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II POST-VIKING BIOLOGY STRATEGY FOR MARS Although Viking has not determined whether life exists on Mars or whether it once existed, the detection of atmospheric nitrogen prevents one from excluding the former possibility. Some of the findings enhance the possibility of current or past life: ancient water flows, the existence of salts, and the near certainty that large amounts of ice are locked in the regolith. Some factors diminish the possibility of current life: the lack of detectable organic com- pounds or reduced carbon, the presence of strong oxidants in the soil, and the low probability for the existence of liquid water under equilibrium conditions. A. Criteria for Sample Selection and Characterization Any biological experiment begins with the selection of samples. If the top few meters of the Martian surface were homogenous over the entire planet, or if variations were randomly distributed, the optimum strategy for the selection of samples would be fairly obvious. But photographs from orbit and from the landing sites have shown that the surface is heterogeneous and that the het- erogeneities are not randomly distributed. Furthermore, data from various experiments strongly suggest that the physical and chemical characteristics of the regolith are not uniform with depth. Inextricably entwined with the question of where to sample is the question of what characteristics of the sample would constitute items of paramount importance to present or past biology and to organic chemical evolution. We submit that the following fall in this category: 1. Does the sample contain detectable organic compounds or reduced carbonl The distribution, state, and abundance of carbon is critical to the possible origin and current existence of Martian life. Carbon is the fourth element in cosmic abundance. Diverse organic compounds of considerable complexity are distributed throughout our galaxy. It is remarkable, then, that on Mars no carbon has been detected definitively except in the atmo- sphere and in the winter polar caps, and none has been detected in a form more reduced than CO. The detection of reduced carbon would not prove current or past life (since it could be deposited by carbonaceous chondrites 12

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or it could be synthesized abiogenically), nor, as already discussed, could the lack of detection disprove it. Nevertheless, since reduced carbon is considered a sine qua non of living systems, samples with detectable elemental carbon or organic compounds clearly have an enhanced probability of containing past or current life. Characterization of the type of compounds and the determination of 12C/13C ratios in carbonate and in organic matter and the 32S/34S ratios of reduced sulfur and sulfate in minerals25 might permit some discrimination between biological and nonbiological genesis. 2. Does or could the sample contain liquid water at high chemical poten- tial!* Liquid water is also generally considered to be an absolute prerequisite of living systems. The properties of liquid water are unique and play a major role in determining the conformation and therefore the function of terrestrial macromolecules. Water vapor does not have these unique properties. Ice has many of the properties of liquid water, but its very high viscosity would greatly restrict biochemical reactions, and at sufficiently low temperatures would preclude them. 3. Does the sample contain water-soluble electrolytes! So universal is the presence of electrolytes in terrestrial biological systems and so important is their role in both macromolecular conformation and enzymatic and physio- logical function, that we consider them a likely essential of all living systems. Especially significant in samples would be ions of Na, K, Mg, Ca, Cl, S, and P. Furthermore, their presence in appreciable concentrations in a sample would enhance possibilities for the existence of liquid water.* 4. Is the composition of the gases in the soil sample out of equilibrium with that in the atmosphere! Biological activity modifies the composition of the surrounding gases. The detection of disequilibria between the bulk atmosphere and the occluded gases in a soil sample would not prove the presence of life, but soil samples exhibiting prolonged disequilibria of chang- ing magnitude would certainly be deemed more likely to contain active organ- isms. This would be especially so if the gases were those that on Earth cycle biologically critical elements: hydrogen, methane, ammonia, hydrogen sulfide, oxygen, nitrogen, carbon dioxide, nitrogen oxides, and volatile amines. *Although water retains many liquid properties, such as molecular rotational mobility down to activities as low as perhaps 0.2, nearly all fully functioning terrestrial life re- quires the chemical potential of liquid water, expressed as activity, to be >0.9. The ex- treme lower limit is about 0.61 for one species of mold, and there are scattered reports of growth ataM,< 0.8. [Water activity =P/PO, where P is the vapor pressure of the water under consideration, and PQ is the vapor pressure of pure bulk water.] The presence of solutes reduces the water activity and consequently the freezing point. For example, solute concentrations that lower the water activity to 0.9, the minimum value for the functioning of most terrestrial organisms, also reduce the freezing point of the aqueous solution to about -11°C. 13

