*2.9. Statistical Analysis*

All results presented are the means of three independent replicates. Data were subjected to statistical analysis by a statistical package SPSS v.17. The mean difference comparison between the treatments was analyzed by *t*-test or the analysis of variance (ANOVA) and subsequently by Tukey HSD test at *p* < 0.05.

#### **3. Results and Discussion**

#### *3.1. Isolation and Identification of Fungal Isolate*

The fungal isolate E3 was isolated and underwent primary identification according to standard keys. Based on morphological and culturable characteristics, the fungal isolate E3 was belonging to *Rhizopus* sp., which was subjected to molecular identification based on amplification and sequencing of the internal transcribed spacer (ITS) gene. The sequence analysis revealed that the fungal strain E3 was highly related to *Rhizopus oryaze* (accession number: NR103596), with similarity percentages of 99%. Therefore, the fungal strain obtained in this study was specifically identified as *Rhizopus oryaze* strain E3 (Figure 1). The obtained ITS sequence was deposited in the gene bank under accession number MW774584.

**Figure 1.** Phylogenetic tree of the fungal strain E3 with the sequences from NCBI. The symbol refers to ITS fragments retrieved from this study. The tree was conducted with MEGA 6.1 using the neighbor-joining method.

*Rhizopus oryaze* are characterized by their platform's highly secondary metabolites such as chemicals (fumaric acid, lactic acid, and ethanol), enzymes, fermentative compounds, and a wide range of by-products [44,45]. This wide range of metabolites increases the possibilities of *R. oryaze* to incorporate into various biomedical and biotechnological applications. To date, this is the first report that utilized *R. oryaze* as a biocatalyst for the green synthesis of MgO-NPs.
