Contribuciones

 

Influence of fungi in the weathering of limestone of Mayan monuments

 

Influencia de los hongos en el deterioro de piedra caliza de monumentos mayas

 

Susana del Carmen De la Rosa–García¹, Otto Ortega–Morales¹, Christine Claire Gaylarde², Miguel Beltrán–García³, Patricia Quintana–Owen⁴, Manuela Reyes–Estebanez¹

 

¹ Departamento Microbiología Ambiental y Biotecnología, Universidad Autónoma de Campeche, México.

² Microbiology Research Laboratory, School of Pharmacy and Biomedical Sciences University of Portsmouth, UK.

³ Universidad Autónoma de Guadalajara, Guadalajara, Jal., México.

⁴ Departamento de Física Aplicada, CINVESTAV Unidad Mérida, Yucatán, México.

 

Recibido 26 de octubre 2010;
aceptado 15 de abril 2011.

 

Autor para correspondencia:
B.O. Ortega–Morales
beortega@uacam.mx

 

Resumen

La roca caliza deteriorada de monumentos mayas de Yucatán, México, fue analizada por medio de microscopia electrónica de barrido acoplada con análisis por rayos X retrodispersados (SEM–EDAX), difracción de rayos X (XRD) y espectroscopia infrarojo transformada de Fourier (FT–IR). Los cambios en la composición química de la superficie causadas por la biopelícula con la consecuente conversión de calcita a yeso, fueron demostrados. Los géneros más abundantes fueron Aspergillus, Penicillium, Fusarium y Paecilomyces. Cepas de Aspergillus niger y Penicillium sp., fueron seleccionadas por su capacidad de producir metabolitos ácidos. Ambos hongos excretaron ácidos orgánicos cuando fueron incubados (glucónico, succínico, málico y oxálico). A. niger fue el productor más activo y también excreto ácido cítrico. El impacto del hongo sobre los cupones liberó calcio de la matriz mineral asociado a la producción de acido oxálico. Sin embargo, el calcio soluble fue considerablemente bajo en los filtrados de cultivos que contenían los cupones de piedra, sugiriendo quelación. El análisis de SEM–EDAX confirmó el papel biodeteriorante de los ácidos micogénicos.

Palabras clave: acidólisis, Aspergillus, biopelículas microbianas, piedra caliza, Penicillium.

 

Abstract

Deteriorated limestone from Mayan buildings in Yucatan, Mexico, was analyzed with scanning electron microscopy (SEM) and microprobe with energy dispersive X–ray analysis (EDAX), X–ray diffraction (XRD) and Fourier–Transformed Infrared Spectroscopy (FTIR). Changes in surface chemical composition, caused by the biofilm layer and the conversion of calcite into gypsum, were demonstrated. Representative fungi include Aspergillus, Penicillium, Fusarium and Paecilomyces. Strains of Aspergillus niger and Penicillium sp., were selected for their ability to produce acidic metabolites. Both fungi excreted organic acids when incubated; ion exchange chromatography identified these acids as gluconic, succcinic–malic (coeluted) and oxalic. A. niger, the most active acid producer, also excreted citric acid. When grown in the presence of limestone coupons, calcium release from the mineral matrix paralleled the production of oxalic acid. However free calcium was considerably lower in filtrates from limestone coupon–containing culture, suggesting its complexation. SEM and EDAX confirmed that calcium oxalate crystals developed on the surface of the stone coupons. The results show that organic–acid–producing fungi may contribute to the deterioration of limestone monuments.

Keywords: acidelysis, Aspergillus, biofilm layers, lime stone, Penicillium.

 

Introduction

Microorganisms particípate actively in the weathering of minerais (Banfield and Hamers 1997). Microbial processes leading to the degradation of mineral may include microbial oxidation and reduction, creation and maintenance of appropriate physicochemical conditions, and production of acidic metabolites (Barker et al., 1997; Gaylarde and Morton, 2002). These microbially–mediated processes are partially responsible for the chemical and physical weathering of rocks, which lead, eventually, to the formation of soils (Eckhardt, 1985).

Microorganisms may also contribute to the deterioration of stone artifacts such as historical monuments and statues (Warscheid and Braams, 2000). The production of organic and inorganic acids by microflora in the biofilm has been generally recognised as the predominant mechanism of stone deterioration (Eckhardt, 1978; Sand et al., 2002). Most authors have tested acid production by isolated microorganisms in laboratory cultures, in the absence of the stone substrate, extrapolating these results to the field situation (Gaylarde et al., 2001; Resende et al., 1996).

