*2.3. Transformation of ZnO Nanoparticles by Extracellular Metabolites of Aspergillus niger*

In our experiments with *A. niger* mycelia, the used concentration was very low, and most, if not all, the ZnO NPs and bulk ZnO were dissolved. Therefore, the experiment with fungal extracellular metabolites, which were also used in other studies of ZnO NPs synthesis [22], was undertaken to find out if these metabolites are able to dissolve ZnO NPs and transform them into Zn biominerals.

To achieve this, the *A. niger* was statically grown for 7 days on Sabouraud growth media in the growth chamber. It was grown the same way as the control group was grown in the previous experiment.

After the 7-day cultivation period, the mycelium was removed from the Erlenmeyer flask and was put for 3 days into 50 mL of sterilized distilled water. After 3-day cultivation, the mycelium was removed, and the acquired solution was filtered through 0.45 µm membrane filter paper. A small amount of the solution was removed and used for the analysis of oxalic acid produced by the fungal mycelium.

An Erlenmeyer flask was filled with 47.5 mL of a solution of extracellular fungal metabolites, and 500 mg of ZnO NPs was added in the form of 2.5 mL of ZnO NP dispersion. After 5 days of static reaction, the solution was decanted and analyzed for the content of oxalic acid and the white sediment was dried out and sent to X-ray powder diffraction (XRD) analysis and analysis by transmission electron microscopy (TEM). The solutions containing extracellular metabolites were analyzed for the content of oxalic acid by isotachophoresis (ZKI-1, Villa Labeco, Spišská Nová Ves, Slovakia) in itp-itp mode. The acquired isotachopherograms were evaluated by a software suite supplied with the analyzer [34].
