*3.2. Biosynthesis and Characterization of AgNPs*

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 CFSs obtained from endophytic bacterium *B. endophyticus* strain H3 were tested in the biosynthesis of AgNPs in 3 mM of AgNO<sup>3</sup> solution. Incubation of 10 mL of CFSs with 90 mL of AgNO<sup>3</sup> for three days resulted in color conversion from light yellow to dark brown, demonstrating the development of nanoparticles in the reaction mix. Differences in the color of AgNPs have been reported due to the formation of the biomolecules responsible for the synthesis of nanoparticles and the reduction of Ag<sup>+</sup> to Ag<sup>0</sup> [9,12,13,25]. The reduction of silver ion (Ag+) of AgNO<sup>3</sup> has been found in many bacterial species, such as the *Bacillus siamensis* strain C1 [14] and *Pseudomonas rhodesiae* [13]. During the reduction of AgNO3, the nitrate ions (NO<sup>3</sup> <sup>−</sup>) are reduced to nitrite (NO<sup>2</sup> – ) by firstly accepting two protons and then releasing two electrons and water. The electrons emitted in the reduction reaction are transferred to the Ag<sup>+</sup> to form the silver element Ag<sup>0</sup> [26,27].

Furthermore, the formation of nanoparticles in the mixture was confirmed by a UV spectrophotometer, which showed a spectrum of surface plasmon resonance (SRP) at 412 nm (Figure 2), which is within the range reported earlier [13,28,29]. Similarly, Ahmed et al. [30] confirmed the formation of biogenic AgNPs in the reaction mixture by the presence of the peak at 418 nm.

TEM and SEM observations showed the nanoparticle to have spherical shape with sizes ranging from 4.17 to 26.9 nm in the reaction mixture (Figure 3). The present observations are consistent with the results from previous reports [13,31,32]. The toxicity of AgNPs depends on the variation of particle size.

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AgNPs have an important influence on fungal cell's viability and ROS generation in a size-dependent manner. It is evident that the surface area, the volume ratio and the interaction of the surface with the particle size can be changed. Furthermore, sedimentation rate, mass diffusion, binding efficiency and sedimentation rate of NPs on biological or solid surfaces are highly influenced by particle size [33]. For example, Carlson et al. found that the 15 nm AgNPs can produce more ROS compared to 55 nm AgNPs in a macrophage cell line [34]. Furthermore, the EDS result showed that the element peak of silver, silica and sulfur are 92.77, 5.53 and 1.70%, respectively, in the reaction mixture (Figure 4). The results of this study are in agreement with the literature related to silver nanoparticles, where the silver ions peak was confirmed at 3 KeV [30,35].

**Figure 2.** UV-vis absorption spectra of green silver nanoparticles (AgNPs) in the reaction mixture.

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**Figure 3.** Characterization of AgNPs biosynthesized by using culture filtrates of *B. endophyticus* strain H3 isolated from onion. (**A**) Transmission electron microscopy and (**B**) scanning electron microscopy.

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**Figure 4.** Characterization of biosynthesized AgNPs by using energy dispersive spectrum (EDS).

‐ ‐ ‐ ‐ ‐ ‐ ‐ − ‐ − − The functional groups of the biosynthesized AgNPs were confirmed by using FTIR analysis. In fact, 6 peaks at 3416, 2924, 1635, 1395, 1062 and 516 cm−<sup>1</sup> were observed in the FTIR spectra of the synthesized AgNPs, which are shown in (Figure 5A). The main peak of 3416 cm−<sup>1</sup> is due to the N–H stretching vibrations; a characteristic peak at 1635 cm−<sup>1</sup> represents C=O carbonyl group and C=C stretching vibrations; the peaks at 2924, 1395 and 1062 cm−<sup>1</sup> represent the C–H stretching vibrations, C=N bond of Amide II, O–H deformation vibrations, and C–N stretching amine vibrations, respectively; the peak at 516 cm−<sup>1</sup> represents C–Br stretching. The existence of such groups in the chlorofluorocarbons (CFCs) from endophytic bacterium confirms the presence of proteins and indicates that these functional groups have a major role in reducing Ag<sup>+</sup> to Ag<sup>0</sup> [13,32,36]. Similarly, various studies reported the presence of functional groups representing different macromolecules, such as nucleic acids, proteins, lipids, carbohydrates and sugars, surrounding the green NPs which prevents the oxidation and deterioration of nanoparticles [30,37,38].

**Figure 5.** Characterization of the biosynthesized AgNPs. (**A**) Fourier transform infra-red (FTIR) spectra. (**B**) X-ray diffraction (XRD) spectra.

The crystalline structure of the biogenic AgNPs was determined through the XRD analysis result, that showed five emission peaks of 2θ = 28.81◦ , 32.41◦ , 46.24◦ , 57.37◦ and 76.77◦ , compatible with crystalline silver planes (101), (111), (200), (220), (311), respectively (Figure 5B). Similar results have been reported in other studies [11,14,38].
