30 th ICPIG, August 28 th – September 2 nd 2011, Belfast, Northern Ireland, UK 16 Identification of antibacterial species in plasma treated liquids K. Oehmigen P 1 P , U C. Wilke UP 1 , K D. Weltmann P 1 P , Th. von Woedtke P 1 P P 1 P Leibniz Institute for Plasma Science and Technology e. V. (INP Greifswald), Felix-Hausdorff-Str. 2, D-17489 Greifswald, Germany Both plasma treatment of E. coli suspension and plasma treatment of the liquid and retrofitting addition to the microorganisms resulted in strong bactericidal effects. To get more insight into action mechanisms, plasma/gas phases were analysed by OES and FT-IR. Interactions and/or reactions with the liquid surface were hypothesized and some assumed low-molecular substances (e. g. nitrate, nitrite, hydrogen peroxide, protons) in the liquid phase were detected by pH- measurements, spectrophotometrical and ion chromatographical methods. Antimicrobial tests were performed using these before mentioned low-molecular substances. Moreover, the microorganism suspensions were treated with different concentrations of ozone. Finally, the results were compared with the plasma treatment and it was concluded that the plasma treatment is more effective in inactivation of E. coli than the individual components 1. Introduction Inactivation of bacteria in liquids by plasma treatment is an important and actual field of investigation. Latest research has shown that microorganism suspensions have not to be treated directly to realize a strong bactericidal effect. It is also possible to treat the liquid by plasma and add it to the bacteria subsequently. [1] These results lead to the conclusion that the inactivating effect of the plasma is mediated mainly by the liquid. More or less stable plasma-generated species may diffuse into the liquid to interact there with the bacteria or to become part of secondary reactions, all together resulting in bactericidal activity of plasma treated liquids. The following investigations and hypotheses should give more insight into the complex chemistry of plasma-liquid interactions and analytical methods. 2. Methods [1, 2] 2.1. Physical Methods The surface dielectric barrier discharge (DBD) arrangement which was specially designed for plasma treatment of microorganisms or cell cultures and liquid samples in petri dishes (Fig. 1), has been described in detail elsewhere. The electrode array is mounted by a special construction into the upper shell of a petri dish (60 mm diameter). The plasma was generated at the surface of the electrode arrangement. The distance between the electrode arrangement and the liquid surface was adjusted at 5 mm, there was no direct contact of the plasma to the liquid. All experiments are performed under atmospheric pressure at ambient air conditions using a pulsed sinusoidal voltage of 10 kV peak (20 kHz) with a 0.413/1.223 s plasma-on/plasma-off time. Energy of 2.4 mJ was dissipated into the plasma in each cycle of high voltage. Optical emission spectroscopy (OES) in the range from 200 up to 900 nm was performed using a compact spectrometer (AvaSpec-2048, Avantes) with an entrance slit of 25 µm and a spectral resolution of 0.6 nm. Due to the small plasma intensity a large exposure time of 10 s and a two scan average was necessary to obtain a valuable spectrum. The Fourier transformed infrared spectroscopy (FT-IR) was performed with the multicomponent FT IR gas analyser Gasmet CR-2000 (ansyco). For data analyzing the software CALCMET was used. 2.2. Biological Methods As test liquid sodium chloride solution (physiological saline; NaCl 0.85 %; 8.5 g NaCl per 1000 ml water) and as test microorganism Escherichia coli NTCC 10538 have been used. E. coli has been kindly provided by Institute of Hygiene and Environmental Medicine, Ernst Moritz Arndt University Greifswald, Germany. Overnight culture of E. coli was diluted using NaCl solution, to get concentrations of 10 9 colony forming units per millilitre (cfu . ml -1 ; stock suspension). In each culture tube 50 µl of the microorganism stock suspension were pipetted. 