searching for tiny life indices in extreme environments...

Searching for tiny life indices in

extreme environments relevant to

Jovian’s Europa: Lessons from

subglacial ice studies at Lake

Vostok (East Antarctica)

Sergey Bulat*

Russian-French GDRI consortium

GDRI consortium • PNPI FSBI, St Petersburg, RU

– Sergey Bulat - PI

• AARI FSBI, St Petersburg, RU – Vladimir Lipenkov - PI

– Valery Lukin

• VNIIOceangeologia, St Petersburg, RU – German Leitchenkov

• LGGE CNRS-UJF, Grenoble, FR – Jean Robert Petit - PI

– Michel Legrand (DOC)

• Université de Bretagne Occidentale, Brest, FR – Mohamed Jebbar

• Station Biologique de Roscoff, Roscoff, FR – Dominique Marie

• Ecologie Microbienne UCB-Lyon I, Lyon, FR – Philippe Normand

Bulat 11 dec-11 It-Rus Astrobiol, Dubna

Main BIO

Culturing

Cell counts

Repetition

Culturing

Working on Earth-known

microbial DNA

4 primary [nucleo]bases:

cytosine, guanine, adenine and

thymine - C, G, A, T

GenBank sequence database

“Dust-free” room at LGGE – class 10,000

It-Rus Astrobiol, Dubna Bulat 11 dec-11

Fine snow/ice dust particles et DNA

Clean (dust-free) room 10,000

Laminar flow hood 100 DNA/RNA BioCap hood 100

Vostok ice for Biology

• Comprehensive Biological controls

– Sham/mock DNA extraction

– Negative PCR

– Ice core wash water

– Lab Environment (dust)

– Vostok drill fluid

– Outer-core (optionally)

REQUIREMENTS:

211 seqs July 2011

Contaminant

databases Bulat 11 dec-11 It-Rus Astrobiol, Dubna

Vostok ice for Biology

• Authentication of findings by ecology (environmental conditions)

REQUIREMENTS:

“Everything is everywhere,

but, the environment selects”

– Baas Becking, 1934


A framework for

biological

studies adopted

for Lake Vostok

DNA extraction

Primary PCR No PCR

products

Bulat 11 dec-11

Absolute minimum -89.2°C

Average ann. Temp. -55.1°C

RADARSAT, CSA

Vostok station (+3488m) (since 1957)

Lake Vostok (-3663m)

1260 km from

the coast

(78°S, 106°E)

Bulat 11 dec-11

Vostok SURFACE

SNOW

Bulat 11 dec-11

Very clean „life-hostile‟ environ

• Low accumulation – 2.1-2.4g /cm2/a

• Low Aeolian dust input – 2 μm mode; 10-15 ppb (summer time)

• Harsh irradiation (UV-C etc.) („ozone hole‟ – Aug-Nov) – + Water-Oxygen free radicals

– Oxidized organics (photochemical reactions at ice-air interface)

• Low freezing temperatures – below -36oC (-55.1oC ann.)

Vostok Surface snow (0 – 130m up to 4 Kyr)

(6-6.5 cm/a – snow / needles!)

Below 3m the snow is pristine

NO FREE (only BOUND) WATER!

It-Rus Astrobiol, Dubna

Vostok MEGA- et Colossal-

snow 2011


220L

CS11 (78,4722S, 106,7796E)

(VostokCollossalSnow2011 (RAE 56)

ОК!

Trouble!

Courtesy of A. Ekaykin et al (RAE56)

Vostok Colossal-snow 2011

Vostok MEGA-snow 2011

• 216km from coastal Progress – PMS11-1

– Crate 23L 8.6L melt

• 29km from coastal Progress – PMS11-2

– Crate 23L 7.6L melt

Bulat 11 dec-11

PMS11-1 7034.925S, 8124.103E H 2376m

PMS11-2 6941.081S, 7633.621E H 865m


Dust particle counts

Vostok MEGA- et Colossal-snow 2011 - RESULTS

Sample N/g M/g (ppb) Masse(0.8-2) Masse (2.2-5 ) Mass >5

Vivendi ELGA-26 1347 7.63 2.79 4.30 0.00

Ultra-pure water 577 1.45 1.45 0.00 0.00

3549 9.2 7.28 1.17 0.00

PMS11-1 4821 12.14 9.09 2.27 0.00

216 km Progress 5375 16.19 10.45 4.11 0.00

PMS11-2 4933 14.6 10.22 3.29 0.00

29 km Progress 4241 11.8 8.80 2.00 0.00

3811 10.7 7.90 1.86 0.00

CS11-15 Vostok 3643 11.94 7.52 3.41 0.00

2.8-3.0 m deep 3754 13.13 7.93 4.07 0.00

Метод Electrical Sensing Zone (Coulter counter, Beckman, США)

CS11-15 – light microscopy

OLYMPUS BH-2

Overall view (x500) Bulat 11 dec-11 It-Rus Astrobiol, Dubna



Cell counts Sample Bacteria / ml

Roscoff sea water 30961

PMS11-1 - 216 km Progress 45


CS11-15 Vostok - 2.8-3.0 m deep 0


PCR - phylotypes


Sample Bacteria / ml


V4-v8 16S rDNA amplicon sequencing

HA-Pseudomonas sp (alpha-proteo) - 80% 99% Ewingella americana (gamma-proteo, Enterobact.)

