Single-cell analysis of microbial production strains in microfluidic bioreactors

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 microcolonies