Biological Context for Exploring Subglacial Lake Environments
Brent Christner, Department of Biological [email protected]; http://brent.xner.net/
3rd SCAR SALE Meeting, 6 - 7 June 2007
Big Sky, Montana
OUTLINEOUTLINE
• 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.
Rationale for Ice Core Rationale for Ice Core Decontamination ProtocolDecontamination Protocol
5 mmscraped
final sample
5 mm removed by washing
5 mm removedby melting
Christner et al. 2005, Icarus, 174:572-584
core diameter removed[p
aram
eter
]core diameter removed
[par
amet
er]
Christner et al. in press; In: Psychrophiles: From Biodiversity to Biotechology, Springer
Cells mL-1
1e+1 1e+2 1e+3 1e+4 1e+5 1e+6
De
pth
(m
)
3520
3540
3560
3580
3600
3620
Core exterior (outer 0.5 cm)Core interior (1.5 cm removed)
Cells mL-1 on core exterior
0 1e+5 2e+5 3e+5 4e+50
100
200
300
400
500A B
Ce
lls m
L-1 o
n c
ore
inte
rio
r
CONCENTRATION OF CELLS ON THE EXTERIOR AND INTERIOR OF ICE CONCENTRATION OF CELLS ON THE EXTERIOR AND INTERIOR OF ICE SAMPLES FROM THE BOTTOM ~100 M OF THE VOSTOK 5G ICE CORESAMPLES 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)
Cells mL-1
0 200 400
Dep
th (
met
ers
belo
w t
he
surf
ace)
0
500
1000
1500
2000
2500
3000
3500
Total organic carbon(ppb)
0 500 1000 1500
Borehole Temperature (oC)-50 -30 -10
VOSTOK 5G ICE CORE (VOSTOK STATION, ANTARCTICA)VOSTOK 5G ICE CORE (VOSTOK STATION, ANTARCTICA)
Christner et al. 2006, L&O 51:2485-2501
ACCRETION ICE I
ACCRETION ICE II
GLACIAL ICE(>420,000 years-old)
3510
3520
3530
3540
3550
3560
3570
3580
3590
3600
3610
3620
0 100 200 300 400 500 600
Cells mL-1 of melt waterD
epth
in
Vo
sto
k co
re (
m)
SYBR Gold(DNA-containing)
Propidium Iodide(DEAD)
SYTO 9(LIVE)
Christner et al. 2006L&O, 51:2485-2501
Significantly higher(p < 0.001) than cell densities >3,572 m
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 51:2485-2501
NONPURGEABLE ORGANIC CARBON AND TOTAL AMINO NONPURGEABLE ORGANIC CARBON AND TOTAL AMINO ACID CONCENTRATIONS IN THE ACCRETION ICE ACID CONCENTRATIONS IN THE ACCRETION ICE
3200
m
3310
m
3539
m
3609
m
3623
m
3750
m
Ice-Ice-waterwaterglacier ice shear layer (deformed)
up
Type I(particle inclusions)
Type II(few inclusions)
Christner et al. 2006
ConstituentDissolved
Organic carbon
(mol L-1)
Cell number
(cells mL -1)
Total dissolved solids (mmol L-1)
Glacial ice (average) 16 120 0.0088Type I accretion ice (average) 65 260 0.061Type II accretion ice (average) 35 83 0.0033
Embayment water† 160 460 34Main lake water† 86 150 1.5Average continental rainfall NA NA 0.15Average marine/coastal rainfall NA NA 0.38Average surface seawater 40-80 0.05-5 x 105 710
Biogeochemical conditions in the surface Biogeochemical conditions in the surface waters of Lake Vostokwaters of Lake Vostok
Christner et al. 2006, L&O, 51:2485-2501.
†Partitioning coefficients based on ice & water chemistry of L. Bonney, Antarctica
MOLECULAR IDENTIFICATION OF BACTERIAL DNA MOLECULAR IDENTIFICATION OF BACTERIAL DNA SEQUENCES IN LAKE VOSTOK ACCRETION ICESEQUENCES 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 phylotypesRubrobacterHydrogenophilus
• Phylotypes related to chemolithoautotrophsHydrogenophilus 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
Christner et al. in press; In: Psychrophiles: From Biodiversity to Biotechology, Springer
PHYSIOLOGY, SURVIVAL STRATEGIES, AND PHYSIOLOGY, SURVIVAL STRATEGIES, AND EVOLUTION OF MICROBES IN SALEsEVOLUTION 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?
