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Biological Context for Exploring Subglacial Lake Environments Brent Christner, Department of Biological Sciences 3.

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Presentation on theme: "Biological Context for Exploring Subglacial Lake Environments Brent Christner, Department of Biological Sciences 3."— Presentation transcript:

1 Biological Context for Exploring Subglacial Lake Environments Brent Christner, Department of Biological Sciences 3 rd SCAR SALE Meeting, June 2007 Big Sky, Montana

2 OUTLINE Limnological conditions in surface waters of Subglacial Lake Vostok. Predicting the biogeochemical contributions and physiology of microbes in subglacial lakes. Adaptations of microorganisms to life in ice and extreme cold. Genetic relationships between bacteria from global subglacial environments.

3 Rationale for Ice Core Decontamination Protocol 5 mm scraped final sample 5 mm removed by washing 5 mm removed by melting Christner et al. 2005, Icarus, 174: core diameter removed [parameter] core diameter removed [parameter]

4 Christner et al. in press; In: Psychrophiles: From Biodiversity to Biotechology, Springer Cells mL 1e+11e+21e+31e+41e+51e+6 Depth (m) Core exterior (outer 0.5 cm) Core interior (1.5 cm removed) Cells mL -1 on core exterior 01e+52e+53e+54e A B Cells mL -1 on core interior CONCENTRATION OF CELLS ON THE EXTERIOR AND INTERIOR OF ICE SAMPLES FROM THE BOTTOM ~100 M OF THE VOSTOK 5G ICE CORE Cell densities on the inside versus the outside of the ice core are statistically different (r = 0.016) and the data do not co-vary with depth (paired t-test, p < 0.050)

5 VOSTOK 5G ICE CORE (VOSTOK STATION, ANTARCTICA) Christner et al. 2006, L&O 51:

6 ACCRETION ICE I ACCRETION ICE II GLACIAL ICE (>420,000 years-old) Cells mL -1 of melt water Depth in Vostok core (m) SYBR Gold (DNA-containing) Propidium Iodide (DEAD) SYTO 9 (LIVE) Christner et al L&O, 51: Significantly higher (p 3,572 m

7 AAs represent 0.01% to 2% of the NPOC; AAs and NPOC concentrations were correlated in the accretion ice Christner et al. 2006, L&O 51: NONPURGEABLE ORGANIC CARBON AND TOTAL AMINO ACID CONCENTRATIONS IN THE ACCRETION ICE

8 3200 m3310 m3539 m 3609 m 3623 m3750 m Ice-water glacier ice shear layer (deformed) up Type I (particle inclusions) Type II (few inclusions) Christner et al. 2006

9 Constituent Dissolved Organic carbon (  mol L -1 ) Cell number (cells mL -1 ) Total dissolved solids (mmol L -1 ) Glacial ice (average) Type I accretion ice (average) Type II accretion ice (average) Embayment water † Main lake water † Average continental rainfall NA 0.15 Average marine/coastal rainfall NA 0.38 Average surface seawater x Biogeochemical conditions in the surface waters of Lake Vostok Christner et al. 2006, L&O, 51: † Partitioning coefficients based on ice & water chemistry of L. Bonney, Antarctica

10 MOLECULAR IDENTIFICATION OF BACTERIAL DNA SEQUENCES IN LAKE VOSTOK ACCRETION ICE Major bacterial lineages: Proteobacteria ( , ,  and  ), Firmicutes, Actinobacteria, and Bacteroidetes (Priscu et al. 1999; Christner et al. 2001, 2006; Bulat et al. 2004) Thermophile-related phylotypes Rubrobacter Hydrogenophilus Phylotypes related to chemolithoautotrophs Hydrogenophilus Thiobacillus/Acidithiobacillus Other notable bacterial phylotypes: Metal-reducing anaerobes? Methylotrophs? THESE DATA PROVIDE THE RATIONALE TO GENERATE HYPOTHESES ON MICROBIAL LIFESTYLES IN THE LAKE, BUT DO NOT CONFIRM PHYSIOLOGY

11 Christner et al. in press; In: Psychrophiles: From Biodiversity to Biotechology, Springer

12 PHYSIOLOGY, SURVIVAL STRATEGIES, AND EVOLUTION OF MICROBES IN SALEs Can cells survive for extended periods in glacier ice and provide viable inoculi to SALEs? How do microbes offset macromolecular damage incurred during transport through the ice? Are there genotypic features which allow microbes to overcome the effects of low temperature? Have microbes adapted to the high pressure and gas concentrations in SALEs? Do cosmopolitan or endemic microbial species exist in subglacial environments?

