## 4 Molecular Analyses
### DNA Analysis
#### 4.2.2 Microbiome Analyses and Applications
Regarding the _Actinobacteria_, the members of the order _Pseudonocardiales_ have been suggested as being the dominant ones [5; 20], with some of the identified genera being _Saccharomycespora_, _Saccharomycespora_ and _Pseudonocardia_[2; 3; 8; 51] (which were also detected in _Glagolita Clozianus_ in this study, see Figure 7). However, also, some species belonging to the genera _Nocardiopsis_, such as _N. xinjiangensis_ and _N. kunsanensis_, which are tolerant to salt, alkalis and desiccation [67; 68] and _Actinopolyspora_ and species of _Yaniella_, as _Y. halotolerans_ have been identified as being part of the core microbiome of the parchments [3; 51]. Figure 6: Shotgun metagenomics showing the relative abundance (%) of bacterial phyla in the microbiomes of ancient Slavonic codices [3; 8] (and _Glagolita Clozianus_ in this study). Regarding the _Firmicutes_, some of the halotolerant members of the genera _Gracilbacillus_, _Halobacillus_[3; 51] and _Salinicoccus_[8] have been identified in previous studies on parchments, as well as in the _Glagolita Clozianus_ in this study (see Figure 7), but also of the genus _Staphylococcus_[2; 3; 8]. Specifically, the species _S. equorum_ exhibits a high salt tolerance (up to 25% (_w/v_)), and it has been shown to be part of the core microbiome of several investigated parchment manuscripts [3; 8] (and in this study, see Figure 7). Several studies have also demonstrated the presence of _Proteobacteria_ in the parchment microbiome, with most of the sequences being related to the _Gammaproteobacteria_ class. [PERSON] et al. [5] reported on the halotolerant aquatic or marine _Gammaproteobacteria_ in a study that was conducted on a scroll from the Vatican Secret Archives. The authors identified a high proportion of members of the order _Vibrionales_, but also relative proportions of _Alteromonasales_, _Aerononadas_, _Oceanospirillales_ and _Chromatiales_. Among them, the most frequent genera were: _Alivibrio_, _Allomonas_, _Photobacterium_ and _Vibrio_, but _also_, there were _Aeromonas_, _Halomonas_, _Marinomonas_, _Pseudoalteromonas_, _Rheinheinera_ and _Shewanella_. In addition, the same authors reported on a relative high proportion of the order _Pseudomonasales_ in this scroll. The presence of _Proteobacteria_ has also been reported in our studies on the ancient Slavonic documents (see Figure 6), where we also detected the dominance of _Gammaproteobacteria_. However, the genera we found do not match the saline bacteria that were found by [PERSON] et al. [5]. Instead, we found sequences that are related to the genera _Pantoea_, _Enhydrobacter_, _Pseudomonas_, _Acinetobacter_ and _Haemophilus_ in our previous studies [3; 8], as well as in the study of the _Glagolita Clozianus_ (this study, see Figure 7). Some of these genera were also identified in other studies by molecular  
Figure 7: Relative abundance of the bacterial taxa (genera or family) showing \(\geq\)0.5% of total bacterial reads in the microbiome of the _Glagolita Clozianus_. _Actinobacteria_: blue (47% of bacteria); _Firmicutes_: orange (46% of bacteria); _Proteobacteria_: green (5% of bacteria). Others: grey (bacteria showing a relative proportion under 0.5% cut-off). means [5; 18], as well as cultivated from parchments in studies that were conducted by other authors [52; 69]. The members of the _Alphaproteobacteria_ and _Betaproteobacteria_ have been also identified in parchments, but with lower proportions, with species of the genera _Bartonella_, _Brevundimonas_ and _Neisseria_[3; 5] (the last one also detected in this study, see Figure 7). It is important to note that some of the halotolerant bacteria that were identified in the parchments pose a potential risk for this material. As some examples, the species of the genus _Nocardiopsis_ produce a large number of bioactive compounds and excrete extracellular enzymes such as proteases and keratinases, which facilitate the utilization of the proteins in nature [70].