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OPEN Antimicrobial effects of airborne

acoustic ultrasound and plasma
activated water from cold
and thermal plasma systems
on biofilms
Clémentine M. G. Charoux1,2, Apurva D. Patange1*, Laura M. Hinds1,2, Jeremy C. Simpson3,
Colm P. O’Donnell2 & Brijesh K. Tiwari1,2

Bacterial biofilms are difficult to inactivate due to their high antimicrobial resistance. Therefore,
new approaches are required for more effective bacterial biofilm inactivation. Airborne acoustic
ultrasound improves bactericidal or bacteriostatic activity which is safe and environmentally friendly.
While, plasma activated water (PAW) is attracting increasing attention due to its strong antimicrobial
properties. This study determined efficacy of combined airborne acoustic ultrasound and plasma
activated water from both cold and thermal plasma systems in inactivating Escherichia coli K12
biofilms. The application of airborne acoustic ultrasound (15 min) alone was significantly more
effective in reducing E. coli counts in 48 and 72 h biofilms compared to 30 min treatment with PAW.
The effect of airborne acoustic ultrasound was more pronounced when used in combination with
PAW. Airborne acoustic ultrasound treatment for 15 min of the E. coli biofilm followed by treatment
with PAW significantly reduced the bacterial count by 2.2—2.62 ­Log10 CFU/mL when compared to
control biofilm treated with distilled water. This study demonstrates that the synergistic effects of
airborne acoustic ultrasound and PAW for enhanced antimicrobial effects. These technologies have the
potential to prevent and control biofilm formation in food and bio-medical applications.

Biofilms are an assemblage of surface-associated microbial cells that are enclosed in a self-produced extracel-
lular polymeric substance matrix; which is composed of polymeric conglomeration of extracellular polysac-
­ NA1. Due to the complexity of their structure, biofilms exhibit higher antibiotic
charides, proteins, lipids and D
resistance to disinfection processes than planktonic c­ ells2. Biofilms can form on both biotic and abiotic surfaces
and are prevalent throughout industrial e­ nvironments3,4. Various techniques for removal of the clustered cells
exist, mostly involving chlorinated sanitizers, organic acids and other chemical products. However, most of
the chemical agents either require high concentration or are ineffective to penetrate into the whole biofilm
­structure5,6. While, some chemical agents like chlorine based products have resulted in health and environmental
issues associated with chemical residues and production of toxic by-products7. Poorly maintained equipment,
unhygienic conditions, and improper cleaning procedures in food manufacturing environments may increase
the tolerance of microbial biofilms to disinfectants. Bacterial populations are known to carry genes that may
protect the bacteria under adverse conditions. For instance, the presence and distribution of qacH and bcrABC
genes in Listeria monocytogenes ensure survival and growth of this strain when subjected to sub-lethal levels of
disinfectants based on quaternary ammonium c­ ompounds8.
Food contact surfaces are a major source of food product microbial contamination during processing, trans-
port and storage. Stainless steel is a common food contact surface for biofilm adhesion in the food i­ndustry9.
Airborne acoustic ultrasound employs non-contact transducers which are capable of transmitting ultrasonic
waves to the product using air as the coupling m ­ edium10. In recent years, the design of airborne acoustic ultra-
sonic devices has been significantly advanced and successfully demonstrated in various food industry applications

1
Department of Food Chemistry and Technology, Teagasc Food Research Centre, Ashtown, Dublin,
Ireland. 2School of Biosystems and Food Engineering, University College Dublin, Dublin, Ireland. 3School of Biology
and Environmental Science, University College Dublin, Dublin, Ireland. *email: [email protected]

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Figure 1.  Microbial count after biofilm detachment of ultrasonic-treated samples. “Black square” 48 h biofilm
“Grey square” 72 h biofilm. C: control; AA: Airborne acoustic ultrasound 15 min. a,bDifferent letters on column
are significantly different (p < 0.05). A, B Different letters on column are significantly different (p < 0.05). Error
bars stand for standard deviation.

