Fungal decontamination of mural paintings by dielectric barrier atmospheric
pressure planar plasma jet
E.C. Stancu1, M.I. Moza2, D. Maxim3, G. Dinescu1*
1
National Institute for Laser, Plasma and Radiation Physics, 077125 Magurele, Bucharest, Romania
*
e-mail: [email protected]
2
Department of Ecology and Environmental Protection, Faculty of Sciences,”Lucian Blaga” University of Sibiu,
Romania
3
Department of Biology, Faculty of Biology,”Alexandru Ioan Cuza” University of Iasi, Romania
Abstract: In this contribution we present results regarding the
antifungal effects of a RF atmospheric plasma jet based on
barrier dielectric discharge. Plasma treatment on mural frescoes
can be effective in the prevention of contamination with fungal
spores but also in decontamination.
Keywords: RF plasma, DBD jet, bio-decontamination
1. General
Like other materials, mural paintings or frescoes are
susceptible to environmental degradation, mainly caused
by micro-organisms attacks especially fungi which can
produce serious deteriorations of the material [1].
Because of this, the frescoes surfaces have to be
protected. Nowadays, various antifungal treatments are
performed, but most of them did not fulfill the
requirements in terms of material structure, stability in
time and environmental aspects. Therefore, new
alternatives to conventional antifungal methods such as
natural biocides [2,3,4,5,6,7] or alternative physical
methods like UV radiation [8,9] are required to be
developed. An alternative approach to traditional methods
is cold plasma technique. Cold plasmas are slightly
invasive techniques which can modify the surface
properties in a desired way, including surface activation
or thin film deposition, without change of bulk properties.
Moreover, these plasmas are environmentally friendly
and for many applications, economically advantageous.
Recently, cold plasmas are used for bio-decontamination
and sterilization of various surfaces like medical
instruments, wastewater, air, food etc. These effects can
be obtained due to the UV radiation, radicals and reactive
species generated by plasma. Previous studies were made
with cold plasma for paper decontamination of
photographs or metal cleaning [10,11]. The plasma
treatment at low pressure becomes difficult when the
materials present special features like size or mass, are
sensitive to thermal damage or particles bombardment.
For these reasons new sources working in cold regime at
atmospheric pressure conditions were developed. These
sources are more easily to be handled and in many cases
economically preferable against the vacuum plasmas.
The main objective of present contribution was to find an
alternative physical method for material decontamination
and the proper conditions for this treatment against fungal
attack. In this respect, we describe experiments and
present results obtained by using a radio-frequency (RF)
cold atmospheric pressure plasma jet based on dielectric
barrier discharge (DBD) configuration for inactivation of
fungal spores. Previously, this plasma source was used to
improve the hydrophilicity of acrylic polymeric resins
used in dental prosthesis, highly important factor for the
stabilization of non-permanent artificial teeth [12].
2. Experimental
2.1. DBD jet used for fungal inactivation
More details about principle of operation of the dielectric
barrier atmospheric pressure plasma jet are described in
ref. [13]. A representative
image of plasma source is
given in Fig.1. Briefly, the
plasma source is of small size
with a rectangular exit
(nozzle of 4 mm x 1 mm
dimensions). The plasma jet
starts from the nozzle and has
a triangular shape, with a
length of 9 to 10 mm. The
power is supplied to the
discharge by a RF generator
Fig.1 Image of DBD
at 13.56 MHz via a matching
source [13].
network. It is easily operated
in argon without significant gas heating at RF power
values in the range of 6-50 W and gas flow rates of 5005000 sccm.
The present experiments have been carried out under the
following working conditions: RF power at 15 W, argon
gas flow rate at 5000 sccm, substrate-nozzle distance at
1.5 mm, no water cooling. For antifungal treatments the
DBD source was kept fixed above a motorized scanning
stage with movement in x-y coordinates. The scanning
route, consisting of parallel paths spaced at 1 mm
distance, was designed to assure a quasi-uniform
treatment. The scanning speed along the scanning path
was 2 mm/s, which corresponds to a surface exposure of
0.8 s/mm2. Selected areas of sample were exposed to
plasma with variable number of scans.
2.2. Sampling and culture preparation
Antifungal treatments were evaluated on a fungal spores
suspension consisting of species of genera: Rhizopus,
Aspergillus, Penicillium, Alternaria, Cladosporium, and
Fusarium. All species were isolated from two Romanian
frescoes and are known as responsible for frescoes
degradation and deterioration. Initially „in vitro“ testing
was made, followed by „ex situ“ treatment applied with
an microbiological screening of the surface.
