天山1号冰川表面雪中细菌群落的季节性变化研究
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摘要
冰川中蕴藏着丰富的微生物资源,这些微生物按年代保存于冰川中、其组成特性的变化是大气及环境变化信息的响应。在冰川沉积过程中,微生物及花粉、孢子、植物片断等随大气环流的运移,与粉尘一起沉降在冰川表面,经历冰川环境的选择,微生物自身的适应性调节等一系列变化,然后随新雪的降临,逐渐埋藏于深层冰川中。因此,表层雪中微生物群落变化特征对于揭示雪坑及深层冰芯中微生物和环境变化的关系具有重要意义。
本研究以天山1号冰川各个月份的表面雪为实验材料,利用分子标记变性梯度凝胶电泳(denaturing gradient gel electrophoresis.DGGE)技术,荧光显微计数、微生物恢复培养和16SrDNA-PCR基因克隆技术,通过对雪样中微生物数量和群落结构的研究,分析了不同季节表面雪中微生物的数量、群落结构特征及其与气候环境因子的关系。主要结果如下：
1、天山1号冰川表面雪中的细菌数量分布具有明显的季节性分布特征。细菌总数分布范围在3.20×103-57.85×103(cell/ml)(Mean,20.56×103; SD,16.17×103；N=22)之间,细菌数量在春季和夏季雪层中较低,变化趋势较平缓；在秋冬季节雪层中较高,波动幅度也较大。11cm-20cm雪层中细菌浓度普遍比Ocm-10cm雪层高。细菌总数与可培养细菌数量正相关,变化趋势一致。月平均气温、Ca2+浓度与表面雪中细菌数量分布也有一定的关系。
2、细菌群落具有季节性分布特征。表面雪中恢复培养得到的细菌隶属于5个系统发育组(Actinobacteria(HGC), Firmicute(LGC),α-proteobacteria,β-proteobacteria,γ-proteobacteria),属于Arthrobacter, Curtobacterium, Corynebacterium, Frigoribacterium, Microbacterium, Rathayibacter, Bacillus Paenibacillus, Sporosarcina, Sphingomonas, Brevundimonas, Massilia, Psychrobacter和两个未定属[unclassified genus(代表菌株TS0211R和TS0914T)],总计15属。Actinobacteria(HGC)组普遍存在于各个季节中,占全部分离菌株的60%,其次是Firmicute(LGC)组占17%。春节和夏季(4月-8月)细菌种类较少,秋冬季节(9月-2月)细菌种类较多。Arthrobacter和Sphingomonas两属普遍存在于各个月份中,其它属细菌只在一些特定的月份分离到。
3、变形梯度凝胶电泳(DGGE)结果显示了细菌DNA的季节性变化特征。各个月份细菌DNA泳道相似性聚类分析显示：Ocm-10cm雪层中细菌群落分布特征规律性较10cm-20cm雪层明显,其春季与夏季细菌DNA相似(5月-8月聚类成簇),秋季与冬季相似(10月-2月聚类成簇)。Shannon指数(H’)显示细菌多样性较高,在不同深度雪层中分别于1月和5月出现峰值。DGGE图谱代表性条带中的DNA经过测序分析后显示：这些序列所代表的细菌同属于可培养细菌隶属的5个系统发育组；其中Arthrobacter属和Bacillus属在DGGE和分离培养中都有发现,Cryobacterium属,Devosia属,Janthinobacterium属,cryptococcus属和两个未定属则只是在DGGE条带中发现。
以上研究结果表明：天山1号冰川表面雪中细菌数量和群落季节性变化特征明显；荧光显微技术、细菌恢复培养和DGGE技术分别从不同角度说明了细菌群落的季节性分布规律,将这些技术综合运用能更为准确的揭示表面雪中的微生物多样性和季节性沉降特征。
Glacier ice is a unique habitat because it preserves microbial life and past climate records chronologically for hundreds of thousands of years. Microbial cells and other biological material, such as pollen gains, spores, plant debris and insects, associated with dust particles are propelled by the wind from neighbouring and distant places, deposited on the ice surface, and gradually, as snow accumulates, embedded into the deeper ice layers. The microbial studies of deep glacier ice suggested that in-depth fluctuations in cell numbers and microbial composition were related to different dust concentrations and climate changes, could be a potential bio-indicator of paleoclimatology. Therefore, the study of microbial community changes in surface snow is important to reveal the dynamics of correlation between microorganisms and environmental changes in deep ice cores.
