Comprehensive characterization of atmospheric pressure plasma impact on vegetative microorganisms in argon and air
Theresa Winter1, Jörn Winter2, Martin Polak2, Ulrike Mäder1,3, Jörg Bernhardt1, Rabea Sietmann1, Jörg
Ehlbeck2, Klaus-Dieter Weltmann2, Michael Hecker1, Harald Kusch1
1
Institute for Microbiology, Ernst-Moritz-Arndt-University, Greifswald, Germany
Leibniz Institute for Plasma Science and Technology (INP Greifswald e.V.), Greifswald, Germany
3
Interfaculty Institute for Genetics and Functional Genomics, Department for Functional Genomics,
Ernst-Moritz-Arndt-University, Greifswald, Germany
2
Abstract: The understanding of the key mechanisms of bacteria-plasma interaction is essential for the enhancement of existing and the development of new decontamination procedures.
Vegetative growing Bacillus subtilis was cultivated in liquid medium and treated with plasma. Different gas admixtures (argon and air) and discharge voltages were applied and revealed complex cellular responses (cell wall-, oxidative and DNA stress) in dependence of
the used gas and applied power.
Keywords: B. subtilis, DBD, plasma-microorganism interaction, transcriptomics
1. Introduction
Low temperature plasma interaction with biological
material is of great interest for different applications,
ranging from decontamination of various surfaces to
treatment of skin diseases [1-4]. For a successful exploitation of plasma in biological settings, an understanding of the mechanisms of interaction of gas discharges with living cells and tissue is essential.
Therefore we addressed this topic with functional genomics to investigate the cellular responses towards
plasma treatment.
2. Material & methods
The cultivation and the plasma treatment were performed inside the discharge chamber displayed in Fig.
1. Inside a V2A-steel cup with a quadratic base area
(edge length 55 mm) and a height of 30 mm a volume
of about 45 ml bacteria suspension is situated. Via a
PID-controlled Peltier element the suspension can be
precisely heated to 37°C. A rotatable hollow cylinder
made of 1 mm thick V2A-steel and several swirling
elements which are mounted on the inside of the cylinder provide the mixing of the suspension. When the
hollow cylinder is rotated inside the suspension a
homogenous thin film forms on its surface whereas
the thickness of this film depends on the rotational
speed ω.
The cup and the hollow cylinder are fitted into a
box-like acryl glass chassis. In the top cover of this
box an aluminum electrode (52 mm x 52 mm x 6 mm)
is mounted on a 1 mm thick borosilicate plate as di
electric and connected to a high voltage source. This
voltage source generates a sinusoidal signal with a
frequency of 7 kHz and peak to peak voltages of up to
20 kV. The measurement of the supply voltage Us is
performed by a digital oscilloscope (Tektronix TDS
3054). The electrical power transferred into the discharge is measured by charge-voltage oscillometry.
The transferred charge is determined by an additional
capacitor (Cp= 9 nF).
Fig. 1: Setup for cultivation and plasma treatment of bacteria.
Beside the air plasma treatment setting, where the
discharge was ignited inside the naturally present
chamber air atmosphere, an additional argon gas
source (Argon 4.6, 99.996 %, AirLiquide) is connected to the chamber. By means of the needle valve
the argon flow rate is set to 1.0 slm. Via the gas outlet
three plasma diagnostic methods, namely temperature
measurements (FOT), optical emission spectroscopy
(OES) and Fourier transformed infrared spectroscopy
(FTIR), were performed. For the temperature measurements a fiber optical sensor (Optocon GmbH,
Fotemp1) was positioned between the dielectric and
the hollow cylinder assuring that no contact to the
liquid occurred. Spectroscopic measurements covering the spectral range from 240 – 900 nm were obtained by the compact spectrometer Avaspec-2048-USB-2 (Avantes). It has an entrance slit of
25 µm, which leads to a spectral resolution of 0.6 nm.
Furthermore, small amounts of gas from the plasma
region were exhausted and analyzed using a FTIR
GASMET CR-2000i (Ansyco) spectrometer with a
diagnostic range from 900 – 4200 cm-1 and a resolution of 8 cm-1.
