大柴旦盐湖嗜盐细菌多样性与功能酶挖掘

Diversity and functional enzymes of halophilic bacteria in the Da Qaidam Salt Lake

摘要:

目的 探究MgSO₄亚型大柴旦盐湖嗜盐细菌的多样性，比较不同培养条件对可培养嗜盐细菌多样性的影响，并对嗜盐细菌的胞外功能酶活性进行筛选。方法 采用免培养Illumina MiSeq高通量测序平台分析大柴旦盐湖细菌的群落组成多样性；选取13种培养基、2个盐度、8组富集培养天数、6个稀释梯度分离嗜盐细菌，通过16S rRNA基因测序与BLAST序列比对确定菌株系统分类学地位。选取分属于18属的45株不同种的代表菌株，采用7种筛选培养基进行蛋白酶、纤维素酶、淀粉酶和酯酶4种功能酶活性的筛选。结果 大柴旦盐湖免培养测序结果中共得到244个细菌操作分类单元(operational taxonomic unit, OTU)；获得明确分类地位的细菌有19门53纲92目133科153属，其中假单胞菌门(Pseudomonadota)和放线菌门(Actinomycetota)为主要的优势菌门。从大柴旦盐湖水泥样本中共分离出593株嗜盐细菌，分属于4门5纲8目12科22属，其中11株可能为潜在新种。Pseudomonadota和芽孢杆菌门(Bacillota)为可培养嗜盐细菌的优势菌门，盐单胞菌属(Halomonas)、枝芽孢菌属(Virgibacillus)和芽孢杆菌属(Bacillus)为主要的优势菌属。本研究比较了不同分离培养条件的分离效果。在10% NaCl浓度下分离得到的嗜盐细菌菌株数明显高于18% NaCl浓度，说明可培养嗜盐细菌中以中度嗜盐细菌居多；优势培养基为寡营养培养基2216E、1/2 R2A、1/10 2216E和1/10 TSA；最佳富集天数为7-30 d；不稀释的样本分离效果最好，其次为稀释梯度10^-1和10^-2。在45株代表菌株中，分别有40.0%、31.1%、40.0%和82.22%的菌株表现出蛋白酶、纤维素酶、淀粉酶和酯酶活性。大柴旦盐湖可培养嗜盐细菌的酯酶活性检出率较高，而纤维素酶活性检出率相对较低。结论 优化分离培养方法可显著提升盐湖可培养嗜盐细菌的多样性。大柴旦盐湖水泥样本中嗜盐细菌多样性较丰富，且具有较高的嗜盐酶活性，为后续进一步研究嗜盐细菌的应用提供了依据。

关键词:大柴旦盐湖;嗜盐细菌;生物多样性;分离培养;高通量测序;功能酶

Abstract:

Objective To explore the diversity of halophilic bacteria in the magnesium sulfate-subtype Da Qaidam Salt Lake, compare the effects of different culture conditions on the diversity of halophilic bacteria, and screen the extracellular functional enzymes of halophilic bacteria.Methods Illumina MiSeq was used to analyze the diversity of bacteria in the Da Qaidam Salt Lake. Thirteen media, 2 salinities, 8 enrichment culture periods, and 6 dilution gradients were selected to isolate halophilic bacteria, and the taxonomic status of the strains was determined by 16S rRNA gene sequencing and BLAST sequence alignment. According to the sequencing results, 45 representative strains of different species belonging to 18 genera were selected and 7 media were used to screen the strains with activities of functional enzymes including protease, cellulase, amylase, and esterase.Results A total of 244 bacterial OTUs were obtained by culture-free high-throughput sequencing in the Da Qaidam Salt Lake. The strains with clear taxonomic status were annotated to 153 genera, 133 families, 92 orders, 53 classes of 19 phyla, with Pseudomonadota and Actinomycetota being the dominant phyla. A total of 593 strains of halophilic bacteria were isolated from the mixed water and mud samples of the Da Qaidam Salt Lake, belonging to 22 genera, 12 families, 8 orders, 5 classes of 4 phyla, of which 11 strains may belong to potential new species. Pseudomonadota and Bacillota were the dominant phyla, and Halomonas, Virgibacillus, and Bacillus were the dominant genera. The number of halophilic bacteria isolated under the culture with 10% NaCl was significantly higher than that with 18% NaCl, indicating that moderately halophilic bacteria were dominant among culturable halophilic bacteria. The media with better isolation performance were 2216E, 1/2 R2A, 1/10 2216E, and 1/10 TSA, all of which are oligotrophic media. The optimal enrichment culture periods ranged from 7 to 30 days. The undiluted samples obtained the best isolation results, followed by dilution gradients of 10^-1 and 10^-2. Among the 45 representative strains, 40.0%, 31.1%, 40.0%, and 82.22% of the strains had activities of protease, cellulase, amylase, and esterase, respectively.Conclusion Optimization of the isolation and culture conditions can significantly improve the diversity of halophilic bacteria that can be cultured in salt lakes. The high diversity and high halophilic enzyme activities of the culturable halophilic bacteria from the Da Qaidam Salt Lake provide a basis for further application of these halophilic bacteria.

Key words:Da Qaidam Salt Lake;halophilic bacteria;biodiversity;isolation and culture;high-throughput sequencing;functional enzymes