高盐盐湖可分离嗜盐耐盐菌的种群多样性及四氢嘧啶产量评价

doi:10.13560/j.cnki.biotech.bull.1985.2021-0201

摘要/Abstract

摘要：

为了解柴达木盆地茶卡盐湖、柯柯盐湖和小柴旦盐湖等三大硫酸镁亚型高盐盐湖可分离嗜盐耐盐菌的种群多样性,采用RM中、高盐培养基筛选分离可培养的嗜盐菌和耐盐菌,扩增16S rRNA基因序列进行种属鉴定和环境因子典范对应分析（CCA）,选取优势菌属构建系统发育树,并采用高效液相色谱法（HPLC）检测次级代谢产物四氢嘧啶（Ectoine）积聚量,筛选高产菌株。结果表明三大盐湖共分离获得嗜盐和耐盐菌113株,优势种属为Bacillus（42株）、Staphylococcus（30株）和Halomonas（26株）,各丰度与盐湖类型相关;其中中度嗜盐菌居多,其次为耐盐菌和弱嗜盐菌。系统发育分析表明Bacillus属存在5个进化分支,由6个种组成,Halomonas属存在6个进化分支,由7个种组成。HPLC检测四氢嘧啶积聚量,获得具有高产四氢嘧啶潜力菌株7株。这些研究结果说明柴达木盆地硫酸镁亚型高盐盐湖可分离嗜盐耐盐菌的优势菌属以Bacillus、Staphylococcus和Halomonas为主,且多为中度嗜盐菌,其高产四氢嘧啶的潜力菌株可用于后续四氢嘧啶发酵应用研究。

关键词: 柴达木盆地, 高盐盐湖, 可培养细菌, 种群多样性, 四氢嘧啶

Abstract:

To fully understand the population diversity of isolated halophilic and halotolerant bacteria in the Chaka Salt Lake,Keke Salt Lake and Xiaochaidan Salt Lake of 3 major magnesium sulfate subtype hypersaline salt lakes,cultivable halophilic and halotolerant bacteria were screened in RM medium with medium and high salinity. The 16S rRNA gene sequences of the isolated strains were amplified for species identification and canonical correspondence analysis（CCA）of environmental factors. The dominant genera were selected to construct individual phylogenetic trees,and the accumulation of secondary metabolite ectoine was measured by high performance liquid chromatography（HPLC）,and finally the strains with high-yield of ectoine were screened. Results showed that a total of 113 halophilic and halotolerant bacteria were isolated from the three major salt lakes. The dominant genera were Bacillus（42 strains）,Staphylococcus（30 strains）and Halomonas（26 strains）. Their abundance was related to the type of the salt lakes. The dominant genera Bacillus and Halomonas were mostly moderately halophilic bacteria,followed by halotolerant bacteria and weakly halophilic bacteria. Phylogenetic analysis demonstrated that Bacillus bacteria had 5 evolutionary branches,composed of 6 species,and Halomonas bacteria had 6 evolutionary branches,composed of 7 species. The accumulation of ectoine was detected by HPLC,and 7 strains with potential of high ectoine production were obtained. These results indicate that the dominant halophilic and halotolerant bacteria from the magnesium sulfate subtype hypersaline salt lakes in the Qaidam Basin are mainly Bacillus,Staphylococcus and Halomonas,and most of them are moderately halophilic bacteria. The strains with high ectoine production could be used for subsequent application research of ectoine fermentation.

Key words: Qaidam Basin, hypersaline salt lake, cultivable bacteria, population diversity, ectoine

张田田, 李永臻, 沈国平, 王嵘, 朱德锐, 邢江娃. 高盐盐湖可分离嗜盐耐盐菌的种群多样性及四氢嘧啶产量评价[J]. 生物技术通报, 2022, 38(1): 168-178.

