This　study　evaluated　the　performance　of　thiosulfate-driven　denitratation　(TDD)　systems.　The　results　demonstrated　that　a　cycle　time　of　4.00　h　was　adequate　for　NO3--N　removal　and　NO2--N　accumulation　(average　NO3--N　removal　efficiencies　(NREs):　95%;　average　NO3--N　accumulation　efficiencies　(NAEs):　79%)　under　pH　range　of　6.50–9.50.　According　to　metagenomic　research,　the　enrichment　of　dominant　denitrifiers　is　essential　for　the　pH　responsiveness　of　the　TDD　system.　Furthermore,　interspecific　microorganism　cooperation　and　the　potential　selective　advantage　of　NO3--N　reducing　bacteria　over　NO2--N　reducing　bacteria　contributed　to　a　higher　NO2--N　accumulation　efficiency.　In　addition,　the　results　revealed　that　the　reduction　in　hydraulic　loading　time　(HRT)　improved　metabolic　activities　(i.e.,　energy　metabolism).　Overall,　this　study　broadened　the　application　potential　of　the　TDD　system　and　enhanced　the　understanding　of　microbial　niches　and　the　metabolic　potential　of　autotrophic　denitrification　from　electron　transfer　and　metagenomic　insights.　©　2022　The　Institution　of　Chemical　Engineers