Community In Pit Mud Research Articles

Mining the factors driving the succession of microbial community in pit mud used for the production of Nongxiangxing baijiu

The pit mud (PM) provides a unique anaerobic environment for the fermentation of Nongxiangxing baijiu. Previous research has indicated a close relationship between PM quality and cellar age. However, the molecular basis of microbial dynamics and associated functions of microbial community remain unclear. Here, we explored the microbial communities of PM samples from three different locations with various temporal gradients. Based on the maturity time of PM, all samples were divided into two stages, P1 (cellar age<20) and P2 (cellar age≥20), and 30 microbial genera were identified as biomarkers. Mantel test demonstrated a significant correlation between acetic acid and biomarkers in P1 stage, whereas in P2 stage, hexanoic acid and lactic acid were significantly correlated with the bacterial community. Furthermore, random forest analysis suggested that the main driving factors of the bacterial community succession were NH4+–N, available phosphorus, and moisture, while the fungal community succession were available phosphorus and acetic acid. PICRUSt2 analysis demonstrated that the bacteria in aged PM had a higher potential to synthesize butyric acid compared with the new pit mud. Results presented in this study may provide a basis for the future artificial adjustment of PM quality.

Microbial community spatial structures in Luzhou-flavored liquor pit muds with different brewing materials.

BackgroundAlthough studies have shown that Bacteroidetes, Clostridiales, and Lactobacillales are the main components of the microbial community in pit mud during the brewing of Luzhou-flavored liquor, little is known about the effect of brewing materials on spatial structures of this microbiome.MethodsHigh-throughput sequencing of the V4–V5 region of prokaryotic 16S rRNA gene was performed to analyze the microbial community diversity and spatial heterogeneity in Luzhou-flavored liquor pit muds with different brewing ingredients. The structural characteristics and heterogeneous spatial distribution of the pit mud microbial communities were examined using bioinformatics and multivariate statistical analysis methods.ResultsOur results showed that Euryarchaeota, Actinobacteria, Bacteroidetes, Chlorobi, Chloroflexi, Firmicutes, Proteobacteria, Synergistetes, Tenericutes, and WWE1 were the dominant phyla in the pit mud microbiome. The Shannon and Simpson indices of the pit mud microbiome with three grains (M3G) in the upper layer were significantly lower than those in middle layer and bottom, whereas those of the pit mud microbiome with five grains (M5G) in bottom were significantly lower than those in middle layer (p < 0.05). There were significant differences in the microbial community compositions between the pit muds with different brewing ingredients and locations in the same pit (p < 0.05). T78 of Anaerolinaceae, Butyrivibrio, Dehalobacter_Syntrophobotulus, Desulfosporosinus, Asteroleplasma, and vadinCA02 of Synergistaceae were significantly enriched in M3G, whereas Prevotella, Vagococcus, Caldicoprobacter, Butyrivibrio, Coprococcus, Dorea, Sporanaerobacter, Tepidimicrobium, TissierellaSoehngenia, RFN20 of Erysipelotrichaceae, Sutterella, 125ds10 of Alteromonadales, Vibrio, and Sphaerochaeta were significantly enriched in M5G. This study provides a theoretical basis for exploring the influence of brewing ingredients in pit muds on the production of Luzhou-flavored liquor and the specific influence of pit mud microorganisms in different locations on liquor production.

Spatial Distribution and Relationship of Volatile Compounds and Microbial Community in Pit Mud

Spatial Distribution and Relationship of Volatile Compounds and Microbial Community in Pit Mud

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.

