Tetrasphaera　has　been　identified　as　the　main　polyphosphate-accumulating　organism　(PAOs)　in　enhanced　biological　phosphate　removal　(EBPR),　but　the　ability　of　Tetrasphaera　to　use　nitrite　as　electron　acceptor　for　denitrifying　phosphorus　removal　(DPR)　is　still　unclear.　This　study　investigated　the　DPR　performance　and　mechanism,　microbial　structures,　and　FNA　inhibition　of　Tetrasphaera-dominated　culture　in　the　long-term　acclimatization　at　different　nitrite　concentrations　(10,　20,　40　and　50　mgN/L).　The　results　showed　that　the　most　optimal　influent　nitrite　concentration　was　20　mgN/L　(C/N　=　6),　and　the　removal　efficiency　of　nitrite　and　phosphorus　reached　above　90　%　and　95　%,　respectively.　Fluorescence　In　Situ　Hybridization-Flow　Cytometry　(FISH-FCM)　showed　that　the　population　abundance　of　Tetrasphaera　was　80.95　%,　in　which　Tet2-892　clade　was　the　most　abundant　and　mainly　responsible　for　DPR　with　nitrite　as　electron　acceptor.　When　the　nitrite　concentration　reached　50　mgN/L　(FNA　=　12.80　µgHNO2-N/L,　pH　=　7.0　±　0.1),　electron　supply　deficiency　and　FNA　inhibition　resulted　in　only　43.0　%　and　68.4　%　of　nitrite　and　phosphorus　removal　efficiency,　respectively.　The　intracellular　amino　acid　and　glycogen　metabolism　were　inhibited　by　FNA　under　this　condition.　A　significant　accumulation　of　N2O　was　observed,　resulting　from　a　combination　of　the　insufficient　electron　supply,　absence　of　nos-Z　gene,　and　long-term　FNA　inhibition.　DPR　dominated　by　Tetrasphaera　with　appropriate　nitrite　as　electron　acceptor　provides　a　new　way　to　achieve　nitrogen　and　phosphorus　simultaneous　removal.　In　addition,　this　study　found　that　Tet2-892　clade　of　Tetrasphaera　was　more　tolerant　to　FNA　compared　to　Accumulibacter　and　GAOs,　which　provides　a　strategy　for　inhibiting　GAOs　activity　to　maintain　the　stability　of　the　EBPR　system.　©　2023　Elsevier　B.V.