Abstract:

【Objective】 To explore the effects of different enrichment time, culture salinity, dilution gradient and medium on the diversity of culturable halophilic bacteria in chloride type Kunteyi high salt lake, to determine the best isolation and culture conditions for halophilic bacteria, and to explore more resources of environmental halophilic bacteria. 【Method】 The mixed water and mud samples were collected from Kunteyi Salt Lake, and 7 media with 2 salinities of 10% and 18% NaCl were selected to isolate the halophilic bacteria by enrichment culture, plate dilution coating and plate zonal delineation methods. The phylogenetic status of the strains was determined by 16S rRNA gene sequencing and BLAST sequence comparison. At the same time, culture-free Illumina MiSeq high-throughput sequencing technology was used to analyze the bacterial community structure diversity in Kunteyi Salt Lake. 【Result】 A total of 39 phyla, 64 classes, 101 orders, 219 families and 703 genera of bacteria were identified in the culture-free samples collected from Kunteyi Salt Lake. Among them, Pseudomonadota, Bacillota, Bacteroidota and Actinomycetota were the main dominant phyla. According to the size, shape, color, gloss, transparency, texture, uplift status, and marginal characteristics of the colonies, 436 strains of halophilic bacteria were isolated, belonging to 3 phyla, 3 classes, 6 orders, 9 families, 28 genera and 77 species, among which 16 strains were potential new species. The three phyla were Bacillota, Pseudomonadota and Actinomycetota, all of which were the dominant phyla in the culture-free results. At the genus level, Bacillus, Halomonas, Halobacillus, Virgibacillus and Salicola were the dominant genera. Tuberibacillus, Rossellomorea and Glutamicibacter were isolated from the salt lake for the first time. The number of strains isolated by 18% NaCl was significantly lower than that by 10% NaCl, and the diversity was low. Sporolactobacillaceae was the unique bacteriaceae isolated under this condition. Among the 7 media, 1/2 RCA medium had the best isolation results. The optimal enrichment time was 0, 7 and 60 d, and the optimal sample dilution gradients were 10^-2 and 10^-3. 【Conclusion】 There were 3 phyla, 3 classes, 6 orders, 9 families, 28 genera and 77 species of halophilic bacteria isolated from Kunteyi Salt Lake. The diversity of culturable halophilic bacteria could be significantly improved by using various culture media and different salinities, enrichment culture time and dilution gradients.

Key words: halophilic bacteria, biodiversity, chloride type salt lake, isolation medium, enrichment culture, gradient dilution, high-throughput sequencing