Biological Context for Exploring Subglacial Lake Environments

Brent Christner, Department of Biological Sciencesxner@lsu.edu; 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 $