José Mirão

Microbial communities analysis assessed by pyrosequencing--a new approach applied to conservation state studies of mural paintings.

The knowledge about the microbial communities present in mural paintings is of utmost importance to develop effective conservation and mitigation strategies. The present paper describes a methodological approach for the detailed characterisation of microorganisms thriving in mural paintings by combining culture-dependent methods that allow the identification of microorganisms capable of growing in the laboratory conditions and to obtain high cell densities for further studies, and culture independent methods, such as denaturing gradient gel electrophoresis (DGGE) and pyrosequencing. The coupled use of culture-dependent methods and DGGE does not give enough information to investigate the diversity and abundance of microorganisms present in wall paintings. Pyrosequencing, a novel molecular technique, used here for the first time in this area of research, allowed the identification of a large number of microorganisms, confirming some already identified by the cultivation-dependent methods such as fungi of the genera Penicillium and Cladosporium, but also providing a great contribution in the identification of several genera and species, not previously identified in these artworks, giving also a detailed overview of contaminants which was not possible with the other approaches. The results obtained on several mural painting samples show a strong relationship between the most deteriorated areas of the paintings and higher microbial contamination.

Weathering of S. Domingos (Iberian Pyritic Belt) abandoned mine slags

Abstract The S. Domingos abandoned mine is a major source of pollutant-metals contamination. Slags from the 19th-20th century contain the most hazardous material. However, the release of pollutant metals depends on the metal-retention capability of weathering products. This work shows that Zn is not effectively retained by these weathering products while As and Pb are partially stored in sulphates. Copper, on the other hand, has a different behaviour and is the most leachable of the elements studied.

Material Characterization and Biodegradation Assessment of Mural Paintings: Renaissance Frescoes from Santo Aleixo Church, Southern Portugal

The aim of this work is the material characterization of wall paintings and biodegradation assessment, including the analysis of microbial growth and the effect of microbial proliferation, in view of their conservation. The methodology was applied to the study of frescoes dated from 1531, located in the ancient parish church of Santo Aleixo, Southern Portugal. The combined use of optical microscopy, scanning electron microscopy coupled with energy dispersive X-ray spectrometry (SEM-EDS) and μ- X-ray diffraction (XRD) showed that the painting palette is composed of red and yellow ochers, malachite, azurite, and bone black. The pigments do not show signs of chemical deterioration, except for malachite, which transformed to black copper oxide (tenorite). The microbiological study allowed the identification of several bacterial strains (e.g., Gram+ cocci, Gram+ bacilli, Actinomycetes sp.), yeast strains, and filamentous fungi of the genera Penicillium, Cladosporium and Aspergillus, among others of the microflora present in the paintings. Their metabolic activity is primarily responsible for the physical disruption of paint layers and underlying mortars. The combined approach using SEM analysis and enzymatic dehydrogenase measurement allowed the evaluation of microflora proliferation and diagnosis of the biodeterioration of the mural paintings. Additionally, the effect of some commercial biocides was evaluated for the predominant strains in order to select the most efficient biocide.

Magnetic fingerprint of southern Portuguese speleothems and implications for paleomagnetism and environmental magnetism

Environmental magnetism of speleothems is still in its early stage of development. Here we report on our investigation of the environmental and paleomagnetic information that has been recorded in speleothems, and what are the factors that control its preservation and reliability. To address these issues, we used a multidisciplinary approach, including rock magnetism, petrography, scanning electron microscopy, stable carbon and oxygen isotope compositions, and major and trace element concentrations. We applied this to a set of samples from different stages of speleothem evolution: present-day dripwater (glass plates), a weathered stalactite, a fresh stalagmite, cave sediments, and terra rossa soils. These samples come from the Penico and Excentricas caves, located in two distinct aquifers of the Algarve region, South Portugal. Our results show that the main magnetic carriers of the speleothems under study are primary (detrital) and consist of maghemite (and magnetite?). Similarities in coercivity and temperature dependence of the studied set of samples suggest that iron oxides are inherited from the terra rossa soils that cap the cave and were transported to the speleothems by dripwater. Hence, they represent a regional environmental signature. Interestingly, a stable and probably detrital remanent magnetization could be isolated in the fresh stalagmite, whereas the weathered stalactite yielded chaotic magnetic directions and very low remanent intensities. We propose that these low intensities can be the result from (i) different remanence acquisition mechanisms between stalagmite and stalactite and/or (ii) iron dissolution by fungal activity. We also suggest that magnetic properties and color and the content in detrital elements in the fresh speleothem inform about environmental processes acting on the interface of rock (soil)-atmosphere, while oxygen isotope composition and alkaline-earth element concentrations inform about calcite-water interaction processes. These results provide a better understanding of how environmental information is recorded in speleothems and what the factors are that control the reliability of the paleomagnetic and paleo-environmental signal.

