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Biological and Medical Applications of Materials and Interfaces

Microplasma Bubbles: Reactive Vehicles for Biofilm Dispersal Renwu Zhou, Rusen Zhou, Peiyu Wang, Bingyu Luan, Xianhui Zhang, Zhi Fang, Yubin Xian, Xinpei Lu, Kostya (Ken) Ostrikov, and Kateryna Bazaka ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b03961 • Publication Date (Web): 08 May 2019 Downloaded from http://pubs.acs.org on May 8, 2019

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ACS Applied Materials & Interfaces

Microplasma

Bubbles:

Reactive

Vehicles

for

Biofilm Dispersal Renwu Zhou,†,‡,# Rusen Zhou,‡,# Peiyu Wang,† Bingyu Luan,§ Xianhui Zhang,§,* Zhi Fang,‖,* Yubin Xian,†,¤ Xinpei Lu,¤ Kostya (Ken) Ostrikov,†,‡ and Kateryna Bazaka†,‡,* †

Institute of Health and Biomedical Innovation and School of Biomedical Sciences, Queensland

University of Technology, Brisbane, QLD 4000, Australia ‡

School of Chemistry, Physics and Mechanical Engineering, Queensland University of Technology,

Brisbane, QLD 4000, Australia § Fujian Provincial Key Laboratory of Plasma and Magnetic Resonance, Institute of Electromagnetics and

Acoustics, Department of Electronic Science, College of Electronic Science and Technology, Xiamen University, Xiamen 361005, China ‖

College of Electrical Engineering and Control Science, Nanjing Tech University, Nanjing 210009, China

¤

State Key Laboratory of Advanced Electromagnetic Engineering and Technology, School of Electrical

and Electronic Engineering, Huazhong University of Science and Technology, Wuhan 430074, China

1 ACS Paragon Plus Environment

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ABSTRACT Interactions between effects generated by cold atmospheric-pressure plasmas (CAPPs) and water have been widely investigated 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 singleorganism system at lab 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 (DBD) at the gas-liquid interface with the aplied voltage of 4.0 kV) act as transport vehicles to efficiently deliver reactive plasma species to target biofilm sites on artificial and living surfaces while keeping healthy water conditions in a multi-species system. Thus-generated air microplasma bubbles and plasma activated water (PAW) both can effectively reduce existing pathogenic biofilm load, by ~83% and 60% respectively after 15 min-discharge at 40 W, and prevent any new biofilm from forming. Generation of underwater microplasma bubbles in a custom-made fish tank for less than a minute per day (20 seconds per-time, twice daily) can introduce sufficient quantities of RONS into PAW to reduce biofilm infected area by ~80-90% and imrpove 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 plasma-induced nitric oxide acts as a critical bioactive species that triggers the release of cells from biofilm and their inactivation.

KEYWORDS: microplasma bubbles · gas-liquid interface · plasma activated water · reactive oxygen and nitrogen species · biofilm dispersal


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Introduction Cold atmospheric pressure plasmas (CAPPs) have been successfully applied in water purification, nanomaterials synthesis, biomedicine, and food processing with high efficiency.1-7 The reactivity of CAPPs is inherently linked to the rich assortment of highlyreactive chemical species that are generated as a result of gas ionization.1,3,6 However, the lifetime of these chemical species generated in the gas phase is relatively short.8,9 For this reason, upon their convective transfer to the gas–liquid interface, only a fraction of these gas-phase plasma-generated particles are able to penetrate the interface and remain in the activated liquid. This may limit the efficiency with which plasma activated water (PAW)

can

target

and

induce

biochemical

changes

in

biological

targets,

e.g.

microorganisms within a biofilm, in a moist environment or in bulk liquids.10 Indeed, although many studies have shown that PAW possesses unique biochemical reactivity to inhibit a wide range of planktonic microorganisms and even spores,9,11,12 and do so with high selectivity,9,13 most of these studies were conducted in vitro using simple systems, e.g. a suspension of pathogenic microorganisms.9,14 Relevant mechanisms that underlie the biochemical activity of agents generated by plasmas in solution against pathogens within biofilm, a self-produced biopolymeric matrix that affords microorganisms protection, remain elusive.15,16 Furthermore, these in vitro findings may not necessarily translate to real-life applications where PAW is expected to kill pathogens within a complex, multiorganism system.17 Indeed, prolonged activation of water can lead to significant localized changes in its pH and concentration gradients of reactive species. This may pose a challenge for applications where target organisms co-exist with other living organisms, e.g. pathogenic microorganisms in a large aquaculture fish tank. In this work, we show that combining cold atmospheric pressure plasma with microbubbles in a form of underwater microplasma bubbles may provide a new avenue to enhance the efficiency of mass transfer of biochemically-reactive species from plasma to microbial targets in solution,2,18-20 and enhance the biological activity and selectivity of thus-activated solutions against pathogen-residing microorganisms. Microbubbles have



