Development and optimization of a silica-bound laccase biocatalyst and its application in hospital wastewater...

Laccase from Trametes hirsuta was immobilized on amino-functionalized silica by adsorption and covalent binding using glutaraldehyde (GLA) and glyoxal (GLX) as cross-linkers. The immobilization process was optimized applying the Box-Behnken methodology. The immobilized biocatalysts were characterized using scanning electron microscopy, Brunauer-Emmett-Teller surface area and Barrett-Joyner-Halenda pore size analyses, and elemental analysis. Covalent immobilization proved to be better than adsorption based on the specific activity, immobilization yield, and stability in hospital wastewater (HWW) of the biocatalysts. The biocatalyst prepared using GLA under optimized conditions (laccase loading: 10 kU/L, pH 5, temperature: 5 °C, immobilization time: 8 h, and GLA amount: 100 mM) demonstrated better stability to pH, temperature, and other denaturants, compared to free laccase. It exhibited good catalytic potential to remove phenolic compound acetaminophen (83%) and other trace organic contaminants (TrOCs) such as mefenamic acid (86%), indomethacin (73%), carbamazepine (62%), ibuprofen (43%), naproxen (37%), and ketoprofen (27%), in a mixture from a real non-treated hospital effluent spiked with 1 µg/L of each of the above compounds. In addition, the measured catalytic parameters (K_m, k_cat and k_cat/K_m) of acetaminophen (free laccase vs. immobilized laccase (AFHMS-GLA-Lac)) are relatively similar. This is one of the first evaluative studies on different immobilization strategies using the Box-Behnken optimization method to develop efficient and stable laccase biocatalysts by immobilization on amino-functionalized silica for real hospital wastewater treatment.