Fungal decontamination by cold plasma : an innovating process

for wood treatment

Charlotte LECLAIRE 1, Elodie LECOQ 2, Geneviève ORIAL 3, Franck CLEMENT 2, Faisl BOUSTA3

1 Cercle des Partenaires du Patrimoine, LRMH, Champs-sur-Marne, France

2 Université de Pau et des Pays de l’Adour, Laboratoire d’Electronique des Gaz et des Plasmas, Pau, France
3 Laboratoire de Recherche des Monuments Historiques, Champs-sur-Marne, France

Abstract

Cold plasma technology offers an environmental alternative method to decontaminate wood surfaces, over the
conventional methods such as biocides applications. Decontamination effect of plasma discharge is evaluated on
a blue-stain fungus Aureobasidium pullulans, responsible for bluish discoloration of wood material causing
esthetical and economical damages. Several gas mixtures and exposure times were assessed. These results
suggest that fungal spores are inactivated after an afterglow exposure of 15 minutes.

1. Introduction

In the last years, biological decontamination by afterglows issued from plasma devices has shown
promising results [1-3]. Efficiency of plasma discharges as a sterilisation process has been proved on
bacteria contamination, in several industrial activities.
Wood material exposed to natural outdoor conditions is subject to biological development. In the case
of fresh maritime pine (Pinus pinaster), bluish discoloration due to blue-stain fungi development
causes esthetical damages, limiting its use according safety issues. Nowadays, preventive chemical
treatments are performed to delay fungal growth, but new environmental issues impose to develop
alternatives to conventional biocides.
According to industrial requirements, flowing afterglow which is a clean dry process offers numerous
advantages and may answer to specific needs regarding preservation of cultural heritage.
The present study focuses on the assessment of atmospheric pressure afterglow efficiency as a curative
treatment. In order to better understand mechanisms involved between the excited gas and
microorganims, efficiency of plasma afterglow is initially assessed directly on fungal cultures of
Aureobasidium pullulans, set on a membrane filter. After recalling plasma characteristics, the main
objective is, in the first place, to set up the experimental device and the characterisation means to find
better plasma parameters to inactivate Aureobasidium pullulans. Finally, first promising results are
presented.

2. Gas plasma

2.1. Definition and characteristics of gas plasmas

Cold plasmas, in reference to their low gas temperature, are partial ionised gases generated from the
introduction of sufficient energy to ionise a molecular or atomic gas. For instance the application
between two electrodes, in the gas, of an electric field leads to such environments. Thanks to the
supplied energy, numerous reactive species are created, resulting from collisions between electrons
and present neutral species. As a consequence, a cold plasma is composed of electrons, ions,
molecular and atomic neutral particles. Moreover, ions and neutral particles are constituted of a very
large number of excited states, themselves responsible of the formation of new elements as photons,
radicals, etc. This complex composition suggests that cold plasmas are potential energy tanks which
may interact with material surfaces in order to obtain physical and/or chemical modifications.
The Dielectric Barrier Discharge (DBD) technology provided for the experiments is a particular way
to generate cold plasmas. In this system, a dielectric coating on one or more electrodes is realised in
order to limit the electrical current and thus allow the generation of electrical discharges in gases at
atmospheric pressure.

2.2. The flowing afterglow

The experimental device uses the flowing afterglow to process on samples. Many advantages of using
afterglows are recognised. First of all, afterglow is only composed of neutral species (atoms,
molecules, radicals and photons), and following the experimental conditions the gas temperature can
be closed to ambient temperature, important to treat heat-sensitive materials. Moreover, studies have
demonstrated that neutral species [4], in large amounts in the afterglow, play the major role in the
sterilisation process. Operating in the discharge itself is thus not required and anyway the little gas gap
(1mm) does not allow to introduce large samples in the reactor. Then, the afterglow can fill large
chamber volumes to process on large pieces, and can be carried out via a tube directly through a
contaminated surface, to be one day directly supplied with a portable equipment. Finally, interactions
between the contaminated surface and neutral species from the afterglow can be considered as a phase
gas chemical process and thereby assures material integrity and protects the material intrinsic
properties. Consequently, decontamination process by afterglows issued from DBD answers to
specifications required regarding preservation cultural heritage field.

