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Wastewater deriving from cellulose and paper manufacturing is one of the most important industrial effluents due to its large-volume production and high pollution load. Effluent deriving from the pulp bleaching stage (Kraft E1 effluent) remains one of the major issues faced by paper mills among all wastewaters generated in each stage of paper-making processes. Kraft E1 effluent was submitted to a sequential chemical (ozonization) – biological (immobilized laccase) treatment. Laccase was obtained from  Trametes versicolour  in liquid medium of culture using 2,5 -xylidine as inducer. Crude laccase extract was immobilized through covalent binding in Montmorillonite KSF and Eupergit ® C supports based on different protocols. Eupergit ® C has shown the best protein immobilization (51%), retention activity (100%), and operational stability (ten oxidative cycles) results. Enzymatic treatments using free and immobilized laccase onto Eupergit ® C were applied to Kraft E1 effluent. After 18-h treatment, total phenol removal reached 20% and 40% in free and immobilized laccase, respectively. Ozone combined to enzymatic processes using reactor assembled with immobilized laccase (31 U g −1 , total mass = 10.0 g) had effect on decolonization efficiency and on total phenols’ removal from Kraft effluent. Ozone treatment was capable of removing 52% of total phenols and 76% of colour from the investigated effluent. Sequential enzymatic treatment has increased total phenols’ removal to 64% within 30-minute treatment and reached 70% removal within 60 minutes. The herein observed additional phenol removal based on enzymatic treatment is an important outcome if one takes into consideration the fraction of total phenols that could not be removed by the ozone process.

Trametes versicolour laccase immobilization by covalent binding and its application in Kraft E 1 effluent pre-treated with ozone