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Immobilization of enzymes on the solid supports can improve the stability as well as catalytic properties of enzymes. In this study, biochar derived from various feedstocks were used as immobilization support considering biochars carbon negative as well as sustainable properties. Partially purified (concentrated) crude laccase was covalently immobilized onto pine wood (BC-PW), pig manure (BC-PM) and almond shell (BC-AS) micro-biochars using optimized 5% w/v glutaraldehyde. Moreover, citric acid pretreatment improved the laccase binding capacity of all the micro-biochars and the highest laccase binding of 40.2 ± 1.8 U g −1 was observed with BC-PM in comparison with raw BC-PM (34.1 ± 1.1 U g −1 ). The enhanced binding of laccase on BC-PM over wood derived biochars was attributed to the higher surface area (46.1 m 2  g −1 ) of BC-PM. Moreover, feedstock selection as well as a method of production influenced the biochar physicochemical properties such as surface area and consequently, different biochars showed various laccase immobilization efficiencies. BC-PW showed better pH, thermal, storage, and operational stability, compared with BC-AS and BC-PM. While applying the laccase bound micro-biochar, complete removal was observed in 2 h under batch mode with 0.5 g of laccase bound BC-PM at an environmentally relevant concentration of 500 µg L −1 in wastewater effluent. About 40% of the laccase activity was retained with all the laccase-bound micro-biochars after 5 cycles of diclofenac treatment. [ABSTRACT FROM AUTHOR]

Although enzymes are gifted with unique and unprecedented catalytic activity and selectivity over a wide range of pollutants, still their stability related issues often hinder their application in real environmental conditions. In this study, agro-industrially produced crude laccase was concentrated using ultrafiltration. Crude laccase was immobilized on pine wood (BC-PW), pig manure (BC-PM) and almond shell (BC-AS) biochar microparticles. Immobilization of laccase was investigated at various laccase activities on micro-biochars and the release (desorption) of the enzyme has been studied. It was observed that for all the biochars, as the initial concentration of laccase increased in the crude solution, the binding capacity and as result immobilization efficiency also increased. BC-PM was found to be the most effective (31.4 ± 3.1 U g −1 ) at 10 U mL −1 of enzyme activity followed by BC-AS (24.3 ± 4.8 U g −1 ) and BC-PW (14.58 ± 3.3 U g −1 ). In addition, the biochars were functionalized with citric acid for possible surface modifications and the effect of biochars for the adsorption of enzymes has been investigated. Isotherm studies of enzyme loading onto biochar established homogeneous monolayer adsorption as the major mechanism. The desorption of laccase from all biochars followed pseudo-second-order model. Immobilized laccase exhibited superior storage ability and shelf-life which were three times higher than free laccase. Finally, the immobilized laccase was used for the degradation of micropollutant, DCF and near 100% removal was obtained within 5 h at an environmentally relevant concentration (500 μg L −1 ). [ABSTRACT FROM AUTHOR]