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Biofouling of crypts of historical and architectural interest at la plata cemetery (argentina)

Available online at Biofouling of crypts of historical and architectural interest at La Plata Cemetery Patricia S. Guiamet , Vilma Rosato , Sandra Gómez de Saravia , Ana M. García , Diego A. Moreno a Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), Departamento de Química, Facultad de Ciencias Exactas, UNLP, CCT La Plata- CONICET. C.C. 16, Suc. 4, 1900 La Plata, Argentina b Facultad de Ciencias Veterinarias, UNLP, Calle 60 y 118 s/n, 1900 La Plata, Argentina c LEMIT, Laboratorio de Entrenamiento Multidisciplinario para la Investigación Tecnológica, 52 e/121 y 122, 1900 La Plata, Argentina d LEMaC, Universidad Tecnológica Nacional, Facultad Regional La Plata, 60 esq. 124, 1900 La Plata, Argentina e Facultad de Ciencias Naturales y Museo (UNLP-CICBA), Avenida 60 y 122, 1900 La Plata, Argentina f Universidad Politécnica de Madrid, Escuela Técnica Superior de Ingenieros Industriales, Departamento de Ingeniería y Ciencia de los Materiales, José Gutiérrez Abascal, 2, 28006 Cemeteries are part of the cultural heritage of urban communities, containing funerary crypts and mon- Received 24 June 2011 uments of historical and architectural interest. Efforts aimed at the conservation of these structures must Accepted 4 November 2011 target not only the abiotic stresses that cause their destruction, such as light and humidity, but also bio- Available online 9 December 2011 fouling by biotic agents. The purpose of this study was to assess the development of biofouling of several historically and architecturally valuable crypts at La Plata Cemetery (Argentina). Samples obtained from the biofilms, lichens, and fungal colonies that had developed on the marble surfaces and cement mortar Cultural heritage of these crypts were analyzed by conventional microbiological techniques and by scanning electron Funerary monument microscopy. The lichens were identified as Caloplaca austrocitrina, Lecanora albescens, Xanthoparmelia farinosa and Xanthoria candelaria, the fungi as Aspergillus sp., Penicillium sp., Fusarium sp., Candida sp. and Rhodotorula sp., and the bacteria as Bacillus sp. and Pseudomonas sp. The mechanisms by which these microorganisms cause the aesthetic and biochemical deterioration of the crypts are discussed.
2011 Elsevier Masson SAS. All rights reserved.
1. Research aims
loss of historical information since in many cases it has become impossible to read the names, dates and inscriptions engraved As the first city in South America to install electric light bulbs, in the headstones. An additional problem, pointed out by the La Plata represented the modernization of Argentina at the end of Commonwealth War Graves Commission, is the disfigurement of the 19th century and as such even aspired to become the capital soldiers' graves by colonies of lichens of the Republic. Its progress was also recognized at the 1889 Paris Efforts to rescue the crypts from further disfigurement and EXPO, which awarded the city two gold medals, as the "City of destruction must begin with a detailed analysis of the invad- the Future" and the "Best Built Project". The city of La Plata had ing microorganisms. The appropriate measures can then be taken been designed by Pedro Benoit, who was also the architect of its to preserve and restore these beautiful and culturally important Cathedral and, in 1886, its public cemetery. Among the latter's funerary monuments.
notable architectonic structures are its portico and many of the family crypts, with their Neoclassic, Neogothic, Art Nouveau, Art Deco, and Egyptian Revival styles. These monuments were primarily built with marble and cement mortar Now, after 2. Material and methods
120 years, they show signs of advanced biodeterioration, including the appearance of biofilms and fungal colonies, both of which are 2.1. Site, visual inspection, sampling and biofilm analysis often quite large and continue to spread. Biofouling has not only resulted in the aesthetic deterioration of the stones but also in the This work was carried out at La Plata Cemetery in Argentina. La Plata has an average annual temperature of around 16.3 oC and the average annual rainfall is 1023 mm. As the city is close to the La Plata River, it tends to be rather humid, with an average annual relative ∗ Corresponding author. Tel.: +34 9 13 36 31 64; fax: +34 9 13 36 30 07.
humidity of about 77%. The air temperature during sampling was E-mail address: (D.A. Moreno).
around 20 oC and the relative humidity 50–55%.
