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

Bulat 11 dec-11 It-Rus Astrobiol, Dubna

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

Bulat 11 dec-11 It-Rus Astrobiol, Dubna

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

Bulat 11 dec-11 It-Rus Astrobiol, Dubna

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

Vostok MEGA- et Colossal-snow 2011 - RESULTS

Bulat 11 dec-11 It-Rus Astrobiol, Dubna

Cell counts Sample Bacteria / ml

Roscoff sea water 30961

PMS11-1 - 216 km Progress 45

PMS11-2 - 29 km Progress 0

CS11-15 Vostok - 2.8-3.0 m deep 0

Bulat 11 dec-11 It-Rus Astrobiol, Dubna

PCR - phylotypes

Vostok MEGA- et Colossal-snow 2011 - RESULTS

Sample Bacteria / ml

PMS11-1 - 216 km Progress 45

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]

Bulat 11 dec-11 It-Rus Astrobiol, Dubna

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)

Bulat 11 dec-11 It-Rus Astrobiol, Dubna

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

Bulat 11 dec-11 It-Rus Astrobiol, Dubna

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

Bulat 11 dec-11 It-Rus Astrobiol, Dubna

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

It-Rus Astrobiol, Dubna

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

It-Rus Astrobiol, Dubna

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

Bulat 11 dec-11 It-Rus Astrobiol, Dubna

Vostok cells / phylotypes

Bulat 11 dec-11 It-Rus Astrobiol, Dubna

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

It-Rus Astrobiol, Dubna

3590m

103-104 cells/ml

Karl et al 1999 Science

It-Rus Astrobiol, Dubna

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]

Bulat 11 dec-11 It-Rus Astrobiol, Dubna

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

Bulat 11 dec-11 It-Rus Astrobiol, Dubna

Bulat 11 dec-11 It-Rus Astrobiol, Dubna

Antarctic polar vortex effect

www.ldeo.columbia.edu

May – August

at Vostok

10 – 50 km

Bulat 11 dec-11 It-Rus Astrobiol, Dubna

Bulat 11 dec-11 It-Rus Astrobiol, Dubna

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

Bulat 11 dec-11 It-Rus Astrobiol, Dubna

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 * *