Fungal strain and fermentation process

Rhizopus oryzae ATCC 20344 (purchased from ATCC) was the strain used for chitin accumulation. Spores of R. oryzae ATCC 20344 were collected from the culture on the potato dextrose agar (PDA) medium (Sigma-Aldrich®). The spore concentration of the collected spore solution was approximately 10⁷ spores/mL. 0.5 mL of the spore solution were inoculated to 100 mL of sterilized potato dextrose broth (PDB) medium (Sigma-Aldrich®) with 8 g L⁻¹ yeast extract (Acumedia^®), and cultivated at 30 °C, 180 rpm for 36 h to prepare the seed. The detoxified solution from “Pretreatment and enzymatic hydrolysis of solid digestate” section was mixed with 3 g L⁻¹ of CaCO₃ and trace elements [28], and sterilized under 121 °C for 15 min to prepare the fermentation medium. 5 mL of the seed was inoculated to 45 mL of the fermentation medium. The fermentation was carried out at 30 °C and 180 rpm for 120 h. Samples were taken during the process to monitor kinetics of substrate consumption, growth, and product production.