The H2O2-H2O Hypothesis of Martian Extremophiles
and Missions to Mars
Dirk Schulze-Makuch1 and Joop Houtkooper2
1SEES,
Washington State University
2Justus-Liebig University, Giessen
On Earth, Extremophilic Life is everywhere
White
Sands
Volcanoes
http://www.usask.ca/communications/ocn/
Soda
Lake
Antarctic Dry Valleys
http://www.homepage.montana.edu/~lkbonney/IMAGES/Antarctic
http://www.abc.net.au/science/news/img/environment/extremophile101103.jpg
Extremophiles on Mars?
SURVIVAL STRATEGIES FOR MICROBIAL LIFE
UNDER MARTIAN ENVIRONMENTAL
CONDITIONS
Evolutionary Controls on Microbes
• Constant environment:
Evolutionary options narrow
to stable forms that persist with
little change through time
=> STABILIZING SELECTION
• Changing environment:
Evolution favors selection for
new forms and functions
=> DIRECTIONAL SELECTION
Martian History: SCENARIO 1
Cold and dry mostly but with
(1) periodic global flooding triggered by episodic
volcanism and meteor bombardment
(2) localized flow from snowmelt or groundwater
eruptions
• Surface features consistent
with catastrophic flooding
•Surface features consistent
with local sapping
or seepage
Martian gullies
SCENARIO 1: Life
• Origin: chemoautotrophs;
evolved into heterotrophs.
• Phototrophs?
• Directional selection for
chemoautotrophy and the
development of alternative
energy sources.
• Strong directional selection
for life cycles alternating
between dormant and
proliferative forms.
Catastrophic Flooding - The Ma'adim
Vallis, (white arrow) a 2.1 km deep
cut into the Southern Highlands that
runs from a topographically lower
area to the Gusev crater
SCENARIO 1: Today
•
Single or simple cells;
dormant forms persist near surface.
•
Living microorganisms within
ground water or lava tubes;
some differ considerably from
ancestral forms.
Martian History: SCENARIO 2
Cold and dry mostly;
Periodic and prolonged presence of surface
water beneath ice sheets.
•periodic eruption of ground
water into long-standing
basins
• atmosphere non-conducive
for repeated rainfall
Charitum Montes area: a possible
water ice coating on the hilly surface.
SCENARIO 2: Cold & dry most of its history, but with periodic &
prolonged surface water beneath ice sheets.
• Origin of life: Chemotrophs
• Evolution of phototrophs,
possibly  heterotrophs
• Stabilizing selection (in general) for
psychrophilic microbes,
surviving for long periods
beneath ice cover
• Extinction of surface phototrophs
and heterotrophs, but persistence
of chemotrophs and some
heterotrophs below ground.
• Possible survival of psychrophilic
phototrophs at or near wet or icy
surface
Psychrophilic Organism
SCENARIO 2: Today
Subterranean microorganisms
survive in ground water.
Liquid ground water
underneath
Thickness of cryosphere
is determined by
•geothermal gradient
•hydraulic conductivity
of cryosphere
Recent evidence supports
SCENARIOS 1 or 2
1. Models: Lengthy cold, dry periods with
impact- induced precipitation (Segura et al.,
2002)
2. Surface water in the past and abundant nearsubsurface water to the present time (Carr,
1989; Boynton et al., 2000)
3. Limited but recent surface flow of lava
and/or water (Malin & Edgett, 2000; Berman
& Hartmann, 2002; Christensen, 2003)
Most recent evidence supports SCENARIOS 1 or 2
4. Main Proposed Martian Shorelines:
•Meridiani shoreline (the maximum extent of a Noachian ocean)
•Arabia shoreline (not likely a complete ancient paleoshoreline)
•Deuteronilus shoreline (the maximum extent of a Late Hesperian ocean)
Meridiani shoreline
Arabia Shoreline
Deuteronilus shoreline
Mean elevation (m)
-1500
-2090
-3792
Basin area (107 km2)
5.35
4.67
2.47
Volume (107 km3)
10.7 – 15.5
7.7 – 11.1
1.4 – 2.0
Mean Depth (km)
1.99 – 2.89
1.64 – 2.38
0.56 – 0.81
From: UPDATING THE EVIDENCE FOR OCEANS ON EARLY MARS, Fairén et al. (LPSC, 2005)
Most recent evidence supports
SCENARIOS 1 or 2
5. Jarosite, indicative of
mildly acidic and
moderately oxidizing water
“Blueberry bowl” at Meridiani Planum
The stability field for Jarosite
Expectations: Scenarios 1 or 2
 Single
or simple cells in ground water.
