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

Several environmental factors affect the biosynthesis processes, the stability of NPs, and their applications. These factors can affect the reducing agents such as enzymes, proteins, carbohydrates, and others present in biomass filtrate, which directly influence the biosynthesis processes [53]. The optimization of these factors has positive impacts on reducing and capping agents, decreasing the biosynthesis time, increasing NP stability, and decrease aggregation [32]. Among these factors, contact time, pH, temperature, and concentrations of precursor are investigated in the current study.

The incubation period of fungal biomass in distilled water to secrete bioactive compounds that acts as reducing and capping agents is considered a critical factor. In the present study, the optimum contacting time between fungal biomass and distilled water was 72 h. At this time, the color intensity drastically increased, referring to the efficacy of active compounds involved in biomass filtrate to reduce the highest amount of Mg(NO3)2·6H2O (Figure 3A). The concentration of Mg ions is considered an important factor that influences the myco-synthesis of MgO-NPs; therefore, different concentrations of precursor were used. In this study, the absorbance at λ<sup>282</sup> was increased gradually by increasing Mg(NO3)2·6H2O concentration until 4 mM, after which the absorbance was decreased (Figure 3B). This indicates the efficacy of active metabolites including enzymes and protein to reduce Mg(NO3)2·6H2O, whereas the MgO-NPs tend to aggregate or agglomerate by increasing precursor concentrations and hence decreasing the absorbance [54]. Compatible with our study, Ibrahim [55] reported that the color intensity of Ag-NPs synthesized by banana peel extract was changed from yellowish-brown to dark reddish-brown by increasing the precursor (AgNO3) concentrations.

Another important factor affecting the myco-synthesis of MgO-NPs is the contact time or the reaction time between biomass filtrate and optimum precursor concentration (4 mM). The color changes to white precipitate offer evidence for the successful fabrication of MgO-NPs, which is monitored by detecting the absorbance at λ282. Our data showed that the highest absorbance was achieved at 36 h of reaction time between fungal biomass filtrate and Mg(NO3)2·6H2O by constant the previous optimum conditions (Figure 3C). At the early stages of reaction time, the SPR peak was broadened due to the slow reduction of metal ions to NPs. At the optimum reaction time, the large number of metal ions were reduced to NPs and identical SPR bands were formed. By increasing the reaction time, the absorbance gradually decreased due to the aggregation of some particles, consequently reducing the color intensity and particle size [55].

In addition, pH is considered an important factor that influences reducing agents, which affects the green synthesis. In the current study, different pH values were adjusted ranging from 6 to 11, and their impact on the reduction process was investigated through measuring the absorbance at λ<sup>282</sup> after the constant of the other parameters. Data analysis showed that the alkaline solution was preferred for the myco-synthesis of MgO-NPs by *R. oryaze* strain E3. The highest absorbance at λ<sup>282</sup> was attained at pH 8, which was evidence for maximum MgO-NPs production (Figure 3D). The lowest absorbance was attained at an acidic solution (pH = 6) with an absorbance value of 0.845 ± 0.01, which indicates that the activity of reducing groups present in biomass filtrate was reduced at acidic medium. Our recently published study showed that the activities of metabolites secreted by *Aspergillus carbonarious* strain D-1 were highly active at alkaline medium for reducing, cap, and stabilizing of α-Fe2O3-NPs and MgO-NPs [56]. The charges of biomolecules present in biomass filtrate can be altered due to differences in pH values and thus, the reducing capacity is affected [57].

Finally, the temperature is considered the main factor effect on the enzymes, proteins, carbohydrates, and other reducing agents present in biomass filtrate and consequently influence the NP synthesis process. In this study, different incubation temperatures (25–40 ◦C) were investigated to detect the preferable incubation temperature by measuring the absorbance intensity at λ282. Data analysis showed that the capacity of metabolites as reducing, capping, and stabilizing agents were accomplished at 35 ◦C (Figure 3E). The intensity of color as indicated by absorbance was decreased at a temperature greater or less than 35 ◦C. This phenomenon could be attributed to the inactivation of reducing agents at low and high incubation temperatures. Rai et al. [58] reported that the incubation temperature of the NP synthesis process can influence the nature, size, and shape of NPs formed. Interestingly, Patra and Baek [53] reported that the synthesis of NPs using physical methods needs an incubation temperature >350 ◦C, whereas chemical approaches require an incubation temperature <350 ◦C; in contrast, green approaches required ambient temperature.

**Figure 3.** Optimizing factors for myco-synthesis MgO-NPs using *R. oryaze* strain E3. (**A**) is contact time between fungal biomass and distilled water, (**B**) is precursor concentrations, (**C**) is contact time between biomass filtrate and optimum precursor concentration, (**D**) is the effect of pH values, and (**E**) is the effect of incubation temperature on the biosynthesis process.