Bradyrhizobium sp(elkani) (alpha-proteo)

Vostok Contaminant Library

Human Normal Microbiome

Clinical specimens including wound,

sputum, urine, stool, blood etc.

Sources of infection – domestic air

conditioning units, ice baths etc.

Pre CONCLUSION

• Vostok [even 29km from coast!] SURFACE

snow contains a few(?) cells / ml -

NO CELL populations [lifeless]


Background contamination?

Bulat 2011 sept-21 GDRI meeting-Irkutsk

BIO-EXPOSURE 2011

Bulat 2011 sept-21 GDRI meeting-Irkutsk

RESULTS

No signals

(v4-v8890bp, v3-v5590bp, v6170bp reg.

16S rDNA) even in one[1st] day!

BIO-EXPOSURE 2011

(only SW2-09 gDNA)

(9 days – each day) No exposure at Vostok

(~107 cells/ml)

CONCLUSION

Intensive solar (UV?)

radiation as effective cell/DNA killing factor?

Vostok ICE CORE

Towards subglacial Lake Vostok

SALE Subglacial Antarctic Lake Environments - >386

Siegert et al., 2005

AGU Chapman conf 2010

Bulat 11 dec-11

Friendly environment?

• Deeply ice buried (in dark) – 4 km

• High pressure – 337-377 bar

• Permanently BUT not very cold – -2.65°C

• Likely oxygen supersaturated - >70 mg/L

up to 700-1300 mg/L

Lake Vostok known since 1994

FREE (LIQUID) WATER!

RADARSAT, CSA

Published in Nature (Kapitsa et al., 1996)


No life

May host life

ISOLATED

Bulat 11 dec-11

Lake Vostok settings

• Huge subglacial lake (Masolov et al., SCAR OSC2008)

– 275 x 65 km in size; 15 500 km2 area; 6 100 km3 water

– Two main basins with the ridge between 150-200 m deep

– Average depth - 300 m; max depth – ~1650 m (south basin)

– Water renewal – 80-100 Kyr

• Buried beneath 2.0-0.8 Myr old 3750-4200 m thick ice sheet

• Isolated from surface biota for >14 Ma – Age of water (melted ice) - ~1 Ma

• No hydrological links to other lakes

RADARSAT, CSA

2.0-0.8 Ma years old


Vostok ice core

Ледниковый лед

Озерный лед

Вода

Фундамент

Уровень моря

4200 м 3750 м

FMВосток

050100150200250Расстояние, км

3623 м

5Г

300

Melting zone

Accretion

North South

Meteoric ice

Lake water body

Accretion (lake) ice

Accr. rate – <10 mm/a

0.8 Ma 2.0 Ma – 3538m

Gases 680m

(78°S, 106°E) 19.02.1990

3650m


3720 m - Jan 21, 2011

Bulat 11 dec-11 30m left!

Bulat 11 dec-11

Accretion

ICE

GAS

contents*

DOC

(ppbC)

Major

ions*

TYPE 1

Sediments

Low

(10-2)

CO2, CH4, O2

10 ± 9 Mg2+ Ca2+

SO42- (S2-)

(no NH4+,

nitrate)

TYPE 2

Very clean

Very low

(10-3)

(no O2, CH4)

5.6 ± 4.6 -

(no NH4+,

nitrate)

Conditions Low aerobic

Anaerobic

Ultra-

Oligotrophic

No heterotrophs

No e-donors?

(Hydrogen?)

Accretion ice BIO features

NO GAS NO DOC

Many oligotrophic lakes

have DOC's in the range of

100 to 500 ppb


Bulat 11 dec-11

Bacterial metabolic pathways

Accretion ice type 1

E-donor E-acceptor Carbon Process Microbes

H2 SO42- CO2 Sulfate reduction

(H2S)

Sulfate-

reducers

H2 O2 (in mineral

inclusions)

CO2 Hydrogen-

oxidation

Hydrogen-

oxidizers

H2 CO2 CO2 Methanogenesis

(CH4)

Methanogenic

archaea

S2-

S0

O2 (NO3-) CO2 Sulfur oxidation

(SO42- - acidic)

Sulfur-

oxidizers

In mineral inclusions

RECALL: Gas content is very low - 0.001 vs. 0.1 g/cm3


Life prospective in lake ice 1

Anaerobic chemolithoautotrophic

piezophilic psychrophiles

Type1 Type2

?

Given a limiting life temperature threshold - -20°C

(Astrobiology, 2006, 6, 677-732)

„Oxygenophilic‟(?)

chemolithoautotrophic

piezophilic psychrophiles

RADARSAT, CSA

NEW microbes in lake water

Superoxide, hydrogen peroxide, singlet

O, hydroxyl radicals etc. vs. carotinoids,

catalase, superoxide dismutase etc.