The seasonal change of microbial numbers and community structure in the surface snow sampled every month from glacier No.l of Tianshan were analyzed by denaturing gradient gel electrophoresis (DGGE), fluorescence microscopy count, microbial culture and the PCR amplification of bacteria 16S rRNA gene. The main results were as follows:
1. The distribution of bacteria in surface snow of glacier No.1 in Tianshan demonstrated obvious seasonal characteristic. Total number of bacteria ranged from 3.20 ×103 to 57.85×103 (cells/ml) (Mean,20.56×103; SD,16.17×103; N= 22). The number of bacteria in the spring and summer is lower than that in autumn and winter; the fluctuation of bacterial quantity is larger in autumn and winter. In general, the concentration of bacteria in the snow of 11-20cm layer is higher than that in snow of 0-10cm layer. The amounts of bacteria and cultured bacteria that are positive correlation ship show the same trend related to seasonal changes. There is a certain relationship among the distribution of bacteria, concentration of Ca2+and average temperature of months.
2. The seasonal change of bacteria communities was obvious. Cultured bacteria belonged to five phylogenetic groups(Actinobacteria(HGC), Firmicute(LGC),α-proteobacteria,β-proteobacteria,γ-proteobacteria).Including:Arthrobacter, Curtobacterium, Corynebacterium, Frigoribacterium, Microbacterium Rathayibacter, Bacillus, Paenibacillus, Sporosarcina, Sphingomonas, Brevundimonas, Massilia, Psychrobacter and unclassified genus(Representative strain TS0211R and TS0914T), total 15 genera. Actinobacteria(HGC) group commonly existed in all seasons, accounting for 60%of all isolates, followed Firmicute (LGC) group accounted for 17%. There were fewer species of bacteria in Spring and summer (April-August) samples than that in fall and winter season (September-February). Arthrobacter and Sphingomonas were isolated from the samples of all of months; other genera only existed in specific months.
3. The results showed the seasonal compositions of bacterial DNA by denaturing gradient gel electrophoresis (DGGE).The cluster analysis of DGGE displayed better regularity in 0cm-10cm snow layer than that in 10cm-20cm snow layer. The structure of bacteria DNA in May to August and October to February displayed high similarity forming two clusters respectively in 0-10cm snow layer. Shannon index (H') showed high bacterial diversity; its peak value appeared in January and May in two snow layer respectively. Sequencing analysis demonstrated Arthrobacter and Bacillus were found in cultivation and DGGE; Crybacterium, Devosia, Janthinobacterium and Cryptococcus were only found in cultivation.
There were obvious seasonal change of bacterial diversity and quantity in surface snow of glacier No.1 in Tianshan. The comprehensive picture of microbial shift can be approached by the combined techniques such as fluorescent microscopy, bacterial cultivation and DGGE.
本研究以天山1号冰川各个月份的表面雪为实验材料,利用分子标记变性梯度凝胶电泳(denaturing gradient gel electrophoresis.DGGE)技术,荧光显微计数、微生物恢复培养和16SrDNA-PCR基因克隆技术,通过对雪样中微生物数量和群落结构的研究,分析了不同季节表面雪中微生物的数量、群落结构特征及其与气候环境因子的关系。主要结果如下：
1、天山1号冰川表面雪中的细菌数量分布具有明显的季节性分布特征。细菌总数分布范围在3.20×103-57.85×103(cell/ml)(Mean,20.56×103; SD,16.17×103；N=22)之间,细菌数量在春季和夏季雪层中较低,变化趋势较平缓；在秋冬季节雪层中较高,波动幅度也较大。11cm-20cm雪层中细菌浓度普遍比Ocm-10cm雪层高。细菌总数与可培养细菌数量正相关,变化趋势一致。月平均气温、Ca2+浓度与表面雪中细菌数量分布也有一定的关系。
2、细菌群落具有季节性分布特征。表面雪中恢复培养得到的细菌隶属于5个系统发育组(Actinobacteria(HGC), Firmicute(LGC),α-proteobacteria,β-proteobacteria,γ-proteobacteria),属于Arthrobacter, Curtobacterium, Corynebacterium, Frigoribacterium, Microbacterium, Rathayibacter, Bacillus Paenibacillus, Sporosarcina, Sphingomonas, Brevundimonas, Massilia, Psychrobacter和两个未定属[unclassified genus(代表菌株TS0211R和TS0914T)],总计15属。Actinobacteria(HGC)组普遍存在于各个季节中,占全部分离菌株的60%,其次是Firmicute(LGC)组占17%。春节和夏季(4月-8月)细菌种类较少,秋冬季节(9月-2月)细菌种类较多。Arthrobacter和Sphingomonas两属普遍存在于各个月份中,其它属细菌只在一些特定的月份分离到。
3、变形梯度凝胶电泳(DGGE)结果显示了细菌DNA的季节性变化特征。各个月份细菌DNA泳道相似性聚类分析显示：Ocm-10cm雪层中细菌群落分布特征规律性较10cm-20cm雪层明显,其春季与夏季细菌DNA相似(5月-8月聚类成簇),秋季与冬季相似(10月-2月聚类成簇)。Shannon指数(H’)显示细菌多样性较高,在不同深度雪层中分别于1月和5月出现峰值。DGGE图谱代表性条带中的DNA经过测序分析后显示：这些序列所代表的细菌同属于可培养细菌隶属的5个系统发育组；其中Arthrobacter属和Bacillus属在DGGE和分离培养中都有发现,Cryobacterium属,Devosia属,Janthinobacterium属,cryptococcus属和两个未定属则只是在DGGE条带中发现。
