## 3 Results
### Diversity of Acetic Acid Bacteria from Kombucha Tea  
The RFLP analysis on 16S rRNA, using _Rsal_ and _Alul_ restriction endonucleases, was conducted as a preliminary test to investigate the diversity of isolates recovered from kombucha tea samples. The mapping analysis based on the size of the fragments obtained by _Rsal_ and _Alul_ showed high homogeneous results, indicating the isolates as members of the same species. This evidence was in agreement with a previous study [16] which grouped _Komagatebacter_ strains according to _Rsal_ and _Alul_ restriction enzymes and observed similarities between the pattern of _K. xylinus_ DSMZ 2004 with the _Komagatebacter_ strains investigated in this study. The low variability of the fingerprinting pattern is also consistent with previous studies on the high similarity of the 16S rRNA gene among the _Komagatebacter_ genus and especially within the _K. xylinus_ species [32; 33]. From the genotyping of strains performed by using (GTG)5 and ERIC fingerprinting techniques, two dendrograms were generated (Figure 2). Based on the cluster cut-off analysis, profiles obtained using the amplification of interspersed tandem repeats GTG (Figure 2) were capable of discriminating between the strains considering a minimum percentage of similarity of 27.9%. A total of five major clades were created with a discrimination power of 0.42, calculated using the D index (Simpson index) [27]. In order to  
Figure 1: Bacterial cellulose yields of selected strains isolated from Kombucha tea. Each value is the mean of three parallel replicates \(\pm\) standard deviation. improve the discrimination power of the analysis, we considered a minimum percentage of similarity of 70%. At this percentage of similarity, the resulted discrimination power was 0.94, defining 20 different biotypes. Among the detected clades, eight of them included just one isolate while seven were formed by two isolates. It was possible to distinguish two major clades, of which, one included nine isolates (clade 18) and another included five isolates (clade 19). The two reference strains, represented by K1G4 (=UMCC 2947) and K2G30 (=UMCC 2756) clustered into two different clades, represented by clades 19 and 7, respectively. The similarity percentage among the two references was 40.2%. The phylogenetic tree obtained from the digitized pattern profile using ERIC rep PCR is represented in Figure 3. Based on the clustering cut-off analysis, ERIC was able  
Figure 2: Dendrograms obtained from UPGMA analysis, using Dice’s coefficient, of the digitized patterns obtained from (GTG)\({}_{5}\) rep-PCR. The similarity threshold for biotypes discrimination was 70%. The cophenetic coefficient is represented by numbers and dots colored as red–orange–yellow–green, based on the branch quality. to discriminate using a minimum similarity percentage of 40%, identifying five major clades, as in the case of (GTG)\({}_{5}\). The clustering cut-off analysis discriminated a total of 19 biotypes, with a discrimination power of 0.95, slightly higher compared to (GTG)\({}_{5}\). A total of six clades were represented by just one isolate, while the remaining isolates were well distributed among the detected clades. Three clades were represented by five isolates (clade 7, 16, and 17) and two clusters by four isolates (clade 8 and 9). The remaining clades were represented by three and two isolates. The number of detected clades was not so different compared to (GTG)\({}_{5}\) and, also in this case, the reference strains (K1G4 and K2G30) were found to be separated into two different groups (clade 15 for K1G4 and clade 12 for K2G30). The similarity of Dice indexes based on the patterns obtained from ERIC rep-PCR was 56.7%; quite similar to that obtained using (GTG)\({}_{5}\) rep-PCR (40.2%). Figure 3: Dendrograms obtained from UPGMA analysis, using Dice’s coefficient, of the digitized patterns obtained from ERIC rep-PCR. The similarity threshold for biotypes discrimination was 70%.