Introduction

Disinfectant pollution has caused wide public concern over the world. Widespread use of chemical disinfectant is a terrible hazard for the environment. Plasma activated water (PAW) has gained increasing attention as a novel and very efficient disinfectant [1,2,3,4]. PAW can be generated by non-thermal plasma and is currently under study as potential alternatives to conventional sterilization techniques applied for agriculture [5, 6], food [4,5,6,7] and medicine [8, 9]. The main advantages of using PAW for sterilization include less adverse impact on the environment and no risk during storage and transportation [10].

Depending on the type of discharge, long-lived reactive species, such as H2O2 and O3 can be transferred from the plasma into the liquid, and they are believed to play dominant roles in the inactivation process [2, 11]. The contribution of nitrogen-containing species, such as NO2−, ONOOH, and O2NOOH [12, 13], also needs to be considered. These species are highly cytotoxic and can react with cellular tissue [14, 15]. Therefore, the antimicrobial properties of PAW were temporarily attributed to the synergetic effect of H+ ions,the H2O2 and NO2−/NO3− remaining in noticeable concentrations in the solution [16].

However, few investigations focus on the effective inactivation of Spoilage bacteria by PAW. Spoilage bacteria has gained an increasing attention [17, 18], and the decay of meat products is mainly caused by Spoilage bacteria. Rotten meat products can emit some gases with peculiar smells, for instance hydrogen sulfide (H2S) and trimethylamine (TMA). TMA is easy to resolve into dimethylamine and formaldehyde. Formaldehyde that has been identified as carcinogen by the World Health Organization can seriously affect the activities of cells and cause great damage to cells. S. putrefaciens is a major spoilage bacterium of meat, and it emits gas with a peculiar smell [19]. Therefore, there is an urgent need to search an effective and natural way to inhibit the bacterial activity.

In this study, an atmospheric-pressure plasma device consisting of 28 air microplasmas was used to generate PAW. PAW was used to sterilize S. putrefaciens cells. The sterilization efficiency of S. putrefaciens cells was evaluated via colony forming unit (CFU) count on Petri dish. In order to analyze the inactivation process of S. putrefaciens cells, the scanning electron microscopy (SEM) was used to observe their structural changes. After PAW treatment, DNA concentration of S. putrefaciens cells has been measured by UV/VIS-spectrophotometer. Moreover, the physicochemical characteristics of PAW, including initial PAW temperature, pH value, and the concentrations of H2O2, NO3−, NO2− and O3 were measured. Finally, the sterilization efficiency of S. putrefacien cells treated by PAW was compared to the one of S. putrefacien cells treated by the H2O2/HNO3 mixture solution.

Experiment Setup

Plasma Device and PAW Generation

A schematic diagram of the microplasma array is shown in Fig. 1. Previously, we have reported a similar structure [20]. The plasma device is mainly composed of 28 microplasma jet units housed in an equilateral triangle quart cup. In each microplasma jet unit, a quartz tube with its inner diameter of 700 μm and its outer diameter of 1000 μm is used to generate the microplasma. The 300 μm thick tungsten wires, acting as a high-voltage electrode, are inserted into the quartz fiber, and the distance between the nozzle and tungsten wire is 3 mm. The 28 microplasmas jet units are uniformly distributed inside the equilateral triangle reactor. The air is fed into the reactor at a flow rate of 1.0 standard liter per minute (SLM). The air flows from the quartz tubes into the water. One copper electrode immersed in the water is connected to the ground. The power supply consists of a signal generator, a power amplifier and a transformer and can generate the pulse voltage with its peak voltage of 6.0–12.0 kV and its frequency of 7.0 kHz. The 40 mL deionized water was activated by changing the water-activated time from 0–30 min.

