Characterisation of biotic interactions between a Dyadobacter strain and the diatom Achnanthidium minutissimum

Abstract

Diatoms are phototrophic, unicellular algae. Due to their large share in CO2 fixation and O2 production, they are very important for Earth's biogeochemistry. Many diatoms are also of economic relevance, due to their capacity to colonise submerged surfaces such as ship hulls. This so called biofouling includes the undesired growth of biofilms, which form by the aggregation of cells and the secretion of a matrix of extracellular polymeric substances (EPS). Depending on the composition of the biofilm matrix, this microenvironment of the embedded cells can be quite different compared to the surrounding environment. Naturally occurring biofilms are often complex communities of microorganisms from different kingdoms of life, such as photoautotrophic diatoms and heterotrophic bacteria. Complex intercellular interactions occur in such communities, but only few signalling or messenger molecules, which constitute these chemical communication pathways, are known. Therefore, the identification of such molecules is of great interest. In this thesis, a biological assay system ("bioassay") was tested to study biotic interactions between the freshwater diatom Achnanthidium minutissimum and a biofilm-dwelling bacterium from Lake Constance. It was intended to elucidate chemical modes of communication between these species and to identify relevant molecules. An additional goal was the investigation of EPS capsule formation by the diatom in reaction to contact with the bacterium. For these purposes, an assay that quantifies the biofilm formation of the diatom was improved and automated. The assay was also used as a reporter system for the fractionation of bioactive compounds from the bacterial supernatant. The supernatant production was upscaled by optimising the bacterium's growth conditions. A combination of pH-dependent liquid-liquid and solid phase extraction was used for the fractionation of the supernatant. Moreover, light and electron microscopy were correlated in an easily adaptable manner to enable the microstructural analysis of EPS capsules. The bioassay with A. minutissimum and the Dyadobacter-related Bacteroidetes strain clarified that its biofilm induction is mediated by soluble, yet hydrophobic, bacterial compounds. These could be extracted, albeit in low purities and amounts. However, reproducibility and user-friendliness of the staining-dependent bioassay could be enhanced by its partial automation. This was achieved by implementing both human- and machine-readable data structures, which improved the planning of experiments, simplified their repetition, as well as increased the efficiency of data evaluation in the context of medium-throughput screenings. Moreover, tests of biofilm quantification via staining-independent parameters allowed to further simplify the measurement of this bioassay. The scanning electron microscopy uncovered novel fibrillar microstructures both on the diatom frustules as well as in the material of the EPS capsule. It can be postulated that this capsule forms by condensation of a mesh of frustule-attached fibrils. Due to a quantifiable, preferential attachment of bacteria cells to the capsules, their relevance for the interaction of diatom and bacteria is confirmed. The fractionation methods tested here highlight problems and necessary improvement options for the purification of bioactive substances in the A. minutissimum bioassay in sufficient quantities for molecular analyses. Moreover, the partial automation of measurement and data processing workflows highlight additional fields of application for this biofilm assay. The workflows developed here may also be beneficial for other biofilm assays.