Polymer-based nutrient release system for small-scale microbial fed-batch cultivations

  • Polymerbasiertes Nährstofffreisetzungssystem zur mikrobiellen Fed-batch-Kultivierung im Kleinkulturmaßstab

Keil, Timm; Büchs, Jochen (Thesis advisor); Leonhard, Kai (Thesis advisor)

Aachen (2020)
Dissertation / PhD Thesis

Dissertation, Rheinisch-Westfälische Technische Hochschule Aachen, 2020

Abstract

Microtiter plates are a well-established platform for microbial screening applications. Conventionally, cultivations in microtiter plates are operated in batch mode. However, batch operation mode provokes disadvantages like catabolite repression, overflow metabolism or elevated osmotic concentrations. Thus, the fed-batch operation mode is usually applied in industrial microbial production processes. Hence, microtiter plate experiments performed in a batch operation mode complicate the transfer of results into the production scale. This thesis investigated a polymer-based fed-batch system for the slow release of nutrients into each well of a microtiter plate. The influence of different media properties on the glucose release characteristics was examined. The pH-value, the osmotic concentration, the temperature and changing nitrogen salt concentrations affected the glucose release rate. The functionality of the fed-batch microtiter plate was investigated using biological model systems. Exemplarily, H. polymorpha showed a 176 times higher glucose-to-GFP yield under fed-batch operation mode compared to batch operation mode. The polymer matrix composition was further improved to allow higher glucose release rates and to avoid influencing effects on release characteristics. A conventional and a fed-batch MTP were used for a comparative screening of a clone library in batch and fed-batch operation mode. A transfer to STR experiments verified that the strain selected under fed-batch conditions indeed outperformed the strain selected under batch conditions. Additionally, the influence of the fed-batch preculture on the reliability of subsequent screening results was investigated. The process with fed-batch operated preculture resulted in a reduced mean standard deviation in the final product quantification. In addition, the clone ranking with fed-batch preculture was considerably improved. The spectrum of applications for the polymer-based release technique was extended for the release of a pH-active agent. Therefore, a model was used to predict the necessary amount of pH-active agent to keep the pH-value in the optimal range for growth of E. coli at reduced buffer concentrations. As a result, the osmotic concentration in the medium was similar to media in large-scale processes. A further application was the release of ammonium into shake flask cultivations of microalgae. This allowed nitrogen-limited fed-batch cultivations in small-scale, which resulted in increased lipid productivity compared to the conventional two-step cultivation process.

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