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- Carlquist, Magnus, et al.
(author)
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Physiological heterogeneities in microbial populations and implications for physical stress tolerance
- 2012
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In: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 11
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Journal article (peer-reviewed)abstract
- Background: Traditionally average values of the whole population are considered when analysing microbial cell cultivations. However, a typical microbial population in a bioreactor is heterogeneous in most phenotypes measurable at a single-cell level. There are indications that such heterogeneity may be unfavourable on the one hand (reduces yields and productivities), but also beneficial on the other hand (facilitates quick adaptation to new conditions - i.e. increases the robustness of the fermentation process). Understanding and control of microbial population heterogeneity is thus of major importance for improving microbial cell factory processes. Results: In this work, a dual reporter system was developed and applied to map growth and cell fitness heterogeneities within budding yeast populations during aerobic cultivation in well-mixed bioreactors. The reporter strain, which was based on the expression of green fluorescent protein (GFP) under the control of the ribosomal protein RPL22a promoter, made it possible to distinguish cell growth phases by the level of fluorescence intensity. Furthermore, by exploiting the strong correlation of intracellular GFP level and cell membrane integrity it was possible to distinguish subpopulations with high and low cell membrane robustness and hence ability to withstand freeze-thaw stress. A strong inverse correlation between growth and cell membrane robustness was observed, which further supports the hypothesis that cellular resources are limited and need to be distributed as a trade-off between two functions: growth and robustness. In addition, the trade-off was shown to vary within the population, and the occurrence of two distinct subpopulations shifting between these two antagonistic modes of cell operation could be distinguished. Conclusions: The reporter strain enabled mapping of population heterogeneities in growth and cell membrane robustness towards freeze-thaw stress at different phases of cell cultivation. The described reporter system is a valuable tool for understanding the effect of environmental conditions on population heterogeneity of microbial cells and thereby to understand cell responses during industrial process-like conditions. It may be applied to identify more robust subpopulations, and for developing novel strategies for strain improvement and process design for more effective bioprocessing.
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| 3. |
- Fernandes, Rita Lencastre, et al.
(author)
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Applying Mechanistic Models in Bioprocess Development
- 2013
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In: Advances in Biochemical Engineering, Biotechnology. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 0724-6145. ; 132, s. 137-166
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Journal article (peer-reviewed)abstract
- The available knowledge on the mechanisms of a bioprocess system is central to process analytical technology. In this respect, mechanistic modeling has gained renewed attention, since a mechanistic model can provide an excellent summary of available process knowledge. Such a model therefore incorporates process-relevant input (critical process variables)-output (product concentration and product quality attributes) relations. The model therefore has great value in planning experiments, or in determining which critical process variables need to be monitored and controlled tightly. Mechanistic models should be combined with proper model analysis tools, such as uncertainty and sensitivity analysis. When assuming distributed inputs, the resulting uncertainty in the model outputs can be decomposed using sensitivity analysis to determine which input parameters are responsible for the major part of the output uncertainty. Such information can be used as guidance for experimental work; i.e., only parameters with a significant influence on model outputs need to be determined experimentally. The use of mechanistic models and model analysis tools is demonstrated in this chapter. As a practical case study, experimental data from Saccharomyces cerevisiae fermentations are used. The data are described with the well-known model of Sonnleitner and Kappeli (Biotechnol Bioeng 28: 927-937, 1986) and the model is analyzed further. The methods used are generic, and can be transferred easily to other, more complex case studies as well.
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| 4. |
- Fernandes, Rita Lencastre, et al.
(author)
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Cell mass and cell cycle dynamics of an asynchronous budding yeast population: Experimental observations, flow cytometry data analysis, and multi-scale modeling
- 2013
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In: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 110:3, s. 812-826
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Journal article (peer-reviewed)abstract
- Despite traditionally regarded as identical, cells in a microbial cultivation present a distribution of phenotypic traits, forming a heterogeneous cell population. Moreover, the degree of heterogeneity is notably enhanced by changes in micro-environmental conditions. A major development in experimental single-cell studies has taken place in the last decades. It has however not been fully accompanied by similar contributions within data analysis and mathematical modeling. Indeed, literature reporting, for example, quantitative analyses of experimental single-cell observations and validation of model predictions for cell property distributions against experimental data is scarce. This study focuses on the experimental and mathematical description of the dynamics of cell size and cell cycle position distributions, of a population of Saccharomyces cerevisiae, in response to the substrate consumption observed during batch cultivation. The good agreement between the proposed multi-scale model (a population balance model [PBM] coupled to an unstructured model) and experimental data (both the overall physiology and cell size and cell cycle distributions) indicates that a mechanistic model is a suitable tool for describing the microbial population dynamics in a bioreactor. This study therefore contributes towards the understanding of the development of heterogeneous populations during microbial cultivations. More generally, it consists of a step towards a paradigm change in the study and description of cell cultivations, where average cell behaviors observed experimentally now are interpreted as a potential joint result of various co-existing single-cell behaviors, rather than a unique response common to all cells in the cultivation. Biotechnol. Bioeng. 2013; 110: 812826. (c) 2012 Wiley Periodicals, Inc.
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| 5. |
- Heins, Anna Lena, et al.
(author)
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Quantitative flow cytometry to understand population heterogeneity in response to changes in substrate availability in escherichia coli and saccharomyces cerevisiae chemostats
- 2019
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In: Frontiers in Bioengineering and Biotechnology. - : Frontiers Media SA. - 2296-4185. ; 7:AUG
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Journal article (peer-reviewed)abstract
- Microbial cells in bioprocesses are usually described with averaged parameters. But in fact, single cells within populations vary greatly in characteristics such as stress resistance, especially in response to carbon source gradients. Our aim was to introduce tools to quantify population heterogeneity in bioprocesses using a combination of reporter strains, flow cytometry, and easily comprehensible parameters. We calculated mean, mode, peak width, and coefficient of variance to describe distribution characteristics and temporal shifts in fluorescence intensity. The skewness and the slope of cumulative distribution function plots illustrated differences in distribution shape. These parameters are person-independent and precise. We demonstrated this by quantifying growth-related population heterogeneity of Saccharomyces cerevisiae and Escherichia coli reporter strains in steady-state of aerobic glucose-limited chemostat cultures at different dilution rates and in response to glucose pulses. Generally, slow-growing cells showed stronger responses to glucose excess than fast-growing cells. Cell robustness, measured as membrane integrity after exposure to freeze-thaw treatment, of fast-growing cells was strongly affected in subpopulations of low membrane robustness. Glucose pulses protected subpopulations of fast-growing but not slower-growing yeast cells against membrane damage. Our parameters could successfully describe population heterogeneity, thereby revealing physiological characteristics that might have been overlooked during traditional averaged analysis.
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