including; drying, defoaming and decontamination. However, to date, it has not been employed for microbial
biofilm disruption.
Plasma is defined as a partially or wholly ionized gas composed of positive and negative ions, electrons,
photons, free radicals and neutrons atoms and m ­ olecules11. Plasma can be categorised into thermal (equilib-
rium) and non-thermal (non-equilibrium) p ­ lasma12. Thermal plasma has all constituent species, electrons, ions
at same temperature, thus in thermodynamic temperature equilibrium state. Whereas, non-thermal plasma is
characterised by higher temperature of electrons compared to other active species within plasma i.e. they are
in non-equilibrium ­state13. The cooling effect of ions and uncharged particles are much effective than energy
transfer from electrons, therefore the gas still remains in low temperature, therefore they are also referred as
cold ­plasma13. Plasma has been mainly used in bio-medical, textile and polymer i­ ndustries14–16. However, recent
studies have demonstrated potential applications of plasma technology in the food ­industry17,18. There is a range
of different approaches to generate plasma discharges, using different geometry configuration. Typical corona
discharge, dielectric barrier discharge (DBD), radio-frequency (RF) discharge, plasma jet (APPJ), microwave
discharge are commonly used. Recently, atmospheric plasma jet is widely used plasma discharge, which uses air
and special electrode design to prevent arcing. Due to its design and configuration, plasma jet has been applied
for ­biomedical19, surface ­decontamination20 or food ­system21. Atmospheric plasma jet is an efficient technology
with high chemical activity that inactivates wide range of m­ icroorganisms20,22.
There are a range of different methods to apply plasma treatment on the targeted product: direct or indi-
rect mode of plasma ­exposure23. Several studies have applied atmospheric pressure plasma systems directly
to ­biofilms24. In the last few years, plasma treated liquids have also shown promising effect. Plasma can also
be used indirectly via plasma activated water (PAW), whereby the targeted product is not subjected directly
to the plasma gas, but is in contact with water which was pre-treated with plasma. Direct treatment has some
limitations, including effects on food quality and changes in surface topography. Treating biofilms via PAW is
an alternative to direct contact, which can overcome these limitations for water-soluble ­products25. Replacing
the chemical liquid products used in the food industry to remove biofilms with PAW rich in reactive species
would be simple to integrate into industrial cleaning protocols. The objective of this study was to investigate the
inactivation efficacy of individual and combined airborne acoustic ultrasound and PAW treatments from both
cold and thermal plasma systems on Escherichia coli K12 biofilms.

Results and discussion
Effect of airborne acoustic ultrasound on bacterial biofilms.  Figure  1 shows the ­Log10 CFU/mL
of biofilms grown for 48 and 72 h on stainless steel coupons and treated using airborne acoustic ultrasound
for 15 min. A slightly higher number of adherent cells were observed in case of untreated controls after 72 h
of incubation compared to 48 h. Regardless of the higher number of cells, significant reductions of 1.17 ± 0.24
­Log10 CFU/mL and 2.19 ± 1.01 ­Log10 CFU/mL were observed for treatment of samples grown for 48 h and 72 h
respectively (p < 0.05). This significant reduction in biofilms after airborne acoustic ultrasound treatment can
be attributed to morphological changes occurring in bacterial cell membranes. Previous studies demonstrated
fragmented and ruptured bacterial cells with 15 min airborne acoustic ultrasound t­ reatment26. Ultrasound cre-
ates stable cavitation and micro-streaming which causes direct damage to the ultrastructure of bacterial cells in
­biofilms27. However, the acoustic intensity used in this study is relatively much lower compared to contact power
ultrasound systems. The microbial inactivation due to airborne acoustic ultrasound can mainly be attributed

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Figure 2.  Microbial count after (72 h) biofilm detachment of samples treated with cold and thermal plasma-
treated-water alone and in combination with ultrasound treatment. a,b,cDifferent letters on column are
significantly different (p < 0.05); ‘nd’ indicates “not detected”; Error bars stand for standard deviation.

to physical effects caused by acoustic pressure and partly due to thermal effects and ultrasonic wave s­ tresses28.
However, the exact mechanism of microbial inactivation for non-contact type ultrasound is not yet fully under-
stood.

