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青藏高原高海拔冰冻圈中的微生物不仅是极端环境下生物生态研究的重点，也是研究全球气候变暖背景下微生物对气候环境变化响应的重要方面。同时现代过程和机制的研究是利用各种替代性指标恢复古气候环境的重要方面。本研究应用先进的流式细胞技术和建立16S rRNA基因文库的方法，研究了珠穆朗玛峰地区、各拉丹东果曲冰川、纳木错扎当冰川、藏东南帕隆4号冰川现代雪冰和冰川融水中微生物数量和群落结构特征。通过对珠峰北坡冰塔林、表层雪中微生物的研究，揭示了珠峰高海拨冰雪环境中微生物的特征；通过对珠穆朗玛峰地区冰碛湖和冰川融水中细菌群落及生理特征的研究，揭示了青藏高原高海拔水体中微生物特征及其与气候环境的关系；通过对各拉丹东果曲冰川48m冰芯中细菌数量的变化以及雪坑中细菌数量及多样性的研究，结合各项冰雪物理化学参数及温度的变化，揭示了冰雪中细菌的季节和年度变化及其与气候环境的关系；通过对不同地区冰川雪中微生物的对比研究，揭示了青藏高原现代雪冰微生物数量和群落结构的分布特征及其对气候环境变化的响应。珠穆朗玛峰北坡6600－8000m表层雪中细菌数量在2.11×104 cells/ ml－9.44×104 cells / ml之间，高于南极地区但与其他高山雪中相似。表层雪中细菌的数量有随海拔升高而增多的趋势，但与离子浓度相关性不大。珠峰北坡冰塔林和表层雪中的细菌16S rRNA序列与土壤、湖泊和河流、动植物体及其他冷冻环境中细菌的相似。6000 m冰塔林冰中细菌的16S　rRNA基因序列分属于α，β，γ-Proteobacteria，Actinobacteria，Firmicutes，CFB, Cyanobacteria 7大类，β-Proteobacteria为其优势类群。6350m表层雪中仅有CFB，β-Proteobacteria和Firmicutes 3大类细菌，以CFB类细菌占绝对优势，占总克隆的93%。6600 m表层雪细菌的16S rRNA基因文库中近一半序列属于β-Proteobacteria (占全部克隆的46%)，其余分属于Actinobacteria(35%)，α-Proteobacteria(13%)，CFB(2%)，Deinococci(2%)类群及未定类(2%)。珠峰东绒布冰川雪坑中微生物的16S rRNA基因序列分属于α、β、γ-Proteobacteria, Actinobacteria, Firmicutes, CFB, Cyanobacteria, Eukaryotic chloroplast, TM7 candidate phylum共 9大类，以γ-Proteobacteria为主要类群，其中Acinetobacter和 Leclercia属是整个菌群中的优势属。微生物的数量和菌群结构具有明显的季节特征，夏季微生物的数量高于冬季；菌群结构具有一些共有属种的同时，在夏、冬季雪中具有各自特有的属种，共有属种推测是青藏高原局地源的微生物，序列同源性分析结果表明夏季较多细菌属种与海洋环境相关，冬季细菌则具有更强的耐冷性。微生物明显的季节变化可能是受珠峰地区夏季和冬季不同水汽来源的影响。各拉丹东48m冰芯中细菌数量与冰芯中的污化层密切相关，在年际尺度上与氧同位素和钙离子浓度都显著相关性，多元线性回归分析表明，冰芯中的细菌数是温度和外来粉尘共同作用的结果，但粉尘对细菌数量的影响略大于温度。冰芯中细菌的数量在年际尺度上不仅可以反映大气中粉尘的变化，也同时可以反映温度的变化。各拉丹东果曲冰川雪坑中细菌的多样性具明显的季节变化，季风期末沉积雪中与季风期前冬半年沉积雪中细菌具有完全不同的群落组成；季风期末细菌的多样性远高于季风期前；季风期前雪中细菌只分离自两类环境：冰冻环境和土壤，季风期末沉积雪中细菌除与冰冻环境和土壤中细菌相似外，还与海洋、植物、动物和人体多种环境相关。不同季节中细菌多样性的显著不同是雪中有机生物对不同季节不同大气环流响应的结果。藏东南帕隆4号冰川中不同海拔雪坑中细菌的数量沿深度具相同的变化趋势，低海拔雪坑中细菌数量普遍较高海拔中的高。雪中细菌属于α、β、γ-Protobacteria， Actinobacteria，CFB，Firmicutes，Acidobacteria , Cyanobacteria , Planctomycetes共9类39个属，14个未定属，其中α-Protobacteria和Actinobacteria为主要类群。藏东南帕隆4号冰川雪中细菌群落在不同季节有所不同，但季节变化不如珠峰和各拉丹东地区明显。夏季细菌的多样性高于冬季，且在夏冬季具不同的种属特征，但冬夏季共有属所代表的克隆占全部克隆的77%，为群落中的优势种群。分离自植物相关环境的Agreia和Rhizobium为主要优势属。扎当冰川雪中细菌归属于α、β、γ、δ-Protobacteria， Actinobacteria，CFB，Firmicutes，Acidobacteria , Cyanobacteria , Chloroflexi，Deinococcus-Thermus，Verrucomicrobia共12类及4个未定类。其中α、β、γ-Protobacteria，CFB和Actinobacteria在群落中所占比例相似（14%－17%），没有数量上的优势类群。扎当冰川雪坑代表2005年冬季－2006年5月的降雪，冬春季降雪中细菌的群落结构相似，但5月中旬的降雪中细菌较多地受纳木错湖水的影响。对各拉丹东果曲冰川、纳木错扎当冰川、珠峰东绒布冰川和藏东南帕隆4号冰川现代冰雪中微生物特征的综合分析表明，青藏高原不同区域雪中细菌的数量和多样性都有不同的分布特征。细菌数量呈现从南到北增加的趋势，位于高原北部的各拉丹东果曲冰川和高原中部的纳木错扎当冰川雪中细菌的数量要明显高于南部的珠峰东绒布冰川和藏东南帕隆4号冰川。受不同的大气环流和不同类型的周边生态环境的影响，不同区域冰川雪中具不同的细菌群落组成，地理位置较近的冰川细菌的群落组成也较相似。但青藏高原冰雪中也存在一些共有的典型种属，分别为α-Protobacteria类的Sphingomonas和Ochrobactrum属，β-Protobacteria 类Polaromonas、Acidovorax、Aquabacterium、Ralstonia属，γ-Protobacteria 类Acinetobacter属，Firmicutes类Bacillus属，CFB类Chitinophaga属。珠峰地区冰碛湖及冰川融水中60条16S rRNA基因序列分属α，β，γ-Proteobacteria，Actinobacteria，CFB, Fibrobacteres, Planctomycetes，Verrucomicrobia, Eukaryotic chroloplast 9大类，CFB类群为优势类群。不同海拔高度冰碛湖和冰川融水中细菌的数量和群落结构不同，5152m冰碛湖中细菌数量最多且多样性最高，6350m冰川融水中的多样性最低。温度对群落的组成具有重要的影响。同样的海拔高度，营养状况影响细菌的群落结构。珠峰地区6350m冰川融水中可培养细菌有较高的多样性，共获得α、β、γ-Protobacteria，Firmicutes，Actinobacteria　5类97株细菌。在形态上以球形和棒形为主，大多具有色素。根据细菌对营养的利用和对温度的响应，冰川融水中可培养细菌分为3大类，一类是广泛地利用多种营养物质，代谢活性强，对温度变化敏感的窄温种，代表菌株为Janthinobacterium sp.， Arthrobacter luteolus；第二类是对碳源的利用较低，但适应低温环境的广温种，如：Cellulomonas sp.，Acinetobacter baumannii；第三类细菌则几乎不利用碳源，对温度的响应复杂，如：Microbacterium sp.，Bacillus megaterium。

