## 4 Discussion  
Bacterial cellulose is receiving a great deal of attention due to several features that make it suitable for many biotechnological applications, such as biomedical devices, food packaging components and engineering materials [1; 2; 35]. Figure 5: Correlation plot of the most abundant clades. Spearman correlation among time, environmental variables and obtained yields. (**a**) All retrieved clades were compared with time and environmental variables. Yield obtained within (**b**) clade 5, (**c**) clade 6 were compared with time and fraction variables. For clade 7 (**d**), includes only isolates sampled from liquid fraction. It is well known from the literature that kombucha tea represents one of the most abundant reservoirs of BC producer bacteria, in particular, strains belonging to the _Komagataeibacter_ genus [10; 12]. In this study, kombucha tea was chosen as a selective source for recovering cellulose producing bacteria. A total of 46 isolates were selected as the main components of the liquid and pellicle fractions of GTK and BTK kombucha tea samples and their phenotypic and genotypic variability was evaluated. RFLP of 16S rRNA gene and an analysis of the interspecific variability of isolates was conducted, and the results were discussed, considering the available genome sequences of strains K1G4 and K2G30. The data obtained by RFLP analysis showed a very low variability in terms of the number of different species isolated from kombucha tea. This result is in agreement with previous studies reflecting both the low species diversity in kombucha tea [10], as well as the difficulty of taxonomic assignment of _Acetobacteraceae_ by using 16S rRNA gene, due to its similarity in high phylogenetic related organisms, as extensively reported in the literature [36]. Considering the analysis of the intraspecific variability within bacteria, usually, genotyping is performed using the amplification of highly conserved tandem repeat regions that are widely dispersed in the genome. The amplification of repeated elements of genomic DNA, such as (GTG)\({}_{5}\) and ERIC sequences, offers some advantages in terms of costs and rapidity, and is well developed for several Gram-negative and Gram-positive bacterial species, such as _Lactobacillus_[37] and _Enterococci_[38] and acetic acid bacteria [39]. In this study, the clustering analysis using the digitized patterns of (GTG)\({}_{5}\) elements revealed the clustering of the 46 isolates into five large clusters considering a cluster cut-off value of 50% in similarity. The discrimination power of this analysis was 0.94 considering a similarity threshold of 70%. At this similarity value, a total of 20 different biotypes were detected. Similar results were obtained using the amplification of ERIC repeated elements. Considering the cluster cut-off analysis, the comparison of the digitized patterns of ERIC rep-PCR showed a clustering of the 46 isolates with a similarity percentage of 50%, the same result obtained using (GTG)\({}_{5}\) rep-PCR. The discrimination power of ERIC fingerprint was 0.95, very similar to the (GTG)\({}_{5}\) rep-PCR, considering a similarity percentage of 70. Based on these results, a total of 19 different clades were identified, compared to the 20 obtained using (GTG)\({}_{5}\) rep-PCR. In both the fingerprint approaches, K1G4 and K2G30, previously identified as _K. xylinus_[14; 34], were considered as reference strains and they were clustered into two different clades. Among the two reference strains, the percentage of similarity was 44.2% in the case of (GTG)\({}_{5}\) rep-PCR and 56.7% for ERIC rep-PCR. These results support the hypothesis that most of the strains analyzed belong to the _K. xylinus_ species and that the discrimination was performed at the strain level. In order to improve the genotyping analysis, a combined model of the two fingerprint assays was performed (Figure 3). The resulting analysis showed a clusterization of the isolates into seven clades based on the cluster cut-off value of 88% of pattern similarity.