Potassium iodide enhances the antimicrobial activity of plasma-activated water

Abstract

Plasma-activated water (PAW) is a promising disinfection strategy that generates a complex mixture of reactive oxygen and nitrogen species (ROS/RNS), including hydrogen peroxide (H₂O₂), nitrate (NO₃ ^-), and transient oxidants, in an acidic aqueous environment. These reactive species contribute to both immediate and extended antimicrobial activity. This study investigates how the addition of low concentrations (<100 μM) of potassium iodide (KI) enhances the bactericidal properties of spark-generated PAW by enabling the in-situ generation of reactive iodine species (RIS), particularly hypoiodous acid (HIO), under acidic conditions. KI addition (10-100 μM) led to a counterintuitive, dose-dependent increase in H₂O₂ concentrations, from ∼1.2 mM in PAW alone to ∼1.8 mM at 30 μM KI, possibly due to iodine-mediated catalytic effects or reduced H₂O₂ degradation. NO₃ ^- levels also increased by ∼17 % with increasing KI. Equivalent concentrations of H₂O₂ + KI failed to replicate the rapid antimicrobial activity observed in PAW + KI, which achieved complete inactivation of Escherichia coli and Listeria monocytogenes planktonic cells within 3 min, compared to over 10 min for PAW alone, indicating the involvement of additional reactive species in KI-enhanced antimicrobial activity of PAW. However, Salmonella enterica planktonic cells exhibited only partial inactivation even with KI, indicating species-specific tolerance under these conditions. 24h biofilms of L. monocytogenes and E. coli were eradicated with PAW + KI in 10 min, whereas S. enterica showed only a 2-log reduction. Scavenger assays revealed that both longer-lived species (H₂O₂) and shorter-lived oxidants such as singlet oxygen are essential for this enhanced killing, while ozone and superoxide appeared dispensable. These findings support a multi-step antimicrobial mechanism: (1) plasma treatment creates a low pH, H₂O₂-rich solution; (2) iodide is oxidised to RIS such as I₃ ^- and HIO; (3) additional PAW-derived oxidants potentiate RIS chemistry; and (4) unionised HIO diffuses across bacterial membranes to induce oxidative damage. PAW-KI remained stable for at least 14 days at 4 °C, with sustained RIS activity and minimal loss of H₂O₂ or NO₃ ^-, suggesting preserved antimicrobial capacity over time. The antimicrobial mechanism likely proceeds through a four-step pathway: plasma-mediated generation of H₂O₂ and NO₃ ^-; oxidation of I^- to I₂ and HIO; potentiation of RIS via PAW-derived ROS/RNS; and subsequent microbial inactivation via membrane damage. Together, these results demonstrate that PAW + KI forms a powerful, in situ RIS-generating system, offering a residue-minimising and environmentally sustainable disinfection platform. Its rapid action, scalability, and reliance on only air, water, electricity, and GRAS-listed KI make it an attractive intervention for food safety, clinical disinfection, and decentralised sanitation settings.

Keywords: Biofilms; Cold plasma; Food pathogens; Hypoiodous acid; Plasma-activated water; Potassium iodide; Salmonella.

Fig. 1

Setup of Spark (A) and Glow (B) discharge to make PAW. The plasma from the different electrical set-up generates a visually different plume. Created in BioRender. Mcclenaghan, L. (2025) https://BioRender.com/d57a154.

Fig. 2

Generation of H₂O₂ (A), NO₃⁻ (B), NO₂⁻ (C) and the change in pH (D) of deionised water following treatment with either a Spark or Glow discharge. The Spark PAW contains H₂O₂ and NO₃⁻, while the Glow PAW contains NO₃⁻ and NO₂⁻ (n = 3).

Fig. 3

The H₂O₂ (A) and NO₃⁻ (B) measurements and pH changes (C) for PAW and PAW + KI. The addition of KI caused an increase in both H₂O₂ and NO₃⁻ species, whereas the pH remained relatively the same throughout. (n = 3).

Fig. 4

The antimicrobial efficacy of PAW and PAW + KI against E. coli (A), L. monocytogenes (B) and S. enterica (C) in planktonic phenotype. Spark PAW only , PAW +10 μM PAW +50 μM , 10 μM KI only , and 50 μM KI only . The antimicrobial efficacy of PAW and PAW + KI against E. coli (D), L. monocytogenes (E) and S. enterica (F) 24 h biofilms grown on polycarbonate coupons. Spark PAW only , PAW +50 μM , and PAW +100 μM . (n = 3).

Fig. 5

CLSM images of S. enterica (top row) and E. coli (bottom row) biofilms following PAW + 100 μM KI treatment for 0-, 3-, and 5-min. Biofilms were stained with LIVE/DEAD stain, where green indicates viable cells and red indicates membrane-compromised or dead cells, Images show increasing cell death over time, particularly in E. coli. Scale bars = 50 μm.

Fig. 6

Impact of chemical scavengers on the antimicrobial activity of PAW, PAW + KI (50 μM) and PAW + KI (100 μM) with scavengers targeting H₂O₂ (sodium pyruvate), singlet oxygen and other longer-living ROS (l-histidine), ozone (uric acid), superoxide anions (O₂⁻) (Tiron) (A). pH adjusted PAW + KI (50 μM) planktonic E. coli kill curve. The pH of the PAW + KI was adjusted post plasma exposure to inadvertently assess whether ionised iodine was the active antimicrobial agent, due to the pKa of HIO being approximately 10.5. No reduction in E. coli was observed when the pH was adjusted to pH of 5, 7 or 10, suggesting that unionised HIO is the main antimicrobial agent in PAW + KI. (n = 3). (B). Assessing the antimicrobial activity of H₂O₂ (1.2 mM) and of H₂O₂ (1.2 mM) in combination with either 50 μM or 100 μM KI in planktonic culture. The presence of KI was shown not to enhance the antimicrobial effect of H₂O₂, suggesting that it is the unique combination of reactive species within PAW which is responsible for this effect (n = 3). (C). Proposed reaction mechanism of the PAW and KI system (D). (I) Cold plasma exposure of water in the presence of ambient air (O₂/N₂) generates H₂O₂, protons (H⁺), NO₃⁻, and a variety of transient ROS/RNS. (II) In the acidic environment of PAW, I⁻ from KI are oxidised by H₂O₂ to I₂. (III) I₂ reacts with excess I⁻ to form triiodide (I₃⁻). (IV) I₂ can also hydrolyse to form HIO, a potent antimicrobial species. (V) Over time, HIO may be further oxidised by H₂O₂ to iodate (IO₃⁻), an inert end-product.

Fig. 7

Antimicrobial activity of PAW + KI after storage at 4 °C for 7 days against planktonic cells of E. coli (A) and L. monocytogenes (B). The stability of H₂O₂ (C) and NO₃⁻ (D) measurements and pH changes (E) for PAW and PAW + KI stored at 4 °C. The addition of KI caused an increase in both H₂O₂ and NO₃⁻ species, whereas the pH remained unchanged throughout (n = 3).