Red stains and associated microbial community of the external white marble of Florence cathedral

Red stains are a common and persistent form of deterioration affecting historic marble monuments and are notoriously difficult to remove. The red colouration has been attributed to the presence of the in- organic pigment minium (Pb3 O4 ) in many works. Previous studies have reported the presence of lead- resistant microorganisms within these stains. However, the potential microbial contribution to minium formation on marble surfaces remains unclear, and the origin of the discolourations, whether biological or inorganic, is still debated. In this study, we investigated the red discolourations on the external white marble of the Cathedral of Santa Maria del Fiore in Florence (Italy), to characterise their nature and the associated microbial community through a multidisciplinary approach. We combined chemical analyses (X-Ray Fluorescence and Raman Spectroscopy) with both culture-dependent and -independent methods. Raman spectroscopy confirmed the presence of minium in the red-stained areas. Targeted metagenomic analysis provided a first insight into the structure and diversity of the microbial community inhabiting the red-stained marble. Moreover, lead-resistant bacteria and yeasts were successfully isolated and iden- tified, providing a basis for investigating their possible role in the origin of these stains.