**3. Results and Discussion**

#### *3.1. Synthesis and Characterization of Se-NPs*

In the current study, the supernatant of *Bacillus megaterium* ATCC 55000 was used to synthesize Se-NPs. The process of selenious acid reduction was monitored, while the cell-free extract changed from colorless to reddish color [53,54]. The UV-visible spectrum of Se-NPs synthesized indicated that it had maximum absorption at (0.860 abs) and 435 nm. DLS was performed to evaluate the particle size distribution, and the average particle size was found to be 45.9 nm, as shown in Figure 1B. On the other hand, the TEM result demonstrated that particles had a spherical shape within a nanoscale range from 29.72 to 74.36 nm with an average of the main diameter of 41.2 nm, as shown in Figure 1C. The XRD pattern for the Se-NPs was presented in Figure 1D. Several peaks were observed at nine theta (degree) as 23.2◦ , 30.5◦ , 41.7◦ , 44.3◦ , 46.4◦ , 52.3◦ , 56.7◦ , 62.5◦ , and 72.6◦ corresponding to the (100), (101), (110), (102), (111), (201), (113), (202), and (210) planes of the standard cubic phase of Se, respectively. The XRD pattern indicated that Se-NPs were in the facecentered cubic (FCC) structure and crystal in nature. The observation of diffraction peaks for the Se-NPs indicated that they were crystalline, while their refining was related to the particles in the nanometer size regime. The strong interaction of the Se-NPs with light was the result of the electrons conducting on the metal surface that were subjected to a collective oscillation when excited by light at specific wavelengths, which is known as surface plasma resonance (SPR) [55,56]. In another study, the culture supernatant of *A. terreus* with SeO<sup>2</sup> (100 µg/mL) produced Se-NPs with an average size of 47 nm [57]. *Bacillus megaterium* (a halophile strain) strongly reduced selenite (up to 0.25 mM) to Se-NPs after 40 h of incubation [58]. A microbial source *Bacillus cereus-*mediated synthesis of Se-NPs showed an absorption maxima at 590 nm, whereas nanoparticles synthesized from lemon leaf extract exhibited a maximum absorption at 395 nm [59]. The band gap energy calculated for chemically formed nano-Se was 2.1 eV, which significantly different from a biological

source (band gaps for nano-Se from *Sulphurospirillum barnessi, Bacillus selenitireducens,* and *Selenihalanaerobacter shriftii* were 1.62, 1.67, and 1.52 eV, respectively [60].

**Figure 1.** Characterization of bacteriogenic Se-NPs produced by *B. megaterium* (**A**–**D**); (**A**) UV-Visible spectrum; (**B**) dynamic light scattering (DLS); (**C**) TEM image; (**D**) XRD.
