Effects of ecological water replenishment on microbial denitrification in aquatic environment of infiltration area

Ecological water replenishment (EWR) is a crucial strategy for solving regional water shortages, yet its ecological impacts warrant further exploration. This study investigated the microbial community succession mechanism in surface water (SW) and groundwater (GW), the denitrification potential of functional bacteria, and their responses to water replenishment events of the South-to-North Water Diversion (SNWD), the largest water transfer project in China. Nitrogen and antibiotic contamination, resulting from prolonged infiltration of reclaimed water, decreased following water replenishment event. During water replenishment, both SW and GW showed an increase of microbial denitrification capacities. Post-replenishment, SW microbial denitrification capacity continued to rise, while GW returned to the baseline levels. Total organic carbon (TOC) and antibiotic resistance genes (ARGs) were the primary factors influenced denitrification before and after water replenishment. Among the denitrification steps, NO₂^--N reduction was most affected, linked to microbial community reassembly and resource utilization alteration after water replenishment. Furthermore, random forest analysis identified potential bacterial indicators and combinations sensitive to water replenishments highlighting key denitrification functional bacteria. These findings offer critical insights for optimizing water resource management and improving EWR effectiveness.