Shotgun Metagenomics of a Water Kefir Fermentation Ecosystem Reveals a Novel Oenococcus Species

Water kefir is a fruity, sour, slightly alcoholic and carbonated beverage, which is made by fermentation of an aqueous sucrose solution in the presence of dried figs and water kefir grains. These polysaccharide grains contain lactic acid bacteria (LAB), yeasts, and sometimes bifidobacteria and/or acetic acid bacteria, which consume sucrose to produce exopolysaccharides, lactic acid, acetic acid, ethanol, and carbon dioxide. Shotgun metagenomic sequencing was used to examine the microbial species diversity present at two time points during water kefir fermentation in detail, both in the water kefir liquor and on the water kefir grains, hence representing four samples. Lactobacillus harbinensis, Lactobacillus hilgardii, Lactobacillus nagelii, Lactobacillus paracasei, and a Lactobacillus species similar to Lactobacillus hordei/mali were present in the water kefir examined, along with Bifidobacterium aquikefiri and two yeast species, namely Saccharomyces cerevisiae and Dekkera bruxellensis. In addition, evidence for a novel Oenococcus species related to Oenococcus oeni and Oenococcus kitaharae was found. Its genome was derived from the metagenome and made available under the name of Candidatus Oenococcus aquikefiri. Through functional analysis of the four metagenomic data sets, it was possible to link the production of lactic acid, acetic acid, ethanol, and carbon dioxide to subgroups of the microbial species found. In particular, the production of mannitol from fructose was linked to L. hilgardii, Candidatus O. aquikefiri, and B. aquikefiri, whereas glycerol production was associated with S. cerevisiae. Also, there were indications of cross-feeding, for instance in the case of amino acid supply. Few bacterial species could synthesize a limited number of cofactors, making them reliant on the figs or S. cerevisiae. The LAB species in turn were found to be capable of contributing to water kefir grain growth, as dextransucrase-encoding genes were attributed to L. hilgardii, L. hordei/mali, and Candidatus O. aquikefiri.