Bactericidal Effects against S. aureus and Physicochemical Properties of Plasma Activated Water stored at different temperatures

Water activated by non-thermal plasma creates an acidified solution containing reactive oxygen and nitrogen species, known as plasma-activated water (PAW). The objective of this study was to investigate the effects of different storage temperatures (25 °C, 4 °C, −20 °C, −80 °C) on bactericidal activities against S. aureus and physicochemical properties of PAW up to 30 days. Interestingly, PAW stored at −80 °C yielded the best antibacterial activity against Staphylococcus aureus, 3~4 log reduction over a 30-day period after PAW generation; meanwhile, PAW stored at 25 °C, 4 °C, and −20 °C, respectively, yielded 0.2~2 log decrease in cell viability after the same exposure and storage time. These results were verified by scanning electron microscope (SEM). The physicochemical properties of PAW stored at different temperatures were evaluated, including pH, oxidation reduction potential (ORP), and hydrogen peroxide, nitrate, nitrite anion and NO radical levels. These findings suggested that bacterial activity of PAW stored at 25 °C, 4 °C, −20 °C decreased over time, and depended on three germicidal factors, specifically ORP, H2O2, and NO3−. Moreover, PAW stored at −80 °C retained bactericidal activity, with NO2− contributing to bactericidal ability in association with H2O2. Our findings provide a basis for PAW storage and practical applications in disinfection and food preservation.

Scientific RepoRts | 6:28505 | DOI: 10.1038/srep28505 in noticeable concentrations in the solution 6,8,19 . Reactive-nitrogen-and -oxygen-based species play an important role in the toxic effect of PAW, and have DNA, RNA, proteins, and lipids as principal targets 20,21 . Moreover, both non-thermal plasma and PAW could catalyze the generation of intracellular ROS, consequently causing oxidative stress in bacterial cells [22][23][24] .
However, active species in PAW are difficult to assess due to short life spans and fast disproportionation in the plasma-liquid systems. Studies evaluating the effect of atomic oxygen on bacterial killing in water are scarce, since excited atomic oxygen only has a life time of ~30 ns 25 . It is also worth noting that trace amounts of ozone (a few ppm) were detected in air 26 . In water, ozone can live for 1 000 s at room temperature 27 . OH, with extreme reactivity, can be easily scavenged by virtually any organic molecule in its close vicinity, which accounts for its short life-span of about 10 −9 s 28 . Exploring PAW stability, Matthew J Traylor 8 pointed out the complexity of PAW solutions, where multiple chemical components exert varying biological effects in differing time scales, after measuring physicochemical properties and bacterial inhibition over a 7-day period.
Storage conditions are important factors affecting the physicochemical properties and bactericidal activity of PAW. However, little information is available concerning the effects of storage conditions such as temperature, lighting, and sealing. The aim of this study was to investigate the physicochemical properties and bactericidal activities of PAW stored at different temperatures. The effects of storage temperatures on pH, ORP, hydrogen peroxide, nitrate and nitrite anion levels and the stability of bactericidal efficiency of PAW were assessed for up to 30 days after generation. PAW's antimicrobial efficacy against S. aureus was examined by colony forming unit (CFU) count, and further verified by SEM. OES was employed to detect the main excited reactive species in PAW. Moreover, PAW's physicochemical characteristics, including pH, ORP, and hydrogen peroxide, nitrate and nitrite anion levels, were recorded. Finally, ESR spectroscopy was used to detect NO radicals in PAW. Understanding the physicochemical properties and bactericidal activity of PAW before and after storage is important to the practical application in inactivating harmful microorganisms in food industries and agriculture.