5 ml of NaCl solution were treated with the DBD plasma for different times (1 - 12 min). Treated samples were split up in two parts (2.45 ml each). One part was pipetted into the culture tube containing 50 µl of the E. coli stock suspension immediately (t < 10 s) after plasma treatment. The other part was added into another tube containing microorganism stock suspension 30 th ICPIG, August 28 th – September 2 nd 2011, Belfast, Northern Ireland, UK 16 30 min after plasma treatment. After 15 min exposure time in the plasma-treated liquids, number of surviving microorganisms was estimated. For plasma treatment of liquids containing suspended microorganisms, 100 µl of E. coli stock suspension were pipetted into 4.9 ml saline solution. The resulting bacteria suspensions were treated with the DBD plasma for different times (1 - 12 min). Sodium nitrate (NaNO 3 ; Merck), sodium nitrite (NaNO 2 ; Merck) and hydrogen peroxide solution (H 2 O 2 ; Merck) were used as test substances to investigate the bactericidal potential of the species generated in water after plasma treatment. The test substances were used as single- component solutions as well as in different combinations. To test its bactericidal efficacy, 1 ml of stock solution of the respective component was pipetted into a culture tube containing 50 µl of the E. coli stock suspension. The lacking volume up to 5 ml was filled up with NaCl solution to get the following final concentrations of the chemicals in 5 ml sample: 50 mg . l -1 nitrate as NaNO 3 , 1.5 mg . l -1 nitrite as NaNO 2 and 2.5 mg . l -1 H 2 O 2 . For acidification to pH 3, 10 µl of hydrochloric acid (54 g . l -1 ; HCl; Merck) was added to the solutions (per 5 ml). Exposure time was 15 min and 60 min, respectively. For the gassing with ozone the ozonisator “Laborozonisator 300” (Erwin Sander Elektro- apparatebau GmbH, Ueltze-Eltze, Germany) was used. Different concentration ranges were configured (A: 100 ppm, B: 470 ppm, C: 660 ppm, D: 1260 ppm, E: 1950 ppm) and blown over the liquid surface (flow: 0.5 slm) for different times. The number of viable microorganisms (cfu ⋅ ml -1 ) was estimated by the surface spread plate count method using aliquots of serial dilutions of microorganism suspensions in saline solution according to the European Pharmacopoeia. Detection limit of this procedure was 10 cfu ⋅ ml -1 . Serial dilution of microorganism suspensions served also as an effective procedure to neutralize the bactericidal activity of reactive species contained. 2.3. Chemical Analytics For pH measurement, a semi-micro pH-electrode (4.5 mm diameter; SENTEK P13, Sentek Ltd., UK) was used. For photometric measurements a UV/VIS Spectrophotometer SPECORD® S 600 (analytic jena GmbH, Jena, Germany) was used. Nitrite concentrations are estimated by a colorforming reaction using a commercially available test kit (Spectroquant ® , Merck). The pH value of the probe has to be adjusted between 2.0 and 2.5. Therefore, samples were acidulated by sulfuric acid (H 2 SO 4 ; Merck). Nitrite reacts with sulfanilic acid and N-(1-naphthyl)-ethylen diamine hydrochloride via azo sulfanilic acid to a magenta colored azo dye whose absorption at 525 nm was measured. Nitrate reaction (Spectroquant ® , Merck) with 2,6-dimethylphenol gives, after a reaction time of ten minutes 4-nitro-2,6-dimethylphenol, an orange colored product, whose absorption was measured at 340 nm. Hydrogen peroxide detection based on the reaction of titanyl sulfate to yellow-colored peroxotitanyl sulfate, which was detected at 405 nm. For acidification to pH 2.0 - 2.5, sulfuric acid was used. For direct photometric analysis, total absorption spectra have been recorded from 200 up to 1000 nm. The ion chromatography was performed by an isocratic ICS-5000 system (Dionex) with a separation column IonPac AS23 and variable wave length and conductivity detectors. As eluent 4.5 mM disodium carbonate and 0.8 mM