**2. The intringuing results of the Vikings program**

The two Viking Landers in the '70 years made the first direct search of traces of present or past biological life on Mars [5] (**Figures 9** and **10**).

The results were so contentious that, after more than 45 years, no unambiguous interpretation was found.

The GCMS instruments (Gas Chromatograph-Mass Spectrometer) on board both Viking Landers [6, 7] were tasked with detecting organic compounds. GC–MS heated many samples of martian soil up to 500°C, but did not detect any trace of complex organic molecules, even if detected an amount of 0,1–1% of H2O and 50–500 ppm CO2 respectively (**Figure 11**) and the enigmatic release of about 15 ppb of CH3Cl (chloro-methane) and up to 20–30 ppb of CH2Cl2 (methylene chloride) (**Figure 12**).

At that time, the two light chloro-derivatives, being released together with some trace of a solvent of sure terrestrial origin such as Freon-E, were considered as a terrestrial contamination, ruling out the occurrence of any form of martian life [8]. In the meantime the H2O and CO2 release upon heating were explained as thermal decomposition of hydrous silicates and carbonates respectively.

#### **Figure 9.**

*Viking 1 on the surface of Cryse Planitia. The white arm in the center is a meteorological sensor; the arm that took samples is visible to the right.*

The measured ratio 37Cl/35/Cl = 0,319 similar to that one of the terrestrial chloride supported to this interpretation. However, Z.D. Sharp [8] found that the ratio 37Cl/35/Cl is quite constant all over the Solar System: actually its value is the same on the Earth, on the chloro-salts enclosed inside the carbonaceous chondrites and also on some Martian meteorites.

Moreover, a suitable inquiry proved that CH3Cl and/or CH2Cl2 never were used during the Viking assembly, so any trace on board was impossible [9].

We could re-discuss the conclusion taken from the Viking GC–MS results on the basis of a couple of reasons.

The first reason is that the Martian soil in any case should be enriched by the organic molecules (that is carbon-containing chemicals) that could be taken by comets and carbonaceous chondrites. The recent discovery of simple and polymeric organic substances inside many Martian meteorites could be an evidence in this regard [10].

weren't just earthly contamination. So microbiologists of the Carnegie Institution for Science's Geophysical Laboratory in Washington, D.C., looked in the most protected parts of Martian meteorites: microscopic mineral grains that had been securely locked in larger crystals for up to 4 billion years. Using a number of analytical techniques, they probed for organic matter through the encasing minerals. Organic chemicals were found in 10 of 11 once-molten meteorites examined, at an abundance of about 20 parts per million. Raman spectroscopy showed that

*The GC on board of the Viking Landers detected only enigmatic presence of chloro-methane and methylene*

*The main analytical results of the CGMS (GasChromatograph-Mass Spectrometer) on board of the Viking*

**Figure 11.**

**Figure 12.**

**143**

*chloride, at that time deemed terrestrial pollutants.*

*New Insights into the Search for Life on Mars DOI: http://dx.doi.org/10.5772/intechopen.97176*

*landers.*

Carbon-containing chemicals such as those that make up the stuff of life on Earth, had been found in rocks that were blasted off Mars millions of years ago by large asteroid impacts and fell to Earth as meteorites (at present about 300 Martian meteorites are known). But no one could be sure the organics in Martian meteorites *New Insights into the Search for Life on Mars DOI: http://dx.doi.org/10.5772/intechopen.97176*

#### **Figure 11.**

*The main analytical results of the CGMS (GasChromatograph-Mass Spectrometer) on board of the Viking landers.*

#### **Figure 12.**

The measured ratio 37Cl/35/Cl = 0,319 similar to that one of the terrestrial chloride supported to this interpretation. However, Z.D. Sharp [8] found that the ratio 37Cl/35/Cl is quite constant all over the Solar System: actually its value is the same on the Earth, on the chloro-salts enclosed inside the carbonaceous chondrites

*Viking 1 on the surface of Cryse Planitia. The white arm in the center is a meteorological sensor; the arm that*

during the Viking assembly, so any trace on board was impossible [9].

*May 18, 1979: water frost on Mars rocks and soil near the Viking 2 lander.*

Moreover, a suitable inquiry proved that CH3Cl and/or CH2Cl2 never were used

We could re-discuss the conclusion taken from the Viking GC–MS results on the

The first reason is that the Martian soil in any case should be enriched by the organic molecules (that is carbon-containing chemicals) that could be taken by comets and carbonaceous chondrites. The recent discovery of simple and polymeric organic substances inside many Martian meteorites could be an evidence in this

Carbon-containing chemicals such as those that make up the stuff of life on Earth, had been found in rocks that were blasted off Mars millions of years ago by large asteroid impacts and fell to Earth as meteorites (at present about 300 Martian meteorites are known). But no one could be sure the organics in Martian meteorites

and also on some Martian meteorites.

basis of a couple of reasons.

regard [10].

