Engineering of Scale-Down Bioreactor Setup: Deciphering Metabolic Phenotype of Corynebacterium glutamicum under Simulated Bioreactor Inhomogeneity

  • Entwicklung eines Scale-Down Bioreaktor-Setups: Entschlüsselung metabolischer Phenotypen von Corynebacterium glutamicum in nachgestellten Bioreaktorinhomogenitäten

Limberg, Michael H.; Oldiges, Marco (Thesis advisor); Wiechert, Wolfgang (Thesis advisor); Blank, Lars Mathias (Thesis advisor)

Aachen (2017)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2017

Abstract

Performance losses during the scale-up from laboratory into production scale and the associated increase of production costs imperil the competitiveness of sustainable biotechnological bulk products. Since the formation of environmental gradients in the large-scale was identified as the major obstacle for process scalability, scale-up simulation in the laboratory scale became one of the most important prediction tools. This work hooks up with the so-called scale-down strategy by providing a flexible state of the art two compartment scale down device, which is designed for easy process parallelization. The system is composed of two connected and fully controlled stirred tank reactors (STR) implemented in a parallel cultivation platform.To validate the STR-STR application for Corynebacterium glutamicum processes, it was compared to an already established scale-down device consisting of a plug-flow reactor (PFR) connected to a STR. Apart from differing side-product levels, very similar results were observed for the metabolic phenotype and bioprocess performance when sole oxygen perturbations were simulated, although the differing setups provide opposed back mixing profiles.A standardized scale-down workflow for the systematic separation and recombination of critical scale-up parameters was established, including state of the art technology for intracellular metabolite, protein and transcript analysis. Within a comprehensive study of 13 different combinations of oxygen, substrate and pH fluctuations, the workflow was used to investigate the scalability of a C. glutamicum based 1,5-diaminopentane production process. Thereby C. glutamicum provided an outstanding level of robustness to oxygen and substrate inhomogeneities. Using omics based methods, it could be shown, that the flexible rearrangement of the central metabolism is the key element to overcome shortage in NAD+ recycling. Furthermore, formed L-lactate served as reversible and flexible external buffer for carbon and redox equivalents. Only acidic and alkaline pH fluctuation of one pH unit compromised C. glutamicum process performance. Especially, acidic pH levels inhibited regulation of the fermentative key pathway and by that the adaption to short term oxygen limitations. This reduced the biomass and product formation in a significant manner. Consequently, pH was identified to be one of the most sensitive parameter for C. glutamicum processes scale-up. Nevertheless, this systematic investigation of intracellular adaption mechanisms provided a comprehensive metabolic characterization of an adaptive and robust microbial strain for large-scale production.