Successive metabolite fluxes shape a domesticated water kefir microbial community

Water kefir is a mildly alcoholic fermented beverage produced by a domesticated consortium of yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) embedded in a polysaccharide biofilm known as the kefir “grain”. Here we present a comprehensive, multi-omics dissection of a model water kefir community to understand how successive metabolite fluxes shape the spatio-temporal organization of this microbial ecosystem. Focusing on one representative domesticated grain, we isolated 18 microbial strains and assembled high-quality genomes for each, then recovered an additional genome for an uncultured LAB (Oenococcus sicerae) from metagenomic and Hi-C data. In total, 19 distinct species (> 99% of community reads) were identified, providing the first near-complete genomic catalog of a water kefir consortium. Time-resolved fermentation experiments revealed that the community progresses through three metabolic phases: an initial yeast-dominated phase fermenting sugars to ethanol and CO2, a middle phase where LAB proliferate on remaining sugars, producing lactic acid and mannitol and a late phase where AAB oxidize yeast-derived ethanol to acetic acid. These successional metabolic fluxes were reflected in measured metabolite dynamics and shifts in community composition. Our findings demonstrate that cross-feeding of metabolites drives a spatio-temporal division of labor in the kefir grain, enabling a stable and resilient fermentation process. This work provides a high-resolution view of a long-term domesticated microbial community and suggests design principles for constructing synthetic kefir consortia for research and industrial applications