摘要
极端环境条件下微生物群落演替与气候演变的关系研究是生命科学和地球科学交叉的一个国际前沿领域。冻土微生物常年处于极端严酷的环境条件下,对环境变化特别敏感,因而,研究冻土中的微生物分布、遗传变化与自然选择对研究过去气候环境形成于变化、评价生物和人类活动对地球环境的影响有重要意义。
本研究分别以乌鲁木齐河源区不同海拔表层土壤和不同层位冻土样品为介质,利用荧光显微计数技术、寡营养恢复培养技术、16SrDNA基因克隆技术、分子标记-变形梯度凝胶电泳(DGGE)、限制性片度长度多态性(T-RFLP)技术等,通过对冻土样品中微生物的数量和物种多样性的研究,分析了不同海拔、不同深度中微生物的群体结构和种类变化,并结合相关的土样理化性质资料,探讨冻土微生物数量、多样性和物种垂直结构的时空变化特征,揭示微生物的来源及其物种的演替规律以及与土壤理化参数的关联度。主要结果如下：
1、在20个代表不同年代的冻土芯样品中,可培养细菌数量与年代、土壤含水量、总C和总N含量都成显著的正相关,与pH值呈显著负相关,表明深坑样冻土中可培养细菌数与环境有紧密联系。而且,可培养细菌数及其在细胞总数中所占比值随年代的增加而下降。海拔样品中可培养细菌数与含水量、总C、总N和pH值相关性不如深坑样。通过分析研究海拔、植被与可培养细菌数之间的相互关系,我们发现海拔高度并不是影响可培养细菌数的决定因素,但不同的植被类型对细菌生长有一定的决定作用：单子叶草本植物>乔木类植物>双子叶草本植物。
2、恢复培养结果显示,从深坑样品中检测到的31株细菌分别属于Firnicutes、Actinobacteria、α-Protebacteria、β-Protebacteria和y-Protebacteria5个系统发育组的20个属;从海拔样品中检测到的23株细菌分别属于Firmicutes、Actinobacteria、α-Protebacteria、β-Protebacteria、γ-Protebacteria和Bacteroidetes 6个系统发育组的16个属。深坑样中Actinobacteria与α-,β-,γ-Protebacteria呈显著负相关,Firmicutes与β-Protebacteria呈显著正相关；海拔样中α-Protebacteria与β-Protebacteria呈显著负相关,与Firmicutes成显著正相关。而且,在深坑样中,γ-Protebacteria与pH呈显著正相关,与可培养细菌总数呈显著负相关；海拔样中Actinobacteria与海拔呈显著正相关。从而推断出,细菌的物种多样性不仅受环境因素影响,而且是种间相互作用的结果。产色素菌的比例较高,深坑样中为98.60%,深层样品中产色素菌含量较表层样品小；海拔样品中为47.06%,随海拔的增加呈减小趋势。进一步分析发现,分离出来的许多菌株都是功能菌,在净化乌鲁木齐河源区的环境及生态建设方面具有很大的应用价值。
3、DGGE对细菌的分析显示,从深坑样中检测到的23条细菌16S rDNA片段分别属于Actinobacteria、α-Protebacteria、β-Protebacteria、δ-proteobacteria、Nitrospira、Acidobacteria和Gemmatimonadete 7个系统发育组；从海拔样中检测到的30条细菌16SrDNA片段分别属于Actinobacteria、a-Protebacteria、P-Protebacteria、8-proteobacteria、Nitrospira、Acidobacteria、Gemmatimonadete、Firmicutes、Thermodesulfobacteria、Chloroflexi和Bacteroidetes 11个系统发育组。对DGGE图谱中细菌物种多样性(shannon指数)和丰富度的分析表明,细菌物种多样性在不同深度及不同海拔样品中差异较小。然而需要指出的是,DGGE方法可以检测出来更多类群的细菌,但有些能恢复培养出来的而并未检测到,因而需要将分子方法与传统的培养技术相结合,才能更充分、更全面地研究冻土微生物。
4、DGGE对古菌的分析显示,从深坑样中检测的21条古菌16SrDNA片段分别属于Crenarchaeota(泉古菌门)的Caldisphaera和Pyrolobus2属和Euryarchaeota：广古菌门)的Methanolinea、Thermogymnomonas、Methanosphaerula和Methanocella4属。海拔样检测到的17条古菌16SrDNA片段分别隶属于Crenarchaeota的Caldisphaera1个属和Euryarchaeota的Methanolinea、Thermogymnomonas和Methanosphaerula3属。对其进行物种多样性分析显示,深坑样的shannon指数基本在0.8-1.0之间,多样性随深度的增加呈上升趋势。海拔样的shannon指数在0.5-1.1之间,多样性随海拔的增加呈上升趋势。
以上研究表明,不同深度及不同海拔梯度样品中微生物的数量、组成和分布有很大差异,而且都在一定程度上与气候环境的指标和时空变化相关,为阐明、推演乌鲁木齐河源区气候环境的演变规律,为当地生态环境建设的调控提供科学参考。
On the condition of extreme environment, the relationship between microbial community succession and climate evolution is an international frontier. Permafrost microorganisms are particularly sensitive to environmental changes, thus, it is significant for the study of the distribution of permafrost microorganisms, genetic change and natural selection to evaluate past climate changes as well as the influence of biological and human activities on the Earth's environment.
