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

Intracellular biopolymers serve versatile functions that allow microbes to adapt to fluctuating environmental conditions. The metabolic interdependence of dual intracellular polymers, namely polyphosphate (polyP) and polyhydroxyalkanoates (PHA), is a defining feature of functionally polyphosphate-accumulating organisms (PAOs), the key agents enabling enhanced biological phosphorus removal (EBPR) for wastewater treatment. However, beyond EBPR systems, the presence and identities of PAOs that possess both polyP and PHA in natural environments such as soil have never been examined due to a lack of available detection tools, despite their potential roles in carbon and phosphorus cycling. This study presents a novel phenotype-targeted approach integrating triple-stained fluorescence-activated cell sorting (FACS) with 16S rRNA gene amplicon sequencing (termed TriFlow-Seq) to simultaneously detect, quantify, and phylogenetically characterize PAOs accumulating both polyP and PHA (referred to as PHA- PAOs). TriFlow-Seq was validated using polymer staining image analysis and Single-Cell Raman micro-spectroscopy. Application to EBPR systems successfully enriched known PHA- PAOs, including Candidatus Accumulibacter, Tetrasphaera , Dechloromonas , Pseudomonas. It also revealed novel candidate PHA-PAOs, particularly within the Rhodobacteraceae family. When applied to soil samples, TriFlow-Seq led to the first discovery of diverse PHA-PAOs dominated by Pseudomonas , Halomonas , and Nannocystis in maize rhizosphere soils. These predominant genera are known rhizosphere inhabitants of essential crops with key plant growth- promoting functions including phosphate solubilization, biofilm formation, and phytohormone production, yet simultaneous polyP and PHA accumulation has not been directly reported in the maize rhizosphere. Our findings revealed unexpectedly high PHA-PAO prevalence and distinct phylogenetic patterns associated with different maize genotypes, suggesting a potentially overlooked role for dual polymer storage in microbial rhizosphere dynamics and function. This study establishes a pioneering approach to investigate dual polyP and PHA-containing PAO identities and their important roles in rhizosphere structure and plant health.