Microorganism in high altitude cryosphere in the Tibetan Plateau is important not only to study microbe in extreme environments, but also to study how the microbe response the globe warmer change. In the mean time, research on modern deposition courses and rulers in the glacier is significant to study climate and environment in the past time. The bacterial community at East Rongbuk glacier at Mount Everest, Guoqu glacier at Geladandong region, Zadang glacier at Namuco region, and Palong No.4 glacier at Eastsouth of Tibet were examined through 16S rRNA gene clone library and flow cytometry approach. Microbial community in snow, serac ice, moraine lake and glacier meltwater on the north slope of Mt. Everest was investigated, microbial features in such high elevation region and their relationships with monsoon and environment were elaborated. Bacterial abundance in 48m ice core and bacterial diversity in snowpit at Guoqu glacier at Geladandong region were studied, aiming at the seasonal and annual change of bacteria in glacier. Our research also compared the bacterial characters in different glaciers located at various regions to study the distribution of snow bacteria across the Tibetan Plateau, and the response rule of bacteria to the climate and environment change. Bacterial abundance in surface snow ranged from 2.11×104 to 9.44×104 cells/mL between 6600 to 8000 m a.s.l. on the north slope of Mt. Everest，increasing with altitude but showed no correlation with chemical parameters. Bacterial abundance in the snow on Mt. Everest was 10-100 fold that in Antarctica, similar to that in other high mount. Bacteria in the cryosphere on Mt. Everest were closely related to those obtained from other habitats(i.e. soil, aquatic environments, plant, animal and human), and suggested that most of the bacteria in the Mt. Everest region were much likely to be exotic, and derived from different sources. Bacterial diversity in the serac ice at 6000 m a.s.l., surface snow at 6350 m and 6600 m a.s.l were various. Bacteria in the serac ice clone library were affiliated with 7 phylogenetic groups, i.e. α, β, γ-Proteobacteria, Actinobacteria, Firmicutes, CFB, and Cyanobacteria. The dominant group was β-Proteobacteria, accounting for 44% of the total clones. Bacteria in the surface snow at 6350 m were classified into the groups CFB, β-Proteobacteria and Firmicutes ,which accounted for 93%，6% and 1% of total clones, respectively. Bacteria belonging to CFB were absolutely dominant, and most of them were closed related to psychrophilic bacteria. Nearly half (46%) of clones from the surface snow at 6600 m clone library belonged to β-Proteobacteria. Others belonged to Actinobacteria (35%), α-Proteobacteria (13%), CFB (2%), Deinococci (2%) or were unclassified (2%). Bacterial 16S rRNA sequences at East Rongbuk glacier at Mt Everest belonged to α、β、γ-Proteobacteria, Actinobacteria, Firmicutes, CFB, Cyanobacteria, Eukaryotic chloroplast, and TM7 candidate phylum. γ-Proteobacteria was dominant in this region, and genera Acinetobacter and Leclercia were predominant genus. Population