The search for LIFE in the subglacial Lake Vostok in Antarctica
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1 The search for LIFE in the subglacial Lake Vostok in Antarctica Sergey BULAT Cryoastrobiology laboratory Petersburg nuclear physics institute - NRC Kurchatov institute bulat@omrb.pnpi.spb.ru
2 Life around us? Searching for extraterrestrial life is one of the aims of astrobiology, and there are several potentially habitable worlds in the Solar System, in addition to Earth. Two of Jupiter s moons (Ganymede and Europa) and two of Saturn s moons (Titan and Enceladus) harbour liquid-water oceans. And Mars probably had vast liquid-water environments during much of its early history. Lazcano 2012 Nature 488: Bulat17 Sept-26 Astro-Art, Croatia
3 'Diving' Tectonic Plates on Jupiter's Moon Europa million years old Radiation dose ~0.9 (leading) until ~90 (trailing hemisphere) kgy per day (from Paranicas et al) Temperatures range from as high as about 140 Kelvin (- 133oC) in dark material at the equator to as low as about 50 Kelvin Bulat17 (-223oC) Sept-26in bright icy patches at the poles NASA/JPL/Ted Stryk Astro-Art, Croatia 25 kgy average sterization dose 10 kgy - Deinococcus radiodurans survives 1 kgy humans die within 1 h (>4 Gy all die within 30 days) K. Retherford/Southwest Research Institute
4 Large subsurface ocean near Enceladus south pole Bulat17 Sept-26 Astro-Art, Croatia Cassini Imaging Science Subsystem courtesy NASA- JPL-Caltech
5 Large subsurface ocean near Enceladus south pole Methanol H 2 Saturn s moon Enceladus spews water vapor and ice particles from fractures in the ice. Scientists have found about 100 of these geysers. Credit: NASA/JPL-Caltech/SSI Bulat17 Sept-26 Astro-Art, Croatia
6 Vostok station environs, Bulat17 Sept-26 EAST Antarctica Astro-Art, Croatia
7 402 lakes 2016 (Siegert, pers. comm.) SALE Subglacial Antarctic Lake Environments CRYOSPHERE Mean heat flux 45-55mW/m 2 (Ritz, 1992) Wright et Siegert, 2012
8 Vostok station (+3488m) (78 S, 106 E) 1260 km from the coast (since dec 1957) 67 kpa 67% oxygen 21 July 1983 Absolute minimum C Average ann. Temp C RADARSAT, CSA Lake Vostok (-3663m)
9 Lake Vostok Discovery First reported at the 23 rd session of SCAR in Rome in 1994 Published in Nature (Kapitsa A.P. et al., 1996) Aug 02, 2011
10 Озерный л Lake Vostok et ice core North M (78 S, 106 E) South Восток F ~4km from coast Ледниковый лед 0.8 Ma 2.0 Ma 3538m 4200 м 3750 м Accretion 3650m Уровень моря Melting zone Gases 680m Фундамент Вода Accretion (lake) ice Accr. rate <10 mm/a
11 RADARSAT, CSA Lake Vostok settings Huge subglacial lake (Popov et al., 2011) 270 x 70 km in size; km 2 area; km 3 water Two main basins with the ridge between m deep Average depth m; max depth ~1220 m (south basin) Ma years old Water renewal Kyr Buried beneath Myr old m thick ice sheet Isolated from surface biota for >14 Ma Age of water (melted ice) - ~1 Ma No hydrological links to other lakes Bulat17 Sept-26 Astro-Art, Croatia
12 Complete drainage pathways of subglacial water around the Dome C and Ridge B regions of East Antarctica (Siegert et al., 2007).
13 Lake Vostok known since 1994 Published in Nature (Kapitsa et al., 1996) RADARSAT, CSA Friendly environment? FREE (LIQUID) WATER! Deeply ice buried (in dark) 4 km High pressure bar Permanently BUT not very cold C Likely oxygen supersaturated mg/l (upper bound mg/l) 320 mg/l Bulat17 Sept-26 (V Lipenkov estimate from frozen water) Astro-Art, Croatia
14 Low temperature limit for terrestrial life (Archaea, Bacteria, Eukarya) Cell division by terrestrial microbes has not been reported below -18 o C (255 K) Cellular metabolic activity has not been demonstrated below -33 o C (240 K), although some biophysical processes may be functional at lower temperatures Rummel et al 2014 Astrobiology 14(11)_ Bulat17 Sept-26 Astro-Art, Croatia
15 Dissolved O2 (mg/l) OXYGEN in lake water < < O Water cycles Time (t/t R )
16 Cold room at LGGE
17 Dust-free room at LGGE class 10,000 Fine snow/ice dust particles et DNA analyses DNA/RNA BioCap hood class 100
18 Centriprep Plus-70 (ml vol.) Biomax membrane 5 kd 135 bp DNA (45.9 nm)
19 Number of microbial species (red) Number of microbial genes (blue-cyan) Microbial inhabitants outnumber our body s own cells by about ten to one
20 Vostok ice for Biology Comprehensive Biological controls Bulat17 Sept-26 REQUIREMENTS: Sham/mock DNA extraction Negative PCR Ice core wash water Lab Environment (dust) Vostok drill fluid Outer-core (optionally) Repeatability Astro-Art, Croatia 309 seqs Contaminant databases July 2017
21 Grenoble IGE/LGGE UGA Lyon l ENS No PCR products DNA extraction Primary PCR Clean cold and certified clean laboratory A framework for biological studies adopted for Lake Vostok Grenoble IGE/LTHE UGA
22 We believe Whatever we find It s going to be special stuff, unique stuff, life forms we ve never encountered before Oxygen-loving? Extra-terrestrial?