been shown to enhance mass transfer from the gas to liquid phase owing to their high specific interfacial area, long residence time and high inner pressure.18 Use of microbubbles to contain species produced by CAPPs may be an effective approach in improving the efficiency of existing reactors. As they burst, microbubbles can also potentially generate mechanical agitation and local heating, leading to localized formation of additional reactive radicals within the aqueous phase in the proximity of the target site, e.g. biofilm.21 This is important because pathogens within a protective biofilm22 may display up to 1,000-fold higher tolerance to conventional antibiotics than their single-cell, planktonic counterparts.23,24 Nitric oxide (NO) is a ubiquitous signalling molecule in organisms with an estimated biological half-life of up to several seconds.25,26 The existence of NO species has been confirmed both in gaseous CAPPs and in PAW.27,28 It has recently been proposed as a promising antibiofilm agent with a low chance of resistance development,24,29,30 which at low concentrations disperses biofilms and at higher doses kills bacteria.25,30-32 Application of NO at large scales is challenging due to poor stability and solubility of NO and/or NO donors in biological media and the lack of controlled and localized methods for NO delivery at large scales.33 Previous studies even proved that the concentration of NO in PAW could be much higher than that in aerobic solutions, where the saturated concentration of NO is usually less than dozens of μM.28 In this paper, we show that CAPP can effectively produce large quantities of NO and other reactive species, whereas microbubbles act as a physical vehicles to transport these species in aqueous solution to the biofilm present on artificial and living surfaces. The aims of this work are (1) to gain a deeper insight into the mechanism of plasmaenabled biofilm removal based on nitric oxide generation and retention in solution using an atmospheric microplasma bubble reactor; (2) to determine the individual and synergistic contributions of thus-generated reactive oxygen and nitrogen species (RONS) to biological activity and more specifically the ability of plasma-activated water to initiate and sustain biofilm dispersal; and (3) to show potential of the new knowledge to lead to real-life applications such as curing biofilm-associated fish skin infections. The


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concentrations of common long-lived species H2O2, NO2-, NO3- and O3 in plasma activated solution are measured by chemical and optical methods. Escherichia coli biofilm, one of the biofilms that causes problems related to food safety and the health of aquatic ecosystems, is used as a microbial model organism to investigate and compare the antibiofilm effects of PAW and equivalent chemical solutions. Moreover, when integrated into a fish tank, the effects of cold atmospheric plasma-generated microbubbles containing biochemically-reactive chemistries on the biofilm removal from the skin of infected fish are investigated.

RESULTS Microplasma bubbles for reactive species generation. As illustrated in Figure 1a and 1b, a custom-made microplasma bubble reactor is developed to generate underwater microplasma bubbles and then is integrated within a fish tank for a specific real-life application. The porous membrane used in this reactor not only serves as the ground electrode for the plasma generation, but also produces smaller bubbles (~400 μm to 2 mm) transferring reactive plasma species. Electrical characteristics of the microplasma reactor are shown in Figure S1 (Supporting Information). Air is used as the feed gas as it is a rich source of oxygen and nitrogen, cheap and environmentally friendly. Furthermore, reactive oxygen and nitrogen species that are formed as a result of such plasma treatment have notable broad-spectrum biocidal activity, yet do not lead to residual toxicity or environmental pollution. Once air is fed through the microplasma jet array and the discharge is generated, microplasma generated species will be delivered by bubbles through the stainless-steel mesh and then transported into aqueous media. The bubble size distribution is shown in Figure S3 (Supporting Information), with the diameter mainly ranging from 400−2200 μm. These bubbles are expected to serve as unique microreactors with a large gas-liquid interface,2,20 which helps the bubbles to survive for a considerable period of time before bursting. This increases the likelihood of short-lived



gaseous species to be transported to the site of biofilm, and delivered to pathogens at concentrations sufficient for biofilm dispersion and microorganism deactivation. Eventual bursting of the bubbles will not only release the reactive species, but also introduce mechanical agitation and local heating to promote biofilm dispersion. Although in this study we do not focus on the adjustment of the bubble size, it is worth pointing out that the size of those bubbles are of high significance in species generation, transport and the overall efficacy of the treatment.2,34 Generally, based on the size, bubbles can be classified as macrobubbles (over 1 mm), microbubbles (over 1 μm), and nanobubbles (below 1 μm).2,3436

The bubble size distribution is shown in Figure S3 (Supporting Information), with the

diameter mainly ranging from 400−2200 μm. These bubbles are expected to serve as unique micro-reactors with a large gas-liquid interface,2,20,36 Smaller is the size, the higher is the internal pressure (leading to more energy release once burst) and the larger gasliquid interface will be. Although they have considerably higher internal pressure, nanobubbles have been observed to have lifetimes up to several days or even weeks in liquid, and are considered important for in-liquid plasma initiation and streamer branching.2,34 Because of their buoyancy, macrobubbles are unstable in liquid, and tend to rise fast to the surface of water, at which point they burst. To date, most applications have focused on the generation and application of microbubbles, since they can be stable in liquids for controlled periods of time, and improve the generation of chemical and physical substances, and importantly due to the technical feasibility of generation of these microbubbles in liquids.37 We then evaluate the efficacy with which reactive species are generated by the same microplasma jet array housed in a glass cup and transported within the liquid phase using microplasma bubbles by quantifying chemical species with known strong antimicrobial activity. To ensure that these are the only species to play a significant role in biofilm removal and pathogen deactivation, we prepare mimicked solutions that contain the same concentration of aforementioned reactive species. Biofilm samples are treated in two ways, via the direct treatment of microplasma bubbles in aqueous solution (Figure 1d) and via first activating water and then applying it to samples (Figure 1e).


