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

http://www.usask.ca/communications/ocn/

http://www.abc.net.au/science/news/img/environment/extremophile101103.jpg

http://www.homepage.montana.edu/~lkbonney/IMAGES/Antarctic

On Earth, Extremophilic Life is everywhere

Antarctic Dry Valleys

White Sands

SodaLake

Volcanoes

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 consistentwith 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 Recent evidence supportssupports SCENARIOS 1 SCENARIOS 1

or 2or 21.Models: Lengthy cold, dry periods with impact- induced precipitation (Segura et al., 2002)

2. Surface water in the past and abundant near-subsurface 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 Most recent evidence supports SCENARIOS 1 or 2SCENARIOS 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)

From: UPDATING THE EVIDENCE FOR OCEANS ON EARLY MARS, Fairén et al. (LPSC, 2005)

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

Most recent evidence Most recent evidence supports supports SCENARIOS 1 or SCENARIOS 1 or

225. Jarosite, indicative of mildly acidic and moderately oxidizing water

“Blueberry bowl” at Meridiani PlanumThe stability field for Jarosite

Expectations: Expectations: Scenarios 1 or 2Scenarios 1 or 2 Single or simple cells in Single or simple cells in

ground water. ground water. Dormant forms (spores) at or Dormant forms (spores) at or near surfacenear surface

Energy sources likely Energy sources likely chemoautotrophy, or a form chemoautotrophy, or a form unfamiliar to life on Earth.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)

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)

Experimental Testing

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 (200nm-400nm)

8.4 to 67 Wm-2 37 Wm-2

Microbial viability in soil and water samples after treatment to environmental stresses

Treatment E. coli D. radioduransMedium Soil (depth and

viabilities [%])Sea Water

Soil (depth and viabilities [%]) Sea Water

Sub-Zero Temperature

0 cm 1.5 % 20% 0 cm 56 % 57%

1 cm 2.5 % 1 cm 71 %

5 cm 2 % 5 cm 64 %

Low-Pressure 0 cm 0 % 73% 0 cm 73 % 100%

1 cm 0 % 1 cm 72 %

5 cm 0 % 5 cm 47 %

Sub-Zero Temperature +

Low-Pressure

0 cm 59 % 20% 0 cm 92 % 100%

1 cm 83 % 1 cm 98 %

5 cm 60 % 5 cm 89 %

UV 0 cm 1.5 % 34% 0 cm 27 % 59%

1 cm 15 % 1 cm 67 %

5 cm 50 % 5 cm 97 %

UV+ Low-Pressure

0 cm 0 % 69% 0 cm 0.3 % 91%

1 cm 0 % 1 cm 0.1 %

5 cm 52 % 5 cm 56 %

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

1D. radiodurans in soil

71 % 72 %

98 %

67 %

0.1 %

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 in 1 cm Deep Soil

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

1E. coli in soil

Sub-zero temperature

Low-Pressure

Sub-zero temperatureand low-pressure

UV irradiation

UV irradiation and low-pressure

2.5 %

83 %

15 %

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.

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

A History of Life on Mars?

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)

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 oxygenPossible Metabolism:

CO2 + 3 H2O + Sunlight → CH2O + 2 H2O2

2 H2O2 → 2 H2O + O2

Extremophiles on Mars?

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. Theoxidant 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.

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 heterotrophicorganisms on Mars, microbes that feed on organic molecules.

Can our Hypothesis Explain the Viking Results?

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).

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?

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.

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?

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 H2O2- centered 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 VikingObservations remarkably well

The hypothesis has consequences for future missions especiallythe 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.

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

Planned Experiments:

-2000

0

2000

4000

6000

8000

10000

12000

0 50 100 150 200 250 300 350

86.7 oC/~ 160 J/g

oC

35 % H2O2: 17.4 mg, heated at 10oC/min

300

250

200

150

100

50

0

mW

J/g

Cameras Mast CameraMast Hand Lens ImagerMars Descent Imager

Spectrometers Alpha Particle X-ray SpectrometerChemistry & CameraChemistry&Mineralogy X-ray Diffraction/X-ray Fluorescence InstrumentSample Analysis at Mars Instrument Suite with Gas Chromatograph, Mass Spectrometer and Tunable Laser Spectrometer

Radiation Detectors Radiation Assessment DetectorDynamic of Albedo Neutron

Environmental Sensors Rover Environmental Monitoring Station

Mars Science Laboratory(to be launched 2009)

to test Habitability

More info on H2O2-H2O hypothesis at Phys. Archives:

http://arxiv.org/ftp/physics/papers/0610/0610093.pdf

More background info: