微生物学报  2017, Vol. 57 Issue (9): 1409-1420
http://dx.doi.org/10.13343/j.cnki.wsxb.20170309
中国科学院微生物研究所,中国微生物学会,中国菌物学会
0

文章信息

赵百锁, 李俊. 2017
Baisuo Zhao, Jun Li. 2017
可培养盐碱菌多样性的研究进展
Biodiversity of culture-dependent haloalkaliphilic microorganisms
微生物学报, 57(9): 1409-1420
Acta Microbiologica Sinica, 57(9): 1409-1420

文章历史

收稿日期:2017-06-22
修回日期:2017-07-11
网络出版日期:2017-07-12
可培养盐碱菌多样性的研究进展
赵百锁1,2, 李俊2     
1. 中国农业科学院研究生院, 北京 100081;
2. 农业部微生物产品质量安全风险评估实验室, 北京 100081
摘要:存在于高盐强碱极端环境的微生物因其独特的生命方式,引起了广泛的关注。根据盐碱环境所含的可溶性盐成分,可分为"NaCl型"和"苏打型(Na2CO3/NaHCO3)"两大类,前者的碱性pH值较低而后者碱性pH值较高。本文总结了盐碱菌适宜生长条件在盐度0.5 mol/L和碱性pH 9.0之上且有效发表的标准菌株,并对这些菌株的生物多样性及生理特性进行了阐述;可培养盐碱细菌的数量及其多样性远远大于盐碱古菌,但是盐碱细菌对高盐度和强碱性pH依赖程度相对较低。盐碱细菌主要组成依次为芽孢杆菌纲(Bacilli,占总数约40%)、γ-变形菌纲(γ-Proteobacteria,30%)、梭菌纲(Clostridia,11%)、δ-变形菌纲(δ-Proteobacteria,6%)和放线菌纲(Actinobacteria,6%),而盐碱古菌主要组成为盐古菌纲(Halobacteria,92%)和甲烷微菌纲(Methanomicrobia,8%)。这些极端微生物在生物地球化学过程中或生态循环中扮演着重要的角色和功能,挖掘和利用盐碱菌具有重要意义。
关键词: 盐碱菌     微生物多样性     盐碱环境    
Biodiversity of culture-dependent haloalkaliphilic microorganisms
Baisuo Zhao1,2, Jun Li2     
1. Graduate School, Chinese Academy of Agricultural Sciences, Beijing 100081, China;
2. Laboratory of Quality & Safety Risk Assessment for Microbial Products(Beijing), Ministry of Agriculture, Beijing 100081, China
Received 22 June 2017; Revised 11 July 2017; Published online 12 July 2017
*Corresponding author: Baisuo Zhao, Tel/Fax: +86-10-82106784; E-mail: bszhao@163.com
Supported by the National Natural Science Foundation of China (31570110, 31370158) and by the Graduate School of Chinese Academy of Agricultural Sciences (0042014011, 1610042017001)
Abstract: Microorganisms growing at high salinity and elevated alkaline environments have gained wide attention in term of their unique properties. According to the soluble composition, saline-alkaline environment can be two groups, i.e. "NaCl type" and "soda type (Na2CO3/NaHCO3)". The alkaline pH of the former is relatively low, and that of the latter is higher. This review summarizes validated haloalkaliphilic type strains for optimal growth requiring Na+ concentrations above 0.5 mol/L and an alkaline pH of 9, and their biodiversity and physiological characteristics. The biodiversity of the halophilic bacteria is far greater than that of halophilic archaea. Halophilic bacteria are mainly composed of Bacilli (40% of the total), gamma-Proteobacteria (30%), Clostridia (11%), delta-Proteobacteria (6%) and Actinobacteria (6%); Halophilic archaea are Halobacteria (92%) and Methanomicrobia (8%). These extremophiles under double stress play essential roles and functions in biogeochemical processes and the ecological function.
Key words: haloalkaliphilic microorganisms     microbial diversity     saline-alkaline environment    

地球上存在着多种类型的盐碱土壤和盐碱湖,在一些特殊的盐碱环境中的盐度可达到饱和且其碱性pH值最高可达11[1]。但是在这些被人类认为很恶劣的极端环境中仍然栖息繁殖着一类独特的生命形式——盐碱菌。近年来,有关生命起源于海洋的一些科学假说推测原始海洋为盐碱环境[2-3],因此认知盐碱菌的生命存在原理可为研究生命起源提供新的线索。目前火星上可能存在液态水的土壤也已被证实为盐碱环境[4],所以盐碱菌极有可能是人类探索外太空生命过程中最先被发现的生命形式。与嗜盐菌或嗜碱菌相比较,盐碱菌不仅将地球生命的极限概念做了延伸,也寓意着茫茫无际的宇宙中可能存在生命不再是空想。本文将从盐碱环境的类型及成因、可培养盐碱菌的类群及其生理特征等方面进行深入分析归纳和述评,以获得抛砖引玉的效果,激励国内更多学者从事与盐碱菌相关的基础理论和应用研究。

1 盐碱环境的类型及形成

根据盐碱环境所含的可溶性盐的组分,可分为“NaCl型”和“苏打型(Na2CO3/NaHCO3)”。一般而言,前者pH值为8-11,所含的氯离子浓度较高,但是碳酸根离子/碳酸氢根离子浓度较低;后者pH值为9-11,所含的碳酸根离子/碳酸氢根离子浓度较高,但是氯离子和硫酸根离子浓度较低[5]。此外,由于镁离子和钙离子在碱性碳酸盐条件下容易形成沉淀,所以它们在盐碱环境中的含量都比较低[6]

盐碱环境主要为盐碱土和盐碱湖。盐碱土分布十分广泛,大部分集中于中东的土耳其、中亚的西伯利亚南部和蒙古、非洲埃及和肯尼亚以及我国东北和西部半干旱或干旱地区。盐碱土的形成过程很复杂,主要是由于气候干旱、长期的地质演化及人类的干预和集中灌溉等[6]。例如地处中温带-寒温带的东北松嫩平原就属于干旱-半干旱气候区。该区域内的盐碱土地总面积达373万公顷,是世界上三大苏打型盐碱土集中分布的区域之一且重度盐碱化面积仍然以每年1.4%的速度扩展[7]。该地区盐碱土壤属于内陆苏打盐碱型,盐分组成以小苏打(碳酸氢钠)/苏打(碳酸钠)为主,有些土壤还含有少量硫酸盐和氯化物。笔者研究组2014年6月至7月从松嫩平原采集了盐碱土50份,分析表明(数据暂未出版),这些样品的总盐度为0.3‰-11.8‰和pH值为8.3-10.5,含较多的Na+,80%以上的样品中HCO-浓度大于Cl-。该地区的总盐度虽然不是很高,但是在秋季干旱时,很多区域盐碱土表面会析出一层白色粉末(小苏打),说明其表层的总盐度很高且达到饱和。松嫩平原盐碱土呈碱性,土壤易吸收大气中二氧化碳(CO2),因此松嫩平原盐碱土对维持该地区生物地球碳循环(无机碳和有机碳之间的平衡)起着重要的作用。

相对于盐碱土的成因,盐碱湖的形成则有自己鲜明的特点。盐碱湖主要是在地貌拓扑、地理化学和干旱气候等共同作用下经过上百万年演化形成的[8-10]。地貌拓扑主要指地表上存在一个相对封闭的持久性“凹型”局部区域。地理化学表明地表土壤中含有丰富的盐类化合物。其中阳离子以钠/钾离子为主,阴离子则以氯离子、硫酸根离子、碳酸根/碳酸氢根离子中的一种或几种混合物为主。在夏季雨水较多的时候,土壤中丰富的盐类被雨水溶解,随后流入“凹型”洼地中,最后形成“堰塞湖”。在干旱/半干旱气候条件下,封闭的湖水在春秋冬季干旱季节中蒸发量则大于夏季雨水的补给。如此年复一年,湖中可溶性盐不断浓缩,逐渐演化形成高盐度且碱性pH较高的盐碱湖,例如东非Rift裂谷的Natron湖和Magadi湖,北美Mono湖和Soap湖。此外,中东Wadi Natrun地区和西伯利亚草原,我国内蒙古草原和松嫩草原,青藏高原地区也分布着诸多大小不一的盐碱湖[6]

科学家已经在这些盐碱环境中发现了多种类群的微生物。相对单一的极端条件,如高盐,强碱、强酸、高温或低温条件,盐碱环境则属于复合型的极端条件,因此生存繁殖于双重极端条件的微生物必须面对高渗透压、低水分活性和高浓度的氢氧根离子等相当恶劣的条件。这些微生物在生物地球化学过程中或生态循环中扮演着关键的角色和发挥着重要的功能。同时,生存于盐碱环境中的盐碱菌种群对盐碱条件的“适应或喜好”是长期自然选择的结果,通过不断的进化来适应复杂的极端盐碱环境。例如盐碱菌与外界盐碱环境密切接触的细胞壁的构造成分和细胞膜的一些成分可发生显著的变化。这些对探讨地质环境演化、地球碳氮循环和重建古环境都具有重要的科学意义。

2 可培养盐碱菌的多样性及其生理特征 2.1 盐碱菌的定义范畴

正如荷兰著名微生物学家劳伦斯·巴斯·贝金(Louren Baas Becking)所言,“一切生物,本应无处不在,但环境施予其选择,从而形成了今天的格局”[11-12]。因此自然环境中的盐碱条件是一个天然的胁迫和富集过程,在该环境中孕育着多种类型的盐碱菌。Soliman和Trüper[13]在1982年首次从埃及Abu Gabara盐碱湖中获得了一株嗜盐嗜碱古菌,即法老嗜盐碱单胞菌(Natronomonas pharaonis DSM 2160T,曾被命名为Natronobacterium pharaonis)。从此之后,嗜盐嗜碱微生物的纯培养取得令人瞩目的成绩。目前在国际主流微生物分类期刊上发表且有效命名的盐碱标准菌株达300多株。但是至今国内外学术界对“嗜盐嗜碱菌”还没有统一的归类依据或定义,究竟哪些微生物属于嗜盐嗜碱菌,不同学者对盐碱菌的定义范畴则有所差别。基于笔者在该领域内长期的学术积累和认识,暂定适宜生长盐度在0.5 mol/L和碱性pH 9.0之上的菌为“嗜盐嗜碱菌”,简称为“盐碱菌”[6]