3. Experiment

3.1. Isolation and identification of fungal strains

To isolate fungi responsible of maritime pine biodeterioration, samples were taken from decayed wood
and deposed on malt agar plates. After 10 days of incubation, subcultures of single colonies were
carried out on fresh agar plates in order to isolate and purify the different fungal strains. Once isolated,
identification could be performed. Mains species identified are moulds : Penicillium sp. , Gliocladium
sp. and Trichoderma sp. and blue-stain fungi : Ceratocystis sp. and Aureobasidium pullulans.

3.2. Samples preparation

The fungus Aureobasidium pullulans is cultured on malt agar medium for ten days at 24°C. A fungal
suspension is prepared adding 20 mL of sterile water to the plate culture and then filtered through a
glass filter in order to recover only fungal spores (mycelium is retained in the filter). An aliquot of the
suspension is next spread on sterile nitrocellulose membrane filters stuck on a glass slide and let to dry
at ambient temperature before being exposed to the afterglow. Initial spores concentration of the
fungal suspension is evaluated. Dilutions in sterile water are then plated on malt agar plates and
incubated 5 days at 24°C. The number of colonies formed are counted, and thus initial concentration is
assessed.

3.3. Afterglow exposure

The DBD reactor is an industrial reactor (AXCYS Technologies) electrically supplied by a generator
which delivers chopped quasi sinusoidal voltage and current waveforms in a frequency of 125 kHz.
Total delivered power was fixed to 900 Watts, and the duty cycle was adjusted to 10% in order to keep
the temperature inferior to 40°C so that thermal effect of the gas is not to be considered.
An adapter is added to the reactor in order to collect the gas exiting from the reactor and guide it in a
quartz tube of 1 cm of diameter. Glass slides are placed at 1 cm from the exit of the tube. The sample
moves thanks to a custom-made conveyor belt so that the treatment is dynamic and the entire surface
can be treated. The duration of the treatment is then defined by the velocity of the conveyor belt. In
this work, three times of treatment were studied : 1, 2 and 15 minutes. Moreover, used gases are
mixtures of nitrogen and oxygen, the oxygen percentage being equal to 0, 5 and 20%.
 Gas in
60
mm
Outer
electrode

Inner
electrode Dielectric
coating

Gas out

Figure 1 - Schematic representation of the industrial

reactor used for the process (AXCYS Technologies).

3.4. Characterisation means

3.4.1. Fluorescence microscopy

Fluorescence microscopy observations are performed directly on membrane filters. A double staining
procedure, with two fluorescent dyes : fluorescein diacetate (FDA) and propidium iodide (PI), gives
information about cells viability [4]. FDA stains viable cells because of the enzymatic activity and the
integrity of membrane cells. On the contrary, PI bounds DNA of dead cells emitting red fluorescence ;
this stain cannot cross the membrane of viable cells. In this way, when spores are observed by
fluorescence microscopy, viable and non viable cells respectively fluoresce green and red [5]. Such
characterisation mean is used as a qualitative one.

3.4.2. Quantitative plate count

To measure growth inhibition after afterglow treatment, colonies on plate cultures are counted to
estimate the number of survivors. Filters are rinsed in 10mL sterile water and suspensions are vortexed
for 1 minute in order to recover the spores. Dilutions in sterile water are then streaked on malt agar
plates. The number of CFU (Colonies Forming Unit) is determined after incubation for 5 days at 24°C.
Inhibition percentage of fungal growth is calculated as indicated in (1) :

% inhibition = CFUcontrol – CFUafter treatment x 100 (1)

CFUcontrol

4. Results

Three experiments were realised. Results of the first run are presented in Table 1. Gas mixtures
containing 0, 5 and 20% of oxygen were tested and the treatment duration was equal to 15 minutes.
Results show that after 15 minutes of afterglow exposure, spores are inactivated for all gas mixtures.

