Discovering Polyphosphate and Polyhydroxyalkanoate-Accumulating Organisms Across Ecosystems: Phenotype-targeted Genotyping via FACS-sequencing

Polyphosphate-accumulating organisms (PAOs) play a central role in enhanced biological phosphorus removal (EBPR) systems by coupling intracellular polyphosphate (polyP) cycling with carbon storage through polyhydroxyalkanoates (PHA). However, in natural environments such as soil and sediments, the term PAOs has been loosely applied to polyphosphate-containing organisms (PCOs) without consideration of their carbon storage capacity or phylogeny. This oversimplification overlooks a critical metabolic trait—surplus co-accumulation of polyP and PHA—that defines functionally active PAOs in engineered systems. This study presents a novel phenotype-targeted approach that integrates triple-stained fluorescence-activated cell sorting (FACS) with 16S rRNA gene sequencing, TriFlow-Seq, enabling the simultaneous detection, quantification, and phylogenetic characterization of functional PAOs capable of dual polyP and PHA accumulation. TriFlow-Seq was applied to microbial communities from full-scale and pilot-scale EBPR systems, as well as maize rhizosphere soils across multiple genotypes and fields. In EBPR systems, the method successfully enriched known PAOs, including Candidatus Accumulibacter , Tetrasphaera , Dechloromonas , and Pseudomonas , while also revealing novel candidate PAOs, particularly within Rhodobacteraceae . In agricultural soils, TriFlow-Seq uncovered a previously unrecognized and diverse PAO community, dominated by Pseudomonas , Halomonas , and Nannocystis . Notably, PAO community composition in the maize rhizosphere exhibited significant genotype-specific patterns, indicating plant genetic background influences the recruitment and enrichment of functional PAOs. These findings highlight the importance of exploring alternative PAOs and their specific metabolic preferences across environmental samples. By providing insights into the detection and characterization of bacteria of interest, flow cytometry combined with phylogenetic analysis offers a promising avenue for advancing our understanding of PAOs and their roles in wastewater treatment and environmental processes.