Plant and microbial interactions under different planting patterns regulate nitrogen removal in constructed wetlands

The performance of constructed wetlands in mitigating nitrogen pollution depends on interactions between plants and microorganisms, however, the microbial mechanisms associated with different planting patterns remain insufficiently understood. We established laboratory-scale constructed wetlands with five monocultures and four mixed plantings of aquatic macrophytes and evaluated nitrogen removal, nitrogen mass balance, and microbial community and functional gene dynamics using 16S rRNA sequencing and quantitative PCR. All planted systems achieved higher total nitrogen removal than the unplanted control. Mixed planting produced slightly greater nitrate and COD removal than monocultures, while ammonium removal was similar across treatments. Mass balance analysis showed that sedimentation and adsorption accounted for more than half of total nitrogen removal, microbial transformation contributed about 28 %, and plant uptake increased from 12.67 % to 16.50 % under mixed planting. Microbial community analyses revealed complementary functional roles: mixed planting increased alpha diversity, shifted beta diversity, and enriched ammonia-oxidizing Nitrosomonas and heterotrophic denitrifiers such as Paracoccus. Taxa associated with dissimilatory nitrate reduction to ammonium, including Geobacter and Lacunisphaera, also increased, indicating strengthened nitrogen-recycling. Quantitative PCR showed a functional division of labor, with amoA increasing under mixed planting, nxrA enriched in monocultures, and denitrification genes more abundant in mixed systems. Co-occurrence networks in mixed plantings were more connected and modular, suggesting greater microbial stability. Overall, planting pattern reshaped the contributions of different microbial guilds rather than uniformly enhancing nitrogen removal, providing mechanistic evidence that plant diversity strengthens microbial functional robustness and informing ecological design of constructed wetlands.