Cooperation within the microbial consortia of fermented grains and pit mud drives organic acid synthesis in strong-flavor Baijiu production

Mud cellars have long been used as anaerobic bioreactors for the fermentation of Chinese strong-flavor Baijiu, where starchy raw materials (mainly sorghum) are metabolized to ethanol and various flavor compounds by multi-species microorganisms. Jiupei (fermented grains) and pit mud are two spatially linked microbial habitats in the mud cellar, yet their metabolic division of labor remains unclear. Here, we investigated the changes in environmental variables (e.g., temperature, oxygen, pH), key metabolites (e.g., ethanol, organic acids) and microbial communities in jiupei and pit mud during fermentation. Jiupei (low pH, high ethanol) and pit mud (neutral pH) provided two habitats with distinctly different environmental conditions for microbial growth. Lactic acid accumulated in jiupei, while butyric and hexanoic acids were mainly produced by microbes inhabiting the pit mud. Biomass analysis using quantitative real-time PCR showed that bacteria dominated the microbial consortia during fermentation, moreover cluster and principal coordinate analysis (PCoA) analysis showed that the bacterial communities of jiupei and pit mud were significantly divergent. The bacterial community diversity of jiupei decreased significantly during the fermentation process, and was relatively stable in pit mud. Lactobacillus dominated the jiupei bacterial community, and its relative abundance reached 98.0% at the end of fermentation. Clostridia (relative abundance: 42.9–85.5%) was the most abundant bacteria in pit mud, mainly distributed in the genus Hydrogenispora (5.3–68.4%). Fungal communities of jiupei and pit mud showed a similar succession pattern, and Kazachstania, Aspergillus and Thermoascus were the predominant genera. PICRUSt analysis demonstrated that enzymes participating in the biosynthesis of acetic and lactic acid were mainly enriched in jiupei samples, while the bacterial community in the pit mud displayed greater potential for butyric and hexanoic acid synthesis. Assays from an in vitro simulated fermentation further validated the roles of jiupei microbiota in acetic and lactic acid production, and these acids were subsequently metabolized to butyric and hexanoic acid by the pit mud microbiota. This work has demonstrated the synergistic cooperation between the microbial communities of jiupei and pit mud for the representative flavor formation of strong-flavor Baijiu.

Production of medium-chain fatty acid caproate from Chinese liquor distillers’ grain using pit mud as the fermentation microbes

Chinese liquor distillers’ grain (CLDG) is an abundant industrial organic waste showing high potential as feedstock for biofuel conversion. In this study, CLDG was used as substrate by microbial community in pit mud to produce medium-chain fatty acids (especially caproate). Simulated and real fermentation were used to evaluate the effect of ethanol and lactic acid being the electronic donors (EDs) during the anaerobic chain elongation (CE). The caproate concentration was achieved at 449 mg COD/g VS, with the corresponding high carbon selectivity at 37.1%. Microbial analysis revealed that the domestication of pit mud increased the abundance of Caproiciproducens (converting lactic acid into caproate) and Lactobacillus (producing lactic acid), leading to enhanced caproate production. The lactic acid conversion facilitated in full utilization of ethanol through CE consumption. The coexistence of EDs benefited the CE system and that this green energy production can be a promising high-performance biofuel donor for sustainable industrial production development.

Metagenome and analysis of metabolic potential of the microbial community in pit mud used for Chinese strong-flavor liquor production

Complex microbiomes of pit mud (PM) play significant roles in imbuing flavors and qualities of Chinese strong-flavor liquor (CSFL) during fermentation. However, understanding both of the taxonomic and functional diversity of the whole microorganisms in PM still remain a major challenge. Here, PM microbiomes were investigated based on metagenomic sequencing, assembly and binning. Metagenomic data revealed that Euryarchaeota was the predominant phylum, followed by Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria. For further functional exploration, 703 metagenome-assembled genomes (MAGs), including 304 novel strains, 197 novel species, and 94 novel genera were reconstructed. Three primary groups of Firmicutes (n = 406), Euryarchaeota (n = 130) and Bacteroidetes (n = 74), particularly genus of them Syntrophomonas, Thermacetogenium and Clostridium, methanogens (Methanobacterium, Methanoculleus, and Methanosarcina), Proteiniphilum and Prevotella, contained most of metabolic potential genes. Additionally, Chloroflexi was firstly reported to have potential to be involved in the caproic acid (CA) production. Bacteroidetes could be the key phylum to synthesize terpenes, and Armatimonadetes, Firmicutes, Ignavibacteriae and Verrucomicrobia may possess the same metabolic potential as well. Overall, this study will significantly improve our understanding of the diverse PM microbiome and help guide the future exploration of microbial resources for modifying PM fermentation processes.

