Physicochemical and antimicrobial characterization of nanobubbles reveals physical disruption is the primary mode of biofilm inactivationNorthage, Naomi (Avtor) Gomilšek, Matjaž (Avtor) Modic, Martina (Avtor) Vengust, Damjan (Avtor) Zorko, Andrej (Avtor) Cvelbar, Uroš (Avtor) Walsh, James L. (Avtor) nanobubblesultrafine bubblesreactive oxygen specieshydroxyl radicalsbiofilmsBiofilm-associated contamination represents a persistent and costly challenge across environmental systems, causing reduced efficacy of disinfectants. Recently, nanobubbles (NBs) have shown promise for biofilm decontamination; yet, their underpinning mode of action remains a topic of debate. In this study, the interaction of air-generated NBs with Escherichia coli and Staphylococcus aureus biofilms was investigated. NBs were generated using a venturi nozzle and characterized using Nanoparticle Tracking Analysis, revealing a NB density of 5.66 × 108 particles/mL and a mean diameter of 84 nm. Application of NB solution to microbial biofilms resulted in a 2.16 log reduction for E. coli and 1.52 log reduction for S. aureus, along with visible morphological changes such as cell collapse, wrinkling, and matrix disruption. ESR spin trapping confirmed hydroxyl radical formation, but intracellular ROS and lipid peroxidation levels were minimal and, in some cases, not significantly different from Milli-Q water controls. After 28 days, NBs remained present and continued to demonstrate antimicrobial activity, biofilm disruption, and some ROS activity. These findings indicate that although hydroxyl radicals are generated, oxidative stress is not the dominant antimicrobial mechanism under the examined conditions, suggesting physical biofilm disruption is the primary mode of action.American Chemical Society20262026-06-10 10:08:01Neznano29951ZDAsl© 2026 The Authors.