*2.4. Optimizing Myco-Synthesis of MgO-NPs*

The MgO-NPs production and distribution were affected by the environmental factors such as pH, contact times, incubation temperature, and precursor concentrations. These factors were investigated at maximum surface plasmon resonance (SPR) detected by using a UV-Vis spectrophotometer (Jenway 6305, Staffordshire, UK). The effect of pH values at 6, 7, 8, 9, 10, and 11 on MgO-NPs sorption properties was investigated. The contact time either between fungal biomass and distilled water to produce biomass filtrate (24, 48, 72, and 96 h) or times between biomass filtrate and precursors (6, 12, 24, 36, 48, and 72 h) were also investigated. The incubation temperature (25 ◦C to 40 ◦C with intervals of 5 degrees) and precursor concentrations (1–5 mM) were assessed. At the end of each parameter, 1.0 mL of the sample was withdrawn and measured at maximum SPR at λmax = 282 nm.

### *2.5. Characterization of Biosynthesized MgO-NPs*

The particle sizes and shapes of biosynthesized MgO-NPs were detected using Transmission Electron Microscopy (TEM) (JEOL 1010, Japan, acceleration voltage of 120 KV). A drop of NP solution was loaded on the carbon-copper grid and underwent vacuum desiccation for 24 h and placed after that onto a specimen holder [32]. The elemental compositions of biosynthesized MgO-NPs were assessed using scanning electron microscopy connected with energy dispersive X-ray (SEM-EDX) (JEOL, JSM-6360LA, Akishima, Japan). The crystallographic structure of biosynthesized MgO-NPs was determined using X-ray diffraction (XRD) analysis by X'Pert pro diffractometer (Philips, Eindhoven, Netherlands). The operating conditions were, 2θ values measured in ranges of 4◦ to 80◦ , X-ray radiation source was Ni-filtered Cu Ka and the operating voltage and current were 40 KV and 30 mA, respectively. The average NP sizes were measured using the Debye–Scherrer equation [33] as follows:

$$\mathbf{D} = \mathbf{K}\lambda / \mathfrak{\beta}\cos\theta \tag{3}$$

where D is average particle size, K is the Scherrer's constant (0.9), λ is the wavelength of X-ray radiation (0.154 nm), and β and θ are the half of maximum intensity and Bragg's angle, respectively. Moreover, the size distribution of MgO-NPs in colloidal solution was investigated by dynamic light scattering (DLS) analysis. The sample was subjected to measurement by Zeta sizer nano series (Nano ZS, Malvern, UK).

On the other hand, the functional groups present in fungal biomass filtrate involved in reduction, capping, and stabilization of MgO-NPs were investigated using Fourier transform infrared (FT-IR) spectroscopy (Agilent system Cary 660 FT-IR model). The MgO-NPs sample was ground with KBR pellets (1% *w*/*w*), pressure was applied to form a disk, and scanning was done in the range of 400–4000 cm−<sup>1</sup> .

Finally, the X-ray photoelectron spectroscopy (XPS) analysis was analyzed by ES-CALAB 250XI<sup>+</sup> instrument (Thermo Fischer Scientific, Inc., Waltham, MA, USA) connected with monochromatic X-ray Al Kα radiation (1486.6 eV). The analysis was conducted under the following conditions: the size of the spot was 500 µm, the samples were prepared under a pressure adjusted at 10−<sup>8</sup> mbar., the energy was calibrated with Ag3d5/2 signal (∆BE: 0.45 eV) and C 1s signal (∆BE: 0.82 eV), and the full and narrow-spectrum passed energies were 50 eV and 20 eV, respectively [34,35].
