*3.2. Optimal Fermentation Strategy and Incubation Period*

The cost and yield of bio-pigment production depend on the fermentation strategy used in production [35]. To date, different incubation periods have been tested for the production of pigment from different fungal species and wastes/pulps. For example, Babitha et al. [36] investigated pigment production by *Monascus purpureus* from jack fruit seed and they found that the maximum pigment production was on the sixth day. Gmoser et al. [48] found that the highest pigment (0.7 mg carotene/g waste) was produced by *Neurospora intermedia* from waste bread on the sixth day. In the study conducted by Padmavathi and Prabhudessai [49], the highest pigment production by *Monascus sanguineus* from potato peel was obtained on the 15th day.

Therefore, pigment production by *A. carbonarius* on PP was evaluated with SSF and SmF for 3, 6, 9, 12, and 15 d. In addition, the effect of incubation time on pigment production was limited to 15 d in this study due to the fact that a short fermentation time is desired in industrial production to obtain a competitive production [14]. Figure 2 illustrates the effect of two fermentation strategies (SSF and SmF) on pigment production over 15 d. SSF exhibited higher yellow pigment production (22.9 ± 1.34 AU/g dfm) than SmF. In addition, it was determined that pigment production mostly increased with increasing incubation time for both fermentation strategies.

Pigments are synthesized as secondary metabolites by the fungus [50] and these metabolites often produce at the stationary phase of fungal growth as a result of nutrient limitations and/or under stress conditions [48]. Furthermore, secondary metabolism is commonly associated with sporulation processes for microorganisms, including fungi [51]. It is known that pigment production can be related to the formation of both sexual and asexual spores for some fungal species [51]. On the other hand, *Aspergillus* species can produce asexual spores, which are called conidia [52], from their conidiophores as a result of differentiation of their aerial hyphae. For example, Teertstra et al. [53] showed melanin pigments being produced in the conidia of *Aspergillus niger*. For these reasons, two possible reasons can explain the increasing of pigment production when the incubation day increases: (i) for both fermentation strategies, the stationary phase began after 6 d (Figure 2) and pigment production increased with increasing incubation day because of decreasing nutrients or stress conditions, (ii) conidia that contain pigments were produced after 6 d. In addition, it is known that conidia are only formed in the air [48,54]. Due to the presence of water in SmF, conidia were not produced by *A. carbonarius* until day 12 (Figure 2). Therefore, SSF was found to be more successful in pigment production than SmF. Consequently, further studies were conducted with SSF for maximum pigment production.

**Figure 2.** Evaluation of pigment production of *A. carbonarius* on pomegranate pulp (PP) at solid-state fermentation (SSF) and submerged-state fermentation (SmF) conditions (experimental conditions: pH: 4.5; shaking speed for SmF: 100 rpm).