**142**

**Figure 10.**

**Figure 9.**

*took samples is visible to the right.*

*Solar System Planets and Exoplanets*

*The GC on board of the Viking Landers detected only enigmatic presence of chloro-methane and methylene chloride, at that time deemed terrestrial pollutants.*

weren't just earthly contamination. So microbiologists of the Carnegie Institution for Science's Geophysical Laboratory in Washington, D.C., looked in the most protected parts of Martian meteorites: microscopic mineral grains that had been securely locked in larger crystals for up to 4 billion years. Using a number of analytical techniques, they probed for organic matter through the encasing minerals. Organic chemicals were found in 10 of 11 once-molten meteorites examined, at an abundance of about 20 parts per million. Raman spectroscopy showed that

they include large, complex carbon compounds rich in benzene-like rings of carbon atoms, included polycyclic aromatic hydrocarbons (PAHs), typical of organic-rich meteorites such as carbonaceous chondrites. Given the way the organic matter was sealed in the rock, "it is carbon from Mars," not terrestrial contamination.

A special case remains that of ALH 84001, (**Figure 13**) a meteorite found in 1984 in the antartic region of Allan Hills. Ejection from Mars seems to have taken place about 16 million years ago. Arrival on Earth was about 13 000 years ago. Cracks in the rock appear to have filled with carbonate materials (implying groundwater was present) between 4 and 3.6 billion-years-ago. Evidence of polycyclic aromatic hydrocarbons (PAHs) have been identified with the levels increasing away from the surface. In the crack were also found deposits of iron as magnetite, that are claimed to be typical of bio-depositation on Earth [11].

In some SEM (Scanning Electronic Microscope) pictures taken inside the carbonate material, small ovoid and tubular structures were found [12], that might be interpreted as nanobacteria fossils (**Figure 14**), but also as sample preparation artifacts, being, at that time, unknown earthy life forms so small. The controversy has never ceased even if, some years after, living colonies of nano-organisms were found on Triassic and Jurassic sandstones from petroleum exploration wells offshore western Australia [13]. These living forms were up to 10 times smaller in diameter (10 nm) compared to 'normal' cellular structures.

The other reason for which the negative response of the two GCMS onboard the Viking Landers has to be rediscussed is linked to the fact that the Viking Labeled Release (LR) experiment, made at ambient temperature (10–15°C), gave a biological result substantially positive (Levin, 1976). LR it has been the only experiment with a clearly positive response, whereas the other two 'biological' experiments, i.e. the Gas Exchange (GEX) experiment [14] and the Pyrolytic Release (PR) experiment (Carbon Assimilation Experiment) [15] gave dubious results, suggesting a lack of biological reactions.

The LR experiment (**Figure 15**) was based on the well-known concept that all

In the Viking LR experiment, the Landers collected samples of Martian soil by means of their robotic arm, injected them with a drop of dilute nutrient solution containing alanine, formic acid, glycine, glycolic acid and lactic acid, and then monitored the air above the soil for signs of metabolic byproducts. Since the nutri-

terrestrial microorganisms metabolize the organic substances releasing CO2.

*The tube-like forms on this highly magnified SEM image of ALH84001 could be fossils of simple Martian*

*organisms that lived over 3.6 billion years or, simply artifacts of sample preparation.*

*New Insights into the Search for Life on Mars DOI: http://dx.doi.org/10.5772/intechopen.97176*

*A scheme of the LR experiment (Labeled Release) on board of Viking Landers.*

**Figure 15.**

**145**

**Figure 14.**

ents were tagged with radioactive carbon-14, if microorganisms in the soil

**Figure 13.** *Inclusions of carbonates inside the Martian meteorite 84001, found in Antartica in 1984.*

#### **Figure 14.**

they include large, complex carbon compounds rich in benzene-like rings of carbon atoms, included polycyclic aromatic hydrocarbons (PAHs), typical of organic-rich meteorites such as carbonaceous chondrites. Given the way the organic matter was

A special case remains that of ALH 84001, (**Figure 13**) a meteorite found in 1984 in the antartic region of Allan Hills. Ejection from Mars seems to have taken place about 16 million years ago. Arrival on Earth was about 13 000 years ago. Cracks in the rock appear to have filled with carbonate materials (implying groundwater was present) between 4 and 3.6 billion-years-ago. Evidence of polycyclic aromatic hydrocarbons (PAHs) have been identified with the levels increasing away from the surface. In the crack were also found deposits of iron as magnetite, that are claimed

