From the archive, originally posted by: [ spectre ]

http://www.airspacemag.com/issues/2007/april-may/exobiology.php

Looking for Life in All the Wrong Places
Weird space critters could be right beneath our planetary probes.

By Christen Brownlee

In 1976, scientists anxiously waited for the first data streaming back
from the Viking 1 and 2 landers, sent to search for signs of life on
Mars. The results were frustratingly inconclusive; for decades
researchers have been debating whether the Vikings detected life. Then
last January, two scientists presented a paper arguing that Mars may
indeed harbor life, but that the landers’ life-detecting equipment may
have killed it. They theorized that Martian microorganisms might
contain a mixture of water and hydrogen peroxide; if so, a Viking
experiment that doused Martian soil samples with water would have
drowned such life-forms.

The idea that Mars may harbor microbes containing hydrogen peroxide is
based in part on the presence of what appears to be that chemical on
Mars’ surface. The theory that microbes may be the origin of that
hydrogen peroxide is not well accepted-not yet, anyway. Most
researchers digging for extraterrestrial life are focused on forms
containing water and carbon-based molecules-the only forms found on
Earth. But a growing number of scientists are speculating that the
solar system may harbor what they call “weird life”-forms that contain
chemicals not traditionally associated with living organisms.

Thanks to the discovery of unusual creatures on Earth, such as
“extremophile” bacteria adapted to the extreme heat of underwater
thermal vents, most astrobiologists accept the possibility that life-
forms on other planets could have unfamiliar appearances or
adaptations. However, most still envision microbes filled with water
and carbon-based, or organic, molecules. It’s not unreasonable, says
David Grinspoon, astrobiology curator of the Denver Museum of Nature
and Science and formerly NASA’s principal investigator for exobiology
research. He points out that such compounds have been detected in
practically every corner of the universe that has been examined.

However, he and other researchers now suggest that an element other
than carbon may serve as the backbone for molecules essential to life-
forms on other planets. One proposed substitute is silicon, which
occupies a place on the periodic table directly under carbon. Vertical
rows on the table represent an element’s most basic behavior, so
carbon and silicon’s close positions suggest that one can be swapped
for another to form molecules with similar characteristics, says
Grinspoon.

Likewise, water isn’t the only solvent that life-forms could use to
enable necessary chemical reactions, says Dirk Schulze-Makuch of
Washington State University in Pullman, one of the scientists who
suggested that Viking may have killed Martian microbes. “Life and
environmental conditions on a planet are intrinsically related,” he
explains; he champions the idea of Martian organisms containing
hydrogen peroxide because it fits with the very cold and dry
conditions on that planet. Depending on its concentration in a
solution, hydrogen peroxide does not freeze until at -70 degrees
Fahrenheit, and when it does freeze, it does not form crystals, which
would destroy cells . And the compound absorbs even minute amounts of
water vapor from the atmosphere, which would benefit a water-dependent
organism in an extremely dry environment like Mars’.

Chemist Steven Benner of the University of Florida in
Gainesville suggests that the molecules that might make up weird life
and enable it to reproduce may differ from terrestrial proteins and
the nucleic acids DNA and RNA. By making some simple chemical tweaks
to these molecules, Benner and his colleagues have crafted new
variations that still work. “You can pick any one of these [molecules]
and easily walk away from its natural structure” while still
preserving functionality, he says. Benner and other researchers have
come up with a variety of new amino acids, the molecules that string
together to form proteins, that don’t exist in nature-at least not on
Earth. His group has also constructed new types of DNA with bases
different from the adenine, thymine, guanine, and cytosine that form
the rungs in the double helix on Earth.

The probes that search for life on other planets use technology that
can detect a range of chemicals beyond water and organic molecules.
The trick is to devise experimental protocols that do not destroy or
miss signs of possible life-a protocol, for example, that does not
douse samples with water if hydrogen peroxide is thought to be a
possible constituent. Recently, Rafael Navarro-Gonzalez of the
University of Mexico in Mexico City and others decided to check the
instrument that Viking used to test Martian soil for organic
molecules, a gas chromatograph-mass spectrometer (GCMS), which
identifies the atomic constituents of a substance. The scientists used
the instrument to test soils from areas on Earth that are similar to
Mars and known to have organic molecules, but it nonetheless gave
negative readings, again casting doubts on Viking’s results. Navarro-
Gonzalez says that the Mars Science Laboratory, presently planned to
launch in two years, will also use a GCMS, but it will follow a
different sample-treatment protocol, one that uses solvents, and is
more likely to reveal organic molecules, if any are present.

Another way to increase the chances for finding new life-forms is to
send probes to areas where they are more likely to be found-that is,
to search creatively. The Mars Science Laboratory will cover a much
greater area than Viking did. And NASA’s Phoenix probe, currently
scheduled to take off this August, will land in a subpolar area of
Mars that is especially cold and higher in atmospheric water vapor-
more favorable than the Viking sites for detecting life, especially
the hydrogen peroxide-containing organisms Schulze-Makuch envisions.
Phoenix will also carry non-chemical tests: two microscopes to study
samples for signs of life.

