1. Introduction

The Phoenix Lander landed on Mars on 25 May 2008. During five months, the instruments on board explored the geology and climate of subpolar Mars and looked for the presence and dynamics of water ice, and explored if life could have ever arisen on Mars. Although the Phoenix mission was not a life detection mission per se, it looked for the presence of organic compounds and other evidence to support or discredit the notion of past or present life.

Therefore, the Phoenix Lander can be regarded as a successor mission to the Viking Landers of 30 years earlier. In 1976, these two spacecraft were the first two to successfully land on Mars. Their main purpose was to answer the question whether extant life on Mars could be detected. At that time, knowledge of the physical conditions on the Martian surface was scant, so that the designers of the Viking biology experiments had to work from what was known about life in dry and cold environments on Earth. Not surprisingly, the results of the Viking biology experiments were puzzling. No complete consensus about the meaning of the Viking results was reached among the researchers involved in the design of the experiments, although the majority opinion was that one or more highly oxidizing compounds in the Martian regolith were mainly responsible for the results (Klein, 1978, 1999). A different opinion was voiced by the researchers reponsible for the Labeled Release experiment (Levin and Straat, 1979, 1981; Levin, 1997, 2010), who favored a pro-life interpretation.

A number of hypotheses have been put forward about the composition of the Martian soil and its apparent oxidizing properties: Evolution of O₂ upon humidification was suggested to result from one or more reactive chemical species such as ozonides, superoxides, and peroxides (Oyama and Berdahl, 1977). Proposed inorganic oxidants include metal oxides such as Fe- and Ti-oxides (Quinn and Zent, 1999) and superoxide ions or radicals (Yen et al. 2000; Zent et al. 2006). Yet, a conclusive identification of the oxidizing compounds and their productive process is still lacking (Atreya et al. 2006; Bullock et al. 1994; Crawford et al. 2003; Horowitz, 1986; Levin, 2001; Ponnamperuma et al. 1977; Quinn and Zent, 1999; Tsapin et al. 2000; Yen et al. 2000).

Given the inconclusiveness of a purely chemical explanation, Houtkooper and Schulze-Makuch (2007) put forward the hydrogen peroxide-water hypothesis, entertaining the possibility that extant life on Mars is based on a mixture of hydrogen peroxide and water in their intracellular fluid. This interpretation would have a number of attractive properties and provide a viable alternative to the chemical explanation of many of the Viking results. The recent findings by the Phoenix Lander, especially that the Martian soil contains substantial amounts of perchlorate salts, shed new light on the possibilities for extant Martian life.

2. The H₂O₂-H₂O Hypothesis

The H₂O₂-H₂O hypothesis (Houtkooper and Schulze-Makuch, 2007) turns out to fit the results of the Viking Biology experiments remarkably well, in contrast to a purely chemical explanation (Schulze-Makuch et al. 2009). Particularly, the evolution of oxygen upon moisturizing the soil in the Gas Exchange (GEx) experiment (Oyama and Berdahl, 1977) and the lack of detectable organics (Biemann et al. 1977) are to be expected if the Martian soil contains organisms of which the intracellular fluid consists of a mixture of hydrogen peroxide and water, instead of only (salty) water as is the case in terrestrial life.

Why would organisms evolve to include hydrogen peroxide in their intracellular fluid? The physical and chemical properties of H₂O₂-H₂O mixtures would be beneficial for life to cope with a harsh Martian near-surface environment. Any organisms on Mars would have been subject to evolutionary pressures, as the Martian surface became colder, dryer, and subject to higher radiation levels, to develop an adaptation to H₂O₂, similar to how life on Earth adapted to high concentration of free molecular oxygen about 2.5 billion year ago.

The Martian atmosphere contains small concentrations of H₂O₂ (Encrenaz et al. 2004; 2008). These are in the 10s of ppb range, and may be created by triboelectricity in dust devils (Atreya et al. 2006), but are not in sufficient quantity to explain the Viking results.

