Thiosulfate-driven　denitrification　coupled　with　anammox　(TDDA)　has　garnered　interest　for　its　efficient　and　innovative　nitrogen　removal　capabilities.　However,　the　intricate　dynamics　of　the　internal　microbial　community　and　the　specific　characteristics　of　anaerobic　ammonium　oxidizing　bacteria　(AnAOB)　remain　incompletely　understood.　This　study　combines　experimental　methods　with　density　functional　theory　(DFT)　calculations　to　address　these　gaps.　The　TDDA　reactor　was　successfully　started-up　with　an　optimal　S2O32--S/NO3−-N　ratio　of　0.6,　achieving　a　nitrogen　removal　efficiency　of　89.6%.　Throughout　this　process,　the　relative　abundance　of　Candidatus　Kuenenia　decreased　by　10.2%,　while　the　relative　abundance　of　Candidatus　Brocadia　increased　by　9.6%.　The　elevated　concentration　of　NO₃--N　inhibited　Candidatus　Kuenenia,　and　simultaneously　stimulated　the　secretion　of　extracellular　polymers,　affecting　Fe　uptake　by　Candidatus　Kuenenia.　To　further　elucidate　substrate　competition,　molecular　docking　simulations　and　DFT　calculations　were　employed.　The　binding　energy,　compared　with　the　electrostatic　potential　energy　of　the　protein　pocket,　clearly　demonstrated　that　Nir　in　AnAOB　has　a　higher　affinity　for　the　substrate　(EAnAOB　=　−163.2　kJ/mol　vs.　ESOB　=　-77.7　kJ/mol).　By　integrating　molecular　dynamics　insights,　this　study　overcomes　experimental　limitations　and　deepens　the　understanding　of　the　mechanisms　within　the　TDDA　system.　©　2024