What’s the probability that life unlike anything we know is thriving
in extraterrestrial obscurity? “The chances that it might exist are
high, but the chances that we’re going to encounter it are probably
low,” says Benner. “Space is a big place.” To plan a search that has a
decent chance of finding whatever may be out there, we will need not
just technology but imagination.

“Fundamentally,” says David Grinspoon, “the universe is much more
creative than we are.”

http://researchnews.wsu.edu/physical/157.html

By Cherie Winner, WSU News Service, 509/335-4846, cwin@wsu.edu ,
Washington State Magazine

Contact: Dirk Schulze-Makuch, WSU School of Earth and Environmental
Sciences, 509/335-1180, dir@wsu.edu

New Analysis of Viking Mission Results Points to Possible Presence of
Life on Mars

PULLMAN, Wash. — We may already have ‘met’ Martian organisms,
according to a paper presented Sunday (Jan. 7) at the meeting of the
American Astronomical Society in Seattle.

Dirk Schulze-Makuch of Washington State University and Joop Houtkooper
of Justus-Liebig-University, Giessen, Germany, argue that even as new
missions to Mars seek evidence that the planet might once have
supported life, we already have data that may show life exists there
now-data from experiments done by the Viking Mars landers in the late
1970s.

“I think the Viking results have been a little bit neglected in the
last 10 years or more,” said Schulze-Makuch. “But actually, we got a
lot of data there.” He said recent findings about Earth organisms that
live in extreme environments and improvements in our understanding of
conditions on Mars give astrobiologists new ways of looking at the 30-
year-old data.

The researchers hypothesize that Mars is home to microbe-like
organisms that use a mixture of water and hydrogen peroxide as their
internal fluid. Such a mixture would provide at least three clear
benefits to organisms in the cold, dry Martian environment, said
Schulze-Makuch. Its freezing point is as low as -56.5 C (depending on
the concentration of H2O2); below that temperature it becomes firm but
does not form cell-destroying crystals, as water ice does; and H2O2 is
hygroscopic, which means it attracts water vapor from the atmosphere-a
valuable trait on a planet where liquid water is rare.

Schulze-Makuch said that despite hydrogen peroxide’s reputation as a
powerful disinfectant, the fluid is also compatible with biological
processes if it is accompanied by stabilizing compounds that protect
cells from its harmful effects. It performs useful functions inside
cells of many terrestrial organisms, including mammals. Some soil
microbes tolerate high levels of H2O2 in their surroundings, and the
species Acetobacter peroxidans uses hydrogen peroxide in its
metabolism.

Possibly the most vivid use of hydrogen peroxide by an Earth organism
is performed by the bombardier beetle (Brachinus), which produces a
solution of 25 percent hydrogen peroxide in water as a defensive
spray. The noxious liquid shoots from a special chamber at the
beetle’s rear end when the beetle is threatened.

He said scientists working on the Viking projects weren’t looking for
organisms that rely on hydrogen peroxide, because at the time nobody
was aware that such organisms could exist. The study of extremophiles,
organisms that thrive in conditions of extreme temperatures or
chemical environments, has just taken off since the 90s, well after
the Viking experiments were conducted.

The researchers argue that hydrogen peroxide-containing organisms
could have produced almost all of the results observed in the Viking
experiments.

Hydrogen peroxide is a powerful oxidant. When released from dying
cells, it would sharply lower the amount of organic material in their
surroundings. This would help explain why Viking’s gas chromatograph-
mass spectrometer detected no organic compounds on the surface of
Mars. This result has also been questioned recently by Rafael Navarro-
Gonzalez of the University of Mexico, who reported that similar
instruments and methodology are unable to detect organic compounds in
places on Earth, such as Antarctic dry valleys, where we know soil
microorganisms exist.

The Labeled Release experiment, in which samples of Martian soil (and
putative soil organisms) were exposed to water and a nutrient source
including radiolabeled carbon, showed rapid production of radiolabeled
CO2 which then leveled off. Schulze-Makuch said the initial increase
could have been due to metabolism by hydrogen peroxide-containing
organisms, and the leveling off could have been due to the organisms
dying from exposure to the experimental conditions. He said that point
has been argued for years by Gilbert Levin, who was a primary
investigator on the original Viking team. The new hypothesis explains
why the experimental conditions would have been fatal: microbes using
a water-hydrogen peroxide mixture would either “drown” or burst due to
water absorption, if suddenly exposed to liquid water.

The possibility that the tests killed the organisms they were looking
for is also consistent with the results of the Pyrolytic Release
experiment, in which radiolabeled CO2 was converted to organic
compounds by samples of Martian soil. Of the seven tests done, three
showed significant production of organic substances and one showed
much higher production. The variation could simply be due to patchy
distribution of microbes, said Schulze-Makuch. Perhaps most
interesting was that the sample with the lowest production-lower even
than the control-had been treated with liquid water.