In order to understand spatial and temporal variations of soil microorganism in the headwaters of the Urumqi river, soil from different altitudes and different depths has been sampled. Here, fluorescence microscopy counting techniques, few nutrients of cultivation techniques,16S rDNA gene cloning, molecular markers-deformation gradient gel electrophoresis (DGGE), restriction fragment length polymorphism (T-RFLP), were used to reveal the microbial community structure and diversity. Soil physical, chemical and other environmental variables are mensured as well. The results obtained here were summarized as follows:
1、In the samples which representing different age, the cultured bacterial amount have a significant, positive correlation with age, soil moisture content, total C, total N concentrations, and have a significant, negative correlation with pH value. It suggested that cultured bacterial amount and the environment are closely linked. Moreover, as age increasd, the cultured bacterial amount in the ratio of total cell number are decreasing. In the samples which at different altitudes, the correlation between the cultured bacterial amount and soil moisture content, total C, total N concentrations, pH value are not as well as the samples at different depths. We found that the altitude does not the determining factor affect the number of bacteria, but different vegetation types have some decisive on the growth of bacteria:monocot herb> arbor plants> dicotyledonous herbs.
2、Cultivation-dependent approaches showed that the 31 strains at different depths belong to following group:Firmicutes、Actinobacteria、α-Protebacteria、β-Protebacteria and y-Protebacteria. Actinobacteria have a significant, negative correlation withα-,β-,γ-Protebacteria; Firmicutes have a significant, positive correlation with P-Protebacteria; y-Protebacteria have a significant, positive correlation with pH, and have a significant, negative correlation with the cultured bacterial amount. The 23 strains at different altitudes belong to following group: Firmicutes、Actinobacteria、α-Protebacteria、β-Protebacteria、γ-Protebacteria and Bacteroidetes.α-Protebacteria have a significant, negative correlation withβ-Protebacteria, and have a significant, positive correlation with Firmicutes; Actinobacteria have a significant, positive correlation with depths. It suggested that the types and diversity of bacteria not only affected by environmental, but also the result of the interaction between them.98.60% pigmented colonie was observed in the samples at different depths, the deep sample is smaller than the surface sample; 47.06% pigmented colonie was observed in the samples at different altitudes, as altitude increasd, the ratio is reduced. Further analysis found that many of the strains isolated were functional bacteria, they have great value at purifing the environmental of the headwaters of the Urumqi river and constructing ecological.
3、DGGE analysis of bacteria showed that the 23 representative bacterial bands at different depths belong to following group:Actinobacteria、α-Protebacteria、β-Protebacteria、δ-proteobacteria、Nitrospira、Acidobacteria and Gemmatimonadete; the 30 representative bacterial bands at different altitudes belong to following group: Actinobacteria、α-Protebacteria、β-Protebacteria、δ-proteobacteria、Nitrospira、Acidobacteria、Gemmatimonadete、Firmicutes、Thermodesulfobacteria、Chloroflexi and Bacteroidetes. Analysis of the bacterial community structure diversity (shannon index) and richness showed that the difference of bacterial diversity at different depths and at different altitudes is small. However, it should point out that, DGGE method can be detected more groups of bacteria, but some can resume training were not detected out, and thus need to combined molecular methods with the traditional culture technique to more fully and more comprehensively study soil microorganisms.
