柴达木盆地昆特依盐湖可培养嗜盐细菌多样性研究

doi:10.13560/j.cnki.biotech.bull.1985.2023-1219

摘要/Abstract

摘要：

【目的】 探究不同富集时间、培养盐度、稀释梯度、培养基等对氯化物型昆特依高盐盐湖可培养嗜盐细菌多样性的影响，确定嗜盐细菌的最佳分离培养条件，挖掘更多的环境嗜盐细菌资源。【方法】 从昆特依盐湖中采集水泥混合样本，选取7种培养基和10%、18% NaCl两个盐度，采用富集培养法、平板稀释涂布法和平板分区划线法分离嗜盐菌；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。同时采用免培养Illumina MiSeq高通量测序技术分析昆特依盐湖细菌群落结构多样性特征。【结果】 免培养昆特依盐湖样本中共鉴定到细菌39门64纲101目219科703属，其中假单胞菌门（Pseudomonadota）、厚壁菌门（Bacillota）、拟杆菌门（Bacteroidota）和放线菌门（Actinomycetota）为主要的优势菌门。根据菌落的大小、形态、颜色、光泽度、透明度、质地、隆起状态和边缘特征等，共分离出436株嗜盐菌，隶属于3门3纲6目9科28属77种，其中16株可能为潜在新种。3门为厚壁菌门（Bacillota）、假单胞菌门（Pseudomonadota）和放线菌门（Actinomycetota），且均为免培养测序结果中的优势菌门。在属水平上，芽孢杆菌属（Bacillus）、盐单胞菌属（Halomonas）、喜盐芽孢杆菌属（Halobacillus）、枝芽孢菌属（Virgibacillus）和卤水杆菌属（Salicola）为优势菌属。肿块芽孢杆菌属（Tuberibacillus）、越南蔷薇菌属（Rossellomorea）和谷氨酸杆菌属（Glutamicibacter）是首次从盐湖中分离得到。18% NaCl分离得到的菌株数量明显少于10% NaCl，且多样性较低，芽孢乳杆菌科（Sporolactobacillaceae）是该条件下分出的特有菌科。7种培养基中，1/2 RCA培养基分离效果最好。最佳富集培养时间为0、7和60 d，最佳样品稀释梯度为10^-2和10^-3。【结论】 从昆特依盐湖中共分离出嗜盐细菌3门3纲6目9科28属77种。应用多种培养基，设置不同盐度、富集培养时间和稀释梯度等可显著提升可培养嗜盐菌的多样性。

关键词: 嗜盐细菌, 生物多样性, 氯化物型盐湖, 分离培养基, 富集培养, 梯度稀释, 高通量测序

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

马想蓉, 马欣, 陈胤勋, 龙启福, 王嵘, 邢江娃. 柴达木盆地昆特依盐湖可培养嗜盐细菌多样性研究[J]. 生物技术通报, 2024, 40(7): 285-298.

MA Xiang-rong, MA Xin, CHEN Yin-xun, LONG Qi-fu, WANG Rong, XING Jiang-wa. Diversity of Culturable Halophilic Bacteria in the Chloride Type Kunteyi Salt Lake in the Qaidam Basin[J]. Biotechnology Bulletin, 2024, 40(7): 285-298.

图/表 12

表1 昆特依盐湖样本中的细菌群落α多样性指数

Table 1 Alpha diversity index of bacterial communities in samples from Kunteyi Salt Lake

样品 Sample	Sobs指数 Sobs index	Chao指数 Chao index	Ace指数 Ace index	Shannon指数 Shannon index	Simpson指数 Simpson index	Coverage指数 Coverage index
KS1	1 018	1 204.62	1 230.55	1.72	0.602	0.99
KS2	968	1 384.02	1 125.13	4.74	0.033	0.99
KS3	1 341	1 502.00	1 418.44	5.89	0.008	1.00
KS4	1 178	1 448.01	1 321.52	3.84	0.127	0.99

图1 昆特依盐湖免培养细菌多样性分析 A：门水平；B：纲水平；C：目水平；D：科水平；E：属水平

Fig. 1 Diversity analysis of culture-free bacteria in Kunteyi Salt Lake A: Phylum. B: Class. C: Order. D: Family. E: Genus