13 InvestigatorsAncient material Age (years) Sheridan et al. 2003; Miteva & Brenchley 2005 Glacial ice; GISP2, Greenland 120,000 Abyzov 1993 Glacial ice; Vostok, Antarctica 200,000 Christner et al. 2003, 2006 Glacial ice; Guliya, China and Vostok, Antarctica >420, ,000 Shi et al. 1997Permafrost3,000,000 Cano and Borucki 1995Amber25,000,000 Greenblatt et al. 1999Amber120,000,000 Vreeland et al. 2000Salt crystal250,000,000 Reports of Viable Microorganisms Revived from Ancient Geological Samples

14 Geochemical Anomalies Attributable to Microbial Activity? Souchez et al. (1995) Very low oxygen concentration in the basal ice from Summit, Greenland, Geophys. Res. Lett., 22: Sowers (2001) The N 2 O record spanning the penultimate deglaciation from the Vostok ice core, J. Geograph. Res., 106: Campen et al. (2003) Evidence of microbial consortia metabolizing within a low latitude mountain glacier, Geology, 31: Flǜckiger et al. (2004) N 2 O and CH 4 variations during the last glacial epoch: Insight into global processes. Global Biogeoch. Cycles Vol 18. Ahn et al. (2004) A record of atmospheric CO 2 during the last 40,000 years from the Siple Dome, Antarctica ice core. J. Geophys. Res., 199, D Tung et al. (2005) Microbial origin of excess methane in glacial ice and implications for life on Mars. PNAS, 102: Spahni et al. (2005) Atmospheric methane and nitrous oxide of the late Pleistocene from Antarctic ice cores. Science, 310:

15 Temperature Growth rate Figure adapted from Brock Biology of Microorganisms 11e; † Sun and Friedmann (1999) Geomicrobiol. J. 16: MAXIMUM: protein denaturation; collapse of the cytoplasmic membrane; thermal lysis OPTIMUM: enzymatic reactions occurring at maximal possible rate MINIMUM: membrane gelling; transport processes so slow that growth cannot occur In contrast to the high temperature maximum for growth, determining the low temperature limit can be experimentally difficult (e.g year doubling times of cryptoendoliths † ) and it is usually extrapolated.

16 Christner 2002 Jakosky et al Rivkina et al. 2000; * Campen et al Carpenter et al Bakermans et al Junge et al Panikov et al Breezee et al * Tison et al * Sowers o -15 o -20 o -40 o * Calculated from ice core gas data; not a direct measurement of microbial activity Liquid conditionsFrozen conditions

17 “Microbial habitat consisting of solid ice grains bounded by liquid veins. Two microbes are depicted as living in the vein of diameter d vein surrounding a single grain of diameter D.” Price, P.B. (2000) A habitat for psychrophiles in deep Antarctic ice PNAS 97:

18 Christner 2002, AEM 68: [ 3 H]THYMIDINE INCORPORATION BY ARTHROBACTER G200-C1 AT -15 o C Bulk ion concentration 20 nmol L -1 n = 3

19 Days Dpm x DNA synthesis Protein synthesis Live cells Dead cells METABOLISM UNDER FROZEN CONDITIONS (-5 o C) BY YEAST ISOLATED FROM 179 M IN VOSTOK 5G Amato and Christner, unpublished data n = 3

20 IDENTIFICATION OF AN ICE ACTIVE PROTEIN FROM A CHRYSEOBACTERIUM SPECIES ISOLATED FROM 3519 M No activityIce-pitting activity ~0.5 mm Kilodaltons 39.3 pH The pits form because the IBP binds to the crystal faces, interfering with their growth. IBPs in other species appear to have a cryoprotective function. Christner and Raymond, unpublished data Peptide sequence from trypsin fragment: VSS(I/L)STDSQ(I/L)SD No match to other IBPs and antifreezes that have been identified thus far!

21 Doug Bartlett, Scripps Institution of Oceanography † Display optimal growth at a pressure above atmospheric pressure Pressure units: 1,000 atmospheres ≈ 101 MPa ARE THERE PIEZOPHILES † IN DEEP ICE AND SALEs? High Pressure Low Temperature Cell membranes becomes waxy and relatively impermeable at low temperature and high pressure Most microbes show reduced growth rates at just a few hundred atmospheres

