Assessment of microbiota present on a Portuguese historical stone convent using high-throughput sequencing approaches.

The study performed on the stone materials from the Convent of Christ revealed the presence of a complex microbial ecosystem, emphasizing the determinant role of microorganisms on the biodecay of this built cultural heritage. In this case study, the presence of Rubrobacter sp., Arthrobacter sp., Roseomonas sp., and Marinobacter sp. seems to be responsible for colored stains and biofilm formation while Ulocladium sp., Cladosporium sp., and Dirina sp. may be related to structural damages. The implementation of high-throughput sequencing approaches on the Convent of Christ's biodecay assessment allowed us to explore, compare, and characterize the microbial communities, overcoming the limitations of culture-dependent techniques, which only identify the cultivable population. The application of these different tools and insights gave us a panoramic view of the microbiota thriving on the Convent of Christ and signalize the main biodeteriogenic agents acting on the biodecay of stone materials. This finding highlighted the importance of performing metagenomic studies due to the improvements and the reduced amount of sample DNA needed, promoting a deeper and more detailed knowledge of the microbiota present on these dynamic repositories that support microbial life. This will further enable us to perform prospective studies in quarry and applied stone context, monitoring biogenic and nonbiogenic agents, and also to define long-term mitigation strategies to prevent biodegradation/biodeterioration processes.

A simple procedure for detecting Dekkera bruxellensis in wine environment by RNA-FISH using a novel probe.

Dekkera bruxellensis, considered the major microbial contaminant in wine production, produces 4-ethylphenol, a cause of unpleasant odors. Thus, identification of this yeast before wine spoilage is crucial. Although challenging, it could be achieved using a simple technique: RNA-FISH. To reach it is necessary to design probes that allow specific detection/identification of D. bruxellensis among the wine microorganisms and in the wine environment and, if possible, using low formamide concentrations. Therefore, this study was focused on: a) designing a DNA-FISH probe to identify D. bruxellensis that matches these requirements and b) determining the applicability of the RNA-FISH procedure after the end of the alcoholic fermentation and in wine. A novel DNA-FISH D. bruxellensis probe with good performance and specificity was designed. The application of this probe using an in-suspension RNA-FISH protocol (applying only 5% of formamide) allowed the early detection/identification of D. bruxellensis at low cell densities (5 × 102 cell/mL). This was possible by flow cytometry independently of the growth stage of the target cells, both at the end of the alcoholic fermentation and in wine even in the presence of high S. cerevisiae cell densities. Thus, this study aims to contribute to facilitate the identification of D. bruxellensis before wine spoilage occurs, preventing economic losses to the wine industry.

An important step forward for the future development of an easy and fast procedure for identifying the most dangerous wine spoilage yeast, Dekkera bruxellensis, in wine environment.

Dekkera bruxellensis is the main reason for spoilage in the wine industry. It renders the products unacceptable leading to large economic losses. Fluorescence In Situ Hybridization (FISH) technique has the potential for allowing its specific detection. Nevertheless, some experimental difficulties can be encountered when FISH technique is applied in the wine environment (e.g. matrix and cells' autofluorescence, fluorophore inadequate selection and probes' low specificity to the target organisms). An easy and fast in-suspension RNA-FISH procedure was applied for the first time for identifying D. bruxellensis in wine. A previously designed RNA-FISH probe to detect D. bruxellensis (26S D. brux.5.1) was used, and the matrix and cells' fluorescence interferences, the influence of three fluorophores in FISH performance and the probe specificity were evaluated. The results revealed that to apply RNA-FISH technique in the wine environment, a red-emitting fluorophore should be used. Good probe performance and specificity were achieved with 25% of formamide. The resulting RNA-FISH protocol was applied in wine samples artificially inoculated with D. bruxellensis. This spoilage microorganism was detected in wine at cell densities lower than those associated with phenolic off-flavours. Thus, the RNA-FISH procedure described in this work represents an advancement to facilitate early detection of the most dangerous wine spoilage yeast and, consequently, to reduce the economic losses caused by this yeast to the wine industry.

Inorganic Nanomaterials for Restoration of Cultural Heritage: Synthesis Approaches towards Nanoconsolidants for Stone and Wall Paintings.

The synthesis of inorganic nanostructured materials for the consolidation of stone and wall paintings is reviewed. To begin, a description of the methods most commonly used to prepare nanoconsolidants is provided, particularly in the frame of colloid chemistry. Some concepts of the carbonation mechanism as well as the transport properties of some of these materials are addressed. An overview of the synthesis methods together with some of the application particularities of the distinct consolidants are presented thereafter. Furthermore, the requisites for efficient consolidants and some drawbacks of the nanoconsolidants are discussed.

Analytical characterization of the palette and painting techniques of Jorge Afonso, the great 16th century Master of Lisbon painting workshop.

