Rubrobacter-related bacteria associated with rosy discolouration of masonry and lime wall paintings

A molecular approach was chosen to analyse the correlation between bacterial colonisation and rosy discolouration of masonry and lime wall paintings of two historically important buildings in Austria and Germany. The applied molecular method included PCR amplification of genes encoding the small subunit rRNA of bacteria (16S rDNA), genetic fingerprinting by denaturing gradient gel electrophoresis (DGGE), construction of 16S rDNA clone libraries, and comparative phylogenetic sequence analyses. The bacterial community of one red-pigmented biofilm sampled in Herberstein (Austria) contained bacteria phylogenetically related to the genera Saccharopolyspora, Nocardioides, Pseudonocardia, Rubrobacter, and to a Kineococcus-like bacterium. The bacterial community of the second red-pigmented biofilm sampled in Herberstein contained bacteria related to Arthrobacter, Comamonas, and to Rubrobacter. Rubrobacter-related 16S rDNA sequences were the most abundant. In the red-pigmented biofilm sampled in Burggen (Germany), only Rubrobacter-related bacteria were identified. No Rubrobacter-related bacteria were detected in non-rosy biofilms. The majority of sequences (70%) obtained from the bacterial communities of the three investigated rosy biofilms were related to sequences of the genus Rubrobacter (red-pigmented bacteria), demonstrating a correlation between Rubrobacter-related bacteria and the phenomenon of rosy discolouration of masonry and lime wall paintings.     

Microbial Community Analysis of Fresh and Old Microbial Biofilms on Bayon Temple Sandstone of Angkor Thom, Cambodia

The temples of Angkor monuments including Angkor Thom and Bayon in Cambodia and surrounding countries were exclusively constructed using sandstone. They are severely threatened by biodeterioration caused by active growth of different microorganisms on the sandstone surfaces, but knowledge on the microbial community and composition of the biofilms on the sandstone is not available from this region. This study investigated the microbial community diversity by examining the fresh and old biofilms of the biodeteriorated bas-relief wall surfaces of the Bayon Temple by analysis of 16S and 18S rRNA gene sequences. The results showed that the retrieved sequences were clustered in 11 bacterial, 11 eukaryotic and two archaeal divisions with disparate communities (Acidobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Proteobacteria; Alveolata, Fungi, Metazoa, Viridiplantae; Crenarchaeote, and Euyarchaeota). A comparison of the microbial communities between the fresh and old biofilms revealed that the bacterial community of old biofilm was very similar to the newly formed fresh biofilm in terms of bacterial composition, but the eukaryotic communities were distinctly different between these two. This information has important implications for understanding the formation process and development of the microbial diversity on the sandstone surfaces, and furthermore to the relationship between the extent of biodeterioration and succession of microbial communities on sandstone in tropic region.     

Monitoring the colonization of monuments by bacteria: cultivation versus molecular methods

Building materials commonly used in wall paintings and monuments (mortar, limestone and sandstone) were inoculated with an artificial consortium composed of 14 microorganisms and incubated for 6 months at 28 degrees C. The colonization of the different materials by the consortium was investigated. Culture-independent techniques revealed the presence of a diversity of bacteria, whereas culture-dependent techniques yielded mainly spore-forming bacteria. The data suggest that plating leads to an overestimation of the number of spore-forming bacteria with respect to quiescent vegetative forms; the latter are less easily cultured, but are readily detected by culture-independent techniques.     

Occurrence of halotolerant/halophilic bacterial communities in deteriorated monuments

The use of traditional microbiological methods provides the isolation of a large number of heterotrophic bacteria on deteriorated monuments. However, the spectrum of isolated bacteria changed when the protocols used in studies of halophilic bacteria were applied to mural paintings, efflorescences or mineral deposits. In fact, enumeration of the heterotrophic viable bacteria indicates that the higher counts were, generally, obtained in media with 10% of salt concentration. Media with magnesium sulphate always yielded higher counts than sodium chloride, particularly in environments where magnesium salts were abundant. The predominance of bacilli communities was remarkable. Four cases, in search of halophilic bacteria, are described.     

Comparing bacterial community fingerprints from white colonizations in Altamira Cave (Spain)

Monitoring bacterial communities is critical for assessing biodeterioration among other processes. This study presents a strategy and an example of comparative analysis of bacterial communities developing in a cave environment, Altamira Cave which contains unique paleolithic paintings. The analyzed question was whether white colonizations discovered throughout the cave corresponded to similar or different bacterial communities. Molecular fingerprints were obtained by PCR–DGGE from DNA and RNA and statistically compared. Results based on DNA analysis showed that a similar bacterial community was present in white colonizations throughout the cave. Fingerprints based on RNA confirmed the similarity of the major metabolically active components of these communities. The proposed procedure confirmed that white colonizations in Altamira Cave were a consequence of the development of a single complex bacterial community, and the method proves to be highly useful for comparative analysis of microbial communities, including biodeteriorating processes and any other comparative analysis of bacterial communities.     

