Cultivating Uncultured Anaerobes From Pit Mud Of Chinese Strong-aroma Type Liquor

The quality of pit mud is closely related to the quality of Chinese strong-aroma type liquor.Pit mud is an important habitat for anaerobic microbes,mainly including archaea and bacteria.The metabolic activities of these anaerobes are indispensable for the formation of aroma and flavor of Chinese strong-aroma type liquor.However,current researches on pit mud microbiota are still mainly focused on the direct analysis of microbial community structures using high-throughput sequencing techniques.Except some important caproate-and butyrate-producing microbes that have been cultivated,a large number of microbes in pit mud are currently uncultivable yet.Culture-independent techniques cannot analyze functions of pit mud microbiota,cultivation is still the most direct method to study the metabolic characteristics and brewing functions.The existence of a large number of uncultured microbes has led to the imperfection of current reference databases,which has seriously hindered the analysis of the classification and function potentials of pit mud anaerobes.Therefore,the rapid establishment of suitable cultivation technology for uncultured microbes in pit mud is necessary to deeply analyze the classification and function potentials of pit mud microbiota and provide theoretical guidance for the production of Chinese strong-aroma type liquor.In this study,pit mud of Chinese strong-aroma type liquor fermentation was used as the research object,and oligotrophic culture method was used.As a result,a cultivation technology at consortia level was established based on enrichment culturing.At the same time,according to the metabolic characteristics of pit mud microbes,targeted enrichment of short-chain fatty acid-utilizing bacteria(including syntrophic bacteria and methanogens,etc.)other than caproate-and butyrate-producing bacteria was performed.Then 16 S r RNA gene amplicon sequencing technology was used to study the community structure of enriched consortia,and metagenomic technology was further applied to analyze the species classification and functional information.Major methanogenic pathways were also reconstructed.The main results of this study are as follows:(1)CGM,NBM and MCI media with different nutrients were used to enrich pit mud anaerobes.16 S r RNA gene amplicon sequencing was applied to analyze the structure of the enriched consortia under three culture conditions,and the oligotrophic MCI medium was determined to be the best medium for enriching pit mud microbiota.A total of 8 phyla,11 classes and 14 families were detected at phylum,class and family level separately,which were more than the results of CGM and NBM media.Besides,bacteria and archaea were simultaneously enriched,including Euyarchaeota,Firmicutes,Bacteroidetes and so on,which are important components of pit mud microbiota.(2)Using MCI medium as the basic medium,three carbon source conditions,acetate,butyrate,butyrate & formate,were used to enrich pit mud microbiota according to the metabolic characteristics of syntrophic bacteria and methanogens.16 S rRNA gene amplicon sequencing was combined to analyze the structure of the enriched microbiota.It can be determined that microbial diversity of the enrichment culture was higher regardless of the carbon source conditions used.High-and low-abundance microbes in the pit mud can be effectively enriched.Using acetate as major carbon source,aceticlastic methanogens including Methanosaeta and Methanosarcina were enriched.For the microbes with abundances no less than 1% at genus level,the enrichment efficiency can reach 33.33% and 50% separately in the two enrichment groups.However,this method cannot effectively enrich syntrophic microbes.Using butyrate as major carbon source,Syntrophomonas was enriched.Relative abundance was up to 1.33% and 1.95% separately in two groups.For archaeal communities,the aceticlastic methanogen Methanosarcina,hydrogenotrophic methanogens including Methanoculleus and Methanobacterium,were successfully enriched.For the microbes with abundances no less than 1% at genus level,the enrichment efficiency can reach 66.67% and 81.82% separately.Using butyrate & formate as carbon sources,for the microbes with abundances no less than 1% at genus level,the enrichment efficiency was 55.56% and 66.67% separately.Syntrophomonas and Norank_f_Syntrophomonadaceae were successfully enriched,with the highest abundance of 13.23% and 1.58%,3.98% and 1.94% in two groups respectively.The major methanogens in pit mud are commonly composed of hydrogenotrophic methanogens including Methanobrevibacter,Methanoculleus and Methanobacterium,which were also successfully enriched.It can be inferred from the comparison that the combination of butyrate and formate can most effectively maintain the structure of the dominant microbial structure in pit mud.(3)Species classification and function potentials of acetate-and butyrate & formate-enriched microbiota were mined using metagenomic analysis.Based on the metagenomic data,26 and 33 high-quality genomic drafts were assembled.The major methanogens of the two enriched consortia were annotated to Methanosaeta concilii and Methanoculleus marisnigri.Major syntrophic bacteria in butyrate & formate-enriched microbiota was annotated to Syntrophomonas wolfei.Major methanogenic pathways were also constructed.The combination of metagenomic technique and oligotrophic culturing can analyze the genomic information of uncultured microbes in pit mud.