1296-2074/$ – see front matter 2011 Elsevier Masson SAS. All rights reserved.

P.S. Guiamet et al. / Journal of Cultural Heritage 13 (2012) 339–344 soredia, etc.), fruiting bodies, spore form and number, and specific substances present in the thallus.
Fungal samples were dispersed in physiological saline solu- tion and a dilution series prepared. A volume of 100 ␮L from each dilution was used to inoculate YGC (yeast extract-glucose- chloramphenicol) agar plates, which were then incubated at 22 oC for two weeks. Moulds were identified based on their morphology yeasts using the API 20 C AUX system (bioMérieux).
Total aerobic heterotrophic mesophilic bacteria were deter- mined by first dispersing the samples in physiological saline solution, which was then used to prepare a dilution series.
Volumes of 100 ␮L from each dilution were plated on nutrient agar, plate count agar, and CPS (casein-peptone-starch) agar and then incubated at 28 oC for one week. Bacteria were subjected to Gram staining followed by biochemical testing using the API 20NE and API 50CH systems (bioMérieux) to obtain a preliminary identification prior to sequencing. Genomic DNA of the selected bacteria was extracted by three freeze-thaw cycles (−75 oC, +55 oC). 16 s rDNA fragments corresponding to nucleotides 5–531 in the Escherichia coli sequence were PCR-amplified using the primers 5F (5'-TGGAGAGTTTGATCCTGGCTCAG-3') and 531R (5'- TACCGCGGCTGCTGGCAC-3'). PCR was performed in a GeneAmp PCR System 2400 (Perkin Elmer) with the following thermocycling program: 5 min denaturation at 94 oC, followed by 30 cycles of 1 min denaturation at 94 oC, 1 min annealing at 55 oC, and 1 min extension at 72 oC, and a final extension step of 7 min at 72 oC. The reactions consisted of Ready MixTM Taq PCR Ready Mix with MgCl2 Fig. 1. Portico of the crypt of Francisco Arrechea (crypt F87), which is of architectural
(Sigma) containing 5 ␮L of DNA and 25 pmol of each primer and interest because of its Art Nouveau style. The yellowish coloration is indicative of were brought to a final volume of 25 ␮L with sterile water. PCR the presence of Caloplaca austrocitrina.
products were analyzed by electrophoresis in 1% (wt/vol) agarose gels in 1 × TBE buffer containing ethidium bromide (0.4 ␮g·mL−1) Visual inspection of crypts of historical and architectural interest and then purified by filtration through Microcon-100 (Millipore) showed clear evidence of significant biofouling (lichens, fungi, and columns. Bacteria were identified by sequencing of the respective dark biofilm patinas). Five sites from four crypts were selected for 500-bp rDNA fragment using the BigDyeTM Terminator v1.1 Cycle Sequencing kit (L-7012, PE Applied Biosystems). Sequences were resolved in an ABI PRISMTM 310 Genetic Analyzer following the manufacturer's instructions and then compared directly to crypt C95 (marble column); all known sequences deposited in the NCBI (National Center crypt C77 (inner side of the marble); of Biotechnology Information) databases using the basic local crypt F87 (front, cement mortar – alignment search tool Megablast.
crypt A9 (front, cement mortar); In addition, acid-forming bacteria were quantified using the crypt A9 (side wall and front).
extinction technique, in which serially diluted cultures were main- tained in glucose broth containing a pH indicator.
Biofilms, fungi, and crustose lichens were sampled (1 cm2) by aseptically scraping the external surfaces of selected monuments 3. Results and discussion
using a sterile scalpel. Foliose lichens were carefully detached with the help of a penknife. Biofilm samples to be further analyzed by 3.1. Visual inspection and biofilm analysis scanning electron microscopy (SEM) were fixed overnight with a 2.5% glutaraldehyde solution in phosphate buffer, washed with dis- The studied crypts were those of Vicente Isnardi (crypt C77), tilled water, dehydrated in an acetone-water series, critical-point Emilio B. Coutaret (crypt A9), Domingo Lastra (crypt C95), and Fran- dried, and sputter-coated with gold. They were then examined with cisco Arrechea (crypt F87). Vicente Isnardi was one of the founders a Jeol scanning electron microscope (JSM-T100) at an accelerating of La Plata University. Emilio B. Coutaret was a well-known archi- voltage of 20 kV.