Stone buildings located in tropical and sub–tropical regions throughout the world are particularly vulnerable to microbial deterioration; the prevailing environmental conditions of temperature and humidity are more suitable for microbial growth and development than those in temperate climates. Studies at Mayan archaeological sites in Yucatan, Mexico, have shown that microbial biofilms, dominated by cyanobacterial populations (Gaylarde et al., 2001; Ortega–Morales et al., 2000, 2005), contributed to the biodegradation of these monuments through active boring by cyanobacteria and probably by supporting growth of organic acid–producing microorganisms (Ortega–Morales et al., 2000). Fungi are a group of heterotrophic organisms that have been detected systematically on degraded stone buildings in tropical and temperate regions (Resende et al., 1996; Warscheid and Braams, 2000; Gaylarde and Gaylarde, 2005). They may have greater deteriogenic potential than bacteria, as they produce and excrete higher concentrations of organic acids (Palmer et al., 1991). In addition, these microorganisms may cause physical biodegradation of stone by the growth of hyphal networks through the pore space system (Urzi et al., 2000).

We studied the deterioration of ancient limestone buildings at the archaeological site of Uxmal, Yucatan, Mexico, and investigated the capacity of the fungal populations to produce acid–linked degradation of the stone.

 

Materials and methods

Stone sampling

Samples were removed from walls of the Anexo Norte building at the archeological site of Uxmal, Yucatan, Mexico. Two stone samples from a severely degraded pillar were collected with an alcohol rinsed chisel and hammer. These surfaces did not show any apparent biofilm coverage to the naked eye. Two samples of sound stone (undegraded, 2 cm below the surface of apparently sound stone) were also removed. Additional samples (n=2) of a powdered whitish material that appeared macroscopically to be salt efflorescences covering indoor walls were also collected. Samples were frozen and conveyed to the laboratory, where they were subdivided for analyses.

Chemical analysis of sound and degraded stone

Degraded stone and whitish powdery material samples were analyzed in duplicate by scanning electron microscopy (SEM) with energy–dispersive spectroscopy (EDS). Briefly, specimens were fixed with 2.5% glutaraldehyde (v/v) for 1 hour, air dried overnight and stored in a vacuum dessicator. The coupons were then fixed to aluminium stubs and examined using a Philips XL 30 instrument operating at 30 kV with EDAX facility.

Fourier Transform Infrared analysis (Spectroscopy) (FTIR) was carried out on lyophilized and powdered (where necessary) stone samples, formed into cylindrical blocks using 3mg stone plus 100 mg KBr. The spectra were obtained in an OPIH345 spectrophotometer between wavelengths 400 and 4000 cm^–1. X–ray powder diffraction of samples was performed with a Siemens D 5005 powder diffractometer, CoKa radiation, scanning speed 2Θ= 2.0°/min, 30 kV and 20 mA current, and Diffract–EVA software.

Isolation of fungi

Subsamples of weathered stone were chosen for microbiological analysis, as previously described (Ortega–Morales et al., 1999). Stone material was ground in a laminar flow hood, using an alcohol–flamed pestle and mortar. Samples were serially diluted in physiological saline, plated on Czapek Dox agar (1% glucose) and incubated in the dark at 25 °C for 14 days. Fungal colonies were counted and most frequent colony types were further purified and identified according to morphological characteristics (Booth, 1971; Domsch et al., 1980; Klinch and Pitt, 1988; Pitt, 1991). The fungal strains were maintained on PDA, al 4 °C.

Dissolution experiments

To identify acidogenic strains, fungal isolates were cultured in Czapek Dox broth containing 0.05 % of bromothymol blue as pH indicator. Cultures were incubated at 25 °C for 8 days. The strains capable of decreasing the pH to the lowest values, as indicated by colour change of bromothymol blue to pale yellow, were selected for the dissolution test.

Intact limestone coupons were obtained from a 15 cm core collected from a nearby commercial quarry (Oxkintok, Yucatan). One cm³ coupons were cut with a diamond saw, sterilized in three cycles in an autoclave and two placed in each of250 ml conical flasks containing 150 ml of Czapek Dox broth. Flasks were inoculated with 10 ml of a suspension of approx. 10⁶ cfu/ml of each selected strain, apart from control flasks. Flasks without limestone coupons were similarly inoculated to check the influence of stone on acid. All flasks were incubated at 25 °C. The rate of limestone dissolution was monitored by taking samples of spent medium from the systems at 0, 4 and 10 days and determining calcium and organic acids in the solution. Growth of the fungi was also monitored by plating on Sabouraud agar.

