Three historical manuscripts, two on parchment (dated 1280 and 1514) and one on paper support (dated 1589–1592), were under study. The three manuscripts were strongly attacked by microorganisms exhibiting dark brownish stains all over the surface except in the adjacent areas to some of the used inks, where a halo around the written text could be observed. In order to understand the origin of these halos, inks and manuscript supports were analyzed using portable energy-dispersive X-ray spectroscopy. Characteristic elements (Fe, Cu, Zn, and Mn) from iron gall inks were identified. Inks surrounded by a halo had in common a high amount of Zn in their composition. Microbiologic assays were performed aseptically on collected samples from the areas of the document with significant contamination and degradation. Samples were inoculated in a selective culture media, and the microorganisms developed were identified according to the macroscopic and microscopic features. For the evaluation of microflora proliferation, scanning electron microscopy was used. Furthermore, in vitro tests were carried out in the presence of zinc sulfate, revealing inhibition capacity for the majority of fungi sampled from these manuscripts.
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