The researchers acknowledge that their hypothesis requires further
exploration. “We might be mistaken,” said Schulze-Makuch. “But it’s a
consistent explanation that would explain the Viking results.”

He said the Phoenix mission to Mars, which is scheduled for launch in
August, 2007, offers a good chance to further explore their
hypothesis. Although the mission’s experiments were not designed with
peroxide -containing organisms in mind, Phoenix will land in a sub-
polar area, whose low temperatures and relatively high atmospheric
water vapor (from the nearby polar ice caps) should provide better
growing conditions for such microbes than the more “tropical” region
visited by Viking. Schulze-Makuch said the tests planned for the
mission, including the use of two microscopes to examine samples at
high magnification, could reveal whether we had the answer all along-
and if we’ve already introduced ourselves to our Martian neighbors in
a harsher way than we intended.

“If the hypothesis is true, it would mean that we killed the Martian
microbes during our first extraterrestrial contact, by drowning-due to
ignorance,” said Schulze-Makuch.

PHYSICAL SCIENCES / CHEMISTRY
The limitations on organic detection in Mars-like soils by thermal
volatilization-gas chromatography-MS and their implications for the
Viking results

Rafael Navarro-González*,, Karina F. Navarro*, José de la Rosa*,
Enrique Iñiguez*, Paola Molina*, Luis D. Miranda, Pedro Morales, Edith
Cienfuegos, Patrice Coll¶, François Raulin¶, Ricardo Amils||, and
Christopher P. McKay**

*Laboratorio de Química de Plasmas y Estudios Planetarios, Instituto
de Ciencias Nucleares, and Institutos de Química and Geología,
Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad
Universitaria, P.O. Box 70-543, 04510 México D.F., Mexico;
¶Laboratoire Interuniversitaire des Systèmes Atmosphériques, Unité
Mixte de Recherche 7583, Centre National de la Recherche Scientifique,
Université Paris 12-Val de Marne and Université Paris 7-Denis Diderot,
61 Avenue du Général de Gaulle 94010, Créteil Cedex, France; ||Centro
de Astrobiología, Consejo Superior de Investigaciones Científicas/
Instituto Nacional de Tecnica Aeroespacial, Torrejón de Ardoz, 28850
Madrid, Spain; and **Space Science Division, Ames Research Center,
National Aeronautics and Space Administration, Moffett Field, CA
94035-1000

Edited by Leslie Orgel, The Salk Institute for Biological Studies, La
Jolla, CA, and approved September 11, 2006 (received for review May
21, 2006)

Abstract
The failure of Viking Lander thermal volatilization (TV) (without or
with thermal degradation)-gas chromatography (GC)-MS experiments to
detect organics suggests chemical rather than biological
interpretations for the reactivity of the martian soil. Here, we
report that TV-GC-MS may be blind to low levels of organics on Mars. A
comparison between TV-GC-MS and total organics has been conducted for
a variety of Mars analog soils. In the Antarctic Dry Valleys and the
Atacama and Libyan Deserts we find 10-90 µg of refractory or graphitic
carbon per gram of soil, which would have been undetectable by the
Viking TV-GC-MS. In iron-containing soils (jarosites from Rio Tinto
and Panoche Valley) and the Mars simulant (palogonite), oxidation of
the organic material to carbon dioxide (CO2) by iron oxides and/or
their salts drastically attenuates the detection of organics. The
release of 50-700 ppm of CO2 by TV-GC-MS in the Viking analysis may
indicate that an oxidation of organic material took place. Therefore,
the martian surface could have several orders of magnitude more
organics than the stated Viking detection limit. Because of the
simplicity of sample handling, TV-GC-MS is still considered the
standard method for organic detection on future Mars missions. We
suggest that the design of future organic instruments for Mars should
include other methods to be able to detect extinct and/or extant
life.

