He Peisheng, Yan Yuan, Lee Jangho, Han I L, Li Guangyu, Sabba Fabrizio, Jia Zhen, Wells George F, Gu April Z
School of Civil and Environmental Engineering, Cornell University, Ithaca, NY, United States.
Department of Civil and Environmental Engineering, Northwestern University, Evanston, IL, United States.
Water Res. 2025 Sep 1;283:123897. doi: 10.1016/j.watres.2025.123897. Epub 2025 May 24.
Despite the recognized crucial role of polyphosphate-accumulating organisms (PAOs) in Enhanced Biological Phosphorus Removal (EBPR) process for phosphorus removal and recovery in wastewater treatment, knowledge gaps remain in the diversity and elasticity of their identity, function, and metabolism. Traditional studies have focused on a few canonical PAOs, such as Candidatus Accumulibacter and Tetrasphaera, but the heterogeneity and variation of their phenotypic traits were rarely touched upon, due to a lack of effective tools. In this study, we proposed a novel concept of the phenotypic prevalence index (PPI), derived from the combination of phenotypic traits-targeted fluorescence-activated cell sorting (FACS) and 16S rRNA gene sequencing, to quantify the prevalence of specific phenotypic traits across identified microbial taxa, as case study of uncovering the phenotypic diversity and heterogeneity within PAOs across six EBPR systems. The results revealed distinct and unexpected phylogenetic composition and enrichment patterns of candidate FACS-sorted PAOs across EBPR samples and, particularly, the new PPI assessment of known PAOs elucidated high phenotypic heterogeneity in these PAO taxa. Surprisingly, the PPI values of Candidatus Accumulibacter were significantly lower than Tetrasphaera and other candidate PAOs, such as Propionivibrio, Paracoccus, Thiothrix, and Dechloromonas, suggesting a possibly less dominant role of Accumulibacter than previously assumed, while highlighting the ecological importance of other PAOs taxa that calls for further investigation. The newly proposed PPI enabled us to elucidate and quantify the intricate phenotypic heterogeneity within any PAOs taxa of interest beyond the phylogenetic variation alone and can be applied in different ecosystems.
尽管聚磷菌（PAOs）在强化生物除磷（EBPR）过程中对废水处理中的磷去除和回收具有公认的关键作用，但在其身份、功能和代谢的多样性及弹性方面仍存在知识空白。传统研究主要集中在少数典型的聚磷菌上，如“聚磷菌属候选菌”（Candidatus Accumulibacter）和四球藻属（Tetrasphaera），但由于缺乏有效的工具，很少涉及它们表型特征的异质性和变异性。在本研究中，我们提出了一个新的表型流行指数（PPI）概念，它源自针对表型特征的荧光激活细胞分选（FACS）和16S rRNA基因测序的结合，用于量化特定表型特征在已鉴定微生物分类群中的流行程度，作为揭示六个EBPR系统中聚磷菌表型多样性和异质性的案例研究。结果揭示了候选FACS分选的聚磷菌在EBPR样本中的独特且出乎意料的系统发育组成和富集模式，特别是对已知聚磷菌的新PPI评估揭示了这些聚磷菌类群中的高表型异质性。令人惊讶的是，“聚磷菌属候选菌”的PPI值显著低于四球藻属以及其他候选聚磷菌，如丙酸弧菌属（Propionivibrio）、副球菌属（Paracoccus）、硫丝菌属（Thiothrix）和脱氯单胞菌属（Dechloromonas），这表明聚磷菌属候选菌的主导作用可能比之前假设的要小，同时凸显了其他聚磷菌类群的生态重要性，需要进一步研究。新提出的PPI使我们能够阐明并量化任何感兴趣的聚磷菌类群中除系统发育变异之外的复杂表型异质性，并且可以应用于不同的生态系统。
