Van pure culturen naar vlokken: een bottom-up benadering voor microbiële ecologiestudies van biologische waterzuivering

The scale of human activities requires a well-engineered system to mitigate the impact of discharging the vast amount of wastewater produced every day. When discharged untreated, the high nutrient content of this waste stream contributes to soil and water eutrophication problems, promoting algal blooms, loss of biodiversity, and other ecological issues. In essence, excessive nutrient availability disturbs the natural C/N/P balance of our ecosystems. By leveraging the natural removal activities that occur in the environment, the activated sludge process has become the foundation of engineered systems in biological wastewater treatment (and resource recovery). The process is based on a mixed microbial community (bacteria/fungi/protists/...) that aggregates into flocs, which can be separated from the treated water. Biological aggregation (into flocs or granules) not only yields a clear effluent, it can intensify sludge separation, and retains functional microorganisms in the system. However, the sludge aggregates are mostly still treated as a black box (in terms of community and biopolymer composition). Pinpointing the dominant players for this aggregation in the dynamic mixed microbial community is not straightforward. Especially the matrix of extracellular polymeric substances (EPS) in which the microorganisms are embedded, is a complex structure that should be more elaborately investigated to be able to link it to functionality. As a result of this lack of insight, flocculation problems are usually still remediated without knowledge of the exact causes. By extension, flocculation itself is mainly approached in a kind of trial-and-error way, because mechanistically controlled flocculation (or granulation) is not (yet) possible. In this work, we show that it is feasible to establish a model consortium of activated sludge with reduced complexity to fundamentally study bioflocculation. By avoiding shifts in the microbial community, the applied experimental conditions have a more direct effect on the observations, because community shifts cannot easily be decoupled from the functional result. Five key microbial species are selected based on their functional or structural roles, as well as their relevance in wastewater treatment: Nitrosomonas europaea, Nitrobacter winogradskyi, Azoarcus communis, Tetrasphaera japonica, and Sphaerotilus montanus. The research combines microscopy, metabolite measurements and molecular quantification of the community to study their interactions and response in axenic and coculture conditions. First, the axenic growth of each member is established and their main functional activities are investigated using multiple approaches. Alginate entrapment enables robust cell retention of nitrifiers and promotes the formation of strong, spherical microcolonies similar to those found in full-scale sludge. Cryosectioning showed a homogeneous biomass distribution in beads, while FISH revealed that not all N-cycle species were present in every microcolony. Flow cytometric fingerprints also indicated a lower phenotypic diversity in entrapped nitrifiers in coculture compared to axenic cultures, suggesting that close proximity within beads is sufficient for interspecies interactions. The characterisation of \textit{Tetrasphaera japonica} revealed that this (alleged) phosphate accumulating organism did not cycle polyphosphate in all tested conditions. This observation highlights the effect of the microbial community and environment factors on expressed functionality, because \textit{Tetrasphaera} has been described to perform this process in mixed communities. The EPS-producing denitrifier (\textit{Azoarcus communis}) facilitated attachment of nitrifier microcolonies and binding to filamentous networks. Quantitatively, multiplex qPCR provided reproducible measurements of community composition in both entrapped and planktonic biomass, supporting detailed monitoring of species dynamics and the effects of operational parameters like shear stress. In parallel, an open-source sequencing batch reactor (SBR) platform was developed for controlled, long-term feast-famine experiments as a stress condition to induce flocculation. Detailed build and operation instructions ensure versatility and transferability. Finally, coculture experiments in the SBR setup showed that the consortium could be reproducibly grown in anaerobic/aerobic/anoxic cycles. With the established model consortium of activated sludge and the developed tools for its characterisation and growth, this work hopes to provide a foundation for mechanistic studies of bioflocculation and interspecies interactions. The open-source reactor platform developed here is also readily transferable to other systems and research questions. Future work should focus on fluorescently labelling additional species (notably the filament) to overcome FISH limitations, further investigating the conditions required for phosphate cycling in \textit{Tetrasphaera japonica}, and addressing oxygen limitation issues caused by biofilm-induced aerator clogging. In-depth characterisation of nitrifier microcolonies is recommended, as well as assessing their survival in microcolony state compared to planktonic precultures. In summary, a five-species consortium was established and monitored in pure and coculture conditions. Nitrifier microcolonies were formed using alginate entrapment, and a stable five-species SBR experiment showed that it was feasible to maintain the model consortium over time. In this way, the thesis contributes to synthetic community research by providing the tools and approaches needed to move towards bottom-up flocculation.