## 4 Molecular Analyses
### DNA Analysis
#### 4.2.2 Microbiome Analyses and Applications
The species of _Gracilibacillus_ are able to produce proteolytic enzymes that are capable of hydrolyzing gelatin [71; 72], and elastases [73], thus cleaving the elastin, which together with collagen, determines the mechanical properties of the tissues. _Staphylococcus quorum_ exhibits efficient enzymatic activities, including proteases and lipases [74; 75]. Additionally, other species, such as _Yaniella halotolerans_ and _Halobacillus_ sp. are able to produce hydrolases [76; 77]. Finally, _Acinetobacter_ spp. are considered to have a great destructive potential for this material [8; 52]. Nevertheless, the most intriguing finding is the repeated identification of _Saccharomyces_ species in almost all of the ancient parchment manuscripts that have been analyzed by several research groups and using different molecular techniques, such as clone libraries and DGGE [52] as well as NGS using the target [5; 20] or the shotgun approach [2; 3; 8] (and this study, see Figure 7). _Saccharomyces_ has previously been described as a common microbial culprit that is detected in numerous ancient parchment documents, showing the so-called'measles-like' parchment discoloration, consisting of purple spots that are linked to localized collagen damage and parchment deterioration [51]. However, later, [PERSON] et al. [20] documented the presence of the archaea _Halobacterium salivarum_ in decayed documents that displayed the same phenomenon of purple spots. Furthermore, through a Raman analysis, the authors confirmed the presence of bacteriorhodopsin, which is the main photosynthetic protein of the archaeal _H. salivarum_, together with bacterioruberin, which is also produced by some of the species of _Actinobacteria_. In view of these findings, they postulated that the phenomenon of purple spots is triggered in the parchment initially by the haloarchae and at a later stage of microbial succession, actinobacteria and fungi are implicated in the damage that occurs due to their ability to attack the collagen in depth. This assumption does not contradict the findings that were made by [PERSON] et al. [51], where the authors postulated that actinobacteria, specifically _Saccharomyces_, together with the _Aspergillus_ species were responsible for the purple stains, but rather, these two assumptions reveal the two distinct stages of microbial succession leading to the same phenomenon of the biodeterioration of the parchment. A very similar phenomenon is the so-called \"red heat\", which is a typical red coloration found as pitting on the flesh-side surface of salted hides after their storage during the leather manufacturing process. Some authors have recently confirmed that both, the presence of purple spots and the red heat, depends on the pioneer colonization of _H. salivarum_, thereby producing the red staining due to bacteriorhodopsin [21]. The sea salt that was used for the leather curing is responsible for the transmission of the halobacteria to the hides and skins. The second group of prokaryotes that was found in the parchment microbiome are the secondary colonizers [5], and their distribution depends on the individual history of each parchment, including the specific environment where the parchment was stored (i.e., libraries, archives and churches) and its handling, history of use and the customs of the time [2; 3; 8]. Recent studies have shown that part of these secondary colonizers are typical bacteria of the human microbiome [2], and their presence on the parchment agrees with the fact that the skin microbiome is the most common component of the urban microbiome [78], and in particular, on handled surfaces. These bacteria belong mainly to two phyla, _Actinobacteria_ and _Firmicutes_. Within the _Actinobacteria_, the most dominant group are the species of _Propioniibacterium_, mainly _P. acnes_, but also of the genera _Rothia_, _Corynebacterium_ and _Brevibacterium_ as well as members of the family _Dermabacteraceae_[2; 3; 8],(and in this study, see Figure 7).