Experimental Section
Plasma Microjet Device and PAW Generation. The air plasma generator is schematically illustrated in Fig. 1(A), and was designed based on the HEDBS structure 29 . The system, mainly consisting of copper electrodes and quartz dielectric, was set at the end of a quartz tube (inlet diameter, 1.5 mm). Air with 260 L/h gas flow rate was injected into the quartz tube, and the high voltage electrode connected to a power source (20 kHz). A homogeneous plasma was generated in the discharge gap of 0.5 mm and a plasma jet reaching 7 mm long ejected through the end outlet of 0.5 mm. All device parts were fixed to each other to prevent accidental displacement.
As shown in Fig. 1(B), PAW was produced by placing the plasma jet beneath the water surface. The distance between the PMJ end and liquid surface was 20 mm. Every 10 ml sterile distilled water was activated by plasma for 20 min to obtain PAW, which was stored in centrifuge tubes. Four different storage temperatures were adopted, including 25 °C, 4 °C, −20 °C, and −80 °C for 1, 3, 7, 15, and 30 days, respectively. For simplified description of the different PAW states, PAW (25), PAW(4), PAW(−20), and PAW(−80) represented PAW stored at 25 °C, 4 °C, −20 °C, and −80 °C after plasma activation, respectively. Meanwhile, PAW-0 referred to water without plasma activation.

Bacterial strains, growth conditions, and PAW treatment. S. aureus (China General Microbiological
Culture Collection Center, CGMCC number 1.2465), was used as a model organism in inhibitory tests. Bacteria were grown at 37 °C in Luria-Bertani (LB) medium to logarithmic phase (absorbance of 0.15 at 600 nm on SPECTROstar Omega plate reader, BMG, Germany), about 10 8 /mL bacteria. Afterwards, 1 ml of the bacterial suspension was centrifuged at 5000 r/min for 10 min, and resuspended in 1 ml distilled water. Then, 100 μl of the resulting S. aureus suspension was added into 10 ml PAW equilibrated at 37 °C (5 min), for 20 min incubation. S. aureus suspension treated with PAW-0 was setup as the negative control. All experiments were repeated three times for statistical analysis. Sterilization Ability of PAW. Colony count assay was used to assess kinetic killing curves and antimicrobial effects of a given compound or device. In brief, after 20 min PAW treatment, tenfold serial dilutions of 100 μl treated suspension were plated on LB agar culture medium, and incubated at 37 °C for about 21 h before CFU count. The sterilization ability of PAW was evaluated by the Log Reduction, calculated as follows: Scanning electron microscope (SEM, Quanta 200FEG) was used to evaluate cell morphology before and after PAW treatment. For SEM sample preparation, cells were pelleted by centrifugation (5000 rpm), and dehydrated by alcohol gradient (30,50,70,80,90, and 100%) until desiccation. The samples were then gilded and observed under an electron microscope.

OES Detection of the Main Excited ROS in PAW.
To identify the main excited reactive species in plasma activated water, OES was employed in the 200-1000 nm range along the axial direction of the PMJ with the AvaSpec-2048-8 Fiber Optic. Plasma discharge formed beneath the water surface and one end of the fiber optics cable was used to acquire light signals at the bottom of the water container (quartz tube), approximately 5 mm away from the exit nozzle 30 .

Evaluation of PAW's Physical and Chemical Properties.
After PAW storage at different temperatures for various times, pH, ORP, and H 2 O 2 contents were immediately detected to evaluate its physical and chemical properties. ORP, an indicator of the ability of a solution to oxidize and related to oxidizer concentration, activity, or strength 31  Statistical Analysis. Data were obtained from at least three independent experiments (n ≥ 3). Values are mean ± standard deviation (SD). Statistical analysis was performed using SPSS statistical package 17.0 (SPSS Inc., USA). Analysis of variance (ANOVA) was used to compare different treatments during PAW storage; significant differences were identified by the Student-Newman-Keuls multiple range test, with a confidence level at P ≤ 0.05. In addition, paired-samples t-test was employed to compare the effects of different treatment conditions on NO radicals in PAW after 30 days of storage. Significant differences were represented by *P < 0.05, **P < 0.01, and ***P < 0.001.

