The Depth-Depended Fungal Diversity and Non-depth-Depended Aroma Profiles of Pit Mud for Strong-Flavor Baijiu

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.

Analysis of Microbial Community Structure and Volatile Compounds in the Pit Mud Used for Manufacturing Taorong-type Baijiu Based on High-Throughput Sequencing

In this study, the pit mud used in manufacturing Taorong-type Baijiu was collected from the upper, middle, lower and bottom layers of pits in Henan Yangshao Liquor Co., LTD. Besides, high-throughput sequencing (HTS) technology was adopted to analyze the microbial community structure of the pit mud. In addition, the volatile compounds in the pit mud were subjected to preliminarily qualitative analysis through headspace-solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS). The results of HTS demonstrated that there were 5, 3, 5 and 5 dominant bacterial phyla (including 11, 11, 9 and 8 dominant bacterial genera) and 3, 3, 3 and 3 dominant fungal phyla (including 4, 7, 7 and 5 dominant fungal genera) in the pit mud from F-S (upper), G-Z (middle), H-X (lower) and I-D (bottom), respectively. The qualitative analysis results of volatile compounds demonstrated that a total of 78 kinds of volatile compounds were detected in the pit mud, including 46, 45, 39 and 49 kinds in the pit mud from F-S, G-Z, H-X and I-D, respectively. Ester and acid were the two main components in the pit mud. Meanwhile, the correlation between microorganisms and main volatile compounds in the pit mud was analyzed. Moreover, Lentimicrobium, Syner-01 and Blvii28_wastewater-sludge group were found for the first time in the pit mud used for manufacturing Taorong-type Baijiu. The findings of this study could provide a theoretical foundation for improving the quality of pit mud and the flavor of Taorong-type Baijiu.

Aminipila luticellarii sp. nov., an anaerobic bacterium isolated from the pit mud of strong aromatic Chinese liquor, and emended description of the genus Aminipila

A novel mesophilic, aerotolerant anaerobic bacterium, designated JN-18T, was isolated from the pit mud of a strong aromatic Chinese liquor. According to a 16S rRNA gene sequence analysis, it had the highest sequence similarity to Aminipila butyrica DSM 103574T (95.69%). The G+C content of its genomic DNA was 43.39 mol%. The cells were Gram-stain-negative, slightly curved rods with flagella. Optimum growth was observed at 37 °C, pH 6.5 and without extra addition of NaCl. Strain JN-18Tutilized amino acids (l-alanine, l-arginine, l-asparagine, l-lysine, l-methionine, l-serine and l-threonine), malate and pyruvate, and used l-arginine and l-lysine to produce acetate, butyrate, H2, and CO2. The major cellular fatty acids of strain JN-18T were C14:0, C16:0 DMA and C18:1 cis-9 DMA. The carbohydrate composition of the cell wall predominantly included galactose, glucose and rhamnose. Based on its phylogenetic, phenotypic, physiological and biochemical characteristics, strain JN-18T was classified as a representative of a novel species within the genus Aminipila , for which the name Aminipila luticellarii sp. nov. is proposed. The type strain is JN-18T (=CCAM 412T=JCM 39126T).

Spatial fungal community diversity profiles in pit mud samples from fermentation cellars used to manufacture Chinese Strong-flavour liquor

Background Few studies to date have sought to characterize the spatial profiles of pit mud microbial communities in fermentation cellars from Chinese strong-flavour liquor distilleries. This study was thus designed to evaluate these eukaryotic communities in pit mud samples via a multidimensional DGGE approach and by assessing associated sample physicochemical properties. Results Penicillium fuscoglaucum, Penicillium glandicola, Aotearoamyces nothofagi, Malassezia restricta, Penicillium lanosocoeruleum, Penicillium crustosum, and Aspergillus tonophilus were detected only in pit mud from the upper cellar wall, while Alternaria alstroemeriae, Trichosporon insectorum, Fusarium equiseti, Calonectria pseudoreteaudii, Penicillium clavigerum, Penicillium compactum, Ascochyta phacae, Metarhizium frigidum, Alternaria burnsii, Fusarium nurragi, and Didymella keratinophila were present only in the middle cellar wall layer. Alternaria zantedeschiae and Ilyonectria cyclaminicola were only present in pit mud samples from the lower cellar wall, while Leptobacillium leptobactrum, Calonectria queenslandica, Aspergillus appendiculatus, and Candida pseudolambica were only detected in pit mud from the cellar bottom. Moisture, pH, PO43−, acetic acid, humus, K+, Mg2+, Ca2+, acetic acid, 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 other layers. Conclusions A clear relationship between fungal community structure and physicochemical variables in different spatial pit mud samples, especially moisture, pH, and NH4+-N were identified as the three most significant factors associated with fungal community through a redundancy analysis.