Blue Pigment Colors from Wall Painting Churches in Danger (Portugal 15th to 18th Century): Identification, Diagnosis, and Color Evaluation

Samples of blue wall paint layers from selected 15th to 18th century religious mural paintings from southern Portugal (Alentejo) have been analyzed using a multi-analytical methodology involving the combination of in situ visible spectro-colorimetry with microanalytical techniques such as optical and scanning electron microscopy and Raman spectroscopy. In situ analyses and micro-sampling were carried out in nine different churches, many in an advanced state of deterioration. The objectives of this study were: (a) to identify and compare the pigments that were used in the blue paint layers across the Alentejo region and through time by analysis of the elemental and mineralogical composition and pictorial techniques, and (b) to correlate the data between the actual color of the paint layer and the state of conservation of the pigments. For the paintings dated from the 16th century forward, the results show a generalized use of smalt blue. To a lesser extent, natural azurite was used despite the geological richness of the region in copper and pyrite ores. In only one painting was an optical blue made of carbon black and lime found. The pigments, pure or mixed with red and yellow ochres, were coarsely ground and used in different concentrations to create three-dimensional effects. These parameters as well as the presence of iron oxides in underlayer paints influence the colorimetric coordinates in the more transparent smalt blue paint layers. The state of conservation of the pigments plays an important role in the alteration of the paint color. A clear example of this is the fading of the smalt blue in several paintings due to lixiviation processes.

On the Use of the Unusual Green Pigment Brochantite (Cu 4 (SO 4 )(OH) 6 ) in the 16th-Century Portuguese- Flemish Paintings Attributed to The Master Frei Carlos Workshop

This paper reports an unusual green pigment, brochantite (Cu4(SO4)(OH)6), on 16th-century Portuguese-Flemish paintings, attributed to the Master Frei Carlos workshop. This green mineral is usually identified as an impurity or alteration product in the green pigments verdigris (Cu(CH3COO)2 nCu(OH)2) or malachite (CuCO3 Cu(OH)2). However, after thorough investigation with a broad range of analytical techniques, it became clear that, in this case, brochantite was applied as a pigment. The abundance, pigment granulometry, and pigment morphology suggest intentional use by this Portuguese-Flemish Master as a natural pigment rather than its accidental use as an alteration product. This seems to be a distinguishable feature to other painters (Flemish and Portuguese) working in Portugal at the beginning of the 16th century. The multi-analytical study of these easel paintings was first performed by physical imaging techniques and material characterization was carried out by optical microscopy, micro-Fourier-transform infrared-spectroscopy, micro-Raman spectroscopy, scanning electron microscopy coupled with energy dispersive X-ray spectrometry, and micro-X-ray diffraction analysis (XRD).

Study of mural paintings using in situ XRF, confocal synchrotron-μ-XRF, μ-XRD, optical microscopy, and SEM-EDS--the case of the frescoes from Misericordia Church of Odemira.

