*2.8. Analytical Techniques*

During the pulp type selection experiments, different pigment extraction protocols were performed to evaluate the effect of wet or dry fermented solids. During the following experiments, pigment extraction protocol was performed on a dry basis. Pigment extraction was performed according to the method reported by Kantifedaki et al. [34] with slight modifications. After the fermentation of every case, solid material was weighed as 0.5 g (on wet or dry basis), transferred into a tube and mixed with 5 mL of ethanol (95%). The mixture was placed in an ultrasonic bath at 60 Hz (Witeg, Wertheim, Germany) for 30 min at 25 ◦C. Then, the mixture was mixed on a rotary shaker at 180 rpm for 1 h at 30 ◦C followed by centrifugation at 6000 rpm for 20 min. After centrifugation, the supernatant was used for pigment estimation by measuring the absorbance with a UV–vis spectrophotometer (DR3900, Hach Co, Loveland, CO, USA) along with utilizing a quartz cuvette. Yellow, orange, and red pigments were determined by measuring the absorbance in three different wavelengths, 400 nm, 475 nm, and 500 nm, respectively, taking into consideration the dilution factor of the sample [38]. In every step, the unfermented substrate was subjected to sterilization, but inoculation was not carried out. These unfermented pulp particles were subjected to pigment extraction. The results were expressed as absorbance unit at the corresponding wavelength per gram (AU/g). In this study, intracellular pigment production was assessed due to the application of pigment extraction to only fermented solids including fungal biomass and pulp mass.