Metabolically active microbial communities of yellow and grey colonizations on the walls of Altamira Cave, Spain

Aims: To determine the major components of total and metabolically active microbial communities of yellow and grey colonizations threatening the conservation of palaeolithic paintings in Altamira Cave (Spain). Methods and Results: Micro‐organisms present in yellow and grey colonizations were determined from DNA analysis with those showing metabolic activity determined from RNA analysis. Microbial community fingerprints were obtained by denaturing gradient gel electrophoresis (DGGE) and 16S rDNA libraries were constructed from PCR amplified products. Proteobacteria was the most frequent bacterial phylum. Other phyla detected from RNA‐based microbial surveys were Acidobacteria, Actinobacteria, Firmicutes, Nitrospirae and Gemmatimonadetes. The detected metabolically active micro‐organisms represented only a fraction of the total bacterial community present in the studied colonizations as compared from DGGE analysis. Conclusions: The major bacterial participants in the development of yellow and grey colonizations in Altamira Cave were determined using RNA‐based molecular techniques. Micro‐organisms showing undetectable activity represent a potential risk for the conservation of these paintings if environmental conditions experience variations. Significance and Impact of the Study: Caves with palaeolithic paintings are affected by microbial deterioration. Assessing the composition of the microbial communities colonizing these caves represents a first stage to understand and control these colonizations.     

The bacterial aetiology of rosy discoloration of ancient wall paintings

The inventory of microorganisms responsible for biological deterioration of ancient paintings has become an integral part of restoration activities. Here, the microbial agent of rosy discoloration on medieval frescoes in the Crypt of the Original Sin (Matera, Italy) was investigated by a combination of microscopic, molecular and spectroscopic approaches. The bacterial community from three rosy-discoloured painting sites was characterized by 16S rRNA gene-based techniques. The eubacterial population was prevalently composed of Actinobacteria, among which Rubrobacter radiotolerans-related bacteria accounted for 63-87% of the 16S rRNA gene pool per sampled site. Archaea, with prevalence of Haloarchaea-related species, were detected in one of the three sites where they accounted for < 0.1% of the total 16S rRNA gene pool. Raman spectroscopy confirmed the identity between R. radiotolerans carotenoids (bacterioruberins) and pigments responsible for colour alteration of frescoes. This investigation provides the first evidence of a causal relationship between heavy contamination by Rubrobacter-related bacterioruberin-producing bacteria and rosy discoloration of ancient wall paintings.     

Microbial communities adhering to the obverse and reverse sides of an oil painting on canvas: identification and evaluation of their biodegradative potential

In this study, we investigated and compared the microbial communities adhering to the obverse and the reverse sides of an oil painting on canvas exhibiting signs of biodeterioration. Samples showing no visible damage were investigated as controls. Air samples were also analysed, in order to investigate the presence of airborne microorganisms suspended in the indoor atmosphere. The diversity of the cultivable microorganisms adhering to the surface was analysed by molecular techniques, such as RAPD analysis and gene sequencing. DGGE fingerprints derived from DNA directly extracted from canvas material in combination with clone libraries and sequencing were used to evaluate the non-cultivable fraction of the microbial communities associated with the material. By using culture-dependent methods, most of the bacterial strains were found to be common airborne, spore-forming microorganisms and belonged to the phyla Actinobacteria and Firmicutes, whereas culture-independent techniques identified sequenced clones affiliated with members of the phyla Actinobacteria and Proteobacteria. The diversity of fungi was shown to be much lower than that observed for bacteria, and only species of Penicillium spp. could be detected by cultivation techniques. The selected strategy revealed a higher microbial diversity on the obverse than on the reverse side of the painting and the near absence of actively growing microorganisms on areas showing no visible damage. Furthermore, enzymatic activity tests revealed that the most widespread activities involved in biodeterioration were esterase and esterase lipase among the isolated bacterial strains, and esterase and N-acetyl-β-glucosaminidase among fungi strains.     