Antimicrobial efficacy of plasma activated water treatment and synergistic effects with air-
borne acoustic ultrasound.  The effect of PAW alone and in combination with airborne acoustic ultra-
sound was assessed on 72 h E. coli biofilms formed on the stainless-steel surfaces. Figure 2 presents the ­Log10
CFU/mL of biofilms grown for 72 h and treated with either cold or thermal PAW alone and in combination
with airborne acoustic ultrasound. It can be observed that dipping a biofilm inoculated coupon into non-treated
water (CW) significantly reduced the cell population from 4.44 ± 0.65 L ­ og10 CFU/mL to 2.62 ± 0.25 ­Log10 CFU/
mL. The reduction in microbial counts for biofilms washing with water alone is mainly due to the weak bio-
film formation of the selected microorganism. The biofilm coupons noted as ‘C’ received no treatment; ‘CW’
were treated with sterile distilled water; ‘cPAW’ were treated with PAW generated from cold plasma; ‘tPAW’
were treated with PAW generated from thermal plasma; ‘AA’ were treated with airborne acoustic ultrasound;
‘AA + cPAW’ were treated with airborne acoustic ultrasound and PAW generated from cold plasma; ‘AA + tPAW’
were treated with airborne acoustic ultrasound and PAW generated from thermal plasma. Biofilms treated with
CW, cPAW and tPAW did not show any significant reductions in the count, whereas combined treatment of cold
plasma generated PAW with airborne acoustic ultrasound showed a significant reduction of approx. 2.2 ± 0.59
­Log10 CFU/mL when compared to control biofilm treated with distilled water. While, E. coli biofilm cells were
completely reduced to undetectable levels when treated with AA + tPAW.
The inactivation of biofilms by a combination of PAW and airborne acoustic ultrasound is mainly due to
the reactive species in PAW and the physical disruption of the biofilms due to ultrasound. The main biological
effects induced due to plasma treatment are attributed to reactive species (reactive oxygen and nitrogen spe-
cies)29. But biofilms by nature are complex biomaterials, (i) the biofilm architecture and (ii) its composition may
limit penetration of reactive species into the biofilm matrix. Many previous studies demonstrated, that increas-
ing treatment time and contact period could improve plasma inactivation efficacy against bacterial ­biofilm30–33.
However, in this study PAW was combined with airborne acoustic ultrasound technology in order to obtain
higher inactivation efficacies. An increase in the reduction factor was observed for the combined treatments,
where exposure to airborne acoustic ultrasound significantly increased the susceptibility of bacterial biofilms to
plasma treatment. These results are in agreement with the study by Yang et al.34, where a significant reduction
in mature Candida albicans biofilms was observed when exposed to contact type ultrasound treatment in com-
bination with antifungal agent Amphotericin B. Similarly, Wang et al.35 also demonstrated that low frequency
ultrasound can enhance the activity of vancomycin against methicillin-resistant Staphylococcus aureus (MRSA)
and methicillin-susceptible Staphylococcus aureus (MSSA) biofilms. However, the inactivation efficacy in these
studies was influenced by antimicrobial/antifungal agents, drug concentration, holding and irradiation time.
The combined treatment of airborne acoustic ultrasound with PAW enhanced the antimicrobial efficacy of
PAW. Regarding the inactivation mechanism, one plausible explanation is that airborne acoustic ultrasound
disrupts extracellular polymeric substances, provoking lesions at the surface of the biofilm, which eases the pen-
etration of the reactive species generated in water into biofilm matrix, causing higher inactivation. Recent review
on PAW proposed that the antibacterial efficacy of PAW is mainly attributed to the synergistic effects of plasma
reactive species, pH and oxidation–reduction potential (ORP)25,36. The exact details and mechanisms for plasma
on biofilms are still poorly understood. The antimicrobial effect of PAW on bacterial biofilms are depended on
the specifically involved plasma species, its composition, its interactions with biofilm matrix components and
its diffusion into the biofilm c­ ells29. Researcher have hypothesised that the interaction of plasma-induced reac-
tive oxygen nitrogen species (RONS) with biofilm matrix components and solutes in the hydrated matrix could
cause oxidation reaction as well as phosphodiesterase activity in cells which leads to physiological alterations and
reduction in biofilm matrix ­area36,37. Eventually loss of matrix reduces the adhesiveness and dispersal of biofilm
cells. While the biofilm is dispersing the remaining cells are exposed to RONS and become more susceptible to be

