Post on 27-Dec-2015
IS or Was There Life in IS or Was There Life in Salts ofSalts of MARSMARS??
ROCCO L. MANCINELLISETI Institute, USA
Rocco MancinelliRocco Mancinelli
This presentation is about Halophiles– What are they?– Where do they live?– Who are they? – How do they live in salt?– Ways in which we test their limits of survival (torture)
• Desiccation• Freezing• Radiation• Fly in Space
This presentation is about Mars– History of water– Evaporites (the salts of Mars)
The possibility of life in the salts of Mars
Rocco MancinelliRocco Mancinelli
HalophilesHalophiles: Organisms that live in environments with salt concentrations ranging from 15% to saturation. QuickTime™ and a
Photo - JPEG decompressorare needed to see this picture.
Cargill salt Cargill salt concentrating ponds, concentrating ponds,
Newark, CA, USANewark, CA, USA
OsmophilesOsmophiles: Organisms that live in environments with a high osmotic gradient.
What are they?What are they?
Rocco MancinelliRocco Mancinelli
SalinibacterFlexib
acter
Planc
tom
y
ces
Rho
docy
clus
Hal
omon
as,
Sal
iniv
ibrio
,
Hal
orho
dosp
iraR
ho
do
spirillu
m
Desu
lfoh
alob
ium
Synechococcus
Gloeobacter
Halospirulina
Chlamydia
ChlorobiumLeptonema
Halanaerobium
Halobacillus
Arthrobacter
Heliobacterium
Thermomicrobium
Ther
mus
Ther
mot
oga Aqu
ifex
Methanopyrus
Methanothermus
Methanobacterium
Therm
ococcus
Methanococcus janneschii
Met
hano
cocc
us v
anne
ilii
Arc
haeo
glob
us
Ther
mop
lasm
aHal
obacte
rium
Methanohalophilus
Pyro
dict
ium Sulf
olobu
s
Desulfurococcus
Thermoproteus
Thermofilum
TrichomonasGiardiaHexamitaVairimorpha
Encephalitozoon
Physarum
Trypanosom
a
EuglenaN
aegl
eria
Ent
amoe
ba
Dic
tyos
teliu
m
Bab
esia
Param
ecium
PorphyraCostariaAchlya
Picosystis, Dunaliella
Cryptomonas
Homo
Coprinus
BACTERIA ARCHAEA
EUKARYA
Halophiles/Halophiles/OsmophilesOsmophiles:• Phylogenetically
diverse & occur in all 3 domains.
• Common in the environment
• Arose more than once.
Who are they & Where do they live?Tree of Life
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How do they live in salt?Adaptation to High Salinity
OrganicOrganic osmotic solutes (e.g., glycine betaine) osmotic solutes (e.g., glycine betaine)
Accumulation of intracellular Accumulation of intracellular KClKCl
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PHAGE
Not well known in the Archaeal halophilesNot well known in the Archaeal halophiles Better known in the Bacterial halophilesBetter known in the Bacterial halophiles Physiological attributes vs. 16S rDNA gene sequencing for identification of Physiological attributes vs. 16S rDNA gene sequencing for identification of
Archaeal halophiles often do not agree: Could viruses be responsible (lateral Archaeal halophiles often do not agree: Could viruses be responsible (lateral gene transfer)?gene transfer)?
Halophiles in NaCl Crystals
1 1 mm
ARCHAEAARCHAEA
0.3 m0.3 m
1 mm1 mm
Rocco MancinelliRocco Mancinelli
Cyanobacteria in Gypsum-Halite
Guerrero Negro, Baja California, Mexico
2 cm2 cm
BACTERIABACTERIA
Rocco MancinelliRocco Mancinelli
DunaliellaDunaliella in Salt Pond in Salt Pond
EUKARYAEUKARYA
ESSA salt pond, Baja, MexicoESSA salt pond, Baja, Mexico
10 10 mm
Pushing them to the limit(survival of the fittest)
electrons produce x-ray & radio burstsprotons produce gamma raysSolar Flares
Radiation
magnetosphere
UVUV visiblevisible infra redinfra red
auroraSolar Wind
Protons, electrons-particles, heavy ions)
cosmic radiationProtons, electrons
-particles, heavy ions)
Mostly protons, lesser Heavier ions, Occurs sporadically
Solar Particle Events
Space Vacuum
Temperature
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Exposure to Space HistoryExposure to Space History
Microbes (viruses, vegetative bacteria, [D. radiodurans], B. subtilis spores) have been flown and exposed to space environment since the 1960’s. All died instantly except B. subtilis spores
LDEF 1984-90 – Survival of B. subtilis spores only in multi-layer or overlain with a substance.
