Additional　organics　are　generally　supplemented　in　the　sulfide-driven　autotrophic　denitrification　system　to　accelerate　the　denitrification　rate　and　reduce　sulfate　production.　In　this　study,　different　concentrations　of　sodium　acetate　(NaAc)　were　added　to　the　sulfide-driven　autotrophic　denitrification　reactor,　and　the　S0　accumulation　increased　from　7.8%　to　100%　over　a　120-day　operation　period.　Batch　experiments　revealed　a　threefold　increase　in　total　nitrogen　(TN)　removal　rate　at　an　Ac−-C/N　ratio　of　2.8　compared　to　a　ratio　of　0.5.　Addition　of　organic　carbon　accelerated　denitrification　rate　and　nitrite　consumption,　which　shortened　the　emission　time　of　N2O,　but　increased　the　N2O　production　rate.　The　lowest　N2O　emissions　were　achieved　at　the　Ac−-C/N　ratio　of　1.3.　Stable　isotope　fractionation　is　a　powerful　tool　for　evaluating　different　reaction　pathways,　with　the　18ε/15ε　values　in　nitrate　reduction　ranging　from　0.5　to　1.0.　This　study　further　confirmed　that　isotope　fractionation　can　reveal　denitrifying　nutrient　types,　with　the　18ε　(isotopic　enrichment　factor　of　oxygen)/15ε　(isotopic　enrichment　factor　of　nitrogen)　value　approaching　1.0　for　autotrophic　denitrification　and　0.5　for　heterotrophic　denitrification.　Additionally,　the　18ε/15ε　values　can　indicate　changes　in　nitrate　reductase.　There　is　a　positive　correlation　between　the　18ε/15ε　values　and　the　abundance　of　the　functional　gene　napA,　and　a　negative　correlation　with　the　abundance　of　the　gene　narG.　Moreover,　18ε　and　15ε　were　associated　with　changes　in　kinetic　parameters　during　nitrate　reduction.　In　summary,　the　combination　of　functional　gene　analysis　and　isotope　fractionation　effectively　revealed　the　complexities　of　mixotrophic　denitrification　systems,　providing　insights　for　optimizing　denitrification　processes.　©　2024