ZHANG Tian-tian, LI Yong-zhen, SHEN Guo-ping, WANG Rong, ZHU De-rui, XING Jiang-wa. Population Diversity of Isolated Halophilic and Halotolerant Bacteria from Hypersaline Salt Lakes and Evaluation of Ectoine Production[J]. Biotechnology Bulletin, 2022, 38(1): 168-178.

图/表 5

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培养基 Culture medium	分离菌株数量Isolated strain number			总计 Total
培养基 Culture medium	茶卡盐湖 Chaka Salt Lake	柯柯盐湖 Keke Salt Lake	小柴旦盐湖Xiaochaidan Salt Lake	总计 Total
5% NaCl	38	18	14	70
10% NaCl	9	22	12	43

培养基 Culture medium	分离菌株数量Isolated strain number			总计 Total
培养基 Culture medium	茶卡盐湖 Chaka Salt Lake	柯柯盐湖 Keke Salt Lake	小柴旦盐湖Xiaochaidan Salt Lake	总计 Total
5% NaCl	38	18	14	70
10% NaCl	9	22	12	43

门Phylum	属Genus	5%氯化钠 5% NaCl	10%氯化钠 10% NaCl	菌株数量Strain number			总计Total
门Phylum	属Genus	5%氯化钠 5% NaCl	10%氯化钠 10% NaCl	茶卡盐湖 Chaka Salt Lake	柯柯盐湖 Keke Salt Lake	小柴旦盐湖 Xiaochaidan Salt Lake	总计Total
Firmicutes	Bacillus	32	10	20（42.6%）	16（40%）	6（23.1%）	42
	Staphylococcus	15	15	8（17%）	17（42.5%）	5（19.3%）	30
	Enterococcus	3	0	0（0%）	3（7.5%）	0（0%）	3
	Planococcus	1	0	0（0%）	0（0%）	1（3.8%）	1
	Oceanobacillus	1	0	1（2.1%）	0（0%）	0（0%）	1
Proteobacteria	Halomonas	11	15	10（21.3%）	3（7.5%）	13（50%）	26
	Pantoea	6	0	6（12.8%）	0（0%）	0（0%）	6
	Idiomarina	1	3	2（4.2%）	1（2.5%）	1（3.8%）	4
总计Total		70	43	47	40	26	113

门Phylum	属Genus	5%氯化钠 5% NaCl	10%氯化钠 10% NaCl	菌株数量Strain number			总计Total
门Phylum	属Genus	5%氯化钠 5% NaCl	10%氯化钠 10% NaCl	茶卡盐湖 Chaka Salt Lake	柯柯盐湖 Keke Salt Lake	小柴旦盐湖 Xiaochaidan Salt Lake	总计Total
Firmicutes	Bacillus	32	10	20（42.6%）	16（40%）	6（23.1%）	42
	Staphylococcus	15	15	8（17%）	17（42.5%）	5（19.3%）	30
	Enterococcus	3	0	0（0%）	3（7.5%）	0（0%）	3
	Planococcus	1	0	0（0%）	0（0%）	1（3.8%）	1
	Oceanobacillus	1	0	1（2.1%）	0（0%）	0（0%）	1
Proteobacteria	Halomonas	11	15	10（21.3%）	3（7.5%）	13（50%）	26
	Pantoea	6	0	6（12.8%）	0（0%）	0（0%）	6
	Idiomarina	1	3	2（4.2%）	1（2.5%）	1（3.8%）	4
总计Total		70	43	47	40	26	113

