Small-scale shaken bioreactors for fed-batch cultivation with parallel online monitoring
- Geschüttelte Kleinkultur-Bioreaktoren im Fed-Batch-Betrieb mit paralleler Online-Prozessüberwachung
Habicher, Tobias; Büchs, Jochen (Thesis advisor); Schwaneberg, Ulrich (Thesis advisor)
Dissertation / PhD Thesis
Dissertation, Rheinisch-Westfälische Technische Hochschule Aachen, 2020
Small-scale shaken bioreactors, such as shake flasks and microtiter plates, are the most frequently used reactor systems during initial bioprocess development. Originally, they were designed to be operated in batch mode. However, batch mode causes adverse effects like overflow metabolism, substrate inhibition or catabolite repression. Fed-batch mode can prevent these effects, and thus, it is predominantly applied in production processes. Consequently, the implementation of fed-batch mode at small scale is crucial to obtain physiological conditions that are comparable to fed-batch production processes. In shake flasks, fed-batch mode was realized with the previously introduced 250 mL membrane-based fed-batch shake flasks. Within this thesis, the system was standardized regarding its dimensions and optimized in design, handling and robustness. Furthermore, the operating principle was successfully scaled-up to 500 mL shake flasks. The application of membrane-based fed-batch shake flasks allowed to introduce carbon- and nitrogen-limited fed batch conditions to overcome catabolite repression and substrate inhibition in a protease producing Bacillus licheniformis strain. In microtiter plates, carbon-limited fed-batch conditions were realized with polymer-based controlled-release fed-batch microtiter plates. Special emphasis was paid to the possibilities for online monitoring of the fed-batch operated small-scale shaken bioreactors. Membrane-based fed-batch shake flasks and polymer-based controlled-release fed-batch microtiter plates have been tailored to be compatible with the RAMOS and µRAMOS device, respectively. In order to have access to additional culture parameters, a fed-batch microtiter plate with polymer rings was designed. The polymer rings make the culture broth optically accessible, enabling online monitoring with the established BioLector device. In conclusion, the presented small-scale shaken bioreactors enable fed-batch operation with parallel online monitoring of the most important culture parameters. This allows to mimic the physiological conditions relevant for fed-batch production processes already during initial bioprocess development, which saves time and cost, and accelerates bioprocess development.