Faisl Bousta

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 caves 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.

An overview of techniques for the characterization and quantification of microbial colonization on stone monuments

Biodeterioration can be defined as any undesired change of the properties of a material caused by biological activity of living organisms. The biodeterioration of stone materials is related to the production of pigments (aesthetic action), to cell metabolism (biochemical action) and to the mechanical action of the biomass colonizing the material during its growth (physical action). Quantification of the sessile biomass and characterization of microbial communities colonizing stone are essential first steps to ensure the diagnosis of biodeterioration processes and to implement control strategies and appropriate treatment. Different destructive and non-destructive approaches can be used to sample stone specimens from monuments: scraping, swab using, and cutting. Different analytical methods can be used depending on the type of microorganism sought: determination of chlorophyll content and color analysis for pigmented microorganisms; measurement of in situ physiological activity of surface microcolonies by applying fluorogenic substrate analogues or confocal laser scanning microscopy observations after CTC staining for active biomass; scanning or transmission electron microscopy observation for biofilm visualization; enzyme-linked immunosorbent assay for the investigation of both microorganisms that can and cannot be cultured; classical microbiological methods, which consist in cultivation of microorganisms on synthetic media; and molecular methods for the study of microbial biodiversity based on the polymorphism of molecular markers using PCR, hybridization, classical or high throughput sequencing. The aim of this review is to present basics of the different biodeterioration mechanisms and to focus on the main techniques that can be used to characterize and quantify the biodeterioration biomass.

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.

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.

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.