Community succession and functional trait reorganization in a continuous aerobic denitrification reactor under repeated hydraulic retention time reductions.

Abstract

Aerobic denitrification enables nitrogen removal under oxygen-rich conditions, yet its microbial response to hydraulic retention time (HRT) disturbance remains unclear. Here, a continuous aerobic denitrification reactor inoculated with activated sludge enriched in aerobic denitrifiers was operated by stepwise shortening of HRT from 108 h to 60 h. 16S rRNA gene amplicon sequencing and metagenomic sequencing were used to characterize microbial succession, functional adaptation, and co-occurrence network reorganization. Nitrogen removal efficiency remained above 80% under a high influent nitrogen load of 300 mg N/L, comprising about 150 mg/L NH₄^⁺-N and 150 mg/L NO₃^⁻-N, despite marked taxonomic turnover. The alpha diversity of the microbial community decreased from 3.01 ± 0.13 to 2.24 ± 0.38, whereas metagenomic gene diversity remained relatively stable at approximately 13.30 ± 0.12, showing that gene-level diversity did not decline in parallel with microbial diversity. Dominant aerobic denitrifiers shifted from Aquimonas to Pseudofulvimonas. Network analysis showed that Aquimonas contributed more to within-module connectivity, whereas Pseudofulvimonas exhibited stronger among-module connectivity under short HRT. Short HRT enriched genes related to biofilm formation, adhesion, fimbriae, membrane proteins, and pilus assembly. These results indicate that community succession, functional trait reorganization, and redistribution of network roles accompanied stable aerobic denitrification under hydraulic disturbance.