23 RESULTS Bacteria in Vostok drill-bit et borehole frozen water
24 3 Max. 575m ~345m ~72-77m 3720 m - Jan 21, 2011 Enter 1 st m Fev 05, nd m Jan 25, 2015
25 Lake Vostok 1 st entry feb 05, 2012 Drill-bit frozen water sample
26 Max. ~50pg gdna 167 cells/ml
27 Water-DNA/PCR Flow-Chart ~20pg gdna? Meta Genomics WATER gdna 16S rdna v3-v5 16S rdna v4-v8 16S rdna Full gene ~40% bacterial divisions ~40% bacterial divisions ~10% bacterial divisions 16S rdna v4 16S rdna v4-v6
28 Vostok frozen water (2 entries) 50 (47 1 st entry) contaminant phylotypes Clone Proteobacteria Alpha- 8 Beta- 7 Gamma- 21 Actinobacteria 6 Firmicutes 7 Bacteroidetes 1 Phylotype
29 w <86% known taxa Unknown bacterium Max. ~50pg gdna 167 cells/ml
30
31 Vostok cells/phylotypes (16S rrna - v2-v5 reg.) - SUMMARY Ice type Sample (m) Cells/ml (% Similarity with closest sequence in GenBank) Snow (50 yr) Contaminants Glacier Sulfate-reducing bacteria kyr Hydrogen-oxidizing bacteria ? Methanogenic archaea? Sulfur-oxidizing bacteria Iron-oxidizing bacteria Contaminants Accretion I Contaminants kyr Contaminants Contaminants + thermophile 3607 ND Thermophile (92%) Uncultured bacterium (410 bp, AF532061) 3607-re BK-re ND (95%) Ilumatobacter fluminis (526 bp, NR_041633) Actino 5G Contaminants Accretion II kyr G Contaminants No more water untreated surface pockets G G Lake water 5G2-water* w2ori 167 Refer to below Lake water frozen on a drill bit Bulat et al., 2004; 2012
32 Lake Vostok 1 st entry feb 05, 2012 Borehole frozen water samples re-corred one year later
33 5G-2[3] Vostok borehole m Uppermost frozen water ice m layer of frozen lake water 3458 m Bottommost frozen water ice 3539 m Type I m Fev m Type II m m Late jan m remained to drill 2014/2015 season - at rate ~2m/day m Fev 05, 2012
34 5G-1N-3425 Vostok water ice 40 cells/ml? Bulat17 Sept-26
35 5G-1N-3429 Vostok water ice 5.5 cells/ml Max. ~30pg gdna
36 5G-1N-3450 Vostok water ice 23 cells/ml No cells Ongoing analyses Dust/microparticles Analytical chemistry Cell concentrations 38 cells/ml Bulat17 Sept-26 Astro-Art, Croatia gdna 16S rrna gene amplifications w specific PCR assay
37 5G-1N S rrna gene Clone v3-v5 3429v Janthinobacterium sp Burkholderiales, Oxalobacteraceae, Beta-Proteobacteria 1.5 L processed
38 Vostok frozen water (2 entries) 50 (47 1 st entry) contaminant phylotypes Clone Proteobacteria Alpha- 8 Beta- 7 Gamma- 21 Actinobacteria 6 Firmicutes 7 Bacteroidetes 1 Phylotype
39 CONCLUSION It seems there is UNKNOWN very tiny LIFE (w population) in the uppermost layer of the Lake Vostok water body w <86% known taxa 3429v3-4 93% - Janthinobacterium sp
40 Papers Bulat S., J.R. Petit (2015) Vostok, Subglacial Lake. In Gargaud M, Irvine W (eds.) Encyclopedia of Astrobiology, Vol. 1, pp. 1-6 (Ch ), Springer Heidelberg New York Dordrecht London Bulat S.A. (2016) Microbiology of the subglacial Lake Vostok: first results of borehole-frozen lake water analysis and prospects for searching lake inhabitants. Phil. Trans. R. Soc. A 374: Bulat17 Sept-26 Astro-Art, Croatia
41 Lake Vostok 2 nd entry jan 25, 2015 Borehole frozen water samples re-corred in a few days
42 3 Max. 575m ~345m ~72-77m ΔT o C hrs open Enter 3720 m - Jan 21, st m Fev 05, nd m Jan 25, 2015
43 New 5G-3N borehole frozen lake water ( ) PNPI Bulat17 Sept-26 Astro-Art, Croatia
44 5G-3N-3709A 1-3 treatment Channels Cold room Clean room
45 3709A 1-3 Counter Coulter Particle abundance ppb Mode is around 7-10 microns that corresponds to kerosene micro-droplets