Earth-unknown !

RESULTS

Bacteria in [deep] lake ice

3608BK - Big ‘Kamina’

6 x 8.5 x 15 (mm)

3608m

Mono-crystalline ice


Vostok cells / phylotypes


Glacier flour

Cells DOC Phylotypes Vostok ice core

Ice Type Sample (m) DOC

(ppbC) Cells / ml

(% similarity, GenBank)

Phylotype

Snow (50 yr) 4.0-4.3 0-0.02

Glacier 122 5.4 ± 1.1 1.9

4.5-760 kyr 2005 6.4 ± 0.7 2.4

2054 4.4 ± 0.7 3-24

3489 ND 0

Accretion I 3561 ND 4-9 (99%) Hydrogenophilus thermoluteolus

30-40 kyr 3607 12.5 ± 0.3 ND (100%) H. thermoluteolus (β-Proteo)

(91%) Uncult bacterium (OP11 div.)

3608

3608BK

ND

ND

0-19

ND

(94%) Ilumatobacter luminis (Actino)

Accretion II 3613 3.0 – 7.0 3

20-30 kyr 3621 3.2 – 20.4 2

3622 2.5 ± 0.7 0.6

3635 4.0 – 9.0 4.7

3650 9.0 – 15.0 3.1 thoroughly treated

3650 ND 4777 untreated surface

3659 ND 12

Sulfate-reducing bacteria

Hydrogen-oxidizing bacteria

Methanogenic archaea

Sulfur-oxidizing bacteria

Both UNCLASSIFIED!

Priscu et al 1999 Science


3590m

103-104 cells/ml

Karl et al 1999 Science


3603m

2-3 x 102 cells/ml

General CONCLUSION

• Vostok [even 29km from coast!] SURFACE

snow - NO CELL populations [lifeless]

• Vostok lake ICE type II contains NO

CELL populations [lifeless]?

• Vostok lake WATER contains(?)

unknown LIFE [close to sediments]


Astrobiology implications

In searching for [tiny] life indices

on icy worlds like Jovian Europa

Lake Vostok [ice] is a unique

TEST area

Work guide for ice samples

delivered to Earth • applying stringent ice sample (and tools to work on)

decontamination procedures in clean conditions to meet trace

chemistry and Ancient DNA analysis standards

• certification of the various environments in contact with the ice

samples for biological content (establishment of contaminant

library)

• implementation of appropriate methods to obtain a signal at as low

as possible a detection level

• verification of findings through their possible metabolic profiles as

deduced from physical/chemical features of the environment under

consideration

• replication of findings at an independent laboratory (or

experiment) within the framework of specialized laboratories


Antarctic polar vortex effect

www.ldeo.columbia.edu

May – August

at Vostok

10 – 50 km


http://www.ldeo.columbia.edu/

Elements detected in Ant fine dust

particles

Much more rare as

compared to Fe!

Particle numbers tested

not enough to recover

Mg, Al, Si, S, Ca, Cr, Mn вместе с

C?, O и Na

Not yet detected

Ni, Ir - refer to A.F. Grachev

Elements detected in Ant fine dust

particles

CS11-1, -15 et PMS11-1 – electron

scanning microscopy JSM-6400 Element content/composition of some Ant snow dust particles (cps/ev)

* Background values

Образец/

Элемент

C

(>26)

O

(>1.5)

Fe Al Si Na Mg K Ca N S P Pt Минерал

CS11-1A-A 0.4 * * 2.4 Кварц?

CS11-1A-B 3.5 * 0.9 6.3 * 0.5 *

CS11-1A-C 2

CS11-1B-A-1 2.3 1 3.4 0.4 * *

CS11-1B-A-2 0.8 * 1.6 2.2 0.21 0.3

CS11-1B-A-3 2.5 1.5 2.7 0.8 * *

CS11-1B-A-4 2.5 * 1 3.2 0.9

CS11-1B-B-1 2.1 0.3 1.2 1.6 0.5 *

CS11-1B-B-2 1.9 1.1 3.5 0.3 0.3

CS11-1B-C-1 0.7 2.7 0.3 0.4

CS11-1B-C-2 * 0.7 1.8 *

CS11-1B-C-3 2.4 4.1 0.4 * *

CS11-1B-C-4 1.9 1.5 3.6 0.5 * 0.4 *

CS11-15B-A-1 0.4 1.9 1.9 *

CS11-15B-A-2 5.8 3 3.5 0.5 1

CS11-15B-A-3 1.4 0.9 1.2 0.4 0.3

CS11-15B-B-1 0.7 0.5 0.3 * *

CS11-15B-B-2 8.4 1 8.5 0.5 *

CS11-15B-B-3 9 0.3 4 4.5 * *

CS11-15B-B-4 6 0.9 2.5 8.5 0.8 2.2 * 0.5

PMS11-1-A 2 * 3.9

PMS11-1-B 2.3 * 1.5 3 0.5 0.3 * *

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