Fig. 1
figure 1

Schematic diagram of the experimental setup used in this study a mimic diagram of microplasma triangle array device, b top view of the plasma device

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Antimicrobial Activity

The isolate of Shewanella putrefaciens (China General Microbiological Culture Collection Center, CGMCC number 1.3667) was used in this study. Fresh solution of Shewanella putrefaciens (S. putrefaciens) was cultured overnight in 100 mL Luria–Bertani (LB) by shaking with 180 rpm at 37 °C for 12 h. S. putrefaciens cells were cultured in LB medium at 37 °C until its density reached 1 × 108 cfu/mL. In order to obtain a countable number of colonies on control plates, S. putrefaciens cells were diluted to 1 × 105 cfu/mL with normal saline solution. As shown in Fig. 2, 40 mL PAW was generated by changing water-activated time and applied voltage. After the discharge for 3–5 s, 20 μL S. putrefaciens solution diluted was immediately added into 300 μL PAW. 20 μL S. putrefaciens solution diluted was transferred to 300 μL sterile water as the control. After 5 min incubation time, the 320 μL mixed solution was spread over the LB agar.

Fig. 2
figure 2

Schematic diagram of the sterilization way

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The effects of initial water temperature and pH values on sterilization efficiency were studied to analyze the inactivation process of S. putrefaciens cells by PAW. Thermostat water bath and ice were used to control initial sterile water temperature. The initial temperature of sterile water value was in the range of 5–100 °C. Nitric acid was used to preset the pH value of water. The pH value of the water was in the range of 2–5.9. 20 μL S. putrefaciens solution diluted was added into 300 μL sterile water. After 5 min incubation time, the 320 μL mixed solution was spread over the LB agar. The sterilization efficiencies of S. putrefaciens cells treated by H2O2, HNO3 solutions and their mixture solution were also evaluated. 20 μL S. putrefaciens diluted solution was added into 300 μL H2O2, HNO3 solutions, and H2O2/HNO3 mixture solution. After 5 min incubation time, the 320 μL mixed solution was spread over the LB agar. Plate colony-counting method was used to count the survival amount microflora. It should be pointed out that all the bacterial-inactivated experiments reported here were repeated three times. The sterilization efficiency of solution was evaluated by Log Reduction, as follows:

$${\text{Log}}\;{\text{Reduction}} = {\text{Log}}\left( {\frac{{{\text{CFU}}_{\text{control}} }}{{{\text{CFU}}_{\text{treated}} }}} \right)$$
(1)

SEM Analysis

After S. putrefaciens cells were treated by PAW, scanning electron microscopy (S-4800, HITACHI, Japan) was used to observe the morphological changes of the S. putrefaciens cells. To perform SEM measurements, S. putrefaciens cells were diluted to 1 × 107 cfu/mL with normal saline solution. 100 μL S. putrefaciens solution was mixed with the 1.5 mL sterile water or PAW. 10 μL the mixed solution was transferred to sterile conductive silicon wafers. The S. putrefaciens solution was washed by PBS and fixed by 2.5% glutaraldehyde solution at 4 °C. The samples were washed twice by using the PBS and fixed by 1% osmic acid. Subsequently, they were eluted by changing concentrations of alcohol dehydration. After coated with a gold layer, they were visualized with SEM [21].

DNA Released from S. putrefaciens Cells Analysis

The concentration of DNA molecules released from S. putrefaciens cells was measured to evaluate the integrity of S. putrefaciens cells. The DNA concentration was calculated by monitoring the absorbance peak of samples at 260/280 nm by UV/VIS-spectrophotometer [22]. Nucleic acids (DNA or RNA) contain conjugated double bonds in their purine and pyrimidine rings, which have a specific absorption peak at 260 nm. The intensity of UV absorbance is proportional to the concentration of nucleic acid [22]. 3 mL of the S. putrefaciens solution was transferred to 10 mL sterile water or PAW. To obtain a clarification solution, their mixture solution was centrifuged at 6000 g for 10 min. Then, 6 mL supernatant was filtered by 0.22 μm filtration membrane. Subsequently, 3 mL solution filtered was used to measure DNA concentration by using UV/VIS-spectrophotometer (TU-1950, PERSEE, China).

pH and Temperature Measurement of PAW

PAW was generated by changing water-activated time and applied voltage. After discharge, both the pH value and temperature of PAW were immediately measured. The pH value and initial temperature of PAW were measured by a pH meter (model Lab-850; SI Analytics Co., Germany) and a mercury thermometer, respectively.

