*3.1. Optimal Fungal Species and Pulp Type*

Elemental analysis of pulps performed before fermentation are shown in Table 1. Results show that each type of pulp had essential nutrient content for fungal growth [41]. In addition to this, after the inoculation of *A. carbonarius* onto different pulps, it was observed that fungus was grown on each different pulp type and covered the surface of the pulps within 3 d. This observation (Figure S3) supports the results of elemental analysis.

Figure 1 shows the results of pigment extraction obtained from wet and dry fermented mass after SSF using *A. carbonarius* cultivated on the pulp of black carrot (Figure 1A), apple (Figure 1B), pomegranate (Figure 1C), and red beet (Figure 1D). The literature describes that nitrogen and carbon sources have effects on secondary metabolite regulation [42]. Therefore, it is possible that different levels and types of pigments could be produced by the fungus grown on the different types of pulps, which have different contents of C and N sources.


**Table 1.** Elemental analysis of unfermented pulps.

C: Carbon, H: Hydrogen, N: Nitrogen, S: Sulphur, C:N: carbon to nitrogen ratio.

**Figure 1.** Evaluation of wet and dry mass pigment extraction methods during solid-state fermentation using *A. carbonarius* cultivated on (**A**) black carrot, (**B**) apple, (**C**) pomegranate, (**D**) red beet pulps (experimental conditions: incubation time, 5 d).

Generally, a high C:N ratio has been reported to induce pigment production for filamentous fungi [43]. For example, Palacio-Barrera et al. [44] studied the effect of C:N ratio on pigment production by *Aspergillus chevalieri* and they found that a high C:N ratio (20/1, glucose/yeast extract) was optimum for inducing pigment production. However, it was not the case in this work for all type of pulps. The highest pigment production by *A. carbonarius* was on pomegranate, red beet and apple pulp, respectively. Nevertheless, the highest initial C:N ratio was red beet (20.63/1), pomegranate (16.87/1) and apple (11.53/1), respectively. This could be attributed to the decreasing pH levels during the fermentation of red beet pulps [45]. In this study, a more complex substrate such as a real industrial pulp was used for pigment production. It is known that pigment production is affected not only by the C:N ratio, but also by other factors such as types of C and N sources and the presence of organic acids

and minerals [46]. For this reason, it is possible to obtain different results in pigment production from synthetic media and from more complex substrates.

On the other hand, the highest pigment extraction was mostly achieved with dry fermented mass compared to wet fermented mass, for apple (Figure 1B), pomegranate (Figure 1C), and red beet (Figure 1D) pulp. This can be explained as the wet mass (0.5 g on wet basis) used in the extraction method contained more water, whereas the dry mass (0.5 g on dry basis) contained less water due to drying. Hence, it can be concluded that the dry mass, as a result of dehydration, may contain more concentrated pigment due to the lack of water. Pigment extraction analysis carried out under SSF conditions showed that the maximum pigment production was determined as 9.21 ± 0.59 AU/g dry fermented mass (dfm) in pomegranate pulp by *A. carbonarius* for 5 d (Figure 1C) at 400 nm. It was also found that the highest absorbance and color were determined at 400 nm as yellow hue for all pulp types. It is known that yellow hydroxyanthraquinone (HAQ) pigments are produced by many species of *Aspergillus* [47]. Hence, the following studies were performed with pomegranate pulp (PP) for optimal pigment production.
