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Impact of mixing insufficiencies on L-phenylalanine production with an Escherichia coli reporter strain in a novel two-compartment bioreactor
Citation Link: https://doi.org/10.15480/882.8809
Publikationstyp
Journal Article
Publikationsdatum
2023-12-01
Sprache
English
Author
Biochemical Engineering, TUM School of Engineering and Design, Technical University of Munich
Polte, Ingmar
Biochemical Engineering, TUM School of Engineering and Design, Technical University of Munich
Frantzmann, Lukas
Biochemical Engineering, TUM School of Engineering and Design, Technical University of Munich
Eichen, Nikolas von den
Biochemical Engineering, TUM School of Engineering and Design, Technical University of Munich
Biochemical Engineering, TUM School of Engineering and Design, Technical University of Munich
Enthalten in
Volume
22
Issue
1
Article Number
153
Citation
Microbial Cell Factories 22 (1): 153 (2023-12-01)
Publisher DOI
Scopus ID
Publisher
Biomed Central
Background: The omnipresence of population heterogeneity in industrial bioprocesses originates from prevailing dynamic bioprocess conditions, which promote differences in the expression of cellular characteristics. Despite the awareness, the concrete consequences of this phenomenon remain poorly understood.
Results: Therefore, for the first time, a L-phenylalanine overproducing Escherichia coli quadruple reporter strain was established for monitoring of general stress response, growth behavior, oxygen limitation and product formation of single cells based on mTagBFP2, mEmerald, CyOFP1, and mCardinal2 expression measured by flow cytometry. This strain was applied for the fed-batch production of L-phenylalanine from glycerol and ammonia in a stirred-tank bioreactor at homogeneous conditions compared to the same process in a novel two-compartment bioreactor. This two-compartment bioreactor consists of a stirred-tank bioreactor with an initial volume of 0.9 L (homogeneous zone) with a coiled flow inverter with a fixed working volume of 0.45 L as a bypass (limitation zone) operated at a mean hydraulic residence time of 102 s. The product formation was similar in both bioreactor setups with maximum L-phenylalanine concentrations of 21.1 ± 0.6 g L−1 demonstrating the consistency of this study’s microbial L-phenylalanine production. However, cell growth was vulnerable to repetitive exposure to the dynamically changing conditions in the two-compartment bioreactor with maximum biomass yields reduced by 21%. The functionality of reporter molecules was approved in the stirred-tank bioreactor cultivation, in which expressed fluorescence levels of all four markers were in accordance with respective process state variables. Additional evaluation of the distributions on single-cell level revealed the presence of population heterogeneity in both bioprocesses. Especially for the marker of the general stress response and the product formation, the corresponding histograms were characterized by bimodal shapes and broad distributions. These phenomena were pronounced particularly at the beginning and the end of the fed-batch process.
Conclusions: The here shown findings confirm multiple reporter strains to be a noninvasive tool for monitoring cellular characteristics and identifying potential subpopulations in bioprocesses. In combination with experiments in scale-down setups, these can be utilized for a better physiological understanding of bioprocesses and support future scale-up procedures.
Results: Therefore, for the first time, a L-phenylalanine overproducing Escherichia coli quadruple reporter strain was established for monitoring of general stress response, growth behavior, oxygen limitation and product formation of single cells based on mTagBFP2, mEmerald, CyOFP1, and mCardinal2 expression measured by flow cytometry. This strain was applied for the fed-batch production of L-phenylalanine from glycerol and ammonia in a stirred-tank bioreactor at homogeneous conditions compared to the same process in a novel two-compartment bioreactor. This two-compartment bioreactor consists of a stirred-tank bioreactor with an initial volume of 0.9 L (homogeneous zone) with a coiled flow inverter with a fixed working volume of 0.45 L as a bypass (limitation zone) operated at a mean hydraulic residence time of 102 s. The product formation was similar in both bioreactor setups with maximum L-phenylalanine concentrations of 21.1 ± 0.6 g L−1 demonstrating the consistency of this study’s microbial L-phenylalanine production. However, cell growth was vulnerable to repetitive exposure to the dynamically changing conditions in the two-compartment bioreactor with maximum biomass yields reduced by 21%. The functionality of reporter molecules was approved in the stirred-tank bioreactor cultivation, in which expressed fluorescence levels of all four markers were in accordance with respective process state variables. Additional evaluation of the distributions on single-cell level revealed the presence of population heterogeneity in both bioprocesses. Especially for the marker of the general stress response and the product formation, the corresponding histograms were characterized by bimodal shapes and broad distributions. These phenomena were pronounced particularly at the beginning and the end of the fed-batch process.
Conclusions: The here shown findings confirm multiple reporter strains to be a noninvasive tool for monitoring cellular characteristics and identifying potential subpopulations in bioprocesses. In combination with experiments in scale-down setups, these can be utilized for a better physiological understanding of bioprocesses and support future scale-up procedures.
Schlagworte
Amino acid production
Coiled flow inverter
Escherichia coli
Population heterogeneity
Reporter strains
Two-compartment bioreactor
DDC Class
570: Life Sciences, Biology
Publication version
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