Cyanobacteria cause black staining of the National Museum of the American Indian Building, Washington, DC, USA

Microbial deterioration of stone is a widely recognised problem affecting monuments and buildings all over the world. In this paper, dark-coloured staining, putatively attributed to microorganisms, on areas of the National Museum of the American Indian Building, Washington, DC, USA, were studied. Observations by optical and electron microscopy of surfaces and cross sections of limestone indicated that biofilms, which penetrated up to a maximum depth of about 1?mm, were mainly composed of cyanobacteria, with the predominance of Gloeocapsa and Lyngbya. Denaturing gradient gel electrophoresis analysis revealed that the microbial community also included eukaryotic algae (Trebouxiophyceae) and fungi (Ascomycota), along with a consortium of bacteria. Energy-dispersive X-ray spectroscopy analysis showed the same elemental composition in stained and unstained areas of the samples, indicating that the discolouration was not due to abiotic chemical changes within the stone. The dark pigmentation of the stone was correlated with the high content of scytonemin, which was found in all samples.     

Changes in the biofilm microflora of limestone caused by atmospheric pollutants

Historic limestone materials in urban environments are continually exposed to air pollutants, including sulfur compounds and hydrocarbons. We investigated the effects of air pollution on the biofilm microflora of historic limestone gravestones located at two locations Massachusetts, USA. Our data showed that the culturable populations of chemolithotrophic and heterotrophic bacteria, and fungi were suppressed in the polluted habitat comparing with the unpolluted location. The diversity of the microflora was also reduced in the surface biofilms on gravestones in the city contaminated by air pollution. However, both the sulfur-oxidizing and hydrocarbon-utilizing microflora were enriched in the biofilms exposed to air pollution. In a laboratory study, low concentrations of the polluting chemicals stimulated growth of these bacteria, and resulted in rapid acid production. Scanning electron microscopy demonstrated that the biofilms of both the sulfur-oxidizing bacteria and the hydrocarbon-degrading microflora penetrated into the limestone. The enrichment of sulfur- and hydrocarbon-utilizing bacteria in the biofilms may contribute to dissolution of the stone. However, further research is required to determine the effects of specific metabolites of these microorganisms on stone deterioration.     

Oxalate patinas on ancient monuments: the biological hypothesis

Summary Whewellite and weddellite, calcium oxalate monohydrate and dihydrate respectively, have been found in the form of thin surface layers on limestone and marble monuments and artifacts of various historical periods at different sites. Experimental results indicate that the formation of both minerals must be attributed essentially to the action of oxalic acid secreted by microorganisms (lichens) which live and proliferate on the stone. Oxalic acid attacks the calcium carbonate of the stone surface giving rise to the precipitation of calcium oxalate.     

Biodeterioration of Mayan archaeological sites in the Yucatan Peninsula, Mexico

Many of the monuments of the Mayan civilization are suffering deterioration caused by environmental factors (high temperatures and relative humidities), increasing contamination from natural and anthropogenic sources, and by the action of micro- and macro-biological communities. Archaeological sites and historical monuments in the Mayan area were constructed with different limestones which offer different resistances to degradation by the various types of contamination. Two different sampling sites were chosen at the archaeological site of Uxmal in the Yucatan Peninsula, Mexico. Heterotrophic bacteria, cyanobacteria and different fungi were isolated and classified taxonomically. The other archaeological site chosen for this study was the fortress of Tulum, located at the side of the Caribbean Sea and exposed to chloride of marine spray and sand erosion. In this case, heterotrophic aerobic and anaerobic bacteria, cyanobacteria and fungi were isolated from the four sampling areas selected. In both archaeological sites crust deposits were observed by using light microscopy, SEM and ESEM. Surface analyses were made by means of EDAX and electron microprobe. Possible mechanisms of stone decay, based on the type of microorganisms isolated, the physico-chemical characteristics of the constructional materials and environmental factors are discussed.     

