Integrated biochemical engineering : strain and process engineering for the production of rhamnolipids

• Integrierte Stamm- und Prozessentwicklung am Beispiel der rekombinanten Rhamnolipidproduktion

Demling, Philipp; Blank, Lars M. (Thesis advisor); Jupke, Andreas (Thesis advisor)

1. Auflage. - Aachen : Apprimus Verlag (2021, 2022)
Book, Dissertation / PhD Thesis

In: Applied microbiology 25
Page(s)/Article-Nr.: 1 Online-Ressource : Illustrationen, Diagramme

Dissertation, RWTH Aachen University, 2021

Abstract

Establishing biotechnological processes within the envisaged circular bioeconomy requires holistic approaches including an integration of all stages of process development. In this scope, the presented thesis illustrates the integration of strain and process engineering using the example of producing the biosurfactant rhamnolipids with recombinant Pseudomonas putida KT2440. A process-hindering challenge during the production of biosurfactants in aerated bioreactors is excessive foaming. To mitigate this challenge, an in situ liquid-liquid extraction was established by selecting a suitable, biocompatible extraction solvent in a multi-step strategy, including the assessment of product recovery and solvent recycling. A refinement of cultivation parameters was required to enhance functionality of the extraction, in turn influencing the performance of the whole-cell biocatalyst. Unraveling interactions of integrated up- and downstream processing resulted in the definition of a joint operational window. Finally, a foam-free fed-batch cultivation of rhamnolipid-producing P. putida KT2440 with integrated in situ extraction was established. Subsequently, different cultivation modes have been evaluated for enhanced production, and strategies for improved phase separation have been explored. As the microbial boundaries of the operational window are restricting process performance, host engineering was approached to enhance the solvent tolerance of P. putida KT2440, thereby enabling the utilization of additional extraction solvents. P. putida KT2440 was successfully adapted to tolerate high concentrations of 1-octanol. Genome re-sequencing and subsequent reverse genome engineering enabled the construction of tolerant strains capable of producing rhamnolipids in the presence of 1-octanol. Another challenge are transient oxygen limitations in industrial fermentations, which P. putida KT2440 masters well without losing production capacity, thereby highlighting its outstanding suitability for industrial-scale production processes. Previous findings were considered to define an operational window to produce rhamnolipids with recombinant P. putida KT2440 in a custom-designed multiphase loop reactor, integrating cultivation and an in situ counter-current liquid-liquid extraction. Performance indicators compared well with previous two-liquid phase cultivations in stirred-tank reactors, emphasizing the robustness of P. putida KT2440 and serving as a proof of concept for the novel reactor type. In conclusion, this thesis advocates integrated bioprocessing and the urgency for a holistic, interdisciplinary perspective on the overall process development. While the specific outcomes of this thesis are related to the presented system of producing rhamnolipids with recombinant P. putida KT2440, basic concepts and proposed solutions can be extrapolated to other challenging cases in bioprocess development. Thereby, competitive industrial bioprocesses are promoted, eventually contributing to establishing the envisaged circular bioeconomy.