INTRODUCTION

Two relatively recent insights into planetary evolution, i.e., the antiquity of organisms on the Earth, and evidence suggestive of a "wetter, warmer" environment on early Mars, coupled with recognition of the ubiquitous presence of organic compounds in the cosmos, lend credence to the speculation that Mars may have developed a biota in its early history. If an origin of life did, indeed, occur on Mars then one can logically ask whether the organisms were able to evolve over the subsequent millennia of martian history, allowing them to retreat into special environmental niches, where metabolism, growth or simply survival is possible. It is this possibility that sets the stage for the search for extant life, the discovery of which would be of first-order significance for the science of biology. As Lederberg pointed out almost 35 years ago, "...'life' until now has meant only terrestrial Life" and, unlike the case for the physical sciences, there are few universal principles that can be applied to biology. Comparison of basic terrestrial biological attributes, such as cellular structure and biochemical constituents, with those of an extraterrestrial life form can lead to deeper understanding of what are truly fundamental attributes of living things.

An added dimension to the search for extant life on Mars is that, from a programmatic point of view, plans for human missions to Mars must take into account whether or not Mars is a "dead" planet. At the present time, with the limited information we have about life on Mars, human missions will almost certainly be encumbered by elaborate and costly measures to assure that both Mars and Earth are protected from biological contamination. If future missions, with appropriate payloads to search for extant life, continue to find no evidence for martian organisms, mission planners will almost certainly be less constrained by considerations of planetary protection. Conversely, if such experimentation reveals the presence of extant life on Mars, it is likely that human missions will be delayed until this biota is characterized.

We begin with the definition of extant life as that biomass that is now either growing or surviving in some dormant state. Three distinct types of evidence for extant life may be postulated. First, growing life could be recognized directly, via the detection of metabolic activity, probably practicable only within an appropriate niche where growth is occurring. The major condition to consider here is that of liquid water, which, while generally absent on the surface of the planet, may have transient or even long-term stability at certain sites. The second type of evidence involves dormant life, which may be spatially or temporally separated from a hospitable niche and in a nongrowing, but surviving stage, from which it could in principle be resuscitated for detection. Finally, we consider the possibility of nonliving indicators of extant life which would be found as geochemical tracers (organic or inorganic remnants or products) in recent environments that are hostile to life, but which would be indicative of life existing in other niches. Such indicators might include biogenic gases, biogenic minerals, or complex organic molecules indicative of living systems. Clearly, then, a major item of importance in the search for extant life is the location of sites that are most likely to favor finding life or an indicator of it. These will include both protected environments or niches favorable to life, or those places where evidence of hidden life may be found near to the surface of the planet.

CRITICAL REVIEW OF VIKING BIOLOGY EXPERIMENTS

To date, the only attempts at probing the surface of Mars for the presence of extant life were carried out on the two Viking landers (discussed earlier) in the late 1970's. Experiments were conducted that would have detected any of the three types of evidence for extant life considered above (metabolically active, dormant, nonliving indicators). The logical, and perhaps only workable, assumption was that the properties of any extant martian life form should be similar to terrestrial living forms. Given the uncertainties of testing for an undefined life form, and the constraints of mission design, the Viking life-detection experiments appear logical and proper. In essence, the rationale involved the detection of organic molecules (bio-indicators), and metabolic activities of photosynthesis or respiration (by metabolically active or dormant organisms). Analysis of the Viking life-detection experiments, when taken together with all of the other Viking results, have generally been interpreted as indicating the absence of extant biology at the two sites that were examined.

Over the intervening years, a number of arguments have been raised regarding both the validity of the Viking data and the conclusions that were drawn from them. Some workers, for example, have maintained that the results of the Viking Labeled Release experiment were consistent with the presence of indigenous organisms on Mars and have argued against the prevailing interpretation of the Viking biology experiments. While results of this and other experiments clearly indicated the occurrence of chemical reactions on Mars, the inability to distinguish biological from chemical processes clouds the issue. This is perhaps a consequence of the unexpected chemical activity of martian soils, suggestive of a variety of chemical oxidants, combined with differential temperature sensitivities of the chemical reactions they catalyze. As argued earlier, further study of the Viking results using simulated martian materials and environments is clearly warranted.

