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Investigating the impact of hydrothermal pretreatment (HTP) on organic solubilization and anaerobic acidification of thickened waste activated sludge (TWAS) was the objective of this study. For this purpose, 15 different hydrothermal pretreatment scenarios were investigated. The HTP temperature, retention time, and pressure for this study extended from 150 to 240 °C, 5 to 30 min, and 69 to 488 psi respectively. Mesophilic batch acidification tests were conducted for all pretreated and raw samples. The highest solubilization percentage after HTP and acidification was observed at HTP condition of "200 °C–10 min–225 psi" and "180 °C–15 min–145 psi" accounting for 48% and 40% respectively. Moreover, The HTP condition of "190 °C–10 min–181 psi" indicated the highest volatile fatty acid (VFA) production yield of 5.38 g/L or 1076.6 mgVFAs/gVSSadded. For all hydrothermally pretreated samples, acetic acid was determined to be the most abundant VFA. There was no significant hydrogen produced during the acidification process. [ABSTRACT FROM AUTHOR]