46 3709A 1-3 amino acid content Metrohm ion chromatograph using a C4 cation and an asupp5 anion columns By Carlo Barbante Institute for the Dynamics of Environmental Processes CNR, Venice, and Department of Environmental Sciences, Informatics and Statistics, Ca Foscari University of Venice, Venice 30123, Italy All tested amino acids demonstrated values below of detection limits Bulat17 Sept-26 Astro-Art, Croatia
47 3709A 1-3 cell concentrations Flow cytofluorimetry using the SYBR Green-I dye and a BD FACSAria device By Dominique Marie Station Biologique de Roscoff Place Georges Teissier ROSCOFF Cedex cells/ml No real populations Bulat17 Sept-26 Astro-Art, Croatia
48 3709A 1-3 rrna gene amplicons Clean room condition PCR for 16S rrna gene regions (cloning and sequencing): V3-v5 weak signal 4 phylotypes (contaminants) V4-v8 signal 4 phylotypes (contaminants) V4-v6 v weak signal V4 v weak signal v3-v5 (w phylotype specific) no signal v3-v5 (Hydrogenophilus-specific) no signal Pseudomonas yamanorum (gamma-) 99.5-Stenotrophomonas maltophilia (gamma-) Serratia myotis (gamma-) 99.9-Acinetobacter johnsonii (gamma-) 99.8-Arthrobacter russicus (actino) 99.8-Microbacterium ginsengisoli (actino) Undibacterium oligocarboniphilum (beta-) Escherichia coli (gamma-)
49 SEM-EDX analyses SEM Zeiss Ultra 55 with Bruker 127eV - resolution at 15kV is 1nm. 3709A 1-3 frozen water (2 nd entry)
50 Aluminosilicates plagioclases /layered silicates Si-O-Al-[C] 6/12 particles
51 Lake Vostok 3 rd entry feb 03, 2015 Borehole frozen water samples re-corred two years later
52 5G-3N-3698 et 3715 treatment Freezing center Cold room - top view 3698m Bulat17 Sept-26 Astro-Art, Croatia 3715m
53 5G-3N-3715 Crystal feathers and channels Bulat17 Sept-26 Astro-Art, Croatia 3715m
54 5G-3N-3698 et 3715 studies Meltwater strong smell of kerosene No particles No chemistry Cell concentrations by flow cytometry DNA analyses (16S rrna gene v4-v6) Ongoing since juin 23, 2017 Bulat17 Sept-26 Astro-Art, Croatia
55 Exciting results seem coming soon? We surely need cleanly collected (with no DRILL FLUID) lake water
56 'Diving' Tectonic Plates on Jupiter's Moon Europa 5G cell/ml million years old Radiation dose ~0.9 (leading) until ~90 (trailing hemisphere) kgy per day (from Paranicas et al) Temperatures range from as high as about 140 Kelvin (- 133oC) in dark material at the equator to as low as about 50 Kelvin Bulat14 (-223oC) oct-16 in bright icy patches at the poles NASA/JPL/Ted Stryk 5MS3, IKI 25 kgy average sterization dose 10 kgy - Deinococcus radiodurans survives 1 kgy humans die within 1 h (>4 Gy all die within 30 days) K. Retherford/Southwest Research Institute
57 10 years FUTURE DIRECTIONS Rapidly frozen lake water (within the borehole) since 2012 Lake water column (680m) Lake sediments (>300m) 4-5 order difference Microbial biomass at BIONTRANS (Jorgensen and Boetius 2007)
58 Acknowlegements LGGE CNRS-UJF, Grenoble, FR Jean Robert Petit Jean Martins et Frederic Charlot (EDX-SEM) Station Biologique de Roscoff, Roscoff, FR Dominique Marie AARI, St Petersburg, RU Vladimir Lipenkov, Alexey Ekaykin ice cores Valery Lukin logistics PNPI Cryoastrobiol labo, St Petersburg-Gatchina, RU D Karlov, M Doronin, M Khilchenko, G Pavlov, O Belova Bulat17 Sept-26 Astro-Art, Croatia
59 Subglacial Lake Entry on the Horizon in Antarctica Subglacial Lake Vostok, the largest known subglacial lake on earth (3769.3m-1.5km) Subglacial environments beneath West Antarctic Whillans ice stream (800m- 2m) Subglacial Lake Ellsworth, West Antarctica (3200m- 160m) Fev 05, 2012 Russia Jan 27, 2013 US > UK
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