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Figure 3.  Confocal laser scanning microscope images of E. coli biofilm on glass slide (a) non-treated and
following (b) 15 min treatment of airborne ultrasound. The cells were stained with STYO 9 (green fluorescence,
live cells)/PI (red/yellow, dead cells). All images were compiled, and noise reduction filter was applied in the
Olympus Fluoview FV1000 software (version 4.1.1.5. https​://www.olymp​us-lifes​cienc​e.com/en/suppo​rt/downl​
oads/ ).

Airborne acoustic ultrasound Cold plasma activated water Thermal plasma activated water
Samples nomenclature* treatment treatment treatment
C – – –
CW – – –
cPAW – 30 min soaking –
tPAW – – 30 min soaking
AA 15 min – –
AA + cPAW 15 min 30 min soaking –
AA + tPAW 15 min – 30 min soaking

Table 1.  Nomenclature of different samples with conditions detailed. *‘C’ were without any treatment; ‘CW’
were treated with sterile distilled water; ‘cPAW’ treated with cold plasma treated PAW; ‘tPAW’ treated with
thermal plasma treated PAW; ‘AA’ treated with airborne acoustic ultrasound, ‘AA + cPAW’ treated with airborne
acoustic ultrasound and cold plasma treated PAW; ‘AA + tPAW’ treated with airborne acoustic ultrasound and
thermal plasma treated PAW.

inactivated by PAW. The reactive species generated in PAW not only affects the cell membrane integrity but also
penetrate into membrane channels and provoke reactive oxygen species (ROS) production endogenously which
account for the intracellular ROS accumulation inside the ­cell31,38. Studies by Lukes et al39 and Xu et al31 also
demonstrated that RONS interact with intracellular components (DNAs, proteins, lipids) and influence its meta-
bolic responses in microorganisms. These interactions cause oxidative stress on cell components thus initiating
lipid and protein peroxidation on the cell membrane, followed by protein and/or DNA damage and cell d ­ eath36.
The effect of these treatments on the biofilms was also observed using confocal microscopy. Confocal images
of treated and control samples are shown in Fig. 3. The population of biofilms was significantly affected by the air-
borne acoustic ultrasound treatment. Following the same trend as the plate count method, the images have shown
that airborne acoustic ultrasound is an effective tool for biofilm removal. The dead cells (red/yellow-fluorescent
stained) increased upon treatment, thus indicating the bactericidal effect of airborne acoustic ultrasound alone
or when used in combination with PAW.

Physicochemical properties of plasma activated water.  Table 1 shows that pH values of the PAW
decreased significantly after cold or thermal plasma treatment. The pH of the solution is an important bacteri-
cidal property of ­PAW40; however the reduction in pH is not the sole cause for the bacterial ­inactivation41. The
reason for acidification of PAW is predominantly due to the nitrate and nitrous acids generated by plasma treat-

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ment. Nitrates and nitrites in PAW are formed through dissolution of nitrogen oxides (­ NOX) formed in the air
into ­water36. As demonstrated in Table 1, the concentration of nitrates and nitrites increased with plasma treat-
ment, with higher levels of reactive nitrogen species (RNS) generated in cold PAW than thermal PAW.
The higher the oxidation ability of a solution to take electrons from cell membranes of the bacteria, the higher
is the cell instability, which causes damage to the outer and inner cell ­membranes42. Both plasma treatment
methods increased the ORP in the treated PAW in comparison to the control. ­H2O2 is considered to be a potent
anti-microbial agent. Several studies have investigated about the importance of ­H2O2 to the antimicrobial activity
of ­PAW18,25. In this study, concentration of H­ 2O2 was generated in PAW. In addition, the conductivities of PAW
were also recorded in this experiment. As shown in Table 1, the conductivity of PAW after plasma activation
was significantly higher compared to the distilled water control. This is due to the generation of active groups
(reactive oxygen and nitrate species) formed in water which improves the conductivity. Furthermore, the con-
ductivity of PAW generated from cold plasma jet was higher than that generated from the thermal plasma jet.
Overall, no significant difference was observed in the physicochemical properties of PAW generated from cold
or thermal plasma, except for RONS concentration, which explains the similar inactivation efficacies observed.

