Investigation of Microorganisms Inactivation on Biodeteriorated Ancient
Paper Using Atmospheric Plasma and Laser Techniques
M. El Shaer*, M. Mobasher*, R. Sadeq**, T. Abdul fattah*, E. Amer*
* Faculty of Engineering, Zagazig University, Zagazig, Egypt
** Faculty of Medicine, Zagazig University, Zagazig, Egypt
Abstract: Atmospheric Plasma discharges have been successfully used for
conservation of metallic and organic artifacts in order to save cultural heritage.
An atmospheric RF plasma needle, operated at a frequency of 27.2 MHz and a
micro-jet operated at 5.7 KHz in helium environment are applied to different
cultures of microorganisms found in old paper. Three types of microorganisms
are chosen to be treated by plasma: E-coli as gram negative, Staphyloccocusaureus as gram positive, and Aspergillus-niger as fungus. The evaluation of
plasma effect on microorganisms is performed by visual inspection to identify
color changes, by counting to determine the culture survival proportion after
plasma treatment, and by electron microscopy to identify morphological changes.
The inactivation of different microorganisms is found to depend on plasma
conditions as power, exposure time and gas flow rate. Comparison with other
sterilization methods is made especially the effect of UV radiation generated by a
pulsed nitrogen laser of wavelength 337 nm. Plasma has the advantage to be safer
than laser, which if not operated carefully may lead to a destruction of the
artifact. Laser becomes more efficient for sterilization than plasma when fungal
fruiting bodies are embedded in the paper texture.
Keywords: paper conservation, plasma sterilization, laser sterilization, plasma
needle, plasma micro-jet
1. Introduction
Saving Biodeteriorated old paper and parchment
is one of important challenges in preserving cultural
heritage. The deterioration of paper materials is
mainly due to the degradation of cellulose caused by
chemical or biological factors as microorganisms.
The most common source of contamination of old
paper is the fungal spores which are identified by
discolored spots or particles resembling dust giving
undesirable aesthetic aspects and leading to
irreversible degradation, [1]. Fungi are spreader in
the environment due to their ability to accommodate
themselves to various environmental conditions.
The microorganisms in paper should be quickly
identified and eliminated to preserve paper from
harmful attacks leading to future destruction. The
treatment of biodeteriorated paper is a complex
chemical and physical process, [2]. New tools
applied to paper treatment as plasma and laser have
the advantage to be applied locally as contact-less
dry processes, [3], [4].
2. Plasma treatment
We use two types of discharges: an RF Plasma
needle having a central electrode surrounded by a
ring electrode at 27.2 MHz and a plasma micro-jet at
5.7 KHz. The plasma needle is operated in helium
with and without an earthed ring electrode
surrounding the central electrode as shown in Fig. 1.
The needle pin, made of tungsten, is 0.3 mm in
diameter and is inserted in a ceramic tube of 2 mm
inner diameter contained in a Teflon tube as shown
in Fig. 2-a. A grounded ring electrode can be placed
surrounding the nozzle outlet; this has the advantage
of making the impedance matching of the system
more stable, as shown in Fig. 2-b.
Matching
Current Probe
RF power
supply
Gas feed
2.1. Microorganisms’ survivals curves
To demonstrate the efficacy of plasma
sterilization; E-coli, Staphylococcus-aureus, and
Aspergillus-niger were let to grow by using organic
compounds. The microbial abatement was measured
before and after plasma treatment by using the
standard plate count method. The Colony forming
units (CFU) are determined for different plasma
conditions and comparisons are made between the
different schemes to identify the most efficient
scheme of operation suitable for paper conservation,
[5].
untreated
treated
Voltage divider
Figure 1. Experimental setup for RF operation
showing the ring electrode surrounding the needle tip.
Figure 4. Photographic view of E-coli colony on a Petri dish
showing point treatment by plasma needle.
a
b
Figure 2. Photographic view of plasma needle (a) without and
(b) with ring electrode
A second type of plasma discharges occurs at a
frequency of 5.7 KHz under a flow of helium gas.
The discharge expands few centimeters in air from a
central copper wire electrode of 0.9 mm diameter
situated inside a glass tube and surrounded by an
earthed ring electrode, as shown in Fig. 3.
