Isolation and Reassembly of Cultivable Bacteria and Yeasts for Kombucha Tea Fermentation

Kombucha tea fermentation is driven by microbial consortia composed of yeasts, acetic acid bacteria (AAB) and lactic acid bacteria (LAB), whose metabolic interactions determine the product’s functional and sensory characteristics. This study focused on the isolation and characterization of cultivable microorganisms from kombucha tea and the reassembly of four defined communities to evaluate their contribution to the chemical composition of the beverage based on the physicochemical parameters and multivariate analysis (PCA) of sugars, organic acids and ethanol. Microbial isolates, identified in this study, belonged to yeast (Saccharomyces cerevisiae and Brettanomyces bruxellensis), AAB (Novacetimonas hansenii, Komagataeibacter europaeus, Komagataeibacter intermedius and Acetobacter pasteurianus) and LAB (Liquorilactobacillus nagelii). Selected strains were combined to reassemble simplified communities. Fermentation trials demonstrated that community composition markedly influenced metabolite production and acidification (acetic acid and ethanol concentration ranged from 0.30 ± 0.08 and 2.29 ± 0.03 g/L, and from not determined to 27.31 ± 3.41 g/L, respectively). Consortia combining yeasts, AAB and LAB most closely reproduced the chemical composition of the original Kombucha tea, whereas simpler yeast–bacteria consortia produced chemically distinct beverages. Overall, these findings enhance our understanding of the ecological roles of kombucha-associated microorganisms and demonstrate that community composition is a key factor in shaping the chemical profile of the beverage. Moreover, the reassembly of defined microbial communities represents a promising strategy for selecting and applying functional microorganisms to valorize agri-food by-products through sustainable fermentation processes. Kombucha-derived communities, due to their ability to grow under acidic conditions, tolerate osmotic stress and metabolize complex sugar mixtures, could be versatile biofactories for the development of new fermented beverages or functional ingredients from low-value agri-food residues, contributing to circular bioeconomy strategies and waste reduction.