In some SEM (Scanning Electronic Microscope) pictures taken inside the carbonate material, small ovoid and tubular structures were found [12], that might be interpreted as nanobacteria fossils (**Figure 14**), but also as sample preparation artifacts, being, at that time, unknown earthy life forms so small. The controversy has never ceased even if, some years after, living colonies of nano-organisms were found on Triassic and Jurassic sandstones from petroleum exploration wells offshore western Australia [13]. These living forms were up to 10 times smaller in

The other reason for which the negative response of the two GCMS onboard the Viking Landers has to be rediscussed is linked to the fact that the Viking Labeled Release (LR) experiment, made at ambient temperature (10–15°C), gave a biological result substantially positive (Levin, 1976). LR it has been the only experiment with a clearly positive response, whereas the other two 'biological' experiments, i.e. the Gas Exchange (GEX) experiment [14] and the Pyrolytic Release (PR) experiment (Carbon Assimilation Experiment) [15] gave dubious results, suggesting a

sealed in the rock, "it is carbon from Mars," not terrestrial contamination.

to be typical of bio-depositation on Earth [11].

*Solar System Planets and Exoplanets*

lack of biological reactions.

**Figure 13.**

**144**

diameter (10 nm) compared to 'normal' cellular structures.

*Inclusions of carbonates inside the Martian meteorite 84001, found in Antartica in 1984.*

*The tube-like forms on this highly magnified SEM image of ALH84001 could be fossils of simple Martian organisms that lived over 3.6 billion years or, simply artifacts of sample preparation.*

**Figure 15.** *A scheme of the LR experiment (Labeled Release) on board of Viking Landers.*

The LR experiment (**Figure 15**) was based on the well-known concept that all terrestrial microorganisms metabolize the organic substances releasing CO2.

In the Viking LR experiment, the Landers collected samples of Martian soil by means of their robotic arm, injected them with a drop of dilute nutrient solution containing alanine, formic acid, glycine, glycolic acid and lactic acid, and then monitored the air above the soil for signs of metabolic byproducts. Since the nutrients were tagged with radioactive carbon-14, if microorganisms in the soil

metabolized the nutrients, they would be expected to produce radioactive byproducts, such as radioactive carbon dioxide (14CO2). 14CO2 was indeed released when an aqueous solution of 14C labelled amminoacids was added, but a much lower amount (i.e by more than one order of magnitude) was released in the case of terrain samples sterilised at 160°C (**Figure 16**). To rule out the possibility that the strong ultraviolet radiation on Mars might be causing the positive results, the Landers collected also soil buried underneath a rock, which again tested positive. The control tests also worked, with the 160°C sterilization control yielding negative results [16].

In 2002 a possible circadian fashion (i.e. having the same periodicity of the Martian day) of 14CO2 release was found, which may be a typical biological signature [17]. A complex statistical analysis [18] reached the same conclusion.

In any case, it is important underline that the same Levin [19] observed that the release of radioactive CO2 could be due also to nonbiological reactants, a real possibility discussed also by Klein [20]. On this subject some lab tests were performed, assuming that the Fe superoxides are built up in the Martian soil by the strong UV radiation. This Fe superoxide could decompose (with the release of 14CO2) the carbon molecules of the LR nutrient solution directly [21] or through the formation of H2O2 [22]. In the presence of water, the superoxide ion reacts to produce Oxygen, perhydroxyl radical, and hydroxyl radical [23]: 2O2-+H2O ! O2+ HO2- + OH-.

This release of oxygen could decompose carbon molecules in the LR experiment, but could also explain the results of the Gas Exchange (GEX) esperiment. In GEX a water solution of many amino-acids and salts, was injected into a sample of martian soil, measuring by GC (Gas Cromatography) any gas emission such as the release of

H2, N2, 02, CH4, Kr, and CO2. Analyses showed that a 1 cm<sup>3</sup> of Martian soil sample produced up to 700 nM of Oxygen after 50 hours, an amount far superior to any

*June 25, 2008: a trench, called 'Dodo-Goldilocks,' showing lumps of water ice in this picture taken by the*

*Phoenix Lander. A big surprise was the discovery of perchlorates by the MECA instrument.*

A possible explanation of the intringuing results of the Viking analyses was found by the Wet Chemistry Laboratory on the Phoenix Mars Lander (**Figure 17**) that, in the summer of 2008, discovered up to 0,6% of Magnesium perchlorate - Mg (ClO4)2 in the North polar sands of Mars [24]. This salt is inert at low temperature, but at high temperature became a strong oxidant able to decompose all carbon

