A novel single-cell tool for absolute in situ quantification of intracellular poly-P and other storage polymers in wastewater systems key organisms

Enhanced biological phosphorus removal (EBPR) is a process widely applied in wastewater treatment and depends on the activity of specific bacteria, capable of accumulate phosphate as intracellular storage compound. Several microbes are known as potential phosphate-accumulating organisms, but only members of the genera Candidatus Accumulibacter and Tetrasphaera are abundant worldwide in EBPR systems. However, while Ca. Accumulibacter presents the conventional phenotype of poly-P accumulating organisms, more insights are needed on the ecophysiology of the genus Tetrasphaera. This study proposes a novel approach to associate the phylogenetic identity of microbial cells to the in situ and absolute quantification of intracellular storage compounds, using a combination of Fluorescence in situ hybridization (FISH) and Raman microspectroscopy. Analyses of the dynamic of intracellular Poly-P and other storage compounds in Ca. Accumulibacter and Tetrasphaera in activated sludge during feast-famine cycling experiments and in situ, in eight different full-scale EBPR plants, revealed their contribution to the process and showed that Tetrasphaera cells were not consistent with the classical poly-P accumulating microbes physiological model, indicating the necessity for further investigation. Moreover, this novel approach can now be used as a valuable tool in environmental microbiology to verify the presence and quantify other storage compounds in microbial communities of different environments.