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Question: Can Halophiles survive in space as
well as bacterial endospores?
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BiopanBiopan - ESA’s pan shaped biological space exposure facility designed to fly in earth orbit
aboard the Russian Foton rocket.
Rocco MancinelliRocco Mancinelli
Hypothesis & Objectives
Hypothesis: Synechococcus (Nägeli) & Haloarcula-G inhabiting evaporitic salt crusts can survive exposure to the space environment– Carotenoids absorb UV– Carotenoids scavenge radicals– Both organisms are desiccation resistant and live
exposed to UV Objectives:
– Determine organism survival in space– Measure DNA damage
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Experimental protocol: pre-flight
Washed mid-log phase organisms diluted to 107 microbes L-1.
40 L aliquots placed onto 7 mm quartz discs and air dried overnight.
2 sets of discs prepared for flight (UV & dark), 1 set remains in lab (control).
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Biopan ready to close
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Biopan Mounted to Satellite
QuickTime™ and aPhoto - JPEG decompressor
are needed to see this picture.
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Organisms exposed to the space environment for 15 days
Total UV dose = 10,000 KJ m-2 (~ 6,000 KJ m-2 UVA 4,000 UVB-C)
Earth surface dose ≈ 6,000 KJ m-2 (UVA)
Biopan 1 & 3 Launched
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Satellite landed
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Biopan after landing
QuickTime™ and aPhoto - JPEG decompressor
are needed to see this picture.
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Experimental protocol: post-flightExperimental protocol: post-flight
Survival: Most Probable Number (MPN), plate counts, live-dead stain, N-fixation and C-fixation rates.
DNA Damage: Modified nick translation (incorporation of 32P).
Live-dead stain
Live
dead
1 1 mm
Rocco MancinelliRocco Mancinelli
BIOPAN 3
0.00
0.50
1.00
N/N
o S
urv
ival
UV Vacuum Vacuum
Haloarcula G Haloarcula GSynechococcus Synechococcus
Rocco MancinelliRocco Mancinelli
Conclusions
Hypothesis true: Synechococcus (Nägeli) inhabiting gypsum-halite crusts and Haloarcula-G survive exposure to the space environment.
DNA damage correlated negatively with survival.
Rocco MancinelliRocco Mancinelli
Ground Time Course TestsGround Time Course Tests
Samples Exposed to UV in Vacuum & Air
• UV: Source is deuterium lamp (0.07 Wm-2
@200 nm; 0.03 Wm-2@ 300 nm).
• Vacuum: Final pressure 10-5 Pa.
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Survival Haloarcula-G: Ground Time Course Experiment (MPN)
T (hrs)
10-1
10
0.001 0.010 0.100 1.000 10.000 100.000 1000.000 10000.000
UV-Air
Vac
UV (0.07 Wm-2) Vac (10-5 Pa)
Su
rviv
al N
/NS
urv
iva
l N/N
00
10-2
10-3
10-4
10-5
Rocco MancinelliRocco Mancinelli
SUMMARYSUMMARY
Terrestrial life can survive away from earth
–Space environment
First non-sporeformers to survive in space
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Metabolism in Permafrost
Incorporation of 14C-labeled acetate by the native bacterial population in Siberian permafrost over a 550-day period. Because of the very low counts curves were calculated for –15 and –20°C. For all data the limit of error (2s) was less than 5%. (Rivkina et al., 2000, AEM 66:3230)
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OBJECTIVEOBJECTIVE
Determine if Determine if halophileshalophiles can survive can survive andand
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EXPERIMENTAL PROTOCOL: Washed mid-log phase organisms diluted to 10Washed mid-log phase organisms diluted to 1077
microbes mLmicrobes mL-1-1. . HalophilesHalophiles (washed in 25% NaCl)(washed in 25% NaCl) D. D. rad,.rad,. E. coliE. coli & & Ps. fluorescensPs. fluorescens (washed in 0.085% (washed in 0.085% NaCl). NaCl). 20 20 l aliquots placed onto quartz discs & air l aliquots placed onto quartz discs & air dried, or placed into eppendorf tubes.dried, or placed into eppendorf tubes.