Surface analysis and materials characterization for the study of biodeterioration and weathering effects on cultural property

Several methods for material characterization and surface analysis such as scanning electron microscopy (SEM), energy dispersion X-ray analysis (EDX), environmental scanning electron microscopy (ESEM), petrographic analyses, Mössbauer spectroscopy (MS), conventional X-ray diffraction (XRD), grazing incidence diffraction (GID), Raman spectroscopy (RS), other spectroscopic techniques like X-ray photoelectron spectroscopy (XPS), reflection electron energy-loss spectroscopy (REELS) and advanced combined applications of synchrotron based μ-X-ray diffraction/μ-X-ray fluorescence (SR-μXRD/μXRF) can be used for assessing weathering and biodeterioration effects on materials (such as stone buildings, metallic artefacts, pigments, mixtures, and processes) of cultural property. Molecular biology techniques to identify the microbial components of biofilms are also described. Different examples of the use of these methods in the field of cultural property preservation are presented.     

Biological nanosilver particles for the protection of archaeological stones against microbial colonization

The inhabitation of microorganisms and their subsequent interaction with mineral matrix of the stone substrate under varied environmental conditions encourages deterioration of stones leading to the loss of strength, durability and aesthetic. This study highlighted the synthesis of nanosilver particles (AgNPs) using the biogenic volatiles of the bacterial strain Nesterenkonia halobia. The antimicrobial activities of AgNPs were evaluated against the gram positive bacterial strain Streptomyces parvullus and fungal strain Apergillus niger. Furthermore, the silver particles were mixed with two types of consolidation polymers and were used to coat the external surfaces of sandstone and limestone blocks. The stones treated with silicon polymer loaded with AgNPs showed an elevated antimicrobial potentiality against A. niger and S. parvullus. Scan electron microscope (SEM) and electron dispersive X-ray spectroscopy (EDX) analysis of treated stones demonstrated the existence of nano-composite structures containing the elemental silver. Polymers functionalized with AgNPs can be used not only as potent biocides but also for the consolidation of the historic monuments and artifacts.     

Algal and Cyanobacterial Biofilms on Calcareous Historic Buildings

Major microorganisms in biofilms on external surfaces of historic buildings are algae, cyanobacteria, bacteria, and fungi. Their growth causes discoloration and degradation. We compared the phototrophs on cement-based renderings and limestone substrates at 14 historic locations (47 sites sampled) in Europe and Latin America. Most biofilms contained both cyanobacteria and algae. Single-celled and colonial cyanobacteria frequently constituted the major phototroph biomass on limestone monuments (32 sites sampled). Greater numbers of phototrophs, and especially of algae and of filamentous morphotypes, were found on cement-based renderings (15 sites), probably owing to the porosity and small pore size of the latter substrates, allowing greater entry and retention of water. All phototrophic groups were more frequent on Latin American than on European buildings (20 and 27 sites, respectively), with cyanobacteria and filamentous phototrophs showing the greatest differences. The results confirm the influence of both climate and substrate on phototroph colonization of historic buildings. Received: 7 March 2002 / Accepted: 8 April 2002     

Biodecay of cultural heritage as a space/time-related ecological situation — an evaluation of a series of studies

Mural paintings and stone monuments are exposed to natural and man-made hazards for time spans sometimes longer than 10,000 years without total decay. On the other hand, a high percentage of the mineral physical heritage suffers severe damage and even total loss within a few decades. Several factors influence this general situation. The mural paintings of Lascaux and Altamira had been stabilized in a protective environment for millennia until they were re-exposed to severe environmental risks. Wall paintings and stone sculptures may remain unaltered for many years through: (1) stabilized and protective environments, (2) development of a protective patina (biofilm, minerals, organics) or, (3) more recent additional paint or mortar layers, which themselves are exposed to environmental hazard instead of the fresco underneath. Natural or artificial coverage, however, will invariably hinder evaluation and appreciation of the object of art. Detrimental microorganisms often only sporadically interact with these precious paintings. Many microbes settling on and in mural paintings are of a unusual metabolic type, namely poikilotrophic organisms. These poikilotrophs are capable of forming a biocoenosis on and in mural paintings, which may establish itself within a few months or years and then remains unchanged in a dormant or non-cultivable state for many years, or even centuries. The same holds true for chemical and physical alterations brought about by microorganisms. After a damaging initiation period of growth, the microflora may turn into a biofilm, which continues to live at extremely low levels of metabolic activity. In this way, detrimental chemical, physical and biological activities may be screened or shielded off for a very long period of time. In order to understand these processes, we have studied poikilotroph sub-aerial biofilms of mural paintings and sculptures excavated after long periods of dormancy. Several methods for the characterization of the physical properties of the colonies of fungi involved in colonization and deterioration of mural paintings and stone monuments are described. The fairy-tale of “The Sleeping Beauty” maybe an excellent metaphor for this phenomenon. The main issue, however, is to understand the physical or mechanical impact of the poikilotroph flora on the materials of the physical heritage studied.     

