河南叶县岩盐可培养中度嗜盐菌的多样性

Biodiversity of culturable moderate halophilic bacteria of rock salt in Yexian county, Henan province

摘要:

【背景】嗜盐微生物因为独特的生理和代谢特征而对高盐环境有着良好的适应能力，在环境污染治理、酶制剂等领域具有很高的应用和研究价值，是一类重要的极端环境微生物资源。【目的】为了更好地认识我国岩盐微生物的多样性，开发和利用嗜盐微生物资源，积累丰富的微生物菌种资源。【方法】在5%和10%的盐度下，使用Alkaline oligotrophic medium (AOM)、Neutral haloarchaeal medium (NHM)、diluted modified marine agar (dmMA)和ISP3 medium (ISP3)四种培养基，分离和纯化河南叶县岩盐矿的卤水和盐土中的嗜盐菌，使用细菌通用引物27F和1492R扩增和测序纯化菌株的16S rRNA基因，使用EzBioCloud和NCBI上的Blast比对进行分子鉴定，使用Mega 5.0进行遗传进化分析。【结果】从河南叶县岩盐卤水和盐土中一共分离和纯化到78株细菌，菌株16S rRNA基因序列显示它们来自3个门：厚壁菌门(Firmicutes)的Bacillus 26株、Halobacillus 30株、Oceanobacillus 10株和Staphylococcus 1株；变形菌门(Proteobacteria)的Sphingomonas 3株和Halomonas 5株；放线菌门(Actinobacteria)的Brevibacterium 3株。Halobacillus和Bacillus的细菌在叶县岩盐可培养中度嗜盐菌中具有较高的丰度。AOM培养基分离出了最多5个属的细菌，并且仅从AOM培养基分离出了Sphingomonas和Brevibacterium的细菌；另外，仅从ISP3分离出了Oceanobacillus的细菌；4种培养基都培养出了Halobacillus和Bacillus的细菌。来自卤水的有Bacillus、Halobacillus、Oceanobacillus、Sphingomonas和Staphylococcus；来自盐土有Bacillus、Brevibacterium、Halomonas。另外，5%和10%两个盐度下都纯化出Bacillus、Brevibacterium、Halobacillus和Sphingomonas；仅在10%的盐度下纯化到Halomonas和Oceanobacillus的细菌；仅在5%的盐度下培养出一株Staphylococcus。【结论】揭示了河南叶县岩盐可培养中度嗜盐菌的多样性，发现Halobacillus和Bacillus在可分离培养的中度嗜盐菌中具有较高的丰度，为深入开展岩盐嗜盐微生物研究积累了较为丰富的微生物菌株资源。

Abstract:

[Background] Halophilic microorganisms with unique physiological and metabolic characteristics for living in hypersaline environment have high application and research value in the fields of environmental pollution control, enzyme preparation and are one type of important microbial resources from extreme environment. [Objective] To uncover species diversity of cultivable moderate halophilic microorganisms in rock salt in China, to develop and utilize halophilic microbial resources and to accumulate strains of halophilic microorganisms, this research was conducted. [Methods] Moderate halophilic bacteria were isolated from brine and salt soil by using alkaline oligotrophic medium (AOM), neutral haloarchaeal medium (NHM), diluted modified marine agar (dmMA) and ISP3 medium (ISP3) under 5% and 10% salinity separately. Bacteria 16S rRNA gene amplification and sequencing were carried out by using bacterial common primers 27F and 1492R. EzBioCloud and Blast in NCBI web were used for the bacterial taxonomy and phylogenetic tree was constructed by Mega 5.0. [Results] In all, 78 strains were isolated from brine and salt soil in rock salt in Yexian county, Henan province. The results of 16S rRNA gene sequences indicated that they ranged three phylum in seven genus including: 26 strains in genus of Bacillus, 30 strains in Halobacillus, 10 strains in Oceanobacillus and one strain of Staphylococcus in phylum Firmicutes; 3 strains in Sphingomonas and 5 strains in Halomonas in the phylum Proteobacteria; 3 strains in Brevibacterium in the phylum Actinobacteria. The genus of Halobacillus and Bacillus had higher abundance in the rock salt from Yexian county, Henan province. At the most, five genus bacteria were isolated by using AOM medium and only by using AOM, Sphingomonas and Brevibacterium were isolated. In addition, Oceanobacillus were isolated from brine by using ISP3. By using the four media, Halobacillus and Bacillus were isolated. Bacillus, Halobacillus, Oceanobacillus, Sphingomonas and Staphylococcus were from brine, and Bacillus, Brevibacterium, Halomonas were from salt soil. Third, under the 5% and 10% salinity, Bacillus, Brevibacterium, Halobacillus and Sphingomonas were isolated and only under 10% salinity, Halomonas and Oceanobacillus were isolated, only one strain Staphylococcus was isolated under 5% salinity. [Conclusion] The community structure and diversity of rock salt in Yexian county, Henan province were uncovered and Halobacillu and Bacillus had higher abundance in the culturable Moderate Halophilic Bacteria genus. This research has provided rich Moderate Halophilic Bacteria resources for developing rock salt bacteria.