Mechanistic insights into the adaptive responses of Escherichia coli to repeated nanoscale zero-valent iron exposure.

With the expanding deployment of nanoscale zero-valent iron (nZVI) in groundwater remediation, the frequency and duration of microbial exposure in aquatic niches are steadily increasing. Despite extensive research on nZVI-induced toxicity, microbial survival strategies and adaptive resistance mechanisms under repeated or prolonged nZVI exposure remain largely uncharacterized, particularly in anaerobic subsurface environments. Herein, using Escherichia coli as a model Gram-negative facultative anaerobe, we systematically explored bacterial responses to repeated nZVI exposure under aerobic and anaerobic conditions. After 20 consecutive nZVI exposure-cultivation cycles, bacterial strains developed stronger resistance under anaerobic conditions than those under aerobic ones. Mechanistically, the resistant strain exhibited increased biofilm biomass and thickness (1.55-2.00-fold those of the original strain, p < 0.001), reduced intracellular Fe2+ accumulation, elevated catalase activity, and enhanced maintenance of membrane potential and lipid homeostasis. Transcriptomic profiling further revealed that nZVI repressed ribosomal and energy metabolic functions, while activating stress-responsive pathways (e.g., two-component systems, transport, global metabolic regulation), thereby mediating resistance via coordinated multimodal mechanisms. Notably, nZVI-resistant strains exhibited cross-resistance to Ag and Cu nanoparticles, together with stable phenotypic inheritance after subculturing and potential intraspecific transferability in conjugation assays. This study reveals the microbial resistance-associated mechanisms under repeated nZVI exposure in a model system, providing a mechanistic basis for assessing the ecological safety of nZVI and optimizing its environmental applications.