and community structure were various seasonally. Abundance of bacteria in summer snow was higher than those in winter. Community structures of bacteria in winter and summer snow were diverse, with common species of both seasons and particular species of each season respectively. Common species possible originated from the Tibet Plateau. Microbe in summer snow affiliated with marine environment, bacteria in winter snow connected with multiplex surrounding and shown more cold resistant feature. Seasonal various of abundance and diversity of microbe were influenced by the seasonal character of climate and atmospheric circulation in Mt. Everest. Bacterial abundance in Geladandong ice core correlated with dirty layers, and significantly correlated with the oxygen isotope and the concentration of Ca2+. Results of multiple regression showed temperature and dust both affected the bacterial abundance in ice core, but the effect of dust were more obvious than that of temperature. As a result, bacterial abundance can reflect the change of dust and temperature in the past time. Seasonal differnence of abundance and diversity of microbe were observed in snowpit. Microbe in the end of monsoon season snow were remarkable different to those in the premonsoon season, with more abundance and high diversity. Bacterial in the premonsoon season only affiliated with soil and frozen environment, but bacterial in the end of monsoon season were connect with marine, plant, animal and other environment besides soil and cryosphere. Bacterial abundance in two snowpits at different altitudes at Palong No.4 glacier changed in similar trend, higher in lower altitude. Bacterial 16S rRNA sequences belonged to α、β、γ-Proteobacteria, Actinobacteria, Firmicutes, CFB, Cyanobacteria, Acidobacteria,Planctomycetes. Actinobacteria and α-Proteobacteria were dominant in this region. Seasonal differnence of abundance and diversity of microbe in Palong No.4 glacier were less obvious than Rongbuk glacier and Guoqu glacier. Bacterial diversity of summer snow was higher than that in winter, and different genus in summer and winter. But Agreia and Rhizobium genus, which isolated from plant, occurred in both summer and winter were the dominant genus, accounted for 77% of the total. Bacterial 16S rRNA sequences in the snow at Zadang glacier belonged to α、β、γ-Proteobacteria, Actinobacteria, Firmicutes, CFB, Cyanobacteria, Chloroflexi, Deinococcus-Thermus, Verrucomicrobia. The number of bacteria belong to α、β、γ-Proteobacteria, Actinobacteria, Firmicutes, CFB were similer, and no dominant group in this region. Snowpit at Zadang glacier deposited snow from winter, 2005 to May, 2006. Bacterial communities of spring and winter snow were similar. Bacteria in snow in May showed more connection with bacteria in Namuco lake. Microbial community in snow at Guoqu glacier, Zadang glacier, East Rongbuk glacier and Palong No.4 glacier were remarkable different. Bacterial abundance in glacier at the northern was higher than that at the southern. Influenced by different atmosphere cycle and ecosystem around the glacier, bacterial communities in every glacier were distinct, and more common feature occurred in nearby glaciers. There were some representative