Vegetation-Associated Enhancement of Azo Dye Removal in Constructed Wetlands Without External Carbon Addition

Constructed wetlands (CWs) are a low-energy alternative for treating dye-containing wastewater; however, the mechanisms enabling azo dye removal without external carbon supplementation remain unclear. This study demonstrates that azo dye reduction can proceed under oxic bulk conditions in CWs through vegetation-induced microscale redox heterogeneity. Lab-scale CWs planted with cattail and papyrus were evaluated for the removal of Reactive Orange 16 (RO16, monoazo) and Reactive Black 5 (RB5, diazo) at influent concentrations of 10–50 mg/L under varying ambient temperature (2–36 °C) and hydraulic retention time (1–15 days). Vegetated CWs consistently outperformed the unplanted system, achieving 60–95% removal for RO16 and up to 98% removal for RB5, whereas the unplanted CW showed substantially inferior performance, with removal efficiencies below 54% for RO16 and below 37% for RB5. Dye-decolorizing bacteria, including Priestia megaterium and Clostridium spp., were isolated exclusively under anaerobic conditions from vegetated CWs despite oxic bulk dissolved oxygen levels. The isolates did not decolorize dyes under aerobic conditions or when dyes were provided as sole carbon sources, indicating that azo dyes functioned as electron acceptors and required additional electron donors. These results suggest that vegetation promotes localized reductive microenvironments and supplies endogenous organic carbon, enabling anaerobic azo bond reduction within otherwise oxic systems. The findings indicate a mechanistic basis for plant–microbe interactions in CWs and support the design of sustainable treatment systems for dye-containing wastewater without external carbon input, particularly in warm regions. This study resolves a long-standing question of how azo dye reduction proceeds in CWs without external carbon input.