Metagenomic Approach Studying The Taxonomic Diversity And Functional Genes Of The Pit-mud Of Chinese Liquor

The brewing of Chinese strong-aromatic liquor(CSAL)is a traditional national industry of China as well as the cultural heritage with a history of thousands of years.The CSAL is produced by using unique bioreactors that are rectangular soil fermentation pits(FPs).There are a large number of culturable and non-culturable microorganisms whose ecological composition and diversity influence and determine the quality of CSAL to a great extent in the pit-mud.In recent years,a large number of studies have been carried out on the microorganisms in pit-mud.But the obtained information about the microbial community of pit-mud is still not enough to reflect the ecological diversity entirely and objectively due to the limitations of the using research techniques and methods.At the same time,the whole genome sequencing and analysis of the pit-mud microbiotas had not been done up to now.Therefore,it is difficult to comprehensively understand the processes and mechanismes of CSAL fermentation.In the present study,the pit-mud from hundreds of years-old FPs was selected as the research object.The FPs are covering the three major production areas of CSAL and have been in use for up to 400 years.The metagenomic approach using a high-throughput whole-genome shotgun sequencing technique was applied to reveal the microbial community structure and functional genes diversity in FPs.The brewing mechanism of CSAL had been investigated based on multiple bioinformatical analytical technologies.It was expected to provide a theoretical basis for improving the yield and quality of CSAL and promote the innovation and upgrading for the traditional brewing technique.In this dissertation,we focused on following researches:(1)The entire microbial community was established based on high-throughput metagenomic data of FPs.The taxonomic results defined that the microbial community in the FPs was primarily composed of Firmicutes(54.6%),Euryarchaeota(15.3%),Bacteroidetes(10.1%),Gamaproteobacteria(5.8%),Optisthokonta(5.7%),Unclassified-Bacteria(2.3%),Chloroflexi(1.1%),Tenericutes(1.0%),Betaproteobacteria(0.9%),Actinobacteria(0.8%),Synergistetes(0.7%),Alphaproteobacteria(0.6%),Spirochaetes(0.3%),Epsilonproteobacteria(0.2%)and Others(0.6%).133 genera were identified as the dominant genera of this fermentative food.This results contribute to a deeper understanding of the FPs microbial composition and shows a new microbial resource for biotechnological applications.(2)The correlation analysis between the relative abundance of microorganisms in microbiota and the characteristic flavor components(ethyl caproate,ethyl lactate,ethyl acetate,ethyl butyrate)of CSAL had been done.The genera,including Lactobacillus,Sedimentibacter,Syntrophomonas,Methanoculleus,Methanobacterium,Bacillus,Clostridium,Galactomyces,Candida,Pichia,Penicillium,and Aspergillus,were identified as the active abundance group in FPs after the correlation analysis.Lactobacillus was closely linked with the biosynthesis of ethyl lactate for CSAL.Clostridium and Bacillus were closely linked with the biosynthesis of ethyl caproate for CSAL.Sedimentibacter was closely linked with the biosynthesis of ethyl butyrate for CSAL.Syntrophomonas,Methanoculleus and Methanobacterium were closely linked with the biosynthesis of ethyl acetate for CSAL.Candida and Pichia had the capacity of producing esters for CSAL.Galactomyces Penicillium and Aspergillus participated in the metabolism of alcohol form the fermentational raw materials of CSAL.(3)Two-way analysis of variance(ANOVA),Principal component analysis(PCA),Hierarchical clustering analysis(HCA),Heat map analysis and Venn analysis were employed to evaluate the structural changes of microbial communities in different characteristics of pit-mud.The results revealed that the microbial community structures changed significantly with different cellar ages and over different geographical regions.(i)The presence of Bacteroidetes was the most distinctive feature that characterized the different FPs ages.(ii)Distinct contents of Gammaproteobacteria and Euryarchaeota were observed at different positions in the FPs.(iii)Euryarchaeota markedly contributed to the generation of the character of the liquors with distinct geographical associations.Meanwhile,Gas Chromatography-Mass Spectrometry(GC-MS)was used to determine the volatile compounds in CSAL.A total of 49 major volatile components were identified in CSAL samples,including 12 alcohols(mainly ethanol,n-butanol,3-methylbutanol and n-propanol),8 acids(mainly acetic acid,caproic acid and butyric acid),17 esters(mainly ethyl acetate,ethyl lactate,and ethyl caproate)and a few of aldehyde and ketone.Ethyl caproate(3867.0±83.7 mg/L)(the maximum value),ethyl lactate(2601.4±280.4 mg/L),and ethyl acetate(2372.7±1.7 mg/L)are the 3 upmost flavor components had been detected in product liquors.The result indicated that the difference of microbial community structures led to the deviation of concentrations of volatile compounds in CSAL.(4)A microbiota gene catalogue of FPs was constructed based on 259 Gb of metagenomic datasets of the pit-mud,from which 1,392,928 ORFs with length of1,292,881,866 bp and a non-redundant pit-mud genes set containing 1,375,660 genes were harvested.Through further annotation using the known functional database and Venn analysis,3,379 known functional gene clusters were identified as the core functional genes set of pit-mud.(5)The metabolic characteristic of functional genes in pit-mud had been identified based on the annotation of KOs from KEGG database and COGs from egg NOG database.The results indicated that Carbohydrate transport and metabolism 6.00%,Energy production and conwersion 6.59%,Amino acid transport and metabolism 6.83%,Cell wall/membrane/envelop biogensis 6.02%,Translation,ribosomal structure and biogenesis 6.35%,Transcription 6.26%,Replication,recombination and repair 6.24%,Inorganic ion transport and metabolism 5.15%,Lipid transport and metabolism 4.78%and Function unknow up to 23.78% constituted the metabolic characteristics of FPs.This functional profile shown that the flavor compounds in CSAL was mainly come form the metabolism of carbohydrate and amino acids.(6)The maps of whole microbial metabolic pathways and the biosynthesis of secondary metabolites of pit-mud microbiotas were drawn.About 17 key-gene clusters determining the yield and quality of CSAL were identified.The potential mechanism had been found that the biosynthesis of host compounds(ethanol,ethyl caproate,ethyl lactate,ethyl acetate and ethyl butyrate)in CSAL relied on the abundance species of Lactobacillus,Clostridium,Saccharomycetales with the abundance functional genes of Co A dehydrogenase,Co A transferase and NAD dehydrogenase.Base on the biosynthesis of flavor compounds,159 catalyticases had been annotated and 27 species had been defined.The metabolic pathways of flavor compounds had been revealed.The results provide a theoretical reference for studying the substance metabolism in CSAL brewing.