Bacterial cultures of B. subtilis 168 were inoculated
with an overnight culture to an optical density of
OD540 0.5 into Luria-Bertani broth (LB) followed by
incubation with constant swirling at 37°C. Bacterial
growth was monitored. At OD540 0.5, plasma treatment was set for 15 min. Plasma was ignited either in
argon gas or air. Different discharge power settings
were applied (Tab. 1) to the cell suspension. Samples
were taken for transcriptomic analysis and for electron microscopy.
By increasing the discharge power, the plasma gas
temperature increases in each case. A steeper increase
is observed for air plasma compared to argon gas because the additional argon influx has a significant
cooling effect. Despite the temperature increase of the
plasma gas no temperature increase of the growth
broth was measured.
Tab. 1: Discharge power settings for Ar and air plasma
In the case of argon plasma dominant atomic lines of
excited Ar are obtained together with bands of excited
OH. Other species were not detected. In the case of
air plasma the 2nd positive system of nitrogen is the
strongest emission component. Furthermore, spectral
lines produced by atomic oxygen were detected at
777.2 nm, 777.4 nm and 777.5 nm (not spectrally resolved here). In contrast to the argon plasma no bands
of excited OH are visible. Fig. 4 shows the FTIR
spectra obtained for Ar and air plasma. In contrast to
the argon plasma, where no IR active species were
detected in the considered spectral range, air plasma
produces O3, N2O and N2O5.
Argon plasma
Supply
voltage [kV]
7.3
10.5
13.2
Plasma
power [W]
0.9
2.5
5.0
Air plasma
Supply
voltage [kV]
16.2
18.1
19.0
20.3
Plasma
power [W]
2.0
3.5
4.9
7.4
3. Results
3.1. Plasma diagnostic: temperature
To evaluate the temperature effect on the microorganisms, the gas temperature in the discharge region was
measured for both gases argon and air (Fig. 2).
3.2. Plasma diagnostics: OES & FTIR
In Fig. 3 the optical emission spectra of the generated
argon and air plasma are displayed for discharge
power settings of 5.0 W and 4.9 W, respectively.
Fig. 3: Optical emission spectra of argon plasma (5 W) and air
plasma (4.9 W).
Fig. 2: Plasma temperature for used gases air and argon.
Fig. 4: FTIR spectra of the exhaust argon and air plasma gas.
3.3. Plasma treatment leads to growth arrest
Cells, treated with plasma displayed a growth arrest
depending on the discharge power and gas. The higher the applied discharge power, the more severe was
the observed growth arrest. During the 15 min of argon plasma treatment all growth curves showed
growth arrest which started immediately after initiating the argon inflow during the plasma treatment.
While a 15 min plasma treatment with a discharge
power of 0.9 W led to almost no growth difference
compared to the control condition (15 min Argon, no
plasma), both the 2.5 W and 5 W discharge led to a
growth curve decline (Fig. 5). The observed growth
arrest of cells, treated with air plasma was minor severe compared to argon plasma treatment. Only the
highest applied discharge voltages of 7.4 W resulted
in a detectable growth decline (Fig. 5).
As a reference, non treated cells were provided which
showed no morphological abnormalities (Fig. 6A) in
contrast to plasma treated cells. On the surface of argon plasma treated cells (2.5 W), dents appear initially 60 min after plasma treatment and did not alter in
their appearance in due course of time (Fig. 6B-C).
Whereas air plasma treated cells (7.4 W) look
speckled 60 min after treatment (Fig. 6X). These
speckles turn into spider web-like filaments between
the cells after 120 min (Fig. 6Y-Z).
3.5. Plasma treatment leads to fast and global genomic response
DNA array technologies provide extensive information about the gene expression profiles. In total, 397
genes were found to be significantly regulated (ratio
<0.5;>2;p-value 0.01) due to argon plasma and 616
genes due to air plasma treatment (Fig. 7).
number of genes
800
600
total
repressed
induced
400
200
0
argon
air
Fig. 7: Number of affected genes by argon and air plasma.