4、DGGE analysis of archaea showed that the 21 representative arehaeal bands at different depths belong to following kind:Caldisphaera、Pyrolobus、Methanolinea、Thermogymnomonas、Methanosphaerula and Methanocella; the 17 representative arehaeal bands at different altitudes belong to following kind:Caldisphaera、Methanolinea、Thermogymnomonas and Methanosphaerula. Analysis of the arehaeal community structure diversity (shannon index) showed that shannon index is between 0.8 to 1.0 in the samples at different depths, the diversity increase with depth increasing; shannon index is between 0.5 to 1.1 in the samples at different altitudes, the diversity increase with altitude increasing.
Above all, these studies have shown that the number of microbes, composition and distribution in the samples at different depths and at different altitudes are very different, and to some extent, were interrelated with the indicators of environment and climate and spatial and temporal changes. They can provide scientific reference to deduce the evolution law of climate and environment in the headwaters of the Urumqi river, and to control the construction of local ecological environment.
引文
白玉.(2007)博士论文：天山冻土微生物的系统多样性分析及生长特性的研究..兰州大学兰州.
冯虎元,马晓军,章高森,等.(2004)青藏高原多年冻土微生物的培养和计数.冰川冻土26(2)：182-187.
蒋建东,张瑞福,何健等.(2005)细菌对环境污染物的趋化性及其在生物修复中的作用.生态学报25(7):1764-1771.
姜培坤,周国模,等.(2003)侵蚀型红壤植被被恢复后土壤微生物量碳、氮的演变.水土保持学报17(1)：112-114.
鞠远江,刘耕年.(2004)孢粉记录揭示的4000a BP来乌鲁木齐河源区气候环境变化.冰川冻土26(2):166-170.
刘光琇,安黎哲,陈桂琛.(2004)乌鲁木齐河上游植被与环境关系研究.兰州：兰州大学出版社.
刘光绣,胡昌勤,张靖溥,沈永平。(2001)青藏高原多年冻土微生物的分离分析及其意义.冰川冻土23(4):419-422.
刘光绣,马晓军,陈拓,等.(2004)冻土微生物研究进展与意义。冰川冻土26(2):188-191。
李曙光,皮昀丹,等.(2007)古菌研究及其进展.中国科学技术大学学报37(8):830-838.
李志忠,海鹰,周勇,等。(2001)乌鲁木齐河下游地区30Ka BP以来湖泊沉积的孢粉组合与古植被古气候.干旱区地理24(3)：201-205.
刘源,高金鹏,徐春和.(2005)紫细菌捕光色素蛋白复合体及光化学反应中心的研究进展.植物生理与分子生物学学报31(6)：567-574.
邱国庆.(1993)中国天山高山多年冻土的形成条件。冰川冻土15(1)：96-102。
杨大群.(2008)博士论文：天山冷环境中微生物系统多样性及分布特征的研究。兰州大学兰州.
袁玉江,邵雪莓,魏文寿,等.(2005)乌鲁木齐河山区树木年轮-积温关系及≥5.7℃积温的重建.生态学报25(4)：756-762。 俞慎和李振高.1999.稻田生态系统生物硝化-反硝化作用与氮素损失[J]。10(5)：630-634。
章高森.(2007)博士论文：青藏高原多年冻土区微生物多样性及其潜在应用的研究。兰州大学兰州.
张新芳.(2006)博士论文：普若岗日冰芯微生物多样性及其与环境关系的研究。中国科学院寒区旱区环境与工程研究所兰州.
Aguilar A, Ingemansson T, Magnien E (1998) Extremophile microorganisms as cell factories:support from the European Union. Extremophiles 2:367-373.
Amato,,HennebelleR.,Magand,O.,Sancelme,M.,Delort,A.M.,Barbante,C.,Boutron,C. ,Ferrari,C.(2007)Bacterial characterization of the snow cover at Spitzberg,Svalbard.FEMS Microbiol Ecol 59(2):255-264.
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. Research in Microbiology 157:741-751.
Bakermans C, Tsapin AI, Souza-Egipsy V, Gilichinsky DA, Nealson KH (2003) Reproduction and metabolism at-10℃ of bacteria isolated from Siberian permafrost. Environ Microbiol 5(4):321-326.
Barbour W M, Hattermann D R and Stacey G (1991) Chemotaxis of Bradyrhizobium japonicum to soybean exudates. Appl. Environ.Microbiol.57,2635-2639.
Binladen J,Wiuf C,Gilbert MT,Bunce M,Barnatt R,Larson G,Greenwood AD,Haile J,HO SY,Hansen AJ,Willerslev E.(2006) Assessing the fidelity of ancient DNA sequences amplified from nuclear genes.Genetics.172(2):733-741.
