2.3.5. Solid-State Fermentation (SSF): Enzymatic Reaction on Paper Mill Water

After 6 days of *Aspergillus oryzae* growth of Petri dishes in PDA medium, a block of mycelium and spores (5 <sup>×</sup> 5 mm<sup>2</sup> area) of the plate culture with the fungus was used to inoculate a 50 mL Erlenmeyer flask containing 20 mL of PDB medium. The cultures were incubated at 28 ◦C, 120 rpm for up to 96 h. One milliliter of the supernatant was used to inoculate 50 mL Erlenmeyer flasks containing 1 g of sterile rice hulls added with 3 mL of a sterile water solution of KH2PO<sup>4</sup> 2 g/L, NaCl 1 g/L, and MgSO<sup>4</sup> × 7 H2O 1 g/L. The SSF was maintained in static conditions, at 28 ◦C for 10 days, and was then inoculated in 100 mL of paper mill water and kept under stirring at 100 rpm, 28 ◦C for 5 days. One milliliter of the supernatant was withdrawn and 657 mg of (NH4)2SO<sup>4</sup> sodium was added. Once solubilized, the suspension was centrifuged (5600 RCF, 10 min). The precipitates were immediately resuspended in 10 mL of paper mill water. In the case of the addition of amylase, 100 U was added to the reaction. Reaction samples were taken starting from T0, every 24 h up to 72 h. All tests were carried out in triplicate for statistical significance. The relative standard deviation value for statistical analysis was also reported. All the collected samples were analyzed by the DNS assay for the quantification of reducing sugars.