图2 昆特依盐湖可培养嗜盐细菌多样性分析 A：门水平；B：纲水平；C：目水平；D：科水平；E：属水平

Fig. 2 Diversity analysis of culturable halophilic bacteria in Kunteyi Salt Lake A: Phylum. B: Class. C: Order. D: Family. E: Genus

图3 不同盐浓度10%（A）和18%（B）分离得到的嗜盐菌属水平多样性

Fig. 3 Genus-level diversity of halophilic bacteria isolated from 10%（A）and 18%（B）salinities

表2 不同培养基可分离嗜盐菌多样性

Table 2 Biodiversity of halophilic bacteria isolated by different media

培养基 Medium	菌株数量 Number of strains	门 Phylum	纲 Class	目 Order	科 Family	属 Genus	种 Species	优势度指数Dominance index
1/10 2216E	39	2	2	3	3	13	17	0.22
2216E	50	2	2	3	3	12	17	0.22
1/2 R2A	76	2	2	2	3	15	34	0.44
1/2 RCA	99	3	3	5	7	16	27	0.35
1/2 TSA	51	2	2	3	5	11	22	0.29
OSM	77	2	2	4	6	12	16	0.21
MGM	44	2	2	2	2	12	17	0.22

图4 不同培养基嗜盐菌在门（A）、目（B）、科（C）、属（D）水平上的差异性

Fig. 4 Differences of halophilic bacteria isolated by different media at the level of phylum（A）, order（B）, family（C）, and genus（D）

表3 不同富集时期分离菌株数量

Table 3 Number of bacterial strains isolated at different enrichment periods

富集时期 Enrichment period/d	菌株数量 Number of strains	比例 Scale/%
0	77	17.66
7	56	12.84
14	53	12.16
21	68	15.60
30	78	17.89
45	56	12.84
60	48	11.01

表4 不同富集时期可培养嗜盐菌多样性统计

Table 4 Culturable halophilic bacterial diversity statistics at different enrichment periods

富集时期 Enrichment period/d	门 Phylum	纲 Class	目 Order	科 Family	属 Genus	种 Species
0	2	2	3	5	16	29
7	3	3	4	6	16	25
14	2	2	2	3	14	20
21	2	2	3	5	13	21
30	2	2	2	4	16	28
45	2	2	3	4	14	22
60	2	2	4	4	13	24

图5 不同富集时期嗜盐菌在门（A）、目（B）、科（C）、属（D）水平上的差异性

Fig. 5 Differences of halophilic bacteria at different enrichment periods at the level of phylum（A）, order（B）, family（C）and genus（D）

图6 不同稀释梯度获得的菌株数

Fig. 6 Number of strains obtained by different dilution gradients

图7 不同稀释梯度嗜盐菌在门（A）、目（B）、科（C）、属和种（D）水平上的多样性

Fig. 7 Diversity of halophilic bacteria isolated by different dilution gradients at phylum（A），order（B）, family（C）, genus and species（D）levels

表5 昆特依盐湖疑似新种

Table 5 Suspected new species in Kunteyi Salt Lake

菌株编号 Strain No.	近缘菌株 Phylogenetically related strain	16S rRNA基因相似度 16S rRNA gene similarity/%
KTR1161	Halomonas vilamensis SV325	98.56
KTR1102	Salibacterium salarium DSM 16461	98.55
KTT1052	Sediminibacillus halophilus T346	98.48
KTF2041	Halobacillus virgiliensis S3	98.48
KTT4031	Bacillus licheniformis CICC	98.46
KTT5052	Bacillus licheniformis ALB	98.45
KTE2041	Piscibacillus halophilus AT3RS18	98.27
KTC1131	Halovibrio denitrificans HGD 3	98.19
KTO0101	Tuberibacillus calidus 607	98.30
KTE1061	Marinobacter aquaticus M6-53	98.09
KTT2052	Halovibrio mesolongii HMY2	97.92
KTF2021	Aquibacillus koreensis BH30097	97.55
KTO1051	Bacillus haynesii INH14A	97.45
KTR3031	Bacillus licheniformis F2	97.38
KTR2043	Virgibacillus byunsanensis ISL-24	97.35
KTF3021	Alkalibacillus aidingensis YIM 98829	96.94

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