Metabarcoding of the kombucha microbial community grown in different microenvironments

Abstract

Introducing of the DNA metabarcoding analysis of probiotic microbial communities allowed getting insight into their functioning and establishing a better control on safety and efficacy of the probiotic communities. In this work the kombucha poly-microbial probiotic community was analysed to study its flexibility under different growth conditions. Environmental DNA sequencing revealed a complex and flexible composition of the kombucha microbial culture (KMC) constituting more bacterial and fungal organisms in addition to those found by cultural method. The community comprised bacterial and yeast components including cultured and uncultivable microorganisms. Culturing the KMC under different conditions revealed the core part of the community which included acetobacteria of two genera Komagataeibacter (former Gluconacetobacter) and Gluconobacter, and representatives of several yeast genera among which Brettanomyces/Dekkera and Pichia (including former Issatchenkia) were dominant. Herbaspirillum spp. and Halomonas spp., which previously had not been described in KMC, were found to be minor but permanent members of the community. The community composition was dependent on the growth conditions. The bacterial component of KMC was relatively stable, but may include additional member-lactobacilli. The yeast species composition was significantly variable. High-throughput sequencing showed complexity and variability of KMC that may affect the quality of the probiotic drink. It was hypothesized that the kombucha core community might recruit some environmental bacteria, particularly lactobacilli, which potentially may contribute to the fermentative capacity of the probiotic drink. As many KMC-associated microorganisms cannot be cultured out of the community, a robust control for community composition should be provided by using DNA metabarcoding.

Figure 1

Profiles of bacterial species of KMC-IMBG1 grown in sterile black tea with sugar (sBTS), non-sterile black tea with honey (BTH) and non-sterile black tea with sugar (nsBTS) identified by binning of 16S rDNA reads.

Figure 2

Profiles of yeast species in KMC-IMBG1 grown in sterile black tea with sugar (sBTS), sterile black tea with honey (BTH) and non-sterile black tea with sugar (nsBTS) identified by binning of ITS reads.

Figure 3

Normalized abundance of the most frequent OTUs of KMC identified by BLASTN in a 16S rDNA and b ITS reads. A1—liquid phase of the culture; A2—cellulose based biofilm. Numbers of identified reads were normalized by the total numbers of reads in the samples.

Figure 4

Statistical analysis of metabarcode datasets based on 16S rDNA (parts a and c) and ITS (pats b and d) amplicons. Dendrograms in a and b of diversity of datasets were built by neighbor–joining algorithm based on distance tables calculated by Eq. 2. Parts c and d represent rarefication curves.