Investigators Ancient materialAge
(years)
Sheridan et al. 2003; Miteva & Brenchley 2005
Glacial ice; GISP2, Greenland 120,000
Abyzov 1993Glacial ice; Vostok,
Antarctica 200,000
Christner et al. 2003, 2006
Glacial ice; Guliya, China and Vostok,
Antarctica
>420,000-750,000
Shi et al. 1997 Permafrost 3,000,000
Cano and Borucki 1995 Amber 25,000,000
Greenblatt et al. 1999 Amber 120,000,000
Vreeland et al. 2000 Salt crystal 250,000,000
Reports of Viable Microorganisms Revived from Reports of Viable Microorganisms Revived from Ancient Geological SamplesAncient Geological Samples
Geochemical Anomalies Attributable to Geochemical Anomalies Attributable to Microbial Activity?Microbial Activity?
• Souchez et al. (1995) Very low oxygen concentration in the basal ice from Summit, Greenland, Geophys. Res. Lett., 22: 2001-2004.
• Sowers (2001) The N2O record spanning the penultimate deglaciation from the Vostok ice core, J. Geograph. Res., 106:31903-31914.
• Campen et al. (2003) Evidence of microbial consortia metabolizing within a low latitude mountain glacier, Geology, 31:231-234.
• Flǜckiger et al. (2004) N2O and CH4 variations during the last glacial epoch: Insight into global processes. Global Biogeoch. Cycles Vol 18.
• Ahn et al. (2004) A record of atmospheric CO2 during the last 40,000 years from the Siple Dome, Antarctica ice core. J. Geophys. Res., 199, D13305.
• Tung et al. (2005) Microbial origin of excess methane in glacial ice and implications for life on Mars. PNAS, 102:18292-18296.
• Spahni et al. (2005) Atmospheric methane and nitrous oxide of the late Pleistocene from Antarctic ice cores. Science, 310:1317-21.
Temperature
Gro
wth
rat
e
Figure adapted from Brock Biology of Microorganisms 11e; †Sun and Friedmann (1999) Geomicrobiol. J. 16:193-202
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. 104-year doubling times of cryptoendoliths†) and it is usually extrapolated.
Christ
ner 2
002
Jako
sky e
t al. 2
003
Rivkina
et a
l. 200
0;
* Cam
pen
et a
l. 200
3
Carpenter et al. 2000
Bakermans et al. 2003
Jung
e et
al. 2
006
Paniko
v et a
l. 200
6
Breezee et al. 2004
* Tiso
n et
al. 1
998
* Sowers 2001
-10o-15o-20o-40o
* Calculated from ice core gas data; not a direct measurement of microbial activity
Liquid conditions Frozen conditions
“Microbial habitat consisting of solid ice grains bounded by liquid veins. Two microbes are depicted as living in the vein of diameter dvein surrounding a single grain of diameter D.”
Price, P.B. (2000) A habitat for psychrophiles in deep Antarctic ice PNAS 97:1247-1251.
Christner 2002, AEM 68:6435-6438
[[33H]THYMIDINE INCORPORATION BY H]THYMIDINE INCORPORATION BY ARTHROBACTERARTHROBACTER G200-C1 AT -15 G200-C1 AT -15 ooCC
Bulk ion concentration 20 nmol L-1
n = 3
Days
0 5 10 15 20
Dp
m x
10
00
10
20
30
40DNA synthesis
Protein synthesis
Live cells
Dead cells
METABOLISM UNDER FROZEN CONDITIONS (-5 METABOLISM UNDER FROZEN CONDITIONS (-5 ooC) C) BY YEAST ISOLATED FROM 179 M IN VOSTOK 5GBY YEAST ISOLATED FROM 179 M IN VOSTOK 5G
Amato and Christner, unpublished data
n = 3
IDENTIFICATION OF AN ICE ACTIVE PROTEIN FROM A IDENTIFICATION OF AN ICE ACTIVE PROTEIN FROM A CHRYSEOBACTERIUMCHRYSEOBACTERIUM SPECIES ISOLATED FROM 3519 M SPECIES ISOLATED FROM 3519 M
No activity Ice-pitting activity
~0.5 mm
20
15
10
Kilo
dalto
ns
3 9.3pH
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!