Organic acids and solubilized calcium determination

Aliquots (20 ml) from the incubated flasks were filtered (0.45 µm Nucleopore filters) and organic acids determined by ion exclusion chromatography, using 0.5 mM HCl eluant at 0.8 ml/min flow through a Dionex 500XL HPICE–AS1 separator column. Identification and quantification of acids were performed by coinjention of standards (Sigma), the free acids or their salts. Succinic and malic acids coeluted in these chromatographic conditions were therefore quantified as a single suc–mal peak. Dissolved calcium was assayed in the same filtered medium by atomic absorption spectrophotometry, using a model GBC 904AA spectrophotometer.

Microscopic and surface analysis of incubated coupons

Coupons were retrieved from the flasks and prepared for SEM analysis, as described above, using gold sputtering to aid the visualization of biological material. Elemental analysis by EDAX was performed on specimens without gold coating in order to determine the chemistry of the coupon surfaces.

 

Results and discussion

Surface elemental analysis of limestone samples

The chemical composition of the sound and degraded limestone samples, as shown by EDAX, is given in Table 1. The sound sample, as anticipated, consisted mainly of calcium and oxygen, the components of the limestone rock (calcite, CaCO₃), with Mg indicating the presence of dolomite (CaMgCO₃). García de Miguel et al. (1995) report a similar composition for limestone samples from the Mayan Pyramid of El Jaguar at Tikal, Guatemala.

The deteriorated samples could be divided into solid, but biofilmed, surfaces and badly degraded, powdery surfaces, with little apparent biofilm. The former contained high amounts of carbon and oxygen, as would be expected for an organic layer; this was confirmed by the presence of a considerable amount of nitrogen. The percentage of calcium was low, indicating that the limestone substrate was hidden below the organic biofilm layer. The powdery stone samples gave calcium, carbon and oxygen values in between the other two. Apparently, the limestone substrate was partially covered, either by microorganisms or, perhaps more likely in view of the absence of nitrogen, by their metabolic products. These could be organic acids or extracellular polymeric materials (EPS).

The presence of sulfur in these powdery samples was unexpected, although a similar phenomenon had been noted at other Mayan sites located in unpolluted countryside (Ortega–Morales et al., 2005). This indicated that some of the limestone had been converted into gypsum (CaSO₄2H₂O) and this was confirmed by XRD (Figure 1). Gypsum is not unusual in urban sites, but the source of the sulfur in this countryside situation is unknown, although cartographic data shows that well water from near by locations contains significant amounts of sulfates.

Sodium was present at reasonable levels in the biofilmed samples (Table 1). Other authors have noted this enrichment of sodium in rock samples associated with microbial communities (Johnston and Vestal, 1989; Ferris and Lowson, 1996). The small amounts of potassium, chloride and silicon observed by EDS are probably derived from the original mortar coating, which would contain wood ash from calcining limestone, and the secondary inclusion of other minerals, as we have previously noted (Ortega–Morales et al., 2005).

FTIR (Figure 2) confirmed that calcite was present in the undegraded and heavily biofilmed stone (strong bands at 1432cm^–1, 872cm^–1 and 708cm^–1, Negrotti et al., 1996) and that the reduction in this component in the powdery stone was due to its conversion into gypsum (bands at 1622cm^–1, 1140cm^–1, 1120cm^–1, 671cm^–1 and 603cm^–1). The presence of gypsum was confirmed by EDAX (Figure 3). Gómez–Alarcón et al. (1994) also noted a diminution in calcium content, with the formation of gypsum, in a degraded limestone monument in Spain.

Fungi detected in the biofilms

The fungi identified from degraded stone surfaces at the ancient Mayan site of Uxmal, Yucatan, Mexico, were Aspergillus fumigatus Fresen, Aspergillus niger Tiegh, Aspergillus terreus Thom, Aureobasidium pullulans (de Bary) G. Arnaud, Cunninghamella sp., Fusarium sp., Paecilomyces sp. and Penicillium sp. These genera are commonly found on stone monuments around the world (Palmer et al., 1991; Resende et al., 1996), and are typical of the soil mycoflora (Domsch et al., 1980). The dominant fungi were A. niger, A. fumigatus and Penicillium sp. Fungal populations ranged from 10² to 10⁵cfu/g, similar to results obtained by other investigators on stone buildings in Europe and Latin America (Resende et al., 1992; Urzi, 1993; Hirsch et al., 1995; Gaylarde et al., 2001).

