Amine-Containing Micropollutants Exposure Reshapes Sludge Anaerobic Digestion via Enzymatic Inhibition and Stress-Mediated Alteration of Methanogenic Pathways

Amine-containing micropollutants (AMPs), a class of structurally diverse polar compounds characterized by one or more amine functional groups, are frequently detected in wastewater sludge. However, the anaerobic transformation of these compounds and their impacts on microbial metabolism during anaerobic digestion (AD) remain poorly understood. In this work, six representative AMPs were selected to cover 16 structurally diverse primary, secondary, tertiary amine, and quaternary ammonium functionalities. α-C hydroxylation and N-acetylation were identified as the dominant initial reactions among the detected transformation products (TP), collectively accounting for 42.6% of all identified TPs. Furthermore, compound-specific differences in metabolic disturbance were observed. Quaternary ammonium compounds, N-dodecyl-N-benzyl-N,N-dimethylammonium chloride (DDBAC) and N,N-Didodecyl-N,N-dimethylammonium chloride (DDDAC) markedly reduced acetate kinase activity by 10.69 and 14.28%, respectively, and resulted in methane production yield reductions of 88.97 and 88.19%. The genome-centric metagenome revealed that exposure to AMPs prompted the reassembly of the microbial community, altered its functional attributes, and disturbed interspecies cross-feeding interactions. Specifically, AMPs triggered a shift in the methanogenic consortium from mixotrophic Methanosarcina flavescens to hydrogenotrophic Methanobacterium sp., owing to the latter's metabolic versatility, vigorous proliferation, and superior energy conservation. These findings indicated that the chemical properties of amine functional groups have effects on anaerobic biotransformation pathways and microbial energy metabolism, providing mechanistic insight into AMPs toxicity and guiding mitigation strategies to enhance the stability and resilience of full-scale AD systems.