Ultrasonication, enzymic hydrolysis and combinations of both pre-treatments were applied to sewage sludge with the aim of enhancing biogas production in the anaerobic treatment. Short-term monitoring of soluble COD (sCOD) and NH4-N (by keeping the pre-treated substrate under anaerobic conditions) was used to compare the pre-treatments. Five ultrasound energy inputs were applied: 3500, 7000, 10,500, 14,000 and 21,000 kJ/kg TS. Enzymic hydrolysis was achieved by promoting endogenous enzyme actions through the incubation of sludge (at 42 C over 48 h), resulting in a solid phase (top) and a liquid phase (bottom), in a process known as inverted phase fermentation (IPF). Ultrasonication produced a greater increase in sCOD (up to 532% for 7000 kJ/kg TS) than IPF (up to 324%). When applying both pre-treatments, if ultrasonication was applied first, sludge settlement occurred instead of the usual phase inversion that occurred when IPF was applied alone. When IPF was applied first, ultrasonication was only applied to the solid phase, as it was not necessary to apply it to the liquid phase on account of its high soluble organic matter. However, ultrasonication was not effective when applied to the solid phase, due to its high solid content. NH4-N increase was notable in all instances of pre-treatments. (c) 2013 Elsevier B.V. All rights reserved.