Boyd WL, Boyd JW (1964) The presence of bacteria in permafrost of the Alaskan Arctic. Can J Microbiol 10:917-919.
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.
Cameron RE, Morelli FA (1974) Viable microoganisms from ancient Ross Island and Taylor Valley drill core. Antarct J US 9:113-116.
Carr EL, Kampfer P, Patel B, et al. (2003) Seven novel species of Acinetobacter isolated from activated sludge [J]. International Journal of Systematic and Evolutionary Microbiology,53,953-963.
Cole JR,Chai B,Marsh TL,Farris RJ,Wang Q,Kulan SA,Chandra S,McGarrell DM,Schmidt TM,Garrity GM,Tiegji JM..(2003)The Ribosomal Database Project (RDP-?):Previewing a new autoaligner that allows regular updates and the new Prokaryotic taxonomy.Nucleic Acids Res31:442-443.
Costantino Vetriani,Hiep V. Tran,Lee J. Kerkhof. (2003) Fingerprinting Microbial Assemblages from the Oxic/Anoxic Chemocline of the Black Sea.Applied and Environmental Microbiology.69 (11):6481-6488.
Cowan DA, Russell N, Mamais A, Sheppard DM (2002) Antarctic Dry Valley mineral soils contain unexpectedly high levels of microbial biomass. Extremophiles 6:431-436.
Dement'ev AA, Golyshin PN, Riabehenko NF, Pustobaev VN, Shliapnikov SV.(1993) Two forms of extracellular low molecular weight Bacillus sp.BCF 247 ribonuclease. Isolation and characteristics of the Protein. Biokhimiia. 58(8):1258-1265.
Deming J (2002) Psychrophiles and polar regions. Curr Opin Microbiol 5:301-309.
Diez,B.,Pedros-Alio,C.,Marsh,T.L.,and Massana,R.(2001)Application of denaturing gradient gel lelctrophoresis(DGGE) to study the diversity of marine picoeukaryotic assemblages and comparison of DGGE with other molecular techniques.Appl Environ Microbiol 67:2942-2951.
Drancourt M and Raoult D (2005) Palaeomicrobiology:current issues and perspectives.Nat Rev Microbiol.3(1):23-35.
Fedorova ND, Shul'ga AA, Peredel'ehuk MP, Kozharinova LV, Golyshin PN, Riabchenko NF, Kirpichnikov MP.(1994) Cloning of the gene for extracellular Bacillus circulans RNAase.Mol Biol(Mosk).28(2):468-471.
Felsenstein J (1985) Confidence limits on phylogenies:an approach using the bootstrap.Evolution 39:783-791.
Fischer SG, Lerman LS. (1983) DNA f ragments differing by single basepair substitutions are separated in denaturing gradient gels:correspondence with melting t heory. Proc Natl Acad Sci USA 80 (6):1579-1583.
Fromin N, Hamelln J, Tarnawskl S(2002) Statistical analysis of denaturing gel electrophoresis(DGE)fingerprinting Patterns.Environ Microbiol 4:634-643.
Franks F (2003) Nucleation of ice and its management in ecosystems. Phil Trans R Soc Lond A 361:557-574.
Friedmann EI (1994) Permafrost as microbial habitat. In:Gilichinsky DA (ed) Viable microorganisms in permafrost. Russian Academy of Sciences, pp 21-26.
Fritsen CH, Adams EE, McKay CM, Priscu JC (1998) Permanent ice covers of the McMurdo Dry Valley Lakes. Antaretica: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.
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(2):476-488.
Gilbert MT, Binladen J, Miller W, Wiuf C, Willerslev E, Poinar H, Carlson JE, Leebens-ack JH, Sehuster SC.(2007) Recharacterization of ancient DNA miscoding lesions:insights in the era of sequencing-by-synthesis.Nucleic Acids Res.35(1):1-10.
Gilichinsky D (2002a) Permafrost. In:Bitton G (ed) Encyclopedia of environmental microbiology. Wiley, New York, pp 2367-2385.
Gilichinsky DA. (2002b)Permafrost model of extraterrestrial habitats. In:Horneck G, Baumstark-Khan C (eds) Astrobiology:the quest for the conditions of life. Springer, Berlin Heidelberg New York, pp 125-142.
Gilichinsky DA, Khlebnikova GM, Zvyagintsev DG, FedorovDavydov DG, Kudryavtseva NN (1989) Microbiology of sedimentary materials in the permafrost zone. Int Geol Rev 31:847-858.