——————————————————————————–
In 1976, the Viking Landers carried out an extensive set of biological
experiments to search for the presence of extant life on the surface
of Mars (1). In addition, a series of molecular analysis experiments
were conducted to search for the presence of organic compounds in the
martian soil (2). The biological tests consisted of three independent
experiments designed to detect Earth-like microorganisms in the top
few centimeters of the martian soil. The gas exchange experiment was
designed to determine whether martian life could metabolize and
exchange gaseous products in the presence of water vapor and in a
nutrient solution (3); the carbon assimilation experiment was based on
the assumption that martian life would have the capability to
incorporate radioactively labeled carbon dioxide and/or monoxide in
the presence of sunlight (i.e., photosynthesis) (4); and the labeled
release (LR) experiment sought to detect heterotrophic metabolism by
the release of radioactively labeled carbon initially incorporated
into organic compounds in a nutrient solution (5). At both Viking
landing sites the three biological experiments yielded positive
responses demonstrating the presence of a highly reactive soil.
Surprisingly, the LR experiment was suggestive of the possible
presence of biological activity in the martian soil. However, the most
puzzling result came from the molecular analysis experiments (2, 6)
performed in the martian soil: three sample analyses from surface
material from the Viking 1 and 2 sites and another from underneath a
rock from the Viking 2 site. In these experiments, soil was subjected
to thermal volatilization (TV)-gas chromatography (GC)-MS; this assay
consisted of a rapid heating of the soil to vaporize small molecules
and break down larger ones into smaller organic molecules, and the
resultant fragments were separated by GC and analyzed by MS.
Unexpectedly, in none of the experiments performed in both landing
sites could organic material be observed at detection limits generally
of the order of parts per billion for molecules larger than two carbon
atoms and of parts per million for some smaller molecules. The
evolution of CO2 and H2O, but not of other inorganic gases, was
observed upon heating the soil sample at 200°C, 350°C, and 500°C. One
important concern was whether the GC-MS instrument worked properly.
Fortunately, experimental data existed that demonstrated the proper
function of the instrument beyond any doubt (7). Traces of some
organic solvents that were used during the cleaning of the instruments
before they were incorporated into the Landers were detected in the
background, such as methyl chloride (15 parts per billion) and
perfluoroethers (1-50 parts per billion). These contaminants were
previously detected in preflight and cruise tests. Therefore, the
detection of these contaminants demonstrated that the instruments
worked well. Consequently, the presence of life in the martian soil
was in apparent contradiction with the results from the TV-GC-MS. The
lack of organics in the TV-GC-MS experiment was used as the most
compelling argument against the presence of extant life on the surface
of Mars.

The reactivity of the martian soil observed in the three biological
experiments (3-5) was subsequently explained by the presence of one or
more inorganic oxidants (e.g., superoxides, peroxides, and
peroxynitrates) at the parts per million level. The lack of organics
in the martian soil could also be explained by their oxidation to
carbon dioxide due to the presence of such oxidants and/or direct UV
radiation damage (8). There have been many suggestions regarding the
nature of the chemical reactivity of the martian soil, but no
laboratory experiment has yet been able to simulate both the gas
exchange (3) and the LR response (5). Instruments built to further
investigate the reactive nature of the martian soil [e.g., Mars
Oxidant Experiment for the ill-fated Russian Mars 1996 mission (8) and
Mars Oxidant Instrument for the European Space Agency ExoMars 2011
(9)] have not yet performed in situ experiments on Mars. Mars Oxidant
Instrument has been successfully tested in the Mars-like soils of the
Atacama Desert, where the oxidative nature of the soil is thought to
be triggered by strong acids (e.g., sulfuric and nitric acids)
depositing from the atmosphere (9).

A recent evaluation of the oxidative destruction mechanisms of
meteoritic organics on the surface of Mars suggests that the end
products are salts of aliphatic and aromatic polycarboxylic acids
(10). Such compounds are refractory organics (e.g., nonvolatile and
thermally stable) under the temperatures reached by the molecular
analysis experiments, and consequently they were missed by the Viking
TV-GC-MS (10). Alternatively, the absence of organics in the soil at
parts per billion levels does not preclude the presence of extant life
in the martian surface. Klein (11) pointed out that the Viking TV-GC-
MS would not detect Escherichia coli at levels of 106 per gram, which
has been confirmed by recent simulations (12).

The search for organics on Mars continues to be a key science goal for
future missions. Because of the simplicity of sample handling, TV-GC-
MS has still been considered the standard method for organic detection
on Mars; for instance, the ill-fated Beagle Lander carried a
combustion-MS, the Thermal Evolved Gas Analyzer instrument on the 2007
Phoenix mission is a thermal analysis and MS, the basic unit on the
Sample Analysis at Mars instrument selected for the upcoming 2009 Mars
Science Laboratory mission is a TV-GC-MS, and the Mars Organic
Detector unit for the 2011 European Space Agency ExoMars mission is a
TV coupled to capillary electrophoresis with a fluorescence detector.
We report here results of studies on several Mars analog soils in
which we compare the detection of organics by TV-GC-MS with total
organic analysis of the samples. We analyzed samples from the dry Mars-
like environments of the Dry Valleys in Antarctica (13) and the
Atacama Desert (14) in Chile and Peru, where environmental conditions
result in soils with low biological and organic content, and the
Libyan Desert in Egypt, which is part of the hyperarid Sahara. For
comparison, we also analyzed samples from wetter desert areas in the
Atacama and Mojave (in the southwestern U.S.) Deserts. We also
analyzed samples of jarosite-containing soils from the Rio Tinto in
Spain (15) and the Panoche Valley in California (16). These soils may
be analogs for the soils detected by the Mars exploration rover at the
Meridiani Planum site on Mars (17). In addition, we analyzed samples
of the National Aeronautics and Space Administration (NASA) Mars-1
martian soil simulant, which is derived from Hawaiian palagonite
(18).

