Expanding Metabolic Online Monitoring Capabilities in 96-Deepwell Microtiter Plates by Combining Optoelectronic O2 and CO2 Sensors.

To increase the information content in early stages of bioprocess development, various technologies have been introduced for monitoring cultivations in microtiter plates (MTPs). Besides spectroscopical measurements, like in the BioLector technology, optical measurements with luminescent sensors, placed directly in the cultivation medium, became widely established. Monitoring the oxygen transfer rate (OTR) is a key technology for bioprocess development, as it allows mass balancing. At the same time, it is a criterion for a rational scale-up of cultivations. Although this technology has recently been transferred to monitoring of OTR in 96-well MTPs, monitoring of other parameters like carbon dioxide transfer rate (CTR) in individual wells was so far impossible. In this study, the preparation and integration of novel CO2-sensitive fluorescent sensors into the micro(µ)-scale Transfer rate Online Measurement device (µTOM) is reported, that rely on spectrally compatible diketopyrrolopyrrole indicator dye. By including 48 CO2 and 48 O2 sensors, the 96-well sensor layout of the µTOM allows for simultaneous measurement of CTR and OTR in separate replicate wells and the calculation of 48 respiratory quotients (RQs). Additionally, to reduce the overall media consumption, liquid filling volumes between 0.2 and 0.5 mL were applied. The first-ever parallel monitoring of the OTR, CTR, and RQ in one MTP during cultivations of Hansenula polymorpha RB11 pC10-FMD (PFMD-GFP) and E. coli BL21(DE3) pRotHi-YFP are presented. The interpretation of the RQ allowed distinguishing between the oxygen-unlimited growth of Hansenula polymorpha on glucose, as well as the accumulation and consumption of ethanol. Furthermore, the RQ was used to identify the sequential consumption of glucose and glycerol by E. coli BL21(DE3) in Wilms-MOPS autoinduction medium. Thus, the additional online parameters, CTR and RQ, extend the possibilities for early-stage bioprocess development at MTP-scale.