Formation of ammonium in saline solution treated by nanosecond pulsed cold atmospheric microplasma: a route to fast inactivation of E. coli bacteria

Abstract

He/N₂ cold atmospheric plasma treatment of E. coli suspension leads to a fast and efficient inactivation process. Significant generation of ammonium is reported. The formation of NH₄⁺ species in saline solution treated by cold atmospheric plasma is proposed for the first time as the main process responsible for the fast bacterial inactivation in pH-buffered solutions, at ambient temperature and physiological pH.

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Most of the traditional sterilization techniques currently used are based on the inactivation of bacteria by heat, long-lived chemical compounds and radiation.¹ Unfortunately, they are subject to several drawbacks: microbial resistance development, damage to the treated scaffold or incomplete sterilization.² Inactivation of bacteria by means of cold atmospheric plasma (CAP) treatment is therefore seen as a complementary and innovative way to decontaminate inorganic and biological surfaces or liquids as this technology avoids the drawbacks cited above.^3–5 Numerous synergies and inactivation mechanisms have been proposed to explain the efficiency of plasma treatments on bacteria suspension, involving the generation of several reactive oxygen and nitrogen species (RONS): hydrogen peroxide, peroxynitrite, ozone or hydroxyl and perhydroxyl radicals among others, with the potential contribution of plasma-issued (V)UV photons.^6–12

NH₄⁺ and their derived nitrogenous products were never considered as species playing a major role in bacteria inactivation by CAP in solution at physiological pH, although NH₄⁺ was already detected in bacteria suspension treated by plasma in the work of Baik et al.⁹ Bactericidal effects of peroxynitrites were also observed by Machala et al., but only played a significant role in acidic conditions.¹²

In this work, a controlled N₂ atmosphere was generated all around the microplasma jet and the plasma treated solution in order to decrease the number of possible chemical reactions for a better understanding of the inactivation mechanism. Indeed, working with complex systems such as atmospheric plasmas in air, real physiological liquids and bacteria is leading to a huge amount of chemical reactions involved.¹³ A phosphate buffered saline (PBS) solution was also used as a model medium to further simplify the systems interacting with bacteria and to further decrease the number of reactions involved as already done in similar studies.^14–16

Interaction between custom-built CAP in a controlled nitrogen atmosphere, PBS or saline solutions and E. coli bacteria using different plasma gas mixtures are presented and discussed, and the prominent role of NH₄⁺ for bacterial inactivation in suspensions is shown with our conditions, i.e. very low oxygen contents and nanosecond pulsed discharges, giving superior bacterial inactivation efficiency when compared to microsecond pulsed discharges.¹⁷

The experimental setup for plasma treatments is depicted in Fig. 1. The dielectric barrier discharge (DBD) plasma reactor used in this study has already been described in a previous publication.¹⁸ Briefly, it consisted of a tungsten filament powered by a pulsed high voltage and inserted in an alumina dielectric tube. A metallic grounded ring was enclosed outside the tube. Process gas was either He at a 2 standard litres per minute (slm) flow rate, He (2 slm)/N₂ (5–10 standard cubic centimetres per minute (sccm)) mixture or He (2 slm)/O₂ (5–10 sccm) mixture. It was fed into the reactor through MKS mass flow controllers. Plasma was generated by applying a 5 kV, 15 kHz, 1.5% duty cycle, positive nanosecond pulsed wave potential between the two electrodes. Control over the atmosphere composition during plasma treatments was obtained by providing a constant N₂ flow (35 slm) inside a poly(methyl methacrylate) enclosure surrounding the DBD plasma reactor without affecting the plasma treatment zone, thus creating a low overpressure in the chamber. The microplasma reactor was installed on a XYZ-flexible programmable support in order to obtain reproducible plasma treatments. Liquid samples to be plasma-treated were poured in a 6-well sampling rack and placed in the enclosure, below the DBD plasma reactor. Each well contained 5 mL of liquid (5 mm deep), with a 2 mm wide, 1 mm deep meniscus formed by the plasma flow. The vertical distance between the end of the alumina dielectric tube and the liquid interface was kept constant at 18 mm. All measurements were done three times and averaged in order to get reliable results.

Fig. 1 Experimental setup.

While the surrounding N₂ atmosphere was kept constant, a set of 90 s mL⁻¹ plasma treatments of E. coli suspensions in PBS media resulted in varying efficiency in terms of bacteria inactivation, depending on the nature of the plasma gas used (Fig. 2). Whereas a long-term partial decrease in culturability was observed with He and He/O₂ plasma treatments (max. 4.6 log reduction in CFU counts after 10 days post-treatment), He/N₂ plasma treatments surprisingly caused a short-term total inactivation of the samples (7.7 log reduction right after plasma treatment; no viable bacteria could be detected).

