EPS restructuring-linked fate of ceftizoxime in nitrifying sludge: degradation pathways, microbial adaptation and...

A systematic understanding of how antibiotics affect nitrification systems-linking removal pathways, toxicity of degradation products, and microbial community shifts-remains limited. Here, four lab-scale sequencing batch reactors were run 175 d to treat domestic wastewater spiked with the third-generation cephalosporin ceftizoxime (CEX) at 5, 10, 15, and 20 mg/L. The nitrifying sludge exhibited functional recovery, with ammonium removal efficiencies stabilizing at 84.1 %, 72.4 %, 45.5 %, and 39.4 %, respectively. In parallel, CEX removal efficiencies declined from 97.2 % to 43.7 % with increasing influent concentration, remaining positively correlated with nitrification performance, and at ≤10 mg/L CEX was completely transformed into non- or low-toxicity products. CEX stress affected extracellular polymeric substances (EPS): total EPS increased and composition shifted toward protein dominance, with the PN/PS ratio rising from 1.03 to 2.13. Phase-resolved measurements showed distinct partitioning of CEX among the aqueous phase, EPS fractions (S-EPS/LB-EPS/TB-EPS), and the intracellular phase, and the EPS-layer-specific LC-MS/MS fingerprints support an EPS-layer-resolved progressive transformation interpretation. Microbial community analysis showed marked declines in ammonia-oxidizing bacteria (e.g., Nitrosomonas 8.8 %→1.4 %) and nitrite-oxidizing bacteria (Nitrospira 3.2 %→0.1 %), accompanied by the enrichment of several heterotrophic genera associated with CEX-stressed conditions, such as Ferruginibacter (3.7 %→22.3 %), Aquabacterium, and Aquicella. LC-MS/MS identified three CEX degradation pathways initiated by β-lactam ring hydrolysis followed by decarboxylation and C-C bond cleavage, yielding lower-molecular-weight products, while E. coli bioassays confirmed effective detoxification at moderate CEX levels. Overall, these results clarify the coupled roles of EPS restructuring, microbial community adaptation, and biodegradation in mitigating the risks of third-generation cephalosporins in nitrification-based wastewater treatment.