摘要
低温环境是一个独特的生态系统,它被认为是天然的“菌种保藏中心”。近年来冷环境下微生物的研究取得了长足的发展,尤其是微生物生态学的研究。天山地区具有寒冷、中纬度和高海拔等特点,是研究低温微生物的理想生境。本文选取了天山2米深的雪坑、1.6米深的活动层冻土和3.0米深的永冻土作为材料,应用PCR-DGGE技术研究了其中微生物的群落结构和分布,分析和讨论了它们之间的关系及其与温室气体(CH_4和N_2O)排放的关系。得出了以下主要结果:
1、从天山永冻土中检测出44条细菌序列,在系统进化树上聚类为7大类群:酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)、芽单胞菌门(Gemmatimonadetes)、绿弯菌门(Chloroflexi)、厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes)。共包含Gemmatimonas、Carnobacterium、Bacillus、Acidobacterium、Arthrobacter、Pseudomonas、Rhodoplanes、Nordella、Herminiimonas、Denitratisoma、Ramlibacter、Flavobacterium、Thermoleiphilum和一些未确定种属的细菌。其中变形菌门包含α、β、γ和ε等4个亚门,是天山永冻土中的优势菌群。在所有检测到的细菌中与冷环境相关的细菌占22.7%(10/44)。此项研究表明天山永冻土特殊的生境孕育了丰富多样的细菌资源。
2、从天山永冻土中检测出28条古细菌序列,在系统进化树上聚类为2大类群:广古菌门(Euryarchaeota)和泉古菌门(Crenarchaeota)。广古菌分为两大支,一是与盐细菌有相关性的广古菌Ⅰ,二是与甲烷菌有相关性的广古菌Ⅱ。泉古菌根据K(o|¨)nneke等(2005)的分类,归为“低温泉古菌”,只在较深的2.5米和3.0米的冻土中检测到。其中广古菌为永冻土中的优势菌群。在所有检测到的古细菌中与冷环境相关的古细菌占67.9%(19/28)。此项研究开启了高山寒区低温古菌的搜寻,为阐明生命低温起源和进化机制提供了更丰富的研究素材。
3、从天山活动层冻土中检测出34条细菌序列,在系统进化树上聚类为4大类群:变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和拟杆菌门(Bacteroidetes)。共包含Burkholderia、Xanthomonas、Sphingomonas、Pararubellimicrobium、Rudanella、Pedobacter、Dyadobacter、Nocardioides和一些未确定种属的细菌。其中变形菌门细菌为活动层冻土中的优势菌群。在所有检测到的细菌中与冷环境相关的细菌占61.8%(21/34),说明了在活动层冻土存在反复性冻融的情况下冷适应细菌能够更好的被保存下来。
4、从天山雪样中检测出40条细菌序列,在系统进化树上聚类为3大类群:变形菌门(Proteobecteria)、放线菌门(Actinobacteria)和拟杆菌门(Bacteroidetes)。共包含Janthinobacterium、Polaromonas、Acinetobacter、Pseudomonas、Rhodobacter、Nocardioides、Flectobacillus、Hymenobacter和一些未确定种属的细菌。其中变形菌门细菌为雪样中的优势菌群。在所有检测到的细菌中与冷环境相关的细菌占47.5%(19/40)。DGGE图谱表明雪样中的细菌种类与大气中的尘埃有着密切的关系,说明了大气的生物输送对雪样细菌的分布起着重要作用,可能成为一种反映气候环境变化的指标。
5、比较雪样、活动层和永冻层中微生物的群落结构特征,发现它们互相之间几乎没有生物交换。微生物来源方式不同和样品年代的不同可能是造成这一结果的主要原因。
6、在永冻层的上限(1.5米)处,发现存在着许多反硝化细菌(假单胞菌)和产甲烷菌,并且氨态氮和硝态氮的比值(NH_4-N:NO_3/NO_2-N)最大,表明浅层冻土中的微生物具有释放温室气体CH_4和N_2O的潜在能力,全球气候变暖已经开始影响到永冻土中的微生物,微生物也即将对全球气候变暖产生重要的反馈作用。
7、冻土和积雪中存在着一些病原菌。如肉杆菌(Carnobacterium)、伯克氏菌(Burkholderia)和黄单胞菌(Xanthomonas),随着冻土和积雪的融化,这些病原菌将会被释放。
以上研究表明天山地区冷环境中存在着丰富的低温微生物资源,天山低温微生物多样性的研究拓展了低温生物学领域的研究范围,揭示了原核生物在高山环境生物地球化学作用中的分布与功能,为进一步的应用研究提供了理论依据和科学支撑。
Low temperature environment is a special complex ecosytem and it is considered as a center for culture collection.Recently,much progress has been made in terms of microbial research in low temperature environment,especially in cold-adapted microbial ecology.Tianshan area represents a unique environment,low temperature, middle latitude and high altitude,is deemed to a perfect habitat for cold-adapted microorganisms.In this study,snow and frozen soils samples were collected from Tianshan,northwestern China.We investigated the distribution and diversity of the uncultured microorganisms using PCR-DGGE technique,analysed the relationship between microbial communities in different types of samples and discussed the capacity of these organisms to release the greenhouse gases N_2O and CH_4.The major results were obtained as follows:
