The enrichment of more functional microbes induced by the increasing hydraulic retention time accounts for the increment of autotrophic denitrification performance.

With pyrite (FeS2) and polycaprolactone (PCL) as electron donors, three denitrification systems, namely FeS2-based autotrophic denitrification (PAD) system, PCL-supported heterotrophic denitrification (PHD) system and split-mixotrophic denitrification (PPMD) system, were constructed and operated under varying hydraulic retention times (HRT, 1-48 h). Compared with PAD or PHD, the PPMD system could achieve higher removals of NO3--N and PO43--P, and the effluent SO42- concentration was greatly reduced to 7.28 mg/L. Similarly, the abundance of the dominant genera involved in the PAD (Thiobacillus, Sulfurimonas, and Ferritrophicum, etc.) or PHD (Syntrophomonas, Desulfomicrobium, and Desulfovibrio, etc.) process all increased in the PPMD system. Gene prediction completed by PICRUSt2 showed that the abundance of the functional genes involved in denitrification and sulfur oxidation all increased with the increase of HRT. This also accounted for the increased contribution of autotrophic denitrification to total nitrogen removal in the PPMD system. In addition, the analysis of metabolic pathways disclosed the specific conversion mechanisms of nitrogen and sulfur inside the reactor.