Dormant forms (spores) at or near
surface

Energy sources likely
chemoautotrophy, or a form unfamiliar
to life on Earth.
Surviving forms: remnants of marine
thermotrophs deriving energy from
geothermal activity or meteor impacts
from Schulze-Makuch and
Irwin (2004):
Life in the Universe: Expectations
and Constraints, Springer)
Experimental Testing
Media Tested
Medium
Soil
Water
1. Salt-water soil
95% JSC Mars-1
stimulant soil
5% saline water
(30g/L NaCl per
liter)
2. Fresh-water
soil
95% JSC Mars-1
stimulant soil
5% saline water
(0.5g/L NaCl per
liter)
3. Sea water
5% JSC Mars-1
stimulant soil
95% Sea Salt
Water
(30g/L Sea Salt
per liter)
Methodology: Quick Facts
•All experiments were run in triplicates
•Measurements at soil depths of 0 cm, 1 cm,
and 5 cm; and in a well-mixed water medium
•No significant growth in controls was observed
•Stresses: Low Pressure, Low Temperatures;
Combined Low Pressure and Low Temperatures
for 10 days
•Experiments with stresses for UV radiation only,
and UV radiation plus Low Pressure, for 24 hours
Environmental
conditions on Mars
Experimental
conditions
Pressure
~ 6mb
833 mb
Temperature
-123oC to 25oC
-35oC
UV radiation
8.4 to 67 Wm-2
37 Wm-2
(200nm-400nm)
Microbial viability in soil and water samples
after treatment to environmental stresses
Treatment
E. coli
Medium
Soil
(depth
viabilities [%])
D. radiodurans
and
56 %
2.5 %
1 cm
71 %
5 cm
2%
5 cm
64 %
0 cm
0%
0 cm
73 %
1 cm
0%
1 cm
72 %
5 cm
0%
5 cm
47 %
Sub-Zero
Temperature +
Low-Pressure
0 cm
59 %
0 cm
92 %
1 cm
83 %
1 cm
98 %
5 cm
60 %
5 cm
89 %
UV
0 cm
1.5 %
0 cm
27 %
1 cm
15 %
1 cm
67 %
5 cm
50 %
5 cm
97 %
0 cm
0%
0 cm
0.3 %
1 cm
0%
1 cm
0.1 %
5 cm
52 %
5 cm
56 %
Low-Pressure
UV+ LowPressure
1.5 %
1 cm
Soil (depth and viabilities [%])
0 cm
Sub-Zero
Temperature
0 cm
Sea
Water
20%
73%
20%
34%
69%
Sea
Water
57%
100%
100%
59%
91%
D. radiodurans vs E. coli in 1 cm Deep Soil
100%
100%
98 %
90%
90%
80%
80%
70%
70%
71 %
72 %
60%
67 %
Sub-zero temperature
83 %
Sub-zero temperature
Low-Pressure
Low-Pressure
60%
50%
50%
Sub-zero temperature
and low-pressure
40%
40%
UV irradiation
30%
30%
20%
UV irradiation and low20% pressure
10%
10%
Sub-zero temperature
and low-pressure
UV irradiation
UV irradiation and lowpressure
15 %
2.5 %
0.1 %
0%
0%
D.
1
radiodurans
in soil
E. coli1in soil
Notes: Percent (%) viability was gathered from 1 cm depth results from both types of soils. Stresses were applied
in Experiments 1-3 for 10 days, in Experiment 4 and 5 (or 24 hours.
D. radiodurans vs E. coli Survival
in Sea-Water Medium
Notes: Stresses were applied in Experiments 1-3 for 10 days, while in Experiment 4 and 5 for 24
hours. Samples from sea water were taken at a depth of 1 cm.
A History of Life on Mars?
