From Biofilms to Preservation: Addressing Microbial-Induced Stone Discoloration at Batalha Monastery

Throughout history, stone has been a preferred material for the construction of iconic structures due to its aesthetic appeal and intrinsic properties. However, like other construction materials, stone is subject to continuous deterioration by microorganisms, making its preservation critical for the protection of cultural heritage. Batalha Monastery (Portugal), a UNESCO World Heritage Site, exhibits notable surface alterations in the stone architectural elements of both the Founder's Chapel and the church, including extensive pink discoloration affecting the walls and columns. The use of both culturedependent and -independent methods for identifying the colonizing microbiota indicated that the main microorganisms forming the observed biofilms were the bacterial genera Halalkalicoccus, Bacillus, Gordonia, Serratia, and Methylobacterium. In this study, one of the bacterial isolates identified in the stone, from the genus Gordonia, was selected to carry out biocolonization tests on limestone, aiming to mimic the discoloration process observed in the monument. Two stone mock-ups were prepared, one kept in darkness and the other exposed to sunlight, to compare the bacterial behavior under different light conditions. Colorimetry and RAMAN spectroscopy techniques were employed, allowing for the in-situ characterization of color changes and pigments produced by the isolate. Furthermore, cleaning tests were also carried out on the mock-ups, in an attempt to remove the carotenes produced. The results from the aforementioned techniques revealed that the bacteria used in the tests, induced significant colorimetric changes in the stone. These changes were more pronounced when the stone was exposed to sunlight compared to those observed under dark conditions. In addition, the results of the cleaning tests demonstrated that the concentrations of the cleaning solution used are highly effective in removing pigments and can safely be suitable for heritage use.In order to prevent the deterioration of this cultural asset and ensure its preservation, we consider this study to be a critical step towards formulating a precise and effective conservation and intervention strategy for the Batalha Monastery.