Searching for tiny life 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 oct-13 2MS3-IKI RAN, Moscow
Main BIO
Culturing
Cell counts
RepetitionCulturing
Working on Earth-known microbial DNA
4 primary [nucleo]bases: cytosine, guanine, adenine and
thymine - C, G, A, T GenBank sequence database
Clean (dust-free) room 10,000Laminar 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 databasesBulat 11 oct-13 2MS3-IKI RAN, Moscow
Vostok ice for Biology
• Authentication of findings by ecology (environmental conditions)
REQUIREMENTS:
“Everything is everywhere, but, the environment selects” – Baas Becking, 1934
Bulat 11 oct-13 2MS3-IKI RAN, Moscow
A framework for biological
studies adopted for Lake Vostok
DNA extractionPrimary PCR
No PCR products
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Absolute minimum -89.2°CAverage ann. Temp. -55.1°C
RADARSAT, CSA
Vostok station (+3488m) (since 1957)
Lake Vostok (-3663m)
1260 km from the coast
(78°S, 106°E)
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Vostok vs. Europa SURFACE
SNOW
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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!
2MS3-IKI RAN, Moscow
Vostok MEGA-snow 2010
Bulat 11 oct-13 2MS3-IKI RAN, Moscow
V500S10 (Vostok500kmSnow2010
(RAE 55)
Vostok MEGA-snow – SURFACE – 500km toward Vostok
Bulat 11 oct-13 2MS3-IKI RAN, Moscow
Vostok MEGA-snow – SURFACE – 3.0 km SW away
VMS10 (VostokMeteoricSnow2010
(RAE 55)
Vostok MEGA-snow 2010
Bulat 11 oct-13 2MS3-IKI RAN, Moscow
RESULTS
Vostok Contaminant LibraryHuman Normal Microbiome
Vostok MEGA- et Colossal-snow 2011
Vostok MEGA-snow 2011• 216km from coastal Progress – PMS11-1
– Crate 23L 5-8L melt• 29km from coastal Progress – PMS11-2
– Crate 23L 5-8L melt
Bulat 11 oct-13 2MS3-IKI RAN, Moscow
Sergey Popob
PMS11-1 7034.925S, 8124.103E H 2376m
PMS11-2 6941.081S, 7633.621E H 865m
Bulat 11 oct-13 2MS3-IKI RAN, MoscowVostok Colossal-snow 2011
220L
CS11 78,4722S, 106,7796E(VostokCollossalSnow2011 (RAE 56)
Vostok MEGA- et Colossal-snow 2011 - RESULTS
Bulat 11 oct-13 2MS3-IKI RAN, Moscow
Cell counts
Sample Bacteria / ml
Roscoff sea water 30961PMS11-1 - 216 km Progress 45PMS11-2 - 29 km Progress 0CS11-15 Vostok - 2.8-3.0 m deep 0
Bulat 11 oct-13 2MS3-IKI RAN, Moscow
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 LibraryHuman Normal Microbiome
Clinical specimens including wound, sputum, urine, stool, blood etc. Sources of infection – domestic air conditioning units, ice baths etc.
Pre CONCLUSION
• Vostok [et 29km from coast!] SURFACE snow contains a few cells / ml - NO CELL populations [lifeless]
Bulat 11 oct-13 2MS3-IKI RAN, Moscow
Background contamination?
Vostok vs. Europa ICE CORE
Towards the Lake Vostok
SALE Subglacial Antarctic Lake Environments - >386
Siegert et al., 2005AGU Chapman conf 2010
Bulat 11 oct-13 2MS3-IKI RAN, Moscow
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 oct-13 2MS3-IKI RAN, Moscow
No life
May host life
ISOLATED
Bulat 11 oct-13 2MS3-IKI RAN, Moscow
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 oct-13 2MS3-IKI RAN, Moscow
Vostok ice core
Ледниковы й лед
Озерны й лед
Вода
Фундамент
Уровень моря
4200 м 3750 м
FMВ ост о к
05 01 0 01 5 02 0 02 5 0Р а с с т оя н и е , к м
3623 м
5Г
3 0 0
Melting zoneAccretion
North South
Meteoric ice
Lake water body
Accretion (lake) iceAccr. rate – <10 mm/a
0.8 Ma 2.0 Ma – 3538m
Gases 680m
(78°S, 106°E)19.02.1990
3650m
Bulat 11 oct-13 2MS3-IKI RAN, Moscow
3720 m - Jan 21, 2011
Bulat 11 oct-13 2MS3-IKI RAN, Moscow 30m left!
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AccretionICE
GAS contents*
DOC (ppbC)
Majorions*
TYPE 1 Sediments
Low (10-2)
CO2, CH4, O2
10 ± 9 Mg2+ Ca2+ SO4
2- (S2-)
(no NH4+, nitrate)
TYPE 2 Very clean
Very low
(10-3)(no O2, CH4)
5.6 ± 4.6 -(no NH4
+, nitrate)
Conditions
Low aerobicAnaerobic
Ultra-Oligotrophic
No heterotrophs
No e-donors?(Hydrogen?)
Accretion ice BIO featuresNO GAS NO DOC
Many oligotrophic lakes have DOC's in the range of 100 to 500 ppb
2MS3-IKI RAN, Moscow
Bulat 11 oct-13
Bacterial metabolic pathways Accretion ice type
1E-donor E-acceptor Carbon Process Microbes
H2 SO42- CO2 Sulfate reduction
(H2S)Sulfate-reducers
H2 O2 (in mineralinclusions)
CO2 Hydrogen-oxidation
Hydrogen-oxidizers
H2 CO2 CO2 Methanogenesis (CH4)
Methanogenic archaea
S2-
S0
O2 (NO3-) CO2 Sulfur oxidation (SO4
2- - acidic)Sulfur-oxidizers
In mineral inclusions
RECALL: Gas content is very low - 0.001 vs. 0.1 g/cm3
2MS3-IKI RAN, Moscow
Life prospective in lake ice 1Anaerobic
chemolithoautotrophic piezophilic psychrophiles
Type1 Type2
?
Given a limiting life temperature threshold - -20°C (Astrobiology, 2006, 6, 677-732)
RESULTS
Bacteria in [deep] lake ice
3608BK - Big ‘Kamina’
6 x 8.5 x 15 (mm)
3608m
Mono-crystalline ice
Bulat 11 oct-13 2MS3-IKI RAN, Moscow
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5G2-3608 et 5G2-3646
2MS3-IKI RAN, Moscow
Big Kamina-2?
Vostok cells / phylotypes - update 2010
Bulat 11 oct-13 2MS3-IKI RAN, Moscow
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.)
36083608BK
NDND
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 bacteriaHydrogen-oxidizing bacteriaMethanogenic archaea Sulfur-oxidizing bacteria
Both UNCLASSIFIED!
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General CONCLUSION•Vostok [et 29km from coast!] SURFACE snow - NO CELL populations [lifeless]
•DEEP lake Vostok ICE contains NO CELL populations [lifeless]?
Bulat 11 oct-13 2MS3-IKI RAN, Moscow
With no cracks!
Astrobiology implications
In searching for [tiny] life indices on icy worlds like Jovian Europa
Lake Vostok [ice] is a unique TEST area
BioLESSONS from Vostok•Work on youngest the least processed at the surface of Europa material (i.e. ocean water ice) flashed out veins or faults (within ridges?) in a radiation shadow
Bulat 11 oct-13 2MS3-IKI RAN, Moscow
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 oct-13 2MS3-IKI RAN, Moscow