Figure 5. Aspergillus-niger culture on Whatman paper showing
dark spots due to local plasma treatment.
Fig. 4 and 5 show plasma treated samples on Petri
dishes for E-coli and Aspergillus-niger, where dark
regions indicate treated spots. Survival curve is
shown in Fig. 6 for E-coli after application of plasma
needle at three separation distances between the
needle tip and the specimen at an RF power of 3.3 W
and a helium flow rate of 5.2 l/min.
The effect of plasma in reducing the number of
microorganisms’ survivors is more pronounced
when the needle tip is closer to the treated sample.
Figure 3. Plasma micro-jet treating an old book cover.
10
d= 1mm
d= 2mm
d= 3mm
Q=5.2 l/min, P=3.3W
Number of Survivors (CFU/ml)
109
108
107
106
105
104
103
106
Number of Survivors "CFU / ml"
10
Survival Curvefor Aspergillusniger
ExposedtoRFPlasma&HgLamp
RF Plasma
Hg Lamp
105
104
103
102
102
0
1
2
3
4
5
0
Exposure time (minutes)
RF Plasma without ring
RF Plasma with ring
LF Plasma
Number of Survivors " CFU / ml"
104
103
102
0
2
4
6
8
10
10
15
20
25
30
35
40
45
50
55
60
65
Exposure Time (minutes)
Figure 6. Survival curves of E-coli at three separation distances
between tip sample.
105
5
12
Figure 8. survival curves for Aspergillus-niger using RF plasma
compared to Hg lamp.
We used an electron microscope to observe the
effects of the plasma on bacterial cell morphology.
In Fig. 9, transmission electron micrographs showed
morphological changes in Staphylococcus-aureus
cells treated with atmospheric plasma at 3.1 W for 2
minutes. Staphylococcus-aureus are characterized by
robust walls; it is clear from the image in Fig. 9.b
that cell walls remain but its content dissolved by
plasma action.
14
Exposure Time (minutes)
Figure 7. Survival curves for Aspergillus-niger at different
mode of plasma operation
In Fig. 7, the operations of the needle and the microjet are compared at different operation modes and
frequencies applied to Aspergillus-niger. At RF of
27.2 MHz, the operation of the needle shows larger
effect without ring rather than using an earthed ring
electrode surrounding the needle. The application of
the micro-jet operated at LF of 5.7 KHz has less
pronounced effect on microorganisms. However the
operation of micro-jet is easier than the needle and
previous investigations show that the operation at LF
occurs at reduced temperature which is profitable for
paper treatment, [6].
In Fig. 8 the effect of RF plasma is compared to the
action of an Hg lamp. It is oblivious that plasma
achieves lowering of bacterial survivors more
rapidly than conventional sterilizing lamp.
a
b
Figure 9. TEM image for Staphylococcus-aureus
(a) untreated (b) treated.
3. Laser treatment
4. Conclusion
Laser has become an interesting tool in paper
decontamination, [7]. We use a nitrogen laser to
sterilize fungal-contaminated paper. The nitrogen
laser operates in a pulsed mode of wavelength 337
nm, pulse duration equal 12 ns and pulse energy of
15 mJ. The laser beam is used to hit the fungal
targets cultivated on a paper specimen. The laser
beam is focused to a (2 cm × 2.5 cm) rectangular
beam using a quartz cylindrical lens of 160 mm
focal length. A sketch of the experiment set up is
shown in Fig. 10.
Plasma and laser decontamination are investigated
for
biodeteriorated
paper
treatment.
The
effectiveness of those two methods has been proven.
Plasma needle at RF is suitable for point treatment
for localized contamination with bacteria while
micro-jet at LF is more suitable for treatment of
large area and temperature sensitive specimens.
Laser becomes more efficient for sterilization than
plasma when fungal fruiting bodies are embedded in
the paper texture. Plasma has the advantage to be
safer than laser, which if not operated carefully may
lead to a destruction of the artifact.
Extensive measurements should be done to clarify
the effect of plasma and laser on paper strength and
whiteness.
Figure 10. Sketch of the laser experiment set up.
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aspergillus-niger mold planted for 6 days after
treatment at laser energy density of 3×10-3 J/cm2,
and after 300 pulses in (a) and 500 pulses in (b).
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Figure 11. Aspergillus-niger on a 2 pieces of papers after
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