Abstract: Pit mud (PM) plays a key role in controlling the quality of Chinese Luzhou-flavor liquor in anaerobic fermentation. Here, microbial community composition and diversity in PM samples using pyrosequencing technique were investigated. A total of 494293 valid sequences were obtained. The reads fell into 796 operational taxonomic units (OTUs) affiliated to 21 phyla. The predominant groups were Firmicutes (66.8% of total reads), Bacteroidetes (16.0%), Euryarchaeota (9.0%), Spirochaetes (2.2%), Actinobacteria (1.8%) and Chloroflexi (1.0%). Microbial diversity increased with cellar age until 25 years old, but kept relatively constant from 25 to 50 years. A large difference between microbial communities was observed in the pit muds with different cellar ages. Lactobacillus predominanted in 1-year pit mud (62.3%), but its abundance decreased remarkably in 10-year and older pit muds. In contrast, the abundances of Petrimonas, Clostridium IV and methanogens increased dramatically. In addition, Archaea in PM were mainly composed of hydrogenotrophic methanogens such as Methanobrevibacter, Methanoculleus, Methanobacterium, while the H₂/acetate-utilizing Methanosarcina was more abundant in PM with cellar age of 25—50 years (3.1%—4.5%). This research supports the practical experience that old fermentation pits produce high quality Luzhou-flavor liquor.

Key words: brewing, cellar age, pit mud, microbial community, pyrosequencing

摘要： 窖池是中国浓香型白酒的根本，其中窖泥微生物则是决定白酒风味与品质的核心。通过454焦磷酸测序法系统研究了不同窖龄窖泥的细菌和古菌的群落组成及多样性。共获得494293个有效序列，可归为796个可操作分类单元（OTUs），分别属于21个门（phylum）。优势分类门（≥1%）分别为厚壁菌门（Firmicutes）66.8%、拟杆菌门（Bacteroidetes）16.0%、广古菌门（Euryarchaeota）9.0%、螺旋体门（Spirochaetes）2.2%、放线菌门（Actinobacteria）1.8% 和绿弯菌门（Chloroflexi）1.0%。窖泥微生物多样性在1～25年间随着窖龄的增加而呈升高趋势，但在25～50年间保持相对稳定。不同窖龄的窖泥中优势种群存在明显差异，其中乳杆菌（Lactobacillus）在1年窖泥中为绝对优势种群（62.3%），而在10年窖泥中，Lactobacillus丰度显著下降（16.1%），Petrimonas，Clostridium IV和甲烷菌等丰度则显著增加。在25～50年窖泥中，这一趋势更加明显，并且一些未培养菌丰度在这一阶段也显著增加。此外，窖泥中古菌主要为氢型产甲烷菌（Methanobrevibacter、Methanoculleus、Methanobacterium），可同时利用氢气和乙酸的甲烷八叠球菌属（Methanosarcina）在25～50年窖龄的窖泥中也有较高的丰度（3.1%～4.5%）。本研究从微生物群落构成角度为“老窖产好酒”的实践经验提供了理论依据。

关键词: 酿酒, 窖龄, 窖泥, 微生物群落, 焦磷酸测序