Biosynthesis of Metal and Metal Oxide Nanoparticles Using Microbial Cultures: Mechanisms, Antimicrobial Activity and Applications to Cultural Heritage.

Nanoparticles (1 to 100 nm) have unique physical and chemical properties, which makes them suitable for application in a vast range of scientific and technological fields. In particular, metal nanoparticle (MNPs) research has been showing promising antimicrobial activities, paving the way for new applications. However, despite some research into their antimicrobial potential, the antimicrobial mechanisms are still not well determined. Nanoparticles' biosynthesis, using plant extracts or microorganisms, has shown promising results as green alternatives to chemical synthesis; however, the knowledge regarding the mechanisms behind it is neither abundant nor consensual. In this review, findings from studies on the antimicrobial and biosynthesis mechanisms of MNPs were compiled and evidence-based mechanisms proposed. The first revealed the importance of enzymatic disturbance by internalized metal ions, while the second illustrated the role of reducing and negatively charged molecules. Additionally, the main results from recent studies (2018-2022) on the biosynthesis of MNPs using microorganisms were summarized and analyzed, evidencing a prevalence of research on silver nanoparticles synthesized using bacteria aiming toward testing their antimicrobial potential. Finally, a synopsis of studies on MNPs applied to cultural heritage materials showed potential for their future use in preservation.

A multi-analytical characterization of fourteenth to eighteenth century pottery from the Kongo kingdom, Central Africa.

Pottery traditions reflect the socioeconomic framework of past cultures, while the spatial distribution of pottery indicates exchange patterns and interaction processes. Material and earth sciences are employed here to determine raw material sourcing, selection and processing. The Kongo kingdom, internationally renowned since the late fifteenth century, is one of the most famous precolonial states in Central Africa. Despite the large number of historical studies relying on African and European oral and written chronicles, there are still considerable gaps in our current understanding of this political unit. Here, we provide new insights into pottery production and circulation within the Kongo kingdom. Implementing a multi-analytical approach, namely XRD, TGA, petrographic analysis, XRF, VP-SEM-EDS and ICP-MS, on selected samples, we determined their petrographic, mineralogical and geochemical signatures. Our results allowed us to correlate the archaeological objects to naturally occurring materials and to establish ceramic traditions. We identified production templates, exchange patterns, distribution of high-quality goods and interaction processes through technological knowledge transmission. Our results demonstrate that political centralisation in the Lower Congo region of Central Africa had a direct impact on pottery production and circulation. We expect our study to provide a sound basis for further comparative research to contextualise the region.

Rapid light carbon releases and increased aridity linked to Karoo-Ferrar magmatism during the early Toarcian oceanic anoxic event.

Large-scale release of isotopically light carbon is responsible for the carbon isotope excursion (CIE) of the Toarcian Oceanic Anoxic Event during the Lower Jurassic. Proposed sources include methane hydrate dissociation, volcanogenic outgassing of carbon dioxide and/or thermogenic methane release from the Karoo-Ferrar magmatic province (southern Africa). Distinct small-scale shifts superimposed on the long-term CIE have been interpreted as rapid methane pulses linked to astronomically forced climate changes. In the Peniche reference section (Portugal), these small-scale shifts correspond to distinct brownish marly layers featuring markedly high mercury (Hg) and magnetic mineral concentration. Total organic carbon and Hg increase are uncorrelated, which suggests input of Hg into the atmosphere, possibly released after the intrusion of the Karoo-Ferrar sills into organic-rich sediments. Enhanced magnetic properties are associated with the presence of martite, washed-in oxidized magnetite, inferred to be due to increased aridity on the continental hinterland. This study provides strong evidence for a direct link between the Karoo-Ferrar magmatism, the carbon-isotope shifts and the resulting environmental changes.

Surface orange patinas on the limestone of the Batalha Monastery (Portugal): characterization and decay patterns.

Samples of orange patinas found on a limestone window tracery and an ornament of the Batalha Monastery have been investigated by X-ray micro-diffractometry (µ-XRD) and low-vacuum scanning electron microscopy coupled with energy dispersive spectrometry (LV-SEM + EDS). The aim of the study was to determine the composition of the layered patinas, assess whether they have been intentionally applied or naturally formed, and study their degradation patterns. Preliminary results revealed that the orange patinas on the window tracery and the ornament showed different compositions and appearance, suggesting distinct formation pathways. Orange patinas on the ornament, which are now showing decay and delamination patterns, mainly consisted of gypsum with hematite as a minor component, implying the possibility of an intentional application of a mixture of ochre and lime as tint plaster. Orange patinas on the window tracery show, instead, the presence of Ca-oxalates, abundant weddellite, and minor whewellite, with minor hematite suggesting the yellowish/orange color as being due to Ca-oxalate patinas imbedding soil dust airborne particles. Such patina was possibly formed naturally either by the chemical attack due to atmospheric air pollutants from traffic exhausts emissions or by bacterial activity. No delamination was observed on the window tracery sample with granular decohesion as the major decay phenomenon. A comparison was made between this patina and the so-called scialbatura, a surface yellowish coating often found by conservators on limestone and marble in ancient monuments in the Mediterranean region.

