Early Detection of Phototrophic Biofilms in show caves: a novel approach

Abstract

The preservation of paleolithic paintings in European caves faces significant challenges due to the proliferation of phototrophic organisms driven by artificial lighting and high CO2 levels. This study focuses on the early detection and characterization of faint green biofilms of Chlorophyta on the Polychrome Panel in El Castillo Cave, Cantabria, Spain (Figure 1). Despite limited biomass availability hindering direct molecular analyses, we propose an alternative enrichment method to study the microbial communities involved in the initial stages of colonization. This methodology involves cultivating a small amount of green biofilm in BG11 medium at 22ºC and low light intensity for three months. Biomass was collected from each culture, and DNA was extracted for sequencing. Sequencing was performed using Next Generation Sequencing (NGS) of the V3 and V4 regions of the 16S rRNA gene, and the region V4 of the 18S rRNA gene. The sequencing of the 16S rDNA region allowed the identification of prokaryotes and the 18S rDNA region identified eukaryotic organisms. Our findings reveal the presence of diverse prokaryotic and eukaryotic communities, including amoebae and their associated microorganisms, as well as the green algae Jenufa and Coccomyxa. This confirms that the original microbial community developed well in the laboratory conditions. This research provides valuable insight into the complex dynamics of microbial colonizations in cave environments and strategies for the early detection of phototrophic communities which may aid in the conservation of cultural heritage sites.