In this work, we present the results of an analytical method developed for detailed pigment identification, stratigraphy, and degradation of the paint layers of mural paintings applied in the study of the 17th century frescoes from the Misericordia Church of Odemira (Southwest Portugal). In situ X-ray fluorescence spectrometry analyses were performed on three panels of the mural paintings and complemented by colorimetric measurements. The different color areas were also sampled as microfragments (approx. 1 mm2) that were studied as taken or mounted in epoxy resin to expose the different paint layers. The microfragments of paint layers and their cross sections were characterized by optical microscopy and scanning electron microscopy coupled with energy dispersive X-ray spectrometry. Furthermore, elemental analysis was obtained with spatially resolved confocal synchrotron radiation μ-X-ray fluorescence spectrometry performed at ANKA synchrotron FLUO beamline. Occasionally, phase analysis by μ-X-ray diffraction was also performed. Results from the different techniques allowed pigment identification and, in some cases, the evaluation of color changes due to degradation processes and, considering the Southern Portugal geology, the identification of their possible provenance. The pigments used were essentially yellow, brown and red ochres, smalt blue, copper green, and black earths, probably from local sources.

Characterization of glue sizing under calcium carbonate ground layers in Flemish and Luso-Flemish painting – analysis by SEM-EDS, μ-XRD and μ-Raman spectroscopy

This work regards the study of painting techniques in Portuguese workshops of the 15th and early 16th centuries, specifically addressing the methodology used on the preparation of ground layers. The influence of Flemish painting in Portugal is evident in stylistic and iconographic themes of that period. As regards the painting materials, we confirmed that this influence also extended to the ground layer technique. The use of a sizing layer with calcium sulphate or garlic to isolate the support from the calcium carbonate layer was verified by SEM-EDS but not confirmed by μ-XRD or μ-Raman spectroscopy. This work is part of a larger project, “The invisible ground layer and its influence in Portuguese paintings from the 15th and 16th centuries: a question to be settled”.

A multiproxy approach to evaluate biocidal treatments on biodeteriorated majolica glazed tiles

The Fishing House located on the grounds of the Marquis of Pombal Palace, Oeiras, Portugal, was built in the 18th century. During this epoch, Portuguese gardens, such as the one surrounding the Fishing House, were commonly ornamented with glazed wall tile claddings. Currently, some of these outdoor tile panels are covered with dark colored biofilms, contributing to undesirable aesthetic changes and eventually inducing chemical and physical damage to the tile surfaces. Phylogenetic analyses revealed that the investigated biofilms are mainly composed of green algae, cyanobacteria and dematiaceous fungi. With the aim of mitigating biodeterioration, four different biocides (TiO2 nanoparticles, Biotin® T, Preventol® RI 80 and Albilex Biostat® ) were applied in situ to the glazed wall tiles. Their efficacy was monitored by visual examination, epifluorescence microscopy and DNA-based analysis. Significant changes in the microbial community composition were observed 4 months after treatment with Preventol® RI 80 and Biotin® T. Although the original community was inactivated after these treatments, an early stage of re-colonization was detected 6 months after the biocide application. TiO2 nanoparticles showed promising results due to their self-cleaning effect, causing the detachment of the biofilm from the tile surface, which remained clean 6 and even 24 months after biocide application.

Characterizing Microbial Diversity and Damage in Mural Paintings

Mural paintings are some of the oldest and most important cultural expressions of mankind and play an important role for the understanding of societies and civilizations. These cultural assets have high economic and cultural value and therefore their degradation has social and economic impact. The present work presents a novel microanalytical approach to understand the damages caused by microbial communities in mural paintings. This comprises the characterization and identification of microbial diversity and evaluation of damage promoted by their biological activity. Culture-dependent methods and DNA-based approaches like denaturing gradient gel electrophoresis (DGGE) and pyrosequencing are important tools in the isolation and identification of the microbial communities allowing characterization of the biota involved in the biodeterioration phenomena. Raman microspectrometry, infrared spectrometry, and variable pressure scanning electron microscopy coupled with energy-dispersive X-ray spectrometry are also useful tools for evaluation of the presence of microbial contamination and detection of the alteration products resulting from metabolic activity of the microorganisms. This study shows that the degradation status of mural paintings can be correlated to the presence of metabolically active microorganisms.