Next Generation Sequencing applied to Cultural Heritage: detection of microorganisms present on the parchment surface of an ancient Gradual of the 13th century

The original integrity of Cultural Heritage (CH) may be threatened by the microbial deterioration. Conservation strategies to prevent biodeterioration needs the identification of microorganisms colonizing the substrate. In the last decades microbial monitoring has relied on culture-dependent methods. The most significant limit is the cultivability of the microorganisms and their following identification. In recent years, the development of next generation sequencing (NGS) approach provided new tools for their identification. We design and apply a diagnostic protocol on an ancient Gradual of the 13th century by using a targeted metagenomic analysis carried out by Nanopore Technologies consisting of: 1) Non-invasive sampling (sterile swab); 2) DNA extraction; 3) whole genome pre-amplification (WGA); 4) PCR amplification of conserved regions ITS (fungi), 16S (bacteria) and 18S (eukaryotes); and 5) amplicon-sequencing with MinION (ONT). Fungal species identified as Serpula lacrymans, Postia, and Alternaria alternata, may be related to wood degradation (in fact the cover of the manuscript is made of wood), while Aspergillus sp. and Penicillium sp. to parchment degradation. For bacteria instead: Cutibacterium acnes, Finegoldia magna, Bacillus mansiliensis, and Staphylococcus epidermidis, all related to the human skin flora, and which therefore could be present due to handling over the years. Their presence could pose a risk for the Gradual preservation since these species can degrade collagen naturally present in the parchment. This work shows that the NGS applied to CH may produce valuable insight, for the diagnostic of potential threats of biodeterioration.