## 4 Molecular Analyses
### DNA Analysis
#### 4.2.2 Microbiome Analyses and Applications
As mentioned in the Introduction section, since ancient times, the salting of animal skins has been used wherever salt was affordable and available to prevent the skins from puttefying [64]. Salting was carried out either dry or in brine tanks, where the skins were left for several days. During the salting process, NaCl gradually penetrated the skin's inner surface, thereby making the parchment's environment extremely salty [65; 66]. In addition, the salt that was used also introduced marine microorganisms that are present in natural sea salt, such as halophilic archaea and halotolerant bacteria, and it acted as a kind of culture/enrichment medium for these microorganisms [5]. Halophilic/halotolerant microorganisms were therefore, the \"pioneers\" in the colonization of parchments, as only these microorganisms can thrive in such extreme salinity conditions. In fact, [PERSON] et al. [5; 20] proposed a model of microbial succession in which the halophilic archaea would be the first colonizers of the parchment, but subsequently, as the humidity increased and salinity decreased, these microorganisms would gradually disappear and give way to the growth of halotolerant bacteria, such as some groups of _Actinobacteria_, _Proteobacteria_, but also _Frimicutes_, which could grow consuming the haloarchaeal remains. In fact, this model fits very well with the observations that other authors have made and with our own results. In the data that were obtained from our studies on the old Slavonic documents, we could still detect traces of the pioneer colonizers, i.e., archaea, in all the documents except for _L. Sinaiticum_ (see Figure 5). The retrieved sequences showed that they are affiliated with the family _Halobacteriaceae_, specifically with the genus _Halobacterium_ and _Halococcus_[3; 8] (also detected in the _Glagolita Clozianus_ in this study). Figure 5: Shotgun metagenomics showing the relative abundance (%) of bacteria, eukaryota, archaea and viruses in the microbiomes of ancient Slavonic codices [3; 8] and in the _Glagolita Clozianus_ (in this study). The low proportion of archaea in the samples may be due to the limited availability of the ancient DNA strands when assessing the colonization process from a dynamic microbial succession perspective. As [PERSON] et al. [22] explain in their review, a metagenome analysis provides a two-dimensional representation of the colonization process on the parchment, which, in contrast, occurred in a three-dimensional perspective, including the layering of the generations of different microbes over the centuries. In this way, the most recent colonizers quantitatively outnumber the older ones, and they are easily detectable due to the well-preserved state of their DNA. Some of these more recent colonizers are halotolerant bacteria [5; 20]. Our results also agree with these observations, and they show the same phyla that were mentioned by these authors in the microbial succession on the parchments (see Figure 6), but, in addition, we could detect members of the phylum _Bacteroidetes_ in two of the samples that were analyzed, although in a very low proportion. The members of this last phylum were also detected in some of our previous studies which were devoted to ancient parchment manuscripts [51]. Figure 6 shows that the relative proportions of the above-mentioned phyla can vary in the different manuscripts that were investigated, even in the samples that were taken from different folios of the same codex, suggesting that this may reflect different stages in the microbial succession in each sampled specimen. As mentioned above, one part of the secondary colonizers of parchments are still the halotolerant bacteria belonging to the phyla _Actinobacteria_, _Firmicutes_ and _Proteobacteria_.