Optical online oxygen monitoring of aerobic cultivations in shake flasks and microtiter plates

Two of the most important process parameters for aerobic cultivations of microorganisms and cells are the oxygen consumption and oxygen content within the cultivation broth. By means of the consumption, mass balances and stoichiometries can be calculated. On the basis of the oxygen content, an oxygen limitation can be identified doubtlessly. Additionally, the propagation of these parameters can be seen as a characteristic physiological fingerprint of an aerobic culture. Both parameters enable a sound and valid process monitoring and characterization. For these reasons, respective sensors and analytics are commonly applied in fermenter scale. However, the first steps of bioprocess development are generally performed in small scale experiments within shake flasks or microtiter plates. This is due to the ability to use smaller reaction volumes while performing a greater number of parallel experiments, which saves time, resources and consequently lowers the overall costs per experiment. Unfortunately, due to the small dimensions, respective sensors and analytics are not easily transferable from fermenter to small scale and the current solutions are not completely satisfying. To augment the process knowledge already in early process development, it is of general interest to develop new tools and improve existing technologies for small-scale bioreactors. Within this work, three different novel systems for online monitoring of aerobic cultures in small scale fermentations have been developed, introduced and characterized.Firstly, a novel shake flask based system for measuring the dissolved oxygen tension (DOT) is introduced. By using dispersed oxygen-sensitive nanoparticles, an easy to use, robust and reliable DOT measurement system was developed, which is applicable for almost all cultivation conditions in shake flasks and for both, soluble complex and synthetic media. Reliable DOT measurements were achieved using oxygen-sensitive nanoparticles added to the cultivation broth at a concentration of 0.1 g L 1. The biocompatibility of the dispersed oxygen-sensitive nanoparticles was demonstrated for H. polymorpha, X. campestris and E. coli by means of RAMOS cultivations. Furthermore, the importance of system calibration under cultivation conditions was demonstrated. Besides that, the ability of the dispersed oxygen-sensitive nanoparticles to withstand autoclaving is an additional advantage for sterile cultivations. This measurement system turned out to be a valuable alternative for existing DOT measurement systems in shake flask cultivations, especially at low filling volumes and high shaking frequencies. In combination with RAMOS data it was possible to determine kLa values online during cultivations of K. lactis, E. coli and H. polymorpha. The determined kLa values were investigated with respect to their corresponding uncertainties caused by systematic errors in OTR, DOT, L_(O_2 ) and p_(O_2)^gas estimations and sensor inertia. All kLa values were in good agreement with an empirical correlation based on the medium osmolality from the literature. Secondly, this oxygen-sensitive nanoparticle-based technique was transferred to the microtiter plate scale and combined with the established BioLector technology. The respective optical fiber of the oxygen analytic was integrated into the BioLector system and displaced via the BioLector movement axes underneath the microtiter plate. Since this system does not require the presence of optodes at the bottom of each well, the measurement system can be applied to various microtiter plates (MTPs). Finally, the cost constraints associated with MTPs possessing immobilized sensor spots (optodes) can be overcome using this technology. Additionally, the costs per cultivation are significantly reduced and the system provides a serious alternative to established devices. Furthermore, kLa values for the applied cultivation conditions were estimated and used to calculate the oxygen transfer rates (OTRs) in MTPs based on the DOT. OTRs calculated for the MTPs and OTRs determined in shake flasks via the RAMOS technology agreed very well with each other.Lastly, a microtiter plate-based µRAMOS system was developed to measuring the oxygen transfer rate (OTR) in every individual well of a 48-well microtiter plate. On the basis of a first four-well prototype a second enhanced prototype covering the whole 48-well microtiter plate was implemented. The necessary valve and sensor technology was thereby successfully integrated in a microfluidic microtiter plate cover. To compromise on sufficient measurement rates and an economically acceptable number of oxygen instruments an 8x48 optical multiplexer (MUX) was developed and utilized. By means of four exemplary RAMOS and µRAMOS cultivations, both systems delivered the same OTR signal quality. The cultivation throughput could be increased 6-fold compared to the established eight shake flask RAMOS system. Additionally, the media consumption was reduced 12.5-fold per cultivation from a typical filling volume of 10 mL in the shake flask to 800 µL in the microtiter plate. In summary, three novel measurement systems were successfully developed, built and characterized. These systems offer a sound determination of the dissolved oxygen tension and oxygen transfer rate respectively and turned out to be predestined small scale analytics for process development and characterization purposes