1.Forty-one representative bacterial bands were selected for sequencing and phylogenetic analysis from permafrost.The phylogenetic trees placed these clones into 7 major groups:Acidobacteria,Actinobacteria,Gemmatimonadetes,Chloroflexi, Firmicutes,Proteobacteria and Bacteroidetes,including genera Gemmatimonas, Carnobacterium,Bacillus,Acidobacterium,Arthrobacter,Pseudomonas,Rhodoplanes, Nordella,Herminiimonas,Denitratisoma,Ramlibacter,Flavobacterium, Thermoleiphilum and unidentified bacteria.The Proteobacteria,consisting of theα,β,γ, andεsubdivision,was a clearly dominant group at all depths studied.Of all bacteria, there were 22.7%(9/44)with highest sequence similarity to their closest relatives recovered from other low temperature environments.From this study,it was proposed that permafrost sediments provide a specific ecological niche for diverse microbial lineages.
2.Twenty-eight representative archaeal bands were selected for sequencing and phylogenetic analysis from permafrost.The phylogenetic trees placed these clones into three phylogenetic clusters within the two kingdoms,Euryarchaeota and Crenarchaeota. Within the Euryarchaeota,methanogen-related groupⅡwas most abundant at shallow depth,whereas halobacterium-related groupⅠdominated at greater depths.A Low-Temperature Crenarchaeota group was only detected at 2.5 and 3.0 m.Of all archaea,there were 67.9%(19/28)with highest sequence similarity to their closest relatives recovered from others low temperature environment.This study help to the exploration of cold-adapted archaea in the cold alpine environment,and provided abundant opportunities to clarify cold origin of life and the mechanisms of evolution.
3.Thirty-four representative bacterial bands were selected for sequencing and phylogenetic analysis from active layers.The phylogenetic trees placed these clones into 4 major groups:Actinobacteria,Firmicutes,Proteobacteria and Bacteroidetes, including genera Burkholderia,Xanthomonas,Sphingomonas,Pararubellimicrobium, Rudanella,Pedobacter,Dyadobacter,Nocardioides and unidentified bacteria.The most abundant and diverse bacteria were members of Proteobacteria.Of all bacteria,there were 61.8%(21/34)with highest sequence similarity to their closest relatives recovered from others low temperature environment.From this study,it was proposed that cold adaptation bacteria can be preservated on repeated freezing and thawing in active layers.
4.Forty representative bacterial bands were selected for sequencing and phylogenetic analysis from snow samples.The phylogenetic trees placed these clones into 3 major groups:Actinobacteria,Proteobacteria and Bacteroidetes,including genera Janthinobacterium,Polaromonas,Acinetobacter,Pseudomonas,Rhodobacter, Nocardioides,Flectobacillus,Hymenobacter and unidentified bacteria.The most abundant and diverse bacteria were members of Proteobacteria.Of all bacteria,there were 47.5%(19/40)with highest sequence similarity to their closest relatives recovered from others low temperature environment.The DGGE pattern showed there were some positive relationship between bacterial species and micro-particles deposited,it suggested the main influence of atmospheric transportation on the microbial distribution in snow and microbial analysis of snow may provide proxy formation for past climates and environments.
5.Compare with the bacterial diversity and abundance in snow,active layer and permafrost.We found the bacteria almost wouldn't exchange from each niche.The main reasons caused were the different bacterial sources and different samples formation time.
