Microbial Composition of SCOBY Starter Cultures Used by Commercial Kombucha Brewers in North America

Abstract

Kombucha fermentation is initiated by transferring a solid-phase cellulosic pellicle into sweetened tea and allowing the microbes that it contains to initiate the fermentation. This pellicle, commonly referred to as a symbiotic culture of bacteria and yeast (SCOBY), floats to the surface of the fermenting tea and represents an interphase environment, where embedded microbes gain access to oxygen as well as nutrients in the tea. To date, various yeast and bacteria have been reported to exist within the SCOBY, with little consensus as to which species are essential and which are incidental to Kombucha production. In this study, we used high-throughput sequencing approaches to evaluate spatial homogeneity within a single commercial SCOBY and taxonomic diversity across a large number (n = 103) of SCOBY used by Kombucha brewers, predominantly in North America. Our results show that the most prevalent and abundant SCOBY taxa were the yeast genus Brettanomyces and the bacterial genus Komagataeibacter, through careful sampling of upper and lower SCOBY layers. This sampling procedure is critical to avoid over-representation of lactic acid bacteria. K-means clustering was used on metabarcoding data of all 103 SCOBY, delineating four SCOBY archetypes based upon differences in their microbial community structures. Fungal genera Zygosaccharomyces, Lachancea and Starmerella were identified as the major compensatory taxa for SCOBY with lower relative abundance of Brettanomyces. Interestingly, while Lactobacillacae was the major compensatory taxa where Komagataeibacter abundance was lower, phylogenic heat-tree analysis infers a possible antagonistic relationship between Starmerella and the acetic acid bacterium. Our results provide the basis for further investigation of how SCOBY archetype affects Kombucha fermentation, and fundamental studies of microbial community assembly in an interphase environment.

Keywords: Brettanomyces; Illumina sequencing; Kombucha fermentation; acetic acid bacteria; microbiome; mycobiome.

Figure 1

Schematic overview of metabarcoding and shotgun metagenomic sequencing library preparation and data analysis pipelines, as performed for Taxonomic Diversity Study. Metabarcoding of Spatial Analysis Study samples was performed using the same pipeline.

Figure 2

Spatial distribution of bacteria and fungi within sectioned Kombucha SCOBY. Quantitative real-time PCR ct-values converted to log-CFU/g against standard curves of Brettanomyces (fungi) and Gluconobacter (bacteria).

Figure 3

Spatial variation in the composition of fungal and bacterial communities within sectioned Kombucha SCOBY, derived by metabarcoding analysis. Percentages of normalized sequencing reads for each taxa are presented within concentric rings according to their spatial position (inner, mid, outer), with separate plots for top and bottom SCOBY layers.

Figure 4

Shotgun sequencing of composite ‘meta’-SCOBY. Results of Kraken2 kmer analysis presented as Krona charts for bacteria and Ascomycota, which made up 99.5% of fungal reads.

Figure 5

Microbial community composition of 103 commercial Kombucha SCOBY determined by metabarcoding analysis and divided into four k-clusters. (a) Comparison of Simpson Diversity Index. (b) Ordination plot of weighed Unifrac distance comparing beta diversity. (c) Two-way hierarchically clustered heatmap showing the relative abundance of bacterial and fungal microbial genera within individual SCOBY, grouped by similarity and divided into four k-clusters.

Figure 6

Differential phylogenic heat tree of ASV relative abundance for each cluster. Tree includes ASVs that differ significantly in median read proportion (log2 ratio of median proportions) between k-cluster groupings of 103 commercial Kombucha SCOBY. Differences depicted as greater abundance in row clusters (green) versus column cluster (brown). Significance was determined using Wilcox rank-sum tests with a false discovery rate correction for multiple comparisons.