This work investigated fungal co-culture as inducer of ligninolytic enzymes and decolourising activity in the Colombian strain Leptosphaerulina sp., an ascomycete white-rot fungus isolated from lignocellulosic material. Aspergillus niger, Aspergillus fumigatus, Aspergillus terreus, Trichoderma viride, Fusarium sp. and Penicillium chrysogenum were tested as Leptosphaerulina sp. inducers. The best fungal combinations in terms of enzyme production, fungal growth and decolourising activity were selected from solid media experiments. Response surface methodology (RSM) was utilised to optimise enzyme production and decolourising activity in liquid media. Solid media assays evidenced T. viride and A. terreus as the best Leptosphaerulina sp. inducers. The RSM identified a triple co-culture inoculated with T. viride (1000 μL) and A. terreus (1000 μL) into a 7-day culture of Leptosphaerulina sp. as the best treatment. This triple combination significantly improved ligninolytic enzymes production and Reactive Black 5 dye removal when compared to the Leptosphaerulina sp. monoculture and previously used chemical inducers. These results demonstrated the potential of fungal co-culture as an environmentally-friendly method to enhance Leptosphaerulina sp. enzymes production and decolourising activity.

In this work, Leptosphaerulina sp. (a Colombian native fungus) significantly removed three Isoxazolyl-Penicillin antibiotics (IP): oxacillin (OXA, 16000 μg L), cloxacillin (CLX, 17500 μg L) and dicloxacillin (DCX, 19000 μg L) from water. The biological treatment was performed at pH 5.6, 28 °C, and 160 rpm for 15 days. The biotransformation process and lack of toxicity of the final solutions (antibacterial activity (AA) and cytotoxicity) were tested. The role of enzymes in IP removal was analysed through in vitro studies with enzymatic extracts (crude and pre-purified) from Leptosphaerulina sp., commercial enzymes and enzymatic inhibitors. Furthermore, the applicability of mycoremediation process to a complex matrix (simulated hospital wastewater) was evaluated. IP were considerably abated by the fungus, OXA was the fastest degraded (day 6), followed by CLX (day 7) and DCX (day 8). Antibiotics biodegradation was associated to laccase and versatile peroxidase action. Assays using commercial enzymes (i.e. laccase from Trametes versicolor and horseradish peroxidase) and inhibitors (EDTA, NaCl, sodium acetate, manganese (II) ions) confirmed the significant role of enzymatic transformation. Whereas, biomass sorption was not an important process in the antibiotics elimination. Evaluation of AA against Staphylococcus aureus ATCC 6538 revealed that Leptosphaerulina sp. also eliminated the AA. In addition, the cytotoxicity assay (MTT) on the HepG2 cell line demonstrated that the IP final solutions were non-toxic. Finally, Leptosphaerulina sp. eliminated OXA and its AA from synthetic hospital wastewater at 6 days. All these results evidenced the potential of Leptosphaerulina sp. mycoremediation as a novel environmentally friendly process for the removal of IP from aqueous systems.

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