Identification of antibacterial species in plasma treated liquids

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 bacterici

Identification of antibacterial species in plasma treated liquids

K OehmigenP

1

P, UC WilkeUP

1 , K.-D WeltmannP

1

P, Th von WoedtkeP

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

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 kVpeak (20kHz)

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 10s 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 109 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 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 (NaNO3; Merck), sodium nitrite

(NaNO2; Merck) and hydrogen peroxide solution

(H2O2; 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 NaNO3,1.5 mg . l-1

nitrite as NaNO2 and 2.5 mg . l-1 H2O2 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

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

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

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 (H2SO4; 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

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

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 (N2O), ozone (O3), carbon dioxide (CO2) and traces of

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

references from literature In figure 2 some possible

reaction channels are pictured [1] Most of them

resulted in generation of protons (H+), nitrate

(NO3

-), nitrite (NO2

-) or hydrogen peroxide (H2O2), 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+, NO3-, NO2-, and H2O2 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

plasma treatment time [min]

m

-1 ] plas ma treated NaCl solution added to E coli with 30 m in delay

plas ma treated NaCl solution directly added to E coli plas ma treated E coli s uspension

109

10 8

10 7

10 6

10 5

10 4

10 3

10 2

10 1

10 0

plasma treated NaCl s olution added to E coli with 30 min delay plasma treated NaCl s olution directly added to E coli plasma treated E coli sus pension

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

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

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 NO2

-and H2O2 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 (NO2 •

) and hydroxyl radical (HO•): [3, 4, 5]

(1) 2 H+ + NO2- ↔ H2NO2+ ↔ H2O + NO+ (2) NO+ + H2O2 ↔ ONOOH + H+

+ 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]

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

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

ozone treatment time [min]

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

ozone treatment time [min]

m

-1 ]

E coli (pure oxygen)

E coli (A)

E coli (B)

E coli (C)

E coli (D)

E coli (DBE)

E coli (E)

(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

Chanphetch, T Watcharachaipong, R Poonkhum,

C Srisukonth, J Gen Appl Microbiol (2002) 48

(4 A)

(4 B)