h-1 and viable cell density of 2.1x10^6 cells/mL for batch cultivation of cell line CCX.E10 in a DASbox bioreactor. A cell-specific lactate production rate 30% higher than the cell-specific glucose consumption rate was observed during the lag phase of cell cultivation. The growth kinetics derived during cell proliferation suggest that cell line CCX.E10 exhibits the Warburg effect. The findings indicate that the operational conditions for cultivated meat production based on CCX.E10 require further improvement to design a commercial scale bioprocess.
A Study on Oxygen Mass Transfer
The Role of Fermentation Products in Stirred Tank Reactors
The oxygen mass transfer coefficient, kLa, is a critical parameter in bioprocess performance, influencing the design of stirred tank reactors. During a fermentation process, the behavior of bubbles and the kLa can be effected by various components present in the broth. This study investigates the effect of organic compounds (ethanol, glycerol and acetic acid), which are produced during a yeast fermentation process (S. cerevisiae). It is concluded that acetic acid appears to decrease the geometric mean bubble diameter, in aqueous solutions. As a surfactant, the acetic acid molecules accumulate in the gas-liquid interface, thereby inhibiting bubble coalescence. This leads to an increase in interfacial area and a 34% rise in kLa has been measured. Glycerol, however, showed no significant impact in water. Ethanol should exhibit a similar trend to acetic acid, as reported in previous literature (Puiman, Elisiário, et al., 2022). Remarkably, when these organic compounds were added to growth medium for a yeast fermentation - which consist of synthetic medium, glucose and vitamins - the effects were negligible. This likely due to the coalescence-enhancing properties of antifoam, which is present in synthetic media. On the other hand, the concentrations of the organic compounds tested were relatively low. In addition, during the fermentation process, the production of organic compounds did not significantly affect the bubble size. A slight increase in kLa was observed, arguably due to a reduction in working volume. Whilst performing the tests, the kLa was measuring with three different experimental determination methods: the dynamic pressure method, the dynamic gassing-out method, and the gaseous oxygen balance method. The dynamic gassing-out method appeared a bit more consistent than the dynamic pressure method, possibly due to pressure stabilization issues. The gaseous oxygen balance method did not provide consistent results throughout the entire fermentation, as the method is sensitive and relies on very accurate gas measurements, especially when the oxygen consumption is low. The dynamic gassing-out method proved to be stable and provided the most results used for comparisons of experiment. However, considering its limitations, it requires validation with a ground truth, such as a validated reliable chemical method. The experimental kLa data was compared with predicted values, using empirical correlations. All predicted values remained within 50% margin to the experimental values (determined with dynamic gassing-out method). To be able to use these prediction models in bioprocess design, further investigation is essential.
...
The oxygen mass transfer coefficient, kLa, is a critical parameter in bioprocess performance, influencing the design of stirred tank reactors. During a fermentation process, the behavior of bubbles and the kLa can be effected by various components present in the broth. This study investigates the effect of organic compounds (ethanol, glycerol and acetic acid), which are produced during a yeast fermentation process (S. cerevisiae). It is concluded that acetic acid appears to decrease the geometric mean bubble diameter, in aqueous solutions. As a surfactant, the acetic acid molecules accumulate in the gas-liquid interface, thereby inhibiting bubble coalescence. This leads to an increase in interfacial area and a 34% rise in kLa has been measured. Glycerol, however, showed no significant impact in water. Ethanol should exhibit a similar trend to acetic acid, as reported in previous literature (Puiman, Elisiário, et al., 2022). Remarkably, when these organic compounds were added to growth medium for a yeast fermentation - which consist of synthetic medium, glucose and vitamins - the effects were negligible. This likely due to the coalescence-enhancing properties of antifoam, which is present in synthetic media. On the other hand, the concentrations of the organic compounds tested were relatively low. In addition, during the fermentation process, the production of organic compounds did not significantly affect the bubble size. A slight increase in kLa was observed, arguably due to a reduction in working volume. Whilst performing the tests, the kLa was measuring with three different experimental determination methods: the dynamic pressure method, the dynamic gassing-out method, and the gaseous oxygen balance method. The dynamic gassing-out method appeared a bit more consistent than the dynamic pressure method, possibly due to pressure stabilization issues. The gaseous oxygen balance method did not provide consistent results throughout the entire fermentation, as the method is sensitive and relies on very accurate gas measurements, especially when the oxygen consumption is low. The dynamic gassing-out method proved to be stable and provided the most results used for comparisons of experiment. However, considering its limitations, it requires validation with a ground truth, such as a validated reliable chemical method. The experimental kLa data was compared with predicted values, using empirical correlations. All predicted values remained within 50% margin to the experimental values (determined with dynamic gassing-out method). To be able to use these prediction models in bioprocess design, further investigation is essential.