Community of extremely halophilic bacteria in historic Dagong Brine Well in southwestern China

The Dagong Brine Well, the earliest man-made brine well in record, located in Zigong, Sichuan, China, had the construction and facilities mainly made of bamboo, wood and stone that are eroded easily by halophiles colonizing in the brine of the well. To better preserve this historic placemark, we initiated an investigation on the microbial community and the diversity of the halophilic bacteria in the brine of Dagong well for the first time. A total of 112 aerobic halophile strains were isolated and characterized according to their phenotype, cellular fatty acids composition and 16S rRNA. Furthermore, the intra specific phylogeny of closely related strains was also screened by PCR fragment length polymorphism of 16S-23S ribosomal RNA intergenic spacer regions (ISR). The result showed that the halophilic isolates found in current study were closely related to the following genera: Planococcus, Halomonas, Halobacillus, Oceanobacillus and Virgibacillus, a lineage of the domain Bacteria. Halomonas and Halobacillus were the dominant genera as represents 27% and 40% of the total isolates, respectively. The distribution of other genera was as follows: Oceanobacillus (18%), Virgibacillus (10%) and Planococcus (5%). The ISR analysis disclosed variation of banding pattern in some isolates related to Oceanobacillus and Halobacillus which was observed with phenotypic and physiological characterizations as well. It was clear that these halophiles have adapted to the special man-made hypersaline environment by the basic physiological evolution during phylogenesis and thus resulted in phenotypic and genotypic diversity in the Dagong Brine Well.