*2.1. Preparation of ZnO Nanoparticle Suspensions and ZnSO<sup>4</sup> Solution*

For the experiment with fungal growth, three Zn forms were used, ionic Zn in the form of a solution of ZnSO4, suspension of ZnO NPs, and bulk ZnO. Bulk ZnO was acquired in the form of powder (p.a. quality, Chemapol, Prague, Czech Republic). ZnO NP dispersion used in the experiment was purchased from Sigma Aldrich, St. Louis, MO, USA (<100 nm particle size (TEM), ≤40 nm Avg. part. size (APS), 20 wt. % in H2O). ZnO NPs used in this work were also used in the article by Kolenˇcík et al. [16], and additional characterization of the nanoparticles can be found there. Right before the experiment, the suspension of ZnO NPs with a concentration of 65 mg·L −1 (1 mmol Zn·L −1 ) was prepared by adding 65 µL of ZnO NP dispersion to a 200 mL volumetric flask that was filled to mark

with distilled water. The suspension was then sonicated for 15 min in an ultrasonic bath. An ionic zinc solution of 65 mg·L −1 (1 mmol Zn·L −1 ) ZnSO<sup>4</sup> was prepared by dissolving 0.2876 g ZnSO4·7H2O (p.a. quality, CentralChem, Bratislava, Slovakia) in 1 L of distilled water.

### *2.2. Cultivation of Aspergillus niger*

Microscopic filamentous fungus *Aspergillus niger* (Tiegh.), strain CBS 140837, originally isolated from the mercury-contaminated soil [33], was grown in Sabouraud growth medium (HiMedia, Mumbai, India) in 250 mL Erlenmeyer flasks using a 7-day static cultivation in a growth chamber (dark, 25 ◦C). Four different types of growth media were created with three forms of Zn at 6.5 mg Zn·L −1 (0.1 mmol Zn·L −1 ), ZnSO4, ZnO NP, and bulk ZnO, and one control without added Zn. The concentration of 6.5 mg Zn·L <sup>−</sup>1 was selected in a preliminary experiment with ZnSO<sup>4</sup> (Supplementary Material Table S1, Figure S1), where a concentration of 13 mg Zn·L −1 in the form of ZnSO<sup>4</sup> prolonged sporulation with negligible effect on dry biomass weight; a concentration of 26 mg Zn·L <sup>−</sup><sup>1</sup> was inhibitory for fungal growth and no compact mycelium was formed after 7 days.

In the case of ZnSO4, and ZnO NPs, 5 mL of either 1 mmol Zn·L <sup>−</sup><sup>1</sup> ZnSO<sup>4</sup> solution or ZnO NP suspension were added to 45 mL of Sabouraud growth medium in Erlenmeyer flask. Bulk ZnO in the form of 0.0033 g of ZnO powder was added to 45 mL of growth medium and 5 mL of sterilized distilled water put into Erlenmeyer flask. The control experiment was done in Erlenmeyer flasks filled with 45 mL of the growth medium and 5 mL of sterilized distilled water. Each of the Zn forms and control had six replicates. All the Erlenmeyer flasks with the growth media were then put into the ultrasonic bath for 15 min.

After the aforementioned procedure, each of the growth media in Erlenmeyer flasks was inoculated with 50 µL of *A. niger* spore suspension and grown in the dark in the growth chamber for 7 days. After a 7-day growth period, the weight of dry biomass, the concentration of Zn in dry biomass, pH in the growth media, and the concentration of Zn in growth media in form of ionic Zn and Zn bound in organic or inorganic colloids was measured.

The biomass grown on the top of the growth media was collected and washed several times with distilled water. Afterwards, it was dried out at 60 ◦C, then weighed, and transferred into PTFE containers and 5 mL of 65% HNO<sup>3</sup> was added to digest the biomass. The PTFE containers were put into high-pressure acid digestion vessels, and the vessels were closed afterwards. Then, the vessels were placed into an oven heated to 150 ◦C for 4 h to digest the biomass.

To discern between Zn bound to colloidal form and ionic Zn, the removed growth medium was centrifuged at 700 g for 1 min, to remove big clusters of residual biomasses bigger than 1000 nm. Then, part of the supernatant was removed and analyzed for Zn concentration (*C*1000). The concentration of ionic Zn (< 1 nm, *C*1) was acquired after the ultrafiltration of the supernatant, 6 mL of supernatant was transferred to ultrafiltration centrifugation units (Sartorius Vivaspin® 6 mL, 3 kDa, Goettingen, Germany) which were centrifuged at 3500 g for 20 min. 0.5 mL of filtrate was collected, stabilized with HNO3, and analyzed for Zn concentration. The concentration in filtrates, supernatants, and digested biomass was measured by flame atomic absorption spectrometry (Perkin-Elmer 1100, Perkin-Elmer, Rodgau, Germany). A concentration of Zn bound to colloidal forms (1–1000 nm, *C*1-1000) was calculated by subtracting the concentration of ionic Zn from the concentration of Zn in colloidal supernatant (*C*1-1000 = *C*<sup>1000</sup> − *C*1).

The dry weight of mycelia and pH was compared for all applications via a two-tail *t*-test at a significance level α = 0.05. Before the *t*-test, data were analyzed for differences in variances by F-test, and then a *t*-test for either equal or unequal variances was used. The statistical evaluation was done with Analysis ToolPak add-in for Microsoft Excel (Redmond, WA, USA).
