Large amounts of phosphorus (P) are present in activated sludge from municipal wastewater treatment plants, where it exists in the form of metal salt precipitates or biologically bound into the biomass as nucleic acids, cell membrane components, and the extracellular polymeric substances or, in special polyphosphate-accumulating organisms (PAOs), as intracellular polyphosphate. Only recently, methods that reliably allow an absolute quantification of the different P-fractions, such as sequential extraction, Raman microspectroscopy, solid-state 31P magic angle spinning (MAS) NMR, and solution state 31P NMR have been developed. This study combines these techniques to obtain a comprehensive P mass-balance of activated sludge from four wastewater treatment plants with enhanced biological phosphate removal (EBPR). The total content of P and various cations was measured by chemical analysis (ICP-OES), and different P fractions were extracted for chemical characterization. Chemically bound P constituted 38-69% of total P, most likely in the form of Fe, Mg, or Al minerals, while organically bound P constituted 7-9%. By using Raman microspectroscopy and solution state 31P NMR and 31P MAS NMR spectroscopy before and after anaerobic P-release experiments, poly-P was quantified and constituted 22-54% of total P in the activated sludges and was found in approx. 25% of all bacterial cells. Moreover, Raman microspectroscopy in combination with fluorescence in situ hybridization (FISH) was used to quantify the species-specific intracellular poly-P of known PAO genera (Tetrasphaera, Ca. Accumulibacter, Dechloromonas) and other microorganisms known to possess high level of poly-P, such as the filamentous Ca. Microthrix. They were all abundant, as measured by quantitative-FISH and amplicon sequencing, and accumulated large amount of poly-P, depending on their cell-size, contributing substantially to the P-removal. Interestingly, in all four EBPR plants investigated, only 1-13% of total poly-P was stored by unidentified PAO, highlighting that most PAOs in the full-scale EBPR plants investigated are now known. HighlightsO_LIExhaustive P mass-balance of main organic and inorganic P-species in four EBPR plants C_LIO_LIQuantification of poly-P of FISH-defined PAO and other species with high P content C_LIO_LITotal P content was 36-50 mgP/gSS of which 31-62% was in biomass and as poly-P C_LIO_LIA high fraction of all cells (25-30%) contained a high content of poly-P C_LIO_LIKnown PAOs contained almost all poly-P in the EBPR plants investigated C_LI

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