Chemical Species in PAW

The atmospheric-pressure air microplasma contains reactive oxygen species (ROS), such as H2O2, HO2, O3, NOX, and O and OH radicals in the gas phase. Long-lived species, such as H2O2, NO3−, NO2− and O3 were formed during the chemical reaction between the plasma and water, and their concentrations were measured by UV/VIS-spectrophotometer (TU-1950, PERSEE, China). For the measurements of H2O2 concentration, 2 mL TiSO4 (5%) solution was immediately transferred to 2 mL PAW after it was treated by the atmospheric-pressure microplasma. 4 mL H2SO4 (4 mol/L) solution was added to the mixed solution. The 3 mL PAW containing TiSO4 and H2SO4 was measured by using UV/VIS-spectrometer at 407 nm [16, 23, 24]. To measure the NO3− concentration, 1 mL PAW was immediately added to 5 mL Nickel Sulfamate solution (5%). Then, 3 mL of the mixed solution was measured by using UV/VIS-spectrophotometer at 219 nm. NO2− concentration was also measured by using UV/VIS-spectrophotometer. Sulphanilamide was used as the diazotizing reagent and N-(1-Naphthy1)-ethylenediamine hydrochloride was used as the coupling reagent. 40 μL of 10 g/L sulphanilamide was added to 2 mL PAW. Then this solution was incubated at room temperature for 2 min. Subsequently, 40 μL of 1.0 g/L N-(1-Naphthy1)-ethylenediamine hydrochloride was added into the mixture solution. After reaction for 20 min at room temperature, the NO2− concentration was measured by using UV/VIS-spectrophotometer at 540 nm [16, 23, 24]. The concentration of O3 dissolved in liquid was determined by the Indigo method. 2 mL sodium indigotin-disulfonate solution (279 mg/L) was transferred to 5 mL phosphate buffer solution (7 g/L KH2PO4, 7 g/L NaH2PO4, pH = 2). Then, 5 mL PAW was added to the mixed solution, then their mixture was diluted to 50 mL by ultrapure water. The O3 concentration was measured by using UV/VIS-spectrophotometer at 610 nm [25, 26].

Results

Sterilization Efficiency

Figure 3 shows the sterilization efficiency as a function of water-activated time and applied voltage. The water-activated time was varied from 0 to 30 min. It can be clearly seen that both the water-activated time and applied voltage have a significant effect on the sterilization efficiency. After 30 min of water-activated time, the average sterilization efficiency can reach 0.13 Log Reduction at 6.0 kV, 0.38 Log Reduction at 7.0 kV and 2.0 Log Reduction at 8.0 kV. With increasing the applied voltage to 10.0 kV, the average sterilization efficiency can reach as high as 2.0 Log Reduction at the water-activated time of 25 min. When applied voltage is 12.0 kV, the average sterilization efficiency can be up to 2.0 Log Reduction at water-activated time of 20 min.

Fig. 3
figure 3

The sterilization efficiency of PAW as a function of applied voltage and water-activated time. The incubation time of S. putrefaciens with PAW is 5 min. The data points and error bars in all figures represent the averages and standard deviations from three independent measurements

