Formulation and characterization of an immobilized laccase biocatalyst and its application to eliminate organic micropollutants in wastewater.

Over the past decades, water pollution by trace organic compounds (ng L(-1)) has become one of the key environmental issues for developed countries. To date there is no effective and sustainable remediation strategy available. Laccases from white rot fungi were found particularly attractive for the removal of some micropollutants such as the plasticizer bisphenol A (BPA), the anti-inflammatory drug diclofenac (DF) and the steroidal hormone 17-α-ethinylestradiol (EE2). Laccase immobilization is a prerequisite for their use in continuous water treatment processes. In this study, laccase from Coriolopsis gallica was immobilized on mesoporous silica spheres in a two-step adsorption-crosslinking process. The initial laccase activity, crosslinker (glutaraldehyde) concentration and extra protein (albumin) concentration were varied following a central composite experimental design and optimized with respect to the immobilization yield, activity and thermal stability of the biocatalysts. After a multi-objective optimization of the biocatalyst formulation, a maximum biocatalyst activity of 383 Ug(-1), determined with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) at pH 4.5, was obtained. Biocatalyst particles were physically characterized by means of scanning electron microscopy, Brunauer-Emmett-Teller surface area and Barrett-Joyner-Halenda pore size analyses revealing few modifications of the surface area and structure during/after the immobilization procedure. The biocatalyst showed a significantly higher thermostability than the free enzyme with a half-life of 31.5 hours and 3.9 hours compared to 6.1 hours and 0.6 hours at 55°C and 75°C respectively. The biocatalyst was able to eliminate in a continuously stirred membrane reactor more than 95% of BPA 10 μM and EE2 10 μM and 70% of DF 10 μM when treated individually and more than 90% when treated as a mixture in aqueous buffered solution (pH 5) for more than 60 reactor volumes. In real wastewater conditions (pH 7.8) the biocatalyst could degrade more than 85% of BPA and EE2 along with 30% of DF when tested in mixture for more than 80 hours, which illustrates the potential of this biocatalyst for the treatment of aquatic micropollutants.