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

针对强化生物除磷(EBPR)活性污泥处理体系中原位代谢状态微观监测滞后及关键功能菌株分离困难的瓶颈问题, 构建融合单细胞拉曼光谱(SCRS)与微流控细胞分选技术的“原位诊断–靶向分选”一体化策略。通过对5个典型稳态好氧微生境进行原位光谱采集与智能阈值扫描分析, 建立磷代谢表型与环境磷负荷的生态响应模型。基于模型确定的生物学阈值, 对富集培养后的污泥实施靶向分选与16S rRNA基因鉴定。结果表明, 原位聚磷菌(polyphosphate-accumulating organisms, PAOs)群落表现出显著的单细胞水平代 谢异质性。通过确定拉曼多聚磷酸盐(Poly-P)与苯丙氨酸峰强度比值(Ratio)的阈值, 可以精准地区分高活性聚磷菌亚群。该亚群的相对丰度及单细胞代谢强度均与环境实时溶解性正磷酸盐(SOP)浓度显著正相关, 证实了聚磷菌群落为适应环境底物波动而采取的数量调控与活性调控双重生态响应机制。以 Ratio>2.6 为筛选阈值, 通过分选获得15株具备高聚磷活性的纯培养聚磷菌, 经鉴定, 涵盖反硝化聚磷菌(Microvirgula aerodenitrificans)、兼性聚磷菌(Pseudomonas protegens)及经典聚磷菌(Acinetobacter spp.)。研究结果实现污水脱磷体系代谢状态的微观定量诊断, 并为难培养功能微生物资源的定向获取提供了精准途径。