Contrasting bacterial community structure in artificial pit mud-starter cultures of different qualities: a complex biological mixture for Chinese strong-flavor Baijiu production.

The complex starter culture for artificial pit mud (APMSC) hosts a wide variety of microbial communities that play a crucial role in Chinese strong-flavor Baijiu production. Based on its organoleptic properties, the quality of APMSC can be divided into normal and inferior quality grades. However, the relationship between the APMSC microbial community and APMSC quality is poorly understood. In this study, the bacterial community structure in normal and inferior APMSC derived from two different production batches was analyzed using denaturing gradient gel electrophoresis and Illumina MiSeq sequencing. Highly similar patterns of bacterial diversity and community structure were observed in the APMSC samples of the same quality, and a significant higher bacterial species diversity (Shannon index and Chao1) was detected in the normal compared to the inferior APMSC samples. Fifteen genera were detected in the APMSC samples, and seven (Caproiciproducens, Clostridium, Lactobacillus, Bacillus, Pediococcus, Rummeliibacillus, and Sporolactobacillus) were dominant, accounting for 92.12-99.89% of total abundance. Furthermore, the bacterial communities in the normal and inferior APMSC had significantly different structure and function. The normal APMSC was characterized by abundant Caproiciproducens and Clostridium and high caproic and butyric acid contents. In contrast, the inferior APMSC was overrepresented by Lactobacillus and Bacillus and lactic and acetic acids. This study may help clarify the key microbes sustaining APMSC ecosystem stability and functionality, and guide future improvements in APMSC production.

Deep sequencing reveals high bacterial diversity and phylogenetic novelty in pit mud from Luzhou Laojiao cellars for Chinese strong-flavor Baijiu.

The pit mud (PM) in fermentation cellar is a complex ecosystem that hosts diverse microbial communities that contribute to the production of Chinese strong-flavor Baijiu (CSFB). However, the microbial ecology of PM, particularly the extent of their phylogenetic novelty remains poorly understood. Here we conducted Illumina MiSeq sequencing to explore the diversity and novelty patterns of PM bacterial communities from Luzhou Laojiao cellars in use for 40 and 400years. High diversity indices were found in the PM with 16 phyla and 105 genera. Interestingly, the compositions of dominant genera of the PM were significantly different than that reported previously for PM sampled from other geographic sites, suggesting greater microbial diversity of PM. The dominant genus of Caproiciproducens, a caproic acid-producing bacterium, is the first reported for Chinese Baijiu production. Our results demonstrate that the PM hosts a large number of novel taxa, with 26% of the total OTUs (operational taxonomic units) distant to cultured counterparts. The class Clostridia within Firmicutes presented the highest proportion of novel OTUs. Most novel OTUs were initially isolated from diverse environments, the most abundant of which came from Chinese Baijiu brewing ecosystems, highlighting the huge culturing gap within the PM, but at the same time suggesting the importance of these OTUs in CSFB production. The data presented in this study significantly increases the number of bacteria known to be associated with CSFB production and should help guide the future exploration of microbial resources for biotechnological applications.

Determination of the fungal community of pit mud in fermentation cellars for Chinese strong-flavor liquor, using DGGE and Illumina MiSeq sequencing.

Chinese strong-flavor liquor (CSFL) is fermented in cellars lined with pit mud (PM). This PM, specific fermented clay, contains microbes that play important roles in CSFL production. However, there is limited information about fungal community structure and cellar-age-related changes in PM. In this study, PM samples were removed from the cellars used for 5 and 100years and characterized using denaturing gradient gel electrophoresis (DGGE) and Illumina MiSeq sequencing. Both methods revealed there were no significant differences in fungal species diversity (Shannon index, Chao1, and observed species) between the 5- and 100-year PM samples (p>0.05), but the communities were more stable in the 100-year PM samples (UPGMA). Illumina MiSeq sequencing allowed identification of 111 fungal genera belonging to 4 phyla (Ascomycota, Zygomycota, Basidiomycota, and Chytridiomycota) in the PM samples, with the predominant phylum being Ascomycota. The results also indicated that the compositions of dominant genera in the PM samples were significantly changed during long-term CSFL fermentation. There were relatively more Rhizopus, Phoma, and Trichosporon in the 5-year PM samples, and Aspergillus and Candida were most highly represented in the 100-year PM samples (p<0.05). Of these, Candida increased its relative abundance significantly in the 100-year samples (p<0.05). Overall, the results provide novel insights into the fungal community associated with CSFL production, and may suggest why fermentation in a cellar with PM that has been in usage for a longer time allows better quality CSFL production.