In this work, a study on a set of paintings from the most significant altarpiece assigned to Master Jorge Afonso (c. 1470-1540) painting workshop is presented. This altarpiece is composed by fourteen paintings made to the church of Convento de Jesus, in Setúbal, Portugal, and was made circa 1517-19/1530, according to art-history. This set of paintings is compared to one of the other most important Portuguese altarpieces from the 16th century: the panels of the Round Church of the Convento de Cristo, in Tomar, made circa 1510-1515. The aim of this study is to characterize the wooden support, pigments, ground layers materials and technique used in Jorge Afonso workshop by means of complementary analyses. A dendrochronological approach was made in order to corroborate (or not) the historical date initially assigned. Infrared photography (IRP) and reflectography (IRR) allowed the study of the underdrawing technique and macro photography (MP) was used to recognize overlapping layers technique. Cross-sections from the paintings were examined by optical microscopy (OM), and analyzed by µ-X-ray diffraction (µ-XRD), Energy Dispersive X-ray Fluorescence spectroscopy (EDXRF), Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy (SEM-EDS), micro-Raman spectroscopy (µ-Raman), micro-Fourier Transform Infrared spectroscopy (µ-FTIR), Pyrolysis Gas Chromatography Mass Spectrometry (py-GC/MS). The characterization of the palette and ground layers and the study of the overlapping of paint layers brought a new insight of the adopted painting techniques by the most important group of painters working in Portugal in the 16th century - the Lisbon workshop, leaded by Master Jorge Afonso.

Use of Monte Carlo Simulation as a Tool for the Nondestructive Energy Dispersive X-ray Fluorescence (ED-XRF) Spectroscopy Analysis of Archaeological Copper-Based Artifacts from the Chalcolithic Site of Perdigões, Southern Portugal.

This work is part of a broader research line that aims to develop and implement a nondestructive methodology for the chemical characterization of archaeological metals based on a protocol that combines energy dispersive X-ray fluorescence spectrometry (ED-XRF) with a Monte Carlo (MC) simulation algorithm. In this paper, the ED-XRF-MC protocol has been applied to estimate the chemical composition of a selected group of 26 copper-based artifacts and fragments recovered at Perdigões, one of the larger Chalcolithic sites of southwest Iberia. All the analyzed artifacts have a multilayered structure composed of the alloy substrate and of a superficial layer common in each metal buried for hundreds of years and consisting of the patina mixed with soil. Due to the difficulty in determining the quantitative composition of these alloys in the presence of this complex patina/encrustation layer, the spectrometric protocol applied in this paper allows to simulate and to determine the composition of the bulk alloy without any prior removal of the overlying corrosion patina layer and soil-derived crust, even in the presence of rough and irregular surfaces, thus preserving the physical integrity of the artifacts. The overall results obtained with the ED-XRF-MC protocol indicates that the artifacts from Perdigões are almost pure coppers with a low amount of arsenic (<3.0 wt%) and reduced concentration of elements such as Pb, Bi, and Sb, in agreement with the third millennium metallurgy known in southwestern Iberia. Also based on previously theoretical-experimental studies, the data presented in this paper show how the applied analytical methodology can be a fast and completely nondestructive analytical tool reliable for routine and large-scale chemical analysis of archaeological metals, thus representing a major advance to be broadly applied within the field of cultural heritage studies.

Natural limestone discolouration triggered by microbial activity-a contribution.

Colour is a major argument that drives the decision of an architect in a specific architecture project and one of the most important characteristics and perceptible aspects of natural building stones. "Blue" limestones are building rocks, with different geological ages, typically used in several countries, and are known for their vulnerability to alteration, which causes colour change and the occurrence of unaesthetic patterns. Owing to this vulnerability, the conservation-restoration works in monuments, or new buildings constructed with "blue" limestone is extremely costly. Considering that the main limitation of this lithological variation is the chromatic change, a multidisciplinary approach was envisaged in this study to allow a closer insight into the chemical and mineralogical alterations and the microbial communities. Results obtained suggest that the inorganic alteration in the "blue" limestone may create favourable conditions for microbial growth and could lead to an increment in deterioration process.

Green mitigation strategy for cultural heritage: bacterial potential for biocide production.