根据上述盐碱菌的定义,从国际微生物系统分类的权威期刊International Journal of Systematic and Evolutionary MicrobiologySystematic and Applied MicrobiologyAntonie van LeeuwenhoekExtremophiles等及在List of Prokaryotic Names with Standing in Nomenclature (LPSN,http://www.bacterio.net/)发表有效的标准菌株,共获得了110株可培养的盐碱菌(表 1表 2),其中包括86株细菌和24株古菌。需要注意的是,盐碱菌对盐碱条件的依赖程度会随着培养条件的变化而改变。例如,同一株盐碱菌在培养基成分、盐度、pH值和温度等不同条件下培养可能会导致该菌对盐碱的耐受及适宜生长条件有所差别。此外,一些盐碱菌在培养过程中需要混合碳酸盐(Na2CO3/NaHCO3)来维持碱性pH,但是碳酸盐缓冲液碱性pH值会随着温度的增加而降低,因此盐碱菌在碱性条件下的一些重要的生理特性需要标注相应的生长温度[14]

表 1. 盐碱古菌生长适宜的Na+浓度、pH值和温度a Table 1. [Na+], pH and temperature optima and ranges for halo-alkalphilic archaeaa
Species [Na+]opt./(mol/L) [Na+]range/(mol/L) pH opt. pH range Temp. opt. Temp. range Accession number Isolated from
Halalkalicocus tibetensis DS12T 3.4 1.4–5.2 9.8 8.0–9.5 40 23–47 AF435112 Lake Zabuye (Tibet, China)
Halorubrum alkaliphilum DZ-1T 4.1 1.8–5.2 9.0 8.0–10.5 38 20–44 AY510708 Soda lake (XinJiang, China)
Halorubrum gandharaense MK13-1T 3.2–3.4 3.0–5.2 9.0–9.5 8.0–11.0 45 15–50 AB563178 Rock salt (Pakistan)
Halorubrum luteum CGSA15T 4.2 2.5–5.2 9.8 7.5–10.5 35 17–41 DQ987877 Chagannor lake (China)
Halorubrum tibetense 8W8T 3.2 1.7–5.2 9.3 8.0–10.5 39 22–45 AY149598 Zabuye lake (China)
Halorubrum vacuolatum M24T 3.5 2.6–5.1 9.5 8.5–10.5 38 20–50 D87972 Magadi lake (Kenya)
Halostagnicola alkaliphila T26T 4.3 3.4–5.1 9.0–9.5 9.0–10.0 37 30–45 HF544345 Bange soda lake (Tibet, China)
Haloterrigena daqingensis X313T 2.0–2.5 1.7–5.5 10.0 8.0–10.5 35 20–50 FJ545273 Saline alkaline soil (Daqing, China)
Methanosalsum natronophilum AME2T 1.4–1.9 1.0–3.3 9.5 8.0–10.2 37 N.–43 HM053965 Hypersaline soda lakes (Kulunda, Steppe)
Methanocalculus alkaliphilus AMF2T 0.6 0.2–1.5 9.5 8.0–10.2 35 N.–41 HM053969 Hypersaline soda lakes (Kulunda, Steppe)
Natrialba chahannaoensis C112T 2.6 1.7–5.1 9.0 8.5–10.5 45 20–55 AJ004806 Soda lake (China)
Natrialba hulunbeirensis X21T 3.4 2.0–5.1 9.0 8.5–10.5 50 20–55 AF262026 Soda lake (China)
Natronolimnobius aegyptiacus JW/NM-HA-15T 3.2–4.6 2.8–sat 9.0–9.5 7.5–10.5 52 30–58 KX857214 Soda lake (Wadi An Natrun, Egypt)
Natrialba magadii MS3T 3.5 2.0–5.2 9.5 8.5–11.0 39 20–50 X72495 Magadi lake (Kenya)
Natronoarchaeum mannanilyticum YSM-123T 4.0–4.5 2.0–5.3 9.0 5.8–9.5 37 20–55 AB501361 Salt made from Japanese seawater (Niigata, Japan)
Natronococcus amylolyticus Ah-36T 3.1 1.5–5.1 9.0 8.0–10.0 43 22–50 D43628 Magadi lake (Kenya)
Natronococcus occultus SP4T 2.8 1.4–5.2 9.8 8.0–N. 38 25–50 Z28378 Soda lake (East African)
Natronococcus roseus CG-1T 4.3–5.1 2.6–5.1 9–9.5 8.0–11.0 37–45 30–50 FR877778 Soda lake Chagannor(Inner Mongolia, China)
Natronolimnobius baerhuensis IHC-005T 3.4 2.6–0.0 9.0 7.0–10.0 37 30–46 AB125106 Soda lakes (China)
Natronolimnobius innermongolicus N-1311T 3.1 1.7–0.0 9.5 7.5–10.0 45 19–54 NR028162 Soda lakes (China)
Natronomonas pharaonis DSM 2160T 3.5 2.0–5.2 9.0 8.0–11.0 45 25–53 CR936257 Magadi lake (Kenya)
Natronorubrum bangense A33T 3.8 2.0–4.3 9.5 8.0–11.0 45 22–55 Y14028 Soda lake (Tibet, China)
Natronorubrum sulfidifaciens AD2T 3.1 2.1–4.8 8.7–9.2 8.0–10.0 44–47 20–55 DQ535889 Salt lake (Xinjiang, China)
Natronorubrum tibetense GA33T 3.4 2.0–5.1 9.0 8.5–11.0 45 22–55 AB005656 Soda lake (Tibet, China)
Natronobacterium gregoryi SP2T 3.0 2.5–5.2 9.8 8.0–N. 37 25–40 D87970 Rift valley (Kenya)
a: Since there are too many references to the model strain, this table does not contain any literature. Please examine the documents of interest. N: Data not shown in the published paper.

