Diversity of Parengyodontium spp. strains isolated from the cultural heritage environment: Phylogenetic diversity, phenotypical diversity, and occurrence.

Parengyodontium album is a fungal species that frequently occurs in the cultural heritage environment. Although three subclades were initially described in the species, no study has sought to determine the occurrence of each subclade in the cultural heritage context. These subclades are easily distinguishable phylogenetically, but their morphological identification is more difficult. Eighteen strains isolated from different cultural sites and initially identified as P. album were studied phylogenetically, morphologically, and in terms of their susceptibility to econazole nitrate 0.2%, an antifungal product used as preservation treatment in cultural heritage domain. The phylogenetic study revealed that all studied strains belonged to P. album subclade 1 or P. torokii (P. album subclade 3) and none belonged to P. album subclade 2. The morphological study revealed the best characteristics to differentiate the three subclades/species, namely, the ability of the strains to grow at 32 C and 35 C on potato dextrose agar (PDA) medium and the shape of conidia. Finally, the strains displayed variable susceptibilities to econazole nitrate, with no apparent link to any particular subclade/species.

Factors inducing bryophyte growth on prehistoric pigments and effect of UV-C treatment.

In La Glacière cave (France), the touristic activity has been conducted to an environmental parameter change that has led to photosynthetic organism proliferation (microalgae, diatoms, cyanobacteria, bryophytes). The present study is focused on bryophyte development occurring in the show cave that was responsible of limestone biodeterioration. In order to understand the colonization process of limestone, we have maintained limestone blocks under optimal Lampenflora growth conditions. Moreover, some limestone blocks were painted with several pigments that were used in the prehistory (e.g., red ocher, bone char). Microorganisms and bryophyte growth were monitored during 1 year, and then, the block samples were treated using UV-C light (254 nm). Thus, obtained results were compared with in situ treatment in La Glacière cave. Results have showed dense bryophyte propagation on the several blocks. However, the growth rate was correlated with the chemical composition of the pigment. In fact, the presence of some chemical elements such as As, Cr, Ti, and Co contributed to reduce bryophyte growth. Finally, moss treatment using UV-C light has demonstrated high efficiency under in situ condition, while a fast recolonization has been observed for samples maintained in laboratory. This difference was explained by the high bryophyte density under laboratory conditions that make UV-C light penetration difficult.

Hydrogeological control on carbon dioxide input into the atmosphere of the Chauvet-Pont d'Arc cave.

Carbon dioxide (CO2) concentration (CDC) is an essential parameter of underground atmospheres for safety and cave heritage preservation. In the Chauvet cave (South France), a world heritage site hosting unique paintings dated 36,000 years BP, a high-sensitivity monitoring, ongoing since 1997, revealed: 1) two compartments with a spatially uniform CDC, a large volume (A) (40,000 to 80,000 m3) with a mean value of 2.20 ± 0.01% vol. in 2016, and a smaller remote room (B) (2000 m3), with a higher mean value of 3.42 ± 0.01%; 2) large CDC annual variations with peak-to-peak amplitude of 2% and 1.6% in A and B, respectively; 3) long-term changes, with an increase of CDC and of its annual amplitude since 1997, then faster since 2013, reaching a maximum of 4.4% in B in 2017, decreasing afterwards. While a large effect of seasonal ventilation is ruled out, monitoring of seepage at two dripping points indicated that the main control of CDC seasonal reduction was transient infiltration. During periods of water deficit, calculated from surface temperature and rainfall, CDC systematically increased. The carbon isotopic composition of CO2, correlated with water excess, is consistent with a time-varying component of CO2 seeping from above. The CO2 flux, which is the primary driver of CDC in A and B, inferred using box modelling, was found to confirm the relationship between water excess and reduced CO2 flux into A, compatible with a more constant flux into B. A buoyancy-driven horizontal CO2 flow model in the vadose zone, hindered by water infiltration, is proposed. Similarly, pluri-annual and long-term CDC changes can likely be attributed to variations of water excess, but also to increasing vegetation density above the cave. As CDC controls the carbonate geochemistry, an increased variability of CDC raises concern for the preservation of the Chauvet cave paintings.

Assessment of fungi proliferation and diversity in cultural heritage: Reactions to UV-C treatment.