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 Micro-Analytical Study of the Scarabs of the Necropolis of Vinha das Caliças (Portugal).

Five scarabs and one scaraboid found in Vinha das Caliças 4 (Beja, Portugal) were analyzed using a micro-analytical methodology in order to determine their mineralogical and chemical composition. Microstructural characterization and chemical analysis revealed that all were composed of a white body of crushed feldspathic sand covered by a lead-rich, alkaline-depleted silicate blue-green glaze showing evident signs of glass deterioration. Variable pressure scanning electron microscopy with X-ray energy dispersive spectrometry, handheld X-ray fluorescence spectroscopy, and micro X-ray diffraction results show that blue-green color of the glaze was produced by using copper ions (Cu2+) in conjunction with the lead antimonate bindheimite, a yellow-colored opacifier. The introduction of small amounts of tin in the structure of bindheimite enabled the production of a ternary Pb-Sb-Sn oxide. Tin, which was most likely added with the copper source (bronze scrapings), is known to facilitate the crystallization of bindheimite. The results are consistent with the five scarabs and one scaraboid being manufactured in Egypt. This study, the first archeometric study of scarabs found in the Iberian peninsula, has greatly contributed to the understanding of the influence of the Eastern and Central Mediterranean world in the Southwestern Iberia during the first millennium B.C.

Mineralogical Characterization of Hispano-Moresque Glazes: A µ-Raman and Scanning Electron Microscopy with X-Ray Energy Dispersive Spectrometry (SEM-EDS) Study.

This work explores the combination of µ-Raman spectroscopy and scanning electron microscopy with X-ray energy dispersive spectrometry (SEM-EDS) for the study of the glazes in 15th-16th century Hispano-Moresque architectural tiles. These are high lead glazes that can be tin-opacified or transparent, and present five colors: tin-white, cobalt-blue, copper-green, iron-amber, and manganese-brown. They are generally homogenous and mineral inclusions are mostly concentrated in the glaze-ceramic interface. Through SEM-EDS, these inclusions were observed and chemically analyzed, whereas µ-Raman allowed their identification on a molecular level. K-feldspars, wollastonite and diopside were the most common compounds, as well as cassiterite agglomerates that render the glaze opaque. Malayaite was identified in green glazes, and andradite and magnesioferrite in amber glazes. Co-Ni-ferrites were identified in blue glazes, as well as Ni-Fe-olivines. Manganese-brown is the color where most compounds were identified: bustamite, jacobsite, hausmannite, braunite, and kentrolite. Through the µ-Raman analysis of different areas in large inclusions previously observed by SEM, it was possible to identify intermediate phases that illustrate the reaction process that occurs between the color-conferring compounds and the surrounding lead glaze. Furthermore, the obtained results allowed inference of the raw materials and firing temperatures used on the manufacture of these tiles.

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.

End-Cretaceous akaganéite as a mineral marker of Deccan volcanism in the sedimentary record.

An enigmatic chloride-rich iron (oxyhydr)oxide has been recently identified together with mercury anomalies in End-Cretaceous marine sediments coeval with the Deccan Traps eruptions. The mineral was observed in Bidart (France) and Gubbio (Italy), suggesting a widespread phenomenon. However, the exact nature and origin of this Cl-bearing mineral remained speculative. Here, we characterized the accurate composition and nanostructure of this chloride-rich phase by using micro-Raman spectroscopy, Transmission (TEM) and Scanning (SEM) Electron Microscopy on Focused Ion Beam foils. We also provide new evidence of its occurrence in Zumaia, a reference KPg section from Spain. Results confirm akaganéite (ß-FeOOH) as the main phase, with chloride content of 3-5 atomic weight %. Akaganéite particles are constituted by the aggregation of nanorods of akaganéite. Internal structures contain empty spaces, suggesting formation in a low-density (atmospheric) environment. This new mineralogical evidence supports the hypothesis that the observed akaganéite was formed in the Deccan volcanic plume and was transported to the Atlantic and Tethysian realms through the stratosphere. Therefore, akaganéite provides a potential new sedimentary marker to identify the imprint of the Deccan eruptions in the stratigraphic record and is evidence of volcanic halogen degassing and its potential role for the Cretaceous-Tertiary mass extinction.

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.

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.

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.

The liturgical cope of D. Teotónio of Braganza: material characterization of a 16th century pluviale.

A 16th century liturgical cope belonging to D. Teotónio of Braganza (collection of the Museum of Évora, ME 172/1) was selected for a material study. The cope is made of a variety of materials that include two different types of metal threads, dyed silk yarns, and vegetable yarns used in the weft. Several samples from different points representing the different metal thread types and colored silk yarns were collected. Stereomicroscopy (optical microscopy) and scanning electron microscopy were used for morphological analysis of the textile fibers and evaluation of metal thread degradation products. Evaluation of mordants and metal thread composition was carried out by energy-dispersive X-ray spectroscopy. Liquid chromatography with diode array and mass spectrometry detection was used for dye identification, which allowed the determination of three different red dye sources and one yellow dye source in the colored silk yarns. Although different fabrics were used in the manufacturing of the cope, similarities identified in the characterization of the materials suggest that a single workshop was involved in its making.

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.