Several biosurfactants with antagonistic activity are produced by a variety of microorganisms. Lipopeptides (LPPs) produced by some Bacillus strains, including surfactin, fengycin and iturin are synthesized nonribosomally by mega-peptide synthetase (NRPS) units and they are particularly relevant as antifungal agents. Characterisation, identification and evaluation of the potentials of several bacterial isolates were undertaken in order to establish the production of active lipopeptides against biodeteriogenic fungi from heritage assets. Analysis of the iturin operon revealed four open reading frames (ORFs) with the structural organisation of the peptide synthetases. Therefore, this work adopted a molecular procedure to access antifungal potential of LPP production by Bacillus strains in order to exploit the bioactive compounds synthesis as a green natural approach to be applied in biodegraded cultural heritage context. The results reveal that the bacterial strains with higher antifungal potential exhibit the same morphological and biochemical characteristics, belonging to the genera Bacillus. On the other hand, the higher iturinic genetic expression, for Bacillus sp. 3 and Bacillus sp. 4, is in accordance with the culture antifungal spectra. Accordingly, the adopted methodology combining antifungal screening and molecular data is represent a valuable tool for quick identification of iturin-producing strains, constituting an effective approach for confirming the selection of lipopeptides producer strains.

On the Chemical Signature and Origin of Dicoppertrihydroxyformate (Cu2(OH)3HCOO) Formed on Copper Miniatures of 17th and 18th centuries.

A corrosion product rarely reported in the literature has been found on the copper support of three miniature paintings of the 17th and 18th centuries. This product, which has been identified as dicoppertrihydroxyformate (Cu2(OH)3HCOO), is an unusual basic copper formate found on copper artifacts. The identification and characterization of dicoppertrihydroxyformate was carried out directly over the corroded surface of the objects, using a nondestructive approach, which combines the integrated use of various microanalytical techniques. Using this approach, it was possible to obtain a set of new reference data about the natural form of Cu2(OH)3HCOO, that will enable its unambiguous identification in other similar objects. In this work, the probable causes that may have contributed to its formation are also discussed.

Tortoiseshell or Polymer? Spectroscopic Analysis to Redefine a Purported Tortoiseshell Box with Gold Decorations as a Plastic Box with Brass.

The study and preservation of museum collections requires complete knowledge and understanding of constituent materials that can be natural, synthetic, or semi-synthetic polymers. In former times, objects were incorporated in museum collections and classified solely by their appearance. New studies, prompted by severe degradation processes or conservation-restoration actions, help shed light on the materiality of objects that can contradict the original information or assumptions. The selected case study presented here is of a box dating from the beginning of the 20th century that belongs to the Portuguese National Ancient Art Museum. Museum curators classified it as a tortoiseshell box decorated with gold applications solely on the basis of visual inspection and the information provided by the donor. This box has visible signs of degradation with white veils, initially assumed to be the result of biological degradation of a proteinaceous matrix. This paper presents the methodological rationale behind this study and proposes a totally non-invasive methodology for the identification of polymeric materials in museum artifacts. The analysis of surface leachates using (1)H and (13)C nuclear magnetic resonance (NMR) complemented by in situ attenuated total reflection infrared spectroscopy (ATR FT-IR) allowed for full characterization of the object s substratum. The NMR technique unequivocally identified a great number of additives and ATR FT-IR provided information about the polymer structure and while also confirming the presence of additives. The pressure applied during ATR FT-IR spectroscopy did not cause any physical change in the structure of the material at the level of the surface (e.g., color, texture, brightness, etc.). In this study, variable pressure scanning electron microscopy (VP-SEM-EDS) was also used to obtain the elemental composition of the metallic decorations. Additionally, microbiologic and enzymatic assays were performed in order to identify the possible biofilm composition and understand the role of microorganisms in the biodeterioration process. Using these methodologies, the box was correctly identified as being made of cellulose acetate plastic with brass decorations and the white film was identified as being composed mainly of polymer exudates, namely sulphonamides and triphenyl phosphate.

Microscopy and Microanalysis of an Extreme Case of Salt and Biodegradation in 17th Century Wall Paintings.

The present study characterizes the main deterioration mechanisms affecting the early 17th frescoes of Casa de Fresco, the only known example in Portugal of a semi-underground leisure room richly decorated with a balcony over a water well. Frescoes from the vault are at risk due to salt weathering and biodeterioration. The aim of the research was identification of the deterioration materials, determination of their origin, and their effect on the frescoes before future intervention. Scanning electron microscopy with an energy-dispersive X-ray detector (SEM-EDS) was used to determine salt morphology and microanalysis. The mineralogical characterization was performed by X-ray powder diffraction, complemented with µ-Raman and µ-Fourier transform infrared spectroscopy. Biological assessment was evaluated with optical microscopy and SEM-EDS. Bacterial and fungal isolation and identification were performed using standard culture media and methods according to Bergey's Manual of Systematic Bacteriology and from the Compendium of Soil Fungi. The results show that Ca and Ca-Mg carbonates from the paint renderings are the predominant salt species affecting the site. Bacterial strains from the genera Bacillus and Pseudomonas and fungal strains from the Cladosporium spp. and Penicillium spp. were isolated in the salt formations, within and between the mortar layers. Azurite, malachite, and smalt paint layers are the most affected by the weathering conditions.

Analytical and microbiological characterization of paper samples exhibiting foxing stains.