以上研究结果表明：天山1号冰川表面雪中细菌数量和群落季节性变化特征明显；荧光显微技术、细菌恢复培养和DGGE技术分别从不同角度说明了细菌群落的季节性分布规律,将这些技术综合运用能更为准确的揭示表面雪中的微生物多样性和季节性沉降特征。
Glacier ice is a unique habitat because it preserves microbial life and past climate records chronologically for hundreds of thousands of years. Microbial cells and other biological material, such as pollen gains, spores, plant debris and insects, associated with dust particles are propelled by the wind from neighbouring and distant places, deposited on the ice surface, and gradually, as snow accumulates, embedded into the deeper ice layers. The microbial studies of deep glacier ice suggested that in-depth fluctuations in cell numbers and microbial composition were related to different dust concentrations and climate changes, could be a potential bio-indicator of paleoclimatology. Therefore, the study of microbial community changes in surface snow is important to reveal the dynamics of correlation between microorganisms and environmental changes in deep ice cores.
The seasonal change of microbial numbers and community structure in the surface snow sampled every month from glacier No.l of Tianshan were analyzed by denaturing gradient gel electrophoresis (DGGE), fluorescence microscopy count, microbial culture and the PCR amplification of bacteria 16S rRNA gene. The main results were as follows:
1. The distribution of bacteria in surface snow of glacier No.1 in Tianshan demonstrated obvious seasonal characteristic. Total number of bacteria ranged from 3.20 ×103 to 57.85×103 (cells/ml) (Mean,20.56×103; SD,16.17×103; N= 22). The number of bacteria in the spring and summer is lower than that in autumn and winter; the fluctuation of bacterial quantity is larger in autumn and winter. In general, the concentration of bacteria in the snow of 11-20cm layer is higher than that in snow of 0-10cm layer. The amounts of bacteria and cultured bacteria that are positive correlation ship show the same trend related to seasonal changes. There is a certain relationship among the distribution of bacteria, concentration of Ca2+and average temperature of months.
2. The seasonal change of bacteria communities was obvious. Cultured bacteria belonged to five phylogenetic groups(Actinobacteria(HGC), Firmicute(LGC),α-proteobacteria,β-proteobacteria,γ-proteobacteria).Including:Arthrobacter, Curtobacterium, Corynebacterium, Frigoribacterium, Microbacterium Rathayibacter, Bacillus, Paenibacillus, Sporosarcina, Sphingomonas, Brevundimonas, Massilia, Psychrobacter and unclassified genus(Representative strain TS0211R and TS0914T), total 15 genera. Actinobacteria(HGC) group commonly existed in all seasons, accounting for 60%of all isolates, followed Firmicute (LGC) group accounted for 17%. There were fewer species of bacteria in Spring and summer (April-August) samples than that in fall and winter season (September-February). Arthrobacter and Sphingomonas were isolated from the samples of all of months; other genera only existed in specific months.
3. The results showed the seasonal compositions of bacterial DNA by denaturing gradient gel electrophoresis (DGGE).The cluster analysis of DGGE displayed better regularity in 0cm-10cm snow layer than that in 10cm-20cm snow layer. The structure of bacteria DNA in May to August and October to February displayed high similarity forming two clusters respectively in 0-10cm snow layer. Shannon index (H') showed high bacterial diversity; its peak value appeared in January and May in two snow layer respectively. Sequencing analysis demonstrated Arthrobacter and Bacillus were found in cultivation and DGGE; Crybacterium, Devosia, Janthinobacterium and Cryptococcus were only found in cultivation.
There were obvious seasonal change of bacterial diversity and quantity in surface snow of glacier No.1 in Tianshan. The comprehensive picture of microbial shift can be approached by the combined techniques such as fluorescent microscopy, bacterial cultivation and DGGE.
引文
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