Real-time dissolved carbon dioxide monitoring II: Surface aeration intensification for efficient CO2 removal in shake flasks and mini-bioreactors leads to superior growth and recombinant protein yields.

Mass transfer is known to play a critical role in bioprocess performance and henceforth monitoring dissolved O2 (DO) and dissolved CO2 (dCO2 ) is of paramount importance. At bioreactor level these parameters can be monitored online and can be controlled by sparging air/oxygen or stirrer speed. However, traditional small-scale systems such as shake flasks lack real time monitoring and also employ only surface aeration with additional diffusion limitations imposed by the culture plug. Here we present implementation of intensifying surface aeration by sparging air in the headspace of the reaction vessel and real-time monitoring of DO and dCO2 in the bioprocesses to evaluate the impact of intensified surface aeration. We observed that sparging air in the headspace allowed us to keep dCO2 at low level, which significantly improved not only biomass growth but also protein yield. We expect that implementing such controlled smart shake flasks can minimize the process development gap which currently exists in shake flask level and bioreactor level results.

Real-time dissolved carbon dioxide monitoring I: Application of a novel in situ sensor for CO2 monitoring and control.

Dissolved carbon dioxide (dCO2 ) is a well-known critical parameter in bioprocesses due to its significant impact on cell metabolism and on product quality attributes. Processes run at small-scale faces many challenges due to limited options for modular sensors for online monitoring and control. Traditional sensors are bulky, costly, and invasive in nature and do not fit in small-scale systems. In this study, we present the implementation of a novel, rate-based technique for real-time monitoring of dCO2 in bioprocesses. A silicone sampling probe that allows the diffusion of CO2 through its wall was inserted inside a shake flask/bioreactor and then flushed with air to remove the CO2 that had diffused into the probe from the culture broth (sensor was calibrated using air as zero-point calibration). The gas inside the probe was then allowed to recirculate through gas-impermeable tubing to a CO2 monitor. We have shown that by measuring the initial diffusion rate of CO2 into the sampling probe we were able to determine the partial pressure of the dCO2 in the culture. This technique can be readily automated, and measurements can be made in minutes. Demonstration experiments conducted with baker's yeast and Yarrowia lipolytica yeast cells in both shake flasks and mini bioreactors showed that it can monitor dCO2 in real-time. Using the proposed sensor, we successfully implemented a dCO2 -based control scheme, which resulted in significant improvement in process performance.