Bioremediation of weathered-building stone surfaces

Atmospheric pollution and weathering of stone surfaces in urban historic buildings frequently results in disfigurement or damage by salt crust formation (often gypsum), presenting opportunities for bioremediation using microorganisms. Conventional techniques for the removal of these salt crusts from stone have several disadvantages: they can cause colour changes; adversely affect the movement of salts within the stone structure; or remove excessive amounts of the original surface. Although microorganisms are commonly associated with detrimental effects to the integrity of stone structures, there is growing evidence that they can be used to treat this type of stone deterioration in objects of historical and cultural significance. In particular, the ability and potential of different microorganisms to either remove sulfate crusts or form sacrificial layers of calcite that consolidate mineral surfaces have been demonstrated. Current research suggests that bioremediation has the potential to offer an additional technology to conservators working to restore stone surfaces in heritage buildings.     

“In vitro” biofilm formation by Penicillum frequentans strains on sandstone,granite, and limestone

The appearance of crusts or patinas on surfaces of stone monuments are indicative signs of weathering. In many cases, microorganisms are mainly responsible for stone decay, giving rise to the formation of characteristic biodeteriorative patinas, called biofilms. In the present work, in vitro biofilm formation on sandstone, limestone, and granite block samples by Penicillium frequentans strains isolated from two Spanish cathedrals is demonstrated. Spore suspensions of P. frequentans strains were inoculated on each block sample of unaltered stone material cited above. Biofilms of 1–2 mm thickness were formed on each of the three rock samples, and analysed by means of Fourier-transform infrared spectroscopy, scanning electron microscopy and X-ray mapping (energy-dispersive X-ray spectrometry, KEVEX system). In the three cases, biofilms were principally composed of: fungal mycelium, mineral particles released from the stony substratum, and newly formed organic salts such as oxalate and citrate. These biofilms enhance and accelerate the deteriorative process of rocks due to the loss of stone material (biopitting and mineral grains captured by mycelium) and to alteration of the mineral crystalline networks (cation release by organic acids). Correspondence to: G. Gómez-Alarcón     

Deterioration of limestone walls in Jerusalem and marble monuments in Rome caused by cyanobacteria and cyanophilous lichens

Hard limestone walls in Jerusalem and marble monuments in Rome were studied. An attempt was made to classify the principal biodeterioration processes in walls facing the prevailing wind and those which were protected from it. In Jerusalem, those that face the prevailing rain-bearing wind become populated by cyanobacteria and cyanophilous lichens. Within c. 100 years white walls are turned to grey or black colour by these organisms. Rain detaches rock particles from the vicinity of the microorganisms at a rate of 1 mm in 200 years. Dark patches on marble monuments in Rome indicate the presence of cyanobacteria. Such biological activity promotes detachment of particles by rain and accelerates weathering leading to pit formation. The estimated rate of pitting is 1 mm per 40 years in the Forum Traianum. To prevent the biodeterioration of limestone and marble monuments the habitats and ecological demands of the various microorganisms must be considered.     

