Microplasma Bubbles: Reactive Vehicles for Biofilm Dispersal

Interactions between effects generated by cold atmospheric-pressure plasmas and water have been widely investigated for water purification, chemical and nanomaterial synthesis, and, more recently, medicine and biotechnology. Reactive oxygen and nitrogen species (RONS) play critical roles in transferring the reactivity from gas plasmas to solutions to induce specific biochemical responses in living targets, e.g., pathogen inactivation and biofilm removal. While this approach works well in a single-organism system at a laboratory scale, integration of plasma-enabled biofilm removal into complex real-life systems, e.g., large aquaculture tanks, is far from trivial. This is because it is difficult to deliver sufficient concentrations of the right kind of species to biofilm-covered surfaces while carefully maintaining a suitable physiochemical environment that is healthy for its inhabitants, e.g., fish. In this work, we show that underwater microplasma bubbles (generated by a microplasma-bubble reactor that forms a dielectric barrier discharge at the gas-liquid interface with the applied voltage of 4.0 kV) act as transport vehicles to efficiently deliver reactive plasma species to the target biofilm sites on artificial and living surfaces while keeping healthy water conditions in a multispecies system. The as-generated air microplasma bubbles and plasma-activated water (PAW) both can effectively reduce the existing pathogenic biofilm load by ∼83 and 60%, respectively, after 15 min of discharge at 40 W and prevent any new biofilm from forming. The generation of underwater microplasma bubbles in a custom-made fish tank for less than a minute per day (20 s per time, twice daily) can introduce sufficient quantities of RONS into PAW to reduce the biofilm-infected area by ∼80-90% and improve the health status of Cichlasoma synspilum × Cichlasoma citrinellum blood parrot cichlid fish. Species generated include hydrogen peroxide, ozone, nitrite, nitrate, and nitric oxide. Using mimicked chemical solutions, we show that the plasma-induced nitric oxide acts as a critical bioactive species that triggers the release of cells from the biofilm and their inactivation.