## 3 Results and Discussion
### Shotgun Metagenomic Analysis of M. gisevii MI-2 and Fermented Kombucha
Using the classic microbiology method (cultivation on selective mediums in vitro) or 16S rRNA gene sequencing, it was possible to identify only a part of microorganisms, usually at the genus level. [PERSON] et al. [68] identified 3 LAB (lactic acid bacteria), 13 AAB (acetic acid bacteria), and 11 species of yeasts from black or green tea kombucha according to 16S rRNA gene sequencing data and 26S metabarcoding analysis and other microorganisms in fermented product remained unidentified. This study revealed the full composition of the symbionts and showed that the biodiversity of _M. gisevii_ can be much wider, and the amounts of substances produced can be modeled by creating the conditions for the action and dominance of more than 250 species of microorganisms. A wide range of bacteria of the genus _Sphingobium_ in _M. gisevii_ MI-2 is worth mentioning. It is known that they are notable for their ability to degrade a variety of complex and toxic organic compounds. _Sphingobium_ bacteria is important in bioremediation efforts to clean up contaminated environments, such as soil and water [69]. Other microorganisms such as heterotrophic \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Kingdom**} & \multicolumn{2}{c}{**Species in _M. gisevii_ MI-2 and fermented—kombucha L4-100**} & \multicolumn{2}{c}{**Species in Kombucha L4-100**} \\ \cline{2-5}  & **Number** & **\%** & **Number** & **\%** \\ \hline Bacteria & 227 & 81.07 & 318 & 82.60 \\ Eucaryote & 44 & 15.72 & 38 & 9.87 \\ Virus & 9 & 3.21 & 28 & 7.28 \\ Archaea & 0 & 0 & 1 & 0.26 \\ Total & 280 * & & 385 * & \\ \hline \hline \end{tabular}  
* A full list of microorganisms is in Supplementary Material S2 (Excel file).  
\end{table} 
Table 6: Number of species in _M. gisevii_ MI-2 and fermented—kombucha L4-100. Figure 1: Core-pan genome in Venn diagram of _M. gisevii_ MI-2 (MG_MI-2) and fermented Kombucha (FP). (**A**)—eucaryote; (**B**)—bacteria; (**C**)—virus. Geminicoccus harenae_[70], obligate intracellular _Rickettsiales_ bacterium [71], _Zymomonas mobilis_ with the ability to ferment sugars into bioethanol [72] or _Burkholderia_ spp. as source of novel antibiotics and bioactive secondary metabolites [73], halophilic black yeast _Hortaea verneckii_[74] or symbionts of _Cococcarpia genus_[75] are present in low abundance in \(M\). _gisevii_ MI-2, but must play an important role in the \(M\). _gisevii_ symbiotic system, which will need to be detailed in the future. Comparison of top species (taxonomy abundance > 0.5% in Kingdom) of bacteria and eucaryotes obtained in metagenomes of \(M\). _gisevii_ MI-2 (MG) and fermented kombucha L4-100 are shown in the Sankey diagram (Figure 2). All the top acetic acid bacteria belong to the family _Acetobacteraceae_. Eukaryote--all yeasts dominated in both metagenomes and belonged to the families _Pichiaaceae_ and _Saccharomycesaceae_. In the metagenome of \(M\). _gisevii_, MI-2 in the first place was \(K\). _xylinus_ (60.4%), and in the second place \(N\). _hansenii_ (16.4%). Meanwhile, the metagenome of the kombucha was dominated by \(N\). _hansenii_ (54.1%) and \(K\). _saccharomyces_ (25.6%). The most common yeast in both cases was the same species, \(B\). _bruxellensis_--75.9% in \(M\). _gisevii_ MI-2 and 94.3% in FP. Results showed that \(P\). _membranificiens_ was not dominant in kombucha. Other microorganisms that have a rather active influence on the fermented product were the bacteria \(K\). _obediens_, \(K\). _europeus_, and \(K\). _intermedius_ and the yeasts--_Pichia californica_, \(Z\). _bailii_, and \(Z\). _paraballi_. According to data from [PERSON] et al. [76], the three main groups of microorganisms in \(M\). _gisevii_ are AAB (_Komagataeibacter_, _Glucobacter_, _Aceptobacter_ spp. ), LAB (_Lactobacillus_, _Lacetococcus_ spp.) and yeasts (_Schizosaccharomyces pombe_, _Saccharomyces ludwigii_, _Kloeckera apiculata_, _Saccharomyces cerevisiae_, _Zygosaccharomyces bailii_, _Toruspora delbrueckii_, _Brettanomyces bruxellensis_).