Purification And Molecular Identification Of Anaerobic Bacteria In Gujing Tribute Pit Mud

GujingTribute pit mud is rich in microbial communities,and these microbial communities directly affect the whole fermentation process by interacting with microbes in the fermented grains and Daqu.By now there is no very mature approach to isolate and culture those complex anaerobic bacteria.Strong-flavor liquor accounted for more than 70% of the Chinese liquor market,but the list of key microbes responsible for liquor flavor is still vague.The study of pit mud microorganisms plays an important role in promoting the development of strong-flavor liquor.In this paper,microbes in the pit mud were investigated with anaerobic culture,colony characterization and 16 S full-length high-throughput DNA sequencing.Some anaerobic bacteria in the yellow water and bottom pit mud were obtained.The list of pure colonies obtained by anaerobic culture was compared with that from high-throughput metagenome sequencing.Bottom pit mud and fermented grains plus the yellow water(located between bottom pit mud and bottom fermented grains)were used as the main part of the culture medium,and many anaerobic colonies were successfully obtained.Then microbes from pit muds with different qualities were cultured in the anaerobic station.A variety of morphological parameters were recorded and statistically classified.Genomic DNA extraction was performed on representative colonies,followed by full length 16 S r DNA amplification and Sanger sequencing.The results showed that most of the colonies belonged to Clostridium and Lactobacillus,but almost none of the colonies could be easily classified into species levels.Pit age and liquor flavor scoring together determine the quality of pit mu d.Nineteen pit mud samples with differential quality were subjected to 16 S full-length high-throughput sequencing technique,and a complex namelist of dominant anaerobic microbes was obtained.The results showed that some anaerobic isolates(Clostridium bornimense,Lactobacillus brevis)were not shown in the above namelist.It suggested that the high-throughput sequencing results were not complete,and the method of culturing anaerobic bacteria in Gujing Tribute pit mud is also needed to be further studied.