Dissolution experiments and organic acid production

Two fungal strains, Aspergillus niger and Penicillium sp., were screened for their effect on limestone dissolution. Previous mineralogical analyses have shown that this limestone material is predominantly composed of calcite, with low amounts of aragonite (Ortega–Morales et al., unpublished results). After four days, Penicillium sp. and A. niger increased dissolved calcium in the culture medium more than eight– and 23–fold, respectively, relative to the abiotic control (Table 2), even though there was no visible growth in the flasks and colony counts were slightly reduced to around 10⁵cfu/ml. At 10 days, fungal numbers had increased to 2 – 4 x 10⁵ cfu/ml and the dissolution rate was increased up to 46–fold for Penicillium sp. and 112–fold for A. niger, compared to the uninoculated control.

Both genera produced gluconic oxalic and suc–mal acids, A. niger being the most active producer and also releasing citric acid. Table 2 shows the levels of oxalic acid, which is well known to complex with calcium. A. niger reduced the final pH in the medium without limestone coupons from 7.1 to 3.1, while the final pH in non–coupon containing flasks was 6.2, close to that for Penicillium sp. (6.8 and 6.4, with and without coupons, respectively).

Braams (1992) found that 41 fungal strains excreted predominantly gluconic, oxalic, citric and fumaric acids, a similar profile to these Uxmal isolates. He did not, however, detect oxalic acid from A. niger, probably because the medium utilized was too rich. It has been shown, for basidiomycetes, that oxalic acid production is linked to low nutrient levels (Dutton et al., 1993). The carbon source (glucose) in Czapek Dox medium was reduced from 3% to 1%, allowing us to detect the high levels shown here.

Nevertheless, we recorded differences in quantitative terms compared with other studies; where up to 4–fold higher concentrations of oxalic acids were observed with similar fungi (Gómez–Alarcón et al., 1994).

The flasks containing limestone specimens showed much lower concentrations of oxalic acid than those without coupons, indicating its removal by reaction or chelation with calcium. A. niger and species of Penicillium, among other fungal genera, have previously been shown to produce calcium oxalate, mainly as the monohydrate form, whewellite, however, small amounts of the dihydrate, weddellite, was also detected (reviewed in Pinna, 1993). The calcium appeared in the form of crystals on the surface of the coupons, in shapes that have been described by Monje and Baran (2002) as typical of calcium oxalate (Figures 4 and 5). EDAX analysis of these crystals showed major peaks of Ca, C and O, confirming their identity (data not shown).

All taxonomic groups of the fungi are able to precipitate calcium oxalate, to a greater or lesser extent (Sterflinger, 2000), although not all species or strains are able to do so (Palmer et al., 1991). This activity, when occurring within stone, leads to increasing internal pressure, the final result being catastrophic failure, or spalling, as was seen in our badly degraded limestone samples from Uxmal. These results suggest that organic acid producing fungi contribute to the biodeterioration of Mayan monuments. This heterotrophic mycoflora could be relying for its carbon source on the organic matter provided by the cyanobacterial populations dominating these epilithic biofilms and which, themselves, contribute to limestone biodegradation (Ortega–Morales et al., 2000, 2005).

The Yucatan Peninsula along with the rest of the world is experiencing rapid climatic change. Current forecasts include increased carbon dioxide release and precipitation. These are key factors that determine microbial activity. Fungi are proved agents intimately involved in biogeochemical transformations at local and global scales through excretion of organic acids (Gadd, 2007). Other metabolites, not analyzed in this study, such as carbonic anhydrase (CA) could also contribute to the dissolution of carbonate rocks (Li et al., 2005). Climate change is likely to have a major impact on built cultural heritage not only through abiotic exacerbation of stone deterioration, but also by increasing microbial metabolic activity (Bonazza et al., 2009).

 

Acknowledgements

The authors would like to thank INAH (National Institute of Anthropology and History) of Yucatan, Mexico, for sampling authorization and assistance during fieldwork. This research work was supported by a PROMEP grant "Comunidades microbianas aeroterrestres asociadas a monumentos mayas como observatorios naturales de cambio climático" and institutional funds from Universidad Autónoma de Campeche.