Conclusion
This study demonstrates the individual and combined antimicrobial effects of airborne acoustic ultrasound
and PAW for the disruption and inactivation of biofilms. Airborne acoustic ultrasound is an effective tool for
biofilm removal and PAW can inactivate cells due to enhanced diffusion of plasma activated water into the
matrix. Shortcomings of current treatment approaches together with resistance among bacterial strains against
key antimicrobial agents i.e. antibiotics could potentially be addressed through use of these novel technologies.
Novel technologies such as plasma and airborne acoustic ultrasound have the potential to effectively eradicate
persistent bacterial biofilms with less energy and short treatment times in food processing environments. How-
ever, further studies are required to better understand the inactivation mechanisms of these treatments, which
affect the physiological and metabolic states of bacteria.

Material and methods
Preparation of coupons for biofilm cultivation.  Stainless steel coupons.  The surface material inves-
tigated in this study was grade 304 stainless steel; coupons (16 mm × 1.2 mm) were obtained from Watermark
Engineering (Tallaght, Dublin 24, Ireland). Prior to inoculation, the coupons were placed in Duran bottles and
sterilised at 121 °C for 15 min.

Glass slides.  For confocal laser scanning microscopic examination, bacterial biofilms were grown on glass
slides (1 × 1 cm). The glass slides were washed with acid (1 M HCl) followed by 70% ethanol as described by Fis-
cher et al.43. After washing, the glass slides were air dried in fume hood on Whatman filter paper for 10–15 min
before autoclaving at 121 °C for 15 min.

Culture preparation and biofilm formation on coupons.  Non-pathogenic Escherichia coli K12
ER2925 was obtained from the microbiological culture stock in Teagasc Food Research Centre (Ashtown, Dub-
lin, Ireland). A loopful of E. coli K12 from the stock culture was transferred into nutrient agar plate (Product
code: CM0003B; Oxoid Ireland c/o Fannin Healthcare, Ireland) and incubated at 37 °C for 24 h. A colony was
then inoculated in 5 mL of Luria Bertani (noted LB, composed of 10 g of tryptone and 5 g of yeast extract per
litre) and incubated for 24 h at 37 °C under shaking conditions (150 rpm). This culture was designated as a work-
ing culture and was stored at 4 °C until use, and a new culture was prepared weekly. On the day of the experi-
ment, the optical density (O.D) was measured using EPOCH2C microplate reader (Biotek, Mason Technology
Ltd, Dublin, Ireland) and adjusted to 0.15 using fresh LB media which corresponded to approx. 8 ­Log10 CFU/
mL. Sterile coupons were placed in sterile 12-well plates and 1 mL of the OD-adjusted culture was added into
each well. The 12-well plates were incubated at 37 °C for 48 and 72 h without medium change or agitation. After
incubation, each coupon was washed twice using phosphate buffer saline BR0014 (PBS; Oxoid Limited, Ireland)
to remove loosely attached and planktonic cells. The coupons were air dried inside a safety cabinet for 20 min.

Airborne acoustic ultrasound and plasma activated water treatments.  Airborne acoustic treat-
ment.  All experiments were carried out using an airborne acoustic ultrasonic system (Pusonics S.L., Madrid,
Spain) operating at a frequency of 26 kHz with a maximum power output of 170 W. The system consists of an
electronic power generator with a dynamic resonance controller, a power amplifier, a high impedance match-
ing box, and a circular stepped-plate transducer. The funnel-shaped acoustic airborne ultrasound construction
provided highly focused acoustic field generated by the vibration of a plate ­transducer44. The biofilm coupons
were placed on a stainless-steel plate positioned at a distance of 42.5 cm from the centre of the transducer at a
maximum acoustic energy density of 10 W/cm2 (Fig. 4). The biofilm coupons were treated with airborne acous-
tic ultrasound for 15 min.