Fig. 2 Bacterial inactivation efficiency of 90 s mL⁻¹ plasma treatments on a 10⁸ CFU E. coli suspension in 2 mL PBS (n ≥ 3). (Flow) means plasma was not initiated. * Means no bacteria detected after He/N₂ treatments.

Several potential causes were considered to explain this drastic change in E. coli culturability and the related efficiency between He and He/O₂ plasmas on the one hand and He/N₂ plasmas on the other hand as discussed below.

Starting with physical effects, UV irradiations were shown to be similar whatever the treatments (Fig. S1, see ESI†). Temperature-induced effects on the bacterial cells were avoided, as maximum temperature reached in liquid samples after 120 s mL⁻¹ plasma treatments was below 30 °C. None of these physical effects are thus responsible for the observed somewhat large differences in bacterial inactivation upon plasma exposure.

Electromagnetic field influences could not be accurately monitored, but were thought to be similar for the different plasma treatment conditions, as electrical signal and plasma parameters were identical and gas mixture compositions were very similar (≥99.5% of He). Although plasma-induced electromagnetic field may provoke some sub-lethal membrane electroporation, only a difference in the nature of the reactive species formed in the plasma phase and subsequently in the aqueous phase could explain this difference of inactivation efficiency in fine.

The potential emission of metallic species (Cu, Zn, Al, W, etc.) by the plasma reactor inner parts was also considered, for they can efficiently inactivate bacterial suspension.^19–21 Therefore, inductively coupled plasma mass spectrometry (ICP-MS, Cu only) for plasma-treated milliQ water samples and time-of-flight mass spectrometry (TOF-SIMS, unreported data) analyses for plasma-treated silicon wafers without liquid were carried out. Regarding ICP-MS measurements, no copper incorporation was detected in treated samples when compared with a blank solution. For TOF-SIMS, treated areas on Si wafers did not show any metallic deposition (Cu, Zn, Al, W, etc.). No metal or metallic compound is therefore responsible for the difference in inactivation efficiency.

Next, the generation of chemical species in solution, thanks to the interaction of the plasma with the liquid interface of the samples, was investigated by measuring the pH of PBS and 0.9% saline solutions (different from bacterial solution treated by plasma nevertheless useful to better understand the nature of reactive species generated and reactions involved for the 3 different plasma gas mixtures used) after plasma treatments varying in duration (0–120 s mL⁻¹) and feed gas composition (He, He/O₂ and He/N₂). On the one hand, plasma treatments of PBS solutions did not result in any significant variation of pH, even for treatment durations longer than 90 s mL⁻¹ and whatever the feed gas composition (Fig. 3A). On the other hand, while He and He/O₂ plasma treatments on 0.9% saline solutions resulted in similar pH drops down to 3.8, He/N₂ plasma treatments induced an unexpected variation of pH: a first phase of pH drop down to 4.3 (approximately for 0–36 s mL⁻¹ treatments), followed by a rise to pH values up to 8.0 (36–120 s mL⁻¹ treatments) (Fig. 3B).

Fig. 3 pH of PBS (A) and 0.9% saline (B) solutions after 0–120 s mL⁻¹ plasma treatments (n = 3). ** Same plasma treatment durations as for bacteria inactivation efficiency tests from Fig. 2.

The pH drop observed during the first phase of He/N₂ treatments was mainly due to chemical reactions between the plasma-generated species at the liquid interface and dissolved gases (O₂, CO₂) in the liquid bulk of the aqueous samples leading to H₂O₂ and other acidic species. Indeed, using a 0.9% saline solution degassed beforehand by N₂ bubbling led to a reduction in the amplitude of this pH drop (Fig. 3B). Therefore, the addition of N₂ in the plasma process gas (helium here), in a pure N₂ atmosphere, was necessarily the cause of the pH rise observed during the second phase. In fact, it was discovered that increasing N₂ concentration in the 0–0.5 vol% range induced a steep increase of pH. Higher concentrations only resulted in attaining a plateau around a pH of 7.7 for 90 s mL⁻¹ treatments (Fig. S2, see ESI†). Different reactive species or a large difference in concentration of some reactive species should therefore be generated and responsible for the higher bacterial inactivation in plasma treated liquids with He/N₂ plasma.

In order to determine these different reactive species formed or any important concentration change for some of the reactive species produced in solutions as a function of plasma gas mixture, ion chromatography and colorimetric analyses were carried out on plasma-treated samples. The concentration of ammonium, nitrate and nitrite ion species generated by the interaction of plasmas with milliQ water and 0.015% saline samples was quantified by ion chromatography (Fig. 4A–C). MilliQ water and 0.015% saline were used instead of 0.9% saline solution, as a 0.9% concentration of Cl⁻ ions (154.7 mM) would prevent the detection of nitrites. In parallel, the concentration of hydrogen peroxide, generally considered as the main indicator of bacterial inactivation efficiency in plasma-treated aqueous solutions, was quantified by a colorimetric method in plasma-treated 0.9% saline and PBS solutions (Fig. 4D).

