Plasma-generated reactive species in water: A sustainable strategy against Aeromonas hydrophila

Waterborne pathogens remain a stubborn problem in aquaculture, with implications for both ecosystem health and food safety. This study tested a plasma-based inactivation approach using plasma-activated water (PAW) generated by dielectric barrier discharge (DBD) plasma. Three plasma source configurations were evaluated - NO_x alone, O₃ alone, and a combined NO_x/O₃ source all run-on ambient air as the feed gas. Each produced a reactive gas mixture of NO, NO₂, N₂O, and O₃, which was diffused into water to yield a solution enriched with reactive oxygen and nitrogen species (RONS). Antimicrobial activity was tested against Escherichia coli, Staphylococcus aureus, and Aeromonas hydrophila - the last being a primary cause of motile Aeromonas septicemia in fish. PAW reduced planktonic bacterial counts by up to ~3 log CFU/mL and disrupted established biofilms. Mechanistic investigation combined IC₅₀ analysis, surface morphology scanning, live/dead fluorescence imaging, flow cytometry, and intracellular RONS quantification. These assays converged on membrane damage, oxidative and nitrosative stress, and loss of cellular integrity as the dominant modes of action. The results suggest plasma-generated PAW is a scalable, without use of harsh industrial chemicals option for pathogen control in aquaculture and water treatment settings.