Jörg Ettenauer

Halophilic microorganisms are responsible for the rosy discolouration of saline environments in three historical buildings with mural paintings

A number of mural paintings and building materials from monuments located in central and south Europe are characterized by the presence of an intriguing rosy discolouration phenomenon. Although some similarities were observed among the bacterial and archaeal microbiota detected in these monuments, their origin and nature is still unknown. In order to get a complete overview of this biodeterioration process, we investigated the microbial communities in saline environments causing the rosy discolouration of mural paintings in three Austrian historical buildings using a combination of culture-dependent and -independent techniques as well as microscopic techniques. The bacterial communities were dominated by halophilic members of Actinobacteria, mainly of the genus Rubrobacter. Representatives of the Archaea were also detected with the predominating genera Halobacterium, Halococcus and Halalkalicoccus. Furthermore, halophilic bacterial strains, mainly of the phylum Firmicutes, could be retrieved from two monuments using special culture media. Inoculation of building materials (limestone and gypsum plaster) with selected isolates reproduced the unaesthetic rosy effect and biodeterioration in the laboratory.

Microbes on building materials — Evaluation of DNA extraction protocols as common basis for molecular analysis

The study of microbial life in building materials is an emerging topic concerning biodeterioration of materials as well as health risks in houses and at working places. Biodegradation and potential health implications associated with microbial growth in our residues claim for more precise methods for quantification and identification. To date, cultivation experiments are commonly used to gain insight into the microbial diversity. Nowadays, molecular techniques for the identification of microorganisms provide efficient methods that can be applied in this field. The efficiency of DNA extraction is decisive in order to perform a reliable and reproducible quantification of the microorganisms by qPCR or to characterize the structure of the microbial community. In this study we tested thirteen DNA extraction methods and evaluated their efficiency for identifying (1) the quantity of DNA, (2) the quality and purity of DNA and (3) the ability of the DNA to be amplified in a PCR reaction using three universal primer sets for the ITS region of fungi as well as one primer pair targeting the 16S rRNA of bacteria with three typical building materials - common plaster, red brick and gypsum cardboard. DNA concentration measurements showed strong variations among the tested methods and materials. Measurement of the DNA yield showed up to three orders of magnitude variation from the same samples, whereas A260/A280 ratios often prognosticated biases in the PCR amplifications. Visualization of the crude DNA extracts and the comparison of DGGE fingerprints showed additional drawbacks of some methods. The FastDNA Spin kit for soil showed to be the best DNA extraction method and could provide positive results for all tests with the three building materials. Therefore, we suggest this method as a gold standard for quantification of indoor fungi and bacteria in building materials.

Molecular monitoring of the microbial dynamics occurring on historical limestone buildings during and after the in situ application of different bio-consolidation treatments

Microbially Induced Carbonate Precipitation is proposed as an environmentally friendly method to protect decayed ornamental stone and introduced in the field of preservation of Cultural Heritage. Recent conservation studies performed under laboratory conditions on non-sterile calcarenite stones have successfully reported on the application of a suitable nutritional solution, inoculated and non-inoculated with Myxococcus xanthus, as a bioconsolidation treatment. Furthermore, this procedure has been applied in situ, very recently, to selected historical buildings in Granada, Spain. For the first time, we evaluate the efficiency and risks of the in situ application of the above mentioned treatments onto two historical buildings in Granada. The evaluation consists of a detailed investigation of the micro-biota actively growing during the seven days of the treatments – short-term monitoring and of that remaining on the stones after six and twelve months of the application – long-term monitoring. A molecular strategy, including DNA extraction, PCR amplification of 16S rRNA sequences, construction of clone libraries and fingerprinting by DGGE (Denaturing Gradient Gel Electrophoresis) analysis followed by sequencing was used to gain insight into the microbial diversity present on the differentially treated stones. The monitoring of M. xanthus was performed by PCR using species-specific primers. Similar dynamics were triggered on both buildings by the application of the nutritional solution (inoculated or non-inoculated). 16S rDNA sequencing revealed the dominant occurrence of members belonging to the Firmicutes and Proteobacteria during the seven days of the treatment, whereas after one year the order Bacillales of the phylum Firmicutes was the predominantly detected microorganisms. M. xanthus could be detected only during the seven days of the treatment. The treatments seem to activate no dangerous microorganisms and furthermore, to select the remainder of a homogeneous group of carbonatogenic bacteria on the stones after a long period of time.

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.

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.

A Combined Approach to Assess the Microbial Contamination of the Archimedes Palimpsest

A combined approach, using molecular and microscopic techniques, was used to identify the microbiota associated with the Archimedes Palimpsest, an unusual parchment manuscript. SEM analyses revealed the microbial damage to the collagen fibers and the presence of characteristic cell chains typical of filamentous bacteria and fungal spores. Molecular analysis confirmed a homogeneous bacterial community colonizing the manuscript. The phyla Proteobacteria and Actinobacteria were associated with this ancient parchment; the sequences were most related to uncultured clones detected in the human skin microbiome and in ephitelium, and to cultivated species of the genera Acinetobacter and Nocardiopsis. Nevertheless, a great variation was observed among the different sampled areas indicating fungal diversity. Blumeria spp. dominated in the healthy areas of the parchment while degraded areas showed disparate fungal communities, with dominant members of the genera Mucor and Cladosporium. In addition, the quantification of the β-actin gene by real-time PCR analyses (qPCR) revealed a higher fungal abundance on degraded areas than on the healthy ones.

Quantification of fungal abundance on cultural heritage using real time PCR targeting the β-actin gene

The traditional methodology used for the identification of microbes colonizing our cultural heritage was the application of cultivation methods and/or microscopy. This approach has many advantages, as living microorganisms may be obtained for physiological investigations. In addition, these techniques allow the quantitative and qualitative assessment of the investigated environment. Quantitative analyses are done by plate count and the determination of abundance by the colony forming unit (CFU). Nevertheless, these techniques have many drawbacks that lead to an underestimation of the cell numbers and do not provide a comprehensive overview of the composition of the inhabiting microbiota. In the last decades, several molecular techniques have been developed enabling many advantages over the cultivation approach. Mainly PCR-based, fingerprinting techniques allow a qualitative detection and identification of the microbiota. In this study, we developed a real time PCR method as a simple, rapid and reliable tool to detect and quantify fungal abundance using the β-actin gene, which is known to appear as a single-copy gene in fungi. To this end, five different indoor thermal insulation materials applied for historical buildings that were previously tested for their bio-susceptibility against various fungi were subjected to qPCR analyses. The obtained results were compared with those obtained from a previous study investigating the bio-susceptibility of the insulation materials using classical cultivation experiments. Both results correlated well, revealing that Perlite plaster was the most suitable insulation material, showing the lowest fungal CFU and qPCR values. In contrast, insulations made of wood showed to be not recommendable from the microbiological point of view. In addition, the potential of qPCR was tested in other materials of cultural heritage, as old parchments, showing to be a suitable method for measuring fungal abundance in these delicate materials.