Biodegradation of B-blocker atenolol was investigated using an enriched nitrifying culture at controlled ammonium concentration and without ammonium addition. Analysis of the kinetics and structural elucidation of biodegradation products showed that atenolol biodegradation was found to be linked to the activity of nitrifying bacteria in the presence of ammonium. Atenolol was degraded cometabolically by ammonia-oxidizing bacteria (AOB), likely due to a broad substrate range of ammonia monooxygenase (AMO). Four products were formed during atenolol biodegradation with ammonia oxidation, including P267 (atenolol acid) and three new products P117 (1-isopropylamino-2-propanol), P167 (1-amino-3-phenoxy-2-propanol), and an unknown product P227 with a nominal molecular mass of 227. In comparison, only P267 and P227 were identified during atenolol biodegradation without ammonia oxidation. Follow-up experiments using atenolol acid as the parent compound indicated the formation of products P117, P167 and P227 in the presence of ammonium. Based on the products identified, a tentative biodegradation pathway of atenolol is suggested, which involves two steps independent of the presence of ammonium: i) microbial amide-bond hydrolysis to carboxyl group and formation of P267 (atetiolol acid) and ii) a possible formation of P227 with its unidentified structure and other two cometabolically induced reactions: iii) breakage of ether bond in the alkyl side chain and formation of P117 and iv) a minor pathway through N-dealkylation and loss of acetamide moiety from the aromatic ring, yielding P167. This study provided an important insight regarding the biotransformation pathways under different metabolic conditions. (C) 2016 Elsevier B.V. All rights reserved.