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Figure 1. The schematic diagram of the experiment setup. Top view (a) and crosssectional view (b) of the electrode of microplasma array device; (c) Plasma contained in bubbles. These bubbles are unstable, and rise fast. Their eventual collapse leads to the rapid dissolution of the active species due to the pressurised interior gas, leading to enhanced mass transfer processes. The condensed ionic clouds around the shrinking gasliquid interface of the collapsing microbubbles may assist in the formation of more stable bubbles by hindering gas dissolution to some extent, effectively increasing residence time of plasma species within aqueous environment. Biofilm samples treated by the microplasma bubbles in aqueous solution (d) and by immersion in PAW and in a mimicked solution containing specific species at the same concentration as that in the PAW (e).

As shown in Figure 2a, longer plasma activation time is more effective in significantly reducing biofilm biomass both in the case of the direct treatment of microplasma bubbles in solution and that of PAW. A biomass reduction of more than 60% can be achieved after 15 min of treatment with plasma activated water, indicating that PAW generated by this



microplasma reactor holds promising biochemical activity for biofilm dispersal. The bacterial killing efficacy of the parallel tests evaluated by colony-forming units (CFU) shown in Figure 2b resemble the result of the biofilm biomass reduction, and thus to simplify the experiments following studies just focus on the biomass reduction. Moreover, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) are employed to confirm and visualize the treatment efficiency of microplasma bubbles and PAW. The obtained images (Figure S6 and S7) reveal the killing efficiency and biofilm dispersal that are consistent to those obtained from staining experiments and CFU analysis.

Figure 2. Biofilm removal efficiency of solution-mediated direct microplasma treatment, PAW and mimicked solutions treatment. (a) The fraction of the removed biofilm as a function of activation time; (b) Bacterial killing efficacy of control (sterile distilled water treatment), bubbles (only air served but without discharge), mimicked solution, PAW, and solution-mediated direct plasma treatment (plasma exposure 15 min); (c) Comparison of individual H2O2, NO2−, NO3−, and O3 solutions, mixed solution and PAW on the biofilm biomass reduction; (d) Comparison of the treatments of PAW and PAW with the addition of different scavenger. Notes: The remaining biofilm values after treatment are normalized with respect to an untreated biofilm sample. The removed biofilm values are calculated by subtracting the remaining values from 100%. The input power was fixed at 40 W, whereas


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the activation time varied to assess the time-resolved effect. Exposure of biofilms was for 15 min in all experiments. When compared to the direct treatment of microplasma bubbles, the PAW treatment shows lower effectiveness with respect to biofilm dispersal at all-time points. The difference in dispersal efficacy between microplasma bubbles and PAW shows a positive correlation with activation time, with approximately 2.5% difference (reduced by 15.5%) at 1 min (activation time) and ~20% difference (reduced by ~30%) in efficacy at more than 10 min of activation. These results can be attributed to the effects that are absent from PAW treatment, such as that of short-lived species (e.g. free radicals), which have been widely acknowledged to play a critical role in attaining satisfactory plasma-associated treatment results,4,27,38 and physical agents, like electric field and shock waves. And in our case, a large number of bubbles that are formed close to the ends of hollow-core fibres during plasma discharge (Figure 1c). These bubbles also may contribute to the dispersal of biomass (~6%, Figure 2a) on their own, although their cell inactivation efficacy is modest. These results indicate that the physical impact of bubbles, particularly the pressure wave and temperature gradient, especially from bubble burst can further facilitate and potentially enhance plasma-enabled biofilm removal. It is worth mentioning that by drawing on the experience of other technologies based on micro and/or nanobubbles, some strategies such as regulating the bubble size (mainly towards the generation of smaller microbubbles with the diameter of several to dozens µm),37 using specific microbubble generators and optimizing the system (e.g. inlet gas flow and, in the case of plasma, applied voltages) to further enhance the pressure inside bubbles and the speed of bubble movement can be used to improve the physical effects from bubbles on biofilm dispersal. Within bubbles especially microbubbles, the counterions, i.e. positive charged species of plasma, are attracted to the gas-liquid interface while electrons stay near the centre of the bubbles. When bubbles shrink, the high interior pressure, presence of a condensed ionic cloud inside and outside of the bubble, and sudden changes in bubble pressure and ions concentration result in the enhanced mass transfer and dissolution of



active species.17 These gaseous species in bubbles rapidly diffuse and permeate into distilled water and drive many chemical reactions similar to that observed at gas-liquid interfaces.37 More details about the dynamics of underwater discharges can be found in Supporting Information (Figure S4).

