Batch Anaerobic Digestion. Batch fermentation assays under mesophilic conditions (37 ± 1°C) were conducted to evaluate the biogas production from raw and pretreated sludge samples. Sealed 120 mL serum bottles were used as anaerobic reactors. A working volume of 60 mL was kept in all batches. WAS sample (substrate) and inocula were introduced to the reactor with the same proportion keeping a VS substrate/VS inocula ratio equal to 1 [22]. The pH of anaerobic medium was adjusted to 7.2. To supply anaerobic condition, all batches were purged with a gas mixture of 75% N2 and 25% CO2 for 2–3 min. Control batch was conducted only with inocula in order to estimate the biogas production resulting from the seeding material. All anaerobic batch tests were conducted in duplicate.