Introduction

The inactivation of microorganisms in water and the removal of biological hazardous contaminants are generally of great interest for both developing and developed countries. Cold plasmas operating at atmospheric pressure contain a variety of microbicidal agents, such as charged species, radicals, ultraviolet (UV) light, excited molecules, and have been successfully used for microbial decontamination [1–4]. However, so far there are insufficient reports about atmospheric pressure cold plasma inactivation of microorganisms under aqueous environment [5–7]. The major difficulties one encounters are: (1) the difficulty to form a stable and uniform cold plasma under aqueous environment. The characteristics of aqueous solution in direct contact with gas-phase plasmas, such as its conductivity can greatly alter the stability and uniformity of atmospheric cold plasmas. (2) the need to maintain the high chemical reaction efficiency of plasma-activated species in water. Before they flow into the aqueous solution, these short-lived and very active species from atmospheric plasmas quench due to the frequent collisions in the gas phase.

Pseudomonas has this ability to metabolise a variety of diverse nutrients and thrive under harsh conditions as a result of their hard cell wall that contains porins. The resistant Pseudomonas can cause food spoilage [8–10]. In this study, we report a hollow-fiber based microplasma array device for generating atmospheric microplasma jets in water. The microplasma array device running with He/O2 mixtures has been successfully utilized for the plasma inactivation of Pseudomonas sp. (Pseudomonas sp HB1) cells in aqueous solution. The inactivation of Pseudomonas sp. cells is evaluated via colony forming unit (CFU) count on Petri dish, and atmospheric microplasma jets are characterized using I–V and optical emission measurements.

Experimental

Plasma Reactor and Multi-glow Mode Discharge

Different with the traditional single-jet configuration where the dielectric tubes of barrier discharges are generally several millimeters in diameter [11–13]. We use microns-thick and hollow quartz fibers as the dielectric tubes, through which to flow the feed gas. Figure 1a shows the schematic of the 6 × 6 microplasma jet array housed in a 0.2 L (45 × 52 × 80 mm) glass container. The inner and outer diameters of the hollow quartz fibers are 250 μm and 1,000 μm, respectively. The tungsten wires (100 μm in diameter) are inserted into these hollow fibers and act as the high-voltage electrodes of microplasma jets. The separation between the ends of tungsten wires and hollow quartz fibers is 1.2 cm. The O2/He gases with their total flow rate of 2.2 standard litre per minute (SLM) are fed into these hollow quartz fibers. The aqueous solution containing Pseudomonas sp. cells acts as the grounded electrode. The power supply is capable of supplying bipolar a.c. output with peak-to-peak voltage (VPP) of 0–30 kV at an a.c. frequency of 10 kHz. Microplasma jets are formed inside the hollow cores of quartz fibers near their ends. Applied voltage and discharge current are simultaneously measured using a Tektronix 2040 digital oscilloscope with a high-voltage (H.V.) probe and a Tektronix P2220 current probe. The discharge current was obtained by measuring the voltage over a small resistor (40 Ω), connected in series to ground. Measurements for the charges across this capacitor and the applied voltage across the discharge chamber result in a Lissajous figure, which was used to calculate the discharge power [14]. The discharge power can increase from 0 to about 80 W when VPP is varied from 0 to 30 kV. Optical emission spectra (OES) are obtained by using a SpectraPro-750i monochromator (Acton Research Corporation) with its resolution of 0.05 nm in the wavelength range of 200–800 nm. Figure 1b shows the photograph of well-aligned He microplasma jets obtained at VPP = 4 kV. Clearly, the microplasmas are generated inside the hollow cores of quartz fibers near their ends. The microplasma jet is formed in the vicinity of the end of each quartz fiber. A great number of microbubbles flow through the aqueous solution containing Pseudomonas sp. cells. Microbubbles as a part of the microplasma jet can contain a variety of microbicidal active agents, including short-lived species, such as charged species (electrons or ions) and reactive radicals. When VPP is larger than 4 kV, the strong discharge can be produced between the tungsten wires and the aqueous solution, resulting in a rapid increase in the temperature of the aqueous solution. When VPP is smaller than 3–4 kV, the microplasma jets are getting much weak due to an insufficient power density. These plasma-activated species are in direct contact with the aqueous solution. This design can lead to an obvious increase in the contact area of microplasma jets with the aqueous solution, thus an improvement in the chemical reaction efficiency of plasma-activated species. The flow rate of the feed gas lower than 1.5 SLM may result in the direct contact between the tungsten wires and aqueous solution, thus the unstable microdischarges in the vicinity of the tungsten wires.

