ABSTRACT: The role of hydraulic retention time (HRT) on S0 production was assessed through metagenomics analyses. Considering comprehensive performance for the tested HRTs (0.25–13.33 h), the optimal HRT was 1 h, while respective sulfide and nitrite loading rate could reach 6.84 kg S/(m3 ⋅d) and 1.95 kg N/(m3 ⋅d), and total S0 yield was 0.36 kg S/(kg (VSS)⋅d). Bacterial community richness decreased along the shortening of HRT. Microbacterium, Sulfurimonas, Sulfurovum, Paracoccus and Thauera were highly abundant bacteria. During sulfur metabolism, high expression of sqr gene was the main reason of maintaining high desulfurization load, while lacking soxB caused the continuous increase of S0 . Regarding nitrogen metabolism, the rapid decrease of nitrite transporter prevented nitrite to enter in cells, which caused a rapid decrease of nitrite removal under extreme HRT. Adjusting HRT is an effective way to enhance S0 production for the application of the simultaneous sulfide and nitrite removal process.