Test sets subjected to freezing at – 20 or – 80 Test sets subjected to freezing at – 20 or – 80 ooC, C, wet and dry. Controls remained at room wet and dry. Controls remained at room temperature. Periodically samples tested for temperature. Periodically samples tested for viability using viability using LiveLive//Dead Dead stain (survivability stain (survivability determined microscopically and fluorometrically) determined microscopically and fluorometrically) and by ability to reproduce.and by ability to reproduce.
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LiveLive--DeadDead Stained Stained E. coliE. coli
11mm
WetWet DryDry
11mm
As a control washed mid-log phase room temperature cultures were stained with live-dead stain either wet or after drying.
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LiveLive--DeadDead Stained Stained Halobacterium Halobacterium NRC-1NRC-1
WetWet DryDry
11mm11mm
As a control washed mid-log phase room temperature cultures were stained with live-dead stain either wet or after drying.
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Survival at -80 Survival at -80 ooC of Frozen C of Frozen wet samples after 3 monthswet samples after 3 months
Halo-G - 51 ± 4% Halobacterium NRC-1 - 15± 2% Deinococcus radiodurans 10 ± 2% Ps. fluorescens - 0 E. coli - 0
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Survival After Drying & Freezing Survival After Drying & Freezing
Escherichia coliPseudomonas fluorescensDeinococcus radioduransHalobacterium NRC-1Halo-G
Org
anis
ms
mL
-1
Cell suspension
Dried —20 oC —80 oC101033
101044
101055
101066
101077
101088
101099
The number of viable organisms mLThe number of viable organisms mL-1-1 cell suspension recovered after being dried, dried and frozen cell suspension recovered after being dried, dried and frozen at -20 at -20 ooC, or dried and frozen at -80 C, or dried and frozen at -80 ooC for 3 months compared to the number of organisms in the C for 3 months compared to the number of organisms in the original cell suspensionoriginal cell suspension
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MARSMARS
Rocco MancinelliRocco Mancinelli
Water Flow during Mars PastWater Flow during Mars Past
•Valley networks in the Valley networks in the Southern HighlandsSouthern Highlands•(MOC, Mars Global Surveyor)(MOC, Mars Global Surveyor) Vast volumes of rapid-moving fluid Vast volumes of rapid-moving fluid
created streamlined islands like created streamlined islands like these in the Chryse Basinthese in the Chryse Basin
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Water Flow during Mars PastWater Flow during Mars Past
Rare recently revealed gullies may be sites of present day, Rare recently revealed gullies may be sites of present day, near surface, liquid water (MGS Image).near surface, liquid water (MGS Image).
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Standing Water during Mars PastStanding Water during Mars Past
•Colorized subframe of MOC image M03-Colorized subframe of MOC image M03-02733 showing layers in Holden Crater02733 showing layers in Holden Crater
Shorelines?Shorelines?
Rocco MancinelliRocco Mancinelli
Evaporites on Mars The OMEGA/Mars Express
hyperspectral imager– Location of the deposits
identified in Valles Marineris and surroundings plotted over the MOLA altimetry. red, kieserite type; green, polyhydrated sulfate; blue, most likely gypsum; pink, other hydrated minerals.
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Evaporites on Mars
MER Rover MER Rover OpportunityOpportunity
• Hematite concretionsHematite concretions
Water & Life on MarsWater & Life on Mars Life may have evolved As Mars lost its atmosphere it became cold and
dry. As water froze, evaporated & retreated into the
subsurface, brine pockets & evaporites formed– Oasis for extant biota– Last refuge for extinct biota
Halophiles/osmophiles are the most likely organisms to survive in a brine pocket, or evaporite.
Rocco MancinelliRocco Mancinelli
SUMMARYSUMMARY Evaporites containing extant and fossilized
microbial communities occur on earth today. Evaporites and/or brine pockets may occur on
Mars. Microbes can metabolize in permafrost. Halophiles/osmophiles can survive in brines,
drying and freeze-thaw cycles, as may have occurred on Mars, better than other organisms.
Halophily/osmophily is not rare and probably evolves easily.
Rocco MancinelliRocco Mancinelli
ConclusionsConclusions If life evolvedIf life evolved on Mars, then on Mars, then osmophilyosmophily
(halophily) probably evolved.(halophily) probably evolved.
Osmophiles are Osmophiles are best suitedbest suited for life in brines for life in brines and evaporites.and evaporites.
A A primary focusprimary focus for the search for extant for the search for extant and extinct and extinct life on Marslife on Mars should be a search should be a search for for osmophilesosmophiles, or their fossilized remnants., or their fossilized remnants.