Another issue raised regarding interpretation of the Viking biology experiments concerns the constraints under which the metabolism experiments were conducted. Incubation conditions (e.g., temperature, light, moisture, duration of incubation) that differed from natural conditions might have had negative effects on indigenous species adapted to local conditions. On the other hand, unnatural incubation conditions could in many cases be viewed as logical attempts to provide more optimal conditions for the recovery of dormant organisms. An even more significant potential shortcoming of the metabolism experiments was the lack of consideration of the full range of potential resources (e.g., energy sources and electron acceptors) that could be utilized by the extant biota. For example, anaerobic respiratory metabolisms have been proposed that can be rationalized for surface, and especially for subsurface, geothermal habitats. The Viking biology payload was selected and developed with very little knowledge about the possible surface chemical and physical resources and conditions to be encountered. An extremely important and valuable lesson derived from these Viking experiments is that the preferred strategy for seeking metabolic evidence of life is first to characterize the conditions and resources in environments where there is reason to believe evidence of metabolically active life may be found.

Geochemical approaches, such as attempts to detect organic molecules typical of life, are more generic, as they do not assume specific types of metabolism. For example, although not a life- detection experiment per se, the pyrolysis GCMS experiment of Viking would have yielded convincing evidence of life if the proper molecules had been detected. This experiment, however, has been criticized for its insensitivity: the lower detection limit was judged to be on the order of 106 bacterial cells equivalents, a number which is frustratingly large. It is fair to say, however, that few sites exist on Earth where positive results would not be obtained. Given the knowledge gained from Viking regarding the water of hydration released during pyrolysis, the advancement in GCMS technology, and new developments in the amplification of specific molecules of life, more sensitive detection of organic compounds should now be possible.

Perhaps the most valid critique of the Viking experiments is that they were conducted at the wrong place (and/or possibly time) to detect biology on Mars. All evidence from experiments done at the two landing sites suggests a cold, arid surface environment, apparently suffused with oxidants capable of degrading organic compounds. Future studies must certainly seek sites that are wet (and thus warm) and/or protected from oxidants if extant life is to be detected. Viking results indicated that if biology exists on Mars it does not imprint an obvious mark on the atmosphere, such as has terrestrial life (e.g., abundances of methane and nitrous oxide dramatically out of equilibrium with an oxygenic atmosphere). There appears not to be a dominant global biogeochemical cycling of major elements. This does not preclude more circumscribed biogeochemical cycles in either local or widespread environments that are more hospitable, isolated from the harsh surface environment. Whether these actually exist on Mars is unknown, but if there are niches capable of supporting martian life, it is of paramount importance that they be identified and probed for the presence of living entities.

POSSIBLE HABITATS FOR EXTANT LIFE

Finding appropriate niches for metabolically active life on Mars is tantamount to finding sources of liquid water, however intermittently water may become available. Since liquid water cannot now exist as a stable phase on the surface of Mars, even as eutectic brine solutions, the critical factor in the search for extant organisms is to bring to bear techniques for the identification of martian sites where liquid water either exists under isolated conditions, or where it can exist transiently for organisms capable of an existence that is punctuated with periods of dormancy with no available liquid water.

In recent years, several scenarios have been advanced for specialized environments on Mars within which biological activity might be maintained. One such example that has been proposed identifies subsurface sources of liquid water as possibly affording environments for extant biology on Mars. In this scenario, geothermal sources produced by volcanic activity could provide water at some depth and, at the same time, provide volcanic materials such as H2, CO, and H2S, which could serve as reductants for nonphotosynthetic, chemoautotrophic metabolism. As discussed above, the Viking biology payload did not include experiments designed to test for this type of metabolism.

The idea of subsurface environments for extant biology is strengthened by evidence suggestive of hydrothermal activity on Mars in the past. However, whether Mars is still geologically active is not yet determined. Nevertheless, as part of the exobiology strategy for extant life, it is crucial to investigate this possibility because of its importance to the question of possible localized hospitable niches on Mars. There may well be subsurface regions where liquid water is available, and where the local conditions might support the growth of an indigenous biota. There may even be small surface features, like vents or fumaroles (undetectable in the Viking imaging and thermal-mapping experiments), where subsurface volcanic sources may be releasing water and reducing gases into the local environment, and providing sources for metabolic activity. In future missions, discovery of such surface features will require very-high-resolution imaging and thermal- mapping capabilities. Also, if methods with high spatial resolution could be developed for the identification of gaseous atmospheric constituents from orbit or at the surface, this technique could be extremely useful in delineating regions that might support the kinds of metabolism envisioned in this scenario.

