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Microorganisms in pit mud are the essential factor determining the style of strong flavor Baijiu. The spatial distribution characteristics of fungal communities and aroma in the pit mud for strong flavor Baijiu from Xinjiang, China, were investigated using Illumina MiSeq high-throughput sequencing and electronic nose technology. A total of 138 fungal genera affiliated with 10 fungal phyla were identified from 27 pit mud samples; of these, Saccharomycopsis, Aspergillus, and Apiotrichum were the core fungal communities, and Aspergillus and Apiotrichum were the hubs that maintain the structural stability of fungal communities in pit mud. The fungal richness and diversity, as well as aroma of pit mud, showed no significant spatial heterogeneity, but divergences in pit mud at different depths were mainly in pH, total acid, and high abundance fungi. Moisture, NH4+, and lactate were the main physicochemical factors involved in the maintenance of fungal stability and quality in pit mud, whereas pH had only a weak effect on fungi in pit mud. In addition, the fungal communities of pit mud were not significantly associated with the aroma. The results of this study provide a foundation for exploring the functional microorganisms and dissecting the brewing mechanism of strong flavor Baijiu in Xinjiang, and also contributes to the improvement of pit mud quality by bioaugmentation and controlling environmental physicochemical factors.

Clostridium beijerinckii is a well-known anaerobic solventogenic bacterium which inhabits a wide range of different niches. Previously, we isolated five butyrate-producing C. beijerinckii strains from pit mud (PM) of strong-flavor baijiu (SFB) ecosystems. Genome annotation of the five strains showed that they could assimilate various carbon sources as well as ammonium to produce acetate, butyrate, lactate, hydrogen, and esters but did not produce the undesirable flavors isopropanol and acetone, making them useful for further exploration in SFB production. Our analysis of the genomes of an additional 233 C. beijerinckii strains revealed an open pangenome based on current sampling and will likely change with additional genomes. The core genome, accessory genome, and strain-specific genes comprised 1567, 8851, and 2154 genes, respectively. A total of 298 genes were found only in the five C. beijerinckii strains from PM, among which only 77 genes were assigned to Clusters of Orthologous Genes categories. In addition, 15 transposase and 12 phage integrase families were found in all five C. beijerinckii strains from PM. Between 18 and 21 genome islands were predicted for the five C. beijerinckii genomes. The existence of a large number of mobile genetic elements indicated that the genomes of the five C. beijerinckii strains evolved with the loss or insertion of DNA fragments in the PM of SFB ecosystems. This study presents a genomic framework of C. beijerinckii strains from PM that could be used for genetic diversification studies and further exploration of these strains.

Clostridium is the key bacteria that inhabits the pit mud in a fermentation cell, for the production of strong-flavor Baijiu. Its activities in the process of Baijiu fermentation is closely related to the niches of pit mud and cells. After multiple rounds of underground fermentation, Clostridium has been domesticated and adapted to the environment. The mechanisms of clostridia succession in the pit mud and how they metabolize nutrients present in grains are not clear. In this study, 15 Clostridium species including three firstly reported ones (Clostridium tertium, Clostridium pabulibutyricum and Clostridium intestinale) in strong-flavor Baijiu pit mud, were isolated from the pit mud. Eighty one percent of these Clostridium strains are motile, and most of them show chemotaxis to organic acids, glutathione, saccharides and lactic acid bacteria. In a simulated Baijiu fermentation system, Clostridium migrated from pit mud to fermented grains with the addition of chemokine lactic acid, resulting in the production of acetic acid and butyric acid. The results help to understand the succession mechanism of Clostridium in pit mud, and provide a reference for regulation of lactic acid level in fermented grains during Baijiu fermentation.