6.Specific-depth distribution of methanogen-related Euryarchaeota groupⅡand denitrifying bacteria of the genus Pseudomonas dominated at 1.5 m depth,accompanied with a distinct peak of NH_4-N:NO_3/NO_2-N ratio,implying the potential capacity of these organisms in near-surface permafrost to release the greenhouse gases N_2O and CH_4.
7.Some pathogenic bacteria were found in permafrost and snow.Such as Carnobacterium,Burkholderia and Xanthomonas.They will be released accompanied with the permafrost degradation and snow thawing.
The data obtained in this study on genetic diversity of uncultured bacteria from the frozen soils and snows in the Tianshan Mountains expand our knowledge on the extent of bacterial diversity in the cold realm and improve our understanding of the biogeographic distribution and function of prokaryotes in alpine environments.
引文
Aguilar A (1996) Extremophile research in the European union : from fundamental aspects to industrial expectations. FEMS Microbiol Rev 18: 89-92
Aljanabi SM, Martinez I (1997) Universal and rapid salt extraction of high quality genomic DNA for PCR-based techniques. Nucl Acids Res 25: 4692-4693
Amann R I, Ludwig W, Schleifer KH. (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59: 143-169
Anisimov O, Reneva S (2006) Permafrost and changing climate: the Russian perspective. Ambio 35: 169-175
Bai Y, Yang D, Wang J, Xu S, Wang X, An L (2006) Phylogenetic diversity of culturable bacteria from alpine permafrost in the Tianshan Mountains, northwestern China. Res Microbiol 157: 741-751
Bakermans C, Ayala-del-Rio HL, Ponder MA, Vishnivetskaya T, Gilichinsky D, Thomashow MF, Tiedje JM (2006) Psychrobacter cryohalolentis sp. nov. and Psychrobacter arcticus sp. nov., isolated from Siberian permafrost. Int J Syst Evol Microbiol 56: 1285-1291
Bassam BJ, Caetano-Anolles G, Gresshoff PM (1991) Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem 198: 217
Bligh EC, Dyer WJ (1995) A rapid method of total lipid extraction and purification. Can J Microbiol 37: 911-917
Bowman WD (1992) Inputs and Storage of Nitrogen in Winter Snowpack in an Alpine Ecosystem. Arctic and Alpine Res 24: 211-215
Bowman JP, McCammon SA, Brown MV, Nichols DS, McMeekin TA (1997) Diversity and association of psychrophilic bacteria in Antarctic sea ice. Appl Environ Microbiol 63: 3068-3078
Brinkmeyer B, Knittel K, Jurgens J, Weyland H, Amann R, Helmke E (2003) Diversity and Structure of Bacterial Communities in Arctic versus Antarctic Pack Ice. Appl Environ Microbiol 69: 6610-6619
Brock TD (1987) The study of microorganisms in situ: progress and problems. Symp Soc Gene Microbiol 41: 1-17
Cameron RE, Morelli FA (1974) Viable microorganisms from ancient Ross Island and Taylor Valley drill core. Antarct J US 9:113-116
Casamayor EO, Massana R, Benlloch S, Ovreas L, Diez B, Goddard VJ, Gasol JM, Joint I, Rodriguez-Valera F, Pedros-Alio C (2002) Changes in archeal, bacterial and eukaryal assemblages along a salinity gradient by comparison of genetic fingerprinting methods in a multipond solar saltern. Environ Microbiol 4: 338-348
Cheng SM, Foght JM (2007) Cultivation-independent and -dependent characterization of Bacteria resident beneath John Evans Glacier. FEMS Microbiol Ecol 59: 318-330
Christner BC, Kvitko BH, Reeve JN (2003) Molecular identification of bacteria and eukarya inhabiting an Antarctic cryoconite hole. Extremophiles 7: 177-183
Cole JR, Chai B, Marsh TL, et al (2003) The Ribosomal Database Project (RDP-II): previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy. Nucleic Acids Res 31: 442-443