Fig. 1
figure 1

a Schematic drawing of the microplasma jet array generated in aqueous solution; bThe photograph of atmospheric helium microplasma jet array formed in aqueous solution at VPP = 16 kV

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Figure 2 shows the temporal characters of the applied voltage and the total current of the atmospheric microplasma jets obtained using He as the feed gas at VPP = 4 kV. The multi-peak appearance of the current waveform is well consistent with the previous reports [15–17]. This suggests that atmospheric dielectric barrier discharge with “multi-glow mode” [18] be generated inside the hollow-core fibers near their ends, and the microplasma jets be formed in the vicinity of the ends of hollow-core fibers. The total current appears as a discharge current superimposed onto a displacement current [15]. The discharge current stands for the effective gas breakdown, and has much shorter duration than the period of applied voltage. In the designs reported previously [7, 16, 19, 20], atmospheric cold plasma jet devices typically consist of a H.V. needle electrode and a dielectric tube. The dielectric tube is generally wrapped with the grounded metal electrode. These designs show the well consistence with the electrode configuration of dielectric barrier discharges. These devices may be used to generate the atmospheric plasma jets when the He or Ar gas is fed into the dielectric tube. In this present design, the aqueous solution surrounding the quartz fibers acts as the grounded electrode. The barrier discharge configuration consists of H.V. tungsten wires, quartz fibers, and the aqueous solution. The pulsed discharge current is formed due to the charge accumulation on the inner surfaces of hollow-core quartz fibers. The surface charges create an electric potential that opposes the applied voltage and, as a result, limit the discharge current and prevent the glow-to-arc transition [15]. The plasma density of microjets can be greatly improved due to an increase in the discharge current.

Fig. 2
figure 2

The applied voltage and the total current of the microplasma jets obtained using He as the feed gas at VPP = 4 kV

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Analytical Methods

A single colony of Pseudomonas sp. was inoculated into the 4 mL Zobell 2216E liquid medium (Bactopeptone: 5 g/L, Yeast powder: 1 g/L, KH2PO4: 4 mL/L, FeSO4: 2 mL/L, PH value: 7.6–7.8), and cultivated into the exponential phase with the rotation speed of 150 rpm at 28 °C. Then, 3.6 mL cultures was transferred to another 360 mL 2216E and fostered to obtain more cultures. 80 mL: of the cultures containing Pseudomonas sp. concentration of 2.5 × 108 CFU/mL was treated by using the atmospheric He or O2/He microplasma jets. Then, the treated cultures was diluted according to a series of gradient using distilled water, from 10−1 to 10−6, separately, while the untreated one was used as the control. For each dilution, we set three parallels. Then the bacterial samples were inoculated on the standard Petri dishes 9 cm in diameter containing approximately 15 mL semisolid 2216E. The static cultivation was kept at 28 °C for 24 h.

Figure 3 shows the photographs of Pseudomonas sp. samples (1) from the untreated cultures; (2) treated by using the 2 % O2 + 98 % He microplasmas within the treatment time of 4 min.; (3) treated by using the 2 % O2 + 98 % He microplasmas within the treatment time of 6 min. Measurement shows that the temperature of the cultures is lower than 35 °C within the treatment time of 10 min, and has no obvious effect on the survival efficiency of Pseudomonas sp. cells. This plasma treatment results in a rapid inactivation of Pseudomonas sp. cells in the cultures. The atmospheric O2/He microplasmas almost killed all the Pseudomonas sp. cells within the treatment time of 6 min. The Pseudomonas sp. concentration of the cultures diluted to 10−6 as a function of processing time is plotted in Fig. 4. Both the processing time and O2/He ratios have a significant effect on the survival efficiency of Pseudomonas sp. cells. Increasing the treatment time to 5–10 min lead to a rapid reduction in the concentration of Pseudomonas sp. cells in the aqueous solution. The He plasma containing 2 % O2 is more effective in killing Pseudomonas sp. cells and its disinfection efficiency reaches as high as 99 % within a treatment time of 4 min. The plasma inactivation measurements were also performed when the Pseudomonas sp. concentration was varied from 268 × 105 CFU/mL to 86 × 108 CFU/mL. The plasma treatment with the treatment time of 5–7 min by the 2 % O2 + 98 % He microplasmas can result in the >97 % disinfection efficiency of the Pseudomonas sp. cells contained in the 268 × 105–86 × 108 CFU/mL suspensions.

Fig. 3
figure 3

Photographs of Pseudomonas sp. samples (a) from the untreated cultures; (b) treated by using the 2 % O2 + 98 % He microplasma jets within the treatment time of 4 min.; (c) treated by using the 2 % O2 + 98 % He microplasma jets within the treatment time of 6 min

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Fig. 4
figure 4

The Pseudomonas sp. concentration of the cultures diluted to 10−6 as a function of processing time

