Tailor-made microbial consortium for Kombucha fermentation: Microbiota-induced biochemical changes and biofilm formation

15Kombucha is a very distinct naturally fermented sweetened tea that has been produced for 16 thousands of years.Fermentation relies on metabolic activities of the complex autochthonous 17 symbiotic microbiota embedded in a floating biofilm and used as a backslop for successive 18 fermentations.Here, we designed a tailor-made microbial consortium representative of the 19 core Kombucha microbiota to drive this fermentation.Microbial (counts, metagenetics), 20 physico-chemical (pH, density) and biochemical (organic acids, volatile compounds) 21 parameters were monitored as well as biofilm formation by confocal laser scanning microscopy 22 and scanning electron microscopy.While 9 species were co-inoculated, 4 (Dekkera 23 bruxellensis, Hanseniaspora uvarum, Acetobacter okinawensis and Lacitilactobacillus nagelii) 24 largely dominated.Microbial activities led to acetic, lactic, succinic and oxalic acids being 25 produced right from the start of fermentation while gluconic and glucuronic acids progressively 26 increased.A distinct shift in volatile profile was also observed with mainly aldehydes identified 27 early on, then high abundances of fatty acids, ketones and esters at the end.Correlation 28 analyses, combining metabolomic and microbial data also showed a shift in species 29 abundances during fermentation.We also determined distinct bacteria-yeast co-occurence 30 patterns in biofilms by microscopy.Our study provides clear evidence that a tailor-made 31 consortium can be successfully used to drive Kombucha fermentations.32 33