Sequence-based Analysis of the Bacterial and Fungal Compositions of Multiple Kombucha (Tea Fungus) Samples AND Sequencing-based Analysis of the Microbial Composition of Water Kefir and Corresponding Grains from Multiple Sources

Kombucha is a sweetened tea beverage that, as a consequence of fermentation, contains ethanol, carbon dioxide, a high concentration of acid (gluconic, acetic and lactic) as well as a number of other metabolites and is thought to contain a number of health-promoting components. The sucrose-tea solution is fermented by a symbiosis of bacteria and yeast embedded within a cellulosic pellicle, which forms a floating mat in the tea, and generates a new layer with each successful fermentation. The specific identity of the microbial populations present has been the focus of attention but, to date, the majority of studies have relied on culture-based analyses. To gain a more comprehensive insight into the kombucha microbiota we have carried out the first culture-independent, high-throughput sequencing analysis of the bacterial and fungal populations of 5 distinct pellicles as well as the resultant fermented kombucha at two time points. Following the analysis it was established that the major bacterial genus present was Gluconacetobacter, present at >85% in most samples, with only trace populations of Acetobacter detected (95% in the fermented beverage, with a greater fungal diversity present in the cellulosic pellicle, including numerous species not identified in kombucha previously. Ultimately, this study represents the most accurate description of the microbiology of kombucha to date. Water kefir is a water-sucrose based beverage, fermented by a symbiosis of bacteria and yeast to produce a final product that is lightly carbonated, acidic and that has a low alcohol percentage. Water kefir is considered a functional food with which a number of health benefits have been associated. These beverages contain a consortium of bacteria and yeast which are introduced via water kefir grains, which consist of a polysaccharide matrix in which the microorganisms are embedded. With that, our aim was to provide a comprehensive sequencing-based analysis of the bacterial population of water kefir beverages fermentate and grains while providing an initial insight into the corresponding fungal population. To facilitate this four water kefirs were sourced from the UK, Canada and the United States. Culture-independent, high-throughput sequencing based analyses revealed that bacterial portion of each water kefir and grain was dominated by Zymomonas, an ethanol-producing bacterium, which has not been detected at such a scale previously. The other genera detected were representatives of the lactic acid bacteria (Lactobacillus [present consistently at considerable levels] and Leuconostoc [less common]) and acetic acid bacteria (Acetobacter and Gluconacetobacter [present at low concentrations]). Our analysis of the fungal component established that it was comprised of the genera Dekkera, Hanseniaspora, Saccharomyces, Zygosaccharomyces, Torulaspora and Lachancea. This information will facilitate the generation of a greater understanding of the health promoting attributes to water kefir beverages and the ultimate identification of the microorganisms responsible for these traits.