Response of nir-type rhizosphere denitrifier communities to cold stress in constructed wetlands with different water levels

Constructed wetlands (CWs) are used to remove nitrogen-containing organic pollutants based on the roles of plant and microbial communities. However, the rhizosphere nitrogen removal process and microbial response to cold stress in CWs in winter remain unclear. In this study, we investigated the potential denitrification rate and microbial mechanisms of two CWs with different water levels in response to decreasing temperatures in winter. Our study sites were located at Beijing Wildlife Rescue and Rehabilitation Center, the low surface water level site (LCW, 0–10 cm) and Cuihu Wetland Park, the high surface water level site (HCW, 40–50 cm). Denitrifying enzyme activity (DEA) and functional microbial diversity were significantly reduced at LCW, but at HCW, the decreases were not as significant as that in LCW due to the insulating effect of ice. There was no significant difference in the composition of functional microbial communities in the rhizosphere of plants from these sites. We observed changes further in the co-occurrence networks of denitrifiers with decreasing temperature. With increasing cold stress, the co-presence links between the same nir gene (nirS-nirS or nirK-nirK) and the mutual exclusion nirS-nirK links gradually increased, indicating that similar denitrifiers tend to accumulate in response to cold stress. In addition, Bradyrhizobium (nirS/nirK), Azoarcus (nirS), Azospira (nirS), Pseudomonads (nirS) and Rhizobium (nirK) as the keystone species are the primary contributors to the denitrification process in winter. Overall, denitrification was stronger in CWs with high water levels (HCW, 40–50 cm) during the freezing period in winter, and the more similar nirK- or nirS- denitrifiers collaborated to maintain denitrification under cold stress. [Display omitted] •The denitrifier community composition was inhibited by temperature in winter.•The denitrifying enzyme activity was much higher in high water level CWs in winter.•The same nir-type denitrifiers closely interacted with each other under cold stress.•Five species of denitrifiers were considered as keystone taxa in winter.