## 3 Results and Discussion
### Molecular Characterization of Microbial Community (Fungi and Bacteria)  
Molecular characterization was conducted via a culture-dependent approach in order to isolate and carry out further investigation on the microbial community present on the artwork and in particular the potential biodeteriorative microorganisms harbored. DNA was extracted from all bacterial and fungal strains of all clusters isolated from the oil on canvas painting; they were identified by 16S rRNA gene and ITS sequencing respectively. Results are shown in Tables S3 and S4. _Bacillus_ was the predominant bacterial genus (clusters I, VII, and XIII). Cluster I was the most abundant, with 20% of bacterial isolates. One member of cluster I, isolated from two sampling points on the reverse side of the painting, showed 96% similarity with _Bacillus_ genus. Another member of cluster I, isolated in correspondence with sample 6R from the reverse side of the canvas was similar to _Bacillus subtilis_ with 100%. _Bacillus_ spp. are Gram-positive spore-forming bacteria, ubiquitous in the environment (soil and water) and dominant over artefact surfaces [31], such as deteriorated historical paper [41; 42]. _Bacillus_ is also responsible for structural changes of carboxymethyl cellulose during biodeterioration processes [43] and it is able to produce amylase, cellulase, protease, and acids, which are known to contribute to archaeological manuscript biodeterioration [44]. Three sampling points (21F, 18F, and 22F) chosen from the obverse side of the canvas allowed 98-99% identification of isolates with _Bacillus thuringiensis_ (cluster I), _B. simplex_ (cluster XIII), and _B. luteus_ (cluster VII). Cluster VII is the second most abundant cluster, with 13.80% of bacterial isolates. Bacterial stains belonging to cluster VII were isolated from sampling points 11F, 13F, 16F, 22F, and 24F. Other identified bacteria on the obverse side belong to _Cellulosinicrobium_, _Paenibacillus_, _Pseudomonas_, _Stentrophomonas_, and _Microoccus_ genera, while isolates affiliated to the Xanthomonadaceae family and _Streptomyces_ were collected from the reverse side of the canvas. _Streptomyces_, isolated from stained areas of the paint layer of oil paintings, have been responsible of bio-pigment production [45]. _Pseudomonas_ sp. can cause different types of surface deterioration, such as pigmentation, efflorescence and patinas [46]. _Paenibacillus_ genus was isolated from human and environmental samples [47]. _Xanthomonas_ shows cellulose structure degradation activity [48]. _Sctonrophomonas_ was isolated and identified from an oil painting on canvas, which showed visible signs of biodeterioration [49]. Gram-positive _Microoccus luteus_ is known to attack cellulosic materials by lytic enzymes and pigmented components [50]. _Pichia occidentalis_ can be used for biological detoxification of lignocellulosic hydrolysate, because it can degrade volatile fatty acids [51]. A Phaeosphaeriaceae species was isolated and identified on aged oil sludge-contaminated soil [52]. Among the fungi, sequence analysis for ITS fragments revealed that the isolates exhibited 99-100% similarity to three main genera, among which the _Penicillium_ genus was the most abundant. Sequences retrieved from sampling points on the obverse side of the canvas showed 99-100% similarity with _Penicillium_ sp. According to the literature, _Penicillium_ is associated with poorly ventilated, moist environments, representing a risk for human health and cultural heritage [39]. Several studies reported the enzymatic activity in biodeterioration of archaeological documents, and cultural heritage in general [53]. _Penicillium chrysogenum_, a member of the most abundant cluster II, was identified from sampling points on both obverse and reverse sides and it is known for its ability to deteriorate cellulose and lignin [54]. The majority of the isolates showed affiliation to _Cephaloteca foveolata_ and to _Apergillus versicolor_, _Aspergillus insuetus_, and other _Aspergillus_ spp.