Biodeterioration of ancient stone materials from the ... - Springer Link

Aerobiologia (2008) 24:27–33 DOI 10.1007/s10453-007-9079-6

ORIGINAL PAPER

Biodeterioration of ancient stone materials from the Persepolis monuments (Iran) Parisa Mohammadi Æ Wolfgang E. Krumbein

Received: 20 July 2007 / Accepted: 26 October 2007 / Published online: 11 January 2008 Ó Springer Science+Business Media B.V. 2008

Abstract The problem of deterioration of art works is particularly relevant in countries like Iran that are rich in cultural heritage. According to UNESCO data, Iran holds the tenth rank in a list of countries possessing the highest number of monuments belonging to the world cultural heritage. Archaeological areas, buildings, mosques, statues, museums and objects are all exposed to different biogenic and abiogenic stresses under generally aggressive climatic conditions. Lichens and fungi are known to actively decompose stone surfaces. This process is both physical and chemical in nature and often reaches deep below the stone surfaces. This is caused by chemical and physical interactions of the microbiota with the fluctuating and often drastically changing environmental conditions. Here, we describe and analyze the mainly physical degradation by invading fungal hyphae between stone crystals and a generally destabilizing stone texture. In addition to physical deterioration, organic acids produced by lichens enhance the chemical decomposing processes. In this work, penetration of hyphal bundles as well as individual fungi was studied, and the biodeteriorating patterns were documented and compared to general physical–chemical weathering phenomena. Several strains of aggressive black yeast-like fungi and P. Mohammadi (&) W. E. Krumbein Geomicrobiology, ICBM, Carl von Ozzietsky St., No. 9–11, 26129 Oldenburg, Germany e-mail: [email protected]

bacteria were isolated and cultivated and will be described in a taxonomical context with many other isolates from different localities using physiological, morphological and molecular data. Keywords Persepolis

Biodeterioration Biopitting

1 Introduction Bioweathering and biodeterioration of stone monuments is one of the principal fields of interest for researchers in the conservation of cultural heritage. Each monument or building located in a specific climatic area can be considered as a special habitat. In addition, in each monument or building, different micro-niches also occur considering outdoor or indoor environments and different expositions. In these positions, different groups of organisms can settle and spread on and into the rock materials. Biodeterioration research has so far focused chiefly on lichens, fungi, algae and bacteria in Europe and the Mediterranean (Tiano et al. 1975; Nimis and Monte 1988; Tretiach 2004). Microorganisms living on inorganic substrates form more or less complex communities structured in biofilms, or microbial mats (Tomaselli 2003). It was estimated that biological weathering is 100–1,000 times greater than inorganic weathering (Aghamiri and Schwartzman 2002). Furthermore, there are many reports that show the effects

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of organisms on stone material are both physical and chemical (Jones and Wilson 1985; Bjelland et al. 2002). Mechanical damage is caused by penetration of the hyphae into the stone, and by the expansion and contraction of the thallus under changes of humidity. Chemical damage, however, is important, and may arise in three ways: by the secretion of oxalic acid, by the generation of carbonic acid, and by the generation of other acids capable of chelating metal ions such as calcium and magnesium (Adamo and Violante 2000; Brady et al. 1999). Biochemical weathering generally takes the form of surface etching on minerals, biopitting, leaching or replacement of minerals, and the production of weathering compounds (Lee and Parsons 1999). Etch marks visible with SEM appear on minerals beneath organisms; in time, the rock crumbles or becomes completely devoid of all minerals other than silicon.

2 Materials and methods 2.1 Geographic position of Persepolis and climate The magnificent ruins of Persepolis, sampling site for this study, contain an impressive display of temples and monuments, stairways and statues with engraved figures (Figs. 1, 2). They are located at the foot of Kuhi-Rahmat in the plain of Marv Dasht about 645 km (400 miles) south of the present capital city of Tehran. The date of founding of Persepolis is assumed to be between 518 and 516 BC, visualizing Persepolis as a show place and the seat of Cyrus the Great’s vast Achaemenian Empire. The geographic position of the area is 29°550 6000 N, 52°540 0000 E (Fars Province). The altitude in the area is 1,740 m. The climate is Figs. 1–2 Figure 1—View of the ruins at Persepolis. Figure 2—Antique engraving figure showing biodeteriorating pattern especially in the left side. Typical fungal biofilms were revealed on the graving figure