Davidson EA, Janssens IA (2006) Temperature sensitivity of soil carbon decomposition and feedbacks to climate change. Nature 440: 165-173
Delbes C, Leclerc M, Zumstein E, Godon J, Moletta, R (2001) A molecular method to study population and activity dynamics in anaerobic digestors. Water Sci Technol 43:51-57
Delong EF, Wu KY, Prezelln BB, PrezlinBB (1994) High Abundance of Archaea in Antartic Marine Pricoplankton. Nature 371: 695-697
Deming JW, Huston AL (2000) An oceanographic perspective on microbial life at low temperature with implications for polar ecology, biotechnology and astrobiology. In: Seckbach J. (Ed). Cellular origins and life in Extreme Habitats, Dordrecht, Kluwer Publishers pp. 149-160
Dmitriev VV, Suzina NE, Rusakova TG, Gilichinskii DA, Duda VI (2001) Ultrastructural characteristics of natural forms of microorganisms isolated from permafrost grounds of eastern Siberia by the method of low-temperature fractionation. Dokl Biol Sci 378: 304-306
Dmitriev VV, Suzina NE, Barinova ES, Duda VI, Boronin AM (2004) An electron microscopic study of the ultrastructure of microbial cells in extreme biotopes in situ. Mikrobiologiia 73: 832-840
Dunbar J, Ticknor LO, Kuske CR (2000) Assessment of microbial diversity in four southwestern United States soils by 16S rRNA gene terminal restriction fragment analysis. Appl Environ Microbiol 66: 2943-2950
Erokhina LG, Spirina EV, Shatilovich AV, Gilichinskii DA (2000) Chromatic adaptation of viable ancient cyanobacteria from Arctic permafrost. Microbiology 69: 730-731
Erokhina LG, Vishnivetskaya TA, Spirina EV, Gilichinskii DA (1999) Accumulation of Phycobilins in Viable Cells of Ancient Cyanobacteria from Arctic Permafrost Grown on Different Nitrogen Sources. Mikrobiologiya 68: 716-720
Felske A, Akkermans AD, De Vos WM (1998) In situ detection of an uncultured predominant bacillus in Dutch grassland soils. Appl Environ Microbiol 64: 4588— 4590
Frank S, Chistoph CT (1998) A new approach to utilize PCR-single-strand-conformation polymorphism for 16S rRNA gene-based microbial community analysis. Appl Environ Microbiol 64: 4870-4876
Frank S, Chistoph CT (2000) Effect of field inoculation with Sinorhizobium meliloti L33 on the composition of bacterial communities in rhizospheres of a target plant (Medicago sativa) and a non-target plant (Chenopodium album) linking of 16S rRNA gene-based single-strand-conformation polymorphism community profiles to the diversity of cultivated bacteria. App Environ Microbiol 66: 3556-3565
Friedmann EI, Gilichinsky DA (1994) Viable Microorganisms in Permafrost, Institute of Soil Science and Photosynthesis, Russian Academy of Sciences, Pushchino pp. 21-26
Fritsen CH, Adams EE, McKay CM, Priscu JC (1998) Permanent ice covers of the McMurdo Dry Valley Lakes, Antarctica: Liquid water content. In J.C. Priscu (ed.), Ecosystem Processes in a Polar Desert: The McMurdo Dry Valleys, Antarctica. Antarctic Research Series, American Geophysical Union 72: 269-280
Fromin N, Hamelln J, Tarnawskl S (2002) Statistical analysis of denaturing gel electrophoresis (DGE) fingerprinting patterns. Environ Microbiol 4: 634-643
Fulco AJ, Fujii DK (1980) Biophysical technique and cellular regulation. In: Kates, M and Kuksis, A. (Ed). Membrane Fluidity, New Jersey, Humana Press pp.77
Ganzert L, Jurgens G, Munster U, Wagner D (2007) Methanogenic communities in permafrost-affected soils of the Laptev Sea coast, Siberian Arctic, characterized by 16S rRNA gene fingerprints. FEMS Microbiol Ecol 59: 476-488
