Hydrolysis of particulate organic matter is frequently the rate-limiting step during AD [227, 228]. Mechanical pretreatment of lignocellulose materials increased the hydrolysis and methane yield by 5-25% [229] and reduced the digestion time by 23-59% [230] compared to nontreated samples. Disintegration of WAS in an agita­tor ball mill Model LME 50 K with fine sand balls (0.5-0.9 mm of diameter and

2.7 kg/L of density) increased chemical oxygen demand (COD) solubilization by 25-31% and enhanced biogas yield by 38% compared to nontreated WAS [231]. Mechanical pretreatment of WAS in a high-pressure homogenizer (pressure up to 600 bar) increased biogas production by 18% [195].

Ultrasonic treatment has been widely studied as a method for enhancement of WAS solubilization and methane yield. The minimum energy level required to dis­rupt the activated sludge cell wall is in the range of 1,000-3,000 kJ/kg TS [197, 203] or 20-30 kJ/L [197, 232]. Ultrasonic pretreatment (£=40,000 kJ/kg SS) of WAS resulted in a fivefold increase in the amount of organic matter solubilization but had no influence on biogas yield [199]. Another study showed the same beneficial trend as solubilization increased by 127% at 42 kHz for 120 min [201]. Biogas and meth­ane yields were observed to increase by 20% [201] and by 50% (for biogas) at £s = 6,950 kJ/kg TS [197].

Mechanical pretreatment is required prior to AD of macroalgae and includes chopping (>5 mm), milling (1-5 mm), or homogenization (<1 mm). Grinding of the

Ulva did not influence the total methane yield but it increased the kinetics of hydro­lysis and methane production rate [129] . The grinding of the feedstock is likely more important for continuous stirred-tank reactor (CSTR) or semi-continuous reactors where the HRT is an important operating parameter.

One study examined the influence of mechanical disintegration plus ultrasonic treatments on A. maxima organic matter solubilization, VS reduction, and methane yield [233]. Ultrasonic treatment (Polytron generator PT20ST, from Brinkmann Instruments) increased the soluble COD (sCOD) 3.8-fold compared to freshly har­vested cyanobacteria. A. maxima COD solubilization increased from 21.3 to 76.7%. Surprisingly, VS reduction and methane yield were 0.9 and 0.85 times the values from fresh (not pretreated) biomass, respectively. Only hydrolytic bacteria and aci — dogens benefited from the larger amount of readily degradable substrate. The VFA concentration increased from 12 g/L in the digester with fresh algae to 46 g/L in the digester with pretreated algae. The lack of improvement in the methane yield might be due to the inhibition of methanogenic organisms from the high VFA concentration. Ultrasound pretreatment (19 kHz, treatment energy 1-5 Wh/L) had no influence on the methane yield from homogenized red macroalga Polysiphonia [123]. While the amount of biogas increased by 25-28%, the methane fraction dropped from 42-49 to 33-37%.