Redox homeostasis governs anaerobic microbial stability: mechanistic insights from selective ROS scavenging under microplastic stress.

Redox homeostasis is fundamental to microbial functions in anaerobic ecosystems. Although microplastics (MPs) induce oxidative stress and reactive oxygen species (ROS) accumulation, the regulatory role and reversibility of oxidative stress in microbial functional stability remain unresolved. Here, we employed Cu/Zn-MOF nanozyme for selective ROS scavenging, combined with metagenomics and biochemical analyses, to elucidate how oxidative stress contributes to PS-MPs induced anaerobic microbial dysfunction. EPR spectroscopy revealed that PS-MPs exposure promoted environmentally persistent free radical accumulation in the digestate (4.26 × 1014 spins/g) and promoted the O2∙- generation, resulting in sustained ROS accumulation (> 120% of the control). This oxidative stress impaired microbial viability and reduced cumulative methane production by 22.7% compared to the control (CK). Metagenomic analysis revealed that PS-MPs decreased the relative abundance of key methanogens (Methanothrix sp. and Methanobacterium sp.) and genes associated with Fe-S cluster assembly, antioxidant defense, VFAs conversion, and methanogenesis. ROS regulation by Cu/Zn-MOF (0.25 mg/g-TS) alleviated these metabolic constraints while PS-MPs remained present. Low dose Cu/Zn-MOF was associated with recovery of Fe-S cluster assembly-related functional potential and methanogenesis-related genes, increased the maximum methane production rate from 14.31 to 21.74 mL CH4/g-VS/d, and enhanced methanogen-centered microbial network connectivity. These findings identify oxidative stress as a reversible regulatory node affecting anaerobic microbial stability and highlight targeted redox regulation as a strategy to enhance the resilience of ROS sensitive biological systems.