Exploration of microbiome diversity of stacked fermented grains by flow cytometry and cell sorting

Sauce-flavor baijiu is one of the twelve flavor types of Chinese distilled fermented product. Microbial composition plays a key role in the stacked fermentation of Baijiu, which uses grains as raw materials and produces flavor compounds, however, the active microbial community and its relationship remain unclear. Here, we investigated the total and active microbial communities of stacked fermented grains of sauce-flavored Baijiu using flow cytometry and high-throughput sequencing technology, respectively. By using traditional high-throughput sequencing technology, a total of 24 bacterial and 14 fungal genera were identified as the core microbiota, the core bacteria were Lactobacillus (0.08-39.05%), Acetobacter (0.25-81.92%), Weissella (0.03-29.61%), etc. The core fungi were Issatchenkia (23.11-98.21%), Monascus (0.02-26.36%), Pichia (0.33-37.56%), etc. In contrast, using flow cytometry combined with high-throughput sequencing, the active dominant bacterial genera after cell sorting were found to be Herbaspirillum, Chitinophaga, Ralstonia, Phenylobacterium, Mucilaginibacter, and Bradyrhizobium, etc., whereas the active dominant fungal genera detected were Aspergillus, Pichia, Exophiala, Candelabrochaete, Italiomyces, and Papiliotrema, etc. These results indicate that although the abundance of Acetobacter, Monascus, and Issatchenkia was high after stacked fermentation, they may have little biological activity. Flow cytometry and cell sorting techniques have been used in the study of beer and wine, but exploring the microbiome in such a complex environment as Chinese baijiu has not been reported. The results also reveal that flow cytometry and cell sorting are convenient methods for rapidly monitoring complex microbial flora and can assist in exploring complex environmental samples.