Biodeterioration of mural paintings in a rocky habitat: The Crypt of the Original Sin (Matera, Italy)

The biological colonization of mural paintings in rocky habitats is a widely recurring phenomenon and should be considered for restoration activities. In order to plan conservation treatment of the Crypt of the Original Sin (Matera, Italy), biodeterioration problems were analysed taking into account their impact on the substrate and their relationship with environmental factors. The mural paintings showed a phenomenology of alteration varying from brilliant green, dark green, brown, and black duff patinas with powdery aspect, to rosy discoloration. These phenomena were linked to different microbial colonizations by Cyanobacteria (Chlorogloea microcystoides, Chroococcus lithophilus, Gloeocapsa spp., Gloeothece rupestris, Pseudocapsa dubia) and green algae (Apatococcus lobatus, Chlorella vulgaris, Chlorococcum sp., Muriella terrestris). The rosy discoloration was connected to pigment production by Actinobacteria related to Rubrobacter radiotolerans. Grey-green patinas, limited to more ventilated areas, were caused by lichen growth (Dirina massiliensis f. sorediata). Vascular plants (Adiantum capillus-veneris) were also detected in wall areas affected by rising damp. To control the growth of microflora, biocide treatment and intervention to reduce humidity and sunlight exposure of mural paintings were suggested. To choose the correct biocide, different products were tested, evaluating their efficiency on Cyanobacteria and algae as well as possible effects on the calcarenite stone.     

Phylogenetic analysis of bacterial populations in waters of the former Texcoco Lake, Mexico

Molecular techniques were used to compare the compositions of the bacterial communities of the 2 following lagoons from the former soda Texcoco Lake, Mexico: the restored Facultativa lagoon and the Nabor Carrillo lagoon. Ribosomal intergenic spacer analysis (RISA) revealed that bacterial communities of the 2 lagoons were different and presented a relatively low diversity. Clone libraries of 16S rDNA genes were constructed, and significant phylotypes were distinguished by restriction fragment length polymorphism (RFLP). A representative clone from each phylotype was partially sequenced. Molecular identification and phylogenetic analyses based on ribosomal sequences revealed that the Facultativa lagoon harbored mainly gamma- and beta-Proteobacteria, low G+C Gram-positive bacteria, and several members of the Halobacteriaceae family of archaea. The Nabor Carrillo lagoon mainly included typical halophilic and alkaliphilic low G+C Gram-positive bacteria, gamma-Proteobacteria, and beta-Proteobacteria similar to those found in other soda lakes. Several probably noncultured new bacterial species were detected. Three strains were isolated from the Nabor Carrillo lagoon, their partial 16S rDNA sequences were obtained. On this basis, they were identified as Halomonas magadiensis (H1), Halomonas eurihalina (H2), and Staphylococcus sciuri (H3). This is the first study that uses molecular techniques to investigate potential genetic diversity in the Texcoco lakes. In this preliminary evaluation, we infer the presence of alkalophilic, halophilic, or haloalkaliphilic bacteria potentially useful for biotechnology.     

Detection of indigenous Halobacillus populations in damaged ancient wall paintings and building materials: molecular monitoring and cultivation

Several moderately halophilic gram-positive, spore-forming bacteria have been isolated by conventional enrichment cultures from damaged medieval wall paintings and building materials. Enrichment and isolation were monitored by denaturing gradient gel electrophoresis and fluorescent in situ hybridization. 16S ribosomal DNA analysis showed that the bacteria are most closely related to Halobacillus litoralis. DNA-DNA reassociation experiments identified the isolates as a population of hitherto unknown Halobacillus species.     

Contribution of the Microbial Communities Detected on an Oil Painting on Canvas to Its Biodeterioration

In this study, we investigated the microbial community (bacteria and fungi) colonising an oil painting on canvas, which showed visible signs of biodeterioration. A combined strategy, comprising culture-dependent and -independent techniques, was selected. The results derived from the two techniques were disparate. Most of the isolated bacterial strains belonged to related species of the phylum Firmicutes, as Bacillus sp. and Paenisporosarcina sp., whereas the majority of the non-cultivable members of the bacterial community were shown to be related to species of the phylum Proteobacteria, as Stenotrophomonas sp. Fungal communities also showed discrepancies: the isolated fungal strains belonged to different genera of the order Eurotiales, as Penicillium and Eurotium, and the non-cultivable belonged to species of the order Pleosporales and Saccharomycetales. The cultivable microorganisms, which exhibited enzymatic activities related to the deterioration processes, were selected to evaluate their biodeteriorative potential on canvas paintings; namely Arthrobacter sp. as the representative bacterium and Penicillium sp. as the representative fungus. With this aim, a sample taken from the painting studied in this work was examined to determine the stratigraphic sequence of its cross-section. From this information, “mock paintings,” simulating the structure of the original painting, were prepared, inoculated with the selected bacterial and fungal strains, and subsequently examined by micro-Fourier Transform Infrared spectroscopy, in order to determine their potential susceptibility to microbial degradation. The FTIR-spectra revealed that neither Arthrobacter sp. nor Penicillium sp. alone, were able to induce chemical changes on the various materials used to prepare “mock paintings.” Only when inoculated together, could a synergistic effect on the FTIR-spectra be observed, in the form of a variation in band position on the spectrum.     