Gilichinsky D, Rivkina E, Bakermans C, Shcherbakova V, Petrovskaya L, Ozerskaya S, Ivanushkina N, Kochkina G, Laurinavichuis K, Pecheritsina S, Fattakhova R,
Tiedje JM (2005) Biodiversity of cryopegs in permafrost. FEMS Microbiol Ecol 53:117-128.
Gilichinsky DA, Rivkina EM, Shcherbakova V, Laurinavichuis K, Tiedje J. (2003) Supercooled water brines within permafrost-an unknown ecological niche for microorganisms:a model for astrobiology. Astrobiology.3(2):331-341.
Gilichinsky DA, Vorobyova E, Erokhina LG, Fyordorov-Day-vdov DG, Chaikovskaya NR (1992) Long-term preservation of microbial ecosystems in permafrost. Adv Space Res 12:255-263.
Gilichinsky DA, Wagener S, Vishnivetskaya TA (1995) Permafrost microbiology. Permafrost Periglac Process 6:281-291.
Golyshin PN, Kombarova SP, Riabchenko NF, Vorob'eva EA, Soina VS, Dement, ev AA,Shliapnikov SV.(1993) Extracellular alkaline ribonueleases produced by Bacilli strains from the permafrost of the Kolyma lowland. Prikl Biokhim Mikrobiol.29(6):844-850.
Grierson P F (1992) Organic-acids in the rhizosphere of Banksia integrifolia L. Plant Soil 144,259-265.
Gubin SV, Maksimovich SV, Davydov SP, Gilichinskii DA, Shatilovich AV, Spirina EV,Iashina SG.(2003)The possible contribution of late pleistocene biota to biodiversity in present permafrost zone.Zh Obshch Biol.64(2):160-165.
Hansen AJ, Mitchell DL, Wiuf C, Paniker L, Brand TB, Binladen J, Gilichinsky DA, Ronn R, Willerslev E..(2006)Crosslinks rather than strand breaks determine access to ancient DNA sequences from frozen sediments. Genetics.173(2): 1175-1179.
Horneck GL(2000)The microbial world and the ease for Mars, The microbial world and the case for Mars.Planet Space Sci 48:1053-1063.
Horowitz NH, Cameron RE, Hubbard JS (1972) Microbiology of the dry valleys of Antarctica. Science 176:242-245.
Insam H., Domsch K.H.(1988)Relationship between soil organic carbon and microbial biomass in chronosequences of reclamation sites. Microb.Ecol.15, 177-188.
Insam H., Haselwandter K. (1989) Metabolic quotient of the soil microflora in relation to plant succession. Oecologia 79,174-178.
James N, Sutherland ML (1942) Are there living bacteria in permanently frozen subsoil? Can J Res 20:229-235.
Jenkinson D S. (1990) The turnover of the organic carbon and nitrogen in soil.Phil.Trans. R. Soc. Lond.329:361-368.
Jones D L, Prabowo A M and Kochian L V. (1996b) Aluminum-organic acid interactions in acid soils.2. Influence of solid-phase sorption on organic acid-Al complexation and A1 rhizotoxicity.Plant Soil 182,229-237.
Jukes TH,Cantor CR(1969) Evolution of protein molecules,In:Munro(ed),Mammalian protein metabolism.Academic Press,NewYork,PP.21-132.
Kashefi K,Lovley D R. (2003) Extending the upper temperature limit for life.Science.301:934.
Katayama T, Tanaka M, Moriizumi J, Nakamura T, Brouchkov A, Douglas TA, Fukuda M, Tomita F, Asano K. (2007) Phylogenetic analysis of bacteria preserved in a permafrost ice wedge for 25,000 years. Appl Environ Microbiol. 73(7):2360-2363.
Khlebnikova GM, Gilichinsky DA, Fedorov-Davydov DG, Vorob'eva EA (1990) Quantitative evaluation of microorganisms in permafrost deposits and buried soils. Translated from Mikrobiologiya 59:148-155.
Kholodii G, Mindlin S, Petrova M, Minakhina S.(2003) Tn5O6O from the Siberian Permafrost is most closely related to the ancestor of Tn21 Prior to integron acquisition.FEMS Microbiol Lett.226(2):251-255.
Kowalchuk GA,de Souza FA,Vanveen JA. (2002) Community analysis of arbuscular mycorrhizal fungi associated with Ammophila arenaria in Dutch coastal sand dunes.Molecular ecology 11:571-581.
Kumar S, Tamura K, Jakobsen IB and Nei M. (2001) MEGA2:molecular evolutionary genetics analysis software.Bioinformatics 12:1244-1245.
