Associations between rhizosphere microbial community structure and antibiotic attenuation in a pilot-scale hybrid constructed wetland

Constructed wetlands are widely used as nature-based solutions for wastewater treatment; however, the role of rhizosphere-associated microbial communities in the attenuation of emerging contaminants remains unclear. In this study, we evaluated the antibiotic removal and microbial community structure in a pilot-scale hybrid constructed wetland treating municipal wastewater by integrating vertical upflow (Sagittaria montevidensis), floating (Salvinia molesta + Lemna gibba), and horizontal subsurface flow (Canna indica) units. Antibiotics from different therapeutic classes were quantified using LC-MS/MS across the treatment compartments. The system achieved high attenuation efficiencies, with removal efficiencies exceeding 98% for the target compounds. The floating macrophyte unit showed the greatest reduction in concentrations relative to the upstream compartments. Microbial community analyses based on 16S rRNA gene sequencing revealed marked shifts in the community structure along the treatment gradient. Alpha diversity indices varied (Shannon index: 1.066-4.954), with higher diversity observed in rhizospheric communities associated with Sagittaria and Canna than in influent wastewater and pre-exposure samples. Beta diversity analysis (Bray-Curtis dissimilarity) showed a clear separation between the wastewater and rhizospheric communities (PERMANOVA, p = 0.001; R² = 0.66). Redundancy analysis and correlation-based approaches indicated that variations in dominant groups were statistically associated with antibiotic attenuation patterns across treatment compartments. These relationships represent ecological covariation patterns and should not be interpreted as direct evidence of microbial biodegradation activity. Overall, the results indicate that hybrid CW promote structured rhizosphere microbial communities that co-vary with antibiotic attenuation, supporting the future integration of microbial ecology into nature-based wastewater treatment optimization.