Single cell analysis of microbial production strains in microfluidic bioreactors

摘要

Industrial biotechnology is concerned with the sustainable production of, for example, fine and bulk chemicals, pharmaceuticals and proteins by utilizing microorganisms for the conversion of renewable carbon sources. Well known examples include the production of amino acids by Corynebacterium glutamicum at a million ton scale per year worldwide, or the recombinant production of insulin by Escherichia coli. Growth and productivity of the underlying host microorganisms are two key performance indicators in biotechnological production processes. Assuming isogenic starting populations, optimal reactor control and mixing, a uniform cell behavior during growth might be expected. However, as confirmed in recent years, isogenic bacterial populations can be physiologically heterogeneous. Obviously, there is a strong demand to unravel microbial population heterogeneity, understand its origin and gain knowledge on its impact on large scale biotechnological production. Therefore, new analytical techniques addressing single-cell behavior are the key for further optimization. In particular, state-of-the-art microfluidic cultivation systems facilitating single-cell resolution and accurate environmental control over long time periods at the same time, offer completely new experimental assays on microbial populations. In contrast to conventional systems, for example, fluorescence activated cell sorting, microfluidic cultivations enable the analysis of cell dynamics by automated time-lapse microscopy with full spatio-temporal resolution. The aim of the present thesis was to develop and establish a new microfluidic platform technology for microbial single-cell analysis in order to address key concerns on population heterogeneity and reactor inhomogeneity in industrial biotechnology. Several unique single-cell cultivation chips were successfully fabricated and validated with a variety of industrially applied microorganisms. Each device contained up to several thousand micrometer sized cultivation structures in parallel intended for high-throughput analysis of single cells and isogenic microcoloniesIn the present research two major single-cell investigations were performed demonstrating the universal applicability and potential of the microfluidic single-cell cultivation technology:(i) Growth analysis of industrially relevant bacteria (in particular E. coli and C. glutamicum) with single-cell resolution was performed. Therefore, isogenic microcolonies were grown in monolayers up to several hundred cells in each growth chamber and imaging was performed by time-lapse microscopy. Compared to a typical 1 liter lab-scale batch cultivation, interestingly a 1.5-fold enhanced growth rate of C. glutamicum wild type cells under constant microfluidic cultivation conditions was found. (ii) Morphological characterization: The cellular response of several C. glutamicum strains under various environmental conditions was investigated in more detail. Studies included artificially induced starvation, occurrence of spontaneously induced stress response of single cells, as well as morphological characterization during growth on different carbon sources. In a multi-scale approach, the elevated single-cell growth rates were investigated in more detail. Therefore, various lab-scale cultivations were performed and results compared with our microfluidic single-cell analysis. This systematic study revealed a maximum growth rate of μ_max=0.6 h^(-1) during microfluidic cultivation compared to μ_max=0.4 h^(-1) during bioreactor, flask and microtiter cultivation. Further single-cell analysis exposed that solely the medium composition was the growth enhancing factor, rather than the continuous perfusion during single-cell cultivation or the analytical method itself. It turned out that the medium compound protocatechuate (PCA), initially added as iron chelator, serves as an additional carbon source and is co-metabolized by C. glutamicum, resulting in higher growth rates when PCA is continuously supplied during microfluidic cultivation. In contrast, the limited amount of PCA is fully consumed during the early process of a typical batch process. Follow-up studies proved that even in conventional batch cultivation systems, the improved growth rates can be realized if PCA is made accessible for a longer time. Short innovation times allowed the fabrication of tailor made systems depending on microbial species and application. In an overview, it is shown, how these systems can be used to cultivate other industrial important organisms such as fungi and yeast. Furthermore, examples are given how the developed system in combination with genetically modified fluorescence sensors can be used to investigate heterogeneity of growth coupled production processes at the single-cell level. The results confirm that cell-to-cell heterogeneity can have significant impact on production processes and need to be further investigated in future. In the present project, novel single-use microfluidic cultivation devices with structures in the sub-micrometer range for trapping and cultivation of individual bacteria were developed and successfully validated. Automated live-cell imaging in combination with accurate environmental control facilitates spatio-temporal analysis of single bacteria with respect to, for example, growth, morphology and single-cell productivity. In a highly interdisciplinary approach, the microfluidic single-cell technology was efficiently utilized to derive cellular information which was not accessible before. The presented findings clearly demonstrate the high potential of microbial single-cell analysis for biotechnological strain and process optimization. The present work established the foundation for further progress in the field.