Polyphosphate　(Poly-P)　is　a　key　compound　in　the　known　metabolism　of　denitrifying　polyphosphate　organisms　(DPAOs).　This　study　investigated　the　efficiency　of　endogenous　denitrification,　metabolic　changes,　and　tolerance　to　free　nitrous　acid　(FNA)　of　DPAOs　at　different　levels　of　Poly-P.　A　high　C/P　ratio　(100:1)　made　DPAOs　transform　from　polyphosphate　accumulating　metabolism　(PAM)　to　glycogen　accumulating　metabolism　(GAM),　and　the　nitrate　(NO3--N)　reduction　rate　of　DPAOs　increased　significantly　in　GAM　mode.　Accumulibacter　IA,　IIC,　and　IID　were　the　main　dominant　DPAOs,　and　their　synergy　and　the　increase　of　PHA　may　be　responsible　for　the　high　NO3--N　reduction　efficiency.　The　DPAOs　under　GAM　mode　had　better　tolerance　to　FNA,　and　N2O　reductase　was　destroyed　due　to　the　FNA　influence,　leading　to　a　large　accumulation　of　N2O.　Flow　cytometry　results　showed　that　FNA　caused　DPAOs　to　over-consume　ATP　and　depolarize,　thereby　inhibiting　their　growth.　And　FNA　disrupted　the　membrane　integrity　and　esterase　activity　of　DPAOs,　which　may　result　in　early　apoptosis.　The　metabolic　transformation　and　FNA　tolerance　of　DPAOs　under　GAM　mode　ensure　the　stable　operation　and　performance　recovery　of　denitrifying　phosphorus　removal　systems,　which　demonstrates　the　DPAOs-dominated　biological　phosphorus　removal　has　wide　application　prospects.　©　2022　Elsevier　B.V.