So, if we suppose that also the soil sampled by Viking were rich of perchlorates, the GCMS analysis, being performed at 500°C, possibly could results in a demolition of all organic molecules (biological or not) during the same analytical process. The assumption of the presence of perchlorates at the Viking landing sites might seem a little hasty, because perchlorate may form preferably at high latitudes [24], whereas the Viking 1 landing site was at equatorial latitudes, and the Viking 2 one at intermediate latitudes. However, we could not exclude this possibility, specially after the discovery of perchlorates inside the Gale Crater (Lat = 5,24° S), landing site of Curiosity [25] and the discovery of perchlorates also in some martian mete-

In the case of analyses performed at low temperature, perchlorates are totally inert and so a positive response, as observed by the Viking LR experiment, may

A further support to a biological interpretation of the Viking LR experiment was given by R. Navarro-Gonzales [27]. In summary, a sample of a Mars-like soil of the driest core of the Atacama desert in Northern Chile (The Yungay Area), containing

terrestrial test, and so not believed to be of biological origin.

*New Insights into the Search for Life on Mars DOI: http://dx.doi.org/10.5772/intechopen.97176*

really suggest the presence of organic substances.

**3. Viking and perchlorates**

compounds.

**Figure 17.**

orites [26].

**147**

**Figure 16.** *The Viking 1 result of Labeled Release experiment on active and sterilized Martian soil (see the text for details).*

#### **Figure 17.**

metabolized the nutrients, they would be expected to produce radioactive

results [16].

*Solar System Planets and Exoplanets*

HO2- + OH-.

**Figure 16.**

**146**

byproducts, such as radioactive carbon dioxide (14CO2). 14CO2 was indeed released when an aqueous solution of 14C labelled amminoacids was added, but a much lower amount (i.e by more than one order of magnitude) was released in the case of terrain samples sterilised at 160°C (**Figure 16**). To rule out the possibility that the strong ultraviolet radiation on Mars might be causing the positive results, the Landers collected also soil buried underneath a rock, which again tested positive. The control tests also worked, with the 160°C sterilization control yielding negative

In 2002 a possible circadian fashion (i.e. having the same periodicity of the Martian day) of 14CO2 release was found, which may be a typical biological signa-

In any case, it is important underline that the same Levin [19] observed that the

This release of oxygen could decompose carbon molecules in the LR experiment, but could also explain the results of the Gas Exchange (GEX) esperiment. In GEX a water solution of many amino-acids and salts, was injected into a sample of martian soil, measuring by GC (Gas Cromatography) any gas emission such as the release of

*The Viking 1 result of Labeled Release experiment on active and sterilized Martian soil (see the text for details).*

performed, assuming that the Fe superoxides are built up in the Martian soil by the strong UV radiation. This Fe superoxide could decompose (with the release of 14CO2) the carbon molecules of the LR nutrient solution directly [21] or through the formation of H2O2 [22]. In the presence of water, the superoxide ion reacts to produce Oxygen, perhydroxyl radical, and hydroxyl radical [23]: 2O2-+H2O ! O2+

ture [17]. A complex statistical analysis [18] reached the same conclusion.

release of radioactive CO2 could be due also to nonbiological reactants, a real possibility discussed also by Klein [20]. On this subject some lab tests were

> *June 25, 2008: a trench, called 'Dodo-Goldilocks,' showing lumps of water ice in this picture taken by the Phoenix Lander. A big surprise was the discovery of perchlorates by the MECA instrument.*

H2, N2, 02, CH4, Kr, and CO2. Analyses showed that a 1 cm<sup>3</sup> of Martian soil sample produced up to 700 nM of Oxygen after 50 hours, an amount far superior to any terrestrial test, and so not believed to be of biological origin.

A possible explanation of the intringuing results of the Viking analyses was found by the Wet Chemistry Laboratory on the Phoenix Mars Lander (**Figure 17**) that, in the summer of 2008, discovered up to 0,6% of Magnesium perchlorate - Mg (ClO4)2 in the North polar sands of Mars [24]. This salt is inert at low temperature, but at high temperature became a strong oxidant able to decompose all carbon compounds.

So, if we suppose that also the soil sampled by Viking were rich of perchlorates, the GCMS analysis, being performed at 500°C, possibly could results in a demolition of all organic molecules (biological or not) during the same analytical process. The assumption of the presence of perchlorates at the Viking landing sites might seem a little hasty, because perchlorate may form preferably at high latitudes [24], whereas the Viking 1 landing site was at equatorial latitudes, and the Viking 2 one at intermediate latitudes. However, we could not exclude this possibility, specially after the discovery of perchlorates inside the Gale Crater (Lat = 5,24° S), landing site of Curiosity [25] and the discovery of perchlorates also in some martian meteorites [26].

In the case of analyses performed at low temperature, perchlorates are totally inert and so a positive response, as observed by the Viking LR experiment, may really suggest the presence of organic substances.
