Impact of mixing insufficiencies on L-phenylalanine production with an Escherichia coli reporter strain in a novel two-compartment bioreactor
Abstract 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 po...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-08-01
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| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12934-023-02165-4 |
| _version_ | 1827707552796770304 |
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| author | Manh Dat Hoang Ingmar Polte Lukas Frantzmann Nikolas von den Eichen Anna-Lena Heins Dirk Weuster-Botz |
| author_facet | Manh Dat Hoang Ingmar Polte Lukas Frantzmann Nikolas von den Eichen Anna-Lena Heins Dirk Weuster-Botz |
| author_sort | Manh Dat Hoang |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-03-10T16:49:10Z |
| format | Article |
| id | doaj.art-d8c47bc640c84cf5a83629758f9de8f2 |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-03-10T16:49:10Z |
| publishDate | 2023-08-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-d8c47bc640c84cf5a83629758f9de8f22023-11-20T11:22:24ZengBMCMicrobial Cell Factories1475-28592023-08-0122111910.1186/s12934-023-02165-4Impact of mixing insufficiencies on L-phenylalanine production with an Escherichia coli reporter strain in a novel two-compartment bioreactorManh Dat Hoang0Ingmar Polte1Lukas Frantzmann2Nikolas von den Eichen3Anna-Lena Heins4Dirk Weuster-Botz5Chair of Biochemical Engineering, TUM School of Engineering and Design, Technical University of MunichChair of Biochemical Engineering, TUM School of Engineering and Design, Technical University of MunichChair of Biochemical Engineering, TUM School of Engineering and Design, Technical University of MunichChair of Biochemical Engineering, TUM School of Engineering and Design, Technical University of MunichChair of Biochemical Engineering, TUM School of Engineering and Design, Technical University of MunichChair of Biochemical Engineering, TUM School of Engineering and Design, Technical University of MunichAbstract 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.https://doi.org/10.1186/s12934-023-02165-4Escherichia coliReporter strainsAmino acid productionTwo-compartment bioreactorCoiled flow inverterPopulation heterogeneity |
| spellingShingle | Manh Dat Hoang Ingmar Polte Lukas Frantzmann Nikolas von den Eichen Anna-Lena Heins Dirk Weuster-Botz Impact of mixing insufficiencies on L-phenylalanine production with an Escherichia coli reporter strain in a novel two-compartment bioreactor Microbial Cell Factories Escherichia coli Reporter strains Amino acid production Two-compartment bioreactor Coiled flow inverter Population heterogeneity |
| title | Impact of mixing insufficiencies on L-phenylalanine production with an Escherichia coli reporter strain in a novel two-compartment bioreactor |
| title_full | Impact of mixing insufficiencies on L-phenylalanine production with an Escherichia coli reporter strain in a novel two-compartment bioreactor |
| title_fullStr | Impact of mixing insufficiencies on L-phenylalanine production with an Escherichia coli reporter strain in a novel two-compartment bioreactor |
| title_full_unstemmed | Impact of mixing insufficiencies on L-phenylalanine production with an Escherichia coli reporter strain in a novel two-compartment bioreactor |
| title_short | Impact of mixing insufficiencies on L-phenylalanine production with an Escherichia coli reporter strain in a novel two-compartment bioreactor |
| title_sort | impact of mixing insufficiencies on l phenylalanine production with an escherichia coli reporter strain in a novel two compartment bioreactor |
| topic | Escherichia coli Reporter strains Amino acid production Two-compartment bioreactor Coiled flow inverter Population heterogeneity |
| url | https://doi.org/10.1186/s12934-023-02165-4 |
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