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B. Where to Sample 1. Sediments. Clearly Mars once had massive quantities of flowing surface liquid water. Flowing b'quid water produces sediments, and it is in these sedi- ments that one would expect a higher probability of the evidence for past life, a higher probability of biologically derived organic compounds, and a higher probability of appreciable concentrations of electrolytes. Obvious candidates for sampling are areas exhibiting past fluvial activity and areas exhibiting se- quential layering, such as the margin of the north residual polar cap. 2. Ice-regolith interfaces. It is at such interfaces that the existence of liquid water would have the highest likelihood. One such promising area is again the margin of the residual polar cap, especially perhaps the margins of those frost- free patches in the residual cap that have temperatures as high as 235°K.5 Other potential locations for liquid water are the regions lying between the surface and the subsurface permafrost. 3. Subsurface sampling. As of April 1977, Viking has conducted organic analyses and biology experiments only on samples from the top 4-6 cm of the regolith. (In March 1977, an inorganic analysis was carried out on a sample from a depth of 20 cm.) Some of the sampled material had been exposed to Mars' intense ultraviolet flux, and all the material presumably contained strong oxidants derived from atmospheric processes—strong oxidants that would probably have destroyed organic compounds if they had been deposited or synthesized. Clearly, sampling at greater subsurface depths is required to re- duce or eliminate the powerful oxidants and thereby enhance the probability of locating organic compounds. Equally clearly, subsurface exploration will likely be required to reach the putative ice-regolith interfaces and sediments just discussed. The chief argument against subsurface sites for living forms has been that the sites would be reached by little or no visible light and, hence, would be incapable of supporting photosynthetic autotrophy. On the other hand, if the results of the current pyrolytic release experiment represent abiogenic organic synthesis, there may be sufficient steady-state quantities of organic compounds below the Martian surface to support heterotrophy or chemical autotrophy. In summary, then, from the biological viewpoint, the prime concern for the next mission to Mars should be exploration of the subsurface in favorable areas containing sediments or layered sequences such as alluvial flows, the margins of polar ice caps, and terrain likely to overlay permafrost. Samples collected from these areas should be characterized with respect to the fol- lowing first-order requisites for current or past living systems: (a) the exis- tence and identification of specific organic compounds ;(b) the distribution of ice and liquid water and of water-soluble electrolytes; (c) examination of 14

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the geochemistry and morphology of sedimentary materials; (d) measure- ments of occluded gases to determine those that are significantly out of equilibrium with the atmosphere; and (e) measurements of the isotopic ratios of carbon in the reduced form, if present, and in carbonate, and measurements of the oxidation state and isotopic ratios of sulfur. C. Instrumental Requirements for Sample Characterization and Considerations of the Strategy for Search and Sampling 1. Instruments. Instruments of the requisite type, sensitivity, and resolu- tion either have flown on the current Viking or appear adaptable to future soft landers without difficulty. A critical requirement for any instrument is that the data it furnishes be directly interpretable and subject to minimal ambiguity. We cite examples for the purposes of illustration—not to represent definitive recommendations. (a) Organic compounds, isotope ratios, and gas analysis. Modifications of the existing Viking GCMS would provide an instrument meeting the basic requirements for all three analyses. We are informed that an instrument could be provided with both broad mass number coverage to aid in the identifica- tion of organic compounds and on command high resolution and accurate peak height analyses over a restricted mass range, as would be desirable for isotopic and gas analyses. The instrument should be capable of carrying out a large number of analyses, and its detection threshold should be at least equal to that in the present GCMS. Alternative, although perhaps somewhat less versatile, approaches would be gas chromatographs or mass spectrometers by themselves. The state of art for both is high. Both have flown on Viking,8'15 and a second-generation instrument incorporating a mass spectrometer is cur- rently under development.26'28 (b) Detection of the amounts and phases of water. A candidate instru- ment has been proposed and tested in the bread board state. It consists of a differential scanning calorimeter (DSC) coupled with a phosphorus-pentoxide- conductivity water detector. Both components are commercially available. Adaptation to flight configuration is deemed feasible.26 Commercially avail- able instruments can detect the heat absorbed in the melting of ~1 X 10~7 g of water. An improvement of two orders of magnitude is probably attain- able.29 (c) Electrolytes. The presence of electrolytes could be easily detected by suspending regolith samples in water and measuring the electrical conduc- tivity. Such measurements combined with inorganic analyses of the sort con- ducted on the current Viking would provide information as to the elemental species involved. Also essential would be at least an approximate measure of pH (±0.5 pH). 15

