Biodeterioration of mural paintings in hypogeal environments is often driven by complex microbial communities. This study investigates the microbial colonization responsible for visible alterations on the mural paintings of the Tomba delle Leonesse, a UNESCO World Heritage Site, with the aim of identifying the microorganisms producing the black spots and olive-green biofilms and assessing their viability. Black-stained areas were sampled using sucrose-enriched traps to evaluate the metabolic reactivity of the resident microorganisms. Once viability was confirmed, a combination of cultivation methods and Nanopore sequencing was used to characterize the microbial taxa involved. Raman spectroscopy and proteomic analyses also revealed melanin production by several isolated fungal strains. The results reveal a viable and metabolically responsive microbial community contributing to biodeterioration. Although these microorganisms may be dormant under current conditions, they retain the capacity to rapidly reactivate in response to environmental changes. This suggests that even small changes-such as increased humidity, nutrient inputs from visitors, or the application of traditional biocides-could trigger uncontrolled microbial proliferation. These findings highlight the importance of adopting precautionary, microbiologically informed conservation strategies. In addition, the identification of enzymes involved in melanin biosynthesis suggests that targeted metabolic inhibitors may offer a promising alternative to traditional biocide treatments.

Aims Hypogeal environments with cultural heritage interest pose a real challenge for their preservation and conservation. The ancient Etruscan Necropolis of Tarquinia, Italy, consists of 200 tombs decorated with extraordinary mural paintings, of great artistic and historical value. Since the beginning of the restoration campaign in 2016, a regular microbiological survey has been performed in the Tomba degli Scudi. The aim of this study was to investigate the nature of an expansion of black spots on the pictorial layers recently observed.Methods and results To determine the origin of the black spots in the atrium chamber of the Tomba degli Scudi, the fungal community was sampled using various techniques: cellulose discs, swabs, and nylon membranes and investigated by a multi-analytical approach. The obtained results suggest that the identified fungal strains (e.g. Gliomastix murorum and Pseudogymnoascus pannorum) are common to many subterranean environments around the world, such as Lascaux cave.Conclusions The continuous and long-term monitoring made it possible to detect alterations at an early stage and assess the harmfulness of different fungal strains. This work is a demonstration of the effectiveness of prevention and monitoring actions within these fragile and valuable environments.

Biomineralization of calcium carbonate by living organisms, including bacteria, has been studied for its potential use in conservation and restoration applications in Cultural Heritage. This study reports the carbonatogenic properties of three bacterial strains (Lysinibacillus fusiformis 3.20, Psychrobacillus psychrodurans 7Mo and Lederbergia lenta Vetro1) isolated from Il Giovane di Mozia sculpture (Mozia), the Etruscan mural paintings of the Tomba degli Scudi (Tarquinia), and a microbial community isolated from the Sant'Eustachio statue (Matera). The crystals precipitated have been investigated through a multi-analytical approach: Field Emission Scanning Electron Microscopy (FESEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The analyses showed the formation of stable vaterite by the bacterial cultures, while the microbial community induced calcite precipitation. This approach aims to support the conservation-restoration efforts, taking into the account the requests of the conservation-restorers and the unique characteristics of each Cultural Heritage artwork.(c) 2023 Consiglio Nazionale delle Ricerche (CNR). Published by Elsevier Masson SAS. All rights reserved.

In this article, we discuss the geological, mineralogical, and geochemical characteristics of the proposed sources of pigments used in the Wadi Sura rock art, southwestern Egypt. Colors used in the paintings include white, yellow, and several reddish hues ranging from pale red to dark reddish brown, rare black, and greenish hues. The results of Raman spectroscopy and pXRF techniques on both raw coloring materials and archaeological pigments show that the ancient artists made extensive use of inorganic clay-based pigment (e.g., kaolinite) associated with anhydrite and gypsum. White raw coloring materials were recorded in the field as thin laminated beds and lenses within the Silurian sandstone bedrock and are also present in paleosol layers and reworked fragments mixed with ocher. Raw materials for red and yellow colors are represented by clay-based mixtures of aluminosilicate with iron oxide, hematite, goethite or magnetite, and gypsum. The amorphous carbon and romanechite, as well as goethite and magnetite, could have been the components of the dark brownish pigments widely used in the rock art of the study area. Surprisingly, lazurite was also recognized among the raw materials, although this blue pigment does not seem to have been used in the Wadi Sura rock art panels (however, bluish and greenish traces as violet reddish hues, along with bluish-greenish yellow colors, are reported in the paintings). Lazurite is not well known in the geology of Egypt and has been detected in this study for the first time. The results of our work indicate that access to the Silurian sandstones, mainly located on the northwestern slope of the plateau, may have been one of the factors for choosing this area of the Gilf Kebir for producing rock art images.

