Assessment of laser treatment on dolostones colonized by
microorganisms and lichens
M. Alvarez de Buergo, M. Gomez-Heras & R. Fort
Instituto de Geociencias (CSIC,UCM), Madrid, Spain
C. Ascaso, A. de los Ríos, S. Pérez Ortega, M. Speranza & J. Wierzchos
Museo Nacional de Ciencias Naturales (CSIC), Madrid, Spain
M. Sanz, M. Oujja & M. Castillejo
Instituto de Química-Física “Rocasolano” (CSIC), Madrid, Spain
ABSTRACT: The preliminary results of the laser assessment for biofilms removal on rock surfaces are shown. The procedure consisted of laser irradiation (Nd:YAG, 1064nm, 10 ns, 200
mJ) on a dolostone colonized by microorganisms (mainly cyanobacteria) and lichens. Samples
were taken to assess the effectiveness of laser treatment on both the lichens thalli and microbial
communities (SEM-BSE and TEM), and also on the stone substrate (SEM-SE+EDS). The results show that lichen thalli were eliminated only partially. The laser fluence applied was not
enough to efficiently eliminate and/or damage the majority of lichen thalli, which is in accordance with previous laboratory results, where it was demonstrated that a higher fluence is necessary to clean and make inactive lichen symbionts. No damage was found in the dolomite crystals after laser radiation. Roughness and color were recorded before and after laser treatment.
Continuous monitoring of the thermal processes during laser irradiation showed that thermal
gradient is extremely high.
1 INTRODUCTION ¬ AIMS
Diverse groups of microorganisms such as heterotrophic bacteria, cyanobacteria, free-living
algae and fungi are responsible for biodeterioration in stones used to construct heritage
buildings and monuments (Caneva et al. 2008). Lichens are also common colonizers of
monumental stone. These biological agents can colonize the surface of the lithic substrate, as
well as the internal zone of the stone, where they develop complex interactions with the mineral
substrate (Ascaso et al. 1998, Caneva et al. 2008). It is known that fungi are the major
biodeterioration agents of stone with significant roles in mineral dissolution and secondary
mineral formation (Gadd 2007).
Methods to control biodeterioration have proven their technical and environmental limitations (Caneva et al. 2008, Doehne & Price 2010). In the field of cultural heritage, laser cleaning
is a well established technique because it provides a fine and selective removal of superficial
deposits and encrustations such as biological and black crust (Cooper 1998, Pouli et al. 2008 &
2012, Maravelaki-Kalaitzaki et al. 2003). However, despite the widespread use of lasers in conservation, few laser cleaning studies have been carried out dealing with the removal of biodeterioration agents, as epilithic lichen and fungi, from stone (de Cruz et al. 2009, Speranza et al.
2012).
The objective of this study is the approaching to the biofilms removal on stone by means of
laser irradiation under a multi and inter-disciplinary perspective considering the laser operation
conditions and the efficacy on the microbial colonization remotion, the modification of the
stone surface, and the recording of the temperature during the process. To our knowledge, this
is the first study dealing with the potential detrimental effect of laser irradiation performed on
the quarry on harmful microorganisms colonizing the lithic substrate that contribute to the biodeterioration of monuments.
2 MATERIALS AND METHODS
The Redueña stone was selected for this study because of its use in traditional construction in
the Central area of Spain. It is a cream-colored dolostone, mainly composed by dolomite and
calcite, with a moderate to high open porosity (10-25%). Some other characteristics of this
stone, as well as its decay pattern can be found in Fort et al. (2008). The quarry in which this
study was developed belongs to a Cretaceous geologic formation from the North of Madrid.
Stone quarries are an ideal natural environment to carry out this type of study since there is a
non-limited supply of sample material and the information obtained is directly transferable to
any nearby monuments built out of the same rock, besides any possible damage to the stone of a
monument is avoided. The microbial colonization of the fronts of this abandoned quarry has
been previously studied (Cámara et al. 2011), obtaining a predominant presence of the lichen
Verrucaria nigrescens and endolithic microorganisms. Also some laser cleaning studies have
been previously carried out dealing with the removal of biodeterioration agents in this same
stone (Speranza et al. 2012).
In situ laser irradiations in the quarry stone front were performed on selected areas, colonized by cyanobacteria and lichens, using, according to the previously mentioned experiments,
the fundamental wavelength (1064 nm) of a Q-switched Nd:YAG laser (CTS Art Laser) that
delivered pulses of 10 ns (FWHM). The laser beam, with maximum energy of 200 mJ, was focused on the surface of the stone delivering fluences up to 1.5 J/cm2 (below the stone ablation
threshold, Speranza et al. 2012) at 30 Hz repetition rate. The fluence was determined as the ratio of the laser pulse energy and the area of the irradiated spot, measured by the print left on an
unplasticized polyvinyl chloride sheet.
Others in situ measurements performed were color and roughness readings on the stone surface before and after laser irradiation. Color was measured by means of a spectrophotometer
(Minolta CM-2002, CIELab system, with CIE Standard Illuminant D65 and a 10° observer angle), obtaining the global color change ∆E*:
1/ 2
2
2
2
E*  L *  a *  b * 


(1)
where L*=Luminosity; a*=green-red coordinate, b*=blue-yellow coordinate, and ∆ reflects
the variation between the values obtained before and after laser irradiation. Surface roughness
was determined by an optical surface roughness meter (TraceIT, Innowep), obtaining the Rz
roughness parameter, which is the arithmetic mean value of the sum of the height of the 5 tallest peaks and the depth of the 5 lowest valleys, expressed in micrometer (µm). These two parameters are used to assess the efficiency of stone cleaning methods (Vazquez et al. 2012).