All the isolated species were harvested on Sabouraud
medium and incubated at 20 degrees. After 5 days, the
colonial appearance was analyzed and the microscopic
investigation was done in order to indentify the genus
that grew on the frescoes. Fungal spores were obtained
after incubation for 10 days, followed by collecting the
spore in sterile distilled water and achieving the spore
suspension.
In vitro experiment. From the spore suspension, in each
Petri dish, it was measured 1 ml of sample and Sabouraud
medium was poured over. After the medium containing
fungal spores solidified, the plasma jet was directly
applied on it. An image during the plasma inactivation of
fungal spores is presented in Fig.2.
with fungal spores suspension and exposed to plasma jet.
After period of incubation, the treated frescoes were
investigated.
Fig.3 Plasma jet applied on
fresco surface.
The results were expressed through digital photos, optical
images and quantitative plate counts analysis. To acquire
digital photos a Canon 350D DSLR camera was used,
together with a 50 mm lens. The work protocol for
sampling and quantitative plate counts analysis is
summarized in Fig.4.
Fig.4 Protocol for sampling and quantitative
plate counts analysis.
Fig.2 Plasma jet applied on Petri
dish with solidified medium
containing fungal spores.
A selected area of 10 mm x 10 mm was exposed to
plasma with a variable number of scans: 1, 5 and 10.
After one week of incubation the samples at room
temperature were subjected to investigation.
Ex situ experiment. The plasma treatments consisting in
10 scans were done on selected areas of frescoes. For this
study various types of frescoes were used: simple fresco;
with calcium caseinate on its surface; with organic dirt
from hands and grease. In Fig. 3 is shown the plasma jet
scanning a fresco surface. Two types of experiments were
performed. In the first experiment, a selected area of a
simple fresco was exposed to plasma jet and inoculated
with fungal spores suspension, leaving it one week period
of incubation.
In the second one, the frescoes surfaces were inoculated
3. Results and discussion
To date, several mechanisms induced by cold plasmas for
microorganisms decontamination have been reported.
Therefore an important role in microorganism
deactivation is playing by heat and UV radiation [14], or
chemical reactions involving active free radicals and
ozone or physical processes caused by electrons or
positive and negative ions (O+, O-, Ar+) in the discharge
[15]. Previously, it was shown that the presented plasma
source has the ability to generate active species like OH,
NH, NO, O in absence of significant heating leading to
modification of surface properties of polymeric foils [13].
In vitro results. Images of fungal spores mixed with
Sabouraud medium in Petri dishes at different exposure
scans (1, 5 and 10 scans) to argon plasma jet are shown in
Fig.5. The treated areas are marked with a black square of
10 mm x 10 mm size. It is obvious that 1 scan and 5
scans of plasma treatments are much less efficient than in
case of using 10 scans for the fungal inhibition in vitro
testing. The inactivation process is enhanced by the
increase of the time exposure to plasma jet. These results
were useful to select the suitable parameters for the next
experiments regarding plasma treatments of frescoes
surfaces before or after inoculation with fungal spores
suspension. Therefore, the follow results were achieved
for 10 scans of plasma exposure.
(a)
(b)
(c)
Fig.5 Photographs of Petri dishes with fungal spores suspension on solidified medium after one week.
The selected area marked with black are plasma treated with (a) 1 scan, (b) 5 scans, (c) 10 scans.
Ex situ results. Fig.6 depicts photographs of Petri dishes
containing fungal spores colony forming units (CFUs) on
selective medium. The samples were taken from selected
untreated and plasma treated areas before and after
inoculation of a simple fresco surface.
The treatment before to inoculation with fungal spores
was conducted to investigate the effect of plasma in the
prevention of contamination with fungal spores. As can
be seen in Fig.6(b), a reduction in the number of colonies
was obtained.
In the case of simple fresco surface, plasma treatment
performed after inoculation with fungal spores suspension
induced a strong reduction of colonies number (Fig.6(c)).
The same results were achieved for other types of
contaminated frescoes namely fresco with calcium
(a)
(b)
caseinate and fresco with organic dirt from hands and
grease. Pictures with Petri dishes with samples taken
from these frescoes are not presented here. Nevertheless,
plasma jet applied on these inoculated frescoes lead also
to antifungal effects.
So, we can conclude that in all cases the fungal spores
activity was inhibited, as result of exposure to the 10
scans with plasma jet. The main contributors to fungal
inhibition can be reactive species generated by plasma.
Since the plasma jet is expanding in the surrounding
room air, the strong oxidizing species are created even if
the jet operating gas is argon, which is inert. Still, some
heating effects should not be completely excluded (the
temperature measured by a thermocouple inserted in the
medium was about 70 deg Celsius at 15 W RF power).
