Comprehensive insights into the impact of pretreatment on anaerobic digestion of waste active sludge from perspectives of organic matter composition, thermodynamics, and multi-omics

• The pretreatment mechanisms were revealed in terms of thermodynamics and multi-omics • The protein secondary structure of WAS became loose after pretreatment • Alkali and thermal pretreatments significantly disrupted the flocculation of WAS • Vitamin metabolism was a vital process enhanced by all pretreatments • Different pretreatments respond differently to specific organic matters in WAS Although various pretreatments have been applied to promote the anaerobic digestion of waste active sludge (WAS), the mechanisms regarding the impact of pretreatment on anaerobic digestion have not been well addressed. In this study, the effects of acid, alkali, and thermal pretreatments on anaerobic digestion of WAS were comprehensively investigated from the perspectives of organic matter composition, thermodynamics, and multi-omics. Results showed acid, alkali, and thermal pretreatments increased the methane production potential of WAS by 53.7%, 98.2%, and 101.8%, respectively, compared with the control. The protein secondary structure was disrupted after pretreatment, with a shift from α-helix and β-sheet to random coil and antiparallel β-sheet/aggregated strands. Thermodynamically, the WAS flocculation process was controlled by the short-range interfacial interactions described by the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory, which was positively correlated (R = 0.97, p < 0.05) with the organic matter solubilization of the WAS. After pretreatment, the flocculation energy barrier of pretreated WAS was 4.1 (acid), 7.0 (alkali) and 7.1 (thermal) times higher than that of the control group, respectively. Multi-omics analysis confirmed that pretreatment promoted amino acids (tryptophan, tyrosine, phenylalanine, aspartate, glutamate) metabolism, energy metabolism (ABC transporters) and vitamin metabolism. Moreover, the comparison of upregulated differentially expressed proteins (DEPs) revealed that for amino acid metabolism, thermal treatment had the best promotion effect; for carbohydrate metabolism, alkali treatment had the best promotion effect; and for lipid metabolism, acid treatment was more advantageous, resulting in different anaerobic digestion efficiencies. This study provides an in-depth understanding of the impact of different pretreatments on WAS anaerobic digestion and has practical implication for the choice of proper pretreatment technology for biosolids.