Biofilm communities survey at the areas of salt crystallization on the walls of a decorated shelter listed at UNESCO World cultural Heritage

The microbial diversity on the walls of the UNESCO World cultural Heritage site Laugerie-Haute east shelter, France, was surveyed in a context of degradation by formation of salt efflorescences. Samples of salt efflorescence and underlying rock were collected in autumn and spring. Sessile microorganisms were studied by cultivation, microscopy, and molecular methods. Concentrations of the order of 103 fungal and 107 bacterial cells per gram of sample were quantified by culturing at both seasons. Higher concentrations (107 to 109 cells per gram) of sessile microorganisms were determined by microscopic counting after DAPI staining with a significant increase in sessile cells in spring. The presence of biofilms was confirmed by electron microscopy with the observation of microorganisms embedded in an abundant matrix in association with rock. Members of Firmicutes, Actinobacteria, and Ascomycota were identified among the cultivable sessile microflora. Bacillus herbersteinensis and Engyodontium album were the only microorganisms to be isolated in both seasons. Sequence analysis of 16S rRNA genes and internal transcribed spacer clone libraries showed dominance of Bacteroidetes, Proteobacteria, and Ascomycota. Bacteroidetes and Actinobacteria were more abundant in autumn, and in spring, respectively. Cyanobacteria were present at both seasons. Bacterial richness increased during spring. Whatever the season, clones affiliated to the 4 bacterial genera Aureimonas, Sphingopyxis, Halomonas, and Crossiella, and to the two fungal genera Engyodontium and Teratosphaeria were present. In conclusion, we demonstrated the presence of abundant biofilm at efflorescence salt on the walls of the shelter; that was increased in quantity and diversity in spring.