Population Diversity And Phylogeny Of Bacteria And Archaea In Different Aged Pit Muds Of Luzhou Laojiao Cellar

Luzhou Laojiao liquor is a typic representative of the Luzhou-flavor liquor, and is famous for its strong pit aroma, coordinationed scent, soft taste and clean aftertaste. Microbes in the pit muds was the source of Luzhou-flavor produced. The flavor of Luzhou Laojiao liquor mainly results from the combined action of microbial flora in pit muds. During the long-term fermentation, the microbial communities were enriched, and the unique functional microbial flora systems were formed, and strains belonged to caproic acid bacteria, methanogenic archaea, and lactic acid bacteria became the dominant groups in pit muds gradually, and produced a series of flavor components which leads to the specific Luzhou-flavor smells. Microbial population in pit muds affect not only the liquor-producing capacity but also the quality and style of Luzhou-flavor liquor, so it is of significance to study the structure and diversity of microbial population in pit mud. In this paper, the microbial community and diversity of bacterial and archaeal population in pit mud collected from different age cellars was discussed by using PCR-DGGE,16S rDNA clone library technique and high-throughput sequencing technique. The results were as the follow:1. In the different aged pit wall mud (PWM) and pit bottom mud (PBM), the diversity of bacterial and functional flora was different, and increased along with the pit age increasing; In the same aged pit mud, the bacterial and functional flora in PWM and PBM were different, but not regular. Along with the pit age increasing, the diversity of archaea comunities in PWM and PBM also increased, the diversity of archaea flora in PWM samples was higher than that in PBM samples in the same aged pit muds.2. In different aged pit mud, the bacteria mainly distributed in Firmicutes, Bacteroidetes, Proteobacteria, Chlorofiexi, Synergistetes, Chlorobi, Planctomycetes, Verrucomicrobia, Actinobacteria, Acidobacteria, Spirochaeta, Lentisphaerae, Dictyoglomi, Cyanobacteria, Deinococcus-Thermus, Tenericutes and so on. Of them, bacteria belong to Firmicutes, Bacteroidetes and Proteobacteria existed in all the samples, hence, they were the dominant bacteria, and account for 52.17%,21.20% and 14.49% in seven clony library respectively.3. The archaea flora in different aged pit mud mainly belonged to Crenarchaeota and Euryarchaeota. Strains in Euryarchaeota were the predominant groups, and consisted of Methanomicrobia, Methanobacteria, and Thermoplasmata; Of them, there were six archaea groups in Methanomicrobia, including genus Methanocorpusculum, Methanosaeta, Methanothrix, Methanoculleus, Methanosarcina and Methanosarcinales; and two groups in Euryarchaeota, i.e. Methanobacterium and Methanobrevibacter; and Thermoplasmatales was the only member in Thermoplasmata. There were a large proportion of uncultured archeaon existed in pit bottom mud (PBM) samples LJ4# and LJ12#, which accounted for 7.13% and 3.76%, respectively, and the proportion increased with the pit mud age. Hence, they may play a positive role during the formation of aromatic matter in the pit mud.4. Analysis of functional flora by PCR-DGGE revealed that:(1) Methanogenic archaea in the pit muds samples formed by five groups, included Methanobrevibacter, Methanosarcina, Methanobacterium, Methanobacteriaceae and Methanosarcinalae;(2) Caproic acid bacteria in the pit muds mainly belonged to genus Clostridium, but consisted of eight species; however, there was about 47.62% strains belonged to uncultured.(3) Lactic acid bacteria in the pit muds consisted of Lactobacillus, Pediococcus, Weissella and Enterococcus, and Lactobacillus acetotolerans was the predominant one.(4) As for methane-oxidizing archaea in the pit muds, methane-oxidizing archaea type I mainly formed by Methylosarcina, Methylococcus, Methylobacter, Methylomicrobium and Methylococcaceae; and the methane-oxidizing archaea type Ⅱ formed by Methylocystis and Methylosinus.