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    Item type:Publication, Access status: with files ,
    Hidden diversity : diversity of cells in industrial bioprocesses
    (Springer, 2023-05)
    Arulrajah, Prasika
    ;
    Heins, Anna-Lena
    Population heterogeneity poses a major challenge in industrial scale bioprocesses, affecting process performance but also leading to more robust phenotypes. To investigate this phenomenon, multiple fluorescent reporter strains are cultivated under simulated industrial scale conditions in multi-compartment bioreactors. This approach significantly raises the quantitative understanding of population heterogenity which is needed before its benefits can help to develop high-yielding, more robust bioprocesses.
    Publicationtype: Journal Article
      63  59
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    Item type:Publication, Access status: with files ,
    Development and characterization of Escherichia coli triple reporter strains for investigation of population heterogeneity in bioprocesses
    (BioMed Central, 2020-01-28)
    Heins, Anna-Lena
    ;
    Reyelt, Jan
    ;
    Schmidt, Marlen
    ;
    Kranz, Harald
    ;
    Weuster-Botz, Dirk
    Background: Today there is an increasing demand for high yielding robust and cost efficient biotechnological production processes. Although cells in these processes originate from isogenic cultures, heterogeneity induced by intrinsic and extrinsic influences is omnipresent. To increase understanding of this mechanistically poorly understood phenomenon, advanced tools that provide insights into single cell physiology are needed. Results: Two Escherichia coli triple reporter strains have been designed based on the industrially relevant production host E. coli BL21(DE3) and a modified version thereof, E. coli T7E2. The strains carry three different fluorescence proteins chromosomally integrated. Single cell growth is followed with EmeraldGFP (EmGFP)-expression together with the ribosomal promoter rrnB. General stress response of single cells is monitored by expression of sigma factor rpoS with mStrawberry, whereas expression of the nar-operon together with TagRFP657 gives information about oxygen limitation of single cells. First, the strains were characterized in batch operated stirred-tank bioreactors in comparison to wildtype E. coli BL21(DE3). Afterwards, applicability of the triple reporter strains for investigation of population heterogeneity in bioprocesses was demonstrated in continuous processes in stirred-tank bioreactors at different growth rates and in response to glucose and oxygen perturbation simulating gradients on industrial scale. Population and single cell level physiology was monitored evaluating general physiology and flow cytometry analysis of fluorescence distributions of the triple reporter strains. Although both triple reporter strains reflected physiological changes that were expected based on the expression characteristics of the marker proteins, the triple reporter strain based on E. coli T7E2 showed higher sensitivity in response to environmental changes. For both strains, noise in gene expression was observed during transition from phases of non-growth to growth. Apparently, under some process conditions, e.g. the stationary phase in batch cultures, the fluorescence response of EmGFP and mStrawberry is preserved, whereas TagRFP657 showed a distinct response. Conclusions: Single cell growth, general stress response and oxygen limitation of single cells could be followed using the two triple reporter strains developed in this study. They represent valuable tools to study population heterogeneity in bioprocesses significantly increasing the level of information compared to the use of single reporter strains.
    Publicationtype: Journal Article
    Scopus© Citations 15  24  21
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    Item type:Publication, Access status: without files ,
    Population heterogeneity in microbial bioprocesses : origin, analysis, mechanisms, and future perspectives
    (Springer, 2018-07-01)
    Heins, Anna-Lena
    ;
    Weuster-Botz, Dirk
    Population heterogeneity is omnipresent in all bioprocesses even in homogenous environments. Its origin, however, is only so well understood that potential strategies like bet-hedging, noise in gene expression and division of labour that lead to population heterogeneity can be derived from experimental studies simulating the dynamics in industrial scale bioprocesses. This review aims at summarizing the current state of the different parts of single cell studies in bioprocesses. This includes setups to visualize different phenotypes of single cells, computational approaches connecting single cell physiology with environmental influence and special cultivation setups like scale-down reactors that have been proven to be useful to simulate large-scale conditions. A step in between investigation of populations and single cells is studying subpopulations with distinct properties that differ from the rest of the population with sub-omics methods which are also presented here. Moreover, the current knowledge about population heterogeneity in bioprocesses is summarized for relevant industrial production hosts and mixed cultures, as they provide the unique opportunity to distribute metabolic burden and optimize production processes in a way that is impossible in traditional monocultures. In the end, approaches to explain the underlying mechanism of population heterogeneity and the evidences found to support each hypothesis are presented. For instance, population heterogeneity serving as a bet-hedging strategy that is used as coordinated action against bioprocess-related stresses while at the same time spreading the risk between individual cells as it ensures the survival of least a part of the population in any environment the cells encounter.
    Publicationtype: Review Article
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