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The morphological changes of S. putrefaciens cells were observed after the PAW treatment. Figure 4 shows SEM images of S. putrefaciens cells treated by PAW at voltage of 12.0 kV. The S. putrefaciens cells treated at the water-activated time of 5, 15 and 20 min were defined as 5-min PAW, 15-min PAW and 20-min PAW, respectively. As shown in Fig. 4a, before S. putrefaciens cells are treated by PAW, their shapes are relatively complete, long and narrow with smooth surface. There is no cell damage or overflow of the content. After PAW treatment, the S. putrefaciens cells exhibit different degrees of damage. The S. putrefaciens cells underwent a transition from initially smooth surfaces to severely deformed surfaces. It was clear that S. putrefaciens cells are destroyed, as shown in the red circles in Fig. 4b, c. Damage of the S. putrefaciens cells walls could be caused by ROS in PAW, which could oxidize unsaturated fatty acids in lipid bilayer of cell wall, cleave peptide bonds and oxidize amino acid side chains [4]. After 20 min PAW treatment, it is difficult to find the relatively complete cells structure, as shown in Fig. 4d. It can be seen that PAW has a detrimental effect on S. putrefaciens cells. S. putrefaciens cells walls or membrane could be destroyed, resulting in the leakage of cell protoplasm and electrolyte, which is considered to be lethal to S. putrefaciens cells.

Fig. 4
figure 4

Morphological changes of S. putrefaciens cells after PAW treatments

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The concentration of DNA molecules released from S. putrefaciens cells was measured to evaluate the integrity of S. putrefaciens cells. The DNA concentration was measured after PAW treatment. As shown in Fig. 5, the DNA concentration is significantly dependent on the water-activated time and applied voltage. The DNA concentration significantly increases at applied voltage of 7.0 kV. The DNA concentration firstly increases with increasing water-activated time and eventually decreases with further increasing water-activated time at voltage of 8.0–12.0 kV. Moreover, the DNA concentration is beyond the detection limit at applied voltage of 6.0 kV. The DNA leakage could be attributed to the disruption of the cell membrane of S. putrefaciens cells during the PAW inactivation process.

Fig. 5
figure 5

The DNA concentration of mixed solution as a function of treatment time and applied voltage. The incubation time of S. putrefaciens with PAW is 5 min. The data points and error bars in all figures represent the averages and standard deviations from three independent measurements

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As shown in Fig. 6a, the pH value of PAW is significantly dependent on the water-activated time and applied voltage. The pH value of PAW is typically in the range of from 5.8 to 2.5, depending on the water-activated time and applied voltage. NOx is formed inside the atmospheric-pressure air microplasma. The chemical reactions between NOx, air and H2O lead to a decline in the pH value of PAW, as shown below [27,28,29,30].

Fig. 6
figure 6

a The pH values of PAW as a function of water-activated time and applied voltage, b the sterilization efficacy of water as a function of pH value. The incubation time of S. putrefaciens with PAW is 5 min. The data points and error bars in all figures represent the averages and standard deviations from three independent measurements

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$${\text{NO}}\left( {aq} \right) + {\text{NO}}_{2} \left( {aq} \right) + {\text{H}}_{2} {\text{O}} \to 2{\text{NO}}_{2}^{ - } + 2{\text{H}}^{ + }$$
(2)
$$2{\text{HNO}}_{2} \to {\text{NO}}^{ \cdot } + {\text{NO}}_{2}^{ \cdot } + {\text{H}}_{2} {\text{O}}$$
(3)
$$2{\text{NO}}_{2} + {\text{H}}_{2} {\text{O}} \to {\text{NO}}_{3}^{ - } + {\text{NO}}_{2}^{ - } + 2{\text{H}}^{ + }$$
(4)
$$4{\text{NO}}^{ \cdot } + {\text{O}}_{2} + 2{\text{H}}_{2} {\text{O}} \to 4{\text{NO}}_{2}^{ - } + 4{\text{H}}^{ + }$$
(5)
$$4{\text{NO}}_{2}^{ \cdot } + {\text{O}}_{2} + 2{\text{H}}_{2} {\text{O}} \to 4{\text{NO}}_{3}^{ - } + 4{\text{H}}^{ + }$$
(6)

The pH value of PAW can have an impact on bacteria growth since the optimum pH value for S. putrefaciens growth is from 6 to 9. The pH value of PAW can affect a series of biological substance of bacteria, such as protein and nucleic acid [29]. However, some bacteria can not be affected by liquid acidity unless a hazardous pH value is reached. For example, for Staphylococcus aureus a hazardous pH value of 2 is needed to initiate inactivation [31]. Figure 6b shows the sterilization efficiency of S. putrefaciens cells as a function of pH value in water. The sterilization efficiency can reach as high as 0.13 Log Reduction at pH value of 2.