Another class of potentially suitable environments is represented by more widespread groundwater or aquifer systems that would be maintained in liquid form by core geothermal heat, but not be involved with surface or near-surface geothermal activity. To find such systems would require drilling, but without further information about the global distribution of water and ice, as well as the areal and depth distribution of the presumed oxidants on Mars, it is difficult now to estimate the depths to which such drilling would be needed, or to locate sites feasible for drilling operations.

An additional potential niche for extant life is illustrated by an ecosystem containing bacteria and algae that can be found within certain rocks found in the cold, dry valleys of Antarctica.

The habitat for these organisms (cryptoendolithic autotrophs) consists of porous, translucent rocks, in which growth of organisms occurs a few mm below their surface when sufficient water is absorbed by the rocks from surface ice and snow as a consequence of warming during sunlit portions of the day. However, arguments have been raised against this scenario, which at least superficially appears to be applicable to Mars. First, that Mars is considerably drier than the dry valleys of Antarctica; second, that, under current martian atmospheric conditions, melting of ice/snow could not supply liquid water to the interior of the rocks; and finally, that the rocks on Mars appear to be opaque rather than transparent. Note that this does not imply that there has not been in the past, or could be in the future, conditions where liquid water could in fact be intermittently supplied to such endolithic-type niches. Some speculative evidence indicates that such might in fact be the case; aspects of the large-scale morphology of the surface suggest that either liquid water or abundant surface ice might have been present, and theoretical calculations of the history of Mars obliquity indicate that conditions at some times might be conducive to the presence of liquid water. Given current Mars conditions, a search for endolithic microbial communities would be a search for dormant microbial biomass in rocks found in regions where water might have been stable in a past geologic epoch.

Still another class of potential sites for extant life consists of those where organisms continue to survive, although growth or metabolism is not apparent. These organisms are distinguished from the temporally dormant organisms by longer-term separation from an environmental niche hospitable to growth. For example, it is known from terrestrial samples that, as evaporites crystallize out of solution, halophilic bacteria can be entrapped within developing salt crystals and it has been suggested that active metabolism may occur within brine inclusions that are sometimes found in such crystals. Furthermore, viable micro-organisms have been isolated from salt crystals that are thought to be 200 Myr old. Assuming these findings to be true, a scenario can be proposed that as Mars lost its surface water over geologic time, organisms retreated into saline environments and that some halophilic organisms may still be surviving inside the resulting evaporite crystals. A strategy that has as its objective the search for such halophilic organisms on Mars must begin with global reconnaissance aimed at locating sites with potential for evaporite deposits.

A somewhat similar scenario for extant biology on Mars is based upon the microbiology of permafrost regions on Earth, where evidence has been presented that organisms can remain viable for very long periods in ice obtained from these sources. Thus, permafrost and ground ice on Mars might be possible sites for extant biology. Current models of ground ice on Mars suggest that it would be unstable at latitudes below 40o, thus restricting to higher latitudes potential targets for testing this scenario. Until more is learned about ice contents of candidate features and their global distribution on Mars, serious attention to this scenario also must begin with global studies (e.g., water distribution and history, and climate variations).

Another class of sites includes those inhospitable to life even in a dormant state, but which might contain nonliving indicators of extant life. For example, environments where water has flowed over the surface of the planet in the relatively recent past are of great interest. If subsurface life is abundant, then these outflows might be expected to have deposited molecules indicative of extant life, either in the form of organic carbon or as minerals characteristic of living systems. While one cannot estimate with precision the age of noncratered fluvial water features, the possibility that some are relatively young, and therefore of potentially high value in the search for nonliving indicators of extant life, should not be dismissed.

As a final point with regard to the search for extant life, we note that routine monitoring of key atmospheric gases indicative of life may pay high scientific dividends. In atmospheres that are otherwise oxidizing in nature, some reduced gases, such as sulfide or methane, are almost exclusively indicators of either living ecosystems or hydrothermal activity (volcanism). Detection of any of these gases would then argue for further monitoring of possible spatial and/or temporal fluctuations in their abundances. Furthermore, analysis of gas inclusions in polar cores could yield data on such reduced gases that would point towards future analyses of their sources and sinks. Analysis of stable-isotope ratios might discriminate between biological and chemical sources for these gases.