NO from microplasma bubbles and in PAW. The treatment of mimicked solutions shows inferior performance when compared to both PAW and solution-medicated direct microplasma bubble treatment, with the difference being statistically significant. The biological effects of plasma activated media are usually inherently attributed to the rich assortment of long-lived reactive oxygen and nitrogen species (RONS), and nitrate (NO3−), nitrite (NO2−), hydrogen peroxide (H2O2) and ozone (O3) with the corresponding typical half-lifetime of years, several days, ~104 s and several to dozens minutes are suggested to play the major role in biochemical activity.39-41 The roles of plasma physical effects and short-lived free radicals in the activated liquids remain a subject of debate. Some of the radicals such as NO and peroxynitrite are otherwise relatively stable with the half-lifetime of up to 5 s and 1 s (at pH 7.4), respectively.25,26,28,40 Peroxynitrite in PAW has been widely studied and recognised as an effective agent capable to inactivate pathogenic microorganisms.9 There is increasing evidence from both theoretical and experimental studies showing that NO present in PAW can originate to a limited extent from solvation of gas phase species, but mainly from bulk and interfacial regions via reactions (1-14), thus sustaining and boosting the existence of NO.28,42 Yet, the exact roles of NO in PAS/PAW in biofilm removal remain unconfirmed. In our study, haemoglobin, a common and effective trapping agent for NO molecules, is used to trap NO radicals in different types of solutions. On the one hand, haemoglobin is added into water prior to subjecting water to direct plasma treatment (before plasma discharge is ignited). On the other, we also add haemoglobin into PAW (immediately after plasma exposure). Both experiments demonstrate of the presence of NO radicals. As detailed in Figure 2a, the addition of 20 μM hemoglobin (the specific dose is chosen based on preliminary optimisation study) adversely affects biofilm dispersal. The effect is


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particularly evident for short plasma processing times. For example, addition of haemoglobin reduces the portion of biofilm removal by around 53% and more than 68% in the first 3 min of activation for direct plasma and PAW treatments, respectively. These results suggest that the biofilm eradication efficiency of plasma based methods may strongly rely on the presence of NO. With the addition of the trapping agent, NO is effectively scavenged, and its concentration decreases rapidly to a low concentration (like < 30 μM), which is not sufficient to induce biofilm dispersal.43 The balance between NO and other species, which is crucial to obtain products with high bio-activity in solution, may also be changed. From literature, NO is expected to act as an important intermediate for generating down-stream and end reactive species in plasma activated solutions via reactions (15-22).9,42 This is also demonstrated by the fact that when discharging for a longer time to saturate the haemoglobin, the extent to which the biofilm dispersion efficacy is reduced by the addition of haemoglobin gradually becomes smaller. NO formation in gas phase: *e + N2/O2 → •N + •O + e

(1)

•N + •O → •NO

(2)

*N2 + •O → •NO + •N

(3)

O2 + •N → •NO

(4)

NO formation at the gas-liquid interface and within the liquid phase: •N + •OH → •NO + •H

(5)

•H + NO2− → •NO + OH−

(6)

N2O3 ↔ •NO + •NO2

(7)

HNO2 + •H → •NO + H2O

(8)

HNO2 + H2O2 → ONOOH ↔ •NO2 + •OH

(9)

NO2−+ H2O2 → ONOO− ↔ •NO + •O2−

(10)

•OH + ONOOH → •NO + O2 + H2O

(11)

•NO2 + •O → •NO + O2

(12)

•NO2 + •N → 2•NO

(13)

•NO2 + O3 → •NO + O2

(14)



Reaction that are likely to consume NO radicals: •NOx + •OH → HNOx+1

(15)

•NO + •NO2 + H2O → 2HNO2

(16)

•NO + •O2−→ ONOO−

(17)

•NO + •HO2−→ ONOOH

(18)

•NO + O3 → •NO2 + O2

(19)

2•NO + O2 → 2•NO2

(20)

4•NO + O2 + H2O → 4HNO2 → 4HNO3

(21)

•NO + •NO2 → N2O3

(22)