表 2. 盐碱细菌生长适宜的Na+浓度、pH值和温度 Table 2. [Na+], pH and temperature optima and ranges for halo-alkalphilic bacteria
Species [Na+]opt./(mol/L) [Na+]range/(mol/L) pH opt. pH range Temp. opt. Temp. range Accession number Isolated from
Alkalibacillus silvisoli BM2T 1.71-2.56 0.85-4.27 9.0-9.5 7.0-10.0 30-37 20-50 AB264528 Non-saline forest soil (Japan)
Alkalibacterium gilvum 3AD-1T 0.85 0-2.99 9.5 7.0-10.0 25-30 0-37 AB690566 Soft and semi-hard cheeses (Japan)
Alkalibacterium putridalgicola T22-1-2T 0.68 0-3.08 9.0 6.5-10.0 37-40 0-45 AB294171 Salted foods (Japan)
Alkalilactibacillus ikkensis GCM68T 0.51-1.37 0-1.71 10.0 8.5-10.7 20 0-25 EU281853 Cold and alkaline environment (Greenland)
Alkalimonas amylolytica N10T 0.51 0-1.20 10.0 7.5-11.0 37 10-42 AF250323 Soda lakes (China)
Alkalimonas delamerensis 1E1T 0.51 0-1.20 10.0-10.5 8.0-11.0 37 10-42 X92130 Soda lakes (China)
Alkalitalea saponilacus SC/BZ-SP2T 0.69 0.35-1.38 9.7 9.0-10.5 35-37 8-40 HQ191474 Soap soda lake (Washington State, USA)
Anaerobacillus arseniciselenatis E1HT 0.51 0-1.88 9.7 8.5-10.7 30-35 18-40 AJ865469 Khadyn soda lake (Russia)
Bacillus alkalisediminis K1-25T 0.85 0.34-2.05 9.0 7.0-12.0 25-28 15-37 AJ606037 Soda lake Chagannor (Inner Mongolia, China)
Bacillus aurantiacus K1-5T 0.51-1.20 0-2.56 9.0-10.0 9.0-12.0 28 10-45 AJ605773 Alkaline soda lakes (Hungary)
Bacillus beveridgei MLTeJBT 1.50 0.50-4.00 9.0 6.5-10.5 40 5-55 FJ825145 Mono Lake sediment
Bacillus lindianensis 12-3T 0.68 0.17-4.27 9.0 8.0-12.0 37 10-45 KP954690 Lindian saline alkaline soils (Heilongjiang, China)
Bacillus locisalis CG1T 1.20-1.71 0.17-4.27 9.0-10.0 8.0-12.0 37 10-45 FR714930 Hypersaline and alkaline lakes (China)
Bacillus oshimensis K11T 1.20 0-3.42 10.0 7.0-10.0 28-32 13-41 AB188090 Soil (Hokkaido, Japan)
Bacillus polygoni YN-1T 0.85 0.51-2.39 9.0 8.0-12.0 29-31 5-47 AB292819 Indigo balls (Ibaraki, Japan)
Bacillus qingdaonensis CM1T 2.05 0.43-3.42 9.0 6.5-10.5 37 25-45 DQ115802 Crude sea-salt sample (Qingdao, China)
Bacillus saliphilus 6AGT 2.74 0-4.27 9.0 7.0-10.0 37 4-50 AJ493660 Mineral pool (Campania, Italy)
Bacillus urumqiensis BZ-SZ-XJ18T 1.08 0.22-4.32 9.0-9.5 6.5-10.0 37 8-41 KM066107 Saline-alkaline lake (Xinjiang, China)
Desulfohalophilus alkaliarsenatis SLSR-1T 3.42 2.14-5.64 9.2 7.7-9.8 44 20-55 JQ582408 Searles Lake, California
Desulfonatronobacter acetoxydans APT3T 1.00 0.30-4.00 9.0-9.5 8.0-10.0 37-40 43 KP223254 Hypersaline soda lake (Siberia, Russia)
Desulfonatronobacter acidivorans APT2T 0.60 0-1.50 10.0 8.5-10.6 35 N.-38 GU289732 Hypersaline soda lakes (Altai, Russia).
Desulfonatronospira thiodismutans ASO3-1T 1.71 0-3.08 9.0 7.0-10.0 30 10-45 AJ493661 Mineral pool (Campania, Italy)
Desulfonatronum thioautotrophicum AHT9T 0.60 0.10-2.30 9.3-10.0 8.3-10.5 40-42 N. FJ469579 Steppe soda lakes (Russia)
Desulfuribacillus alkaliarsenatis AHT28T 0.60 0.20-2.00 10.0 9.0-10.5 35 N.-43 HM046584 Soda lakes (Kulunda)
Ectothiorhodospira variabilis WN22T 0.85-1.37 0.34-3.42 9.0-9.5 7.5-10.0 30-35 20-45 AM943121 Steppe soda lakes (Russia)
Egibacter rhizosphaerae EGI 80759T 1.71-2.05 1.37-4.27 9.0-10.0 7.0-11.0 30-35 25-50 KR605111 Rhizosphere of Tamarix hispida (Xinjiang, China)