Results and Discussion
All samples were analyzed for total organic matter, 13C, C/N ratio,
and their response in TV-GC-MS at 500°C (Viking protocol) and 750°C. A
summary of the results is listed in Table 1. The total organic matter
varied from 10 to 1,500 µg of C per gram of soil depending on the
environment. In all cases, the 13C values varied from -28.93 to –
20.06, a typical range for organic matter produced by C3
photosynthesis (19). Similarly, the C/N ratio for most samples is
typical of soil organic matter, 9-30 (20), except in Antarctica and La
Joya, where the ratio is 1. Surprisingly, the production of benzene, a
major organic compound resulting from TV-GC-MS was not correlated with
the amount of organic matter present originally in the soil. The
samples from the Dry Valleys of Antarctica (cold desert) and the arid
core regions of the Atacama (temperate desert) and the Libyan (hot
desert) contain very low levels of organics from 20 to 90 µg of C per
gram of soil. Antarctic sample 726 is of particular interest because
it was one of the prelaunch test samples for the Viking mission.
Interestingly, this was the only terrestrial sample testing by Viking
that did not contain organics detectable by the TV-GC-MS (21) yet did
give a positive result for the LR experiment (22). Subsequent analysis
has shown that this soil contains primarily metamorphosed coal,
kerogen (John R. Cronin, personal communication), and some low levels
of amino acids (23). We also found that TV-GC-MS of this sample, even
at temperatures higher than used by Viking (up to 750°C), yielded no
detectable organics. Other soils from the Antarctic show low total
organic levels that would also be undetectable by the Viking GC-MS.

Table 1. Total organic matter (TOM) present in different Mars
analogs soils and its detection by TV-GC-MS

The arid core regions of the Atacama Desert (Yungay, Chile) contain
Mars-like soils in the surface that have extremely low levels of
culturable bacteria, low organic concentrations (20-40 µg of C per
gram of soil), and the presence of a nonchirally specific oxidant
(14). The level of organics in these soils (see Table 1) would be
undetectable by TV-GC-MS at Viking temperatures but detectable at
higher temperatures (750°C). The organics present in these soils are
dominated by carboxylic acids and polycyclic aromatic hydrocarbons (as
determined in the extracts by the NMR and IR). Soils from the Libyan
and La Joya Deserts also contain very low levels of organics (20-70 µg
of C per gram of soil) that are undetectable by TV-GC-MS. Samples from
the wetter regions of the Atacama, which contain 400-440 µg of C per
gram of soil, are easily detectable by the Viking TV-GC-MS protocol
(see Table 1).

Soil samples from jarosite-containing soils also contain high levels
of organics (140-1,500 µg of C per gram of soil; see Table 1). In
contrast to the desert soils, this organic material was not readily
detectable by using the Viking TV-GC-MS protocol. TV at higher
temperatures (750°C) results in the detection of low levels of benzene
in comparison with samples from Las Juntas, where the levels of
organics are considerable lower. Fig. 1 shows that CO2 is expected to
be the major thermodynamically stable carbon species at 750°C when
organic matter is subjected to thermal treatment in the presence of
ferric sulfate and pyrite (<95%), two minerals present in the Rio
Tinto sediments; if pyrite is the main component (>95%) in the mineral
matrix, then carbon disulfide (CS2) is the major thermodynamically
stable carbon species. TV of the organic material (1,050-1,500 µg of C
per gram of soil) present in the Rio Tinto sediments produces carbon
dioxide as the most important carbon species. Fig. 2 demonstrates that
the oxidation of the organic matter to carbon dioxide is catalyzed by
the iron species present in the inorganic matrix and goes to
completion at temperatures 350°C in the TV chamber. If the organics
from the Rio Tinto sediment are extracted with organic solvents and
then the dry residue is subjected to TV-GC-MS in the absence of
mineral matrix, a variety of organics are detected (see Fig. 3).
Organic molecules larger than seven carbon atoms do not elute from the
chromatographic column. The organics detected in the extracts were
indigenous from the Rio Tinto sediment and not from contamination
during the processing of samples because blanks run in parallel
indicated the lack of organics in the blanks. We find that organic
detectability is reduced by a factor of >1,000 by TV compared with
extraction by organic solvents. The attenuation in detectability
between liquid extraction and TV for the Panoche soils has also been
reported elsewhere (24).

Fig. 1. Thermodynamically stable carbon species equilibrating at
750°C in an iron matrix containing various quantities of oxidized
[ferric sulfate: Fe2(SO4)3] and/or reduced (pyrite, FeS2) species.
Less than 1,000 µg of organic C per gram of soil was initially present
as stearic acid (C18H36O2). These iron species are present in the
sediments of the Rio Tinto with similar levels of organic matter.
Organic compounds are thermodynamically unstable in the presence of
iron, and the carbon species that are thermally stable contained only
one carbon atom at 750°C.