The long-lived species constituents of PAW, namely NO3−, NO2−, H2O2 and O3 generated in 0.2 L of distilled water as a result of plasma treatment at 40 W for 15 min are determined to be 1.5 mM, 0.2 mM, 1.3 mM and 12 μM, respectively, with a pH value of around 3.5 in the activated water. In order to get more insights into the separate effects of these species on biofilm reduction, chemical solutions containing equivalent dose are separately prepared, and applied to inactivate biofilms. Since it has been widely confirmed that pH and temperature of PAW has certain influences on their bactericidal efficiency, the pH values of all solutions are carefully adjusted by HCl (0.01 M) to be equivalent to 3.5, while temperature of all solutions is kept at 28 C. The degree of biofilm dispersal for each chemical species is depicted in Figure 2c. As expected, with its high oxidization potential of up to 1.763 V and relatively higher concentration,9 H2O2 is the most effective agent among the long-lived reactive species. At the specific concentration mimicking that in PAW, solutions containing H2O2 disperse approximately 21% of the biofilm, while NO3− has almost no effects on biofilm biomass reduction and only a reduction of ~4% is witnessed when using NO2− (because of low concentration, although NO2− in PAW has been suggested with antimicrobial effects particularly in acidic conditions).9 With a significantly lower concentration in PAW, O3 still disperses more than 11% of the biofilm, due to the highest oxidation potential (2.075 V) among these long-lived species,44 which makes O3 one of the most commonly and widely used disinfectants. However, the significant difficulty of dissolving O3 in solutions usually poses a challenge, and thus efforts to increase its


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concentration in solutions including the plasma activated ones are important to further improve the treatment efficacy and extend application feasibility of those solutions.45 The mimicked cocktail solution, i.e. a mixture of the four chemicals at the same concentrations as that in PAW (marked as Mix), shows a significant dispersal enhancement when compared to the sum of the separate reductions, but there still exists a noticeable difference in its reduction performance when compared to the dispersal achieved using PAW treatment. These results align well with those by other reports in literature, and highlight the possibilities of generation of other biochemically-active products or intermediates during plasma activation that are yet to be identified, especially the peroxynitrites (ONOOH/ONOO−) with higher antimicrobial activity via reactions (9 &10).9,42 The peroxynitrites are expected to enhance the content of NO in the solutions mainly via reactions (8-11) as well as (12-14). Once the concentration of thus-generated NO reach a value sufficient to initiate biofilm dispersion and cell detachment, it would then promote other species to interact with bacterial biofilms by making the cells more accessible.26,32,33 This is further confirmed by an experiment where 10 μM of commercial NO donor, spermine NONOate (SperNO), is added to a mix mimicked solution. SuperNO can spontaneously release •NO into aqueous solution at the molar ratio of 1:2. The gap between thus-created mix solution and PAW treatment is then narrowed considerably (Figure 2d). Again, the addition of NO scavenger hemoglobin (20 μM) leads to a considerable decrease in biofilm dispersal ability, both in the PAW and NO-enriched mix solutions. Electron paramagnetic resonance (ESR) is then employed to give direct evidence on the existence of NO in PAW by using MGD as the NO trapping agent. This methods eliminates the interference from other species, such as NO2, NO3− and NO2− (a detailed description of the method is provided in Supporting Information). As illustrated in Figure S5, the concentration of NO in PAW is dependent on the plasma processing time, and only seconds of plasma activation is enough to yield a detectable amount of NO.



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Figure 3. (a)-(d) Initial symptoms of the biofilm-infected fish skins of blood parrot cichlid and advanced symptoms after 1–3 week plasma treatment. (e) Variations of the infected area on the fish skin with the plasma treatment time.

Microplasma bubbles for fish-disease healing.

Pathogenic biofilms pose a

significant threat to health of humans and animals, and are difficult to prevent or remove. For example, in some Asian countries, integrated livestock-fish aquaculture intentionally introduce animal excreta and urine into fish ponds to stimulate growth of plankton and other microorganisms eaten by the fish. As a result, pathogenic microorganisms, such as Escherichia coli, may become incorporated into biofilms found in closed aquaculture systems, causing recurring exposure to potential disease agents and increasing the


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likelihood of contamination of farmed fish. This presents both an economic issue due to fish morbidity and mortality as well as a public health issue. Biofilm bacteria are not easily eliminated by sanitizers alone, and while it is possible to use heat treatment or mechanical scrubbing to physically remove biofilm from artificial surfaces, removal of pathogenic biofilms from skin of infected fish is more challenging.46,47 Furthermore, indiscriminate and excessive use of broad-spectrum synthetic antibiotics may lead to microbial resistance.48,49 Thus, we investigate the efficacy of reactive chemistries obtained using the system for biofilm removal in a real life application (integrated the microplasma bubbles reactor into a custom-made 50 × 24 × 40 cm fish tank for the healing of biofilm-infected fish skin disease). The detailed experimental process can be found in Experimental Section. As shown in Figure S8, the pH values of the treated water experience a slight decrease from 6.72 to 6.64 during the first 20 s of plasma activation. Further increases in the treatment time to 30 s will result in a notable reduction in the pH (6.35). This is because NOx generated in the microplasmas reacts with water to produce nitric and nitrate acids.50 An increase in conductivity of around 100 μS/cm is observed in 30 s of plasma discharge, with more than half of the increase happening in the last 10 s of the treatment. Plasma activation is expected to increase the conductivity of thus activated water because of the ionization of gas leading to the introduction of ions into solution. The temperature of the solution stays virtually unchanged, at 28 °C during the plasma treatment. Based on these results, in order to maintain the suitable growing environment of fish, the cold plasma treatment time is fixed at 20 s each time. The plasma treatment is performed for three weeks and the treated fish are monitored each week. Figure 3a shows the images of a pathogen biofilm-infected fish with skin disease, where two sides of the fish show a welldeveloped biofilm. Figure 3b-3d shows the changes in the health status of biofilm-infected fish skin in response to plasma treatment over three weeks. From Figure 3e showing the infected area (1)-(3) on the fish as a function of the plasma treatment time, it is evident that all of the areas with visible biofilm on fish skin greatly reduce in size with regular plasma treatment. There is no biofilm formed on the sides of the fish tank over the weeks of observation, even though no cleaning of the surfaces is performed. However, for the