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Aerobiologia (2008) 24:27–33

Semiarid–Semitropic with a seasonal contrast stronger than in the Mediterranean area described in many biodeterioration studies (Krumbein and Urzi 1991). The mean of annual temperature is 15.8°C. The mean of annual precipitation is 341.1 mm with about 47.8 rainy days per year. Average of relative humidity is 41%. Number of days with snow or sleet and sunshine are 2.6 and 139.96 days per year, respectively. Another important phenomenon is the enormously high mean of annual dust days (64.1 days per year). The number of cloudy and thunderstorm days is 36.2, and 11.3 days per year, respectively (http://www.weather.ir/english/ monthly&annual/admin2.asp?CODE = 135). 2.2 Sampling Eight representative stone samples of three areas (International Gate, Hundred Column Hall, Xerxes palace) located at differently exposed places and heights from soil surface of the antique site of Persepolis (Iran) were taken aseptically in August 2004. Samples were immediately placed into sterile Petri dishes and taken to the laboratory for studying biofilms, and rock/ biofilm interaction between stones and microorganisms. Several strains of aggressive black yeast-like fungi and bacteria were isolated and cultivated and will be described in a taxonomical context with many other isolates from different localities using physiological, morphological and molecular data which will be published in a further publication. 2.3 Optical microscopic and stereoscopic observation An optical microscope (Axioscope II Zeiss) was used to detect and study lichens and biofilms on the stone


surfaces. Digital photos were taken using a digital camera Olympus 3030 with analytical software analySIS (Soft Imaging Systems, Mu¨nster). A dissecting stereomicroscope (Zeiss equipped with a Winder M35 camera) was used to study alterations of stone surfaces and biofilms which were present on the stones. Photos were taken for documentation.

2.4 Scanning electron microscopy SEM was used to study biofilm characteristics and structures. With this aim, samples were initially fixed in 4% glutaraldehyde buffer solution, later washed with 0.1 M phosphate buffer, dehydrated through an ethanol series (30, 50, 70, 80, 90, and 100%) and finally critical point dried. Before observing the samples, they were gold coated at 10-3 mm Hg in a sputtering apparatus (Blazers). Observations were done using an Environmental Hitachi scanning electron microscope S-3200.

2.5 Thin sections Marble and limestone (partially carbonate cemented sandstone) samples taken at Persepolis were used to analyze and demonstrate the main features of biogenic formation of pitting and other wear-down structures on the stone surface by the preparation and study of thin sections. Small pieces of stones encrusted with biofilm were fixed in 4% glutaraldehyde for 1 h. Vacuum treatment was also performed. The samples were then washed 3 times in 0.1 M phosphate buffer. Dehydration was accomplished in a graded ethanol series (30, 50, 70, 80, 90, and 100%) modified after Golubic et al. (1970). The specimens were embedded in a lowviscosity resin according to Spurr (1969). With infiltration of resin, vacuum treatment was applied. Finally, the samples were embedded in beamer capsules and polymerized. For the preparation of thin sections, the hardened blocks were cut by using a saw (Leitz model 1600) in sections vertical to the stone surface, washed in ethanol, mounted on ground (600 grit) microscope slides (46 9 27 mm) embedded in Spurr’s resin and polymerized. The samples were polished with silicon carbide powder in a series of 320, 400, 600 and 1,000 grit. Photo-micrographs of the sections were made using a Zeiss Axioscope II.

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2.6 PAS staining The Periodic acid Schiff (PAS) reaction was used for staining the mycobiont of lichens or free-living fungi to visualize the hyphae penetration inside the rock. The PAS technique (Witlach and Johnson 1974) was performed on the biofilms on the stone surfaces as well as on thin sections. Schiff’s reagent containing hydrochloric acid (HCL) with dissolve calcium carbonate to some extent, thus the hyphae will be easily visible microscopically.

2.7 Maceration technique In order to study features of corrosion on the stone surfaces, it is necessary to remove biofilms from the surfaces without disturbing them. For this reason, seven samples, which showed different kinds of biofilms, were chosen. They were immersed in ‘‘EAU DE JAVELLE’’ solution. This solution can dissolve away the organic material (Schneider 1976). After maceration, the remaining stones were washed several times in distilled water. The remaining cells easily detached from the surfaces without any mechanical pressure applied. After drying in the air, they were coated with gold and examined under the scanning electron microscope. EAU DE JAVELLE comprises 4 g CaOCl2 in 20 ml H2O, with 10 g K2CO3 in 100 ml H2O (Schneider 1976).