Fungi are present in natural and non-touristic caves due to the presence of organic matter provided mainly by insects or animals such as bats. In show caves, however, tourist infrastructure and the visitors themselves are an important source of organic matter. In addition, photosynthetic biofilms provide a high amount of carbon and nitrogen sources for fungi. This study was conducted to identify the fungal communities present in caves along with the potential use of UV-C treatment against their proliferation. Thus, fungal communities proliferating in biofilms in six French and Swiss show caves were analyzed using high throughput sequencing. The results show 385 species recorded, some of them previously described in cases of fungal outbreak. This preliminary study also aimed to test the use of UV-C light as an environmentally friendly method to treat fungal proliferation. Six fungal strains, from three different sources (Lascaux cave, La Glacière cave, a church in Vicherey, France), were cultivated in an agar dish. Spores, mycelia and the entire colony were irradiated using several UV-C intensities. Results showed that four of the six fungi spores and mycelium died following a low-intensity UV-C treatment (2 kJ m-2, 160 s), though Ochroconis lascauxensis and Penicillium bilaiae spores showed higher resistance. Finally, it was demonstrated that the fungal colony could resist the UV-C light due to a shadow effect. The structure of the fungal colony was affected from the periphery to its inner part. However, after four 30 kJ m-2 treatments (39 min irradiation) all strains there definitively eradicated. Further studies will be necessary to examine the potential of UV-C light under cave conditions as a preventive and curative treatment.

Biofilm biodiversity in French and Swiss show caves using the metabarcoding approach: First data.

In recent decades, show caves have begun to suffer from microorganism proliferation due to artificial lighting installations for touristic activity. In addition to the aesthetic problem, light encourages microorganisms that are responsible for physical and chemical degradation of limestone walls, speleothems and prehistoric paintings of cultural value. Microorganisms have previously been described by microscopy or culture-dependent methods, but data provided by new generation sequencing are rare. The authors identified, for the first time, microorganisms proliferating in one Swiss and in four French show caves using three different primers. The results showed that both photosynthetic and non-photosynthetic bacteria were the dominant taxa present in biofilms. Microalgae were heavily represented by the Trebouxiophyceae, Eustigmatophyceae and Chlorophyceae groups. Twelve diatoms were also recorded, with dominance of Syntrichia sp. (96.1%). Fungi were predominantly represented by Ascomycota, Zygomycota and Basidiomycota, fully half of the sampled biofilms where Fungi were detected. Comparing microbial communities from bleach-treated caves to those in untreated caves showed no significant difference except for a low-level change in the abundance of certain taxa. These findings provided by Illumina sequencing reveal a complex community structure in the 5 caves based on the assembly of bacteria, cyanobacteria, algae, diatoms, fungi and mosses.

Bleaching of biofilm-forming algae induced by UV-C treatment: a preliminary study on chlorophyll degradation and its optimization for an application on cultural heritage.

Green microalgae colonizing stone surfaces represent a major problem for the conservation of heritage monuments, since they lead to biodegradation and aesthetic issues. Previous studies in La Glacière show cave (France) have demonstrated that UV-C may have a strong effect on microalgae, thus leading to chlorophyll bleaching, which was increased when biofilms were maintained under VIS-light condition unlike to those maintained in the dark. To understand the physiological mechanisms underlying this response and in order to optimize in situ treatment, 30 kJ m-2 UV-C exposure times were applied to Chlorophyta Chlorella sp. and chlorophyll degradation kinetics were then monitored. UV-C irradiation was enough to inhibit photosynthesis and to directly kill all algal cells. Results also showed that chlorophyll a was degraded faster than chlorophyll b and that 14 h were necessary for complete degradation of all the present chlorophyll. In addition, our results highlighted the importance of visible light exposition after UV-C treatment which leading to chlorophyll bleaching. Irradiated algae cultivated in the dark were still green 5 days after treatment while cultivated samples in the light lost their green color after 14 h. An efficient UV-C treatment applicable to show caves and other heritage monuments was proposed.

UV-C as an efficient means to combat biofilm formation in show caves: evidence from the La Glacière Cave (France) and laboratory experiments.

