Multidisciplinary characterization of fungi and cyanobacteria involved in biodeterioration of the Tomba delle Leonesse, Tarquinia, Italy

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.