Interactions of plasma-activated water with biofilms: inactivation, dispersal effects and mechanisms of action

Abstract

Biofilms have several characteristics that ensure their survival in a range of adverse environmental conditions, including high cell numbers, close cell proximity to allow easy genetic exchange (e.g., for resistance genes), cell communication and protection through the production of an exopolysaccharide matrix. Together, these characteristics make it difficult to kill undesirable biofilms, despite the many studies aimed at improving the removal of biofilms. An elimination method that is safe, easy to deliver in physically complex environments and not prone to microbial resistance is highly desired. Cold atmospheric plasma, a lightning-like state generated from air or other gases with a high voltage can be used to make plasma-activated water (PAW) that contains many active species and radicals that have antimicrobial activity. Recent studies have shown the potential for PAW to be used for biofilm elimination without causing the bacteria to develop significant resistance. However, the precise mode of action is still the subject of debate. This review discusses the formation of PAW generated species and their impacts on biofilms. A focus is placed on the diffusion of reactive species into biofilms, the formation of gradients and the resulting interaction with the biofilm matrix and specific biofilm components. Such an understanding will provide significant benefits for tackling the ubiquitous problem of biofilm contamination in food, water and medical areas.

Fig. 1. Generation of plasma-activated water.

Discharge over the water surface with indirect contact of the plasma plume with the water surface (a) and direct contact of the plasma plume with the water surface (b).

Fig. 2. Example of plasma water reactor.

Plasma bubble formation in a water reactor (a) and formation of reactive species in a plasma bubble (b). When a large number of bubbles form during plasma generation, the mass transfer of active species from the gas (bubbles) to the liquid is significantly enhanced compared to a plasma discharge at the surface of the liquid. This process leads to higher activity of the resulting PAW.

Fig. 3. Concentrations of four kinds of representative, long‐lived aqueous reactive species (RS; H₂O₂, NO₃⁻, NO₂⁻ and O₃) in plasma‐activated water induced by the air plasma.

Each point represents the mean of nine values ± standard deviation. The significance level ***p < 0.001 for each RS concentration at every plasma inducement time, except for H₂O₂ at the plasma inducement time of 15 min (**p < 0.01). Reprinted with permission from ref. .

Fig. 4. Reactive oxygen species (ROS) pathways.

Pathways of: a H₂O₂, b O₃, c ∙OH, d HO₂, e O, f ¹0₂, g O₂^*−. Reactions show the possible formation and destruction of ROS in discharge with water.

Fig. 5. Reactive nitrogen species (RNS) pathways.

Pathways of: a NO, b NO₂⁻, c NO₃⁻, d ONOOH. Reactions show the possible formation and destruction of RNS in discharge with water.

Fig. 6. Oxygen diffusion within a microcolony.

Oxygen contours and local gradients in a cross section of a 160-µm-thick biofilm. The positions of the cell clusters, shown as orange areas, were determined by microscopic observation. Numbers within the figure and on the right margin indicate the local oxygen concentrations (mM). Reprinted with permission from ref. .

Fig. 7. Proposed mechanism of interaction of PAW with biofilms relying on active species to disrupt the matrix leading to effective biofilm dispersal.

Under non-plasma conditions an oxygen gradient will form in a microcolony (a). Interaction of PAW with microbial biofilms will generate a range of active RONS that can penetrate into the interior of microcolonies and kill biofilm cells. RONS will also lead to the disruption of the biofilm matrix and thus releasing cells from the biofilm interior (b).