Comparative genome analysis of Clostridium beijerinckii strains isolated from pit mud of Chinese strong flavor baijiu ecosystem

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 flavours 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 (COG) 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 (GIs) were predicted for the five C. beijerinckii genomes. The existence of a large number of MGEs 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.

Adaptability of a caproate-producing bacterium contributes to its dominance in an anaerobic fermentation system

The transformation of diverse feedstocks into medium-chain fatty acids (MCFAs) by mixed cultures is a promising biorefinery route because of the high value of MCFAs. A particular concern is how to maintain the microbial consortia in mixed cultures to achieve stable MCFA production. Chinese strong aroma-type liquor ( Baijiu ) fermentation system continually produces caproic acid for decades through a spontaneous inoculation of anaerobes from pit mud into fermented grains. Therefore, illuminating the dominant caproate-producing bacterium (CPB) in pit mud and how the CPB sustains in the spontaneous fermentation system will benefit to reveal the microbiological mechanisms of the stable caproate production. Here, we examined pit mud samples across four Chinese strong aroma-type Baijiu producing areas and found that a caproate-producing Caproicibacterium sp. was widely distributed in these distilleries with relative abundance ranging from 1.4% to 35.5% and an average abundance of 11.4%. Through controlling carbon source availability, we achieved different simplified caproate-producing consortia and found that the growth advantage of Caproicibacterium sp. was highly dependent on glucose. Then two strains, named Caproicibacterium sp. LBM19010 and Caproicibacterium sp. JNU-WLY1368, were isolated from pit mud of two regions. The metabolic versatility of this bacterium utilizing starch, maltose, glucose and lactate reflected its adaptability to the fermentation environment where these carbon sources coexist. The simultaneous utilization of glucose and lactate contributed to the balance between cell growth and pH homeostasis. This study reveals that multiple adaptation strategies employed by the predominant CPB promotes its stability and dominance in a saccharide- and lactate-rich anaerobic habitat. IMPORTANCE Chinese strong aroma-type liquor ( Baijiu ) fermentation environment is a typical medium-chain fatty acid producing system with complex nutrients. Although several studies have revealed the correlation between microbial community composition and abiotic factors, the adaptation mechanisms of dominant species to abiotic environment are still unknown in this special anaerobic habitat. This study identified the predominant CPB in Chinese strong aroma-type Baijiu fermentation system. Metabolic versatility and flexibility of the dominant CPB with a small-size genome indicated that this bacterium can effectively exploit available carbon and nitrogen sources, which could be a key factor to promote its ecological success in a multi-species environment. The understanding of growth and metabolic features of CPB responsible for its dominance in microbial community will not only contribute to the improvement of Chinese strong aroma-type Baijiu production but also expand its potential industrial applications in caproate production.

The Effect of Spatial Location of Fermentation Pit on Prokaryotic Community Diversity in Pit Mud for Chinese Strong-Flavor Baijiu Production