Egicoccus halophilus EGI 80432T 0.51-0.85 0-1.54 9.0 6.0-10.0 30 20-40 KR605110 Saline alkaline soil (Xinjiang, China)
Fuchsiella ferrireducens Z-7101T 3.30-3.80 1.40-4.20 9.8 8.5-10.2 30-37 25-45 JX566886 Soda lake (Altai, Russia)
Geomicrobium halophilum BH1T 1.71-2.56 0.85-4.27 9.0 6.0-10.0 30 20-40 AB449106 Forest soil (Japan)
Gracilibacillus alcaliphilus SG103T 0.51 0-2.56 9.0 7.0-10.0 39 13-48 AB854047 Fermented Polygonum indigo liquor (Japan)
Halalkalibacillus halophilus BH2T 1.71-2.56 0.85-4.27 9.0 5.5-10.0 30-37 20-40 AB264529 Non-saline garden soil (Saitama, Japan)
Halobacillus alkaliphilus FP5T 1.71 0.85-4.27 9.0 6.0-10.0 37 25-45 AM295006 Salt lake (Fuente de Piedra, Spain)
Halolactibacillus alkaliphilus H-5T 0.51 0.09-5.13 13.0 7.5-13.0 28 15-45 EF554593 Xiarinaoer soda lake (Inner Mongolia, China)
Halolactibacillus halophilus M2-2T 0.51 0-4.10 9.0 6.5-9.5 30-37 5-40 AB196783 Decaying, marine algae, living sponge
Halolactibacillus miurensis M23-1T 0.51 0-4.27 9.5 6.5-10.0 37-40 5-45 AB196784 Decaying, marine algae, living sponge
Halomonas aquamarina DSM 30161T 1.71 0-3.42 9.0 7.5-10.0 37 5-50 AJ306888 Pacific Ocean
Halomonas campaniensis 5AGT 1.71 0-2.70 9.0 7.0-10.0 37 10-43 AJ515365 Malvizza (Campania, Italy)
Halomonas campisalisATCC 700597T 1.50 0.20-4.50 9.5 6.0-11.0 30 4-50 AF054286 Alkali Lake (Washington, USA)
Halomonas cupida DSM 4740T 1.50 0.20-4.50 9.5 6.0-12.0 30 4-50 L42615 Marine habitats
Halomonas daqingensis DQD2-30T 0.85-1.71 0.17-2.56 9.0 8.0-10.0 30 10-50 EF121854 Oilfield soil (Heilongjiang, China)
Halomonas kenyensis AIR-2T 0.50-1.20 0.04-2.20 9.5 7.5-10.6 36-40 15-55 AY962237 Soda lakes (Mongolia)
Halomonas korlensis XK1T 1.03-1.71 0.09-4.27 9.0 6.0-10.0 30 4-43 EU085033 Saline and alkaline soil (Korla, China)
Halomonas mongoliensis Z-7009T 0.70-1.70 0.16-2.20 9.0-9.6 8.0-10.5 36-40 15-50 AY962236 Soda lakes (Mongolia)
Halomonas zhaodongensis NEAU-ST10-25T 0.51 0-2.56 9.0 6.0-12.0 35 4-60 JQ762286 Zhaodong saline alkaline (Heilongjiang, China)
Halorhodospira abdelmalekii DSM 2110T 3.08 0.86-5.13 9.2 N. 44 N. X93477 Soda lake Wadi Natrun in Egypt
Marinilactibacillus psychrotolerans M13-2T 0.64 0-2.91 9.0 6.0-10.0 37-40 0-45 AB083406 Temperate and subtropical (Japan)
Marinobacter alkaliphilus ODP1200D-1.5T 0.60 0-3.59 9.0 6.5-10.8 30-35 10-45 AB125941 alkaline serpentine mud from the Ocean
Methylonatrum kenyense AMT 1T 0.50 0.30-4.00 10.0 8.3-10.5 30 N. DQ789390 Hypersaline chloride-sulfate lakes (Altai, Russia)
Methylophaga alcalica M39T 0.68 0.01-1.71 9.0-9.5 7.0-10.5 25-29 4-35 AF384373 Saline soda lake (Mongolian)
Methylophaga murata Kr3T 0.50-1.50 0.05-3.00 9.0 6.0-10.0 30 0-42 AY694421 Deteriorating marble (Moscow, Russia)
Methylophaga natronica Bur2T 0.50 N.-1.71 9.0 7.0-11.0 25-29 4-37 AY128533 Bulamay soda lake (Russia)
Natranaerobius thermophilus JW/NM-WN-LFT 3.90 3.10-4.90 9.5 8.3-10.6 53 35-56 DQ417202 Soda lake (Wadi An Natrun, Egypt)
Natranaerobius trueperi JW/NM-WN-LUT 3.70 3.10-5.40 9.5 7.8-11.0 b43 a26-52 EU338490 Soda lake (Wadi An Natrun, Egypt)
Natronovirga wadinatrunensis JW/NM-WN-LHT 3.90 3.10-5.30 9.9 8.5-11.5 b45-48 a24-52 EU338489 Soda lake (Wadi An Natrun, Egypt)