Fig. 4 Ammonium (A), nitrates (B), nitrites (C) and hydrogen peroxide (D) concentrations in milliQ water (hollow dots, dotted lines), 0.015% saline (filled dots, dashed lines), 0.9% saline (hollow dots, dash-dotted lines) and PBS samples (filled dots, plain lines) after 0–120 s mL⁻¹ plasma treatments (n = 3). ** Same plasma treatment durations as for bacteria inactivation efficiency tests from Fig. 2.

Ammonium concentrations increased linearly with plasma treatment duration, but in a much higher proportion for He + 0.50% N₂ plasma (76.5 and 45.4 μM after 120 s mL⁻¹ in milliQ and 0.015% saline, respectively) than for He (24.1 and 16.5 μM) and He + 0.50% O₂ plasmas (7.4 and 2.8 μM). Nitrate concentrations followed the same linear behaviour, although the He plasma treatment resulted in higher NO₃⁻ generation (134.2 and 97.4 μM after 120 s mL⁻¹ in milliQ and 0.015% saline, respectively) than He + 0.50% O₂ (51.7 and 63.7 μM) and He + 0.50% N₂ plasmas (46.6 and 75.9 μM). Nitrite concentrations varied according to a two-step process: first, an increase up to 39.5–11.4 μM for treatments up to 36 s mL⁻¹, followed by a decrease down to 1.9–12.7 μM after 120 s mL⁻¹ treatments. This decrease was explained by a spontaneous disproportionation of nitrites into nitrates and nitric oxide under acidic conditions, self-catalysed by nitrates.¹² H₂O₂ concentrations increased linearly with plasma treatment duration, in higher proportion for He plasma (878 and 873 μM after 120 s mL⁻¹ in 0.9% saline and PBS, respectively) than for He + 0.50% N₂ (486 and 461 μM) and He + 0.50% O₂ plasmas (311 and 262 μM). Interestingly, traces of chlorate ions could be detected in plasma-treated 0.015% saline (Fig. S3, see ESI†) and would indicate the generation of hypochlorite ions. Indeed, hypochlorite ions slowly degrade into chlorate ions at physiological pH.²²

These results indicate that ammonium ions (pK_a = 9.25) are crucial species playing a role in the fast bacterial inactivation process, as the higher production of this species by He/N₂ plasmas was related to the fast decreased viability in PBS suspension, as well as an increase of pH in saline solution.⁹ Synthesis of ammonium in plasma-treated solutions proceeds via a two-step mechanism: dissociation in plasma phase of vibrationally excited water molecules yields hydrogen gas, synthesis of ammonium then follows by reaction of hydrogen with nitrogen present in the plasma phase and acid–base reaction in the liquid phase (reactions 1 to 5).²³

H₂O* + H₂O → ˙H + ˙OH + H₂O (1)

˙H + H₂O* → H₂ + ˙OH (2)

˙OH + H₂O* → H₂O₂ + ˙H (3)

N^*₂ + 3H₂ → 2NH_3(g) (4)

NH_3(aq) + H⁺ ⇌ NH_4(aq)⁺ (5)

In order to understand how NH₄⁺ ions formed in liquids can generate a fast inactivation of bacteria, wet chemistry treatments involving main species formed by plasma treatments were performed on E. coli suspensions in PBS media (Fig. 5). The exposure time, the nature and concentration of the chemicals and chemical mixtures added were all chosen to be as close as possible to plasma treatment ones. Some reactive species were added in higher concentration to show that they are not playing a role in our fast (less than 1 h) plasma disinfection process.

Fig. 5 Bacterial inactivation efficiency of 35 min chemical treatments on a 10⁸ CFU E. coli suspension in 2 mL PBS. * Means no bacteria detected after treatment.

Interestingly, exposure to hydrogen peroxide concentration up to 4.85 mM for 35 minutes did not induce any significant bactericidal effect. The same observation could be made for the exposure of bacteria to ammonium and nitrite ions (both 500 μM), as well as for the mixture of hydrogen peroxide 485 μM + nitrite 500 μM. Indeed, the amount of peroxynitrite generated through this latter reaction is certainly negligible at pH 7.4.²⁴

Exposure to hypochlorite brought drastically different outcome, depending on its concentration or the chemical it was mixed with. Whereas NO₂Cl (produced by the reaction of hypochlorous acid with nitrite) was harmless at a concentration of 450 μM, monochloramine (produced by the reaction of hypochlorite with ammonium) and hypochlorite alone prompted an efficient inactivation of E. coli bacteria after 35 min: respectively 5.2 and 8.0 log reduction at concentration as low as 45 μM. No viable bacteria were detected at concentration of 450 μM. At 5 μM concentrations, none of these two latter species were inducing an inactivation effect. In our conditions, critical inactivation concentration for HClO is estimated to be in the 15–30 μM range and is slightly higher for NH₂Cl (30–50 μM). Other species need to be added in much higher concentration to show a fast inactivation effect.