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control group where fish are kept in the tank with air fed in but without plasma discharge, little changes on those infected areas but some stains in the tank wall are witnessed and the fish seem in deeper depression. These results suggest that this plasma-based method is extremely effective to control biofilm formation and development on artificial and living surfaces, and limiting biofilm growth in infected fish, with nitric oxide playing a key role. This is because in the environment, bacteria is often in the transition between planktonic and biofilm lifestyles.46 Given the correct environmental cues, bacteria in biofilm may initiate coordinated dispersal of the biofilm, and revert to the planktonic form.24

A

B

D

C

Figure 4. Role of the physical mechanism about the effect of microbubbles and the plasma-derived NO in biofilm dispersal. (a) Biofilm detachment by bursting of the bubble, which not only releases the reactive species, but also introduces mechanical agitation and local heating to promote biofilm dispersion. (b) Biofilm protects bacteria from reactive


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species. (c) Plasma-induced NO causes bacteria to be released from the biofilm. (d) Plasma-generated RONS kill bacterial cells released from biofilm mediated by nitric oxide.

Based on the experimental results and relevant literature,24,32,48,51,52 the following mechanism of plasma treatment-induced biofilm dispersal is proposed. As depicted in Figure 4b-4d, the mechanisms responsible for biofilm tolerance to plasma-generated species include reduced diffusion of plasma-induced RONS, release of protective enzymes capable of destroying or inactivating RONS in the biofilm matrix, and formation of physiologically distinct bacterial subpopulations (e.g., persister cells) resulting from nutrient and oxygen gradients.51,52 Plasma-derived •NO species diffuse into the biofilm and interact

with

cell

receptors

that

mediate

dispersal

by

increasing

bacterial

phosphodiesterase activity, with a consequent reduction of the intracellular second messenger and biofilm regulator cyclic-di-guanosine monophosphate (c-di-GMP).51 This prevents c-di-GMP from interacting with proteins at the transcriptional, translational, or post-translational level and leads to changes in cell surface and physiological processes associated with dispersal and motility.52 Owing to the reversal of the genetic programme that drives biofilm development, one consequence of NO-mediated dispersal is that both the biofilm and the dispersed cells lose their high level of resistance to antimicrobials, making them more susceptible to inactivation by bactericidal PAW. Moreover, plasma-derived RONS exhibit nitrosative and oxidative action alone and upon reaction with each other to form even more oxidative/nitrosative secondary species, including peroxynitrite and dinitrogen trioxide.8,9,42 These reactive species may then synergistically interact with matrix components (eDNA and polysaccharides), dissolved solutes in the hydrated matrix of the biofilm, increasing the penetration of plasma-active species into the biofilm matrix, and accelerating biofilm dispersal. The physical mechanism about the effect of microbubbles on biofilm removal was illustrated in Figure 4a. For the microbubbles reaching the close vicinity of a biofilm, the pressure waves generated through the self-collapse of those bubbles can eventually blow away fixed biomass from the artificial or living surface, and break up the EPS matrix of biofilms. Indeed, the pressure



waves generated by bursting of microbubbles can further induce the bursting of other yetbursting bubbles, which is similar to a chain reaction with the entry of microbubbles into the solution. Consequently, keep on removing fixed biomass and disrupting the EPS matrix until the whole biofilm structure is collapsed and ultimately detached from the surface. After biofilm dispersal, these planktonic bacterial cells were exposed to the bactericidal PAW, which is rich in highly reactive oxygen and nitrogen species. As depicted in Figure S10 (Supporting Information), these generated species may interact with microbial membrane proteins, DNA and metabolic enzymes, ultimately disrupting vital cellular functions and structures leading to potent antimicrobial efficacy.