Fig. 2. Percent oxidation of the initial organic carbon to carbon
dioxide catalyzed by the iron species present in the Rio Tinto during
the TV step at various temperatures in an inert atmosphere. The total
organic matter content in the Rio Tinto sediment (RT04-01) was
determined to be 1,200 µg of C per gram of soil by titration of 1 g of
soil with permanganate and subsequent analysis by GC-MS. The degree of
oxidation of the organic matter during the TV step of 20-40 mg of
sediment was derived from the amount of carbon dioxide detected by TV-
GC-MS. Because of the inhomogeneous distribution of the organics in
the sediment and the small amount of sample used for TV-GC-MS, the
degree of oxidation of some samples exceeds 100%.

Fig. 3. Reconstructed ion gas chromatograms of the volatile
fraction released during flash thermal volatilization at 750°C of a 50-
mg sample of RT04-01 before (a) and after (b) removal of the mineral
matrix. Peaks: 1, nitrogen; 2, carbon dioxide; 3, water; 4, methanol;
5, 1-propene; 6, sulfur dioxide; 7, 1,3-butadiene; 8, acetonitrile; 9,
2-propanone; 10, 1-pentene; 11, cyclopentene; 12, benzene; 13,
toluene.

Another iron-containing soil used as a Mars analog is the NASA Mars-1
martian soil simulant. The main component of this soil is weathered
basalt known as palagonite from a cinder cone south of Mauna Kea,
Hawaii. This volcanic soil has visible and near-IR spectral properties
that are very similar to martian surface materials as determined by
remote sensing (18). In addition, the major inorganic elements in the
soil roughly match the bulk composition of the soils at the Viking
landing sites (18). Because this soil is from Hawaii, it is not
surprising that it contains organic material at 1,200-1,400 µg of C
per gram of soil (Table 1) and microorganisms. Like the jarosite-
containing soils, no organics are detected with the Viking TV-GC-MS
protocol. If the endogenous nonvolatile organics present in the Mars-1
soil simulant are thoroughly removed by organic solvent extraction,
and then the dried soil is doped with stearic acid in different
concentrations, the detectability of this organic material is greatly
reduced when processed by TV due to the catalytic oxidation of the
organics by the iron oxides present in the soil (see Fig. 4).

Fig. 4. Reconstructed ion gas chromatograms of the volatile
fraction released during flash TV. The sample consisted of stearic
acid doped in an organic-free NASA Mars-1 martian soil simulant at
750°C in an inert atmosphere composed of helium: 50 (a), 10 (b), 5
(c), 1 (d), 0.5 (e), and 0.1 (f) mg of C per gram of simulant. Peaks:
1, formic acid; 2, 1-butene; 3, 2-pentene; 4, benzene; 5,
methylbenzene; 6, ethylbenzene; 7, methylethylbenzene. For simplicity,
only the major peaks are labeled in the chromatograms. The NASA Mars-1
martian soil simulant was thoroughly washed with methylene chloride/
methanol (2:1) over 24 h to remove the organics in a Soxhlet
apparatus. The gas chromatograms of this martian soil simulant after
solvent cleaning did not show organics by TV-GC-MS.

The results in Table 1 show two limitations of the Viking TV-GC-MS for
the detection of organic material. First, when organics are present as
low-level refractory substances, the temperatures reached by Viking
(up to 500°C) may be inadequate to release the organics. This
limitation of the Viking instrumentation was recognized but
unavoidable (2), and its implications for detection of organics have
been explored (10, 14). There is a second effect seen in the data in
Table 1 that appears to be due to an interaction of iron in the soil
with the organics during TV. The results of the jarosite and
palagonite soils suggest that during TV there is an oxidation reaction
of the organics catalyzed by the iron in the sample. To investigate
this effect we have constructed a chemical model and an associated set
of experimental simulations to determine the effect of iron compounds
on 1,000 µg of C per gram of soil from stearic acid on a silica matrix
during thermal heating. Fig. 5 shows the results of both the
theoretical model and the experimental simulations. The thermochemical
model predicts that the thermally stable oxidized carbon species at
750°C are CO2 and CO. The lower and upper dotted lines indicate the
predicted conversion to CO2 and the sum of CO2 and CO, respectively.
In the experimental simulations, CO was not detected by TV-GC-MS
possibly because it was readily oxidized to CO2 by the water molecules
absorbed in the mineral matrix from the ambient humidity. However, the
oxidation of stearic acid to CO2 falls within the predicted range,
indicating that the organics are readily oxidized by TV-GC-MS, with
samples containing >0.01% iron in the form of oxides or sulfate salts.
A similar result is obtained at 500°C. If the samples contain higher
levels of stearic acid (1,000 µg of C per gram of soil), then the
oxidation of the organics does not go to completion in the TV step,
and several organic fragments are detected by TV-GC-MS. Therefore, the
degree of attenuation of organic detection by iron compounds in the
soil is not linearly dependent. Consequently TV-GC-MS per se is not an
adequate tool for the study of organics in soils with low levels of
organics and high iron content, as is expected on Mars. If the organic
material is separated from the inorganic matrix by water or organic
solvent extraction and then the dried residue is subjected to TV-GC-
MS, a variety of organic compounds are detected (see Fig. 3).

