Development of a kombucha metagenome catalogue including a one-year longitudinal sampling

Abstract: Kombucha microbiome exhibits dynamic community structures that drive fermentation. While biofilm and liquid phases interact throughout fermentation, their temporal microbiome succession and functional relationships remain underexplored. High–throughput shotgun metagenomic sequencing was performed on samples collected longitudinally from kombucha biofilms and liquid phases over a year–long fermentation (n=73). To evaluate temperature effects, the first half of the fermentation was performed at 20 °C, then shifted to 30 °C. To explore alternative ways of production, various sugar sources were tested, including simple sugar, mashed beans, hydrolysed bread cream, and ground bread. The dataset was integrated with public data (n=53) and enriched with a metadata curation (N_metadata=14), collecting information like fermentation conditions, sugar source, and geographical origin. Thus, a comprehensive catalogue of kombucha microbiome (n=126) was built. Our findings revealed differences in α– and β–diversity depending on the metadata considered, as well as different distribution patterns of microbes and key functional genes associated with cellulose production, flavours and health benefits. Explainable machine learning models classified microbiome across metadata, highlighting microbial markers of kombucha features. Additionally, phylogenetic analyses unveiled strain–level microbial diversity, while functional genomics linked taxa with functional metabolic pathways. Critically, our analysis yielded to 789 metagenome–assembled genomes, incrementing existing kombucha microbiome resources and highlighting previously undescribed taxa. This first longitudinal catalogue of kombucha offers a comprehensive view of biofilm–liquid microbiome dynamics during one year of fermentation. Our findings enhance our understanding of microbial ecology in kombucha fermentations, offering new insights into microbial contributions to kombucha quality and potential health benefits, providing genomic-resolved resources for supporting precision fermentation approaches.