The initial PAW temperature was measured immediately by using mercury thermometer after plasma treatment. Clearly, the initial PAW temperature is significantly dependent on the water-activated time and applied voltage, as shown in Fig. 7a. The effect of water temperature on sterilization efficacy was studied. Figure 7b shows the sterilization efficiency of S. putrefaciens cells as a function of initial sterile water temperature. The sterilization efficacy can reach as high as 0.10 Log Reduction at temperature of 100 °C. This result may be due to the fact that sterile water temperature changed to room temperature during the process of incubating bacteria. Thus, sterilization efficiency at temperature of 100 °C is not so high as expected. However, the sterilization efficacy of S. putrefaciens only is 0.02 Log Reduction at water temperature of 60 °C. This indicates that the initial PAW temperature is not an important parameter for antimicrobial activity. Thus, the temperature of PAW below 50 °C does not significantly result in the inactivation of S. putrefaciens.

Fig. 7
figure 7

a The temperature of PAW as a function of water-activated time and applied voltage, b the sterilization efficiency of water as a function of different temperature value. The incubation time of S. putrefaciens with PAW is 5 min. The data points and error bars in all figures represent the averages and standard deviations from three independent measurements

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Chemical Properties of PAW

H2O2 is thought to be an important oxidant in PAW and a steady product. Depending on water temperature, its half-life period ranges from 8 h to about 20 days [31]. To avoid the effect of holding time on the H2O2 concentration of PAW exposed to the air, the H2O2 concentration was immediately measured by using the UV/VIS-spectrophotometer. Figure 8 shows the H2O2 concentration of PAW as a function of water-activated time and applied voltage. The H2O2 concentration can be up to 4.6 mmol/L at applied voltage of 12 kV, when water-activated time is 30 min. The H2O2 concentration in the PAW was related to the sterilization efficiency of S. putrefaciens. The H2O2 molecule may act as one oxidant in the solution, and they can react with organisms [32].

Fig. 8
figure 8

The H2O2 concentration of PAW as a function of water-activated time and applied voltage. The data points and error bars in all figures represent the averages and standard deviations from three independent measurements

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In addition, reactive nitrogen species (RNS) in PAW, such as nitrites and nitrates, were believed to play crucial roles in the sterilization process [16, 31]. The NO3− concentration was immediately measured by using the spectrophotometer. Figure 9a shows the NO3− concentration of PAW as a function of the water-activated time and applied voltage. The NO3− concentration of PAW is significantly dependent on the applied voltage ranging from 6.0 to 12.0 kV and the water-activated time ranging from 0 to 30 min. The NO3− concentration can be up to 1.6 mmol/L in the PAW at applied voltage of 12.0 kV when water-activated time is 30 min. The chemical reactions related to NO2− and NO3− can be described by reactions (2) and (4)–(9).

Fig. 9
figure 9

a The NO3− concentration of PAW as a function of water-activate time and applied voltage, b the NO2− concentration of PAW as a function of water-activate time and applied voltage. The data points and error bars in all figures represent the averages and standard deviations from three independent measurements

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In this study, NO2− concentration was also measured, as shown in Fig. 9b. The NO2− concentration initially increases with increasing water-activated time and eventually decreases with further increasing water-activated time at applied voltage of 6.0–12.0 kV. The NO2− concentration of PAW can be up to 8.9 μmol/L. This result indicates that the NO2− concentration decreases with increasing applied voltage from 6.0 to 12.0 kV. Due to the increasing of H2O2 and O3 concentration at applied voltage of 6.0–12.0 kV, NO2− can be oxidized into NO3−, ONOOH, O2NOOH, etc. These complicated oxidation reactions can occur in the plasma zone. The relevant chemical reactions can be described by reactions (7)–(11) [16, 27, 33, 34]. The decay mechanisms of NO2− under this experimental condition are still under study.