tect who assisted Pedro Benoit with his plans for La Plata Cathedral, including the design for the Virgin's column, which is located in the garden surrounding the Cathedral. He also built the clubhouse of the La Plata Jockey Club. According to the La Plata Cemetery's Lichen samples were observed by stereoscopic and optical records, Domingo Lastra was simply an employee worker. How- microscopy and identified using a specialized classification sys- ever, his funerary monument is interesting not only because of its tem to the diagnostic characteristics of the different method of construction and the quality of the materials used, but species; growth form, color, thallus, size, surface structures (isidia, also because of its incomplete marble column, meant to symbolize

P.S. Guiamet et al. / Journal of Cultural Heritage 13 (2012) 339–344 Fig. 2. Biofilm on the marble column of crypt C95.
that Lastra was unable to complete his task in this world. Francisco Fig. 4. Scanning electron micrograph of a sample from crypt F87, showing the pres-
Arrechea was probably a prosperous merchant or landowner. His ence of inorganic and organic materials (fragment of an insect).
crypt is noteworthy because of its Art Nouveau style.
Lichens and dark biofilms were readily observed on crypts of historical and architectural interest at La Plata Cemetery. Biofilms in other monuments in La Plata, such as the Cathedral. However, were well established on crypts with E-SE exposures, as they other similarly constructed crypts exposed to the same conditions receive light for only a short time during the morning and are did not show signs of biodeterioration, most likely due to regular exposed to the rain and humid winds coming from the La Plata cleaning and maintenance.
River. In some cases, biofilms occurred under very specific condi- Analysis of the biofilms by scanning electron microscopy tions. For instance, on the column of crypt C95 (a biofilm had revealed both fungal hyphae and bacteria embedded in a matrix grown remarkably well on the south-facing aspect. A dark biofilm of extracellular polymeric substances. Moreover, bdelloid rotifers growing on crypt C77 was particularly prominent on the inner side were also found in the crypts (Gorbushina and Petersen of the marble flowerpot Crypt A9 was found to be in very that the remains of arthropod exoskeletons usually poor condition, obviously due to a lack of maintenance. Moreover, occurred in association with growing fungi. The authors concluded its location favored microbial colonization. Although this crypt is that the ingestion of fungal mycelium by arthropods, as a source situated in front of a small open square, the door faces south and of nutrition, is accompanied by the transportation of fungal spores the building is tucked between two other crypts, under the shade in their intestinal tracts. Subsequent excretion of the spore mass of a tree. The fac¸ade of crypt F87, which is covered in shade in the results in further contamination at sites where the excreted mate- afternoons, was colonized by lichens. The location of monuments rial is deposited. Moreover, arthropods can directly cause substrate in shady places facilitates the growth of lichens and other microor- deterioration, through bioabrasion.
ganisms by reducing exposure to the sun's harmful UV rays and by allowing the increased retention of water in the micropores of the edifice's substrate. This was seen even in south-facing crypts and Lichens identified as Caloplaca austrocitrina Vondrák, Riha, Arup and Søchting Xanthoria candelaria (L.) Th. Fries, Lecanora albescens (Hoffm) Branth and Rostr., and Xanthoparmelia farinosa were found on cement mortar, especially on crypt F87. Approx- imately 95% of the affected surface was covered by Caloplaca austrocitrina and less than 5% by other lichens. Lichens belonging to the Caloplaca genus are known to colonize natural stone. Nimis et al. Latium and Tretiach et al. Sardinia, identified about 300 different species of lichens on archaeological remains.
In that study, almost all Caloplaca sp. were found on calcareous rock with a SW, S, E, or SE exposure. The exception was Caloplaca ochracea, which occurred on northern exposures, specifically, in shaded sites near the soil. Caloplaca was also found to have colo- nized the carbonate rock (limestone) of the Jeronimos Monastery (Lisbon), where lichen thalli of Thyrea, Aspicilia, and Verrucaria were detected as well of Caloplaca sp., among others, were also identified on the pavement mosaics of the Romanesque town of Italica (Seville). Direct colonization of the tesserae was shown to have been preceded by colonization of the mortar Deru- elle et al. the nitrophiles Caloplaca citrina, Xanthoria Fig. 3. Dark biofilm on a marble flowerpot across from crypt C77.
candelaria, and Lecanora albescens on the Basilica of Notre-Dame de P.S. Guiamet et al. / Journal of Cultural Heritage 13 (2012) 339–344 Microorganisms found in various crypts at La Plata Cemetery (Argentina).
