Threshold-dependent sulfamethoxazole biodegradation in activated sludge: linking extracellular interfacial reorganization to intracellular metabolic activation.

Abstract

Antibiotic removal in activated sludge is highly variable, yet the mechanisms governing activation of effective biodegradation remain poorly understood. Here, we combined interpretable machine learning trained on 176 literature-derived cases with controlled microcosm experiments, extracellular polymeric substance characterization, metagenomics, and transformation-product profiling to investigate threshold behavior in sulfamethoxazole (SMX) biodegradation. A threshold-dependent transition from persistent discharge to effective biodegradation emerged at an influent SMX concentration of approximately 0.2 mg/L, with sufficient acclimation (∼15 d) required for degradative capacity to develop, whereas removal remained limited below this concentration. Microcosm experiments showed that SMX removal remained below 20% at 0.2 mg/L throughout acclimation, whereas removal at 2.5 mg/L increased rapidly after day 15 and reached 99.70% by day 18. Allylthiourea inhibition further showed that SMX removal at 2.5 mg/L decreased from 97.38% to 19.35%, accompanied by a 93% reduction in the apparent degradation rate constant, indicating a major contribution from nitrifier-linked co-metabolism under threshold-exceeding conditions. Threshold exceedance was further associated with extracellular interfacial reorganization, enrichment of oxidative metabolic functions and SMX-relevant catabolic potential, and broader shifts in transformation-product profiles. These findings reveal that effective SMX biodegradation is governed by an activation-limited response, providing a mechanistic explanation for the persistent discharge of antibiotics under low-exposure conditions and offering a new conceptual framework for understanding and improving micropollutant attenuation in biological wastewater treatment.