## 4 Discussion  
The antifungal properties of the soil F1 isolate were examined in the current investigation. Using the cross-streak and agar well diffusion procedures, significant antifungal activity against _C. albicans_ and _A. niger_ were established. The F1 isolate was identified as _B. toyonenisis_ based on its morphology, biochemical characteristics, and 16 S ribosomal RNA sequencing (NCBI GenBank accession number OQ071612). This Gram-positive isolate belonged to _Bacillus_, which is frequently cited as a source of beneficial bioactive compounds. The genus _Bacillus_ are known as a bio-factor in antifungal secondary metabolites that combat many pathogenic fungal strains [58]. Previously, a strain of _B. toyonenisis_ obtained from the spontaneous cocoa beans fermentation showed a significant fungistatic activity against _Aspergillus carbonarius_[59]. In another study, _B. toyonenisis_ exhibited antifungal activities against many plant fungal pathogens [60]. Moreover, it showed many antimicrobial properties [61] and targeted activity against _B. cereus_ and _Listeria monocytogenes_ through its toyonin production ability [20]. A soil-recovered strain of _B. toyonenisis_ has also shown promising insecticidal activities in former studies [62]. In the current study, the respective metabolite was found to be formed extracellularly, and ethyl acetate was used to extract it with a considerably higher yield (0.28 mg/mL). A silica gel thin layer chromatogram was used to identify the active fraction (PF18) with the highest antifungal activity, and bioautography testing against _C. albicans_ served as confirmation. The metabolite's primary utility in pharmacotherapy is that it demonstrated fungicidal activity [33]. We identified this compound as 6-methoxy-1H-Indole-2-carboxylic acid using 1D- and 2D-NMR and LC-PDA-MS spectral studies. This compound contains the indole carboxylic acid moiety, which had been previously reported for its antifungal and antimicrobial activities [63] from various microorganisms and algae [23]. Furthermore, 4-[(1E)-3-methylbuta-1,3-dienyl]-1H-indole-3-carbaldehyde (indiacene A) and -[(1E,3E)-4-chloro-3-methylbuta-1,3-dienyl]-1H-indole-3-carbaldehyde (indiacene B)-phenyl indels harboring the same moiety were extracted and purified from the culture broth of the  
Figure 8: Antifungal metabolite concentration using optimized conditions, as compared the unoptimized conditions. Myxobacterium _Sandaracinus amylolytica_[25]. Moreover, 3-methyl-1 Hindole- 2-carboxylic acid and 1H-indole-3-carboxylaldehyde were previously isolated from the culture broth of _Micromonospora_ sp. and showed antagonistic activity against many pathogens, including _Staphylococcus aureus_, _Enterococcus ficeum_, and _Escherichia coli_[64]. Even though 1H-Indole-2-carboxylic acid derivatives have been reported in the literature, this is the first report denoting its production by _B. toyonensis_ OQ071612. The method through which secondary metabolites are produced by microorganisms is complex [65]. The quality and quantity of the produced metabolites can be affected by slight modifications to the medium components and/or circumstances. Hence, the best sources of carbon and nitrogen were first identified using the OFAT method. Carbon and nitrogen are essential components needed for the growth of bacteria and the synthesis of metabolites [64]. In bacterial fermentation operations, carbohydrates are typically employed as carbon sources, but nitrogen sources can be either organic or inorganic [66; 64]. In this investigation, the best media components for maximizing metabolite production were identified to be starch and peptone. Starch is agreed to be an important medium component for antifungal compounds production from microorganisms. Maximum growth and antibiotic production was found to occur when starch was used as the only C source [67]. This is in accordance with [PERSON] et al. (2016), who reported that starch was the optimum carbon source for the _Streptomyces platensis_ 3-10 growth [68]. [PERSON] et al.