New Horizons in Micro/Nanoplastic-Induced Oxidative Stress: Overlooked Free Radical Contributions and Microbial Metabolic Dysregulations in Anaerobic Digestion

Excessive production of reactive oxygen species (ROS) induced by micro/nanoplastics (MPs/NPs) is highly toxic to microbes. However, the mechanisms underlying ROS generation and metabolic regulation within anaerobic guilds remain poorly understood. In this study, we investigated the effects of environmentally relevant levels of polypropylene (PP)-MPs/NPs on oxidative stress and microbial ecology during anaerobic digestion (AD). Electron paramagnetic resonance spectroscopy revealed that PP-MPs/NPs elevated the concentrations of environmentally persistent free radicals (EPFRs) and derived hydroxyl radicals (^•OH). EPFRs were identified as the primary contributors to ^•OH generation, as evidenced by a high Spearman correlation coefficient (r = 0.884, p < 0.001) and free radical-quenching studies. The formation of ^•OH enhanced ROS production by 86.2-100.9%, resulting in decreased cellular viability and methane production (by 37.5-50.5%) at 100 mg/g TS PP-MPs/NPs. Genome-centric metagenomic and metatranscriptomic analyses suggested that PP-MPs/NPs induced the reassembly of community structures, re-evolution of functional traits, and remodeling of interspecies interactions. Specifically, PP-MPs/NPs induced a shift in methanogen consortia from hydrogenotrophic Methanofollis sp. to acetoclastic and hydrogenotrophic Methanothrix soehngenii, primarily because of the latter's diverse ingestion patterns, electron bifurcation complexes, and ROS-scavenging abilities. Downregulation of genes associated with antioxidative defense systems (i.e., sodN, katA, and osmC) and ROS-driven redox signal transduction pathways (c-di-AMP and phosphorylation signaling pathways) provided insights into the mechanisms underlying ROS-induced microbial metabolic dysregulation. Our findings enhance the understanding of microbial ecological and metabolic traits under MPs/NPs stressors, facilitating the control of MPs/NPs toxicity and the stabilization of AD processes.