*3.1. Interactions of Aspergillus niger with ZnO and Aqueous Zn*

Fungal mycelia of *Aspergillus niger* were grown statically in Erlenmeyer flasks with Sabouraud growth media spiked with either ZnSO4, ZnO NPs, or bulk ZnO and control without added Zn. The overproduction of protons (H+) by microscopic filamentous fungus *A. niger* during the 7-day cultivation period generated a substantial decrease of pH from 5.6 to 2.4. It was even more intensified in the presence of Zn where the final pH values decreased to 2.0, 2.1, and 2.2 for ZnSO4, ZnO NPs, and bulk ZnO, respectively (Figure 1).

α **Figure 1.** pH of the growth media after the experiment; a and b represent the statistically similar means between different groups at α = 0.05 (two-tail *t*-test).

Zn was determined primarily as dissolved in the growth media (Figure 2) regardless of the Zn form applied. Only a negligible fraction was bound to colloidal particles. Thus, the distribution of Zn in colloidal, dissolved and bioaccumulated fractions were similar for ZnSO4, ZnO NPs, and bulk ZnO (Figure 2). The bioaccumulation of Zn by *A. niger* was very effective for all Zn forms (Figure 2) and represented up to 76% of the total Zn added to the cultivation system.

**Figure 2.** Distribution of Zn in the experimental system (**a**) and concentration of Zn in (**b**) particles sized 1 to 1000 nm, (**c**) dissolved fraction, and (**d**) dry biomass.

∙ − The applied concentration of 6.5 mg Zn·L <sup>−</sup><sup>1</sup> had no adverse effects on fungal growth and a positive effect of Zn on the growth of the fungal biomass was observed (Figure 3). The application of all Zn form resulted in a higher dry weight of mycelia compared to control. The highest mean dry weight of mycelia was observed for ZnO NP application. The weight was significantly higher than both dry weights in bulk ZnO application and in the control experiment without additional Zn. ∙ −

α **Figure 3.** The dry weight of biomass of *Aspergillus niger* after the experiment; a, b and c represent the statistically similar means between different groups at α = 0.05 (two-tail *t*-test).

α