Fig. 5. Oxidation of a 1,000 µg of C from stearic acid with iron
species present in silica by flash TV at 750°C in an inert atmosphere
composed of helium. Symbols correspond to experimental data, and
dotted lines are predicted. Open circles and triangles are Fe2O3 and
Fe2(SO4)3, respectively. Solid symbols indicate values of oxidation
with sulfuric acid.

Because carbon dioxide was replaced by hydrogen in some Viking TV-GC-
MS experiments, we have investigated whether hydrogen would have
counterbalanced the oxidizing power of the iron species present in the
martian soil. The iron content in the soil of Mars was determined by x-
ray fluorescence spectroscopy, and based on this it was inferred that
Fe2O3 composes 19% of the soil at both Viking landing sites (25). Our
thermodynamic analysis shows that at the Viking temperatures (200-
500°C), the reduction of hematite (Fe2O3) by hydrogen is
thermodynamically favored; however, in the gas phase the dissociation
of molecular hydrogen to atomic hydrogen (a necessary step to cause
the reaction) is extremely slow at temperatures of <1,500°C (26).
Hematite is known to catalyze its own reduction to wüstite (FeO) via
magnetite (Fe3O4) in the presence of hydrogen according to the
following reactions:

and

This process takes place at temperatures of <1,000°C (27-29), but the
reduction is kinetically controlled by hydrogen pressure (29) and
temperature (27, 28, 30). We have experimentally studied the oxidation
of hydrogen to water by the hematite present in the NASA Mars-1
martian soil simulant in the temperature range from 200°C to 1,200°C.
The hydrogen pressure in the TV chamber was 6.4 atm (1 atm = 101.3
kPa), 13 times higher than that used in the Viking experiments (0.5
atm) (2). Fig. 6a shows the evolution of water vapor from heating the
NASA Mars-1 martian soil simulant in helium and hydrogen atmospheres
by TV-MS. At temperatures between 200°C and 650°C, there is a broad
peak in both experiments that originates from the dehydration of the
mineral phases of the soil simulant. However, at temperatures of

>650°C there is a significant enhancement in the production of water

in the presence of hydrogen, reaching a maximum at 930°C. This water
originates from the oxidation of hydrogen catalyzed by hematite. This
result is consistent with previous studies on the reduction of pure
hematite, where the highest reduction rates occur at temperatures of
910°C (28).

Fig. 6. MS ion current curves for water vapor as a function of
temperature for the NASA Mars-1 martian soil simulant (a) and jarosite
from the Panoche Valley (b). Solid lines show values for experiments
run in a helium atmosphere, and dotted lines show values for
experiments run in a hydrogen atmosphere.

We also studied the oxidation of hydrogen to water by jarosite. Our
thermodynamic analysis shows that, at the Viking temperatures (200-
500°C), the oxidation of hydrogen to water by jarosite is
thermodynamically favored. Fig. 6b shows the evolution of water vapor
from heating jarosite from the Panoche Valley in helium and hydrogen
atmospheres by TV-MS. In both experiments, there are three peaks at
305°C, 405°C, and 790°C that originate by the stepwise dehydration of
jarosite, KFe3(SO4)2(OH)6. Each step involves the loss of two hydroxyl
units, resulting in the formation of an oxide and the evolution of a
water molecule (31). In the presence of hydrogen, there are two
additional water peaks caused by the reduction of jarosite centered at
540°C and 940°C, respectively. The first reduction corresponds to the
decomposition of jarosite into magnetite, iron(II) sulfide, potassium
sulfate, and water vapor, according to the following reaction:

The second reduction is due to reaction of magnetite with hydrogen
according to Eq. 2.

The above experiments clearly demonstrate that shifting from carbon
dioxide to hydrogen atmospheres in the Viking TV-GC-MS did not
overcome the oxidizing power of the Fe2O3 present in the martian soil
at both Viking landing sites. For jarosite-rich soils, such as those
found in the Meridiani Planum site, only a slight neutralization
effect occurs as a result of heating to 500°C in the presence of
hydrogen.

