Eubacterial and archaeal community characteristics in the man-made pit mud revealed by combined PCR-DGGE and FISH analyses

Abstract

The aim of this study was to investigate the shift of eubacterial and archaeal communities in various man-made pit muds (PMs) with different pit ages (the use time of cellar) by combining denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH) approaches. Analysis of the diversity index calculated from the eubacterial and archaeal DGGE profiles showed that the 4-year-PMs exhibited higher Shannon–Wiener index than that in other man-made PMs. Results of eubacterial DGGE pattern of the 16S rDNA gene fragment analysis illustrated that nine families including Leuconostocaceae, Clostridiaceae, Lactobacillaceae, Moraxellaceae, Enterococcaceae, Lachnospiraceae, Comamonadaceae, Sphingomonadaceae and Ruminococcaceae were identified, and Clostridiaceae was the dominant, accounting for 46.2%. Specially, Ruminococcaceae was only found in 4-year-PMs. Also, archaeal 16S rDNA gene fragment identification demonstrated that Methanobrevibacter, Methanobacterium and Methanoculleus were the majority of archaea. Validation of the result was performed by FISH experiment, and FISH analysis showed that the number of live eubacteria and archaea in the man-made PMs increased with pit age. The proportions of Methanobacteriales and Methanomicrobiales were decreased from 45.1% and 54.9% to 22.7% and 27.9%, respectively, after the cellars were used for 2years continuously. However, no significant change was observed in 3- and 4-year-PMs. Knowledge of the eubacterial and archaeal communities studied in the man-made PMs is essential to exploit the metabolic regulation during fermentation process and consequently develop appropriate strategies for liquor quality improvement.