A first insight on the biodegradation of limestone - the case of the World Heritage Convent of Christ

The present study is a multidisciplinary approach applied to architectural stone materials of the Convent of Christ in Tomar (Portugal) in order to understand and mitigate the active decay processes. The structure and appearance of the stonework from the Convent of Christ are strongly affected by stains, biofilms and structural degradation. To investigate these phenomena, a multianalytical approach comprising X-ray microdiffraction, scanning electron microscopy, microRaman and microinfrared spectroscopy was applied to the examination of altered outdoor stone areas being detected calcium oxalates, carotenoids and microbial proliferation. The presence of these alteration products seems to be correlated with the microbial activity of bacteria, microalgae, cyanobacteria and filamentous fungi. This work showed that the application of complementary methodologies is an efficient strategy to characterise the stone decay, and constitute a starting point for successful conservation intervention plans that are urgent to ensure the preservation and safeguard of this emblematic monument.

DETECTION OF SPORES AND HYPHAE OF ARTWORKS’ BIODETERIOGENIC FILAMENTOUS FUNGI BY RNA-FISH

Filamentous fungi are a threat to the conservation of Cultural Heritage. Thus, detection and identification of viable filamentous fungi are crucial for applying adequate Safeguard measures. RNA-FISH protocols have been previously applied with this aim in Cultural Heritage samples. However, only hyphae detection was reported in the literature, even if spores and conidia are not only a potential risk to Cultural Heritage but can also be harmful for the health of visitors, curators and restorers. Thus, the aim of this work was to evaluate various permeabilizing strategies for their application in the detection of spores/conidia and hyphae of artworks’ biodeteriogenic filamentous fungi by RNA-FISH. Besides of this, the influence of cell aging on the success of the technique and on the development of fungal autofluorescence (that could hamper the RNA-FISH signal detection) were also investigated. Five common biodeteriogenic filamentous fungi species isolated from biodegradated artworks were used as biological model: Aspergillus niger, Cladosporium sp, Fusarium sp, Penicillium sp. and Exophialia sp. Fungal autofluorescence was only detected in cells harvested from Fusarium sp, and Exophialia sp. old cultures, being aging-dependent. However, it was weak enough to allow autofluorescence/RNA-FISH signals distinction. Thus, autofluorescence was not a limitation for the application of RNA-FISH for detection of the taxa investigated. All the permeabilization strategies tested allowed to detect fungal cells from young cultures by RNA-FISH. However, only the combination of paraformaldehyde with Triton X-100 allowed the detection of conidia/spores and hyphae of old filamentous fungi. All the permeabilization strategies failed in the Aspergillus niger conidia/spores staining, which are known to be particularly difficult to permeabilize. But, even in spite of this, the application of this permeabilization method increased the analytical potential of RNA FISH in Cultural Heritage biodeterioration. Whereas much work is required to validate this RNA-FISH approach for its application in real samples from Cultural Heritage it could represent an important advance for the detection, not only of hyphae but also of spores and conidia of various filamentous fungi taxa by RNA-FISH.