22 Clone from deep-sea sediment Methylobacterium sp. UMB 3 Methylobacterium sp. UMB 26 Methylobacterium sp. V3 Methylobacterium sp. GIC 46 Methylobacterium adhaesivum Methylobacterium sp. UMB 28 Methylobacterium organophilum Methylobacterium sp. zf-IVRht8 Methylobacterium sp. IS11 Methylobacterium rhodinum Methylobacterium sp. G Methylobacterium sp. TD4 Methylobacterium sp. GIC52 Methylobacterium extorquens Methylobacterium zatmanii Methylobacterium sp. zf-IVRht11 Methylobacterium sp. G296-5 Methylobacterium radiotolerans Methylobacterium fujisawaense Sphingomonas sp. Arctic Sphingomonas sp. Antarctic Sphingomonas sp. G296-3 Sphingomonas sp. Muzt-J22 Sphingomonas sp. SIA181-1A1 Sphingomonas sp. SO3-7r Sphingomonas paucimobilis Sphingomonas sp. CanClear1 Sphingomonas sanguis Sphingomonas sp. M3C1.8k-TD1 Sphingomonas parapaucimobilis Sphingomonas echinoides Sphingomonas sp. FXS25 Sphingomonas sp. V1 Sphingomonas sp. G Sphingomonas anadarae Clone from deep-sea octacoral Sphingomonas sp. TSBY 64 Sphingomonas sp. TSBY 38 Sphingomonas sp. eh2 Sphingomonas aurantiaca Sphingomonas aerolata Sphingomonas sp. UMB 19 Sphingomonas sp. J05 Clone from Antarctic soil Sphingomonas sp. TSBY-61 Sphingomonas faeni Clone from subsurface aquifer Sphingomonas sp. Antarctic IS01 Sphingomonas sp. TSBY-49 Red = permanently cold or frozen environments Red Bold = from glacier/basal ice Blue = from Lake Vostok accretion ice Proteobacterial outgroups Christner et al. in press In: Psychrophiles: From Biodiversity to Biotechology, Springer Phylogenetic analysis of Alphaproteobacteria from glacier environments using maximum likelihood 1220-nucleotides of the 16s rRNA gene sequence 0.1 fixed substitutions per nucleotide position

23 Clone from deep-sea sediment Methylobacterium sp. UMB 3 Methylobacterium sp. UMB 26 Methylobacterium sp. V3 Methylobacterium sp. GIC 46 Methylobacterium adhaesivum Methylobacterium sp. UMB 28 Methylobacterium organophilum Methylobacterium sp. zf-IVRht8 Methylobacterium sp. IS11 Methylobacterium rhodinum Methylobacterium sp. G Methylobacterium sp. TD4 Methylobacterium sp. GIC52 Methylobacterium extorquens Methylobacterium zatmanii Methylobacterium sp. zf-IVRht11 Methylobacterium sp. G296-5 Methylobacterium radiotolerans Methylobacterium fujisawaense Sphingomonas sp. Arctic Sphingomonas sp. Antarctic Sphingomonas sp. G296-3 Sphingomonas sp. Muzt-J22 Sphingomonas sp. SIA181-1A1 Sphingomonas sp. SO3-7r Sphingomonas paucimobilis Sphingomonas sp. CanClear1 Sphingomonas sanguis Sphingomonas sp. M3C1.8k-TD1 Sphingomonas parapaucimobilis Sphingomonas echinoides Sphingomonas sp. FXS25 Sphingomonas sp. V1 Sphingomonas sp. G Sphingomonas anadarae Clone from deep-sea octacoral Sphingomonas sp. TSBY 64 Sphingomonas sp. TSBY 38 Sphingomonas sp. eh2 Sphingomonas aurantiaca Sphingomonas aerolata Sphingomonas sp. UMB 19 Sphingomonas sp. J05 Clone from Antarctic soil Sphingomonas sp. TSBY-61 Sphingomonas faeni Clone from subsurface aquifer Sphingomonas sp. Antarctic IS01 Sphingomonas sp. TSBY fixed substitutions per nucleotide position Proteobacterial outgroups Phylogenetic analysis of Alphaproteobacteria from glacier environments using maximum likelihood 1220-nucleotides of the 16s rRNA gene sequence Purple = Greenland (GISP2) Orange = Antarctica (Vostok, Siple, Taylor Dome, Taylor Valley) Green = Himalayan Blue = New Zealand Christner et al. in press In: Psychrophiles: From Biodiversity to Biotechology, Springer Glacier ice samples collected without the use of a drilling fluid

24 CONCLUSIONS The accreted ice is a proxy to estimate biogeochemical conditions in surface waters of Subglacial Lake Vostok. Variation in the accretion ice implies that ecological conditions are not spatially or temporally uniform in SLV. The search for viable microbial ecosystems in SALEs need not be exclusive to those with thermotectonic or hydrothermal activity. The low temperature limit for metabolic activity is probably lower than -40 o C. Territory for further microbiological studies: How do microbes deal with the high pressure, extreme cold, low nutrient, and potentially high O 2 concentrations? $ National Science Foundation: EAR and OPP $


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