Development and application of a microfluidic batch cultivation device
| Autor: | Kaganovitch, Eugen |
|---|---|
| Přispěvatelé: | Kohlheyer, Dietrich, Büchs, Jochen |
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: | |
| Zdroj: | Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen (2021). doi:10.18154/RWTH-2021-09799 = Dissertation, Rheinisch-Westfälische Technische Hochschule Aachen, 2021 |
| DOI: | 10.18154/rwth-2021-09799 |
| Popis: | Dissertation, Rheinisch-Westf��lische Technische Hochschule Aachen, 2021; Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen (2021). = Dissertation, Rheinisch-Westf��lische Technische Hochschule Aachen, 2021 Microfluidics has enabled various research projects in the field of microbial single-cell analysis. In particular, single-use microfluidic cultivation devices in combination with automated time-lapse imaging provide powerful approaches for the analysis of microbial phenomena at the single-cell level. High spatiotemporal resolution facilitates individual cell identification and tracking, delivering detailed insights into cellular physiology. Furthermore, a high level of environmental control enables studies on phenotypic population heterogeneity, which describes the phenomenon that individual cells of an isogenic population exhibit certain differences among each other or in comparison to the rest of the population. The occurrence of certain characteristics within a subset of cells within the population leads to the emergence of phenotypes. These characteristics might relate to the production of a certain protein, for example, and can be highly relevant to the fate of a microbial population. Furthermore, population heterogeneity may influence the efficiency of biotechnological fermentations, in which the production of a certain compound is of major importance. Here, the emergence of a phenotype which is not producing the compound, but instead consumes nutrients, has a negative impact on the bioprocess. Hence, the development of new single-cell analysis tools is essential in order to better understand the emergence and functionality of population heterogeneity. Precise control over environmental parameters, such as temperature, pH, or the concentration of certain compounds in the medium, is necessary in order to exclude these parameters as the cause of the emergence of phenotypes. Alternatively, by linking the observed cellular behavior to the variation of one environmental parameter while leaving other parameters constant, the role of this parameter as a trigger for population heterogeneity can be studied. The focus of the present work was the development and application of a novel microfluidic device for the batch cultivation of microorganisms in spatially defined and isolated cultivation volumes up to several hundred picoliters. So far, most microfluidic cultivation devices relied on continuous medium flow in order to guarantee nutrient supply for the cells and, therefore, allowed for single-cell studies under constant environmental conditions. However, no or only limited information on cell physiology in the stationary phase could be derived using these continuous perfusion approaches. The overcoming of this technical limitation is of high importance for biological and biotechnological applications, since the entry into the stationary phase may serve as a trigger for the emergence of new phenotypes. Based on proven polydimethylsiloxane (PDMS)-chip technology, our new batch cultivation device enables the analysis of bacterial cultures in fixed picoliter-scale volumes for over 200 hours with spatial and temporal single-cell resolution. The fabrication process relies on a well-established soft lithography protocol and, in combination with a simple device operation, contributes to cheap and straightforward device application. The present work covers two applications of the batch cultivation device. The growth of Escherichia coli cells under batch cultivation conditions was analyzed in our first proof of concept experiments. Here, we quantified the cell numbers in the stationary phase in dependence on the carbon source amount which was available in the cultivation volume. In the second application, we studied the population heterogeneity of Bacillus subtilis during starvation. B. subtilis forms a phenotype under nutrient depletion, which initiates the production of antimicrobial peptides before sporulation. These peptides can kill sensitive sister cells. While the physiological relevance of population heterogeneity of B. subtilis is still matter of current research, the production of antimicrobial peptides is supposed to delay sporulation, as the released nutrients from dead cells are consumed by the rest of the population for continued growth. By correlating toxin production with cellular stress response, we were able to analyze bacterial cannibalism on a single-cell level directly for the first time. Published by RWTH Aachen University, Aachen |
| Databáze: | OpenAIRE |
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