Profiling prokaryotic community in pit mud of Chinese strong-aroma type liquor by using oligotrophic culturing

Pit mud microbiota plays a key role in flavour production for Chinese strong-aroma type liquor. However, the pit mud microbiota cannot be cultured in laboratory. In this study, an oligotrophic medium with acetate as carbon source was used to enrich pit mud microbiota. The 16S rRNA gene amplicon sequencing was applied to examine the microbial dynamics of the enrichment consortia. Both methanogens and bacteria were simultaneously enriched. Euryarchaeota, Bacteroidetes and Firmicutes were the top 3 enriched phyla, and 31 genera were successfully enriched. More specifically, 11 genera (65%) out of the 17 dominant genera in pit mud were successfully enriched, including Petrimonas, Proteiniphilum, Anaerocella, Hydrogenispora, Methanosarcina, Fermentimonas, LNR_A2-18, Sedimentibacter, Lutispora, Syntrophomonas and Aminobacterium. Furthermore, 20 rare genera in the analyzed pit mud samples were also enriched. Aceticlastic Methanosaeta and Methanosarcina were found to be dominant methanogens in the enrichment consortia. Metagenomic sequencing was then applied to the enriched microbial consortia to explore the metabolic potentials of pit mud microbes. Aceticlastic methanogenesis pathway of Methanosaeta was reconstructed. Furthermore, 26 high-quality metagenome-assembled genomes (MAGs) were obtained based on the metagenomic binning analysis. Moreover, nutrients in pit mud were found to be crucial to sustain the methanogenesis of the enriched microbial consortia. These results suggested that the enrichment approach by using oligotrophic culturing can effectively cultivate the pit mud microbiota. Combined with metagenomics, the oligotrophic culturing will be greatly helpful to decipher the community composition and metabolic potentials of pit mud microbiota.

Evolving the core microbial community in pit mud based on bioturbation of fortified Daqu.

Directional stress is an effective measure to change the community structure and improve the bioactivity of pit mud (PM). In this study, the addition of fortified Daqu to artificial PM (APM) was intended to disturb the microbial community and further affect metabolites. To evaluate the effect of fortified Daqu on culturing APM, the microbial communities of APM with or without the addition of fortified Daqu were investigated by fluorescence in situ hybridization and Illumina MiSeq. The results indicated that microbes (Clostridium sp., Clostridium kluyveri, hydrogenotrophic methanogens, and acetotrophic methanogens) related to the production of key aroma compounds increased notably when fortified Daqu was added. In particular, the hydrogenotrophic and acetotrophic methanogens increased by 6.19- and 4.63-fold after 30days of culture. Subsequently, metabolites (organic acids, volatile compounds) were also analyzed by HPLC (high-performance liquid chromatography) and HS-SPME-GC-MS (headspace solid phase microextraction - gas chromatography - mass spectrometry). The results showed that the content of butyric acid and hexanoic acid was significantly higher when fortified Daqu was added to APM. In addition, the proportion of esters and phenols was also higher than in APM without fortified Daqu. A survey of the microbial compositions of APMs with or without added fortified Daqu indicated that the microbial community evolves into a functional community favoring liquor brewing. We have developed a novel process by disturbing the community diversity.

Differences of the structure, succession and function of Clostridial communities between jiupei and pit mud during Luzhou-flavour baijiu fermentation