sodium hydrogencarbonat was used. The flow was 0.25 ml ⋅ min -1 . For data analyzing the software Chromeleon 7 (Dionex) was used. 3. Results and Discussion Direct plasma treatment of 5 ml E. coli suspension resulted in inactivation of this microorganism within a few minutes. However, addition of NaCl solution to E. coli immediately after plasma treatment of the microorganism-free liquid showed similar inactivation kinetics. Even a 30 min delayed addition resulted in a reduction of viable microorganisms (see Fig. 1). [2] These results lead to the assumption that the inactivating effect of the plasma treatment is mainly mediated by the liquid phase. But which species caused this effect? Therefore the plasma/gas phase were analysed by OES and FT-IR. Only dinitrogen oxide (N 2 O), ozone (O 3 ), carbon dioxide (CO 2 ) and traces of nitric/pernitrous acid (HNO 3 /ONOOH) and the second positive, as well as, the first negative system of nitrogen were found. [1] These detected compounds may interact and/or react with the liquid surface and diffuse into deeper layers. To get an insight into the kind species which could be generated in the liquid, a multiplicity of reactions were hypothesized based on several 30 th ICPIG, August 28 th – September 2 nd 2011, Belfast, Northern Ireland, UK 16 references from literature. In figure 2 some possible reaction channels are pictured. [1] Most of them resulted in generation of protons (H + ), nitrate (NO 3 - ), nitrite (NO 2 - ) or hydrogen peroxide (H 2 O 2 ), respectively. Consequently, analytics of plasma treated distilled water was performed. For this purpose, well established spectrophotometrical tests for nitrate, nitrite and hydrogen peroxide were used. Furthermore, the pH was measured. Increasing concentrations of H + , NO 3 - , NO 2 - , and H 2 O 2 were detected dependent on plasma treatment time. After 30 min plasma treatment 113 mg ⋅ l -1 nitrate, 1.5 mg ⋅ l -1 nitrite and 18 mg ⋅ l -1 hydrogen peroxide were detected in 5 ml distilled water. The pH decreased down to 2.78. [2] detection limit 1,00E+00 1,00E+01 1,00E+02 1,00E+03 1,00E+04 1,00E+05 1,00E+06 1,00E+07 1,00E+08 1,00E+09 0 2 4 6 8 10 12 plasma treatment time [min] number of viable microorganisms [cfu . ml -1 ] plasma treated NaCl solution added to E. coli with 30 min delay plasma treated NaCl solution directly added to E. coli plasma treated E. coli suspension 10 9 10 8 10 7 10 6 10 5 10 4 10 3 10 2 10 1 10 0 plasma treated NaCl solution added to E. coli with 30 min delay plasma treated NaCl solution directly added to E. coli plasma treated E. coli suspension Fig. 1: Inactivation kinetics of E. coli as a result of plasma treatment of bacteria-containing sodium chloride (NaCl) solution () as well as addition to E. coli of plasma- treated NaCl solution immediately (ж) or 30 min after plasma treatment () [1] Additionally, total spectra of plasma treated water and sodium chloride solution were recorded. Two absorption maxima were detected. The one at 227 nm corresponds with nitrous acid and the other at 302 nm was described in the literature as peroxynitrite (ONOO - )/pernitrous acid (ONOOH). [3] Because nitric acid has an absorption maximum at 305 nm, it could not be identified surely. For greater clarity, ion chromatography (IC) was used as more sophisticated analytical method. The used IC setup is appropriate for the detection of inorganic ions in complex liquids. The analytes were detected both by UV-absorption and conductivity. Although nitrate and nitrite were detected, also other peaks were found in the chromatogram which cannot be identified readily. Fig. 2: Possible reaction channels of plasma/gas-liquid interactions [1] To find out if the detected species nitrate, nitrite and hydrogen peroxide as well as acidification have bactericidal effects, they were added in several different combinations to E. coli. Used concentrations have been identical to that found in water after 10 min plasma treatment [2]. Numbers of surviving