Antimicrobial efficacy and cytotoxic assessment of plasma-activated water generated by a dielectric barrier discharge microbubble system

Abstract

The growing threat of antimicrobial resistance has intensified the search for alternative disinfection strategies. Plasma-activated water (PAW), enriched with reactive oxygen and nitrogen species (RONS), has emerged as a promising non-antibiotic antimicrobial approach. In this study, PAW was generated using a bench-scale dielectric barrier discharge (DBD) reactor coupled with a microbubble diffusion system, operating with compressed ambient air at 8–9 kV, frequencies of 40 kHz, 1.25 W, air flow rate of 5 L/min with activation times of 7 and 14 min. In the generated PAW, ROS were produced in higher abundance than RNS, with hydrogen peroxide (H2O2) and ozone (O3) representing the dominant species contributing to the antimicrobial activity, alongside detectable levels of nitrite (NO2–), nitrate (NO3–), and nitrous acid (HNO2). Physicochemical characterization included measurements of pH, oxidation-reduction potential (ORP), conductivity, and total dissolved solids (TDS), as well as qualitative analysis of RONS using UV–Vis spectrophotometry (190–400 nm), for detection of diagnostic wavelengths, with quantitative assessment based on colorimetric methods for H2O2, NO2–, and NO3–, and a photometric assay for O3. Antimicrobial activity was evaluated against Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 25922), and Candida albicans (ATCC 10231) using PAW generated at 7 and 14 min of activation. In addition, cytotoxicity was evaluated in L929 fibroblasts following ISO 10993-5 criteria, and cell viability remained above the 70% threshold after exposure to PAW. Morphological assessments were performed in both L929 and B16F10 cells using fluorescence microscopy to examine potential differential cellular responses. These findings indicate that longer activation times enhanced antimicrobial activity while preserving fibroblast viability, supporting the potential of PAW as an effective and safe alternative for biomedical applications.