Continuous monitoring of the thermal processes during laser irradiation with an infrared microbolometer-based videocamera (Optris PI infrared camera) was performed, working within
the 7.5 to 13 μm spectral range.
Scanning electron microscopy (SEM) was used to study both the biocolonization (DMS 960
Zeiss, in back scattered mode BSE) as well as the effects on the stone substrate (JEOL JSM
6400 with an Oxford-INCA energy-dispersive X-ray spectrometer EDS, secondary electrons
mode, SE) on samples from the selected area of the quarry front.
Samples for transmission electron microscope (TEM) study were prepared following the process described in de los Ríos and Ascaso (2002), and observed in a Zeiss EM910 equipment.
The procedure followed in the field was the selection of square-rectangular areas of approx.
40-50 cm side, facing South. Color and roughness were first measured in situ, and some samples of biocolonized dolostone surface taken for microscopic analyses. Secondly the surfaces
were water sprayed and laser irradiated. Color and roughness were measured again, and some
samples taken to compare with the previous ones before laser treatment. Temperature monitoring was performed during the whole process of laser irradiation.
3 RESULTS & DISCUSSION
Laser-treated rock areas showed signs of visual superficial cleaning, especially the areas harboring epilithic cyanobacteria biofilms, as the color measurements will prove too. However, at
a microscopic level, the effects were not so positive. The laser produced a specific damage
within the lichen thallus (Figure 1a, asterisks). There are areas with hyphae totally damaged and
in the proximity appear zones composed by cells with appearance of a high cellular integrity
(Fig. 1a, arrows). The images obtained by TEM, shows the maintaining of the structure of the
fungal cell walls as well as the integrity of the organelles present in its cytoplasm (Fig. 1b). In
this experiment, the laser treatment induced the destruction of the majority of the algal cells in
the algal layer of the lichen thallus, since this is what is shown by the appearance of empty areas in the SEM-BSE images. Probably some fungal cells are destroyed but many remain, as
shown by the images obtained. Fungal cells remaining in the thallus after laser irradiation show
good cell integrity, which we have known through the use of TEM. In a previous experiment in
laboratory conditions the irradiation with a Q-switched Nd:YAG laser revealed as a promising
and effective procedure for the control of important biodeterioration processes produced by epilithic and endolithic microorganisms (Speranza et al 2012). Moreover, a certain resistance to
the thermal laser effect was observed in the fungal cell walls. It is well documented that the extent of laser cell damage depends on both the fluence and time of exposure to laser light, and
that this could be enhanced by the presence of pigments (Maravelaki-Kalaitzaki et al. 2003,
Vural et al. 2007).
Figure 1c shows a cross section SEM image of a dolostone sample after laser irradiation. No
damage to the dolomite crystals can be seen, which is due to the fact that laser irradiation fluence did not reach the stone ablation threshold fluence (Speranza et al. 2012).
20µm
*
*
*
1µm
Figure 1. Microscopic images of the stone surface obtained after laser irradiation: 1a) SEMBSE, 1b) TEM, 1c) SEM-SE.
Table 1 shows the results of color and roughness measurements, before and after laser treatment of the biocolonized stone surface, and, in the case of color, compared to a reference value
(fresh rock) for obtaining the global color change. The ∆E* values obtained indicate that the laser treatment makes the stone color surface closer to that of the reference one, meaning that the
laser irradiation was at least efficient in removing part of the biofilm, but not all. With respect
to roughness there is a trend towards diminishing the roughness of the biocolonized surface after being irradiated with laser, although it highly depends on the type of biofilm, in this specific
case, lichen thalli or cyanobacterial communities.
The maximum temperature attained in the higher albedo cleaned areas is around 63.4ºC,
while in the lowest albedo zones, corresponding to the dark Verrucaria nigrescens reach up to
84.3ºC. In absolute terms, temperature does not go up to values that could generate thermal decay per se, but the high temperature gradients (reaching up to the equivalent of well over 500ºC
per second) generate significant thermal shocks that could lead to an increase of cracks density
in the first few outer millimeters.
Table
1. Color and roughness results measured in situ on the stone surface.
__________________________________________________________________
Sample
Color
________________________
Roughness
____________________
Field area
L* a* b* ∆E*
Rz (µm) ∆Rz (%)
__________________________________________________________________
Reference
61 3.1 15.2 ------Biocolonized
40 1.5 7.0 22
35
--Laser
irradiated
48 2.5 10.6 14
33
-2
__________________________________________________________________
4 CONCLUSIONS
The preliminary results show that laser fluence applied in the field was not enough to efficiently eliminate lichen thalli, as observed by TEM and SEM-BSE. This is in accordance with
previous laboratory results, where it was demonstrated that a higher fluence is necessary to
clean and make inactive lichen symbionts. It was also proved that no damage was found in the
dolomite crystals after laser radiation. Laser treatment did not generate an undesirable increase
in the stone surface roughness. Color parameters measured on the stone surface after laser irradiation were closer to those of the fresh cut of the rock, but not clearly enough. While maximum temperatures attained during the laser treatment hardly exceed 55ºC, thermal gradients
can be extremely high (5 ºC/ 0.01 s), especially in low albedo lichen species such as Verrucaria
nigrescens.
Acknowledgements: to GEOMATERIALES programme (Ref. S2009-MAT1629), to CONSOLIDER programme (CSD2007-00058) and to Fabio dal Monte (CTS) for providing the laser
equipment and performing the onsite experiments.
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