青藏高原尕斯库勒盐湖可培养嗜盐耐盐微生物的群落结构与分离方法对比研究

COMMUNITY STRUCTURE OF CULTURABLE HALOPHILIC AND HALOTOLERANT MICROORGANISMS IN GASIKULE SALT LAKE ON THE QINGHAI-TIBET PLATEAU AND COMPARISON OF DIFFERENT ISOLATION METHODS

嗜盐耐盐微生物在生物医药、生物酶等领域有着重要的应用潜力。为更好地利用青藏高原的盐湖微生物资源,以青藏高原尕斯库勒盐湖为研究对象,借助Illumina MiSeq测序平台分析盐湖微生物的群落组成,并采用寡营养培养、富集培养和梯度稀释等方法对嗜盐耐盐微生物进行大规模分离与培养。结果表明,高通量测序获得明确分类地位的细菌有30门50纲81目162科417属,古菌6门7纲9目15科39属。细菌优势属类群以矿生菌属(Fodinibius)和纤维单胞菌属(Gracilimonas)为主。古菌优势属类群以盐红菌属(Halorubrum)为主;纯培养法共获得502株嗜盐耐盐微生物,其中嗜盐耐盐细菌472株,分布在4门5纲7目9科13属,优势属为芽孢杆菌属(Bacillus)和盐单胞菌属(Halomonas),其中巴纽尔斯菌(Balneolota)门Fodinibius属可培养细菌是首次从盐湖中分离得到;嗜盐古菌30株,分布在1门1纲1目3科5属,优势属为Halorubrum。对比不同培养条件下的分离结果发现,1/2 TSA培养基和1/2 RCA培养基更有利于嗜盐耐盐细菌的分离,最佳富集天数为7天和30天,稀释梯度10-1和10-2可与未稀释样本互为补充来增加可培养嗜盐耐盐细菌的多样性;1/102216E培养基更适合于古菌的分离,最佳富集天数为7天和14天,且样本不稀释分离效果更好。研究结果可为青藏高原盐湖可培养微生物的分离方案优化提供理论依据和数据支撑。

Halophilic and halotolerant microorganisms have important application potential in many fields such as biomedicine and biological enzymes.To make better use of the microbial resources of salt lakes on the Qinghai-Tibet Plateau,the culture-free community composition of microorganisms in Gasikule Salt Lake was analyzed with the help of the Illumina MiSeq sequencing platform;and the methods of oligotrophic culture,enrichment culture,and gradient dilution were used to isolate and culture halophilic and halotolerant microorganisms.The culture-free high-throughput sequencing results showed that there were 30 phyla,50 classes,81 orders,162 families,and 417 genera of bacteria;and 6 phyla,7 classes,9 orders,15 families,and 39 genera of Archaea.The dominant genera of bacteria were Fodinibius and Gracilimonas,and the dominant genus of Archaea was Halorubrum.A total of 502 halophilic and halotolerant microorganisms were obtained by pure culture,including 472 strains of bacteria distributed in 4 phyla,5 classes,7 orders,9 families,and 13 genera.The dominant genera were Bacillus and Halomonas.The culturable bacteria of the genus Fodinibius of the phylum Balneolota were isolated from salt lakes for the first time.Thirty strains of halophilic Archaea were obtained by pure culture,distributed in 1 phylum,1 class,1 order,3 families,and 5 genera;and the dominant genus was Halorubrum.Comparing the isolation results under different culture conditions,we found that the 1/2 TSA medium and 1/2 RCA medium were more conducive to the isolation of halophilic and halotolerant bacteria,and the optimal enrichment duration were 7 days and 30 days,respectively.Dilution gradients of 10-1 and 10-2 could complement the undiluted samples to increase the diversity of culturable halophilic and halotolerant bacteria.The 1/102216E medium was more suitable for the isolation of Archaea,the optimal enrichment days were 7 and 14,respectively,and the samples were better separated without dilution.This study provided a theoretical basis and data support for the optimization of culturable microorganism isolation scheme in salt lakes on the Qinghai-Tibet Plateau.