Gas Mixture N2/O2 control 100/0 95/5 80/20

Agar plate cultures

Percentage inhibition - 100% 100% 100%

Table 1 : Agar plate cultures after different gas afterglow exposures (first experiment).
Observations by fluorescence microscopy of exposed filters confirm agar plate cultures results : dead
spores could be observed, whereas viable spores were not detected (Figure 2b). On the contrary, the
presence of viable spores assessed on control filters (Figure 2a), correlated with the high number of
colonies counted on plates, confirms the decontaminating properties of afterglow on fungal spores.

(a) (b)

Figure 2 - Fluorescence microscopy images : (a) untreated control

(b) spores exposed to the afterglow for 15 min.

For the two following runs, the same process and experimental conditions were used. Three exposure
times (1, 2 and 15 minutes) and gas mixtures (N2/O2 80/20, 95/5 and 100/0) were tested. Figure 3
represents the number of survivors after different treatment duration. Results show a reduction of the
initial spores population as a function of exposure time for all gas mixtures, first significant results are
obtained after 2 minutes of exposition. After 15 minutes of afterglow treatment, inactivation of fungal
spores can be considered as total (inhibition percentage = 100%) and confirmed the first run results.

1,E+06
control 1 min 2 min 15 min
Numbers of survivors (CFU / mL)

1,E+05

1,E+04

1,E+03

1,E+02

1,E+01

1,E+00
100/0 95/5 80/20
Gas mixture (N2/O2)

Figure 3 - Evolution as a function of exposure time and gas composition of the

population of spores submitted to the afterglow. (Mean values of three agar plates).

Results obtained by fluorescence microscopy confirm the evolution revealed by the numeration on
agar plates. However, they were not totally conclusive, since glass slides preparation was not
optimised. Nevertheless, few viable spores were observed on untreated filters and short exposure-
times treated samples, whereas the number of dead spores increase as a function of exposure time.
 a b

c d

Figure 4 - Fluorescence microscopy observations of fungal spores after

different exposure times : (a) control, (b) 1min, (c) 2 min, (d) 15 min.

5. Discussion

This work showed first very promising results. Efficiency of afterglows to inactivate fungal spores has
been shown for treatment duration of 15 minutes. Moreover, for shorter exposure times (2 minutes), an
effect has been assessed too, denoting a great interest of such environments.
Quantitative plate counts provide results which can be reproduced, regarding low bar errors, and give
information relative to the percentage of growth inhibition according to exposure time. More
experiments would supplement first data and so the shorter time necessary to inhibit all fungal spores
would be determined.
Moreover, according to first results, the nature of gas mixture do not seem to be a preponderant
parameter in the decontamination process. No significant differences were proved increasing the
percentage of oxygen to 0 to 5 and 20%. Some more runs with different treatment duration and gas
mixtures may reveal the most effective plasma parameters.
Otherwise, fluorescence microscopy observations only give few qualitative information as far as
spores viability is concerned, due to a problem of glass slides preparation. This characterisation mean
is now being improved in order to avoid this problem.

6. Conclusion and perspective

To conclude, this study emphases on the efficiency of a new curative anti-fungal treatment realised at
atmospheric pressure.
Further experiments may improve these first promising results. Influence of several parameters has to
be studied such as the exposure time and the gas mixture. Moreover, new characterisation means could
give more information about spores viability, for example, scanning electron microscopy may show
the aspect of spores after plasma exposure.
Furthermore, to better understand mechanisms, reactive species composing the afterglow have to be
analysed more precisely.
Finally, best plasma parameters once defined, assessment of the efficiency of the afterglow will be
investigate on contaminated wood.

Acknowledgements

This work is financially supplied by the French national research program ANR-PLASMAPAL,
realised with the support of public institutions and companies partners : BEYNEL-MANUSTOCK (M.
Gerard VIERGE, coordinator), FP BOIS, ACXYS Technologies, ARNAUD SA, Projection Plasma
Système, FCBA, LRMH-CPP, LEGP, USBB, LAPLACE.
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