Experimental evolution of Bacillus inaquosorum reveals multi-omics adaptation for polystyrene nanoplastic bioremediation

• Experimental evolution enhanced B. inaquosorum growth on nanoplastics • Parallel mutations emerged in metal transport, stress, and metabolic genes. • Transcriptomics revealed 24 plastic-responsive genes across five functional modules. • Genes clustered into five functional modules: attachment, hydrolysis, oxidation, sulfur mobilization, and export. • These modules reveal candidate pathways for nanoplastic bioremediation. Plastic pollution poses a major ecological threat, yet the genetic mechanisms enabling microbial nanoplastic degradation remain poorly understood. Here, we applied experimental evolution to a soil-derived Bacillus inaquosorum strain exposed to additive-free polystyrene nanoplastic over ∼210 generations. Three replicate lines were serially transferred in minimal medium containing glucose and nanoplastics, with controls evolved on glucose alone. Plastic-selected populations reached up to 22-fold higher cell densities on nanoplastic relative to the ancestor while maintaining fitness in glucose-only medium. Whole-genome resequencing revealed parallel mutations in genes linked to metal transport ( mntH, czrA ), oxidative stress regulation (clpC ), and central metabolism ( pyk ). Transcriptomic profiling identified 24 nanoplastic-responsive genes, including hydrolases, oxidoreductases, sulfur metabolism enzymes, and transporters, which clustered into functional modules representing attachment, polymer hydrolysis, monomer oxidation, sulfur mobilization, and export. Integration of genomic and transcriptomic data revealed regulatory mutations aligned with plastic-inducible pathways, suggesting coordinated adaptation. These findings demonstrate that B. inaquosorum adapts to polystyrene nanoplastic through multi-level genetic and regulatory changes that promote metabolic plasticity, stress tolerance, and metabolite transport. This study provides the first integrated multi-omics view of microbial evolution under pristine nanoplastic selection and identifies candidate pathways for biotechnological applications in plastic bioremediation. .