Formation of a Mixed-Species Biofilm Is a Survival Strategy for Unculturable Lactic Acid Bacteria and Saccharomyces cerevisiae in Daqu, a Chinese Traditional Fermentation Starter

The existence and function of unculturable microorganisms are necessary to explain patterns of microbial diversity and investigate the assembly and succession of the complex microbial community. Chinese traditional alcoholic fermentation starter contains a complex microbial community harboring unculturable species that control the microbial diversity and have distinct functions. In this study, we revealed the presence, functions, and interactions of these unculturable species. Results of microbial diversity revealed by culture-dependent and metagenomic sequencing methods identified unculturable species and the potential functional species. Unculturable Saccharomyces cerevisiae and Lactobacillus sp. had a strong ability to form biofilms and co-existed as a mixed-species biofilm in the starter community. Using a hydrolase activity assay and fortified fermentation, we determined that the function of S. cerevisiae and Lactobacillus sp. to produce ethanol and flavor compounds. Widespread microbial interactions were identified among the biofilm isolates. S. cerevisiae was the main component of the biofilm and dominated the metabolic activities in the mixed-species biofilm. The environmental adaptability and biomass of Lactobacillus sp. were increased through its interaction with S. cerevisiae. The mixed biofilm of S. cerevisiae and Lactobacillus sp. also provides a tool for correlating microbial diversity patterns with their function in the alcoholic fermentation starter, and may provide a new understanding of fermentation mechanisms. Formation of a mixed-species biofilm represents a strategy for unculturable species to survive in competition with other microbes in a complex community.