Gavrish EIu, Krauzova VI, Potekhina NV, Karasev SG, Plotnikova EG, Altyntseva OV, Korosteleva LA, Evtushenko LI (2004) Three new species of Brevibacteria- Brevibacterium antiquum sp. nov., Brevibacterium aurantiacum sp. nov. and Brevibacterium permense sp. nov. Mikrobiologiia 73: 218-225
Gerday C et al (2000) TIB TECH 18: 103-107
Gilichinsky DA, Vorobyova EA, Erokhina LG, Fyordorov-Davydov DG, Chaikovskaya NR, Fyordorov-Dayvdov DG (1992) Long-term preservation of microbial ecosystems in permafrost. Adv Space Res 12: 255-263
Gilichinsky DA, Wagener S (1994) Viable Microorganisms in Permafrost. In: Gilichinsky DA (Ed.) Institute of Soil Science and Photosynthesis, Russian Academy of Sciences, Pushchion pp. 7-20
Gounot AM and Russell NJ (1999) Physiology of cold-adapted microorganisms. In: Margesin R and Schinner F (Ed). Cold-adapted organisms: ecology, physiology,enzymology, and molecular biology. Berlin, Springer pp. 33-35
Grant WD, Tindall BJ (1986) The alkaline saline environment. In: Herbert RA, et al (ed.) Microbes in Extreme Environments. London: Academy Press pp. 25-54
Head IM, Saunders JR, Pickup RW (1998) Microbial evolution, diversity and ecology: A decade of ribosomal RNA analysis of uncultivated microorganisms. Microb Ecol 35: 11-21
Hengstmann U, Chin KJ, Janssen PH, Liesack W (1999) Comparative phylogenetic assignment of environmental sequences of genes encoding 16S rRNA and numerically abundant culturable bacteria from an anoxic rice paddy soil. Appl Environ Microbiol 65: 5050-5058
Horneck GL (2000) The microbial world and the case for Mars, The microbial world and the case for Mars. Planet Space Sci 48: 1053-1063
Horton TR, Bruns TD (2001) The molecular revolution in ectomycorrhizal ecology: Peeking into the black2boxl. Molecular Ecol 10: 1855-1871
John WD, Alice JJ (1996) Methods for assessing soil quality. SSSA special publication number 49, Soil Science Society of America, Inc. Madison, Wisconsin, USA. pp. 203-272
Kaeberlein T, Lewis K, Epstein SS (2002) Isolating "nncultivable" microorganisms in pure culture in a simulated natural environment. Science 296: 1127-1129
Kaneko T, Hasegawa S, Matsumoto N (1978) Numerical taxonomic studies of bacteria isolated from Arctic and subarctic marine environments. Loutit M W, Miles J A R. Microbial Ecology, Berlin: Springer Verlag pp. 26-30
Khlebnikova GM, Gilichinsky DA, Fedorov-Davydov DG, Vorobyova EA (1990) Quantitative evaluation of microorganisms in permafrost deposits and buried soils. Microbiology 59: 106-112
Khmelenina VN, Makutina VA, Kalyuzhnaya MG, Rivkina EM, Gilichinsky DA, Trotsenko Y (2002) Discovery of viable methanotrophic bacteria in permafrost sediments of northeast Siberia. Dokl Biol Sci 384: 235-237
Konneke M, Bernhard AE, de la Torre JR, Walker CB, Waterbury JB, Stahl, DA (2005) Isolation of an autotrophic ammonia-oxidizing marine archaeon. Nature 437: 543-546
Margesin R and Schinner F (1994) Properties of cold-adapted microorganisms and their potential role in biotechnology. J Biotechnol 33: 1-14
Marilley L, Vogt G, Blanc M (1998) Bacterial diversity in the bulk soil and rhizosphere fractions of Lolium perenne and Trifolium repens as revealed by PCR retriction analysis. Plant Soil 198: 219-224
Master ER, Mohn WW (1998) Psychrotolerant bacteria isolated from Arctic soil that degrade polychlorinated biphenyls at low temperatures. Appl Environm Microbiol 64: 4823-4829
Moodley K (2004) Microbial Diversity of Antarctic Dry ValleyMineral Soil