3 Results 3.1 Analysis of macroscopic structures on Persepolis samples The surface hue and color of all samples differed from the freshly broken and original stone. Most samples were found to be covered extensively by epilithic and endolithic lichens. Tiny black-brown spots representing microcolonies of black yeast-like melanised ascomycetes were observed in almost all samples. In Fig. 2, black fungal biofilm on the graving figure can be seen. Many of the tiny black or brown spots are easily confused with dust particles or metal deposits. Kondratyeva et al. (2006) recently hinted at the traditional and continuous spreading of microorganisms with aerial dust. Endolithic thallium

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Fungal hyphae of epilithic and endolithic crustose lichens or individual fungal hyphae as well as photobionts penetrate inside the stone and form biopits (Figs. 4, 5). Extensive penetration of fungal hyphae inside the stone was noted in seven out of eight samples. Biomineralized layers were found on four samples and the thickness of these coverages was from a few lm to 800 lm. Petrographic thin sections were used to document the biogenicity and depth of penetration of the biopits and other biodeteriorative phenomena inside the stones induced by the penetration of lichens and fungi. Fruiting bodies of lichen in the form of individual hyphae or bundle

of hyphae were growing on and inside the stones and inter-crystalline space, and clusters of fungi were found inside the stones. Thin sections of stone sample showed mesopit lining of endolithic fruiting bodies (Fig. 4). Examination after the PAS reaction showed that the extent of hyphae penetration into the stone by epilithic and endolithic lichens was considerable. The deepest penetration was up to 2 mm and sometimes even deeper penetration was seen. The modular hyphae were found in the upper area of stone revealing an extensive network of hyphae branching in all directions. With increasing distance from the stone surface hyphae became narrower. A perithecium of an endolithic lichens was seen in the petrographic stained thin section and showed semispherical pits (Fig. 5). Black fungal cells and photobionts were present on both the surface and inner layer of samples up to a depth of about 0.1 and 10 mm, respectively. In two samples, bundles of fungal hyphae growing in a certain depth were

Figs. 3–6 Figure 3—Endolithic thallium on the sample created biopitting. Arrow indicates photobionts. Figure 4—Thin section of a stone sample shows pits lining, small black arrows show a cluster of cells. Figure 5—Show PAS-stained thin section of the stone sample. Endolithic lichen perforated the stone with a thallus (T) and a perithecium (P) fruiting body,

which created a mesopit. White material situated on top of the thallus presents an external layer (EL) formed by lichen activity. Figure 6—Thin section of a stone sample treated by PAS-stain. Biomineralization products have been formed and fungi hyphae can be seen above and below these mineral layers (calcite)

on the stones created biopitting. The green color on and in subsurfaces of three samples indicated the presence of photobionts (Fig. 3).

3.2 Analysis of thin sections and PAS stained stone samples

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arranged parallel to the surfaces. They seemed to be arranged at a layer of optimal humidity. External material covered the cortical and modular hyphae as shown in Fig. 5. As a result of reaction between metabolites excreted by lichens and the minerals of the Persepolis samples, appreciable amounts of biomineralization products have been formed and deposited on lichen thallium and on the surface of stones (Fig. 6). These layers are usually calcium carbonate with considerable amounts of oxalates and gypsum admixed.

3.3 Analysis of scanning electron microscopy (SEM) observations SEM micrographs showed the biodeteriorated stone surface beneath the microbiota (Figs. 7–10). In the microscopic analysis, the fungal mycelia on and within the rock were clearly established. Different shapes and sizes of bacteria and algal cells were present in some samples (Fig. 7).