 

References

Banfield, J.F., R.J. Hamers, 1997. Processes at minerals and surfaces with relevance to microorganisms and prebiotic synthesis. Reviews in Mineralogy 35:81–122.         [ Links ]

Barker, W.W., S.A. Welch, J.F. Banfield, 1997. Biogeochemical weathering of silicate mineral. In: Banfield, J.F., K.H. Nealson (eds.), Interactions between microbes and minerals, Reviews in Mineralogy vol. 35. The Mineralogical Society of America, Washington, DC. pp. 391–428.         [ Links ]

Bonazza, A., P. Messina, C. Sabbioni, C.M. Grossi, P. Brimblecombe, 2009. Mapping the impact of climate change on surface recession of carbonate buildings in Europe. Science of the Total Environment 407:2039–2050.         [ Links ]

Booth, C. 1971. The genus Fusarium. Commonwealth Mycological Institute KEW, Surrey, England.         [ Links ]

Braams, J, 1992. Ecological studies on the fungal microflora inhabiting historical sandstone monuments. Doctoral thesis. University of Oldenburg, Germany.         [ Links ]

Domsch, K.H., W. Gams, T.H. Andersen, 1980. Compendium on soil fungi. Vol 3. Academic Press, London. UK.         [ Links ]

Dutton, M.V., C.S. Evans, P.T. Atkey, D.A. Wood, 1993. Oxalate production by basidiomycetes, including the white–rot species Coriolus versicolor and Phanaerochaete chrysosporium. Applied Microbiology Biotechnology 39:5–10.         [ Links ]

Eckhardt, F.E.W., 1978. Micro–organisms and the weathering of a sandstone monument. In: Krumbein, W.E. (ed.), Environmental Biogeochemistry and Geomicrobiology. Ann Arbour Science, Mich. USA. pp 675–686.         [ Links ]

Eckhardt, F.E.W., 1985 Solubilization, transport and deposition of mineral cations by microorganisms: Efficient rock weathering agents. In: Drever, J.I. (ed.). The chemistry of weathering, Dordretch, Holland. pp 161–173.         [ Links ]

Ferris, F.G., E.A. Lowson, 1996. Ultrastructure and geochemistry of endolithic microorganisms in limestone of the Niagara Escarpment. Canadian Journal of Microbiology 43:211–219.         [ Links ]

Gadd, G.M., 2007. Geomycology: biogeochemical transformations of rocks, minerals, metals and radionuclides by fungi, bioweathering and bioremediation. Mycological Research 111:3–49.         [ Links ]

García de Miguel, J.M., Sánchez–Carrillo, J.J. Ortega–Calvo, J.A. Gil, C. Saiz–Jimenez, 1995. Deterioration of building materials from the Great Jaguar pyramid at Tikal, Guatemala. Buidilg and Environment 30: 591–598.         [ Links ]

Gaylarde, C., G. Morton, 2002. Biodeterioration of Mineral Materials. In: Britton, G. (ed.), Encyclopedia Environmental Microbiology, Vol. 6. John Wiley & Sons, New York. pp. 516–528.         [ Links ]

Gaylarde, C.C., P.M. Gaylarde, 2005. A comparative study of the major microbial biomass of biofilms on exteriors of buildings in Europe and Latin America. International Biodeterioration & Biodegradation 55:131–139.         [ Links ]

Gaylarde, P.M., C.C. Gaylarde, P.S. Guiamet, S.G. Gomez de Saravia, H.A. Videla, 2001. Biodeterioration of Mayan Buildings at Uxmal and Tulum, Mexico. Biofouling 17:41–45.         [ Links ]

Gómez–Alarcón, G., M.L. Muñoz, M, Flores, 1994. Excretion of organic acids by fungal strains isolated from decayed sandstone. International Biodeterioration & Biodegradation 34:169–180.         [ Links ]

Hirsch, P., F.E.W. Eckhardt, R.J. Palmer Jr, 1995. Fungi active in weathering of rock and stone monuments. Candian Journal of Botany 73:S1384–S1390.         [ Links ]

Johnston, C.G., J.R. Vestal, 1989. Distribution of inorganic species in two Arctic cryptoendolithic microbial communities. Geomicrobiology Journal 7:137–153.         [ Links ]

Klinch, M. A., J.I. Pitt, 1988. A laboratory guide to common Aspergillus species and their teleomorphs. Commonwealth Scientific and Industrial Research Organization. Australia pp 115.         [ Links ]