Natranaerovirga hydrolytica APP2T 1.00 0.20-3.50 10.0 8.2-10.6 30 45 GQ863487 Hypersaline soda lakes (Altai, Russia)
Natranaerovirga pectinivora AP3T 0.60 0.20 -2.50 9.7 8.5-10.5 28 43 GQ922846 Hypersaline soda lakes (Altai, Russia)
Natroniella acetigena Z-7937T 2.57 1.71-3.42 9.7 8.1-10.7 37 N.-42 X95817 Lake Magdi (Kenya)
Natronobacillus azotifigens 24KS-1T 1.00-1.50 0.20-4.00 9.5-10 7.5-10.6 36-38 N. EU143681 Soda soils (Mongolia)
Nesterenkonia aethiopica DSM 17733T 0.51 0.51-2.05 9.0 7.0-11.0 30-37 25-40 AY574575 Soda lake (Ethiopian)
Nesterenkonia populi GP10-3T 0.85-1.71 0.51-2.56 9.0 8.0-12.0 37 10-42 KP057085 Taklimakan desert (Xinjiang, China)
Nocardiopsis kunsanensis HA-9T 1.71 0.51-3.42 9.0 N. 37 N. AF195412 Saltern, Kunsan (Korea)
Nocardiopsis lucentensis A5-1T 1.71 0.51-4.28 9.0 5.0-N. 37 N. X97888. Salt marsh (Alicante, Spain)
Oceanobacillus indicireducens A21T 0.85 0-1.71 10.0 7.0-12.0 39 18-48 AB623011 Fermented Polygonum indigo liquor (Japan)
Oceanobacillus oncorhynchi 20AGT 1.71 0.09-3.42 9.0 6.5-9.5 37 10-40 AJ640134 Sulfurous spring (Mondragone, Italy)
Oceanobacillus polygoni SA9T 0.51 0.51-2.05 9.0 7.0-10.0 35 5-48 AB750685 Fermented Polygonum indigo liquor (Japan)
Planococcus rifietensis M8T 1.80 2.56 9.0 6.0-10.0 37 5-42 AJ493659 Sulfurous Spring (Campania, Italy)
Rhodovulum tesquicola A-36sT 0.51 0.05-1.71 9.0 7.5-10.0 25-35 N. EU741685 Soda lakes(Siberia)
Salinicoccus alkaliphilus T8T 1.71 0-4.28 9.0 6.5-11.5 32 10-46 AF275710 Baer Soda Lake (China)
Salinicoccus kekensis K164T 1.37 0.34-3.42 10.0 6.5-11.5 37 4-50 GU363531 Keke Salt Lake (Qinghai, China)
Salinicoccus luteus YIM 70202T 1.71 0.17-4.28 9.0 7.0-11.0 30 4-45 DQ352839 Desert soil (Egypt)
Salinivibrio costicola 8AGT 1.71 0.34-4.72 9.0 7.0-10.5 30.0 10-40 AJ640132 Saltish spring (Campania, Italy)
Salsuginibacillus halophilus halo-1T 3.25 1.54-5.13 9.0 5.0-10.0 37 18-50 EU581835 Soda lake (Inner Mongolia, China)
Spirochaeta sphaeroplastigenens JC133T 0.85-1.28 0.34-2.05 9.5 8.0-10.0 35-37 15-40 HE806187 Alkaline soda lake (Lonar, India)
Streptomyces alkalithermotolerans AC3T 0.68 0-2.74 9.5-10.0 7.5-11.0 28-30 16-55 HG934299 Lonar soda lake (India)
Thialkalivibrio halophilus HL 17T 2.00 0.20-5.00 9.0 7.5-9.8 35 N. AY346464 Hypersaline alkaline lake (Siberia, Russia)
Thialkalivibrio nitratireducens ALEN 2T 0.50 0.20-1.50 9.5-10.0 8.0-10.5 N. N. AY079010 Hypersaline alkaline lake (Wadi Natrun, Egypt)
Thioalkalimicrobium aerophilum AL 3T 0.50 0.20-1.50 9.5-10.0 7.5-10.6 30 N.-41 AF126548 Fazda soda lake (Wadi Natrun, Egypt)
Thioalkalivibrio thiocyanoxidans ARh 1T 0.50-1.00 0.30-4.30 10.2 8.5-9.5 30 N. AF151432 Soda lakes (Siberia)
Thioalkalivibrio versutus AL 2T 1.00-2.00 N.-4.30 10.0 7.5-10.6 35 N.-47 AF126546 Soda lakes (Siberia)
Tindallia californiensis APOT 0.51-0.85 0.17-3.42 9.5 8.0-10.5 37 10-48 AF373919 Mono Lake in California
Tindallia texcoconensis IMP-300T 1.28 0.43-3.27 9.5 7.5-10.0 35 35-45 DQ234901 Soda lake Texcoco in Mexico
a: Since there are too many references to the model strain, this table does not contain any literature. Please examine the documents of interested. b: Data obtained in our research group. N: Data not shown in the published paper.