Total aerobic bacteria Acid-forming bacteria Crypt C95 (marble column) Crypt C77 (marble flower pot, internal side) Crypt A9 (side wall) L'Epine (a calcareous monument in Marne, France). The presence of also identified by Wollenzein et al. the causative agents of these species indicated that nutrient enrichment of the stone sub- the deterioration of marble and other calcareous materials used strate had favored the lichens' growth. Overall, the recent increase in the building of culturally important monuments in many local- in lichen colonization may be related to the widespread use of fer- ities in the Mediterranean area. Gaylarde and Gaylarde in a tilizers and above all to the newly implemented method of spraying comparative study of the microbial biomass of biofilms occurring by pulverization. This was reflected by the broader extent of lichen on the exteriors of buildings in Europe and Latin America, found populations, augmenting the disfigurement on the west-facing sur- that fungi, although not an important contributor to the biomass faces, which are more exposed to the dominant winds.
invading stone, seemed to preferentially colonize painted surfaces Bech-Andersen and Christensen that in cement- rather than other substrates (cement, mortar, concrete). Perfettini based materials the cement paste was preferentially attacked by et al. a strain of Aspergillus that produced gluconic and lichens, leaving the aggregates unaffected. More recent studies oxalic acids during the degradation of cement. After 8 months of showed that Caloplaca austrocitrina can penetrate both cement and contact with the substrate, these acids had induced the dissolution concrete. This species also produces oxalic acid, whose chemical of portlandite (without the leaching of calcium) and had increased action causes a loss of calcium from mortar substrates a the porosity of the cement while reducing its bending strength.
study of stone monuments in Rome, Seward the dif- In more recent laboratory studies, ordinary Portland cement ferential actions of lichen species in biodeterioration. Furthermore, pastes were severely attacked during bioleaching using Aspergillus not all species are harmful. While the sporadic growth of Lecanora niger cultures, especially by fungal biogenic organic (acetic, butyric, muralis has been related to the mechanical decay of the substrate, lactic, and oxalic) acids and to a lesser extent by respiration- species such as Lecanora dispersa, which also account for significant induced carbonic acid However, this study was conducted coverage, do not cause evident modifications. Monte under atypical conditions, in which large amounts of water were that Lecanora campestris and Lecanora rupicola preferentially grow present, and cannot be related to the conditions to which most on W to SW exposures whereas Xanthoria calcicola grows mainly buildings are exposed.
on south-facing surfaces. These preferences are consistent with The turgor pressure exerted by the hyphae of plant pathogens the fact that some species are stenoic, thriving only within a very such as the rice blast fungus Magnaporthe grisea has been calculated specific range of pH, humidity, luminosity, and nitrogen supply, on synthetic surfaces, including the plastic poly(vinyl chloride) while others are euroic and thus tolerant of a wider range of condi- measurement of turgor pressure indicated that the tions their role as primary colonizer seems doubtful, hyphae of this fungus can generate pressures in excess of 8.0 MPa, lichens nonetheless cause reduced substrate cohesion and there- which is sufficient to allow fungal penetration of marble (tension fore biocorrosion. In the crypts sampled in the present study, the resistance of 3.9 MPa), thereby causing mechanical damage at the surfaces were rough, eroded, and uneven due to the chemically targeted destruction of the cement matrix but not the aggregates, which were unaffected. Mechanical damage, by contrast, results from penetration of the substrate by lichen hyphae. The penetration depth of the thallus depends on the lichen species and the nature of Total aerobic bacteria and acid-forming bacteria counts are the substrate may also cause substrate damage by the shown in Based on these counts, cement mortar was production of acidic substances, such as carbon dioxide, lichenic more highly colonized than marble. From the isolated bac- acids, and oxalic acid. A full review of lichens as agents of damage teria, three isolates were selected for sequencing based on can be found in Lisci et al. the Gram staining and API results. These bacteria were sub- While chemolithotrophic microorganisms have often been sequently identified as Bacillus sp. and Pseudomonas sp. Their described in association with damaged inorganic materials, more sequences were deposited in GenBank under the accession num- recent studies have emphasized the significance of chemoorgan- bers JN837477 (97% similarity with JN208185), JN837478 (98% otrophic bacteria and fungi, together with photoautotrophs, as similarity with HM352366), and JN837479 (96% similarity with the primary colonizers of building stones. The activities of AY269246). Bacillus sp. (spore-forming bacteria) and Pseudomonas these primary colonizers precondition the building for attack by sp. (desiccation-tolerant bacteria) are able to survive in non- chemolithoautotrophs and thus initiate the process of biological favorable conditions and can be easily found in environmental Heterotrophic bacteria have frequently been isolated on stone monuments and are known to cause biodeterioration et al. several species of the genera Bacillus on weathered Aspergillus sp. and Penicillium sp. were the predominant groups sandstones of a church. Vuorinen et al. that Finnish probably because they are ubiquitous air-borne fungi and easily granite is slowly degraded by cultures of Pseudomonas aeruginosa, cultivable. Fusarium sp., Candida sp., and Rhodotorula sp. were also resulting in morphological alteration of the stone surface and the detected among the fungi that colonized La Plata Cemetery's mar- elution of minerals from the stone.
