Comprehensive survey of kombucha microbial communities of diverse origins and fermentation practices

Abstract

Kombucha is a unique, naturally fermented sweetened tea produced for thousands of years, relying on a symbiotic microbiota in a floating biofilm, used for successive fermentations. The microbial communities consist of yeast and bacteria species, distributed across two phases: the liquid and the biofilm fractions. In the fermentation of kombucha, various starters of different shapes and origins are used, and there are multiple brewing practices. By metabarcoding, we explored here the consortia and their evolution from a collection of 23 starters coming from various origins summarizing the diversity of kombucha fermentation processes. A core microbiota of yeast and bacteria has been identified in these diverse kombucha symbiotic consortia, revealing consistent core taxa across symbiotic consortium of bacteria and yeasts from different starters. The common core consists of five taxa: two yeast species from the Brettanomyces genus (B. bruxellensis and B. anomalus) and bacterial taxa Komagataeibacter, Lactobacillus, and Acetobacteraceae, including the Acetobacter genus. The distribution of yeast and bacteria core taxa differs between the liquid and biofilm fractions, as well as between the "mother" and "daughter" biofilms used in successive fermentations. In terms of microbial composition, the diversity is relatively low, with only a few accessory taxa identified. Overall, our study provides a deeper understanding of the core and accessory taxa involved in kombucha fermentation.

Keywords: bacteria; cores; fermentations; symbiotic; taxa; yeasts.

Figure 1.

Generation of the SCOBYs and the fermentation series and collection of the samples analyzed by metabarcoding. Starters are sorted in the table at left, according to the presence or not of a cellulosic biofilm. They are denominated by acronyms in line with the provider names, letter colors indicate the technological origin of the starters (red: industrial brewery; blue: household; black: unpasteurized commercial beverage; gold: commercial starter). The steps for generating comparable initial SCOBYs and the three sequential repitchings to establish the fermentation series are represented. The samples are denominated (red labels) according to the fraction from which the cells were harvested (fermented liquid, L; daughter biofilm, D; mother biofilm, M) and according to the repitching cycles 1–3..

Figure 2.

Yeast taxonomic profiles represented by the normalized sequence count of taxa in each sample. Compositions were resolved at the genus level (A) or at the species level (B) and are represented for 46 samples from the liquid fractions across the three cycles of the fermentation series and in 30 samples from the mother and daughter biofilms of cycle 3. Sequence counts are normalized based on the sample that had the lowest number of sequences. Taxa represented by more than 0.2% of the total sequences are distinguished by the color scheme on the right of the plots. Taxa represented by <0.2% of the total sequences were merged in the “Other” categories. Empty columns correspond to the absence of data for the corresponding samples. Pc1 to Pc5 correspond to the clades defined from the analyses of sequences affiliated to the Pichia genus. Samples were sorted according to (i) the starter name, (ii) the fermentation cycle, (iii) the fraction, and (iv) the profile similarities in the major taxa resolved at the species level (visual assessment). The colored circles beneath the starter names represent their technological origin (red: industrial brewery; blue: household; black: unpasteurized commercial beverage; gold: commercial starter).

Figure 3.

Similarities in the yeast compositions across successive repitchings. (A) The abundances of yeast taxa in the samples along Axis 1 are represented by the normalized sequence counts for B. anomalus (yellow, maximum = 12 006 sequences), B. bruxellensis (red, maximum = 11 959 sequences), aggregated accessory genera (black, maximum = 9172 sequences). (B) and (C) Spatial ordination along Axis 1 and Axis 2 based on Bray–Curtis dissimilarity matrices computed on the sample compositions in yeast species. For better visibility, the samples were displayed separately: (B) samples from AK, BB1, BB2, BK, EB, FE1, FE2, FZ, HK, UR, and VK starters, and (C) samples from CP, KF1, KF2, KOK, LK, MK, and RI starters. The yellow and red dotted ellipses encircle the samples whose compositions are dominated by B. anomalus or B. bruxellensis, respectively. Samples are colored according to the original starter name as defined by the color schemes on the right, and the marker shape indicates the fermentation cycle. The colored lines and polygons connect the samples from the same starter. The colored circles next to the starter names represent their technological origin (red: industrial brewery; blue: household; black: unpasteurized commercial beverage; gold: commercial starter).

Figure 4.

Similarities in the yeast compositions in the different SCOBY fractions of the third fermentation cycle. The spatial ordination along Axis 1 and Axis 2 was based on Bray–Curtis dissimilarity matrices computed on the sample compositions in yeast genera. For better visibility, the samples from BB1, BB2, FE1, HK, and RI starters are displayed on the separate lower panel. Samples are colored according to the original starter name as defined by the color schemes on the right, and the marker shape indicates the SCOBY fraction (L: liquid; D: daughter biofilm; M: mother biofilm). The colored lines and polygons connect the samples from the same starter. The colored circles next to the starter names represent their technological origin (red: industrial brewery; blue: household; black: unpasteurized commercial beverage; gold: commercial starter).

Figure 5.

Bacteria contents and taxonomic profiles of 69 samples. Taxa were revealed by the V3–V4 libraries resolved at the genus level. Sequence counts were normalized based on the sample that had the lowest number of sequences. Taxa represented by more than 0.2% of the total sequences are distinguished by the color scheme on the right. Taxa overall represented by <0.2% of the sequences were merged in the “Other” category. (A) Abundance of each taxa in 69 available samples. (B) Abundances of the six major taxa (Acetobacter, Acetobacteraceae, Gluconobacter, Komagataeibacter, Lactobacillus, and Oenococcus) in the samples of the liquid fractions (left) or in the mother and daughter biofilms (right). In this plot, the data of the samples of AK, FE2, LK, and UR starters were excluded because they lack detection of the Lactobacillus genus. (C) Taxonomic profiles are represented by the normalized sequence count of taxa in 42 samples from the liquid fractions across the three cycles of each fermentation series and 27 samples of the mother and daughter biofilms of cycle 3. Empty columns correspond to the absence of data for the corresponding samples. Samples were sorted according to (i) the starter name, (ii) the fermentation cycle, (iii) the fraction, and (iv) the profile similarities in the major taxa resolved at the species level (visual assessment). The colored circles beneath the starter names represent their technological origin (red: industrial brewery; blue: household; black: unpasteurized commercial beverage; gold: commercial starter).

Figure 6.

Comparisons of the bacteria genus compositions in the liquid fractions across successive repitchings. The spatial ordination along Axis 1 and Axis 2 was based on Bray–Curtis dissimilarity matrices. For better visibility, the samples from AK, BK, EB, FE2, FZ, HK, and UR starters are displayed on the separate left panel. Samples are colored according to the original starter as defined by the color schemes on the right, and the marker shape distinguishes the samples according to the fermentation cycle. The colored lines and polygons connect the samples from the same starter. The barplots at left and above indicate the distribution along Axis 2 or Axis 1 of the abundances represented by the normalized sequence counts in the corresponding samples for Acetobacter (green, maximum = 5369 sequences) and Lactobacillus (purple, maximum = 7015 sequences) genera, respectively. The colored circles next to the starter names represent their technological origin (red: industrial brewery; blue: household; black: unpasteurized commercial beverage; gold: commercial starter).