To address the current bottlenecks of lagging microscopic metabolic monitoring and difficulties in isolating key functional strains of the enhanced biological phosphorus removal (EBPR) process, we developed an integrated “in situ diagnosis-targeted sorting” strategy based on single-cell Raman spectroscopy (SCRS) and microfluidic cell sorting technologies. In situ spectral acquisition and intelligent threshold scanning analysis were performed on sludge from five typical steady-state aerobic batches, establishing an ecological response model linking the phosphorus metabolism phenotype with the environmental phosphorus load. Subsequently, based on the biological thresholds determined by the model, targeted sorting and 16S rRNA gene identification were conducted on the enriched sludge. The results revealed significant single-cell metabolic heterogeneity within the in situ community of polyphosphate-accumulating organisms (PAOs). By defining a threshold for the Raman peak intensity ratio of polyphosphate (Poly-P) to phenylalanine (Ratio), high-activity functional subpopulations could be precisely distinguished. Both the relative abundance of this subpopulation and its single-cell metabolic intensity showed a significant positive correlation with the real-time soluble orthophosphate (SOP) concentration in the aerobic tank, confirming a dual ecological response mechanism of “breadth mobilization” (abundance regulation) and “depth intensification” (activity regulation) adopted by PAOs to adapt to environmental substrate fluctuations. With the threshold Ratio >2.6, 15 pure functional strains exhibiting high polyphosphate accumulation activity were successfully isolated. Identification showed they encompassed a denitrifying PAO (Microvirgula aerodenitrificans), a dominant facultative PAO (Pseudomonas protegens), and classic PAOs (Acinetobacter spp.). The study achieves a microscopically quantifiable diagnosis of the wastewater phosphorus metabolic status and provides a precise technical pathway for the targeted exploration of uncultured functional microbial resources. 

single-cell Raman spectroscopy, high-throughput Raman, polyphosphate-accumulating organisms (PAOs), in situ phenotypic diagnosis, substrate-induced mechanisms, single-cell sorting and cultivation