Growth of phototrophic biofilms from limestone monuments under laboratory conditions

In the current study, five phototrophic biofilms from different Southern Europe limestone monuments were characterised by molecular techniques and cultivated under laboratory conditions. Phototrophic biofilms were collected from Orologio Tower in Martano (Italy), Santa Clara-a-Velha Monastery and Ajuda National Palace, both in Portugal, and Seville and Granada Cathedrals from Spain. The biofilms were grown under laboratory conditions and periodically sampled in order to monitor their evolution over a three-month period. Prokaryotic communities from natural samples and cultivated biofilms were monitored using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments in conjunction with clone sequencing and phylogenetic analysis. DNA-based molecular analysis of 16S rRNA gene fragments from the natural green biofilms revealed complex and different communities composition with respect to phototrophic microorganisms. The biofilms from Orologio Tower (Martano, Italy) and Santa Clara-a-Velha Monastery (Coimbra, Portugal) were dominated by the microalga Chlorella. The cyanobacterium Chroococcidiopsis was the dominating genus from Ajuda National Palace biofilm (Lisbon, Portugal). The biofilms from Seville and Granada Cathedrals (Spain) were both dominated by the cyanobacterium Pleurocapsa. The DGGE analysis of the cultivated biofilms showed that the communities developed differently in terms of species establishment and community composition during the three-month incubation period. The biofilm culture from Coimbra (Portugal) showed a remarkable stability of the microbial components of the natural community in laboratory conditions. With this work, a multiple-species community assemblage was obtained for further stone colonisation experiments.     

The ATP-bioluminescence method for a rapid evaluation of the microbial activity in the stone materials of monuments

The examination of the state of conservation of works of art in stone includes the assessment of the presence of microbiological agents on the surface of the decayed monuments. These microorganisms can accelerate, via their metabolic activity, the decay process of the stone surface. At present this assessment is made with the traditional techniques for the microbiological examination of the soil, provides results only after a delay of 30 days. A bioluminescent ATP assay should provide rapid quantitation of actively growing organisms on the surface of a stone monument, and the applicability of this technique was verified on some samples of sandstone (Pietraforte) collected from a historic building (the Strozzi Palace) in Florence. These samples were evaluated for the amount of the ATP and the total number of microorganisms. The results obtained suggest that the bioluminescent assay could be suitable for detecting and quantitating the presence of microorganisms in a sample of stone.     

Contributions of in situ microscopy to the current understanding of stone biodeterioration.

In situ microscopy consists of simultaneously applying several microscopy techniques without separating the biological component from its habitat. Over the past few years, this strategy has allowed characterization of the biofilms involved in biodeterioration processes affecting stone monuments and has revealed the biogeophysical and biogeochemical impact of the microbiota present. In addition, through in situ microscopy diagnosis, appropriate treatments can be designed to resolve the problems related to microbial colonization of stone monuments.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.     

Enhanced visualization of microbial biofilms by staining and environmental scanning electron microscopy

Bacterial biofilms, i.e. surface-associated cells covered in hydrated extracellular polymeric substances (EPS), are often studied with high-resolution electron microscopy (EM). However, conventional desiccation and high vacuum EM protocols collapse EPS matrices which, in turn, deform biofilm appearances. Alternatively, wet-mode environmental scanning electron microscopy (ESEM) is performed under a moderate vacuum and without biofilm drying. If completely untreated, however, EPS is not electron dense and thus is not resolved well in ESEM. Therefore, this study was towards adapting several conventional SEM staining protocols for improved resolution of biofilms and EPS using ESEM. Three different biofilm types were used: 1) Pseudomonas aeruginosa unsaturated biofilms cultured on membranes, 2) P. aeruginosa cultured in moist sand, and 3) mixed community biofilms cultured on substrates in an estuary. Working with the first specimen type, a staining protocol using ruthenium red, glutaraldehyde, osmium tetroxide and lysine was optimized for best topographic resolution. A quantitative image analysis tool that maps relief, newly adopted here for studying biofilms, was used to compare micrographs. When the optimized staining and ESEM protocols were applied to moist sand cultures and aquatic biofilms, the smoothening effect that bacterial biofilms have on rough sand, and the roughening that aquatic biofilms impart on initially smooth coupons, were each quantifiable. This study thus provides transferable staining and ESEM imaging protocols suitable for a wide range of biofilms, plus a novel tool for quantifying biofilm image data.     