Kuske,C.R.,Ticknor,L.O.,Miller,M.E.,Dunbar,J.M.,Davis,J.A.,Barns,S.M.,and Belnap,J.(2002)Comparison of Soil Bacterial Communities in Rhizospheres of Three Plant Species and the Interspaces in an Arid Grassland.Applied and Environmental Microbiology 68:1854-1863.
Kozlovsky AG, Zhelifonova VP, Adanin VM, Antipova TV, Ozerskaia SM, Kochkina GA, Grafe U.(2003a) The fungus Penicillium citrinum Thom 1910 VKM FW-800 isolated from ancient permafrost sediments as a producer of the ergot alkaloids agroclavine-1 and epoxyagroclavine-1. Mikrobiologiia.72(6):816-821.
Kozlovsky AG, Zhelifonova VP, Antipova TV, Adanin VM, Ozerskaia SM, Kochkina GA, Sehlegel B, Dahse HM, Gollmick FA, Grafe U.(2003b) Quinocitrinines A and B, new quinoline alkaloids from Penicillium citrinum Thom 1910, a permafrost fungus J Antibiot(Tokyo).56(5):488-491.
Kozlovsky AG, Zhelifonova VP, Antipova TV.(2005) Fungus penicillium citrinum, isolated from permafrost sediments, as a producer of ergot alkaloids and new quinoline alkaloids quinocitrinines.Prikl Biokhim Mikrobiol.41(5):568-572.
Lane DJ(1991)16S/23S rRNA sequencing.In:Stackebrandt E and Goodfellow M (ed.), Nucleic acid techniques in bacterial systematics.John Wiley & Sons, Ine., New York, pp.115-148.
Latour X., Philippot L., Corberand T., Lemanceau P. (1996)The establishment of an introduced community of fluorescent pseudomonads in the soil and in the rhizosphere is affected by the soil type. FEMS Microbiol.Ecol.30,163-170.
Lock GSH.(1990) The Growth and decay of Ice. Cambridge University press, Cambridge, p434.
Lydolph MC,Jacobsen J,Arctander P,Gilbert MT,Gilichinsky DA,Hansen AJ,Willerslev E,Lange L.(2005) Beringian paleoecology inferred from permafrost-preserved fungal DNA.Appl Environ Microbiol.71(2):1012-1017.
Matsumoto GI,Friedmann EI,Gilichinsky DA.(1995) Geochemical characteristics of organic compounds in a permafrost sediment core sample from northeast Siberia,Russia.Proc NIPR Symp Antarct Meteorites.8:258-267.
Marota I,Rollo F.(2002) Molecular paleontology.Cell Mol Life Sci.59(1):97-111.
Mindlin S, Minakhin L, Petrova M, Kholodii G, Minakhina S, Gorlenko Z, Nikiforov V.(2005)Present-day mercury resistance transposons are common in bacteria
Preserved in permafrost grounds since the Upper Pleistocene.Res Microbiol. 156(10):994-1004.
Millet M,Wortham H, Sanusi A and Mirabel P. (1997) Low molecular weight organic acids in fogwater in an urban area:Strasbourg (France). Sci. Tot. Environ.206, 57-65.
Miteva VI, Sheridan PP & Brenchley JE (2004) Phylogenetic and Physiological Diversity of Microorganisms Isolated from a Deep Greenland Glacier Ice Core. Applied and Environmental Microbiology 70:202-213.
Morozova D, Mohlmann D, Wagner D. (2007) Survival of Methanogenic Archaea from Siberian Permafrost under Simulated Martian Thermal Conditions. Orig Life Evol Biosph.37:189-200.
Muller S.(1943) Permafrost or permanently frozen ground and related engineering problems. Strategic Studies,62.United States Army,Office Chief of Engineers, Military Intelligence Div. Strategic Eng. Study 62,PP.231.(Also 1974, Ann Arbor,Mich,Edwards Bros.)
Muyzer,G,Wall,DE.,Uitterlinden,A.G.(1993) Profiling of complex microbizl population by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA.Appl Environ Microbiol 59(3):695-700.
Nienow JA, Friedmann EI (1993) Terrestrial lithophytic (rock) communities.In: Antarctic Microbiology, Ed.by Friedmann EJ, Wiley Liss, Inc.NewYork 343-412.
Ohtonen R., Vare H. (1998) Vegetation composition determines microbial activities in a boreal forest soil. Microb.Ecol.36,328-335.