microorganisms were estimated after 15 and 60 min incubation time (see Fig. 3). [1] In the experiments, maximum E. coli reduction by 3.5 log was found using a combination of NO 2 - and H 2 O 2 at pH 3. One possible explanation of this result is the spontaneous reaction of nitrite with hydrogen peroxide in acid media to toxic species like ONOOH, nitrogen dioxide radical (NO 2 • ) and hydroxyl radical ( HO • ) : [3, 4, 5] (1) 2 H + + NO 2 - ↔ H 2 NO 2 + ↔ H 2 O + NO + (2) NO + + H 2 O 2 ↔ ONOOH + H + (3) ONOOH ↔ NO 2 • + HO • Fig. 3: Number of viable E. coli suspended in sodium chloride solution without and with addition of different combinations of nitrate, nitrite, hydrogen peroxide and hydrochloric acid (HCl); exposure times of 15 min (hatched columns) and 60 min (grey columns) [1] 30 th ICPIG, August 28 th – September 2 nd 2011, Belfast, Northern Ireland, UK 16 However, direct action of chemical species is by far not so effective compared to the bactericidal effect of the plasma-treated liquid or plasma treatment of bacteria suspensions, respectively. Consequently, there must be other reactive species which occur additionally in the result of plasma/gas-liquid-interaction as it is hypothesized in the schematic depicted in figure 2. The bactericidal effect of ozone is well known. [6, 7, 8] This antimicrobial effect of ozone treatment of bacteria suspensions was tested in comparison to the DBD plasma treatment in air. E coli suspensions in physiological saline were treated with different concentrations of ozone (~100 – 2000 ppm; 0.5 slm). As it is demonstrated in Fig. 4, there is a bactericidal effect of ozone, but it was much more ozone needed than it was produced by plasma treatment to reach the same inactivation of E. coli compared to direct surface-DBD treatment. 0 500 1000 1500 2000 2500 3000 0 5 10 15 20 25 30 ozone treatment time [min] ozone concentration [ppm] ozone concentration (E) ozone concentration (D) ozone concentration (C) ozone concentration (B) ozone concentration (DBE) ozone concentration (A) detection limit 1,00E+00 1,00E+01 1,00E+02 1,00E+03 1,00E+04 1,00E+05 1,00E+06 1,00E+07 1,00E+08 1,00E+09 0 5 10 15 20 25 30 ozone treatment time [min] number of viable microorganisms [cfu . ml -1 ] E. coli (pure oxygen) E. coli (A) E. coli (B) E. coli (C) E. coli (D) E. coli (DBE) E. coli (E) 10 9 10 8 10 7 10 6 10 5 10 4 10 3 10 2 10 1 10 0 Fi g. 4: by means of FT-IR measured ozone concentrations (A) and their corresponding antimicrobial kinetics (B) The experiments showed clearly that the detected species are less effective in microorganism inactivation than the plasma treatment itself. 4. References [1] K. Oehmigen, J. Winter, Ch. Wilke, R. Brandenburg, M. Hähnel, K D. Weltmann, Th. von Woedtke, Plasma Process. Polym. DOI: 10.1002/ppap.201000099. [2] K. Oehmigen, M. Hähnel, R. Brandenburg, C. Wilke, K D. Weltmann and Th. von Woedtke, Plasma Process. Polym. (2010) 7. [3] A. Daiber, V. Ullrich, Chemie in unserer Zeit (2002) 6. [4] M. Anbar, H. Taube, J. Am. Chem. Soc. (1954) 76. [5] P. Pacher, J. S. Beckman, L. Liaudet, Physiol. Rev. (2007) 87. [6] A. Dyas, B. J. Boughton, B. C. Das, J. Clin. Pathol. (1983) 36. [7] L. Restaino, E. W. Frampton, J. B. Hemphill, P. Palnikar, Appl. Environ. Microbiol. (1995) 61. [8] B. Thanomsub, V. Anupunpisit, S. Chanphetch, T. Watcharachaipong, R. Poonkhum, C. Srisukonth, J. Gen. Appl. Microbiol. (2002) 48. (4 A) (4 B) . After 15 min exposure time in the plasma- treated liquids, number of surviving microorganisms was estimated. For plasma treatment of liquids containing suspended microorganisms, 100 µl of E. coli. become part of secondary reactions, all together resulting in bactericidal activity of plasma treated liquids. The following investigations and hypotheses should give more insight into the complex. 16 Identification of antibacterial species in plasma treated liquids K. Oehmigen P 1 P , U C. Wilke UP 1 , K D. Weltmann P 1 P , Th. von Woedtke P 1 P P 1 P Leibniz Institute for Plasma