Background Chinese strong-flavor baijiu (CSFB) accounts for more than 70% of all Chinese liquor markets. Diverse microbes in pit mud found in the fermentation pit play a key role in CSFB production. However, the effect of spatial location on the diversity and structure of the microbial community in pit mud is still poorly understood Results Prokaryotic microbes in different pit mud（4- and 40-year）were analyzed by using Illumina MiSeq sequencing of 16S rRNA gene. The samples were collected from pit mud that was 4 and 40 years old in the top, middle, and under parts of the cellar walls and at the bottom of them. The results showed there was no significant difference (p>0.05) in the physicochemical factors, the Observed OTU, and α-diversity between the pit mud of two different ages. The index of the 4-year-old pit mud was lower (except for total acid, which was higher than) than that of the 40-year-old pit mud. The pH, total acid, Observed OTU, Chao 1, ACE in the 4-year-old pit mud (in the top and middle part of the cellar wall) and the 40-year-old pit mud(in the top part of the cellar wall) had significant (p<0.05) difference. At the phylum level, Firmicutes (56.70%) and Bacteroidetes (26.56%) accounted for more than 80% of the overall level. For the 4-year-old pit mud sample, the dominant genuses at each location were Proteiniphilum, Lactobacillus, and Caproiciproducens, while for the 40-year-old pit mud, there were 10 common genuses accounted for more than 50% of the dominant genuses in different pit mud belong to Clostridia. The RDA analysis results showed that 85.71% of the dominant bacteria positively correlated with pH, and especially the most correlated with the total acid and available phosphorus, so its content was significantly related to the dominant bacteria (p<0.01). Therefore, it inferred that the total acid and available phosphorus were the main physicochemical factors that affected the spatial distribution of prokaryotic microbial communities in the pit mud of cellars from the liquor distillery. Conclusions Comparing the young pit mud and the old pit mud, the structure and physicochemical factors of the prokaryotic microbial community in pit mud from Henan liquor-making company changed, and the changes occurred in the spatial location of different pits. Clostridia, which accounted for more than 50% of all types of pit mud, were responsible for the effect of total acid and available phosphorus on the microbial community. Based on the above, it provides a theoretical basis for Henan liquor-making companies in maintaining pit mud. Keywords: fermentation pit mud; spatial heterogeneity; microbial community; physicochemical factors; Chinese strong-flavor Baijiu.

Mining the evolutionary patterns of pit mud microbiome and driving factors under the impact of long-term strong-flavor baijiu production

Mud cellar creates a unique microenvironment for the fermentation of strong-flavor baijiu (SFB). Recent researches and long-term practice have highlighted the key roles of microbes inhabiting pit mud in the formation of SFB’s characteristic flavor. A positive correlation between the quality of SFB and cellar age was extracted from practice, however, the evolutionary patterns of pit mud microbiome and driving factors remain unclear. Here, based on the variation regularity analysis of microbial community structure and metabolites of samples from cellars of different ages (∼30/100/300 years), we further investigated the effects of lactate and acetate (main microbial metabolites in fermented grains) on modulating pit mud microbiome. Esters (50.3%-64.5%) dominated the volatile compounds identified in pit mud, and contents of the four typical acids (lactate, hexanoate, acetate and butyrate) increased with cellar age. Bacteria (9.5-10.4 lg copies/g) and archaea (8.3-9.1 lg copies/g) mainly constituted pit mud microbiota, respectively dominated by Clostridia (39.7%-81.2%) and Methanomicrobia (32.8%-92.9%). An upward trend with cellar age characterized the relative and absolute abundance of the most predominant bacterial/archaeal genus, Caproiciproducens / Methanosarcina . Correlation analysis revealed significantly ( P < 0.05) positive relationships between the two genera and major metabolites. Anaerobic fermentation with acetate and lactate as carbon sources enhanced the enrichment of Clostridia, and furthermore, the relative abundance of Caproiciproducens (40.9%) significantly increased after 15-day fed-batch fermentation with lactate compared with the initial pit mud (0.22%). This work presents a directional evolutionary pattern of pit mud microbial consortia and provides an alternative way to accelerate the enrichment of functional microbes. Importance The solid-state anaerobic fermentation in a mud cellar is the most typical feature of strong-flavor baijiu (SFB). Metabolites produced by microbes inhabiting pit mud are crucial to create the unique flavor of SFB. Accordingly, craftspeople have always highlighted the importance of pit mud microbiome and concluded by centuries of practice that the production rate of high-quality baijiu increases with cellar age. To deepen the understanding of pit mud microbiome, we determined the microbial community and metabolites of different-aged pit mud, inferred the main functional groups and explored the forces driving the microbial community evolution through metagenomic, metabolomic and multivariate statistical analyses. The results showed that the microbial consortia of pit mud presented a regular and directional evolutionary pattern under the impact of continuously batch-to-batch brewing activities. This work provides insight into the key roles of pit mud microbiome in SFB production and supports the production optimization of high-quality pit mud.