Agro-industrial-Produced Laccase for Degradation of Diclofenac and Identification of Transformation Products

A widely used anti-inflammatory drug, diclofenac (DCF), is recalcitrant in many environmental compartments and poses threat to several aquatic and terrestrial organisms. Enzymatic degradation of emerging contaminants which are often micropollutants, has gained interest for the past few years. However, production of enzymes often incurs high costs. In this study, ligninolytic enzyme laccase was produced by white rot fungi Tremetes versicolor (ATCC 20869) using agro-industrial residues, apple pomace (AP), pulp and paper solid waste (PPSW), and alfa fibers as substrates. Various known inducers for laccase production, such as tween 80 (0.1% (w/w)), veratryl alcohol (3 mM Kg^–1), CuSO₄ (3 mM Kg^–1), and phenol red (3 mM Kg^–1) were used to enhance laccase production. A maximum laccase activity of 49.16 ± 4.5, 52.4 ± 2.2, and 14.26 ± 0.8 U/gds (units/gram dry substrate) was obtained from apple pomace, PPSW, and alfa plant fibers, respectively, at optimal experimental conditions. Further, the kinetics of the laccase mediated degradation of DCF was studied. At environmentally relevant concentration of DCF (500 μg L^–1), laccase-catalyzed degradation followed first-order kinetics. At environmentally relevant concentrations pH of 4.5 and temperature of 50 °C was found to be optimal for the effective degradation of DCF with laccase. 3′-Hydroxydiclofenac, 4′-hydroxydiclofenac, and 5-hydroxydiclofenac were identified as the major transformation products during the initial 5 h of degradation. However, after 24 h of degradation, neither DCF nor any transformation products were identified so that the proposed degradation mechanism involved hydroxylation followed by ring opening and final mineralization to CO₂, NH₃, and H₂O and HCl.