$${\text{NO}}_{2}^{ - } + {\text{H}}_{2} {\text{O}}_{2} + {\text{H}}^{ + } \to {\text{ONOOH}} + {\text{H}}_{2} {\text{O}}$$
(7)
$${\text{NO}}_{2}^{ - } + {\text{O}}_{3} \to {\text{NO}}_{3}^{ - } + {\text{O}}_{2}$$
(8)
$${\text{NO}}_{2}^{ - } + {\text{H}}_{2} {\text{O}}_{2} + {\text{H}}^{ + } \to {\text{NO}}_{3}^{ - } + {\text{H}}_{2} {\text{O}}$$
(9)
$${\text{HNO}}_{2} + {\text{H}}^{ + } \to {\text{H}}_{2 } {\text{NO}}_{2}^{ + } \to {\text{NO}}^{ + } + {\text{H}}_{2} {\text{O}}$$
(10)
$$2{\text{HNO}}_{2} \to {\text{NO}}^{ \cdot } + {\text{NO}}_{2}^{ \cdot } + {\text{H}}_{2} {\text{O}}$$
(11)

Figure 10 shows the O3 concentration of PAW as a function of the water-activated time and applied voltage. The O3 concentration of PAW is significantly dependent on the applied voltage ranging from 6.0 to 12.0 kV and the treatment time ranging from 0 to 30 min. The concentration of O3 dissolved in PAW increases rapidly in the first minute of water-activated time, and then shows a much slower increase. When water-activated time is 30 min, the O3 concentration of PAW can reach 0.13 mmol/L at 12.0 kV, 0.11 mmol/L at 10.0 kV, 0.09 mmol/L at 8.0 kV, 0.07 mmol/L at 7.0 kV and 0.04 mmol/L at 6.0 kV. O3 as a long-lived species is produced in the gas phase. O3 can transfer from the gas phase into the liquid and participate in the chemical reaction process and inactivation of bacteria in the PAW. In this work, the concentration of O3 in the effluent gas reaches almost 200 PPM. However, the O3 concentration of PAW was relatively lower compared with the one of the effluent gas. O3 can react with NO· and NO ·2 species in the gas phase or react with nitrites in the liquid, and residual O3 can dissolve into PAW [27, 35, 36].

Fig. 10
figure 10

The O3 concentration of PAW as a function of treatment time and applied voltage. The data points and error bars in all figures represent the averages and standard deviations from three independent measurements

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The sterilization efficiencies of S. putrefaciens cells by H2O2, HNO3 and H2O2/HNO3 mixture solution were measured to analyze the inactivation process of S. putrefaciens cells. The solution of S. putrefaciens was mixed with sterile water as control group, and their concentration is shown in Fig. 11. As shown in Fig. 11, the measurements indicate that HNO3 solution does not significantly contribute to the inactivation of S. putrefaciens cells. The sterilization efficiencies of S. putrefaciens cells treated by HNO3 and H2O2 solutions are 0.14 Log Reduction and 0.46 Log Reduction, respectively. Compared to the HNO3 and H2O2 solutions, the H2O2/HNO3 mixture solution is more effective in killing S. putrefaciens cells. The sterilization efficiency can reach as high as 1.16 Log Reduction. This indicates that H2O2 and HNO3 mixture in the solution is important for the sterilization in the PAW.