In fact, from the standpoint of exobiology, no search for evidence for life on Mars would be complete without a thorough investigation of the martian polar caps and layered deposits. Since these deposits are collection and preservation zones for material from all around the planet, they may be among the most efficient places on the planet to search for evidence for life. (Recent studies have recovered culturable micro-organisms from polar ice deposits on Earth.) A major objective for such a search would be to examine an outcrop of exposed layered deposits within one of the polar caps. The examination of old, previously deposited material could provide important information concerning physical and chemical environments that existed during Mars' past history, and the stratigraphy of these deposits could provide information on how the martian environment changed through time. Key observations to be made would include a thorough examination of ice and dust deposits at a microscopic scale for morphologic evidence for biologic activity, as well as detailed chemical analyses of the polar deposits for possible preserved biosignatures.

From the discussion above, it is evident that several hypothetical alternative niches for life on Mars have been suggested in the exobiological literature. As of this writing, however, these remain to be located and characterized. Thus, the initial thrust of the strategy for extant life on Mars must be to determine whether or not these environments actually exist. Only with the acquisition of this fundamental information will it be reasonable, from the point of view of extant biology, to probe such putative environments with landed instrumentation.

OBJECTIVES FOR FLIGHT EXPERIMENTS

From the perspective of experimental strategy, the search for extant life can be broken down according to the nature of both the putative life form and its likely habitat. The objective in each case is the location and characterization of sites where either the biota itself exists or a signature characteristic of it may be found. This leads to definition of several types of site.

Sites where active life may exist
The approach in this case can be divided into three phases. The first phase involves remote sensing through imaging, and spectral and thermal analysis using the highest spatial resolution possible, in order to discover whether sites might exist that could support a living system (i.e., warmer, wetter, possessing appropriate chemical resources). The second phase involves landed instrumentation, targeted to sites thought to be compatible with a biota on Mars. The purpose of analyses during this phase is to seek geochemical evidence in support of the presence of biota, and especially to characterize further sites selected on the basis of remotely obtained information during the first phase. Geochemical analyses would seek information on the presence of organic carbon, and if found, its elemental and isotopic composition, as well as specific molecular identities. Inorganic geochemical analyses would be performed to permit recognition of the relative abundances of elements which might have been altered by metabolic processes. Measurements pertinent to characterization of possible biological niches would include analysis of water abundance, temperature, elemental composition (including biogenic elements), electron donors and acceptors which might drive metabolism, hydrated minerals, chemically reactive atmospheric constituents, and "oxidants". Should these measurements confirm the possibility of potential environmental niches for biology, the third phase would then follow, requiring sampling from these sites and carrying out critical biological experiments designed to test for metabolic activities or to recover organisms adapted to those particular environments. For this phase, sample return missions would provide the greatest flexibility and data return, but sophisticated large landers incorporating well-conceived biological payloads could perform some of the crucial experiments in situ. This overall strategy is similar to that taken by biologists (microbial ecologists) trying to characterize life in terrestrial environments.

In consideration of the possibility that life forms might inhabit sites which are only intermittently wet, observations that aid in understanding when and where liquid water might have been present on the martian surface over geological time would also be useful.

Habitats that might support dormant life
The approach here follows the assumption that dormant life (at least microbial) might be dispersed globally, but would only survive in the absence of oxidants. Particular locations of interest for survival of dormant organisms include permafrost and aqueous mineral deposits, such as evaporites. On landed missions to any site, geochemical evidence of a dormant biota in samples free of oxidants would be sought. More speculatively, methods for growth-based amplification of dormant organisms could be attempted, though broad assumptions about their metabolic capability would have to be made.

Sites that might yield geochemical information about extant life in another location
Two different approaches are envisioned. The first is to locate sites where liquid water may have been in relatively recent contact with subsurface water reservoirs. This would involve global reconnaissance to determine surface features consistent with flowing water in the geologically youngest regions. Such sites might represent places where geochemical evidence of subsurface life might be sought, as described above. The second approach is to sample polar ice as a global trap for biosignatures. These could consist of gases, which might integrate biological metabolites (e.g., oxidized or reduced gases) produced at specific, dispersed and/or temporally intermittent (and thus difficult to locate) sites associated with metabolically active microbial communities, or solid particles, which might bear chemical or morphological evidence of biotic activity.

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