H₂O₂ shows superior properties as an antifreeze, a source of energy and oxygen, and as a scavenger of water vapor.

The H₂O₂-H₂O eutectic mixture (61.2% H₂O₂) freezes at -56.5^oC and tends to supercool below that temperature (Giguère and Secco, 1954). The mixture is hygroscopic because of its low water activity and is also a source of oxygen and energy. The biological compatibility of hydrogen peroxide has to be considered even though H₂O₂ is mostly known as a desinfectant. H₂O₂ is used for a variety of purposes, such as a messenger molecule in the biochemistry of Earth's organisms (Rhee, 2006). H₂O₂ is produced in the peroxisomes of most eukaryotic cells (De Duve, 1969) and the microbe Acetobacter peroxidans uses it in its metabolism (Tanenbaum, 1956). While H₂O₂ is often employed in defense mechanisms, most organisms which come into contact with H₂O₂ protect themselves with scavenging enzymes such as catalase. The most eye-catching use of H₂O₂ is in insects such as the bombardier beetle Brachinus crepitans, which produces a 25% solution of H₂O₂ to produce steam explosions in a chitinous chamber in its posterior, which can be directed at a pursuing predator (Eisner, 2003).

Ambient conditions on Mars are often deemed adverse to the existence of life. Mars is considered to be too cold and too dry, the soil too oxidizing, the atmosphere too thin, the solar radiation containing too much UV and there is much hard radiation from solar and cosmic origin because of the lack of a magnetosphere. However, some Earth organisms show a remarkable resistance to UV and to various types of irradiation, and adaptations to oxidative stresses and irradiation involve many of the same resistance responses in microorganisms.

The physical properties of mixtures of H₂O₂ and H₂O mean that the low temperatures and the low water content of the atmosphere are conditions that allow the organisms with such mixtures in their intracellular fluid not to freeze and to be able to scavenge water vapor from the atmosphere. While H₂O₂-H₂O based life seems to be possible under Martian ambient conditions, H₂O₂ as a cellular component has its disadvantages as well: H₂O₂ is a reactive compound, although less so at lower temperatures, and the organisms may need an active stabilizing mechanism to keep the organic cellular components intact. The organisms would also need a mechanism to produce their own H₂O₂. For this, the H₂O₂-producing bacterium Lactococcus lactis and Brachinus crepitans provide an analog.

The H₂O₂-H₂O hypothesis implies that the addition of water vapor at a relatively high temperature could only be withstood by the organisms for a short time, as they perished due to hyperhydration (Houtkooper and Schulze-Makuch, 2007). Thus, the evolution of oxygen in the GEx experiment is explained by the high oxidative content of the organisms. The lack of detected organics by the Viking GCMS could be the result of autooxidation of the organisms as these were gradually heated. Especially heating together with perchlorate salts would result in combustion to CO₂ and H₂O of any organics present. And indeed, the production of CO₂ was detected by the Viking GCMS (Navarro-Gonzalez et al. 2006).

3. Results from the Phoenix Lander

Major results of the Phoenix Lander mission are the presence of water ice (Smith et al. 2009) with an ice table being within a few cm of the Martian surface in the subpolar region and the presence of calcium carbonate (Boynton et al. 2009). In addition, the Phoenix mission characterized the soluble chemistry of the Martian soil, revealing the alkalinity of the soil with a pH of 7.7 +/- 0.5 and the detection of surprisingly large amounts (0.4-0.6 wt %) of perchlorate salts in the Martian subpolar soil (Hecht et al. 2009). The presence of perchlorates was primarily detected by the Wet Chemistry Laboratory of the MECA package on board the Phoenix Lander (Kounaves et al. 2009), and was also confirmed by the Thermal and Evolved-Gas Analyzer (TEGA).