and common genus existed at all researched glaciers in Tibetan Plateau, e.g. Sphingomonas and Ochrobactrum genus(α-Protobacteria)，Polaromonas, Acidovorax, Aquabacterium, Ralstonia genus(β-Protobacteria)， Acinetobacter genus (γ-Protobacteria)， Bacillus genus (Firmicutes)，Chitinophaga genus (CFB). The bacterial diversity and abundance in two moraine lakes and two glacial meltwaters (5140, 5152, 5800 and 6350 m above sea level, respectively) in the remote Mt Everest region were examined. In total, 247 clones were screened by RFLP and 60 16S rRNA gene sequences were obtained, belonging to the following groups: Proteobacteria (8% alpha subdivision, 21% beta subdivision, and 1% gamma subdivision), CFB (54%), Actinobacteria (4%), Planctomycetes (2%), Verrucomicrobia (2%), Fibrobacteres (1%), and Eukaryotic chroloplast (3%) respectively. The high dominance of CFB distinguished the Mt. Everest waters from other mountain lakes. The highest bacterial abundance and diversity occurred in the open moraine lake at 5152 m, and the lowest in the glacial meltwater at 6350 m. Low temperature at high altitude is considered to be critical for component dominancy. At the same altitude, nutrient availability plays a role in regulating population structure. Total 97 bacterial isolates were obtained from 6350 m glacial meltwaters in the Mt. Everest region. Phylogenetic analysis suggested they affiliated to 5 groups: α、β、γ-Proteobacteria, Actinobacteria, Firmicutes. Morphological properties of isolates were characterized by rod and cocci shape. Most of isolates had pigment. Isolates utilized different kinds of carbon sources and grown in different rates under 25℃ and 15℃, and could be divided into 3 groups. The first group included Janthinobacterium sp. and Arthrobacter luteolus. They were sensitive to temperature change, used most kinds of carbon sources and metabolized actively. The second group utilized less carbon sources, but adapted to low temperature, such as Cellulomonas sp.and Acinetobacter baumannii. The third group (Microbacterium sp.，Bacillus megaterium) hardly utilized carbon source, and their response to the temperature were complicated.

第1章 绪论…….…………………. ….………………….………………………1
第一节 南北极冰芯中细菌群落结构特征…...……...….………………….1
第二节 高山冰川细菌群落结构特征....……………………………………9
第三节 青藏高原冰芯微生物与气候环境的关系….…………………….12
第四节 本研究的目的……………………………………………………..19
第2章 研究方法………………………………………………………………...21
第一节 研究区域及野外样品的采集…………………………...….……..21
第二节 流式细胞技术检测细菌的数量………………………...………...23
第三节 细菌16S rRNA 基因文库的建立……………………...…………25
第四节 细菌的培养与生理研究…………………….…………………….26
第五节 氧同位素、营养盐、离子等环境指数的测量…………………..29
第3章 青藏高原冰雪微生物特征及其与气候环境的关系…………………...31
第一节 珠穆朗玛峰地区冰雪微生物及其与气候环境的关系…………..31
3.1.1 珠峰北坡6600－8000 m表层雪中细菌数量的变化….………....31
3.1.2 珠峰东绒布冰川冰雪微生物群落及其季节变化……………….32
3.1.3 珠峰北坡冰塔林和表层雪中细菌群落特征…………………….42
第二节 各拉丹东地区冰雪中细菌群落结构及其与气候环境的关系…..46
3.2.1 各拉丹东果曲冰川冰芯中细菌数量与气候环境变化的关系…...46
3.2.2 各拉丹东果曲冰川雪中细菌群落及其季节变化特征………..….52
第三节 藏东南帕隆4号冰川细菌与气候环境变化的关系………………59
第四节 纳木错扎当冰川细菌与气候环境变化的关系…………………..68
第五节 青藏高原冰川雪细菌的空间分布特征…………………………..77
第4章 珠穆朗玛峰冰川融水细菌群落及其与环境的关系………………….81
第一节 珠峰地区冰碛湖及冰川融水细菌群落特征……………………..81
第二节 珠峰6350m冰川融水中可培养细菌及其生理特征………….......90
第五章 结论与展望…………………. ….………………….…………………101
参考文献……………………………………………………………………………105
附录：攻读博士学位期间发表的论文……………………………………………113
致谢…………………………………………………………………………………115