Li, W., L–J. Yu, Q–F. He, Y. Wu., D–X. Yuan, J–H. Cao, 2005. Effects of microbes and their carbonic anhydrase on Ca²⁺ and Mg²⁺ migration in column–built leached soil–limestone karst systems. Applied Soil Ecology 29:274–281.         [ Links ]

Monje, P.V., E.J. Baran, 2002. Characterization of Calcium Oxalates Generated as Biominerals in Cacti. Plant Physiology 128:707–713.         [ Links ]

Negrotti, R., M. Realini, L. Toniolo, 1996. The frescoes of the oratory of S. Stefano (Lentate Sul Seveso–Milan): Comparison between the indoor air quality and decay. In Rieder, J. (ed.), Proc. 8^th International congress on deterioration and conservation of stone. Berlin. II. 1647–1653.         [ Links ]

Ortega–Morales, B.O., C.C. Gaylarde, G.E. Englert, P.M. Gaylarde, 2005. Analysis of salt–containing biofilms on limestone buildings of the Mayan culture at Edzna, Mexico. Geomicrobiology Journal 22:261–268.         [ Links ]

Ortega–Morales, O., G. Hernández–Duque, L. Borges–Gómez, J. Guezennec, 1999. Characterization of epilithic microbial communities associated with Mayan stone monuments in Yucatan, Mexico. Geomicrobiology Journal 16:221–232.         [ Links ]

Ortega–Morales, O., J. Guezennec, G. Hernández–Duque, G.C. Gaylarde, P.M. Gaylarde, 2000. Phototrophic Biofilms on Ancient Mayan Buildings in Yucatan, Mexico. Current Microbiology 40:81–85.         [ Links ]

Palmer, Jr, R.J., P. Hirsch, 1991. Photosynthesis–based microbial communities on two churches in Northern Germany: Weathering of granite and glazed brick. Geomicrobiology Journal 9:103–118.         [ Links ]

Pinna, D., 1993. Fungal physiology and the formation of calcium oxalate films on stone monuments. Aerobiologia 9:1557–167.         [ Links ]

Pitt, J.I., 1991. A laboratory guide to common Penicillium species. CSIRO Divition of Food Science. Australia.         [ Links ]

Resende, M.A., C.A. Leite, T. Warscheid, T.W. Becker, W.E. Krumbein, 1992. Microbiological investigations on quartzite and soapstone of historical monuments in Minas Gerais, Brazil. In: Latorre, W.C., C.C. Gaylarde (eds.), FIRST LABS, Proc. First Latin American Biodeter. Symp., Campos do Jordao, TecArt Editora, Brazil, Sao Paulo. pp. 17–22.         [ Links ]

Resende, M.A., G de C. Rezende, E.V. Viana, T.W. Becker, T. Warscheid, 1996. Acid production by fungi isolated from historic monuments in the Brazilian state of Minas Gerais. In: Gaylarde, C.C., E.L.S. de Sá, P.M. Gaylarde (eds.), Biodegradation & Biodeterioration in Latin America , Porto Alegre, Brazil: Mircen/UNEP/UNESCO/ICRO–FEPAGRO/UFRGS, pp. 65–67.         [ Links ]

Sand, W., P–G. Jozsa, R. Mansch, 2002. Weathering, Microbiol. In: Britton, G. (ed.), Environmental Microbiology, Vol. 6. John Wiley & Sons, New York. pp. 3364–3375.         [ Links ]

Sterflinger, K., 2000. Fungi as geologic agents. Geomicrobiology Journal 17:97–124.         [ Links ]

Urzi, C., 1993. Interactions of some microbial communities in the biodeterioration of marble and limestone. In: Guerrero, R., C. Pedros–Alio (eds.), Trends in Microbiology, Society for Microbiology, Madrid, pp. 667–672.         [ Links ]

Urzi, C., F. de Leo, S. de How, K. Sterflinger, 2000. Recent advances in the molecular biology and ecophysiology of meristematic stone–inhabiting fungi. In: Ciferri, O., P. Tiano, G. Mastromei (eds.), Of Microbes and Art. The role of microbial communities in the degradation and protection of cultural heritage. Kluwer Academic/Plenum Publisher, New York. pp. 3–21.         [ Links ]

Warscheid, T., J. Braams, 2000. Biodeterioration of stone: a review. International Biodeterioration Biodegradation 46: 343–368.         [ Links ]