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Results and Discussion

OES Spectra of Microplasma Jets

The typical OES spectra of these microplasma jets obtained using 100 % He or 2 % O2 + 98 % He as the feed gas are shown in Fig. 5a, b, respectively. The major helium emission lines observed in both 100 % He and 2 % O2 + 98 % He plasmas are from transitions of 23P–73D at 371 nm, 21S–31P at 501 nm, 23P–33D at 587 nm, 21P–31D at 667 nm, 23P–33S at 706 nm, and 21P–31S at 728 nm [21, 22]. He* atoms from the microplasma jets are mainly formed due to the collisions of energetic electrons with He atoms [23, 24]. Emissions at 316 and 357 nm from N2 second positive system (C3Πu–B3Πg) and at 656 nm from Hα were also clearly observed. The N2* molecules from the microplasma jets are mainly produced via the energetic collisions of electrons with N2 molecules (e + N2 (X2Σ +g )→e + N2 (C3Π, B3Π)) or excited by the long lived helium metastable (19.8 eV) via the Penning ionization process (He(23) + N2 (X2Σ +g )→He + N2 (C3Π, B3Π)) [23–25]. The strong OH emissions from both 100 % He and 2 % O2 + 98 % He plasmas are assigned to transitions of A2Σ+–X2Π (Δν = 1) at 285 nm and A2Σ+–X2Π (Δν = 0) at 309 nm.22 Since water vapour molecules can diffuse into the microplasma jets, OH and H radicals can be formed by the Penning ionization process of water vapour (He(23) + H2O → He + H + OH) or by electronic impact dissociation (H2O + e− → H + OH + e−). The O emission at 777 nm observed for the 2 % O2 + 98 % He plasma was assigned to the transition of 2s22p33 s–2s22p33p. In the He microplasma jets containing a small amount of O2, both energetic electrons and excited He* can ionize O2 molecules [23–25], which can be responsible for the formation of O radicals.

Fig. 5
figure 5

The typical OES spectra of these microplasma jets obtained using (a) 100 % He and (b) 2 % O2 + 98 % He as the feed gas in aqueous solution

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Plasma-Activated Species for Inactivation of Microorganism

So far, all the plasma induced species, such as charged species (electrons and ions), reactive species, UV photons (generated due to the radiation of excited species) were supposed to play the role in the sterilization process by low temperature plasmas [26–29] In this method, the aqueous solution containing Pseudomonas sp. cells was subject to all possible agents generated by the plasmas, including charged particles, reactive neutrals, and UV photons. UV radiation in the 200–300 nm wavelength range with doses of several milliwatt-seconds per square centimeter are known to cause lethal damage to cells [30]. However, the atmospheric-pressure plasma jet, as the most effective type of cold plasmas for sterilization, usually produces very little UV [4]. It is believed that UV does not play a major role in the inactivation process by atmospheric-pressure cold plasmas [4, 12, 27, 31]. Charged particles can play a significant role in the rupture of the outer membrane of bacterial cells [4, 28]. The electrostatic force caused by charge accumulation on the outer surface of the cell membrane could overcome the tensile of the membrane and cause its rupture. In this study, the microplasma jets are in direct contact with the Pseudomonas sp. cells in the aqueous solution. These short-lived charges can diffuse into the aqueous solution before they recombine with other species during frequent collisions. Especially, the plasma sheath formed near the plasma/liquid interface can cause the potential difference between the gas-phase plasma and the Pseudomonas sp. cells from this interface. Therefore, the charge accumulation on the outer surface of Pseudomonas sp. cells may occur due to the existence of plasma sheath, which can rapidly cause their ruptures.

Among various plasma-activated neutrals, O and OH radicals were supposed to play a crucial role in the plasma inactivation of microorganism [7, 32, 33]. Especially, O radicals were believed to play the most important role in this inactivation process [1, 20, 34, 35]. These reactive species have strong oxidative effects on the outer structures of cells. However, no emission from O radicals was observed from the He plasma in this present study. Strong emission from OH radicals indicates that OH radicals can be very effective in killing the resistant Pseudomonas sp. cells in aqueous solution. In deed, OH radicals with oxidation potential of 2.8 V was supposed to be most reactive and toxic among all reactive oxygen species (ROS) [7]. OH radicals can attack unsaturated fatty acids in the cell membranes [31]. The unsaturated fatty acids give the membrane a gel-like nature, and the membrane containing unsaturated fatty acids acts as a barrier against the transport of ions and polar compounds in and out of the cells. The He plasma containing a small amount of O2 exhibits more effective inactivation of Pseudomonas sp. cells in aqueous solution, which can be related to the generation of O radicals in the microplasma jets. Protein molecules, which are basically linear chains of amino acids, are susceptible to oxidation by the O radicals very active during chemical reaction [27]. Proteins also play the role of gateways that control the passage of various macromolecules in and out of cells. In deed, OH radicals can be formed by the reaction of excited O radicals with water vapour (H2O + O* → 2OH) [7, 36, 37]. This process may lead to an increase in the concentration of OH in aqueous solution, thus improve the inactivation efficiency of the resistant Pseudomonas sp. cells in aqueous solution.

Conclusions

The hollow-fibers based microplasma array device may help to overcome problems in the cold plasma inactivation of microorganisms from aqueous solution. In this design, the short-lived species, such as very active radicals and charge species from microplasma jets can be in direct contact with the microorganisms in aqueous solution, which can result in the highly effective inactivation of resistant Pseudomonas sp. cells. Analysis indicates that OH radicals and charged species play the crucial roles in the plasma inactivation of Pseudomonas sp. cells by this study.