Results and Discussion
Bactericidal efficiency of PAW samples under different storage temperatures. S. aureus inhibition. The colony count assay was employed to assess the bactericidal effects of PAW samples stored at different temperatures for various days. Bactericidal ability of PAW increased with decreasing temperature, with −80 > −20 > 4 > 25 (°C) obtained in descending order for bacterial inhibition. Before storage, fresh PAW resulted in a log reduction of approximately 5 for S. aureus. As shown in Fig. 2, bacterial growth declined to 3.7 and 1.8 at −80 °C and 25 °C, respectively, after 1 day of storage. PAW stored at −80 °C retained higher antibacterial activity compared to the other temperatures, leading to a log reduction of 3~4 for S. aureus; at other temperatures, PAW reduced total bacterial growth by 1.8, 2.2, 2.9 logs compared with untreated control, which decreased to about 1 log after 30 days of storage. Generally speaking, significant differences in bacterial killing were obtained between PAW(−80) and PAWs stored at other temperatures. These findings indicated that loss of bactericidal activity in PAW(−80) was reduced compared with that of other temperatures, indicating PAW(−80) retained efficient content, and consequently bactericidal activity. The differences for various storage temperatures might be explained by great variations in the physicochemical properties of PAW in these conditions. These results were consistent with a previous research assessing acidic electrolyzed water (AEW), and revealing that storage at −18 °C may be a better method to retain the available chlorine concentration as well as bactericidal activity compared to storage at 25 °C 35 .
SEM assessment of S. aureus. Scanning electron microscope (SEM) study further verified the stronger bactericidal ability of PAW(−80) compared to that of other temperatures. Typical SEM images of S. aureus before and after 20 min of PAW treatment following 30-day storage are shown in Fig. 3. Interestingly, the bacteria underwent a transition from initially smooth surfaces to surfaces with distortion, shrinkage and rupture of the outer layer after PAW treatment. The level of damage depended on storage temperature, and was more severe for −80 °C ( Fig. 3(E)) compared to 25 °C (Fig. 3(B)), 4 °C ( Fig. 3(C)), and −20 °C (Fig. 3(D)). The bacteria underwent a transition from smooth (Fig. 3(A)) to severely deformed (Fig. 3(E)) surfaces. These results were consistent with bacterial number reduction (Fig. 2), indicating the superiority of PAW stored at −80 °C over samples kept at other temperatures.

