Toward better understanding of denitrifying phosphorus accumulating organisms (DPAOs) via Single-cell Raman spectroscopy: A review

Denitrifying phosphate-accumulating organisms (DPAOs) hold considerable promise for simultaneous nitrogen and phosphorus removal in wastewater treatment. However, challenges in detecting their in-situ activity and cultivating uncultured DPAOs from natural environments have limited strain identification. The traditional ‘culture-first’ approach has largely hindered the discovery of core efficient DPAO strains, and their stability and adaptability in real wastewater systems remain poorly understood. Additionally, population-level mechanistic studies often neglect cellular heterogeneity, resulting in an incomplete understanding of nitrogen and phosphorus removal mechanisms. Single-cell Raman spectroscopy (SCRS) emerges as a promising, non-destructive technique that allows for rapid, precise identification of target microorganisms, offering valuable insight into their ecological niches and metabolic processes. By combining SCRS with metabolic phenotyping, cultivation, and genomics, a ‘screen-first’ approach can be established to assess and identify DPAO strains directly in situ . This review highlights the current state of DPAO strain resources, their metabolic pathways, and the principles and applications of single-cell technologies, with a focus on how SCRS can reveal DPAO functions and accelerate strain discovery and resource exploration. • SCRS offers substantial potential for the precise identification and cultivation of in situ DPAOs. • SCRS enables in-depth exploration of metabolic diversity and functional heterogeneity in situ DPAOs at the single-cell level. • SCRS can serve as an indicator for characterizing EBPR phosphorus removal performance.