Earth's microbial biosphere extends down through the crust and much of the subsurface, including those microbial ecosystems located within cave systems. Here, we elucidate the microbial ecosystems within anthropogenic 'caves'; the Iron-Age, subterranean tombs of central Italy. The interior walls of the rock (calcium-rich macco) were painted ~2500 years ago and are covered with CaCO3 needles (known as moonmilk). The aims of the current study were to: identify biological/geochemical/biophysical determinants of and characterize bacterial communities involved in CaCO3 precipitation; challenge the maxim that biogenic activity necessarily degrades surfaces; locate the bacterial cells that are the source of the CaCO3 precipitate; and gain insight into the kinetics of moonmilk formation. We reveal that this environment hosts communities that consist primarily of bacteria that are mesophilic for temperature and xerotolerance (including Actinobacteria, Bacteroidetes and Proteobacteria); is populated by photosynthetic Cyanobacteria exhibiting heterotrophic nutrition (Calothrix and Chroococcidiopsis); and has CaCO3 precipitating on the rock surfaces (confirmation that this process is biogenic) that acts to preserve rather than damage the painted surface. We also identified that some community members are psychrotolerant (Polaromonas), acidotolerant or acidophilic (members of the Acidobacteria), or resistant to ionizing radiation (Brevundimonas and Truepera); elucidate the ways in which microbiology impacts mineralogy and vice versa; and reveal that biogenic formation of moonmilk can occur rapidly, that is, over a period of 10 to 56 years. We discuss the paradox that these ecosystems, that are for the most part in the dark and lack primary production, are apparently highly active, biodiverse and biomass-rich.

During recent conservation work carried out on the Etruscan mural paintings in the Tomba degli Scudi, (Tarquinia, 4th C. BCE), the study of the execution techniques led to an amazing discovery: human figures were composed using templates of single body parts. In the Tomba degli Scudi, the perfect overlap of limbs, chests, and heads was confirmed after processing rectified photogrammetric images of the wall paintings with photo editing software. The study also highlighted that such templates were probably made of rigid material and each one had three different sizes, in order to represent the hierarchical organization of the painted characters. In the past, scholars have demonstrated that templates were used to realize a number of Etruscan wall paintings. However, they had reported that only whole figures were reproduced. The notion that such templates consisted of single anatomical parts purposely created to portray figures in different poses never occurs in their studies. In fact, the use of modular templates, also called patroni and antibola, is acknowledged only from the Middle Ages onwards both in western and eastern art. Earlier evidence had never been found. This article aims to predate the use of modular shapes by about ten centuries, thus changing our knowledge of technologies whose tradition spans from the Classical Antiquity to the Middle Ages. Consequently, a new approach to the study of transfer techniques of preparatory drawings in Etruscan wall paintings is established. Furthermore, this article shows that, as in the Middle Ages, the size of such templates was scaled up or down to create hierarchies among the painted characters.

A white deposit covering the walls in the Stanza degli Scudi of the Tomba degli Scudi , Tarquinia, Italy, has been investigated. In this chamber, which is still preserved from any kind of intervention such as cleaning and sanitization, ancient Etruscans painted shields to celebrate the military power of the Velcha family. Scanning electron microscopy analysis has revealed the presence of characteristic nanostructures corresponding to a calcite secondary mineral deposit called moonmilk. Analysis of the microbial community identified Proteobacteria , Acidobacteria and Actinobacteria as the most common phyla in strong association with the moonmilk needle fibre calcite and nanofibers of calcium carbonate. Employing classical microbiological analysis, we isolated from moonmilk a Streptomyces strain able to deposit gypsum and calcium carbonate on plates, supporting the hypothesis of an essential contribution of microorganisms to the formation of moonmilk.

Mural paintings in the hypogeal environment of the Tomba degli Scudi in Tarquinia, Italy, show a quite dramatic condition: the plaster mortar lost his cohesion and a white layer coating is spread over almost all the wall surfaces. The aim of this research is to verify if the activity of microorganisms could be one of the main causes of deterioration and if the adopted countermeasures (conventional biocide treatments) are sufficient to stop it. A biocide treatment of the whole environment has been carried out before the conservative intervention and the tomb has been closed for one month. When the tomb was opened again, we sampled the microorganisms present on the frescoes and we identified four Bacillus species and one mould survived to the biocide treatment. These organisms are able to produce spores, a highly resistant biological form, which has permitted the survival despite the biocide treatment. We show that these Bacillus strains are able to produce calcium carbonate and could be responsible for the white deposition that was damaging and covering the entire surface of the frescoes. Our results confirm that the sanitation intervention is non always resolutive and could even be deleterious in selecting harmful microbial communities.

In 2010 the Italian-Egyptian Environmental Cooperation launched a safeguarding project for the preservation of the caves with prehistoric rock art located in the Gilf Kebir plateau in southwestern Egypt. The project was part of the cooperation program developed to establish the Egyptian Gilf Kebir National Park (GKNP) protected area. Given their bad state of preservation, the Italian conservation project focused on the Caves of Swimmers and Archers, located along the Wadi Sura. Although only very few studies of this kind have been carried out in the Saharan region, our work in the Gilf Kebir can be considered a pilot study, the results of which should be evaluated in the long term. Results obtained to date and reported in this paper provide analytical petrographic studies of the bedrock, a complete photographic and geodetic survey of the two sites, data from climate monitoring, along with a preliminary consolidation of some of the most at-risk areas of the two caves. Finally, laboratory experimentation led us to select the most suitable materials for the consolidation of the rock, shifting in the direction of nano-technology instead of ethyl silicate use because of the longer cross-linking process of the latter in hyper-arid environments; the use of the latter can in fact result in extremely long and expensive field seasons. These results will be valuable for the continuation and extension of the project, which is currently suspended due to safety concerns in the region.