*3.6. Characterization of Fungal Biomass*

To characterize fungal biomass, SEM is the one of most appropriate techniques. Figure 5 shows the SEM image of the fermented *A. carbonarius* on PP under optimal conditions. Fermented *A. carbonarius* on PP (Figure 5A) has more conidia whereas *A. carbonarius* grown on YMB has more hyphae (Figure 5C). This result supports our hypothesis that pigments were produced in conidia. Furthermore, Figure 5B shows the pigmented conidia of fermented *A. carbonarius* on PP. Unfermented PP (Figure 5D) shows a perforated structure.

It is known from the literature that *Aspergillus* species can produce melanin [30]. However, Pihet et al. [60] studied melanin production in *Aspergillus fumigatus* and SEM analysis results show that conidia that do not have smooth walls contain melanin. Therefore, this could support our second hypothesis that *Aspergillus carbonarius* could produce melanin from pomegranate pulp.

GC×GC-TOFMS is a reliable method for the detection of organic compounds in complex matrices [61], and its results are given in Table 3 for the extracted solution from fermented *A. carbonarius* on PP under optimal conditions. It is known that pomegranate contains sugars such as xylose, arabinose, and glucose [62]. GC×GC-TOFMS results show that fermentation was conducted successfully due to occurring alcohols (xylitol, sorbitol, and arabinitol) [63] and fatty acids (linoleic, oleic, and erucic acids) (Table 3) [64]. Ergosterol, which is a secondary metabolite of *Aspergillus spp*., might come from its fungal cell membrane [65,66]. Furthermore, squalene and β-amyrin detected by GC×GC-TOFMS could be in the pathway of ergosterol synthesis [67].

**Figure 5.** SEM images of (**A**) fermented *A. carbonarius* on PP under optimal conditions, (**B**) pigmented conidia of fermented *A. carbonarius* on PP under optimal conditions, (**C**) *A. carbonarius* grown on YMB, (**D**) unfermented PP.

Furthermore, 2,6-diisopropylnaphthalene detected by GC×GC-TOFMS (Table 3) could be caused by the usage of pesticides in pomegranate growth [68]. In a similar way, it is though that γ-tocopherol, which is an antioxidant, may be caused by the composition of pomegranate [69].

Due to the detection of 1,8-dihydroxynaphthalene by GC×GC-TOFMS (Table 3), our findings suggest that *A. carbonarius* might produce melanin from the polymerization of 1,8- dihydroxynaphthalene (DHN) from PP through the DHN pathway [30]. Furthermore, phthalic acid, mequinol, and galangin are known to be potent inhibitors of the DHN pathway [70–72]. Therefore, it is concluded that inhibitors could not surpass the melanin production. A potential metabolic pathway of melanin production by *Aspergillus carbonarius* from pomegranate pulp is illustrated in Figure S3.

To date, several species of *Aspergillus* such as *A. niger*, *A. flavus*, *A. tamarii*, *A. terreus*, *A. tubingensis*, *A. sydowii* and *A. fumigatus* were investigated for melanin production [30,31,60]. However, there is very little information about melanin production by *A. carbonarius*. Babitskaya et al. [55] studied melanin production by *A. carbonarius* on Czapek's medium and they determined that melanin belongs to the DHN pathway. Another study on antioxidant properties of melanin produced by *A. carbonarius* was conducted by Shcherba et al. [73]. In accordance with these studies, it was thought that *A. carbonarius* could produce melanin from pomegranate pulp, which is an economical way, and that the produced melanin contains antioxidant. When evaluated together with SEM results, it was determined that the localization of this melanin was conidia.


**Table 3.** Chemical compounds determined by GC×GC-TOFMS


**Table 3.** Cont.

S: similarity.

Fungal melanin has attracted interest due to its potential usage in many industries including nanotechnology, biomedicine, dermo-cosmetics, andmaterials science [56]. However, *Aspergillus carbonarius* is also known as a producer of ochratoxin A (OTA) [74]. For this reason, further research on melanin production, OTA level and physical OTA removal methods such as filtration and pressure needs to be undertaken. It is known that OTA biosynthesis in *A. carbonarius* is driven by polyketide synthase (pks) and nonribosomal peptide synthetase genes (NRPS) [75]. The inactivation of these genes could be another way to eliminate the ability of this fungus to produce OTA [76] for a techno-economic way of producing pigment from pomegranate pulp.

In addition, GC×GC-TOFMS results show that the production of fatty acids such as linoleic, erucic and oleic acids by *A. carbonarius* could be another option for by-product valorization for the food processing industry [7].
