Response surface methodology (RSM) was applied to optimise laccase production by a new strain of Pycnoporus sanguineus, isolated from the Uruguayan forests, grown on bark shavings under semi-solid-state fermentation conditions. Enhancement of laccase production was studied, with a full 2⁴ factorial design with two replicates and four central points. Operating under the optimal conditions obtained (pH 3.0, yeast extract 10 g L⁻¹, glucose 5 g L⁻¹ and bark shavings 3 g), a laccase activity of nearly 3000 U L⁻¹ was achieved. Subsequently, the effect of different potential inducers on laccase production (Cu⁺², Mn⁺² and lignin) was tested using the one-factor-at-a-time approach. It was found that the addition of 0.5 mM Cu⁺² into the cultures considerably increased laccase production and, thus, maximum activity values of about 16 000 U L⁻¹ were achieved. Further, the optimised culture broth (containing mainly laccase) showed high capacity to decolourise different recalcitrant synthetic dyes such as Acid Red 88 (AR88), Reactive Black 5 (RB5) and Lanaset Grey G (LG). After selecting enzyme concentration and time of reaction, almost 70% of AR88 could be decolourised in the absence of redox mediators. However, mediators were necessary to bleach RB5 and LG. Methyl syringate was the most effective mediator for AR88 (reaching a decolouration degree over 90%) and RB5 (nearly 70%). Around 80% of LG could be decolourised in the presence of violuric acid. These promising results make this enzyme a very useful tool for developing wastewater treatments.

Synthetic dyes in watercourses resulting from industrial effluent discharges cause serious ecological impacts, besides carcinogenic and mutagenic effects on human health. Thus, it is important to develop effective methods to remove the dyes from industrial wastewaters, and also to carry out adequate toxicity studies to establish their safety. Azo dyes are the main class of industrial dyes and important environmental contaminants. We have examined the decolorization of two azo dyes (Acid Red 88 and Acid Black 172) by a native Trametes villosa laccase immobilized on thiolsulfinate-agarose as well as the effect of different redox mediators in the reactions. The method was effective for the decolorization of both dyes. The immobilization method did not affect the capacity of the biocatalyst for dye degradation. Therefore, the insoluble enzyme removed 97% of the color of AR88 and 92% of AB172 in 24 hours at 22 °C in the presence of the selected redox mediators, vanillin (0.1 mM) and violuric acid (1.0 mM), respectively. In addition, the immobilized enzyme kept 78% of its initial capacity for decolorization of AR88 after three cycles of use. The ecotoxicological evaluation of the solutions showed a great variation depending on the biological systems used. In the phytotoxicity test, the decolorization products were not toxic to plants, whereas in Daphnia magna and Microtox® bioassays an acute residual toxicity was found. The last outcome shows the importance of using a battery of bioassays to determine the remaining ecotoxicity of the treated effluents before their discharge into the aquatic environment.

Laccase from Dichostereum sordulentum was entrapped in hydrogels formed using lignocellulosic residues from bioethanol production and ionic liquids. The active hydrogel was efficient in ethinylestradiol removal.