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.

Kombucha is an acidic non-alcoholic fermented tea beverage that has surged in popularity over the past decade. A key driver of this popularity is the perception that kombucha contains microbes that contribute to gut health, a perception that is reinforced by package claims such as “live” and “probiotic”. We surveyed retail-available non-alcoholic (soft) and alcoholic (hard) kombucha and selected 39 products, of which 74.4% included at least one of these claims. Based upon Nuclear Magnetic Resonance (NMR) evaluation of the chemical composition, approximately one-third of soft kombucha products were re-categorized as “soft-aberrant” on the basis of their alcohol by volume (ABV) exceeding 0.5%. Across all three categories, the majority (82.1%) of products were “live”, containing culturable yeast and/or bacteria. However, there were no significant differences in median maximum colony-forming unit (CFU) counts between these categories, nor was there a difference according to package claims. Interestingly, only 6.3% of soft and 10% of soft-aberrant kombucha products exceeded ${10}^6$ CFU/mL, the threshold that would deliver at least one billion cells in one package. None of the sampled hard kombucha exceeded this threshold. Metabarcoding analyses of microbial communities revealed differences in composition between categories and according to package claims. Notably, the subset of products with “probiotic” claims that listed Bacillus coagulans as an ingredient were enriched in read counts for Bacillus.