IS or Was There Life in Salts of MARS? ROCCO L. MANCINELLI SETI Institute, USA.

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


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?


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


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


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


Cyanobacteria in Gypsum-Halite

Guerrero Negro, Baja California, Mexico

2 cm2 cm

BACTERIABACTERIA


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


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.


Question: Can Halophiles survive in space as

well as bacterial endospores?


BiopanBiopan - ESA’s pan shaped biological space exposure facility designed to fly in earth orbit

aboard the Russian Foton rocket.


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


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).


Biopan ready to close


Biopan Mounted to Satellite

QuickTime™ and aPhoto - JPEG decompressor

are needed to see this picture.


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


Satellite landed


Biopan after landing

QuickTime™ and aPhoto - JPEG decompressor

are needed to see this picture.


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


BIOPAN 3

0.00

0.50

1.00

N/N

o S

urv

ival

UV Vacuum Vacuum

Haloarcula G Haloarcula GSynechococcus Synechococcus


Conclusions

Hypothesis true: Synechococcus (Nägeli) inhabiting gypsum-halite crusts and Haloarcula-G survive exposure to the space environment.

DNA damage correlated negatively with survival.


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.


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


SUMMARYSUMMARY

Terrestrial life can survive away from earth

–Space environment

First non-sporeformers to survive in space


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)


OBJECTIVEOBJECTIVE

Determine if Determine if halophileshalophiles can survive can survive andand


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.


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.


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.


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


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


MARSMARS


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


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).


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?


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.


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.


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.


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.

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IS or Was There Life in Salts of MARS? ROCCO L. MANCINELLI SETI Institute, USA.

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