Given the apparent fast rise of life on Earth as soon as favorable conditions
prevailed
• The origin of life seems plausible
(very similar conditions on early
Earth, Venus, and Mars)
Once life became entrenched on Mars and surface conditions became
hostile, life could have retreated to the subsurface or adapted using a novel
biochemistry by natural selection
Or, organisms could have been delivered
by meteorites from early Earth
(recent studies indicate that bacteria
can survive space travel for millions of
years given minimal shielding)
(Davies, 1996; Horneck and Rettberg, 2002)
Extremophiles on Mars?
that use H2O2 incorporated into their Intracellular Fluids ?
•Lowers freezing point significantly (eutectic at -56.5oC)
•Mixtures of high H2O2 concentrations supercool and don’t form ice crystals
•Hygroscopic properties and ability to scavenge water from the atmosphere
•Source of oxygen
Possible Metabolism:
CO2 + 3 H2O + Sunlight → CH2O + 2 H2O2
2 H2O2 → 2 H2O + O2
Could Martian Extremophiles explain the Viking Results?
•Problem of Inorganic Explanations
a. The inorganic strong oxidant has never been identified
b. The sensitivity of the GC-MS has been put in doubt
c. No inorganic reactant or set of reactants can explain the Viking
responses
•Problem of Biological Explanation
a. There is no Earth organism that has
been found to mimic the Viking
responses
b. There are at least some chemical
reactions occurring
Can the H2O2-H2O Hypothesis Explain the Viking Results?
Lack of organic molecules
The organics have been oxidized to nonvolatile salts of benzenecarboxylic acids,
and perhaps oxalic and acetic acid (Benner et al. 2000).
Upon death of the organisms, the organics spontaneously are oxidized by H2O2
with no or very little organic residue. Non-biology bound organic molecules are
oxidized chemically and/or consumed by organisms. The release of 50-700 ppm
of CO2 by the GC-MS may indicate that oxidation of organic material took place
(Navarro-Gonzalez et al. 2006).
Can our Hypothesis Explain the Viking Results?
Lack of oxidant
There is some yet unidentified mechanism producing H2O2 or other oxidants. The
oxidant might be present in form of a compound that has no analog on Earth.
The H2O2 in the H2O2-H2O mixture is part of the biochemistry of the putative
Martian organisms. It would provide sufficient oxidizing potential to explain the
Viking results.
Can our Hypothesis Explain the Viking Results?
In the Gas Exchange Experiment Martian soil samples were incubated
with carbon dioxide, inert gases, and water (no radioactive labeling).
The instrument sampled the atmosphere of the incubation chamber for the
concentration of several gases including carbon dioxide, oxygen, nitrogen,
hydrogen, and methane.
The mission scientists believed that metabolizing organisms would either
consume or release at least one of the measured gases.
* In the humid mode, Martian surface samples were incubated in the presence of
carbon dioxide and water vapor (based on the assumption that substrates might not be
limiting on Mars, but that biological activity is dormant in these samples until enough
water becomes available in the environment).
*The second mode (heterotrophic mode) assumed the presence of heterotrophic
organisms on Mars, microbes that feed on organic molecules.
Can our Hypothesis Explain the Viking Results?
Release and Partial Resorption of O2, CO2, and N2 in the GEx experiment
Evolution of O2 on humidification was suggested to involve one or more reactive
species such as ozonides, superoxides, and peroxides. CO2 production in the
wet mode can be interpreted to be related to the oxidation of nutrient organic
compounds and N2 release can be interpreted to be related to an initial N2
desorption from soil by water vapor and subsequent resorption in liquid water
(Oyama et al. 1977).
The release of O2 (and possibly CO2 to a lesser degree) can be interpreted as the
result of an energy-producing metabolism. Upon humidification it could point
also to the decomposition of dying Martian biota, as could the increase of N2.
The decrease of N2 can be understood as biological fixation, a possibility also
entertained by Oyama et al. (1977).
Can our Hypothesis Explain the Viking Results?
The Pyrolytic Release Experiment looked for evidence of photosynthesis
by incubating Martian soil with light, water, and a carbon-containing
atmosphere of either carbon monoxide or carbon dioxide.
The gases were enriched with radioactive carbon-14. If photosynthetic
organisms were present on Mars, then these organisms would use
some of the carbon as biomass by carbon fixation.
After several days of incubation, the experiment removed the gases,
heated the remaining soil to a sterilizing temperature of 650 C,
and collected the product to count radioactivity. If gaseous carbon 14
had been converted to biomass by carbon fixation, it would be vaporized
during heating, and the radioactive counter would detect it.
