Biological Responses of <i>Bacillus subtilis</i> toward Nanoplastics under Nutritional Stress in Freshwater Ecosystems

Nanoplastics (NPs) are widely distributed in freshwater ecosystems, and most freshwater ecosystems are poor in nutrients. This work thus focused on the responses of bacteria to NPs under nutritional stress. It is found that polystyrene (PS) NPs with various sizes (20 and 80 nm) and surface functionalities (carboxyl-, amine-modified, and naked) ultimately exhibited toxicity in nutrient-poor media. Even amine-modified PS NPs of 20 nm at microgram per liter concentrations (e.g., 2 μg/L) could reduce bacterial growth after prolonged exposure (e.g., 144 h) during starvation. Furthermore, in the presence of natural organic matter, NPs decreased bacterial cell numbers in natural freshwater more than in experimental nutrient-poor media. Transcriptome sequencing and other biological analyses indicated that the adaptive secretion of extracellular polymeric substances (EPS) initially protected cells from NPs, while the upregulation of the fatty acid biosynthesis pathway facilitated energy storage for promoting cell growth. However, bacterial defense mechanisms were eventually downregulated, and EPS secretion became unsustainable. Consequently, cell deformation, membrane injury, and reactive oxygen species overproduction led to irreversible cell death. This study addresses the adverse effect of NPs to bacteria under nutrient-poor conditions and highlights the potential ecological risk of NPs at environmentally relevant concentrations in freshwater systems.