Skeleton bones in museum indoor environments offer niches for fungi and are affected by weathering and deposition of secondary minerals

Large skeleton specimens are often featured as iconic open displays in Natural History Museums, for example, the blue whale ‘Hope’ at the Natural History Museum, London. A study on Hope's bone surface was performed to assess the biodeterioration potential of fungi. Fungi were isolated, and a fungal internal transcribed spacer (ITS) clone library survey was performed on dust and bone material. Mineral particles derived from bone and dust were analysed using energy dispersive X-ray spectroscopy, variable pressure scanning electron microscopy (SEM) and high vacuum SEM. Results showed that bone material, although mainly mineral in nature, and therefore less susceptible than organic materials to biodeterioration phenomena in the indoor environments, offers niches for specialized fungi and is affected by unusual and yet not so well-documented mechanisms of alteration. Areas of bone surface were covered with a dense biofilm mostly composed of fungal hyphae, which produced tunnelling and extensive deposition of calcium and iron-containing secondary minerals. Airborne halophilic and xerophilic fungi including taxa grouping into Ascomycota and Basidiomycota, capable of displacing salts and overcome little water availability, were found to dominate the microbiome of the bone surface.     

Detection of Indigenous Halobacillus Populations in Damaged Ancient Wall Paintings and Building Materials: Molecular Monitoring and Cultivation

Several moderately halophilic gram-positive, spore-forming bacteria have been isolated by conventional enrichment cultures from damaged medieval wall paintings and building materials. Enrichment and isolation were monitored by denaturing gradient gel electrophoresis and fluorescent in situ hybridization. 16S ribosomal DNA analysis showed that the bacteria are most closely related to Halobacillus litoralis. DNA-DNA reassociation experiments identified the isolates as a population of hitherto unknown Halobacillus species.     

Microbial communities on the monuments of Moscow and St. Petersburg: biodiversity and trophic relations

Stone monuments situated in the Alexander Nevsky Abbey, the Summer Garden, and the Smolenskoe Cemetery, St. Petersburg, and marble and limestone sculptures and tombstones situated in the Novodevichy Convent and the Donskoy Monastery, Moscow, were investigated for their microbial contamination. The architectural objects studied date to the 12th century. The monuments in the Alexander Nevsky Abbey were found to be severely contaminated with micromycetes belonging to 24 genera (primarily of the class Deuteromycetes). The analysis of the samples taken from the monuments of the Donskoy Monastery by the serial dilution technique showed that they were contaminated with bacteria at a density of (1-1.7) x 10(5) cells/g. This value, however, turned out to be 1 to 2 orders greater when the bacterial population was evaluated by the luciferin-luciferase method. We succeeded in identifying 12 bacterial genera; however, this number may be increased in the course of further studies. Some preventive measures to control the biodeterioration of stone heritage are discussed.     

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.     

Microbial Communities on the Monuments of Moscow and St. Petersburg: Biodiversity and Trophic Relations

Stone monuments situated in the Alexander Nevsky Abbey, the Summer Garden, and the Smolenskoe Cemetery, St. Petersburg, and marble and limestone sculptures and tombstones situated in the Novodevichy Convent and the Donskoy Monastery, Moscow, were investigated for their microbial contamination. The architectural objects studied date back to the 12th century. The monuments in the Alexander Nevsky Abbey were found to be severely contaminated with micromycetes belonging to 24 genera (primarily of the class Deuteromycetes). The analysis of the samples taken from the monuments of the Donskoy Monastery by the serial dilution technique showed that they were contaminated with bacteria at a density of (1–1.7) × 10⁵ cells/g. This value, however, turned out to be 1 to 2 orders greater when the bacterial population was evaluated by the luciferin–luciferase method. We succeeded in identifying 12 bacterial genera; however, this number may be increased in the course of further studies. Some preventive measures to control the biodeterioration of stone heritage are discussed.     

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.     

Bacterial community structure and function in a metal-working fluid

The diversity of bacterial populations colonizing spatially and temporally separated samples of the same metal-working fluid (MWF) formulation was investigated. Analyses were performed with a view to improve strategies for bioaugmentation of waste MWF in bioreactor systems and prevention of in-use MWF biodeterioration in engineering workshops. Significantly, complementary phenotypic, genotypic and in situ methods revealed that the bacterial communities in operationally exhausted MWFs had low diversity and were similar in species composition from different locations and uses. Of the 179 bacterial isolates studied, only 11 genera and 15 species were identified using fatty acid methyl ester (FAME) analysis, with culture independent analyses by 16S rDNA denaturing gradient gel electrophoresis (DGGE) and fluorescent in situ hybridization being congruent with these FAME data. In order to gain some insight into functional role of detected populations, we correlated the MWF chemical composition and potential pollution load with bacterial abundance and community composition detected within samples.