Plasma activated water treatments.  Cold plasma treatments were carried out using a system sourced
from the National Centre for Plasma Science and Technology at Dublin City University (Glasnevin, Dublin
9, Ireland).This system is elaborately described in previous ­publication21. Plasma generated within this region
exited through a small gap in the ground electrode entering ambient conditions in the plume region where it
came into contact with treatment samples (Fig. 5). Plasma was generated at 30 kV using ambient air as the work-
ing gas.

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Figure 4.  Schematic of the airborne acoustic ultrasound system.

Figure 5.  Schematic diagram of PAW generation by cold plasma jet system. (AFM) air flow monitor; (C) gas
introduction; (PS) power supply.

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Figure 6.  Functional principle of thermal atmospheric plasma system. HV: High voltage generator.

Thermal plasma treatments were carried out using a plasma beam system (Diener electronic GmbH & Co.
KG, Ebhausen, Germany) operating at 20 kHz. The system was composed of three main units: a supply unit
including a high voltage generator, plasma current and gas control devices; gas and power conductors in a flex-
ible tube; and a plasma torch. The high voltage generator produced a voltage up to 10 kV, which was required
to create the electric field to initiate and sustain the electrical plasma discharge. The electrical conductor for the
supplied voltage and the process gas tubing were supplied to the plasma torch via a flexible tube, and the plasma
particles (ions, electrons, excited atoms and molecules) were generated from the air supplied to the plasma torch.
The temperature of the plasma gas would reach up to 200 °C; therefore, a condenser and cooling coil was used
to maintain the temperature of the activated gas generated by the plasma at ambient temperature. Cold water
(4 ± 0.5 °C) was circulated through the condenser using external refrigerating system (Lauda Ecoline, RE104).
The functional principle of this system is shown in Fig. 6.
For PAW generation from thermal and cold plasma jet, the distance between the nozzle of the jet and water
surface was around 7.2 cm. For generation of PAW, a Duran bottle containing 100 mL of sterile deionised water
was treated with cold or thermal plasma for 15 min. Individual biofilm coupons were immersed in 10 mL of
PAW for 30 min at 4 °C during treatments. Sterile distilled water was used instead of PAW for control treatments.

Combined treatments.  To determine the synergistic effects of combined treatment of airborne acous-
tic ultrasound and PAW, biofilm coupons were first exposed to airborne acoustic ultrasound as described in
Sect. 2.3.1 followed by treatment with PAW as described in 2.3.2. Different treatment combinations were carried
out as listed in Table 2.

Microbiological analysis.  The population of E. coli on control and treated coupons was enumerated
using the plate count method. The biofilm cells were detached from coupons using glass beads as described by
­Vatanyoopaisarn45. The coupon was placed in sterile 10 mL of maximum recovery diluent (MRD, Oxoid Ireland
c/o Fannin Healthcare, Ireland) with 10 sterile glass beads (18,406, Sigma-Aldrich, Ireland). After soaking for
10 min, the tube was subjected to vortex-mixing for 1 min at maximum speed, yielding a suspension of E. coli
that adhered on the coupon surface. Decimal dilutions were then carried out and spread on nutrient agar plates.
After 24 h incubation at 37 °C, the colony forming unit per millilitre (CFU/mL) was calculated.

Confocal laser scanning microscopy.  A live-dead BacLight bacterial viability and counting kit L34856
(Thermofisher Scientific, Ireland) was used. This included SYTO 9, a green fluorescent nuclear and chromosome
counterstain, and propidium iodide (PI), a red-fluorescent stain which is not permeant to live cells, and thus was
used to detect dead cells. The staining procedure was based on the instructions from the kit manufacturer with
slight modifications. The working solution of fluorescent stains was prepared by adding 1.5 μL of SYTO 9 stain

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Treatments
Parameters Control Cold plasma Thermal plasma
pH 6.71 ± 0.03 2.85 ± 0.18 2.53 ± 0.04
ORP (mV) 406.07 ± 12.7 565.40 ± 1.9 510 ± 0.6
Conductivity (µs/cm) 4.5 ± 0.5 333.00 ± 13.44 518.0 ± 22
Hydrogen peroxide (µM) nd 14.7 ± 3.9 13.2 ± 5
Nitrate (µM) nd 71.7 ± 5 13.09 ± 6
Nitrite (µM) nd 33.5 ± 0.7 7 ± 0.2