Fig. 11
figure 11

The inactivation efficiencies of S. putrefaciens cells as a function of the concentration of H2O2, HNO3 and H2O2/HNO3 mixture solutions. The incubation time of S. putrefaciens with the solution is 5 min. The data points and error bars in all figures represent the averages and standard deviations from three independent measurements

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In order to compare the sterilization efficiency of H2O2/HNO3 mixture solution with the one of PAW, H2O2 and HNO3 were added into sterile water. Both the HNO3 and H2O2 concentrations of their mixture solution are similar to the ones of the PAW used to sterilize S. putrefaciens cells. As shown in Fig. 12, the sterilization efficiencies of PAW produced at water-activated time of 15, 20 and 30 min are higher than the ones of the H2O2/HNO3 mixture solution. This result indicates that more reactive species in PAW can participate in the inactivation process of bacteria, compared to the H2O2/HNO3 mixture solution. The sterilization efficiency of PAW produced at water-activated time of 5 min is similar to the one of H2O2/HNO3 mixture solution. This result indicates that the synergistic effect of H2O2 and H+ ions plays a crucial role in the sterilization process at water-activated time of 5 min.

Fig. 12
figure 12

Comparion of inactivation efficiency of S. putrefaciens in PAW and H2O2/HNO3 mixture solution. The incubation time of S. putrefaciens with the solution is 5 min. The data points and error bars in all figures represent the averages and standard deviations from three independent measurements

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Discussion

To evaluate the sterilization efficiency of S. putrefaciens cells by PAW, deionized water treated by atmospheric-pressure microplasma arrays was used to sterilize S. putrefaciens cells. Reactive species formed in PAW were measured by UV/VIS-spectrophotometer. Other physicochemical properties, such as pH value and initial PAW temperature were analyzed. Antimicrobial ability of PAW was improved at an increasing applied voltage and water-activated time, as shown in Fig. 3. The ROS and RNS in PAW can play important roles in controlling the sterilization efficiency of S. putrefaciens cells by PAW treatment.

As shown in Fig. 6a, the pH values of PAW are typically in the range of 2.5–5.8, depending on the applied voltage and the water-activated time. Antimicrobial activity of acidic solution was analyzed. As shown in Fig. 6b, the inactivation process of S. putrefaciens cells is affected by the pH value of culture when it is varied from 2 to 5.9. Change in pH value appears to affect the activity of microorganisms by inducing changes both in the cell walls and the structure of the compound [31]. Low pH value is more favorable for the reactive species to penetrate cell walls. The presence of reactive species reduces the resistance of bacteria in acidic solution [32, 33]. Moreover, pH plays a significant role in the chemical reaction of PAW. The ROS in acidic solution may provide a warranty of relatively good germicidal efficacy [34].

The reactive species have been formed in PAW, and the pH value can have an obvious effect on the chemical reactions during the inactivation process of S. putrefaciens cells in PAW. Specifically, a linear increase in the H2O2 concentration of PAW was observed with increasing applied voltage from 6.0 to 12.0 kV and water-activated time from 0 to 30 min, as shown in Fig. 8. Meanwhile, a linear increase in the NO3− concentration of PAW was observed with increasing water-activated time from 0 to 30 min at applied voltage of 7.0–12.0 kV, as shown in Fig. 9a. However, the NO3− concentration was relatively lower at voltage applied of 6.0 kV, compared with the one at applied voltage of 7.0–12.0 kV. The anomalous behavior is ascribed to non-uniform plasma at 6.0 kV, and the amount of NO2− oxidized to NO3− is much less at 6.0 kV, due to the fact that the pH value of PAW is higher than 4 at voltage applied of 6.0 kV [27, 37]. NO2− concentration is highly related to water-activated time and applied voltage, as shown in Fig. 9b. The behavior of NO2− in PAW is ascribed to NO2− reduction to NO and its oxidation to stable NO3−. Moreover, the chemical reaction between H2O2 and NO2− occurs in acidic solution, leading to the formation of ONOOH [27, 37,38,39]. ONOOH is a strong oxidant which reacts with biological molecules through various mechanisms. ONOOH is particularly toxic to cells due to its ability to diffuse through cell walls. ONOOH is also able to initiate lipid peroxidation, causing cell wall damage [13].