2.2 可培养盐碱细菌多样性及其生理特征

截止撰写本论文时,所有文献报道的可培养盐碱细菌的标准菌株在门分类水平上依次为厚壁菌门(Firmicutes,占总数约50%),变形菌门(Proteobacteria,37%),放线菌门(Actinobacteria,9%),其余分布在螺旋体门(Spirochaetes)和拟杆菌门(Bacteroidetes) (图 1)。在纲分类水平上,依次为芽孢杆菌纲(Bacilli,占总数约40%),γ-变形菌纲(γ-Proteobacteria,30%),梭菌纲(Clostridia,11%),δ-变形菌纲(δ-Proteobacteria,6%)和放线菌纲(Actinobacteria,6%)。在科分类水平上,数量最多的为芽孢杆菌科(Bacillaceae),占盐碱古菌总数约31%,其次分别为盐单胞菌科(Halomonadaceae),脱硫杆菌科(Desulfobacteraceae)和外硫红螺旋菌科(Ectothiorhodospiraceae),分别占约11%、6%和6%。而乳杆菌科(Lactobacillaceae)、葡萄球菌科(Staphylococcaceae)和交替单胞菌科(Alteromonadaceae)等所占比例小于5%。可培养盐碱细菌在属分类水平上分布比较分散,数量最多的为芽孢杆菌属(Bacillus)和盐单胞菌属(Halomonas),分别占总数约12%和11%。

图 1 基于16S RNA基因序列构建的可培养盐碱菌系统发育树 Figure 1 Phylogenetic tree based on 16S rRNA gene sequences between halo-alkaliphilc microorganisms in this paper.

表 2所示,在盐碱细菌中约37%菌株适宜生长盐度高于1.7 mol/L (相当于10% NaCl),盐碱细菌中仅有22株生长适宜pH值能达到10,占总数约26%。笔者[15]曾从美国华盛顿州Soap Lake盐碱湖(盐度约为1.5%-14%且pH约为9.8) 中分离获得了一株专性厌氧盐碱细菌Alkalitalea saponilacus SC/BZ-SP2T。该菌能在Na+浓度为0.35-1.38 mol/L (相当于2%-8% W/V NaCl)和pH 7.5-10.5中进行生长繁殖。其适宜生长条件为:Na+浓度为0.44-0.69 mol/L (相当于2.6%-4.0% W/V NaCl)和pH 9.7。SC/BZ-SP2T菌株是在拟杆菌门(Bacteroidetes)中的海洋滑动菌科(Marinilabiliaceae)分离获得的唯一1株盐碱菌,暗示着该菌的盐碱适应机制可能不同于其他类群的盐碱菌。此外,该菌的全基因组测序完成图及其注释表明(数据未出版),其携带1个木聚糖酶(endo-1, 4-β-xylanase)基因,1个葡萄糖醛酸苷酶(α-glucuronidase)基因和1个阿拉伯呋喃糖苷酶(α-N-arabinofuranosidase)基因,这与该菌能以半纤维素为唯一碳源和能源相对应,因此该菌分泌的与半纤维素转化相关的木聚糖酶系在造纸工业中(如用生物漂白制浆去除半纤维素)有广阔的应用前景。同时,该菌能代谢来源于山毛榉木(beechwood)、桦木(birchwood)和燕麦(oat spelts)中的木聚糖(xylan),终产物主要为丙酸(占总羧酸的90%以上),这说明该菌在发酵食品与饲料天然防腐剂中潜在的应用前景。最近笔者研究组[16]从新疆乌鲁木齐附近的盐湖淤泥中分离获得了1株好氧性嗜盐嗜碱的乌鲁木齐芽孢杆菌(Bacillus urumqiensis)。该菌生长盐度范围很宽,可以在0.22-4.32 mol/L Na+(相当于1.3%-25.3% NaCl,W/V)中生存繁殖,其生长最适盐度为1.08 mol/L Na+ (相当于6.3 % NaCl,W/V)。同时,该菌在pH 6.5-10.0范围内都能生长,pH适应范围较宽,在pH 8.5-9.5之间生长良好。由于芽孢杆菌属(Bacillus)为芽孢杆菌科(Bacilliceae)中最大的一个属且在自然界中分布尤为广泛,共包含了300多个标准菌株(http://www.bacterio.net/index.html),因此分离的盐碱芽孢杆菌开拓了对芽孢杆菌多样性的新认识。

值得注意的是,盐碱菌中某些极为特殊的类群不仅仅喜好高盐碱条件,而且在温度上也有较高的依赖性。2007年,笔者曾经所在的研究组Mesbah博士[17]首次报道了1株严格厌氧的嗜热盐碱细菌(Natranaerobius thermophilus DSM 18059T)。N. thermophilus能在高盐、强碱性pH值和高温条件下生长繁衍。它生长范围为:盐度3.1-4.9 mol/L Na+ (相当于18.1%-28.7% NaCl,W/V)、pH 8.3-10.6和35-56 ℃;最佳生长条件为:3.9 mol/L Na+ (相当于23% NaCl,W/V)、pH 9.5和53 ℃。该菌生长所依赖的适宜温度为目前可培养盐碱菌中最高的(50 ℃以上),因此属于盐碱菌定义范畴中一类极为特殊的菌群,也被称之为“多极端微生物”。