Omelon,C.R.,Pollard,W.H.,and Ferris,F.G..(2006)Environmental controls on microbial colonization of high Arctic cryptoendolithic habitats.Polar Biology 30:19-29.
Omelyansky VL(1911) Bakteriologicheskoe issledovanie Sanga mamonta Prilegayushchei pochvy Bacteriological investigation of the Sanga mammoth and surrounding soil. Arkhiv Biologicheskikh Nauk.16:335-340.
Ostroumov V. (1995) A physical and chemical characterization of martian permafrost as a possible habitat for viable microorganisms. Adv Space Res 15:229-236.
Panikov NS, Flanagan PW, Oechel WC, Mastepanov MA, Christensen TR.(2006) Microbial activity in soils frozen to below-39℃. Soil Biol Biochem 38: 785-794.
Petrova MA, Mindlin SZ, Gorlenko ZhM, Kaliaeva ES, Soina VS, Bogdanova ES. (2002)Mercury-resistant bacteria from permafrost sediments and prospects for their use in comparative studies of mercury resistance determinants.Genetika. 38(11):1569-1574.
Podlipaeva IuI, Shmakov LA, Gilichinskii DA, Gudkov AV.(2006) Heat shock protein of HSP70 family revealed in some contemporary freshwater Amoebae and in Acanthamoeba sp.from cysts isolated from permafrost samples.Tsitologiia.48(8): 691-694.
Rivkina E, Gilichinsky D, Wagener S, Tiedje JM, McGrath J (1998) Biogeochemical activity of anaerobic microorganisms from buried permafrost sediments. Geomicrobiol J 15:187-193.
Rivkina EM, Friedmann EI, McKay CP, Gilichinsky D.(2000) Metabolic activity of permafrost bacteria below the freezing point. Appl Environ Microbiol 66:3230-3233.
Rivkina EM, Laurinavichius K, McGrath J, Tiedje J, Shcherbakova V, Gilichinsky D. (2004) Microbial life in permafrost. Adv Space Res.33(8):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.2007 Apr 11 online first.
Rskin L,Stromley JM, Rittmann BE, Stahl DA.(1994) Groupspecific 16S rRNA hybridization probes to describe natural communities of methanogens. Appl Environ Microbiol 60:1232-1240.
Ryan P R, Delhaize E and Randall P J. (1995). Malate efflux from root apices and tolerance to aluminum are highly correlated in wheat.Aust. J. Plant Physiol.122, 531-536.
Sanguinetti CJ,Dias Neto E,Simpson AJ. (1994) Rapid silver staining and recovery of PCR products separated on polyacrylamide gels.Biotechniques,17:914-921.
Saitou N, Nei M.(1987) The neighbor-joining method:a new method for reconstructing phylogenetic trees.Mol Biol Evol 4:406:425.
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.
Soina V, 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.
Staley JT.(1968)Prosthecomicrobium and Ancalomicrobium:new prosthecate freshwater bacteria.J.Bacteraol.95:1921-1942.
Steven B, Leveille R, Pollard W, Whyte LG.(2006) Microbial ecology and biodiversity in permafrost. Extremophiles.10:259-267.
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(2):513-523.
Steven B, Pollard W, McKay CP, Greer CW, Whyte LG.(2004) Diversity of culturable bacteria isolated from permafrost and ground ice from the Canadian High Arctic. In:10th International symposium on microbial ecology.
Stone R.(1999) Permafrost comes alive for Siberian researchers. Science. 286(5437):36-37.
Taberlet P, Coissac E, Pompanon F, Gielly L, Miquel C, Valentini A, Vermat T, Corthier G, Brochmann C, Willerslev E.(2007) Power and limitations of the chloroplast tmL(UAA) intron for plant DNA barcoding.Nueleic Acids Res.35(3):14.
ThomPson JD,Gibson TJ,Plewniak F,Jeanmougin F and Higgins DG(1997)The Clustal X Windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools.Nucleic Acids Res.24:4876-4882.
Torsvik V(2002)Prokaryotic diversity-magnitude, dynamics, and controlling factors. Science 296:1064-1066.
Vinokurova NG, Ivallushkina NE, Kochkina GA, Arinbasarov MU, Ozerskaia SM. (2005 Production of mycophenolic acid by fungi of the genus Penicillium link. Prikl Biokhim Mikrobiol.41(1):95-98.
Vishnivetskaya TA, Kathariou S.(2005) Putative transposases conserved in Exiguobacterium isolates from ancient Siberian permafrost and from contemporary surface habitats.Appl Environ Microbiol.71(11):6954-6962.