(c)
Fig.6 Photographs of Petri dishes with samples taken from selected areas of simple fresco: (a)
untreated, (b) plasma treated before inoculum, (c) plasma treated after inoculum.
In Fig.7 are presented results of the counting of the CFUs
on selective medium. In each case ((a), (b), (c) in Fig.7),
the plasma treatment induced a reduction of the number
of CFUs for all types of treated frescoes, compared to the
control sample. Indeed, the counting was performed in
order to calculate the inactivation rate of fungal spores by
plasma jet.
The inactivation rate (defined as (1-CFU treated/CFU
control) ×100%) showed that 94% of the fungal spores
taken from simple fresco surface were inactivated after 10
scans by plasma jet. In case of fresco with organic dirt
from hands and grease, the inactivation rate reached 91%
after plasma treatment and 87% for fresco with calcium
caseinate. Therefore, the plasma is effective in action
preventing fungal contamination.
Fig.7 Colony forming units from untreated and
treated areas of fresco (a) simple, (b) with dirt
and grease from hands, (c) calcium caseinate.
4. Conclusions
In this contribution we have shown that a cold plasma jet
can be used to inactivate fungal spores from frecoes
surfaces. By increasing the number of scans, the plasma
treatment becomes more efficient, the fungal spores were
inactivated after 10 scans (equivalent to a continuous
treatment of 8 s/mm2). For all types of frescoes, plasma
induced a reduction in the number of colonies. Via CFU
counting, we found that more than 85% of fungal spores
were inactivated after plasma treatment. Also, the plasma
is effective in the prevention of fungal contamination.
Acknowledgments
The financial support of the Ministry of Education,
Research, Youth and Sports under the project number PN
09.39.04.01/2013 (Nucleu Programme) is gratefully
acknowledged. In particular, the authors transmit their
special gratitude and thanks to Dr. Monica Mironescu
(Microbiology Laboratory, Faculty of Agricultural
Sciences, Food Industry and Environmental Protection,
”Lucian Blaga” University of Sibiu) and to biologist
Oana Mirela Chachula (National Centre for Scientific
Investigation, Bucharest) for all the support.
References
[1] O. Ciferri, Applied and Environmental Microbiology,
3, 65 (1999).
[2] D. Maxim, L. Bucşa, M.I. Moza, O.M. Chachula,
Annals of the Romanian Society for Cell Biology, 2, 17
(2012).
[3] M. Mironescu, C. Georgescu, Acta Universitatis
Cibiniensis Series E: Food Technology, 2, 14 (2010).
[4]. M. Mironescu, C. Georgescu, I.D. Mironescu, Annals
of the Romanian Society for Cell Biology, 2, 15 (2010).
[5] M. Mironescu, C. Georgescu, L. Oprean, Journal of
agroalimentary processes and technologies, 3, 15 (2009).
[6] M. Mironescu, C. Georgescu, Journal of
agroalimentary processes and technologies, 1, 14 (2008).
[7] M. Mironescu, I.D. Mironescu, C. Georgescu, Annals
of the Romanian Society for Cell Biology, 2, 15 (2010).
[8] M.I. Moza, M. Mironescu, I.D. Mironescu, Annals of
RSCB, 2, 17 (2012).
[9] S. Iseki, O. Takayuki, A. Akiyoshi, I. Masafumi,
K. Hiroyuki, H. Yasuhiro, H. Masaru, Applied Physics
Letters, 15, 96 (2010).
[10] E.G. Ioanid, A. Ioanid, D. Rusu, European Journal of
Science and Theology, 4, 6 (2010).
[11] E.G. Ioanid, D. Rusu, S. Dunca, C. Tanase, Annals
of Microbiology, 60 (2010).
[12] A.T. Farcasiu, E.C. Stancu, M.D. Ionita, O.C.
Andrei, G. Dinescu, Revista Romana de Stomatologie, 4
(2011).
[13] M.D. Ionita, M. Teodorescu, C. Stancu, E.C. Stancu,
E.R. Ionita, A. Moldovan, T. Acsente, M. Bazavan, G.
Dinescu, Journal of Optoelectronics and Advanced
Materials, 3, 12 (2010).
[14] M. Laroussi, F. Leipold, International Journal of
Mass Spectrometry, 1-3, 233 (2004).
[15] T Maisch, J. Baier, B. Franz, M. Maier, M.
Landthaler, Proceedings of the National Academy of
Sciences of the United States of America, 104 (2007).