The Wet Chemistry Laboratory (WCL) detected the perchlorates by using an "ionspecific" electrode for different anions, apart from ClO₄-, also for NO₃^-. However, the presence of a significant concentration of ClO₄- overwhelms the response for NO₃^-, so that the biologically significant nitrate anion could not be detected (West et al. 1999; Kounaves et al. 2009). The detection of perchlorates may also shed new light on the interpretation of the Viking biology experiments.

4. The Role of Perchlorates: Anti-freeze and Water Activity

As an analog to Mars, perchlorate salts have also been detected on Earth in hyperarid soils (Fig. 1). For instance, Ericksen found that the soils of the Atacama desert contain 0.03-0.6 wt% perchlorate (Ericksen, 1981). Perchlorate is thought to be produced by atmospheric electricity or photooxidation (Miller et al. 2006). For Mars, the scarcity of atmospheric oxygen could be a problem.

An interesting property perchlorate salts share with hydrogen peroxide is their effectiveness as anti-freeze. Whereas the eutectic for H₂O-H₂O₂ freezes at -56^oC, the water- magnesium perchlorate eutectic temperature is as low as -70^oC. Therefore, as Mg and Na are prevalent cations in the Martian soil, their perchlorate salts might well be employed as an antifreeze by hypothetical Martian microbes. Although perchlorates are known as powerful oxidants, there is no perchlorate known to release oxygen upon moisturizing at the Viking experimental temperature of about 10^oC as was observed during the GEx experiments of the Viking mission. Thus, we still consider H₂O₂ a more likely choice for the agent causing the rapid evolution of O₂ in the GEx experiment. In addition, H₂O₂ has the advantage that it can be produced from atmospheric constituents only. What could be another role of perchlorates in addition to allow liquidity at lower temperatures? Like some terrestrial plants living in extremely arid conditions, Martian organisms may employ perchlorate salts to scavenge water from the atmosphere. Both H₂O₂-H₂O mixtures and concentrated solutions of perchlorate salts have very low water activity (down to approximately 0.5 at the respective eutectics). The presence of perchlorate salts on their surface would enable organisms to pump water into their cells at relatively modest energy cost.

5. Discussion

Xerophilic organisms on Earth are often also halophilic, occurring in hypersaline solutions (Grant, 2004). Organisms on Earth evolved a number of organic (e.g., glycerol) and ionic (KCl) solutes to obtain osmotic balance with the environment. The adaptation to H₂O₂ as a solute which is compatible with the cell content, would for Martian organisms enable growth at very low water activity, comparable to that in concentrated perchlorate solutions. The diurnal temperature cycle on the Martian surface is concomitant with a water saturation cycle in the local atmosphere. At low temperatures shortly before and after sunrise, the moisture level is close to saturation. For example, ground fogs were observed by the Phoenix lander. This means any Mg-perchlorate would attract water. The clumpiness of the soil, which posed such a problem for getting a proper sample into the TEGA instrument of Phoenix (Fig. 2), as well as what seemed drops of liquid water on one of Phoenix' struts (Fig. 3), support that notion.

The fact that the upper few cm of the soil is crusted (Smith et al. 2009), together with the nightly precipitation of water ice crystals (Whiteway et al. 2009) has also been found in a parallel study of the migration of moisture in the soil of the hyperarid core of the Atacama desert (Davis et al. 2009). The occurrence of substantial amounts of perchlorate salts, both in the Atacama and at the Phoenix landing site, raises the question of the mechanisms producing perchlorate in both places. The presence of nitrates could not be ascertained on Mars, but for the Atacama a correlational study of the presence of perchlorates, nitrates and biomass might elucidate possible mechanisms.

The presence of perchlorates at the Phoenix landing site, at a level of about 0.5% is surprising, because there is no straightforward chemical production mechanism for these compounds to account for the measured concentrations. The production of perchlorates by putative organisms is therefore an alternative possibility - this could be a case of organisms modifying their environment to serve their needs. The presence of perchlorates improves the habitability of the Martian surface, particularly for xerophilic organisms employing a mixture of hydrogen peroxide and water in their intracellular fluid.