A significant challenge of process online-monitoring in small scale shaken bioreactors is the miniaturization of process analytical tools. Off-gas analysis takes a special role within those tools as it is non-invasive to the liquid broth and provides the production and consumption rates of gaseous components that take part in cell metabolism. In this study, a flexible and robust off-gas analysis tool for surface-aerated bioreactors, measuring OTR, CTR and RQ was developed. In the measurement system TOM (Transferrate Online Monitoring), sensors for off-gas analysis were placed in a bypass system that avoids the shaking of the electronics and sensors. It was combined with the established method of recurrent dynamic measurement phases as presented before for the RAMOS. The newly developed measurement system showed high accuracy, precision and reproducibility among individual flasks, especially regarding CTR measurement. Performance was evaluated with a non-biological model system. It was then applied to the microbial model systems S. cerevisiae, G. oxydans, and E. coli putting focus on the benefits of CTR and RQ measurements. Secondly, the TOM was used to monitor shake flask cultivations on a self-balancing orbital high-speed shaker that is capable to be operated at up to 750 rpm shaking frequency at 25 mm shaking diameter and 600 rpm at 50 mm, showing the robustness of the measurement system. In exemplary cultivations, a maximum kLa value of 650 h-1 was reached at 10 mL filling volume in a 250 mL glass shake flask. This is an increase of about 50%, compared to current commercial orbital shakers. Shaking at 25 mm shaking diameter turned out to be the best trade-off between oxygen supply and machine load at high shaking frequencies. Lastly, ethylene detecting electrochemical sensors were integrated to the measurement setup to monitor the formation of the plant hormone ethylene in combination with the consumption of oxygen in parsley suspension cell cultivations. A calibration method is presented that is not prone to baseline drifting and that considers target gas oxidation at the sensor. In this way, changes in ethylene transfer rate as low as 4 nmol/L/h can be resolved. Ethylene release could be detected when parsley cells were treated with defense priming compounds salicylic acid and methyl jasmonate and when challenged with the microbial pattern Pep13.
Andreas Schulte (Wed,) studied this question.