Morita RY (1975) Psychrophilic bacteria. Bacteriol Rev 39: 144-167
Muyzer G, Wall DE (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes coding for 16S rRNA. Appl Environ Microbiol 59: 695-700
Myers RM, Fischer SG, Lerman LS, Maniatis T (1985) Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Res 13: 3131-3145
Nakagawa S, Takai K, Inagaki F, Hirayama H, Nunoura T, Horikoshi K, Sako Y (2005) Distribution, phylogenetic diversity and physiological characteristics of epsilon-Proteobacteria in a deep-sea hydrothermal field. Environ Microbiol 7: 1619-1632
Neidleman SL (1990) Enzyme reactions under stress conditions. Crit Rev Biotechnol 9: 273-286
Nichols D, Bowman J, Sanderson K, Nichols CM, Lewis T, McMeekin T, Nichols PD (1999) Developments with Antarctic microorganisms: culture collections, bioactivity screening, taxonomy, PUFA production and cold-adapted enzymes. Curr Opin Biotechnol 10: 240-246
Nienow JA, Friedmann EI (1993) Terrestrial lithophytic (rock) communities. In: Antarctic Microbiology, Ed. by Friedmann EJ, Wiley Liss, Inc. New York pp. 343-412
Nozhevnikova AN, Simankova MV, Parshina SN, Kotsyurbenko OR (2001) Temperature characteristics of methanogenic archaea and acetogenic bacteria isolated from cold environments. Water Sci Technol 44: 41-48
Ogram A (2000) Discussion soil molecular microbial ecology at age 20: Methodological challenges for the future. Soil Biol Biochem 32: 1499-1504
Paul B, Spooner B (2001) Soil fungi: Diversity and detection. Plant and Soil 232: 147-154
Perreault NN, Andersen DT, Pollard WH, Greer CW, Whyte LG (2007) Characterization of the Prokaryotic Diversity in Cold Saline Perennial Springs of the Canadian High Arctic. Appl Environ Microbiol 73: 1532-1543
Pikuta EV, Marsic D, Bej A, Tang J, Krader P, Hoover RB (2005) Carnobacterium pleistocenium sp. nov., a novel psychrotolerant, facultative anaerobe isolated from permafrost of the F in Alaska. Int J Syst Evol Microbiol 55: 473—478
Potier P, Drevet P, Gounot AM, Hipkiss AR (1990) Temperature-dependent changes in proteolytic activities and protein composition in the psychrotrophic bacterium Arthrobacter globiformis S 55. J Gen Microbiol 136: 283-291
Prins RA, de Vrij W, Gottschal JC, Hansen TA (1990) Adaptation of microorganisms to extreme environments. FEMS Microbiol Rev 75: 103-104
Qiu G, Huang Y, Li Z (1983) Alpine permafrost in Tianshan, China. In: Proceedings of the 4th International Conference of Permafrost. National Academy Press. Washington D.C.USA pp. 1021-1023
Ramana KV, Singh L, Dhaked RK (2000) Biotechnological application of psychrophiles and their habitat to low-temperature. J Sci Ind Res 59: 87-101
Rappe MS, Connon SA, Vergin KL, Giovannoni SJ (2002) Cultivation of the ubiquitous SAR11 marine bacterioplankton clade. Nature 418: 630-633
Rivknia E, Gilichinsky D, McKay et al (2001) Permafrost Response on Economic Development, Environmental Security and Natural Resources Amsterdam: Kluwer pp. 487-496
Rivkina EM, Laurinavichius K, McGrath J, Tiedje J, Shcherbakova V, Gilichinsky D (2004) Microbial life in permafrost. Adv Space Res 33: 1215-1221
Rivkina EM, Shcherbakova V, Laurinavichius K, Petrovskaya L, Krivushin K, Kraev G, Pecheritsina S, Gilichinsky D (2007) Biogeochemistry of methane and methanogenic archaea in permafrost. FEMS Microbiol Ecol 61: 1-15
Rondon MR, August PR, Bettermann AT, Brady SF, Grossman TH, Liles MR (2000) Cloning the Soil Metagenome: a Strategy for Accessing the Genetic and Functional Diversity of Uncultured Microorganism. Appl Environ Microbiol 66: 2541-2547