ble and cement mortar crypts. The fungal-mediated deterioration In the crypts examined in this study, acid-forming bacteria of stone was previously reviewed by May et al. were were isolated from marble and from cement mortar. These bacteria P.S. Guiamet et al. / Journal of Cultural Heritage 13 (2012) 339–344 are likely to play a role in the biodeterioration of these historical the material (discoloration, salt efflorescences, etc.) and should be monuments through the chemical and physical processes that are specific for the biodeteriorating organisms but not harmful to peo- integral to biofilm formation ple, other animals, or plants.
3.5. Bioreceptivity of the building materials According to Guillitte describes a material Mature biofilms cover crypts of historical and architectural that can be colonized by living organisms but without neces- interest at La Plata Cemetery (Argentina). These biofilms have sarily undergoing biodeterioration. The bioreceptivity of stone is caused the biodeterioration of monuments of different periods, determined by its structure and chemical composition, and the styles, and materials.
intensity of microbial contamination by climatic conditions and Lichens (Caloplaca austrocitrina, Lecanora albescens, Xan- anthropogenic eutrophication of the atmosphere thoparmelia farinosa, Xanthoria fallax), fungi (Aspergillus sp., given the same climate, the influence of the substrate on micro- Fusarium sp., Penicillium sp., Candida sp., Rhodotorula sp.), and bial colonization can be examined. Interestingly, our data ( bacteria (Bacillus sp., Pseudomonas sp.) were identified based on show that in spite of similarities in microclimate, microbial growth morphological and biochemical characteristics and in the case of on marble and cement mortar differed. While the cement mor- bacteria by rDNA sequencing as well. These organisms are not a tar was almost completely covered by lichens, the marble was complete list of those present on the crypts. Phototrophs (algae less colonized. In addition, bacterial and fungal growth on cement and cyanobacteria) were not sought, but there is no doubt that mortar was greater than on marble. A possible explanation for they are present as primary colonizers of building stones. The this is that cement mortar has a higher porosity than marble. In use of PCR-based molecular tools on fresh biofilms, rather than laboratory tests performed at the LEMIT ("Laboratorio de Entre- culture-dependent techniques, would have allowed many more namiento Multidisciplinario para la Investigación Tecnológica", La microorganisms to be detected.
Plata, Argentina), the water absorption rate of samples of Car- rara marble was 0.924% (indicating a low porosity), whereas that of cement mortar was 6–9% (indicating a high porosity). Thus, regardless of the heterogeneity of cement mortars in terms of their The authors thank the "Universidad Politécnica de Madrid" composition, cement/water relationship, and age, they are much (Spain) for its financial support (grants AL07-PID-020 and AL08- more porous than marble. This ability to absorb and retain rela- P (I + D)-08). P.S. Guiamet, V. Rosato, and S. Gómez de Saravia tively large amounts of water provides favorable growth conditions gratefully acknowledge the "Consejo Nacional de Investigaciones for microorganisms.
Científicas y Técnicas" (CONICET) and the "Universidad Nacional de La Plata" (UNLP) "Proyecto de Incentivos" 11 N 578.
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