Physicochemical properties of PAW during storage. OES Detection of Main Excited ROS in
PAW. OES was employed to investigate the main excited active species generated in PAW. In the end-on spectra of the PAW, PMJ operated beneath the water surface at an operating current of 35 mA and voltage of 100 V (Fig. 4(A)). OES results are dominated by the second positive system of at 300-420 nm, 400-520 nm, and 560-730 nm, respectively, as well as N with O emission lines at 730-900 nm. O and N generated from discharge are mainly formed through dissociation of O 2 and N 2 , respectively, which are excited from the ground state by electron impact (1) and (2) 29 .
OES data indicated that excited atomic oxygen and nitrogen were produced in water by the plasma. Atomic oxygen is a chemically reactive species which can cause damage in biological molecules 36 . Atomic oxygen is readily converted into other reactive oxygen species, such as O 3 , ·OH and H 2 O 2 , due to high activity through chemical reactions [37][38][39] . Nitrites, nitrates, S-nitrosothiols and nitrosamines are metabolites of NO and mediators of the related cytotoxic effects, namely inhibition of mitochondrial respiration DNA damage leading to gene mutation, protein alteration and loss of function, necrosis, and apoptosis 40 .
PH and ORP in PAW samples. Physicochemical parameters of PAW showed significant differences at all storage temperatures compared with the control group, PAW-0. Solution pH and ORP were obtained during the storage process (Fig. 5). After plasma activation for 20 min, PAW's pH decreased to 2.3 from 6.8, while ORP reached about 540 mV from the initial 250 mV. On the other hand, there was no significant change among the four different storage temperatures for the same time. As shown in Fig. 5, pH values of all samples only slightly changed with increasing storage time, and remained essentially stable during the 30-day storage in all four conditions; the pH of PAW remained around 2.0 regardless of storage temperature.
ORP is regarded as an important factor influencing microbial inhibition. A high ORP can damage the outer and inner membranes 41 . Thus, it is of great significance to explore changes in PAW ORP for monitoring bactericidal efficacy. Figure 5    respectively. As for different temperatures, changes followed a similar trend; they decreased slightly during the initial seven storage days by 15.6%, 13.3%, 17.1%, and 17.5%, respectively, and were reduced by 15.6%, 13.3%, 10.0%, and 6.2%, respectively, from days 7 to 30.
The synergetic effects of acidic pH and ROS always provide a warranty of relatively good germicidal efficacy 6 . In addition, high ORP indicates a solution with high oxidative strength 14 . There was no obvious change between different temperatures, demonstrating that they are not pivotal factors in sterilizing against S. aureus among different storage conditions.   increasing temperature. Furthermore, significant differences in NO 2 − concentrations between PAW(25) and PAW(−80) were obtained (p < 0.05). In addition, the post-discharge reaction between hydrogen peroxide and nitrite ions occurring in water after treatment with plasma determined the formation of peroxynitrite: NO 2 − +H 2 O 2 +H + →ONOOH + H 2 O 48 . Peroxynitrite chemistry was shown to significantly participate in the antibacterial properties of PAW 6,8,18,19 . NO 3 − amounts in PAW samples significantly decreased after storage, while NO 2 − and H 2 O 2 amounts dropped faster after storage at the other three temperatures compared with −80 °C, indicating that PAW(−80) is more favorable to keep NO 2 − , H 2 O 2 , and consequently, bactericidal activity. For solutions treated by liquid-phase plasmas, the antimicrobial properties of PAW were tentatively attributed to the synergetic effects of H 2 O 2 and nitrite remaining in noticeable concentrations. This would be good news for users in practical applications, because PAW(−80) can avoid active ingredient loss during storage.
NO radical amounts in PAW samples. The direct spin trapping reaction between Fe 2+ (MGD) 2 and NO produces the spin adduct NO-Fe 2+ (MGD) 2 that is characterized by a tripling ESR spectrum, with a peak intensity ratio of 1:1:1. As shown in Fig. 8(A,B), NO was detected in PAW but virtually absent in the PAW-0 control. NO-Fe 2+ (MGD) 2 signals detected in PAW demonstrated that NO radical is generated in solutions activated by plasma. After 30-day storage, relative NO-Fe 2+ (MGD) 2 signal intensities for the same experimental conditions in PAW (25), PAW(4), PAW(−20) and PAW(−80) achieved 75.84, 78.34, 67.91, 85.28 μmol/L, respectively, according to the standard concentration of NO donor. There were no significant differences among treatment groups, indicating that NO radical is not a key factor affecting sterilization ability of PAWs stored at different temperatures. However, potential antimicrobial effects of NO have also been reported. T. Lai 49 observed that exogenous NO can induce generation of ROS and cause oxidative damage to proteins during the germination process, resulting  As shown in is affected by temperature. Nitrite production may thus be transiently encountered and biologically active in PAW.
Our results indicated that bactericidal activity could be preserved at −80 °C during PAW storage. Consequently, −80 °C has the potential to keep freshness and product sanitization in melted PAW. Compared with PAW samples stored at other temperatures, physicochemical parameters of PAW(−80) had significant changes ( Table 2). This is good news for PAW users, since in practice PAW can be prepared and stored at −80 °C until needed, avoiding loss of constituents during storage. Furthermore, melted PAW can be treated as common water because of reduced adverse impact on the human body as well as the environment.

Conclusion
We demonstrated that PAW(−80) is more efficient than PAWs stored at other temperatures (25 °C, 4 °C, and −20 °C) for sterilization. With regard to pH, ORP, and NO 3 − , minimal changes of PAW were observed. H 2 O 2 and nitrite concentrations presented the same trend, decreasing with increasing storage temperature. Evaluation of NO radicals by ESR showed that short-lived species slightly contribute to the differences in sterilization efficiency of PAW stored at different temperatures. This suggested that PAW(−80) can preserve bactericidal activity, and H 2 O 2 and NO 2 − levels, leading to S. aureus inhibition. This work unravels the importance of storage temperature on physicochemical properties and bactericidal efficiency of PAW, providing a basis for practical application of PAW in inactivating harmful microorganisms in medicine and foodstuff. In the future, other storage conditions should be assessed, e.g. light, agitation, and packaging.

Statement of Novelty.
Plasma-activated water (PAW) as a novel disinfectant can be used to inhibit harmful microorganisms in food industry and medical settings. However, the short life of active species in PAW is an important limitation for practical applications. Here, physicochemical properties and bactericidal activities of PAW stored at 25, 4, −20, −80 °C, respectively, were assessed. Results showed PAW stored at −80 °C retained efficient inhibitory activity against S. aureus. Moreover, NO 2 − contributed to the antimicrobial property, in combination with H 2 O 2 . These findings provide a basis for PAW storage and practical applications in food and medical industry.