Temperature-phased anaerobic digestion with a 50–70 °C pre-treatment is widely proposed for sludge. Here, such a sludge pre-treatment (65 °C) was studied against the physical, enzymatic and biodegradation processes. The soluble and particulate fractions were analysed in terms of biochemical composition and hydrolytic enzymatic activities. Two kinetics of organic matter solubilisation were observed: a rapid transfer of the weak-linked biopolymers to the water phase, including sugars, proteins or humic acid-like substances, to the water phase, followed by a slow and long-term solubilisation of proteins and humic acid-like substances. In addition, during the heat treatment a significant pool of thermostable hydrolytic enzymes including proteases, lipases and glucosidases remains active. Consequently, a global impact on organic matter was the transfer of the biodegradable chemical oxygen demand (COD) from the particulate to the soluble fraction as evaluated by the biological methane potential test. However, the total biodegradable COD content of the treated sludge remained constant. The heat process improves the bio-accessibility of the biodegradable molecules but doesn't increase the inherent sludge biodegradability, suggesting that the chemistry of the refractory proteins and humic acids seems to be the real limit to sludge digestion.

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