Shedding Light on the Formation and Structure of Kombucha Biofilm Using Two-Photon Fluorescence Microscopy

Kombucha pellicles are often used as inoculum to produce this beverage and have become a signature feature. This cellulosic biofilm produced by acetic acid bacteria (AAB) involves yeasts, which are also part of the kombucha consortia. The role of microbial interactions in the de novo formation and structure of kombucha pellicles was investigated during the 3 days following inoculation, using two-photon microscopy coupled with fluorescent staining. Aggregated yeast cells appear to serve as scaffolding to which bacterial cellulose accumulates. This initial foundation leads to a layered structure characterized by a top cellulose-rich layer and a biomass-rich sublayer. This sublayer is expected to be the microbiologically active site for cellulose production and spatial optimization of yeast-AAB metabolic interactions. The pellicles then grow in thickness while expanding their layered organization. A comparison with pellicles grown from pure AAB cultures shows differences in consistency and structure that highlight the impact of yeasts on the structure and properties of kombucha pellicles.

Automated summary

Kombucha pellicles, produced by acetic acid bacteria and yeasts, show a layered structure where yeast cells act as scaffolding for bacterial cellulose accumulation. The sublayer rich in biomass is crucial for cellulose production and optimizing yeast-AAB metabolic interactions. Yeasts significantly impact the structure and properties of kombucha pellicles compared to those grown from pure AAB cultures.