PUB - Publikationen an der Universität Bielefeld

The research and biotechnological use of microbial co-cultures has been of great interest for several years, as they offer advantages over the frequently used monocultures and open up new application opportunities. Especially interactions based on synthrophy and commensalism, i.e. mutual or unilateral positive dependencies, can be an asset for biotechnological processes. However, since the analysis of the interaction between two or more different microbial organisms in co-culture is highly challenging due to the complex dynamics on different temporal and spatial scales, there are only few tools and methods available that allow accurate analysis of these interactions.
To achieve high temporal and spatial resolution, microfluidic cultivation devices together with automated microscopy and image analysis are ideal tools to analyze co-culture interactions. In addition, microfluidic cultivation also provides the ability to precisely control and maintain constant environmental conditions. However, until now, microfluidics has been mainly used for single-cell analysis of monocultures.
In this thesis, different chip designs and model consortia were developed to analyze and control the interaction of synthetic co-cultures. In a first study, it was shown that unilateral dependence can be studied in a model consortium consisting of an L-lysine producing Corynebacterium glutamicum strain and an L-lysine auxotrophic C. glutamicum variant in spatially separated but nanochannel-connected cultivation chambers at the single-cell level. L-lysine secreted by the L-lysine producer could be taken up by the L-lysine auxotrophic strain and cell growth was induced. The chamber geometry played an important role, since the cultivation experiments were performed with constant medium flow to keep the environmental conditions constant. This resulted in a constant wash-out of a fraction of the produced L-lysine with the medium flow. The effect on L-lysine concentration inside the cultivation chambers was investigated in a computer-based simulation. The simulation revealed that the L-lysine production rate of the selected C. glutamicum strain (C. glutamicum DM1800) was sufficient to achieve a growth-inducing L-lysine concentration in the chamber half of the L-lysine-auxotrophic strain. However, the L-lysine concentration on the L-lysine consumer side was approximately 75% lower than on the L-lysine producer side caused by a constant wash-out of a fraction of the produced L-lysine and the barrier structure between the two chamber compartments. As an example for further application of this microfluidic cultivation platform, also a contact-based interaction was investigated. Using genetically modified and fluorescence-labeled Pseudomonas putida and Escherichia coli strains, bacterial conjugation and the direct cell contact required for it, were demonstrated at the single-cell level with the developed microfluidic devices.
For control of the consortium, a genetically modified C. glutamicum L-lysine producer was applied (DM1727 pEKEx2-lysC -ßFBR), which secretes a larger amount of L-lysine only by IPTG induction. In comparison with the L-lysine producer DM1800, approximately equal growth rates were obtained with the inducible L-lysine producer in co-culture with the L-lysine-auxotrophic strain. Additionally, to achieve a minimal invasive control, modified IPTG (NP-photocaged IPTG) was used, which is only activated by short UV-A illumination. Significantly different growth rates of the L-lysine-auxotrophic strain could be measured by using different exposure times compared to no UV-A exposure. The L-lysine-auxotrophic strain showed highly heterogeneous growth behavior at low L-lysine concentrations, which could be demonstrated in monocultures for growth characterization at the single-cell level with different L-lysine concentrations supplied to the medium.
As a perspective for contact-based applications, a fluorescence-producing model consortium consisting of a modified E. coli and P. putida strain was used to study bacterial conjugation with high spatial and temporal resolution. Using an appropriate microfluidic cultivation and image analysis strategy, conjugation efficiency under different conditions could be measured and compared at the single-cell level.
These application examples show that microfluidic cultivation platforms have great potential to be used for the analysis of microbial co-cultures in order to better understand and apply them for biotechnological processes. To this end, there are opportunities in chip design as well as in development of synthetic consortia for the characterization of different co-cultures.