Table 2.  The pH, conductivity, ORP and RONS of PAW compared to untreated controls. Data are expressed as
the mean value of three independent experiments, ‘nd’ indicates not detected.

and 1.5 μL of propidium iodide stain to 1 mL of sterile 0.85% sodium chloride (NaCl). This working solution was
prepared and used the same day. Each untreated and treated biofilm slide was covered with 200 µL of prepared
staining solution and incubated at room temperature in dark for 15 min. The biofilm slides were carefully washed
with 1 mL of NaCl solution twice to remove any dye residues. The biofilm samples were then placed on a 35 mm
diameter glass-bottomed dish (ibidi GmbH, Martinsried, Germany) containing one drop of the mounting solu-
tion obtained from the live/dead BacLight bacterial viability kit L7007 (Thermofisher Scientific, Ireland). The
slides were directly examined on a confocal laser scanning microscope (Olympus Fluoview FV1000) equipped
with a 60x / 1.35 NA oil immersion objective. At least 3 randomly chosen microscopic fields were examined
for each sample. A noise reduction filter was applied to the images in the Olympus Fluoview FV1000 software.

Physicochemical properties of plasma activated water.  The physicochemical properties (includ-

ing pH, electrical conductivity, ORP, RONS) of PAW were measured immediately after plasma treatment. For
these measurements, 30 mL of sterile distilled water was treated with either cold or thermal plasma for 5 min.
The pH, electrical conductivity and ORP of each sample was measured using a pH meter (Hanna, pH 213,
UK), an electric conductivity meter (Jenway, 4070, UK), and a redox sensitive electrode (Hanna, HI3230B, UK)
respectively. Measurements of hydrogen peroxide ­(H2O2) generated in the PAW were determined by titanium
oxysulfate calorimetric method; briefly 10 μL of T­ iOSO4 (Sigma-Aldrich, Ireland) was added to 100 μL of PAW
and then incubated in the dark for 10 min. The titanium sulfonate reagent reacts with hydrogen peroxide present
in PAW to produce coloured pertitanic acid, which was measured on spectrophotometric plate reader (BioTek
Epoch 2C, Mason technology Ltd., Dublin, Ireland) at 390 nm. The levels of nitrite in PAW were measured using
Griess reagent (N-(1-Naphthyl) ethylenediamine dihydrochloride) (Sigma-Aldrich, Ireland), where 50 µl of the
reagent was added to 50 µL PAW and incubated in dark for 30 min. After incubation, absorbance of samples
was measured at 548  nm. When detecting nitrate concentration, PAW was pre-treated with sulfamic acid to
eliminate nitrite interference. Nitrate concentration was then assessed by 2,6-dimethyl phenol (DMP) using the
Spectroquant nitrate assay kit (Merck Chemicals, Darmstadt, Germany) with manufactures instructions. During
each reactive species assessment, a known standard was included in each plate to convert absorbance values into
known concentrations.

Statistical analysis.  All experiments were performed in three independent experiments with three tech-
nical replicates each. Analysis of variance (ANOVA) and Tukey’s test was carried out using SAS (Version 8.0)
and considered statistically significant at p < 0.05. The parametric assumptions were verified before performing
ANOVA: normal distribution (Shapiro–Wilk test), homogeneity of variances (Levene test) and data independ-
ence (by experimental design).

Received: 3 April 2020; Accepted: 21 September 2020

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Acknowledgements
This research was carried out with the financial support of the Irish Department of Agriculture, Food and the
Marine (project no 14F845) and Science Foundation Ireland (SFI) under grant number 17/CDA/4653.

Author contributions
C.C, A.P and L.H performed experiments. J.S performed and helped with confocal imaging. C. C and A. P. wrote
the manuscript. L.H, J.S, C.O and B.T edited the manuscript. All authors reviewed the manuscript.

Competing interests 
The authors declare no competing interests.

Additional information
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