This work comprises the use of a multi-analytical approach combined with microbiological studies to characterize six paper samples, containing foxing stains, from the 20th century, regarding their cellulose matrix, fillers, and sizing materials, and to evaluate possible paper degradation that might have occurred during the foxing stains. Photography under different illuminations and optical microscopy were used for morphological characterization of the paper samples and foxing stains. Scanning electron microscopy coupled energy dispersive spectroscopy (SEM-EDS) was of particular importance for defining the presence of fiber disorder and disruption on the surface of some of the stains, and localized accumulations of mineral-like particles on the surface of others. SEM-EDS, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FT-IR), and energy dispersive X-ray fluorescence (EDXRF) were used for the identification of mineral fillers, whereas sizing agents were analyzed using ATR-FT-IR. EDXRF results showed that no differences, within the standard deviation, were found in iron and copper contents between the foxed and unfoxed areas. Fungi belonging to the genus Penicillium spp. were found in all the paper samples. Unfoxed areas presented lower contamination than the foxed areas.

On the Use of the Unusual Green Pigment Brochantite (Cu4(SO4)(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).

White spots on Smoke rings by Bruce Nauman: a case study on contemporary art conservation using microanalytical techniques.

The artwork "Smoke Rings: Two Concentric Tunnels, Non-Communicating" by Bruce Nauman represents a case study of corrosion of a black patina-coated Al-alloy contemporary artwork. The main concern over this artwork was the widespread presence of white spots on its surface. Alloy substrate, patina, and white spots were characterized by means of energy-dispersive X-ray fluorescence and scanning electron microscopy with energy-dispersive spectroscopy. Alloy substrate was identified as an aluminum alloy 6,000 series Al-Si-Mg. Patina's identified composition confirmed the documentation provided by the atelier. Concerning the white spots, zircon particles were found on patina surface as external elements.

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.

Characterization of glue sizing layers in Portuguese wood paintings from the 15th and 16th centuries by SEM secondary electron images and µ-FTIR.

According to treatises on 15th and 16th century paintings, artists dedicated particular attention to the sizing layer-consisting mainly of animal glue applied onto the wood support before further application of the ground layer. The stability of a painting mainly depends on the presence of a very cohesive sizing layer. However, the study of these layers has not received special attention from researchers. In this article we present a methodology for characterization of the sizing layer both chemically, by IR spectroscopy (Fourier transform infrared spectroscopy), and morphologically, by scanning electron microscopy (SEM). Secondary electron images obtained by SEM allow precise characterization of such layers. Painting reconstructions were used as references in development of the method to study the sizing layer in real painting samples. Presented herein are examples of this study on 15th and 16th century Portuguese paintings, particularly on the Triptych of S. Simão, from the Aveiro Museum, and S. Pedro, belonging to the Mercy of Tavira.

Spectroscopic characterization of a masterpiece: the Manueline foral charter of Sintra.

The foral charter attributed by D. Manuel I of Portugal, in 1514, to the village of Sintra was studied using Energy Dispersive X-ray Fluorescence spectrometry, Raman and Infrared micro-spectroscopies. A complete characterization of the pictorial materials used in the production of this masterpiece allowed the identification of iron gall inks used in the written text; pigments such as malachite, azurite, lead white, cinnabar, yellow ochre, gold, silver and carbon black in the illuminations and letterings; filler and binder used in the production of coloring materials and inks. Gum and calcium carbonate were the most recurrent binder and filler identified in this study. Silvering and gilding were mostly obtained by applying ground silver and gold on parchment.

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.

Extracting natural dyes from wool--an evaluation of extraction methods.

The efficiency of eight different procedures used for the extraction of natural dyes was evaluated using contemporary wool samples dyed with cochineal, madder, woad, weld, brazilwood and logwood. Comparison was made based on the LC-DAD peak areas of the natural dye's main components which had been extracted from the wool samples. Among the tested methods, an extraction procedure with Na(2)EDTA in water/DMF (1:1, v/v) proved to be the most suitable for the extraction of the studied dyes, which presented a wide range of chemical structures. The identification of the natural dyes used in the making of an eighteenth century Arraiolos carpet was possible using the Na(2)EDTA/DMF extraction of the wool embroidery samples and an LC-DAD-MS methodology. The effectiveness of the Na(2)EDTA/DMF extraction method was particularly observed in the extraction of weld dye components. Nine flavone derivatives previously identified in weld extracts could be identified in a single historical sample, confirming the use of this natural dye in the making of Arraiolos carpets. Indigo and brazilwood were also identified in the samples, and despite the fact that these natural dyes were referred in the historical recipes of Arraiolos dyeing, it is the first time that the use of brazilwood is confirmed. Mordant analysis by ICP-MS identified the widespread use of alum in the dyeing process, but in some samples with darker hues, high amounts of iron were found instead.