CONCLUSION In summary, underwater microplasma bubbles, produced by atmospheric plasma array, are acting as transport vehicles to deliver RONS from plasma to liquid (PAW) for more effective biofilm dispersal. The anti-biofilm activity of reactive chemical species in PAW is clearly demonstrated and the relative significance of those reactive species in biofilm inactivation is determined. Results show that the corresponding E. coli biofilm biomass reductions with direct microplasma bubbles and PAW treatment are up to 80% and 60% (15 min discharge). The differences highlight the importance of bubbles as well as those other agents derived from plasma discharges. Plasma-derived active species in PAW, especially nitric oxide, are effective in biofilm dispersal. When integrated into a fish tank, cold atmospheric plasma-generated bubbles containing highly biochemically-reactive chemistries can control bacterial biofilms on the skin of infected fish. The findings also provide much needed insights into the fundamental aspects of plasma-liquid physics and chemistry required for the understanding of the biochemical activity of PAW and translation of this novel decontamination strategy into real life applications. Meanwhile, with further improvements to the reactor design and optimization of the whole system, the method discussed in this study may be further scaled up and extended into other in-liquid bio-


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decontamination applications such as the treatment of ballast water and ship surfaces under waterline.

EXPERIMENTAL SECTION Specification of the Microplasma Bubble Reactor. An atmospheric-pressure microplasma array is designed to generate underwater microplasma bubbles with diagrams shown in Figure 1a and 1b. Briefly, the plasma reactor consists of 29 microplasma jet units housed in a glass cup (inner diameter: 8 cm). Each discharge tube consists of two parts: (1) an inner Al2O3-coated electrode and (2) an external quartz tube. The separation between the external quartz tube and the inner electrode is approximately 80 μm. Air is supplied into the discharge tubes from the bottom of the device at the flow rate of 2.0 standard liter per minute (SLM). A stainless-steel mesh with an aperture of 350 μm (diameter: 8 cm) is soldered a copper support ring and electrically grounded, and is positioned about 10 mm above the discharge tubes in water (Figure 1b). When the gas passes through the separation distance, the plasma is produced by dielectric barrier discharge and microplasma bubbles are generated through the stainless-steel membrane. The bubble size distribution are obtained based on figures taken by a high-speed camera coupled with a long-distance lens. The power supply (TCP-2000K, Najing Suman Electronic, China) capable of supplying bipolar AC output with the peak voltage (VP) of 020 kV at an AC frequency of 8.0 kHz is used.

Measurement of Electrical and Optical Characteristics. The applied voltage and discharge current are measured by using a Tektronix 2040 digital oscilloscope with a highvoltage probe and a current probe, shown in Figure S1a. The Lissajous figure of the microplasma array in water is obtained to calculate the discharge power by measuring the charges across the capacitor (2.0 μF) in series to ground and the applied voltage across the discharge chamber (Figure S1b). In this study, all plasma treatments are performed by using the atmospheric microplasma array at VP= 4.0 kV, corresponding to the discharge



power of 40 W. The optical emission spectra (OES) from the discharge region are obtained, using a SpectraPro-750i monochromator (Acton Research Corporation) with a resolution of 0.5 nm in the wavelength range of 200 to 900 nm. The rotational (TRot) and vibrational (TVib) temperatures of N2 molecules in the atmospheric-pressure air microplasmas are determined by comparing the simulated spectra of the C3B3 (=2) band transition of N2 with the experimental recorded spectra (Figure S2, Supporting Information).

Preparation of the Biofilm Sample and PAW Treatment. A single E. coli colony is inoculated into approximately 200 ml LB liquid medium (tryptone, 2 g; yeast extract, 1 g; NaCl, 2 g; distilled water, 200 g; pH 7.2) and is cultivated to a stationary phase on a shaking table with a rotation speed of 200 rpm at 37°C for 48 h. The activated cell culture is centrifuged at a rotation speed of 3,000 rpm for 10 min at 4 °C in a refrigerated centrifuge (Hanil Continent-512R), and the deposit obtained is washed twice with sterile saline solution (0.85%), and finally suspended in sterile saline solution to a final cell concentration of approximately 108 to 109 CFU/mL. Stainless steel pieces (1×1 cm2) washed with 70% ethanol and sterile saline are dried at room temperature. Prepared pieces are immersed in test-culture suspension (25 mL) and incubated at 37 °C for 24 h to form biofilms.6,53 It should be pointed out that 24 h usually is sufficient for cells to attach to the surface, and form a thin biofilm structure.6,53 A schematic illustration of the experimental process is shown in Figure 1d and 1e. For the direct microplasma bubble treatment (in the glass cup containing 0.2 L of sterile distilled water), the biofilm sample is immersed in solution during processing. On the other hand, sterile distilled water of 0.2 L is activated for 15 min to obtain PAW solution which contains a variety of microbicidal active agents. After PAW generation or chemical solution preparation, the solution is poured into the dish where a biofilm cultured on a 1×1 cm2 stainless steel plate is placed. The duration for the PAW treatment and chemical solution treatment for the biofilm is also 15 min. For better comparison, the presence of biofilm in the control group (treated by the same volume of sterile distilled water) and the group of bubbles only (air fed at the same flow but without voltage applied) are also examined.


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Again it should be noted that a single biofilm handling time of 15 min is chosen to simplify experiments and focus on the discussion of the effects of microplasma bubbles and PAW.

