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

ZnO NPs are relatively easily dissolvable in soils, especially in more acidic ones [9,37,38]. The dissolution of ZnO NPs is also enhanced by soil organisms, including filamentous fungi,

via exudation of various acidic and chelating metabolites. The microbially induced acidification led to the dissolution of ZnO NPs, and bulk ZnO [39] and, therefore, nearly all of the Zn in the solutions of growth media was dissolved regardless of the Zn form.

Since Zn is an essential micronutrient [40,41] and our selected concentration was below the threshold of growth inhibition, all forms of Zn at the applied concentration had a positive effect on the dry weight of biomass after 7 days of cultivation. It was reported that the Zn concentrations as low as 0.065 mg Zn·L −1 enhanced the fungal growth [42]. The minimal inhibitory concentration of Zn for microscopic filamentous fungi were reported to be as high as 100 mg Zn·L −1 in the literature [43,44].

### *4.2. Transformation of ZnO Nanoparticles by Fungal Extracellular Metabolites*

Filamentous fungi are capable of releasing extracellular metabolites that chemically deteriorate natural mineral Zn phases in soils and sediments via processes of protonation and chelation. However, under specific conditions (e.g., high biogenic chelate concentrations, alkalic pH), the bioextracted Zn can be reprecipitated from the soil solution to form new mineral phases. It has been reported that fungi react to the presence of ZnO NPs by increasing the production of the extracellular metabolites in order to detoxify and immobilize excessive Zn, and thus the transformation of ZnO NPs occurs in the soil environment [39,41]. Immobilization of Zn dissolved by fungi is facilitated by precipitation with oxalates [39].

We used extracellular metabolites of *A. niger* to transform ZnO NPs into a zinc oxalate biomineral. *A. niger* is well known for the high rate of extracellular metabolite production. Oxalic acid is the most produced of organic acids by the fungus when grown on Sabouraud growth media [30].

In our experiment, at the given applied volume and concentration of oxalic acid applied, only a partial transformation of ZnO NPs to zinc oxalates was observed. No significant change in the size of particles was observed. It is most probable that the transformation occurred on the surface of the nanoparticles, creating a surface of Zn oxalate with the core still being ZnO, or the newly formed zinc oxalates did not have time to grow to larger sizes in five days. If a larger volume of the organic acids was applied, both larger amounts of ZnO NPs might have been transformed, and the size of the particles could have seen a bigger change.

A change in Zn minerals into Zn oxalates had been observed before, albeit the minerals were not nanoparticles [32,39,45]. When silicate and sulfide minerals containing Zn were transformed by fungal extracellular metabolites, the resulting crystals of zinc oxalates had various shapes and sizes, with most of the crystals measuring between 50 and 100 µm [39]. When ZnO microparticulate powder was transformed into zinc oxalates by *Aspergillus* species, the biominerals had a different habitus compared to the minerals formed abiotically. The size of these biominerals was above 50 µm.

The mineral size and shape are also dependent on the place of formation. Minerals of zinc oxalate associated with a fungus have a different shape compared to both biomineral and abiotically formed Zn oxalate. However, the biominerals formed farther away from the fungal mycelium had a shape that was more similar to the abiotically formed Zn oxalate [32]. Since our experiment was only done with extracellular metabolites, the smaller change in the size and shape upon partial transformation may also result from the same processes that led to the increase in similarities between the biomineral formed further away from the mycelium and the abiotically formed mineral.

*Aspergillus* spp. and other fungi can biomineralize inorganic nanoparticles [46] and transform these nanoparticles into more stable oxalate compounds [25,47]. They play a large role in the cycling of elements in the soil environment, and their abilities can also be used for the bioremediation of contaminated areas [48].
