Identification of antibacterial species in plasma treated liquids

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
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ICPIG, August 28
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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)