Searching for tiny life indices in extreme environments...
Transcript of Searching for tiny life indices in extreme environments...
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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
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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
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Working on Earth-known
microbial DNA
4 primary [nucleo]bases:
cytosine, guanine, adenine and
thymine - C, G, A, T
GenBank sequence database
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“Dust-free” room at LGGE – class 10,000
It-Rus Astrobiol, Dubna Bulat 11 dec-11
Fine snow/ice dust particles et DNA
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Clean (dust-free) room 10,000
Laminar flow hood 100 DNA/RNA BioCap hood 100
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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
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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
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A framework for
biological
studies adopted
for Lake Vostok
DNA extraction
Primary PCR No PCR
products
Bulat 11 dec-11
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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
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Vostok SURFACE
SNOW
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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
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Vostok MEGA- et Colossal-
snow 2011
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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
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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
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PMS11-1 7034.925S, 8124.103E H 2376m
PMS11-2 6941.081S, 7633.621E H 865m
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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)
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CS11-15 – light microscopy
OLYMPUS BH-2
Overall view (x500) Bulat 11 dec-11 It-Rus Astrobiol, Dubna
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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
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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.
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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?
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Bulat 2011 sept-21 GDRI meeting-Irkutsk
BIO-EXPOSURE 2011
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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)
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CONCLUSION
Intensive solar (UV?)
radiation as effective cell/DNA killing factor?
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Vostok ICE CORE
Towards subglacial Lake Vostok
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SALE Subglacial Antarctic Lake Environments - >386
Siegert et al., 2005
AGU Chapman conf 2010
Bulat 11 dec-11
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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
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No life
May host life
ISOLATED
Bulat 11 dec-11
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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
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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
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3720 m - Jan 21, 2011
Bulat 11 dec-11 30m left!
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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
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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
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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)
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„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 !
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RESULTS
Bacteria in [deep] lake ice
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3608BK - Big ‘Kamina’
6 x 8.5 x 15 (mm)
3608m
Mono-crystalline ice
Bulat 11 dec-11 It-Rus Astrobiol, Dubna
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Vostok cells / phylotypes
Bulat 11 dec-11 It-Rus Astrobiol, Dubna
Glacier flour
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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!
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Priscu et al 1999 Science
It-Rus Astrobiol, Dubna
3590m
103-104 cells/ml
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Karl et al 1999 Science
It-Rus Astrobiol, Dubna
3603m
2-3 x 102 cells/ml
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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
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Astrobiology implications
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In searching for [tiny] life indices
on icy worlds like Jovian Europa
Lake Vostok [ice] is a unique
TEST area
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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
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Bulat 11 dec-11 It-Rus Astrobiol, Dubna
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Antarctic polar vortex effect
www.ldeo.columbia.edu
May – August
at Vostok
10 – 50 km
Bulat 11 dec-11 It-Rus Astrobiol, Dubna
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Bulat 11 dec-11 It-Rus Astrobiol, Dubna
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Elements detected in Ant fine dust
particles
Much more rare as
compared to Fe!
Particle numbers tested
not enough to recover
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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
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Bulat 11 dec-11 It-Rus Astrobiol, Dubna
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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 * *