Clostridia inhabiting in jiupei and pit mud plays key roles in the formation of flavour during the fermentation process of Luzhou-flavour baijiu. However, the differences of Clostridial communities between jiupei and pit mud remains unclear. Here, the species assembly, succession, and metabolic capacity of Clostridial communities between jiupei and pit mud were analysed by high-throughput sequencing and pure culture approaches. The ratio of Clostridial biomass to bacterial biomass in the pit mud was relatively stable (71.5%-91.2%) throughout the fermentation process. However, it varied widely in jiupei (0.9%-36.5%). The dominant Clostridial bacteria in jiupei were Clostridium (19.9%), Sedimentibacter (8.8%), and Hydrogenispora (7.2%), while Hydrogenispora (57.2%), Sedimentibacter (5.4%), and Caproiciproducens (4.9%) dominated in the Clostridial communities in pit mud. The structures of Clostridial community in pit mud and jiupei were significantly different (P=0.001) throughout fermentation. Isolated Clostridial strains showed different metabolic capacities of volatile fatty acids in pure culture. Spatial and temporal heterogeneity of Clostridial communities existed in the baijiu fermentation pit, which was closely related to the main flavour components of Luzhou-flavour baijiu.

Analyzing bacterial community in pit mud of Yibin Baijiu in China using high throughput sequencing.

“Yibin Baijiu” (YB) is a special Chinese strong-aroma Baijiu (CSAB) that originated in Yibin, a city in western China. YB is fermented in cellars lined with pit mud (PM), the microbiota in which may affect YB quality. In this study, high throughput sequencing of the 16S rRNA gene was used to demonstrate the bacterial community structure and diversity in PM of YB. In addition, the physicochemical characteristics of PM were also analyzed, including moisture content, pH, and available phosphorous, ammonia nitrogen, and humic acid levels. Results showed that Firmicutes was the dominant phylum in all PM samples with abundance > 70.0%, followed by Euryarchaeota (11.3%), Bacteroidetes (6.5%), Synergistetes (3.0%), Actinobacteria (1.4%), and Proteobacteria (1.2%). Furthermore, 14 different genera with average relative abundance of > 1% were detected. The Chao1 and Shannon indexes did not vary significantly between the sub-layer and middle-layer PM (P > 0.05). However, Linear discriminant analysis Effect Size (LEfSe) analysis showed that the relative abundance of Lactobacillus in the sub-layer PM was significantly higher than in middle-layer PM. pH differed significantly (P < 0.05) between the two groups. Canonical correspondence analysis revealed that bacterial community in PM correlated significantly with available phosphorous content and pH. Our study provides basic data for further elucidating the diversity of microbiota in the PM of YB and the potential mechanism of Baijiu production.

Analysis of bacterial communities in pit mud from Zhijiang Baijiu distillery using denaturing gradient gel electrophoresis and high- throughput sequencing

Analysis of bacterial communities in pit mud from Zhijiang Baijiu distillery using denaturing gradient gel electrophoresis and high- throughput sequencing

Profiling the Clostridia with butyrate-producing potential in the mud of Chinese liquor fermentation cellar

Butyrate and its derivates pertain to the key aroma contributors of strong-flavour baijiu, a kind of Chinese liquors, that is produced from grains by solid-state multispecies anaerobic fermentation in a mud cellar. Microbes inhabiting in the fermentation pit mud largely determines baijiu's flavour and quality. In order to shed light on the microbial functional groups driving butyrate production in pit mud, clone library analysis was firstly performed and the results demonstrated that Clostridia (relative abundance: 50%) and Bacilli (37%) were major groups possessing butyrate kinase (buk) pathway and Clostridia (98%) dominated butyryl-CoA:acetate CoA-transferase (but) pathway. According to Clostridial specific-16S rRNA gene sequencing analysis, we found the resilience character of Clostridial community in pit mud. Amongst Clostridial groups, 32.0% of the sequences were grouped into Clostridiales incertae sedis, followed by Heliobacteriaceae (18.3%) and Clostridiaceae 1 (8.4%). Moreover, Hydrogenispora, Sedimentibacter and Clostridium were the top three abundant genera. Relative abundance of Hydrogenispora was higher in the late days of fermentation, while Sedimentibacter exhibited higher proportion in the early days. Different from the previous studies using universal bacterial primer sets, Hydrogenispora was first reported as one dominant genus in pit mud. As for the reported potential butyrate producer Clostridium, nineteen species were obtained and ten of them were first isolated from the pit mud. Amongst them, buk was identified in eleven species by PCR analysis, while but was identified in the other seven, indicating the species-specific butyrate synthesis pathways of Clostridium. This study provides a perspective on targeting and isolating specific functional microbes in baijiu microbiota with the gene sequence-based medium prediction method.

