Multiple roles of Ca2+ in the interaction of ciprofloxacin with activated sludge: Spectroscopic investigations of extracellular polymeric substances

Highlights: • Ca²⁺ markedly inhibited binding of ciprofloxacin to sludge. • Proteins in EPS are critical in providing binding sites for ciprofloxacin and Ca²⁺. • Ca²⁺ acted mainly through complexation and cation competition. • Ternary complex formed among Ca²⁺, ciprofloxacin, and C=O in carboxyl group and O–H • C=O in amide I is key group for Ca²⁺ competition inducing ciprofloxacin release. Calcium ion is an important cation influencing the binding of recalcitrant organic contaminants with activated sludge during wastewater treatment process, but there is still unknown about its role in amphoteric fluoroquinolones binding. Binding experiments show that Ca²⁺ markedly inhibited binding of ciprofloxacin (CIP) onto sludge, causing 7–203 times of CIP release. Multi-spectroscopic examinations indicate that tryptophan-like and tyrosine-like proteins in extracellular polymeric substances (EPS) were dominant components for CIP binding by static quenching and forming CIP-proteins complexes. Addition of Ca²⁺ into EPS and CIP binding systems induced increase of association constants (from 0.024–0.064 to 0.027–0.084 L/μmol) and binding constants (from 0.002–0.039 to 0.012–0.107) and decrease of binding sites number (from 0.893–2.007 to 0.721–1.386). Functional groups of EPS and secondary structure of proteins were remarkably changed upon reactions with CIP and Ca²⁺. Calcium ion interacted with EPS and CIP binding system in two distinct ways: Ca²⁺ shielded CO in amide I in EPS for CIP binding, whereas strengthened binding between CIP and functional groups including CO in carboxyl groups in extra-microcolony polymers and OH in extra-cellular polymers by forming ternary complexes. Cation competition for CO in amide I is responsible for Ca²⁺ induced CIP release from the sludge. Results suggest the highly potential release of CIP from high saline wastewater and cation-conditioned sludge which needs further monitoring and evaluation.