7th Asia-Pacific Conference on Plasma Physics, 12-17 Nov, 2023 at Port Messe Nagoya

The importance of cold plasma-generated short-lived reactive species, especiallysuperoxide anion radicals in antibiofilm activities

Binbin Xia1, Heema Kumari Nilesh Vyas1, Renwu Zhou2, Tianqi Zhang1, Jungmi Hong1, Joanna G.
Rothwell3, Scott A. Rice4, Dee Carter3, Kostya (Ken) Ostrikov5, Patrick J. Cullen1 and Anne
Mai-Prochnow1
1School of Chemical and Biomolecular Engineering, The University of Sydney, 2 State Key
Laboratory of Electrical Insulation and Power Equipment, Center for Plasma Biomedicine, Xi’an
Jiaotong University, 3 School of Life and Environmental Sciences, The University of Sydney, 4Agriculture and Food, Microbiomes for One Systems Health, Commonwealth Scientific andIndustrial Research Organisation, 5 School of Chemistry and Physics and QUT Centre for
Biomedical Technologies, Queensland University of Technology
e-mail (speaker): anne.mai-prochnow@sydney.edu.auIntroduction: Microbial biofilms are responsible for thecontamination of food, filters, membranes, andequipment as well as causing diseases. Thus, thesecommunities of microorganisms that live attached to asurface are a risk to human health and cost millions ofdollars in equipment repair and cleaning. A new reliable
decontamination method that does not cause resistance isneeded. Cold plasma or plasma-activated water (PAW)
exhibits powerful disinfectant activity. However, theoptimal generating conditions, such as the choice of gas
used to produce PAW, remain unclear.
Methods: Our study utilizes PAW generated with abubble spark discharge (BSD) reactor connected todifferent gas sources (argon, nitrogen, oxygen, andatmospheric air). Biofilms of Escherichia coli that weregrown on stainless-steel surfaces were treated in situ to
optimize the biofilm removal efficacy. Antimicrobialactivity was measured using colony forming units (CFU)
counts, microscopic imaging, and intracellular ROS
measurements. The PAW was characterized by OES,
scavenger assays and electron paramagnetic resonance.
Results: We demonstrate that PAW generated using
oxygen (PAW-O2) is the most effective and cancompletely remove E. coli biofilms on stainless steel
surfaces (Fig. 1).
Fig. 1: Reductionof E. coli biofilms
using PAWgenerated inargon, nitrogen,air, and oxygen
assessed by CFU

OES measurements support the experimental
observations on the generation of reactive species suchas OH*, O2-, NO*, and excited atoms in the gas phase of
BSD plasma, which then leads to the solvation anddiffusion of RONS at the bubble-water interface andformation of activated water. In the oxygen BSD plasma,various forms of reactive oxygen species can begenerated including excited oxygen molecular andatomic species, ions and ozone, superoxide anion
radicals and hydrogen radicals through the interaction
with water or its derivatives such as OH- or HO2 [1].
Moreover, for the first time, we show that a short-lived
reactive oxygen species, the superoxide anion radical(•O2-), plays an important role in the inactivation of E.
coli biofilms. Using scavengers and electron
paramagnetic resonance spectra the superoxide anion
radical is detected and shown to cause microbial biofilm
killing (Fig. 2)

Fig. 2: (I) Superoxide detection using electron paramagnetic
resonance spectra A MQ water control; B. PAW-O2 C. PAW-O2+Tiron. (II) The addition of the superoxide scavenger Tiron
prevents bacterial killing of PAW

Conclusion: The results of our study show that PAW
generating input gas is important for RONS generation.
This has implications for biofilm removal strategies,
including drinking and wastewater pipes, filter, andmembrane cleaning, but also equipment decontaminationand sterilization of food products or medical devices
where conventional disinfection is not suitable [2].
References[1] Bolouki, et al. (2021) Applied Sciences 11(13)[2] Xia, et al. (2023) Journal of Environmental ChemicalEngineering