Pit mud, a specific fermented soil, is an essential material for the fermentation of Chinese strong-flavour liquor. However, few studies to date have sought to characterize the spatial profiles of pit mud fungal communities in fermentation cellars from Chinese strong-flavour liquor distilleries. In this analysis, differences in fungal community structures and physicochemical properties in pit mud samples from different spatial positions within fermentation cellars were analyzed, revealing unique characteristic multidimensional pit mud fungal community profiles. Penicillium roqueforti, Pichia kudriavzevii, Aotearoamyces nothofagi, Penicillium robsamsonii, Alternaria arborescens, Trichosporon insectorum, Seltsamia ulmi, Trichosporon coremiiforme, Malassezia restricta were dominant in the pit mud samples form the upper cellar wall, whereas Metarhizium frigidum, Calonectria pseudoreteaudii, Penicillium clavigerum, Fusarium equiseti, Simplicillium chinense, Aspergillus intermedius, Trichosporon coremiiforme, Fusarium circinatum, Alternaria radicina, Aspergillus heterocaryoticus were predominant in the middle cellar wall. Alternaria radicina, Cladosporium chasmanthicola, Alternaria helianthiinficiens, Penicillium argentinense, Antarctomyces psychrotrophicus, and Trichosporon inkin are majorly present in the down cellar wall layer. Bipolaris axonopicola, Ramgea ozimecii, Penicillium argentinense, Calonectria queenslandica, Metarhizium robertsii, and Penicillium roqueforti were identified as the dominant fungi in pit mud samples from the cellar bottom. Additionally, Alternaria destruens and Alternaria doliconidium are present at notably high levels in all layers of pit mud samples. Moisture, pH, PO43−, acetic acid, humus, K+, Mg2+, Ca2+, butyric acid, and caproic acid levels in these different pit mud positions exhibited a rising incremental pattern from the upper wall layer to the bottom layer, whereas lactic acid levels were significantly lower in the bottom pit mud layer relative to these other layers. Moisture, pH, and NH4+-N were identified as the three most significant factors associated with fungal community composition through a redundancy analysis. Overall, these findings may offer a theoretical foundation for future efforts to improve or standardize artificial pit mud.

Microbiota in the pit mud (PM) plays a crucial role in the production of Chinese strong-flavor liquor (CSFL), the most popular distilled liquor in China. However, previous studies used total microbes, instead of viable ones, for the characterization of the microbial community in this environment. In this study, we used propidium monoazide (PMA) combined with quantitative polymerase chain reaction (qPCR) and 16S rRNA gene sequencing to verify the effect of non-viablee bacteria on the characterization of PM bacteria. After PMA concentration optimization, 50 μM PMA was chosen to pretreat 5 and 20 years PMs. The qPCR results showed that there were 50.78 and 71.84% of non-viable bacteria in the 5-year PM and 20-year PM, respectively. Both copy numbers of total bacteria and viable bacteria were significantly higher in 20-year PM than those in 5-year PM. Nevertheless, in terms of bacterial diversity and composition analyses at the operational taxonomic unit (OTU), phylum, class, and genus levels, 16S rRNA gene sequencing results displayed no significant differences between total bacteria and viable bacteria in both PM types. In conclusion, it is necessary for non-viable bacteria to be considered in determining absolute biomass of bacteria in PM, but not necessary in the analysis of diversity and composition of PM bacteria. To the best of our knowledge, our study is the first attempt to analyze viable bacteria in the PM of CSFL and provides useful information on how to accurately characterize a microbial community in a PM environment.

Strong flavor baijiu (SFB), also called Luzhou-flavor liquor, is the most popular Chinese baijiu. It is manufactured via solid fermentation, with daqu as the starter. Microbial diversity of the SFB ecosystem and the synergistic effects of the enzymes and compounds produced by them are responsible for the special flavor and mouthfeel of SFB. The present review covers research studies focused on microbial community analysis of the SFB ecosystem, including the culturable microorganisms, their metabolic functions, microbial community diversity and their interactions. The review specifically emphasizes on the most recently conducted culture-independent analysis of SFB microbial community diversity. Furthermore, the possible application of systems biology approaches for elucidating the molecular mechanisms of SFB production were also reviewed and prospected.