Development of non-invasive diagnostic methods for monitoring biodeterioration of monuments

A large part of the world’s most precious cultural heritage and artworks are made of stone with a finite life, and they are slowly but irreversibly disappearing. Biofilms living at rock-atmosphere interface are heterogeneous, complex consortia whose ability to alter properties of the substratum is defined biodeterioration. This phenomenon received serious attention by scientists only within the last three decades. The present work is aimed to advance the understanding of mechanisms involved in microbial biodeterioration: new investigation tools for the investigation of the biofilms are indeed required, so that the needs of small amount of sampling material to be analyzed in non-invasive and highly reproducible assay can be satisfied. Three sampling campaigns were carried out at the archaeological sites of Oplontis, Pompeii and at Phlegrean Phields. In a series of in vitro colonization experiments, the pioneer attitude of the fungi Fusarium solani and Alternaria tenuissima as well as the cyanobaterium Oculatella subterranea, was tested and monitored for a short-term period. Through the use of many variants of microscopy included CLSM and computer image analysis it has been possible to depict fine structure and architecture of the studied microrganisms, in a controlled environment where the realistic conditions of the respective sampling points have been reproduced. A novel approach for the study of subaerial biofilms via the construction of qPCR primers and fluorescent internal probes is also proposed, based on a deep survey on microrganisms occurring over stone monuments in European countries. A further proposed tool is the characterization of microbial diversity through the use of flow cytometry; phototrophic components of sampled biofilms were analyzed with flow cytometry, which allowed the sorting of the two Genera Cyanidium and Galdieria. Species identification was later obtained with the use of novel-designed species-specific primers targeting plastidial gene rbcL.