From these results above, two main conclusions can be drawn. Only HClO and NH₂Cl are playing a direct role in the fast plasma inactivation process in our conditions looking at the concentration of reactive species generated by plasma treatments. And assuming that HClO generated is similar between He and He/N₂ plasmas because of similar oxygen and Cl contents in the environment and liquids for both conditions and similar plasma characteristics, HClO is always produced at subcritical concentrations for inactivation in our plasma conditions i.e. at concentration lower than 20 μM. However, in the case of He/N₂ plasma alone, enough NH₂Cl is generated from the reaction between ClO⁻ and NH₄⁺ to reach the critical concentration of fast and full inactivation, which is estimated from experiments with chemicals to be in the 5–20 μM range for the sum of both reactive species. In conclusion, a combination of HClO and NH₂Cl is therefore needed to achieve a fast and efficient bacteria inactivation with the concentrations involved in our plasma conditions. Only the He/N₂ plasma generates sufficient amount of NH₂Cl to reach the critical concentration of the HClO and NH₂Cl mixture.

Consequently, the quick and effective inactivation of bacteria at physiological pH observed for He/N₂ plasma treatments was proposed to be directly linked with this higher production of ammonium species formed in He/N₂ plasma-treated liquids. Indeed, it is highly probable that, in such conditions, ammonium ions react with other plasma generated compounds (e.g. hypochlorite confirmed by the presence of chlorate by ion chromatography), to yield bactericidal nitrogenous species (e.g. monochloramine).²⁵ The assumption for HClO formation leading to NH₂Cl formation by reaction with NH₃/NH₄⁺ is supported by the fact that less ammonium species was detected in 0.015% saline solution (i.e. NH₄⁺ is consumed) than in milliQ samples, devoid of chloride ions, after He + 0.50% N₂ plasma treatment. This scenario and other ones are currently under investigation.

Conclusions

A quick and efficient inactivation of buffered E. coli suspensions was achieved with He/N₂ cold atmospheric plasma treatments under N₂ controlled atmosphere at physiological pH, whereas He and He/O₂ treatments only led to limited and time-delayed progressive inactivation.

The role of parameters such as the emission of harmful metallic species by the plasma reactor or the heating of the samples was investigated before being ruled out. The efficiency of He/N₂ plasma treatments, over He and He/O₂ treatments, is attributed to an increased production of NH₄⁺ in liquid phase. This is the first time that such highly efficient bacterial inactivation is directly linked to NH₄⁺/NH₃ preferentially generated by the interaction of He/N₂ cold atmospheric plasma with a PBS bacterial suspension at physiological pH. The high and fast inactivation efficiency observed seems to be due to the preferential formation of chloramine species (NH₂Cl) from the reaction of NH₄⁺/NH₃ with hypochlorite species in combination with the remaining HClO/ClO⁻ species.

Other species (e.g. ˙OH radicals, peroxynitrites, NO) and physical effects (UV) always produced are thought to play a minor or indirect role in the fast inactivation process observed here and they do not provide such highly efficient bacterial inactivation process if a critical amount of NH₄⁺ is not reached, in the conditions used here (He/N₂ plasma, atmosphere devoid of O₂, nanosecond pulsed discharges).

We think that these peculiar conditions of disinfection are of high interest for water and physiological aqueous solutions microbial disinfection as they provide a fast bacterial disinfection at physiological pH and ambient temperature without using any wet chemistry process but only electricity (a few hundred mW), He and N₂. This work is further supporting cold atmospheric plasma processes to become a standard disinfection tool in the future due to its simplicity of use and its moderate cost.

Acknowledgements

The authors would like to acknowledge, from LIST, A. Robert, M. Gerard, A. Moschetta and O. Bouton for technical support, D. Collard and C. Walczak for microbiological manipulations and support, F. Barnich for ion chromatography analyses, Dr G. Frache and N. Desbenoit for TOF-SIMS analyses and J. Ziebel for ICP-MS analyses. This work was supported by the European Laboratory LEA-LIPES and by the Fonds National de la Recherche Luxembourg.

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Footnote

† Electronic supplementary information (ESI) available: Methods for ICP-MS, temperature and pH, ion chromatography and colorimetric analyses; OES, pH and ion chromatography data. See DOI: 10.1039/c5ra01109d

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