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
Clone from deep-sea sedimentMethylobacterium sp. UMB 3Methylobacterium sp. UMB 26Methylobacterium sp. V3Methylobacterium sp. GIC 46
Methylobacterium adhaesivumMethylobacterium sp. UMB 28
Methylobacterium organophilumMethylobacterium sp. zf-IVRht8
Methylobacterium sp. IS11Methylobacterium rhodinumMethylobacterium sp. G296-15Methylobacterium sp. TD4Methylobacterium sp. GIC52
Methylobacterium extorquensMethylobacterium zatmanii
Methylobacterium sp. zf-IVRht11Methylobacterium sp. G296-5Methylobacterium radiotolerans
Methylobacterium fujisawaenseMethylobacterium fujisawaense
Sphingomonas sp. ArcticSphingomonas sp. Antarctic
Sphingomonas sp. G296-3Sphingomonas sp. Muzt-J22
Sphingomonas sp. SIA181-1A1Sphingomonas sp. SO3-7r
Sphingomonas paucimobilisSphingomonas sp. CanClear1
Sphingomonas sanguisSphingomonas sp. M3C1.8k-TD1Sphingomonas parapaucimobilisSphingomonas echinoides
Sphingomonas sp. FXS25Sphingomonas sp. V1Sphingomonas sp. G296-14
Sphingomonas anadaraeClone from deep-sea octacoral
Sphingomonas sp. TSBY 64Sphingomonas sp. TSBY 38Sphingomonas sp. eh2Sphingomonas aurantiaca
Sphingomonas aerolataSphingomonas aerolataSphingomonas aerolata
Sphingomonas sp. UMB 19Sphingomonas sp. J05Clone from Antarctic soilSphingomonas sp. TSBY-61Sphingomonas faeniClone from subsurface aquifer
Sphingomonas sp. Antarctic IS01Sphingomonas sp. TSBY-49
Red = permanently cold or frozen environmentsRed Bold = from glacier/basal iceBlue = from Lake Vostok accretion ice
Proteobacterialoutgroups
Christner et al. in pressIn: Psychrophiles: From Biodiversity to Biotechology, Springer
Phylogenetic analysis of Alphaproteobacteria from Phylogenetic analysis of Alphaproteobacteria from glacier environments using maximum likelihoodglacier environments using maximum likelihood
1220-nucleotides of the 16s rRNA gene sequence1220-nucleotides of the 16s rRNA gene sequence
0.1 fixed substitutions per nucleotide position
Clone from deep-sea sedimentMethylobacterium sp. UMB 3Methylobacterium sp. UMB 26Methylobacterium sp. V3Methylobacterium sp. GIC 46
Methylobacterium adhaesivumMethylobacterium sp. UMB 28
Methylobacterium organophilumMethylobacterium sp. zf-IVRht8
Methylobacterium sp. IS11Methylobacterium rhodinumMethylobacterium sp. G296-15Methylobacterium sp. TD4Methylobacterium sp. GIC52
Methylobacterium extorquensMethylobacterium zatmanii
Methylobacterium sp. zf-IVRht11Methylobacterium sp. G296-5Methylobacterium radiotolerans
Methylobacterium fujisawaenseMethylobacterium fujisawaense
Sphingomonas sp. ArcticSphingomonas sp. Antarctic
Sphingomonas sp. G296-3Sphingomonas sp. Muzt-J22
Sphingomonas sp. SIA181-1A1Sphingomonas sp. SO3-7r
Sphingomonas paucimobilisSphingomonas sp. CanClear1
Sphingomonas sanguisSphingomonas sp. M3C1.8k-TD1Sphingomonas parapaucimobilisSphingomonas echinoides
Sphingomonas sp. FXS25Sphingomonas sp. V1Sphingomonas sp. G296-14
Sphingomonas anadaraeClone from deep-sea octacoral
Sphingomonas sp. TSBY 64Sphingomonas sp. TSBY 38Sphingomonas sp. eh2Sphingomonas aurantiaca
Sphingomonas aerolataSphingomonas aerolataSphingomonas aerolata
Sphingomonas sp. UMB 19Sphingomonas sp. J05Clone from Antarctic soilSphingomonas sp. TSBY-61Sphingomonas faeniClone from subsurface aquifer
Sphingomonas sp. Antarctic IS01Sphingomonas sp. TSBY-49
0.1 fixed substitutions per nucleotide position
Proteobacterialoutgroups
Phylogenetic analysis of Alphaproteobacteria from Phylogenetic analysis of Alphaproteobacteria from glacier environments using maximum likelihoodglacier environments using maximum likelihood
1220-nucleotides of the 16s rRNA gene sequence1220-nucleotides of the 16s rRNA gene sequence
Purple = Greenland (GISP2)
Orange = Antarctica (Vostok, Siple, Taylor Dome, Taylor Valley)
Green = HimalayanBlue = New Zealand
Christner et al. in pressIn: Psychrophiles: From Biodiversity to Biotechology, Springer
Glacier ice samples collected without the use of a drilling fluid
CONCLUSIONSCONCLUSIONS
• 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 oC.
• Territory for further microbiological studies: How do microbes deal with the high pressure, extreme cold, low nutrient, and potentially high O2 concentrations?
$ National Science Foundation: EAR and OPP $