Peracetic acid (PAA) oxidation, which is a kind of chemical method for sludge pretreatment, has been verified to be valid for promoting sludge anaerobic digestion performance. However, the methane production is still limited at certain levels by this method, because excess PAA has negative effects on methanogens. This work selected a freezing method combined with PAA to form a composite sludge pretreatment technology for synergistically improving the biomethane production. According to the experimental data, the methane yield was largely enhanced from 166.4 ± 5.6 mL/g volatile suspended solids (VSS) in the control to 261.5 ± 7.3 mL/g VSS by the combined freezing (−10 °C) and PAA (0.08 g/g TSS) pretreatment, with a 57.2% increase rate. Kinetic analysis showed that the methane production potential, methane production rate, and hydrolysis rate were promoted, respectively, from 159.4 mL/g VSS, 17.18 mL/g VSS/d, and 0.104 d−1 to 254.9 mL/g VSS, 25.69 mL/g VSS/d, and 0.125 d−1 by the freezing + PAA pretreatment. Mechanism analysis revealed that the freezing + PAA pretreatment destroyed both extracellular polymeric substances (EPS) and microbial cells in the sludge, resulting in the increase in hydrolysis efficiency. Gene analysis showed that the hydrolytic microbes (Hyphomicrobium and norank_f_Caldilineaceae), acidogens (e.g., Petrimonas, Tissierella, and Mycobacerium) and methanogens (Methanosaeta, Methanosarcina, and Methanobacterium) were all enriched by the freezing + PAA pretreatment, with the total abundances calculated to be 10.65% and 22.07% in the control and pretreated reactors, respectively. Considering both technical and economic factors, the freezing + PAA method is feasible for sludge pretreatment.
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