Submerged macrophyte promoted nitrogen removal function of biofilms in constructed wetland

Biofilm is one of the important factors affecting nitrogen removal in constructed wetlands (CWs). However, the impact of submerged macrophyte on nitrogen conversion of biofilms on leaf of submerged macrophyte and matrix remains poorly understood. In this study, the CWs with Vallisneria natans and with artificial plant were established to investigate the effects of submerged macrophyte on nitrogen conversion and the composition of nitrogen-converting bacteria in leaf and matrix biofilms under high ammonium nitrogen (NH₄⁺-N) loading. The 16S rRNA sequencing method was employed to explore the changes in bacterial communities in biofilms in CWs. The results showed that average removal rates of total nitrogen and NH₄⁺-N in CW with V. natans reached 71.38% and 82.08%, respectively, representing increases of 24.19% and 28.79% compared with the control with artificial plant. Scanning electron microscope images indicated that high NH₄⁺-N damaged the leaf cells of V. natans, leading to the cellular content release and subsequent increases of aqueous total organic carbon. However, the specific surface area and carrier function of V. natans were unaffected within 25 days. As a natural source of organic matters, submerged macrophyte provided organic matters for bacterial growth in biofilms. Bacterial composition analysis revealed the predominance of phylum Proteobacteria in CW with V. natans. The numbers of nitrifiers and denitrifiers in leaf biofilms reached 1.66 × 10⁵ cells/g and 1.05 × 10⁷ cells/g, as well as 2.79 × 10⁵ cells/g and 7.41 × 10⁷ cells/g in matrix biofilms, respectively. Submerged macrophyte significantly increased the population of nitrogen-converting bacteria and enhanced the expressions of nitrification genes (amoA and hao) and denitrification genes (napA, nirS and nosZ) in both leaf and matrix biofilms. Therefore, our study emphasized the influence of submerged macrophyte on biofilm functions and provided a scientific basis for nitrogen removal of biofilms in CWs.