H2O2, as a stronger oxidant, can oxidize molecules of cell walls. And H2O2 can be decomposed into ·OH radical, which promotes the sterilization effect of S. putrefaciens cells [31]. HNO3, as a long-lived secondary product, is not only a stronger oxidant, but also a reactive species in acidic solution. They are considered to be important constituents in the sterilization process. To evaluate antimicrobial activity of HNO3 and H2O2 species in PAW, H2O2, HNO3 solutions and H2O2/HNO3 mixture solutions were used to inactivate S. putrefaciens cells. As shown in Fig. 11, H2O2/HNO3 mixture solutions exhibit much higher sterilization efficiency compared with the H2O2 or HNO3 solution. The sterilization efficiency of H2O2 was obviously improved due to the presence of nitric acid. Cords et al. [40] studied the effects of pH on bactericidal and sporicidal action of H2O2 and indicated a greater H2O2 activity in the acidic solution. Alzamora [41] reported the effect of H2O2 solutions on the inactivation of E. coli in acidic solutions. Therefore, the synergistic effect of H2O2 and H+ ions may play a main role for sterilization process in the PAW. Figure 12 shows that with increasing water-activated time, the sterilization capacity of PAW is higher than the one of H2O2/HNO3 mixture solution. This result indicates that HNO2, ONOOH and O2NOOH, etc. can be produced by microplasma in PAW, and these reactive species may be able to participate in the inactivation process of S. putrefaciens in PAW.

Pavlovich et al. [42] reported that O3 is the dominant species for bacterial inactivation under these conditions. However, in this current study, the contribution of O3 to the inactivation of bacteria is likely negligible. As shown in Fig. 10, the concentration of O3 dissolved in the PAW is relatively low. Similar results have been reported in the literature [27]. Therefore, O3 cannot play an important role for antimicrobial effect of PAW in this study.

Those species (H2O2, NO3−, NO2− and ONOOH) may be in contact with cell wall and cell membrane of S. putrefaciens cells. They have a direct impact on the cells of microorganisms and especially on their outermost membrane, which can cause membrane damage and cell leakage [18]. Montie et al. [43] suggested that membrane lipids of Gram negative and Gram positive bacteria may be the most vulnerable macromolecule of the cell because of their location near the cell surface and their sensitivity to reactive oxygen species. As shown in Fig. 4, the integrity of the S. putrefaciens cells is damaged. Hence cell permeability is destroyed by those species. The broken cell membrane and cell wall led to the leakage of cell protoplasm, electrolyte and DNA, etc. As shown in Fig. 5, the DNA concentration firstly increases with increasing water-activated time and eventually decreases with further increasing water-activated time at voltage of 8.0–12.0 kV. It was reported that oxidants such as free radicals and hydrogen peroxide resulted in the multiple forms of damage, including base modifications of guanosine and double-strand breaks [44]. With increasing water-activated time, the reactive oxygen and nitrogen species (RONS) accumulation could exceed antioxidant ability of DNA and result in adverse effects on DNA [45]. Thus, the DNA concentration is reduced. However, it is difficult to detect the DNA in the solution at applied voltage of 6.0 kV. This result indicates that the RONS concentration at applied voltage of 6.0 kV is relatively lower than the RONS concentration at applied voltage of 7.0–12.0 kV. Although the sterilization efficiency of S. putrefaciens cells by PAW can be up to 0.13 Log Reduction at applied voltage of 6.0 kV, the cellular structure cannot be destroyed by RONS. Therefore, it is difficult to detect DNA in the solution at applied voltage of 6.0 kV.

Conclusion

In summary, we demonstrate that the sterilization efficiency of S. putrefaciens cells by PAW is dependent on applied voltage and water-activated time, and the sterilization efficiency of S. putrefaciens cells can be up to 2.0 Log Reduction. The synergistic effect of H2O2 and H+ ions in the solution plays an important role in sterilization process in PAW. After PAW inactivation, the cell membrane of S. putrefaciens is disrupted, resulting in the release of the cytoplasm into the surrounding medium. The reactive species in the PAW can attack the protein and DNA molecules in cytoplasm and inactivate the S. putrefaciens cells. This study shows that the PAW generated by atmospheric-pressure air plasmas has enormous potential in the application as an antimicrobial agent.