Classification of bacteria	Strain code	Salinity range /（mol·L^-1）	Optimal salinity /（mol·L^-1）	Classification of halophiles	Ectoine accumulation /（mg·g ^-1CDW）	Strain code	Salinity range /（mol·L^-1）	Optimal salinity /（mol·L^-1）	Classification of halophiles	Ectoine accumulation /（mg·g ^-1 CDW）
Bacillus	C10	0.5-2.5	0.5	Moderate halophile	80.08	K15	0-2.5	1.5	Moderate halophile	147.75
	C11	0-2.0	2.0	Moderate halophile	220.56	K17	0.5-2.5	1.0	Moderate halophile	10
	C12	0-3.0	0.5	Moderate halophile	64.47	K26	0-2.5	1.5	Moderate halophile	ND
	C15	0.5-3.0	0.5	Moderate halophile	324.69	K27	0-2.5	0.5	Moderate halophile	18.20
	C18	0-1.5	0.5	Slight halophile	ND	K28	0-2.0	0.5	Slight halophile	32.26
	C20	0-1.5	0	Halotolerant	ND	K29	0-1.5	0.5	Slight halophile	30.81
	C22	0-1.5	0	Halotolerant	ND	K30	0-2.5	1.0	Moderate halophile	18.50
	C23	0-2.0	0	Halotolerant	ND	K31	0-2.0	0.5	Slight halophile	77.60
	C24	0-2.5	0	halotolerant	31.93	K32	0-2.0	1.0	Moderate halophile	100.35
	C25	0-3.0	1.0	Moderate halophile	80	K37	0-2.5	1.5	Moderate halophile	135.84
	C26	0 -2.0	0	halotolerant	ND	K39	0-2.5	0.5	Moderate halophile	23.88
	C27	0-3.0	1.0	Moderate halophile	ND	K40	0-3.0	0	Halotolerant	ND
	C29	0-3.0	0	Halotolerant	ND	K41	0-3.0	0.5	Moderate halophile	10.91
	C30	0-2.0	0	Halotolerant	ND	K42	0-3.0	0	Halotolerant	ND
	C33	0-3.0	0.5	Moderate halophile	50.95	K48	0-3.0	0	Halotolerant	ND
	C37	0-2.5	0	Halotolerant	ND	X5	0.5-3.0	1.0	Moderate halophile	70.09
	C42	0.5-3.0	1.0	Moderate halophile	65.46	X10	0-2.5	0.5	Moderate halophile	19.82
	C43	0.5-3.0	1.5	Moderate halophile	133.33	X11	0-2.0	0.5	Slight halophile	23.99
	C50	0-1.5	0	Halotolerant	ND	X20	0-2.0	0	Halotolerant	ND
	C52	0-1.0	0	Halotolerant	ND	X22	0-2.5	0	Halotolerant	ND
	K11	0-2.5	1.5	Moderate halophile	275.20	X33	0-3.0	0	Halotolerant	ND
Halomonas	C8	0-2.5	0.5	Moderate halophile	30.85	X4	0-3.0	1.0	Moderate halophile	125.18
	C9	0-3.0	1.5	Moderate halophile	ND	X6	0-3.0	1.0	Moderate halophile	53.98
	C13	0-3.0	0.5	Moderate halophile	122.38	X9	0-3.0	0.5	Moderate halophile	41.80
	C17	0.5-3.0	0.5	Moderate halophile	18.66	X21	0-3.0	1.0	Moderate halophile	100.39
	C34	0-3.0	0.5	Moderate halophile	15.71	X26	0-3.0	1.0	Moderate halophile	369.10
	C39	0-3.0	0	Halotolerant	ND	X27	0-3.0	1.5	Moderate halophile	239.51
	C40	0.5-3.0	2.0	Moderate halophile	11.25	X28	0-3.0	1.0	Moderate halophile	296.56
	C41	0-3.0	0.5	Moderate halophile	32.35	X31	0-3.0	1.5	Moderate halophile	41.37
	C44	0-3.0	2.5	Moderate halophile	21.82	X32	0-3.0	0.5	Moderate halophile	65.23
	C51	0-2.5	0	halotolerant	ND	X34	0-3.0	0.5	Moderate halophile	57.61
	K5	0-3.0	0.5	Moderate halophile	9.98	X35	0-3.0	1.0	Moderate halophile	99.65
	K14	0-2.0	1.5	Moderate halophile	96.81	X36	0-3.0	1.0	Moderate halophile	208.56
	K38	0-3.0	0.5	Moderate halophile	46.30	X39	0-3.0	0.5	Moderate halophile	41.26