Discovering Polyphosphate and Polyhydroxyalkanoate-Accumulating Organisms Across Ecosystems: Phenotype-Targeted Genotyping via FACS-Sequencing

Intracellular biopolymers serve versatile functions, allowing microbes to adapt to fluctuating environmental conditions. The metabolic interdependence of dual biopolymers polyphosphate (polyP) and polyhydroxyalkanoates (PHA) is a defining feature of polyphosphate-accumulating organisms (PAOs), the key agents enabling enhanced biological phosphorus removal (EBPR). Beyond EBPR, PAOs accumulating both polyP and PHA (PHA–PAOs) in natural environments remain unexplored due to limited detection tools, despite hypothesized roles in nutrient cycling. This study presents a novel phenotype-targeted approach integrating triple-stained fluorescence-activated cell sorting with 16S rRNA gene sequencing (TriFlow-Seq) to detect, quantify, and phylogenetically characterize PHA–PAOs, validated by fluorescence imaging and single-cell Raman spectroscopy. TriFlow-Seq of EBPR samples confirmed known PAOs, including Candidatus Phosphoribacter, Tetrasphaera, and Ca. Accumulibacter, and revealed new candidate PHA–PAOs within the Rhodobacteraceae family. In maize rhizosphere, TriFlow-Seq led to the discovery of diverse PHA–PAOs, Pseudomonas, Halomonas, and Nannocystis . These genera participate in plant-growth-promoting functions including phosphate solubilization and phytohormone production, yet their simultaneous polyP and PHA accumulation has never been reported. Our findings revealed high PHA–PAO prevalence and distinct phylogenetic patterns associated with different maize genotypes, suggesting an overlooked role of biopolymer costorage in rhizosphere dynamics. This study establishes a pioneering approach to investigating PHA–PAO identities and roles across ecosystems.