Comparative studies of bacterial biofilms on steel surfaces using atomic force microscopy and environmental scanning electron microscopy

Environmental scanning electron microscopy (ESEM) and atomic force microscopy (AFM) were compared as tools for the observation of bacterial biofilms developed on carbon steel and AISI 316 stainless steel surfaces under stagnant conditions. Biofilms were generated in batch cultures of two different isolates of marine sulphate reducing bacteria (SRB) and in cultures consisting of mixed populations of acidophilic bacteria, known as "acid streamers";. Imaging of single SRB cells on mica was also carried out to reveal the surface topography of individual bacterial cells at nanometre resolution. Following the removal of biofilms, the stainless steel surfaces were profiled using AFM to determine the degree of steel deterioration. ESEM and AFM studies of bacterial biofilms in-situ, gave both qualitative and quantitative information on biofilm structure at high resolution. The use of AFM image analysis software allowed estimation of the width and height of bacterial cells, the thickness and width of exopolymeric (EPS) capsule and bacterial flagella, as well as characterisation of the surface roughness of the steel, including measurements of depth and diameter of individual pits. Exposure of stainless steel specimens to acid streamers resulted in a significant increase in the surface roughness of the steel, compared to specimens placed in sterile medium.     

Calcium oxalate films on stone monuments — Microbiological investigations

Summary The origin of calcium oxalate films on stone monuments is investigated and discussed in relation to atmosphere-polluting microorganisms that, under certain conditions, can produce oxalic acid. A list of the fungi microorganisms that are presumably involved in the formation of oxalate films is reported.     

Characterization of cyanobacteria isolated from biofilms on stone monuments at Santiniketan,India

Cyanobacterial biofilms occurring on the exterior of three stone monuments at Santiniketan, India were analyzed. Species of Scytonema and Tolypothrix were the major components of these biofilms. Identification was obtained by morphometric procedures and 16S rRNA gene sequencing. Biofilms cultured for prolonged periods revealed the presence of several other cyanobacteria belonging to 14 different genera. Cyanobacteria on stone in the tropical environment of India formed a distinct cluster that was quite different from that of cyanobacteria reported for a similar substratum in temperate regions. Absorption spectra of the organisms from Santiniketan showed a high quantity of scytonemin, mycosporine-like amino acids, and carotenoids. All of the organisms survived in a desiccated state and rapidly revived after wetting. The organisms were heterocystous and nitrogenase activity was reactivated within 24?h of wetting by which time heterocysts in their filaments had also appeared.     

Composition and Distribution of Elements and Ultrastructural Topography of a Human Cardiac Calculus

Trace elements (TEs) may contribute to the formation of calculi or stones or be involved in the aetiopathogenesis of stone diseases. The compositions and spatial distribution of elements from the inner nucleus to outer crust of the cardiac calculus were investigated by energy-dispersive X-ray fluorescence (EDXRF) spectrometer. The surface topograph, distribution map of elements, elemental and chemical compositions were also determined by environmental scanning electron microscope (ESEM)–energy-dispersive X-ray (EDX) analysis. Twenty-five elements were identifiable from 18 positions on the cardiac calculus by EDXRF spectrometer, in which the highest concentrations of toxic TEs (Ni, Pt, Hg, Sn, Pb, W, Au, Al, Si) and higher levels of essential TEs (Ca, Sr, Cr, P) were detected. A moderate positive Pearson’s correlation between TEs concentrations of Mg, Ca or P and location differences from centre to periphery in the cardiac calculus was observed. A positive correlation was also found for Ca/Zn and Ca/Cu, indicating the gradual increase of calcium concentration from inner nucleus to outer crust of cardiac calculus. The drop-like nodules/crystals on the surface of petrous part of cardiac calculus were observed from ESEM analysis. ESEM–EDX analysis determined the calculus to be predominantly composed of calcium hydroxyapatite and cholesterol, as indicated by the petrous surface and drop-like nodules/crystals, respectively. This composition was confirmed using a portable Raman analyser. The spatial distribution analysis indicated a gradual increase in Mg, P and Ca concentrations from the inner nucleus to the outer crust of the cardiac calculus. The major chemical compositions of calcium hydroxyapatite and cholesterol were detected on this cardiac calculus.