One of the aims of the treatment of domestic and industrial wastewater is the removal of phosphorus prior to discharge into the environment. Since phosphorus concentrations in wastewater exceed the requirement of bacterial growth, biological phosphorus removal is based on the ability of a group of microorganisms, named “Polyphosphate Accumulating Organisms” (PAO), to form large quantities of polyphosphate in their cells. In this study, we are focussing on the bacteria actively involved in the phosphorus removal in a lab scale bioreactor operated with aerobic granular sludge technology. This process based on dense microbial biofilms is a cost-effective and land-saving alternative to the conventional wastewater treatment with activated sludge. The identification of the microorganisms belonging to PAO relies on their distinct phenotype assessed with fluorescent probes targeting the intracellular polyphosphate structures. Sequencing of the 16S rRNA gene amplicons of PAO selected with flow-cytometry will be used to reveal the phylogenetic affiliation of the different actors involved in the phosphorus removal. This is of particular significance since low abundant microorganisms may play an important role in the phosphorus removal performance of aerobic granular sludge. We hypothesize that the maintenance of different populations involved in the same functional process is explained by multiple ecological niches present in such biofilm structures.