In situ long-term monitoring of microbial patinas on the external marble of Florence Cathedral after treatments with essential oils

• Controlling the biological patina on stones is challenging. • Essential oils (EOs), like thyme and oregano, showed biocidal properties. • EOs treatments were assessed for long-term impact on Florence Cathedral's marble. • (In situ) treatment evaluation through ATP content, colorimetry, and microscopy. • Thyme with oregano EOs solution at 2 % was the best long-lasting treatment. The control of biological patinas on cultural heritage stones is a challenging task as treatments are often not long-lasting, and the microorganisms naturally tend to colonize any surface that meets their living requirements. Historically, the most widely used strategy to combat the biodeterioration of stone cultural heritage has involved the application of synthetic and aggressive biocides. However, recent developments have shifted towards more sustainable conservation methods, including the use of natural biocides. Among these, essential oils (EOs) have gained attention due to their well-documented antimicrobial properties. In previous work, we treated selected areas of the external marble of Florence Cathedral, which were affected by extended darkening, with thyme and oregano EOs. The efficacy of these treatments was assessed in the short- and mid-term using a multidisciplinary approach. In this work, we report the long-term evaluation (after four years) of these treatments, employing on-site microscopy, non-invasive colorimetric, and ATP (adenosine triphosphate) assays. This study shows that treatments performed with different solutions have varying efficacy over time, and that the solutions containing both thyme and oregano essential oils (OT) are those showing the best long-term effectiveness on microbial activity. This extended monitoring period is crucial for understanding the durability of the treatments and their effectiveness in controlling biodeterioration over time.