*2.7. Instrumental Analysis*

The morphological characterization of dried fermented mass obtained from optimal conditions was observed by SEM (Zeiss Supra 55, Oberkochen, Germany). Images were taken by applying an electron beam with an acceleration voltage of 5 kV. In addition, SEM analyses were performed on the unfermented optimum type of pulp and *A. carbonarius* growing in PDB to compare morphological changes.

The elemental analysis of each type of pulp was performed before fermentation due to evaluating C:N ratio utilization in the selection of pulp type experiments. Moreover, it was performed both before and after fermentation for the optimum pulp type, which provides higher pigment extraction. Elemental analysis was performed using a TruSpec Micro (LECO, St. Joseph, MI, USA) elementary analyzer (for C, H, N, and S weight percentages).

Two-dimensional gas chromatography (GC×GC) was used for the qualitative analysis of the extract. Approximately 10 mg of pulp, without any pre-treatment, was weighed and dissolved with 1.0 mL of hexane:acetone (1:1) solvent mixture. Liquid nitrogen used for cold pulses was automatically filled. The Agilent 7890 B (Palo Alto, CA, USA) Gas Chromatograph System was equipped with a LECO Pegasus® BT 4D mass spectrometer (Leco, St. Joseph, MI, USA) dual-stage, quad jet thermal modulator and with a split/splitless injector. The GC primary column had 30 m × 0.25 mm id. × 0.25 μm film thickness Rxi®-17Sil MS (Restek Corp., Bellefonte, PA, USA). The GC secondary column had 0.75 m <sup>×</sup> 0.25 mm id. <sup>×</sup> 0.25 <sup>μ</sup>m film thickness Rxi®-5Sil MS (Restek Corp, Bellefonte, PA, USA) mounted in a separate oven installed within the main GC oven. The carrier gas was helium and set 1 mL/min. Injection speed was 3 μL/s and the inlet purge time was 60 s. A 1 μL injection was made in splitless mode with an inlet temperature of 250 ◦C. The temperature program of the first column was as follows: 40 ◦C kept for 4 min, then raised at 8 ◦C/min up to 310 ◦C kept for 20 min. The temperature of the second oven was programmed with an offset of 10 ◦C and the modulator temperature offset was 25 ◦C relative to the first GC oven temperature. The second-dimension separation time (modulation time) was 5 s divided into a hot pulse time of 1.50 s and a cold pulse time between the stages of 1 s. The transfer line from the secondary oven into the mass spectrometer was maintained at 280 ◦C. The ion source was operated at 250 ◦C. The electron energy was −70 eV. The data acquisition rate was 200-scans/s, covering a mass range of 50–550 m/z.