Fig. 5: Growth curves of argon (left) and air (right) plasma experiment. At OD540 0.5, argon or air plasma treatment was set for 15
min and the growth was monitored for further 120 min.
3.4. Plasma leads to morphological changes
To visualize morphological changes in due course of
plasma treatment, argon and air plasma treated samples were taken for electron microscopy analysis.
Fig.6: Putative morphological effects of plasma treatment. Cells
were grown aerobically in LB medium at 37 °C and argon or air
plasma treated at an OD540 0.5. On Fig. A, non treated cells are
displayed. (B-C) Dents on the cell surface of argon plasma
treated cells (2.5 W) are visible (indicated by arrows). (X) 60 min
after air plasma treatment (7.4 W) cells look speckled and after
120 min (Y- Z) spider web-like filaments appear.
To support the analysis of our gene expression data
from transcriptome level, we used the functional gene
categorization from SubtiWiki orthology [5]. This
scheme classifies genes in an acyclic multihierarchical tree graph according to their function. For a
well-arranged visualization, we adapted Voronoi
treemaps for an intuitive display of large omics data
sets derived from the argon and air plasma experiment, with their relative expression data and functional classification (see Fig. 8) [6].
Fig. 8: (A) Gene expression of argon plasma and (B) air plasma
treated B. subtilis compared with untreated cells. Each cell in the
graph displays a single gene locus that belongs to other functionally related elements in parent convex-shaped categories. These
are again summarized in higher-level categories. For color coding- please read text below.
Expression data were visualized in the treemaps using
a color gradient: For illustration of gene expression
level, colors of the range green (lower than average)
yellow (equal to average) and red (higher than average) were applied to Voronoi cells. With this method,
similarities but of major interest for us- differences
between the two applied plasma treatments can be
visualized.
In both cases, the major carbon metabolism is down
and the general stress response up regulated (Fig. 9).
The responses to oxidative stress, DNA repair and
electron transport are induced in both cases but alter
in their extent between the two gases. Only under argon plasma stress, genes belonging to the functional
category of chemotaxis and motility, flagellar proteins
and biosynthesis of teichoic acids are clearly up regulated (Fig. 9C-D). On the other hand, a clear up regulation of genes, belonging to the category of biosynthesis/ acquisition of aromatic amino acids and of
cysteins as well as of heat shock proteins (Fig. 9C-D)
can be observed under air plasma stress.
4 Disscussion
We have performed a comprehensive proteomic (data
not shown) and transcriptomic analysis to investigate
the complex cellular response of microorganisms towards low temperature plasma. The construction of an
innovative plasma source enabled us to reproducibly
cultivate B. subtilis in liquid medium with or without
plasma treatment [7]. In an analysis about the effects
of plasma on microorganisms the central questions
relate to the cellular targets of the treatment and to the
nature of the plasma agents which induce cellular
responses.
Clear indications for a destructive effect on the cell
envelope were found not only by electron microscopy
but also in the transcriptomic results. Cells face also
oxidative stress, in different extent, which can be related to reactive species found with OES and FTIR.
For pure argon plasma excited OH molecules were
detected whereas in the case of air plasma O atoms
and O3, N2O and N2O5 molecules were verified as
plasma components. Under both gas settings, DNA
damage and repair systems are induced in response to
plasma treatment. But the heat shock response is only
activated during air plasma treatment, for which a
quite serve temperature increase was measured.
This growth chamber apparatus may in future experiments easily be adapted to cultivate and analyze
other pro- or eukaryotic species. An interesting task
will be the comparison of soil adapted microorganisms to those colonizing or infecting the human host
with regard to the plasma stress response. Another
future line of investigation will be the modification of
plasma parameters and their effects on the microbial
proteome and transcriptome. This opens up the opportunity to systematically investigate the microbe-plasma interaction in greater detail.
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Fig. 9: Gene expression of selected functional groups. Top: carbon metabolism of (A) argon plasma and (B) air plasma treated B.
subtilis compared with untreated cells. Bottom: Functional groups
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