Rodrigues DF, Goris J, Vishnivetskaya T, Gilichinsky D, Thomashow MF, Tiedje JM (2006) Characterization of Exiguobacterium isolates from the Siberian permafrost. Description of Exiguobacterium sibiricum sp. nov. Extremophiles 10: 285-294
Russell NJ, Fukunaga NA (1990) comparison of thermal adaptation of membrane lipids in psychrophilic and thermophilic bacteria. FEMS Microbiol Rev 75: 171-182
Saitou N, Nei M (1987) The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406-425
Schinner F, Daxenbichler G, Hallinan M (1992) Extracellular protease-producing psychrotrophic bacteria from high alpine habitats. Arctic Alpine Res 24: 88-92
Shcherbakova VA, Chuvilskaya NA, Rivkina EM, Pecheritsyna SA, Laurinavichius KS, Suzina NE, Osipov GA, Lysenko AM, Gilichinsky DA, Akimenko VK (2005) Novel psychrophilic anaerobic spore-forming bacterium from the overcooled water brine in permafrost: description Clostridium algoriphilum sp. nov. Extremophiles 9: 239-246
Shi T, Reeves RH, Gilichinsky DA, Friedmann EI (1997) Characterization of viable bacteria from Siberian permafrost by 16S rDNA sequencing. Microb Ecol 33: 169-179
Shivaji S, Majumdar KC, Sundareswaran VR (1994) Bacteria and yeast Schirmacher Oasis, Antarctica: Taxonomy, biochemistry and molecular biology. Proc NIRP Symp Polar Biol 7: 173-184
Siebert J, Hirsch P (1988) Characterization of 15 selected coccal bacteria isolated from Antarctic rock and soil samples from the McMurdo-Dry Valleys (southern Victoria Land). Polar Biol 9: 37-44
Sinclair J (1991) Global warming: a vicious circle. Our Planet 3: 4-7
Soina VS, Vorobiova EA, Zvyagintsev DG, Gilichinsky DA (1995) Preservation of cell structures in permafrost: a model for exobiology. Adv Space Res 15: 237-242
Soina VS, Mulyukin AL, Demkina EV, Vorobyova EA, El-Registan GI (2004) The structure of resting bacterial populations in soil and subsoil permafrost. Astrobiology 4: 345-358
Stevenson BS, Eichorst SA, Wertz JY, Schmidt TM, Breznak JA (2004) New Strategies for Cultivation and Detection of Previously Uncultured Microbes. Appl Environ Microbiol 70: 4748-4755
Steven B, Briggs G, Mckay CP, Pollard WH, Greer CW, Whyte LG (2007) Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and culture-independent methods. FEMS Microbiol Ecol 59: 513-523
Sun J, Qin D, Ren J, Li Z, Hou S (2002) A Study of Water Chemistry and Aerosol at the Headwaters of the Urumqi River in the Tianshan Mountains. J Glaciol Geocryol 24: 186-191
Takai K, Inagaki F, Nakagawa S, Hirayama H, Nunoura T, Sako Y, Nealson KH, Horikoshi K (2003) Isolation and phylogenetic diversity of members of previously uncultivated epsilon-Proteobacteria in deep-sea hydrothermal fields. FEMS Microbiol Lett 218: 167-174
Tebbe CC, Vahjen W (1993) Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast. Appl Environ Microbiol 59: 2657-2665
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: Improving sensitivity of progressive multiple sequence alignments through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673-7680
Tiedje JM, Asuming-Brempong S, Marsh TL, Flynn SJ, Nusslein K (1999) Opening the black box of soil microbial diversity. Appl Soil Ecol 13: 1109-1122
Torsvik V (2002) Prokaryotic diversity-magnitude, dynamics, and controlling factors. Science 296: 1064-1066
Vainshtein MB, Gogotova G, Hippe H (1995) A sulfate-reducing bacterium from permafrost. Microbiology 64: 436—439
Vallaeys T, Topp E, Muyzer G (1997) Evaluation of denaturing gradient gel electrophoresis in the detection of 16S rDNA sequence variation in rhizobia and methanotrophs. FEMS Microbiol Ecol 24: 279-285
