Phenotypic prevalence index: Diversity of polyphosphate-accumulating organisms (PAOs) and insights in unknown PAOs revealed by combined fluorescence-activated cell sorting (FACS) and 16S rRNA gene sequencing.

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.