Can our Hypothesis Explain the Viking Results?
Synthesis of organic material in PR experiment
No consistent explanation has been provided, but attempts to explain the
observations include instrument malfunction, incorporation of 14CO into carbon
suboxide polymer preformed on the Martian surface, and reduction of 14CO by
H2O2 in the surface material.
Some of the putative organisms were able to metabolize and synthesize organic
compounds before they died being overwhelmed by water.
Can our Hypothesis Explain the Viking Results?
In the Labeled Release Experiment a sample of the Martian soil was
inoculated with water and nutrient solution. The nutrients such as glucose
were manufactured with radioactive carbon 14.
If any heterotrophic organisms feeding on simple organic compounds
would be present such as animals and many non-photosynthetic bacteria
on Earth, they would consume the nutrients and release carbon 14
containing carbon dioxide, which would be easily detected by a
radioactivity counter.
Can our Hypothesis Explain the Viking Results?
Responses in the Labeled Release experiment
Laboratory test on Earth using inorganic oxidants and clay minerals simulated
many of the key findings, but not the decrease of responses after storage at
elevated temperatures (Klein 1999).
Limited metabolism (Levin and Straat 1977) before the organisms died due to
hyperhydration, osmotic pressure, and/or heat shock.
Earth Analogs for H2O2-using Martian Extremophiles?
•Microbes such as Neisseria sicca, Haemophilus segnis, Actinomyces viscosus utilize H2O2
•Microbes such as Streptococcus and Lactobacillus sp. produce H2O2
•The microbe Acetobacter peroxidans uses H2O2
in its metabolism (H2O2+H2 → 2H2O)
•The cell organelles involved in the H2O2centered oxidative processes, the peroxisomes,
are found in a great majority of eukaryotic cells
Streptococci and Staphylococci bacteria (organisms
that produce and utilize hydrogen peroxide
•The Bombadeer beetle has in its posterior chamber a mix of 25 % solution of H2O2
•Diverse physiological responses such as cell
proliferation, differentiation, and migration
are mediated by H2O2
•Biological redox reactions catalyzed by H2O2
involve the oxidation of cysteine residues on
proteins
www.abc.net.au/science/news/img/bombeet1.jpg
Bombardier beetle spraying an irritant 25 % solution of
hydrogen peroxide from its posterior chitinous chamber
The H2O2-H2O hypothesis for Martian life explains the Viking
Observations remarkably well
The hypothesis has consequences for future missions especially
the upcoming Mars Phoenix Mission
TEGA instrument
MECA instrument
2 Microscopes
Meteorological Station
TEGA is a combination high-temperature furnace and mass spectrometer
instrument that scientists will use to analyze Martian ice and soil samples.
MECA is a combination of several scientific instruments including a wet
chemistry laboratory, optical and atomic force microscopes, and a thermal
and electrical conductivity probe.
Planned Experiments:
Reproduce TEGA Measurements:
- monitor heating profile
- monitor release of oxygen
- measure oxygen isotope ratio continuously
Use - various H2O2 solutions
- JSC-1 Martian analog soil
- H2O2 solution with stabilizer (phenacetin)
- Fe and Ti oxides
12000
300
mW
86.7 oC/~ 160 J/g
10000
250
J/g
200
8000
150
6000
100
4000
50
2000
0
0
0
-2000
50
100
150
200
oC
35 % H2O2: 17.4 mg, heated at 10oC/min
250
300
350
Mars Science Laboratory
(to be launched 2009)
Cameras
Mast Camera
Mast Hand Lens Imager
Mars Descent Imager
Spectrometers
Alpha Particle X-ray Spectrometer
Chemistry & Camera
Chemistry&Mineralogy X-ray Diffraction/X-ray Fluorescence Instrument
Sample Analysis at Mars Instrument Suite with Gas Chromatograph, Mass
Spectrometer and Tunable Laser Spectrometer
Radiation Detectors
Radiation Assessment Detector
Dynamic of Albedo Neutron
to test Habitability
Environmental Sensors
Rover Environmental Monitoring Station
More info on H2O2-H2O hypothesis at Phys. Archives:
http://arxiv.org/ftp/physics/papers/0610/0610093.pdf
More background info: