Real-time monitoring of culture medium- and species-specific transformation of zinc oxide nanoparticles in bacterial systems using coupled CyElFFF-ICPMS

Zinc oxide nanoparticles (ZnO NPs) have emerged as promising nanoagrochemicals due to their high utility and efficiency. For the first time, we employed an online-coupled cyclical electrical field-flow fractionation and inductively coupled plasma mass spectrometry (CyElFFF-ICPMS) system for rapid (<10 min) simultaneous characterization and quantification of ZnO NPs and Zn(II) ions in aqueous matrices, achieving a detection limit of 0.03 μg/mL and 92 % recovery. CyElFFF-ICPMS analysis demonstrated that ZnO NPs remain well-dispersed in ultrapure water but formed surface coronas in yeast mannitol broth prior to precipitation, demonstrating culture medium-specific stability profiles. Crucially, we observed divergent transformation mechanisms between beneficial bacteria. Gram-positive bacteria ( Bacillus subtilis ) stabilized ZnO NPs via biomolecular corona formation, while Gram-negative bacteria ( Sinorhizobium fredii ) promoted extracellular ZnO NPs aggregation and dissolution via protein-rich extracellular polymeric substance secretion, as validated by 3D excitation-emission matrix fluorescence spectroscopy. Intracellular transformation diverged markedly after 6 h: B. subtilis reduced internalized Zn(II) to ZnO nanocomposites through activated the detoxification mechanism, contrasting with S. fredii 's extensive intracellular dissolution and extracellular precipitation. These findings demonstrate that bacterial species-specific interactions critically govern the stability, transformation, and intracellular fate of ZnO NPs, providing essential mechanistic insights imperative for rational design of nanoagrochemicals and their environmental risk assessment.