Vishnivetskaya T, Kathariou S, McGrath J, Gilichinsky D, Tiedje JM (2000) Low-temperature recovery strategies for the isolation of bacteria from ancient permafrost sediments. Extremophiles 4: 165-173
Voget S, Leggewie C, Uesbeck A. Raasch C, Jaeger KE, Streit WR (2003) Prospecting for Novel Biocatalysts in a Soil Metagenome. Appl Environ Microbiol 69: 6235-6242
Walter KM, Zimov SA, Chanton JP, Verbyla D, Chapin FS (2006) Methane bubbling from Siberian thaw lakes as a positive feedback to climate warming. Nature 443: 71-75
Wilkinson SC, Anderson JM (2001) Spatial patterns of soil microbial communities in a Norway. Microb Ecol 42: 248-255
Williams JG, Kubelik AR, Livak KJ (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18: 6531-6535
Winding A, Hendriksen NJ (1997) Biolog substrate utilization assay for metabolic fingerprint of soil bacteria: incubation effects. In: Insam H, Rangger A. eds. Microbial communities: Functional versus structural approaches. Heidelberg: Springer pp. 192-205
Wynn Williams DD (1999) Evolution on Planet Earth: Origins and achievements. Trends Ecol Evol 14: 379-381
Xiang S, Yao T, An L, Xu B, Wang J (2005) 16S rRNA Sequences and Differences in Bacteria Isolated from the Muztag Ata Glacier at Increasing Depths. Appl Environ Microbiol 71: 4619-4627
Yoshimura Y, Kohshima S, Takeuchi N (2000) Himalayan ice-core dating with snow algae. J Glaciol 46: 335-340
Zamolodchikov DG, Karelin DV, Chestnykh OV (2004) Measurements of carbon balance in permafrost ecosystems: advances and problems. Dokl Biol Sci 397: 333-335
Zhang G, Busse HJ, Ma X, Niu F, Feng H, An L, Cheng G (2008) Hymenobacter psychrotolerans sp. nov., isolated from permafrost region. Int J Syst Evol Microbiol 58: 1215-1220
Zhang G, Ma X, Niu F, Dong M, Feng H, An L, Cheng G (2007) Diversity and distribution of alkaliphilic psychrotolerant bacteria in the Qinghai-Tibet Plateau permafrost region. Extremophiles 11: 415-424
Zhang X,Yao T,Tian L,Xu S,An L(2008)Phylogenetic and Physiological Diversity of Bacteria Isolated from Puruogangri Ice Core.Microb Ecol 55:476-488
Zhong W,Cai Z(2004)Methods for studying soil microbacterial diversity.Chin J Appl Ecol 15:899-904
Zhou J,Davey ME,Figueras JB,Rivkina E,Gilichinsky D,Tiedje JM(1997)Phylogenetic diversity of a bacterial community determined from Siberian tundra soil DNA.Microbiology 143:3913-3919
Zvyagintsev DG(Ed.)(1992)Microorganisms in Permafrost.Proc 1st Int Conf Cryopedology.Pushchino,Russian
Zvyagintsev DG,Gilichinsky DA,Khlebnikova DA,Davydov DG,Kudryavtseva NN(1990)Comparative characteristics of microbial cenoses from permafrost rocks of different age and genesis.Microbiology 59:332-338
金会军,李述训,王绍令,赵林.气候变化对中国多年冻土和寒区环境的影响[J].地理学报,2000,55(2):161-171
张锐,林念炜,赵晶,曾润颖,朱仁斌,孙立广,刘晓东.南极阿德雷岛地表沉积物中细菌多样性及对环境的响应[J].自然科学进展,2003,13(10):1067-1072
李会荣,孙嘉康,陈丽珊,俞勇,陈波,任大明.南极菲尔德斯半岛表层土壤样品中细菌多样性的系统发育分析[J].极地研究,2005,17(4):245-254
李会荣,俞勇,曾胤新,陈波,任大明.北极太平洋扇区海洋沉积物细菌多样性的系统发育分析[J].微生物学报,2006,46(2):177-183
苏玉环,李会荣,李筠,俞勇,陈波.北极太平洋扇区深海沉积物的细菌多样性研究[J].高技术通讯,2006,16(7):752-756
肖昌松,刘大力,周培瑾.南极长城站地区土壤微生物生态作用的初步探讨[J].生物多样性,1995,3(3):134-138
冯瑞华.慢生型大豆根瘤菌的遗传多样性的研究[[J].应用与环境生物学报,2000,6(2):176-181
席劲瑛,胡洪营,钱易.Biolog方法在环境微生物群落研究中的应用[J].微生物学报,2003,43(1):138-141
李桂菊,庄新国.多年冻土区沉积物中甲烷的生成[J].天然气地球科学,2004,15(5):516-518
俞勇,李会荣,陈波,曾胤新,何剑锋.北极高维度海域海冰嗜冷菌系统发育多样 性及其低温水解酶分析[J].微生物学报,2006,46(2):184-190
张宝涛,王立群,伍宁丰,石鹏君.PCR-DGGE技术及其在微生物生态学中的应用[J].生物信息雪,2006,4(3):132-134
杨元根,Paterson E,Campbell C.Biolog方法在区分城市土壤与农村土壤微生物特性上的应用[J].土壤学报,2002,39(4):582-589
