窖泥是厌氧微生物的重要栖息地,厌氧微生物菌群的代谢活动对浓香型白酒的香气和风味形成起着重要作用。但大多数窖泥厌氧微生物尚未能在人工实验室培养条件下培养,这严重阻碍了对窖泥厌氧菌群功能的解析。采用CGM、NBM和MCI 三种营养成分丰富度不同的培养基,以典型窖泥作为菌群接种源,探索富集菌群的种类与动力学变化。扩增子测序分析表明,寡营养的MCI培养基对于富集窖泥主体菌群是较为有效的,可以同时富集得到产甲烷菌群和细菌菌群,主要包括古菌的广古菌门(Euyarchaeota)、细菌的拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes)。使用基于MCI培养基的寡营养技术能有效富集窖泥中高丰度和低丰度厌氧菌,如普雷沃氏菌科(Prevotellaceae)、理研菌科(Rikenellaceae)、Dysgonomonadaceae、太阳杆菌科(Heliobacteriaceae)、瘤胃菌科(Ruminococcaceae)和甲烷八叠球菌科(Methanosarcinaceae)等;同时对扩增子测序无法检测的稀有微生物,包括甲烷鬃菌科(Methanosaetaceae)、消化链球菌科(Peptostreptococcaceae)、肠杆菌科(Enterobacteriaceae)、Oligosphaeraceae、Cloacimonadaceae、纤维杆菌科(Fibrobacteraceae)和Pedosphaeraceae等也具有良好的富集作用。结合宏基因组学技术,寡培养技术展现出在解析窖泥微生物菌群的物种组成、菌群结构和代谢潜能等方面的重要价值,为深入剖析窖泥未培养厌氧菌对窖泥依赖型白酒风味物质合成贡献起到重要作用。
Pit mud is an important habitat for anaerobic microbes that are involved in the production of aromas and flavors for strong-flavor Baijiu. However, most of the anaerobes have not been cultured under artificial culture conditions, which severely hinders the understanding of the functions of anaerobes in pit mud. To explore the types and dynamics of the enriched microbiota, CGM, NBM, and MCI culture media with different nutrients were used. Typical pit muds from a Chinese strong-flavor type liquor distillery were used as inocula. Combined with amplicon sequencing technique, the results showed that oligotrophic MCI medium was the most effective medium to enrich the dominant microbes in pit muds, including Euyarchaeota, Bacteroidetes and Firmicutes at the same time. The oligotrophic culturing based on MCI medium not only enriched the high- and low-abundance anaerobes in pit muds such as Prevotellaceae, Rikenellaceae, Dysgonomonadaceae, Heliobacteriaceae, Ruminococcaceae and Methanosarcinaceae, but also effectively enriched rare anaerobes such as Methanosaetaceae, Peptostreptococcacea, Enterobacteriaceae, Oligosphaeraceae, Cloacimonadaceae, Fibrobacteraceae, and Pedosphaeraceae. More detailed microbial structures and species composition of the enriched microbiota were further revealed by metagenomics sequencing. In this study, combined with high-throughput sequencing approaches, oligotrophic culturing technique shows high values on illuminating the classification and genomic structures as well as the metabolic potentials of pit mud microbiota.
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