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A macerated Persepolis stone sample showed pitting. These pits were brought about by penetration of bundles of hyphae or penetration of fruiting bodies of lichen thalli inside the stone. These samples revealed homogenously pitted areas as well as heterogeneously distributed pitted regions. Two shapes of pits were observed in the Persepolis samples. The first shape is round to ellipsoidal with sharp margin and rough bottom (Fig. 9). The second is a meandric forming pattern on the samples (Fig. 10). The roundish pits were found to branch inside so that under one large superficial hole several smaller internal holes are found. The diameter of the largest pit was about 800 lm (mesopit). The smallest pit was about 10 lm in diameter (micropit). Continued solution of vulnerable sites causes fusion of biopits. In this way, grooves or channels are forming. These structures were first defined as bioerosion fronts by Krumbein (1969). Small biopits inside a large pit indicate the recolonization of these pits by algal cells or individual hyphae (Figs. 9, 10). Another biodeterioration pattern produces etching figures in

Figs. 7–10 Figure 7— Etching pattern is clear, dividing bacterial cells are visible. Figure 8— Macerated sample revealed imprint of fruiting bodies, granular rock debris, micro and meso pits. Figure 9— A typical mesopit left after the fruiting body was removed by maceration, in the lower right side a hiding fruiting body of endolithic lichens and micropits inside the pits are visible. Figure 10—Meandric pattern of biopitting; mesopit related to thallus

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the form of shallow imprints on the stone surfaces resulting from the activity of the thallus of lichen or imprints of bacterial cells and colonies (Figs. 7, 8). These patterns could only be seen in connection with these organisms. Macropits were not seen in Persepolis stones.

4 Discussion The deterioration of stones in buildings and monuments through the action of biological organisms has been recognized over a long period, but the topic has received increasing attention over the past few years. The presence of biological agents in Persepolis stones was studied using light microscopy and scanning electron microscopy, as Brock and Madigan (1997) has stressed that any study in microbial ecology must begin with the microscope. Altogether, the analysis of thin sections, micrographs and SEM-micrographs clearly demonstrates that morphogenesis of pitting at Persepolis is closely related to the morphology and action of the rock-dwelling microflora, and that material losses are progressive. The lichens are the predominant microbiota. It was shown that epilithic lichens seem to be more aggressive than endolithic lichens based on the higher biomass of the thallus. Furthermore, endolithic lichen and fungal growth can be used to describe the ecophysiological adaptations of them to the environmental extremes of the rock as Bungartz et al. (2004) have reported. No sand-blasting or surface roughness changes by, e.g., acid rain or other aggressive atmospheric influences were documented. Another factor observed in lichenic weathering of Persepolis stones is external polysaccharide substances covering cortical and modular hyphae in a kind of biofilm material. It was proved that in a dry state it can produce high adhesion strengths, leading to a reduction of cohesion and adhesion between the structural components. Many of these substances are aggressive or active on the surface or degraded by acid-producing bacteria (Krumbein and Scho¨nborn-Krumbein 1987). These materials also reduce the physiological demand for water (Gorbushina et al. 1999). Furthermore, these materials can catch particulate aerosols and contribute to accelerated biodeterioration of rocks. Why were green patinas in the subsurface of Persepolis stones also observed? The presence of

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photosynthetic bacteria has been reported from many parts of the world, such as hot and cold deserts (Friedmann 1980). It was mentioned that a response to a harsh environment conditions such as temperature and UV-radiation is rock penetration. Some species of cyanobacteria can be stimulated by higher light intensity to change from epilithic to endolithic growth (Wessels and Bu¨del 1995). It seems, however, that biopits are often no longer colonized by microorganisms. This is probably caused by changes in microclimatic and macroclimatic conditions, through the influence of excavation and perhaps air pollution. Even global climate change could perhaps be a reason for a decrease in inhabited pitted areas. Similar observations were made with the macro and mesopitting structures on the monuments in Rome, where increased air pollution was held responsible for the extinction of the biodegrading microflora existing in the early nineteentth century and documented by casts made from some of the monuments (Caneva et al. 1995). From documentations of the Germany archaeologist who started the excavation of the Persepolis monuments in 1931, it could be explained that the most of biodecay phenomena had developed during more than 70 years after excavation. Similar time frames were reported by Sterflinger and Krumbein (1997) for the French excavations at Delos and Gorbushina (personal communication) for the Caesarea excavations in Israel. Sand and aerosols, air pollution and secondary invasions of bacteria, algae, fungi and endo-epilithic lichens, finding in the pits favorable niches for developing in abandoned biopits, could contribute to subsequent pit enlargement. Acknowledgement The financial support of the Science, Research and Technology Ministry of Iran is gratefully acknowledged. I also wish to express my gratitude to Dr. A. A. Gorbushina for technical help and to the EU BIODAM project (http://biodam.biogema.de/index.html) for contribution during this study.

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