Ultra-violet C (UV-C) treatment is commonly used in sterilization processes in industry, laboratories, and hospitals, showing its efficacy against microorganisms such as bacteria, algae, or fungi. In this study, we have eradicated for the first time all proliferating biofilms present in a show cave (the La Glacière Cave, Chaux-lès-Passavant, France). Colorimetric measurements of irradiated biofilms were then monitored for 21 months. To understand the importance of exposition of algae to light just after UV radiation, similar tests were carried out in laboratory conditions. Since UV-C can be deleterious for biofilm support, especially parietal painting, we investigated their effects on prehistoric pigment. Results showed complete eradication of cave biofilms with no algae proliferation observed after 21 months. Moreover, quantum yield results showed a decrease directly after UV-C treatment, indicating inhibition of algae photosynthesis. Furthermore, no changes in pigment color nor in chemical and crystalline properties has been demonstrated. The present findings demonstrate that the UV-C method can be considered environmentally friendly and the best alternative to chemicals. This inexpensive and easily implemented method is advantageous for cave owners and managers.

UV-C as a means to combat biofilm proliferation on prehistoric paintings: evidence from laboratory experiments.

A laboratory investigation of UV-C effects was conducted over a 62-h period: a much higher dose than in classic UV-C treatment was applied to five pigments and two painting binders used by prehistoric humans. Colorimetric parameters were compared to a control to see if UV-C can change pigment and binder color. Infrared spectroscopy, scanning electron microscopy, inductively coupled plasma and X-ray crystallography were also carried out to confirm colorimetric measurement. In order to understand how microorganism may physically deteriorate paintings, limestone blocks were painted and monitored until their complete colonization by algae, cyanobacteria, fungi and/or mosses. The results show that UV-C has no effect on mineral compounds. Conversely, it is noteworthy that binder color changed under both UV-C light conditions as well as in visible light. Concerning painted blocks, a fast proliferation has been observed with deterioration of the paintings. These results show the high importance of treating biofilm as soon as possible. Moreover, these findings may be a promising avenue inducing cave managers to use friendly UV-C light to treat contaminated cave paintings and also in the prevention of biodeterioration by lampenflora.

Browning phenomenon of medieval stained glass windows.

In this work, three pieces of historical on-site glass windows dated from the 13th to 16th century and one archeological sample (8th century) showing Mn-rich brown spots at their surface or subsurface have been characterized by optical microscopy and Scanning Electron Microscopy coupled with Energy Dispersive X-ray spectroscopy. The oxidation state of Mn as well as the Mn environment in the alteration phase have been characterized by X-ray absorption spectroscopy at the Mn K-edge. Results show that the oxidation state of Mn and therefore the nature of the alteration phase varies according to the sample considered and is correlated with the extent of the brown alteration. The larger the brown areas the more oxidized the Mn. However, by contrast with literature, the samples presenting the more extended brown areas are not similar to pyrolusite and contain Mn mainly under a (+III) oxidation state.

Factors driving epilithic algal colonization in show caves and new insights into combating biofilm development with UV-C treatments.

The proliferation of epilithic algae that form biofilms in subterranean environments, such as show caves, is a major problem for conservators. In an effort to reduce the use of chemical cleansers when addressing this problem, we proposed investigating the effects of UV-C on combating algal biofilm expansion in a cave located in northeastern France (Moidons Cave). First, the biofilms and cavity were studied in terms of their algal growth-influencing factors to understand the dynamics of colonization in these very harsh environments. Next, colorimetric measurements were used both to diagnose the initial colonization state and monitor the UV-C-treated biofilms for several months after irradiation. The results indicated that passive dispersal vectors of the viable spores and cells were the primary factors involved in the cave's algae repartition. The illumination time during visits appeared to be responsible for greater colonization in some parts of the cave. We also showed that colorimetric measurements could be used for the detection of both thin and thick biofilms, regardless of the type of colonized surface. Finally, our results showed that UV-C treatment led to bleaching of the treated biofilm due to chlorophyll degradation even one year after UV-C treatment. However, a re-colonization phenomenon was colorimetrically and visually detected 16months later, suggesting that the colonization dynamics had not been fully halted.

Invasion of the French paleolithic painted cave of Lascaux by members of the Fusarium solani species complex.

A major fungal invasion was discovered in the prehistoric painted cave of Lascaux in France in Sep 2001. At least three species of the Fusarium solani complex were isolated and identified with a portion of the translation elongation factor 1alpha gene (EF-1alpha), a portion of the nuclear large subunit rDNA (LSU) and nuclear ribosomal intergenic spacer region (ITS). This study represents the first time that Fusarium species have been reported from a cave containing prehistoric paintings. Significant interspecific molecular variability was observed, suggesting that there might have been repeated introduction of the species, possibly carried by water from soils above the cave.