2.3 可培养盐碱古菌的多样性及其生理特征

可培养盐碱古菌标准菌株的数量及其在微生物系统发育地位上的多样性远小于盐碱古菌。可培养盐碱古菌现有的标准菌株在微生物系统发育地位上全部被归类为广古菌门(Euryarchaeota) (图 1)。在纲、目和科分类水平上,大约92%的盐碱古菌隶属于盐古菌纲(Halobacteria)、盐古杆菌目(Halobacteriales)和盐古杆菌科(Halobacteriaceae),仅有约8%的盐碱古菌隶属于甲烷微菌纲(Methanomicrobia),甲烷微菌目(Methanomicrobiales)和甲烷微菌科(Methanocalculaceae)。在属分类水平上,数量最多的为盐红菌属(Halorubrum),占盐碱古菌总数约20%;其次为盐无色菌属(Natrialba)、盐碱球菌属(Natronococcus)、盐碱湖菌属(Natronolimnobius)和嗜盐碱红菌属(Natronorubrum),均占约12%;最后为拉氏栖盐湖菌属(Halostagnicola)、盐陆生菌属(Haloterrigena)、小巧甲烷卵圆形菌属(Methanocalculus)、甲烷盐菌属(Methanosalsum)、盐碱单胞菌属(Natronomonas)和Halalkalicocus菌属,均占约4%。

在盐碱古菌中(表 1),约有96%的菌株适宜生长盐度高于1.7 mol/L (约等同于10% NaCl),因此盐碱古菌对盐依赖及耐受程度要远高于盐碱细菌。但是,仅有1株盐碱古菌生长适宜pH值能达到10,占总数的4%,所以盐碱古菌对碱性pH的依赖性及耐受程度要低于盐碱细菌。最近笔者从埃及盐碱湖中分离获得了1株好氧性嗜热盐碱古菌(暂命名为Natronolimnobius aegyptiacus,已投稿)。该菌株对盐碱的耐受和适宜范围也很宽,在盐度2.6 mol/L Na+至饱和(相当于15.2%-30.4% NaCl,W/V)及pH 7.5-10.3都能生长繁殖,在盐度3.3-4.6 mol/L Na+ (相当于19.3%-26.9% NaCl,W/V)及pH 9.0-9.5生长良好(数据暂未发表)。该菌的温度生长范围(30-58 ℃)和适宜生长温度(52 ℃)在盐碱古菌中也是最高的。因此,盐碱古菌N. aegyptiacus和盐碱细菌N. thermophilus可为研究生命在3个极端条件下的存在本质提供极为珍贵的材料和潜在生物技术应用价值。

3 总结与展望

可培养盐碱菌主要来源于盐碱土或盐碱湖、盐湖或碱湖、工业发酵盐碱液以及人工海盐制品等极端环境,但是还可以从中性的土壤或植物根际土等非极端环境中分离获得(表 1表 2)。相对单一的嗜盐菌或嗜碱菌,盐碱菌的分离培养过程较特殊和复杂,尤其是以Na2CO3/NaHCO3作为碱性pH缓冲液来分离培养盐碱菌。目前我国科学家对可培养盐碱菌的标准菌株报道还很少,采用磁性纳米技术等新技术方法分离捕获盐碱菌非常迫切[18-19]。在我国内蒙古、黑龙江、吉林、青海、新疆和西藏等地区分布着很多类型的盐碱土和盐碱湖[20-21],必定孕育着丰富的盐碱菌。诸如新疆吐鲁番地区众多特殊地质环境的盐/碱湖为分离盐碱菌提供了良好的契机,可能会分离到一些更具有科学价值和应用前景的盐碱菌。

盐碱菌生长于高盐度和碱性双重胁迫条件下,既要适应胞外高渗透压和低水分活度条件又要调控胞内外pH以达到平衡,因此这类菌的嗜极机理应有其独特之处,但是相对于嗜盐菌或嗜碱菌适应机制研究,有关盐碱菌适应性的科学报道仍然很少。根据笔者掌握的资料,目前盐碱菌中尚缺乏有效的分子遗传工具或成熟的遗传转化技术,因此对其分子机理的研究带来极大的障碍[6]。据我们了解,尚未发现一种成熟的技术可以对野生型盐碱菌目的基因进行突变,所以还需要开展这方面的研究,以寻求有效的遗传操作工具。近年来,微生物基因组、转录组和蛋白组学新技术取得了飞速发展,其中验证了之前预测的一些与耐盐碱相关的基因,发现了一些已知盐碱基因的新功能或新的耐盐碱基因,这为研究盐碱菌的嗜极机理及其应用提供了新的思路[22-25]。目前,笔者研究组正在应用传统的分子生物学手段和组学新技术以“点”和“面”结合,对盐碱细菌N. thermophilus和盐碱古菌N. aegyptiacus从嗜盐、嗜碱和嗜热多极端环境适应机制进行全面系统的科学研究。相信不久的将来,我国具有自主知识产权的可培养盐碱菌的分离培养及其应用,以及它们在盐碱嗜机理方面的科学研究必将蓬勃发展,该领域内的科学成果将为现代生物技术的快速发展提供新的思路和材料,也为极端生命存在的本质提供一些新的理解、诠释和理论补充。

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