Physicochemical Properties of PAW. The pH values of PAW solutions are gauged with a pH meter (Lab-850) as the solutions are exposed to air microplasmas. Once the plasma treatment is completed, the temperature of each plasma-treated solution is measured using a mercury thermometer. The measurements show that the temperature of the PAW is usually lower than 40 C. For reactive species in PAW, the NO2- concentration is determined by the well-known Griess assay. The NO3- concentration is also measured via Griess assay, through the conversion to NO2- by the use of vanadium (III) chloride. The H2O2 concentration was obtained by color forming reactions with titanium oxysulfate and spectrophotometric measurements. The concentration of ozone is measured by a portable ozone colorimeter (HACH PCII). An electron spin resonance (ESR) spectrometer (EMX-10/12) is used to text •NO existed in PAW after traped by N-methyl-Dglucamine dithio-carbamate (MGD) (99%, J&K Scientific, Ltd., China) through a combination of FeSO4·7H2O (AR, National Medicine Group Chemical Reagent Co., Ltd).

Quantification of the Remaining Biofilm Assay. To quantify the remaining static biofilm after treatment, crystal violet (CV) staining method is used to underestimate the antimicrobial activity of PAW. The absorbance is measured at 590 nm on a microplate reader (Synergy HT, Biotek Instruments, Inc.) and is corrected by subtracting the absorbance of the untreated biofilm. To assess the viability of biofilm after treatments, the remaining viable bacteria are also determined as colony count assay. The treated samples are sonicated (Bransonic 5100E-MT) in saline solution for 5 min and centrifuged for 1 min to detach the bacterial cells. Then, appropriate dilutions are prepared in sterile saline, plated onto the LB medium and incubated at 37 °C for 48 h. Results are prepared and the microbial counts are expressed as log CFU/cm2.



Confocal Laser Scanning Microscopy and Scanning Electron Microscopy. For confocal laser scanning microscopy (CLSM), biofilms mounted on standard glass coverslips are stained using the baclightTM live/Dead Bacterial viability kit (L-7012; Invitrogen Molecular Probes, Life Technologies, Thermo Fisher Scientific Pty Ltd, Australia) and analyzed with a FLUOVIEW FV3000RV confocal microscopy (Olympus, Waltham, MA). The morphologies of bacterial biofilms are examined with a ZEISS Sigma Scanning electronic microscope at 10-15 kV. Prior to imaging, the samples are gently rinsed twice with phosphate-buffered saline (PBS, pH 7.4) and fixed with 2.5% glutaraldehyde. The samples are then dehydrated using ethanol in 30, 50, 70, 90 and 100% concentrations. To prevent sample charging, the dried samples are coated with a thin (several nm-thick) layer of gold using a Leica Gold Coater.

Microplasma Bubble Reactor Applied in a Fish Tank. To investigate the efficacy of reactive chemistries obtained using our system for biofilm removal in a real life application, the microplasma bubble reactor is seamlessly integrated into a home-made fish tank with the length, width and height of 50 cm, 25 cm and 40 cm, respectively. Eight naturally infected adult blood parrot cichlid fish (Cichlasoma synspilum × Cichlasoma citrinellum) with weight of ~ 80 g and length of ~12 cm and similar infected area mainly on the head, fins and tail, are selected and introduced into the tank. The infecting species is identified as E. coli using a rapid test kit. The fish are purchased from local flower and bird market (Xiamen City, China). Twice daily, a discharge lasting for 20 seconds is used to study whether it will contribute to biofilm removal and positively contribute to improving fish health status (see the video in the Supporting Information). As control, eight infected fish are kept in the same tank in the absence of plasma treatment. Water is renewed every morning before the first discharge. The physicochemical properties of plasma activated pond water (temperature, pH values and the concentration of plasma-generated long-lived chemical species) are recorded and detailed in Figure S8 and S9. This is important due to the strict standards of water quality for freshwater fisheries,54 mainly including (1) pH value: 6.5-8.5; (2) dissolved oxygen content: more than 16 h of one day must be higher


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than 5.0 mg/L and the rest time shall not be less than 3.0 mg/L; (3) number of E. coli cells: no more than 5000 CFU/L.

Supporting Information Available. The supporting information is available free of charge on

the

ACS

publications

website

at

http://pubs.acs.org.

Physico-chemical

characterizations of microplasma bubble reactor and thus-generated PAW, SEM and CLSM images of treated biofilms, as well as a video for the microplasma bubble reactor used in a real-life application.

Corresponding Author *E-mail: [email protected] (K.B.), [email protected] (F.Z.) and [email protected] (X.Z)

Author Contributions # R.W.Z

and R.S.Z contributed equally to this work.

Notes The authors declare no competing financial interest. Acknowledgments. This work was partially supported by the National Natural Science Foundation of China (Grant No. 51877184) and the Australian Research Council (ARC). We acknowledge support by the QUT Postgraduate Research Award (QUTPRA).

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Underwater microplasma bubbles, produced by a microplasma-bubble reactor, are acting as transport vehicles to efficiently deliver reactive plasma species to target biofilm sites on artificial and living surfaces, for biofilm dispersal.


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