Our results influence the interpretation of the Viking TV-GC-MS data.
The fact that no organic molecules were released by this analytical
treatment during the analysis of the Mars soils does not demonstrate
that there were no organic materials on the surface of Mars because it
is feasible that they were too refractory to be released at the
temperatures achieved or were oxidized during the TV step by the iron
present in the soil. The release of 50-700 ppm of CO2 by TV from 200°C
to 500°C in the Viking analysis (2) may indicate that an oxidation of
organic material took placed. The water that evolved in the
volatilization experiments (0.01-1.0%) could be associated with the
oxidation of hydrogen present in the organic matter by the iron oxides
as well as water present in the soil. The detection of CO2 evolving
from the heating of martian samples in the TV-GC-MS experiments
required a major change in the experimental procedure of the
instrument. In all samples analyzed by TV-GC-MS experiments on the
Viking 1 Lander and in two of nine experiments with two samples of the
Viking 2 Lander, the martian soil was heated in a 13CO2 atmosphere.
H2, which was the carrier gas for the gas chromatograph, was not used
to avoid the possible catalytic or thermally induced reduction of
organic material possibly present in the sample (2). However, in an
effort to lower the detection limit for the most volatile components,
H2 was used in two sample experiments (2). The source of the H2 was
the gas chromatograph carrier gas, and the net hydrogen pressure in
the sample oven was 0.5 atm. Our thermodynamic analysis shows that, at
the Viking temperatures (200-500°C), the reduction of iron oxides by
hydrogen is thermodynamically favored; however, our experimental data
indicate that the reaction is kinetically controlled and does not
occur at temperatures of <650°C. Therefore, it seems unlikely that
hydrogen could have neutralized the oxidizing power of the Fe2O3
present in the martian soil. The CO2 released from the thermal
treatment of the martian soil could have also originated from an
inorganic source, such as carbonates (2); however, carbonate minerals
do not seem to be important in the martian environment (32). Thermal
IR spectra of the martian surface indicate the presence of small
concentrations (2-5 wt %) of carbonates, specially dominated by
magnesite, MgCO3 (33). Because magnesite starts to decompose into
magnesium oxide (MgO) and CO2 at 490°C (34) and considering that the
amount of CO2 released in the martian soil did not change from 350°C
to 500°C (2), we can conclude that the effect of magnesite in the
martian soil at Viking Landing Site 2 was negligible. Certainly most
of the CO2 and H2O detected by the Viking TV-GC-MS was derived from
desorption from the soil as suggested (2). We are demonstrating that
some fraction could have been derived from oxidation of organics.
Therefore, the question of whether organic compounds exist on the
surface of the planet Mars was not conclusively answered by the
organic analysis experiment carried out by the Viking Landers.
Furthermore, it is important that future missions to Mars include
other analytical methods to search for extinct and/or extant life in
the martian soil. The Thermal Evolved Gas Analyzer instrument on
NASA’s 2007 Mars Scott Phoenix mission is a TV-MS for the analysis of
water, carbon dioxide, and volatile organics (35). The Sample Analysis
at Mars Instrument Suite for the upcoming NASA 2009 Mars Science
Laboratory mission will include laser desorption MS for analysis of
insoluble refractory organics, solvent extraction followed by chemical
derivatization coupled to GC-MS, and TV-GC-MS for the analysis of
soluble and insoluble organics, respectively. The Mars Organic
Detector for the European Space Agency ExoMars mission scheduled for
launch in 2011 or 2013 will include a TV chamber connected to a cold
finger for the sublimation of amino acids and polycyclic aromatic
hydrocarbons, which will then be analyzed by capillary electrophoresis
using a fluorescence detector (36).

For further detail, see Supporting Materials and Methods, which is
published as supporting information on the PNAS web site. Total
organic matter was determined by titration with the oxidation of
permanganate and by its oxidation to carbon dioxide followed by GC
(model no. HP-5890; Hewlett-Packard, Palo Alto, CA) MS (model no.
HP-5989B; Hewlett-Packard) analysis. Elemental analysis was done with
a model EA1108 analyzer (Fisions, Loughborough, U.K.) at 1,200°C. TV-
GC-MS was performed with a coil filament-type pyrolyzer (Pyroprobe
2000; CDS Analytical, Inc., Oxford, PA) coupled to GC-MS (model nos.
HP-5890 and HP-5989B). Organics from the soil were extracted by a
Soxhlet system with methylene chloride/methanol (2:1) over 8 h, and
the dried residue was analyzed by 1H NMR (Eclipse 300-MHz
spectrometer; JEOL, Ltd., Tokyo, Japan), Fourier transform IR
spectroscopy (Tensor 27 spectrometer; Bruker, Billerica, MA), and by
TV-GC-MS with a ribbon element probe for direct deposition. The carbon
isotope analysis was performed with MS (Delta Plus XL analyzer;
Finnigan, Breman, Germany) equipped with a Flash 1112EA elemental
analyzer. Hydrogen oxidation of soil analogs was carried out by
replacing helium with hydrogen in the oven of the TV-MS analysis.
Thermochemical modeling was carried out with the FactSage software
package.

{To whom correspondence should be addressed. E-mail:
nava@nucleares.unam.mx}

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