Vorobyova E, Minkovsky N, Mamukelashvili A, Zvyagintsev D,Soina V, Polanskaya L, Gilichinsky D.(2001) Micro-organisms and biomarkers in permafrost. In: Paepe R, Melnikov VP (eds) Permafrost response on economic development, environmental security and natural resources. Kluwer Academic Publishers,New York, pp 527-541.
Vorobyova E, Soina V, GorlenkoM,Minkovskaya N, Zalinova N,Mamukelashvili A, Gilichinsky DA, Rivkina E, Vishnivetskaya T.(1997) The deep cold biosphere: facts and hypothesis. FEMS Microbiol Rev 20:277-290.
Wagner D, Lipski A, Embacher A, Gattinger A. (2005) Methane fluxes in permafrost habitats of the Lena Delta:effects of microbial community structure and organic matter quality. Environ Microbiol.7(10):1582-92.
Willerslev E, Hansen AJ, Binladen J, Brand TB, Gilbert MT, Shapiro B, Bunce M, Wiuf C, Gilichinsky DA, Cooper A. (2003) Diverse plant and animal genetic records from Holocene and Pleistocene sediments. Science.300(5620):791-795.
Wardle D.A., Verhoef H.A., Clarholm M. (1998)Trophic relationships in the soil microfood-web:predicting the responses to a changing global environment. Global Change Biol.4,713-727.
Watanabe K, Kodma Y, Harayama S. (2001) Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community
fingerprinting[J]. Microbiol Methods 44:253-262.
Willerslev E,Hansen AJ,Binladen J,Brand TB,Gilbert MT,Shapiro B,Bunce M,Wiuf C,Gilichinsky DA,Cooper A.(2003) Diverse plant and animal genetic records from Holocene and Pleistocene sediment.Science.300(5620):791-795.
Willerslev E,Hansen AJ,Poinar HN.(2004) Isolation of nucleic acids and cultures from fossil ice and permafrost.Trends Ecol Evol.19(3):141-147.
Willerslev E, Hansen AJ, Ronn R, Brand TB, Barnes I, Wiuf C,Gilichinsky D, Mitchell D, Cooper A.(2004) Long-term persistence of bacterial DNA. Curr Biol 14:R9-R10.
Williams PJ,Smith MW.(1989) The frozen earth:fundamentals of geocryology. Cambridge University Press,Cambridge,PP306.
Wynn Williams DD (1999) Evolution on Planet Earth:Origins and achievements. Trends Ecol Evol 14:379-381.
Wynn Williams DD (2000) Cyanobacteria in deserts-life at the limit? In:The Ecology of Cyanobacteria:Their Diversity in Time and Space.(ed.s) Whitton BA,Potts M,341-366.
Xia J H and Roberts J K M 1994 Improved cytoplasmic pH regulation, increased lactate efflux, and reduced cytoplasmic lactate levels are biochemical traits expressed in root-tips of whole maize seedlings acclimated to a low-oxygen environment. Plant Physiol.105,651-657.
Xiaojun MA, Tuo CHEN, Gaosen ZHANG, Rui WANG. (2004)Microbial community structure Along an Altitude Gradient in Three Different Localities. Folia Microbiol.49 (2),105-111.
Zak D.R., Grigal D.R., Gleeson S., Tilmen D. (1990)Carbon and nitrogen cycling during old-field succession:constraints on plant and microbial biomass. Biogeochemistry 11,111-129.
Zak D.R., Tilman D., Parmenter R.R., Rice C.W., Fisher F.M., Vose J., Milchunas D., Martin C.W.(1994)Plant production and soil microorganisms in late-successional ecosystems:a continental scale study. Ecology 75,2333-2347.
Zhang S, Hou S, Ma X, Qin D & Chen T (2007) Culturable bacteria in Himalayan glacial ice in response to atmospheric circulation. Biogeosciences 4:1-9.
Zhang Yun, Kong Zhaochen, Yang Zhenjing et al. (2004) Vegetation changes and environmental evolution in the Urumqi river head, central tianshan mountains since 3.6 ka BP:a case study of Daxigou profile. Acta Botanica Sinica 46(6): 655-667.
Zhelifonova VP,Antipova TV, Ozerskaia SM, Ivanushkina NE, Kozlovskii AG(2006) The fungus penicillium variabile sopp 1912 isolated from permafrost deposits as a producer of rugulovasines.Mikrobiologiia.75(6):742-746.