In　this　study,　thiosulfate-driven　denitratation　(TDD)　system　was　successfully　established　under　optimal　S/N　molar　ratio　of　1.00,　with　nitrite　accumulation　efficiency　(NAE)　of　82.24　±　17.09%.　This　work　highlighted　that　thiosulfate　significantly　preferred　the　reduction　of　nitrate　than　nitrite.　However,　after　the　depletion　of　thiosulfate,　the　in-situ　formed　intermediate　product　element　sulfur　(S0)　served　as　the　main　electron　donor,　and　significantly　favored　the　reduction　of　nitrite　than　nitrate,　which　constrained　nitrite　accumulation　and　nitrate　removal.　In　addition,　metagenomic　sequencing　revealed　that　the　functional　denitratation　species　might　be　Thiobacillus_sp._65-29,　but　the　occurrence　of　Nir-annotated　species　would　decrease　nitrite　accumulation.　Under　S/N　ratio　of　1.00,　the　decreased　abundant　Nir-annotated　species　(e.g.,　Thiobacillus_sp.),　as　well　as　the　down-regulated　quorum　sensing　interactions　between　Nar-　and　Nir-annotated　species　were　key　microbial　metabolisms　of　high　NAE　in　the　TDD　system.　Overall,　this　work　provides　new　sight　into　the　metagenome-base　functional　species　and　metabolic　potential　of　thiosulfate-driven　denitratation.　©　2021　Elsevier　Ltd