Prokaryotic Communities in Pit Mud from Different-Aged Cellars Used for the Production of Chinese Strong-Flavored Liquor

Chinese strong-flavored liquor (CSFL) accounts for more than 70% of all Chinese liquor production. Microbes in pit mud play key roles in the fermentation cellar for the CSFL production. However, microbial diversity, community structure, and cellar-age-related changes in pit mud are poorly understood. Here, we investigated the prokaryotic community structure and diversity in pit-mud samples with different cellar ages (1, 10, 25, and 50 years) using the pyrosequencing technique. Results indicated that prokaryotic diversity increased with cellar age until the age reached 25 years and that prokaryotic community structure changed significantly between three cellar ages (1, 10, and 25 years). Significant correlations between prokaryotic communities and environmental variables (pH, NH4(+), lactic acid, butyric acid, and caproic acid) were observed. Overall, our study results suggested that the long-term brewing operation shapes unique prokaryotic community structure and diversity as well as pit-mud chemistry. We have proposed a three-phase model to characterize the changes of pit-mud prokaryotic communities. (i) Phase I is an initial domestication period. Pit mud is characterized by abundant Lactobacillus and high lactic acid and low pH levels. (ii) Phase II is a transition period. While Lactobacillus abundance decreases dramatically, that of Bacteroidetes and methanogens increases. (iii) Phase III is a relative mature period. The prokaryotic community shows the highest diversity and capability to produce more caproic acid as a precursor for synthesis of ethyl caproate, the main flavor component in CSFL. This research provides scientific evidence to support the practical experience that old fermentation cellars produce high-quality liquor.

PCR-DGGE analysis on microbial communities in pit mud of cellars used for different periods of time

Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyse microbial community evolution in the pit mud of cellars used for different periods of time in production of Chinese Luzhou-flavour liquor. The pit mud was collected from the cellars and the microbial DNA was extracted from the microbes in the pit mud. The Bf 968 primer was used for PCR-DGGE to analyse the variable region 6 (V6) to variable region 8 (V8) of the microbial 16S rDNA. It was found that the band number, dominance, diversity and similarity of the 16S rDNA were clearly different in the DGGE patterns, because of the great diversity expressed by the different microbial communities in the different-aged cellars. It is concluded that mutual collaboration and constraint exist between the different microbial communities in the different-aged cellars, and this relationship leads to an evolutional change in the structures and in the numbers of the microbial communities in the pit mud of the cellars. Changes become more obvious with increasing age of the liquor cellars. Copyright © 2012 The Institute of Brewing & Distilling

PLFA Fingerprint Method for Assessing Microbial Communities of Strong Aromatic Liquor Pit Mud

In the view of the specificity of microbial communities in pit mud during brewing process,different types of soil samples and common strains in liquor brewing were investigated to assess the fingerprints of phospholipid fatty acid(PLFA) of microbial communities in Luzhou flavor pit mud.The results suggested the composition and content of PLFA significantly affected by physiological and environmental conditions which resulted in the significant differences(P0.05) of PLFA fingerprints among the samples(the ratio of fungal/bacterial PLFA in garden and roadside soils were 0.30 and 0.66,respectively,which were both less than 1,the ratio in pit mud however was 1.17).The content of special PLFA changed significantly(P0.05) when five pure cultured microorganisms were added.The content of a14:0 and a16:0 in pit mud adding SICC1.13 and CICC20633 were 1.44 and 9.92 times higher than those in blank respectively,while 18:2ω6,9 in SICC3398 and SICC31482 were 82.58 and 90.33 times higher than that in the blank.Furthermore,cy17:0 was identified in the pit mud adding DH5α.Principal component analysis(PCA) was performed to evaluate the changes of special PLFA’s composition and content in pit mud after adding the microorganisms.The results of this study demonstrated that PLFA fingerprint method was a fast biochemical culture-independent method,which was beneficial to characterize the fingerprints of microbial communities during liquor process.