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(d) Identification and characterization of sediments in fluvial areas. Samples will need to be examined and characterized with respect to deter- minable major features. One feature would be particle size within horizontal laminae or beds. Another would be the size and shape of the rock or mineral grains. The angularity of fragments would be suggestive of the degree of transport to which the material had been subjected and might allow some characterization of mineral composition or source rock. The imaging systems required to conduct these examinations are considered technically feasible.26 2. Mobility and sampling in depth. We stress the importance of obtaining samples from sedimentary areas, from ice-regolith interfaces, and from areas overlaying permafrost. To locate these and to escape the uv-oxidant condi- tions at the surface require subsurface sampling. However, the problem of specifying an optimum strategy for lander delivery, search, and sample acqui- sition is formidable. (a) Delivery. Potentially interesting sites will be selected from orbital photography and other orbital measurements. But sites of maximum biological interest are among the more hazardous with respect to soft landings. One solu- tion to this quandary is to provide the lander with mobility. But how much mobility? The requirements of safety and the size of the landing error ellipse probably dictate mobilities of many kilometers. But the operational feasibility of achieving such mobilities is moot. The alternative route is hard landers. The penalties here are the restrictions on size, weight, and complexity of the instrumental payload (see below). (b) Search. A tremendous gap exists between the topographical features that are resolved and interpretable at orbital altitudes and the surface features that are actually found by landers. It is quite conceivable that a site chosen from orbit for highly interesting topographical features such as fluvial activity would upon landing appear indistinguishable from the VL-1 and VL-2 sites, both at the landing site and for a hundred kilometers around. Perhaps candi- date sites for sampling will be made self-evident by the visual observation of geological features such as a sedimentary outcropping, but more probably they will not. The likelihood, then, is that specific subsurface sampling sites will have to be chosen stochastically. (c) Subsurface sampling. Similar problems arise with respect to defining the depths from which samples should be acquired. A few millimeters of rego- lith would greatly attenuate the uv flux if the regolith were static.293 It is not static, but to our knowledge neither the time scale nor the depth of mix- ing has been estimated. There are even more serious obstacles to estimating the depths required to reduce or eliminate the powerful surface oxidants, since they may well consist in part of diffusible compounds such as hydrogen peroxide. Finally, we do not at present know whether the subsurface sedi- 16

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ments or regolith-ice interfaces that we wish to locate lie 6 cm or 60 m below the surface. The technical problems and attendant costs of subsurface sampling by soft landers are formidable, and both are exponential functions of the specified depth. // is essential, then, that NA SA conduct studies to estimate or to devise approaches to estimating the required subsurface depths. Perhaps estimates can be derived from data from the current Viking. Perhaps information on the distance to the subsurface ice could be gained from radar or other obser- vations from Earth. Or perhaps the only approach to obtaining useful esti- mates will be in situ measurements on the Martian surface ofregolith proper- ties such as the frequency dependence of conductivity. The last possibility would lead to difficult choices: Should the exploratory phase which acquires and characterizes subsurface samples for biologically relevant properties be preceded by a mission that is designed in part to esti- mate such physical attributes as the distance to the ice-regolith interface? Or should the biological exploratory phase be initiated without this prior information? If initiated without the prior information, should the delivery mode be penetrators which could sample at uncontrollable depths down to several meters, or should the delivery mode be a mobile soft lander provided at major additional cost with the ability to sample at controllable sites to controllable depths of perhaps several meters? 3. Penetrators and other hard landers. As mentioned, hard landers offer one approach to sampling in-depth areas that are topographically interesting but too hazardous for soft landers. One type of hard lander, the penetrator, has been evaluated in considerable detail and offers promise.30 The pene- trator is a missile-shaped object that impacts the surface at high speed and penetrates to depths of 1 m to 15 m, leaving an afterbody on the surface. The forebody contains most of the scientific payload; the afterbody contains a transmitter and, if desired, an imaging system. A "nominal" penetrator could carry a payload weighing 7 kg and occupying 4500 cm3. The payload is subjected to a deceleration of about 1000 X g. The payload capacity (along with limitation in power) puts severe constraints on instrumentation, but the deceleration forces do not. (One preliminary study has indicated that a Viking-type mission could carry perhaps nine such penetrators in lieu of a soft lander. However, the two types of landers are not mutually exclusive, even on a Viking-type mission. The types and numbers of landers transported depend, of course, on the propulsion systems available.) In spite of the payload restrictions, the instrumentation on penetrators can be remarkably sophisticated. With respect to measurements critical to biology, it could probably carry (1) a DSC-P2O5 instrument for detecting amounts and phases of water, (2) apparatus for measuring soil electrical conductivity, and 17

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(3) various instruments to conduct inorganic and elemental analyses. A critical but still moot question is what could be carried in the way of organic analyti- cal instrumentation. The adaptation of a GCMS or a mass spectrometer itself seems unlikely. However, the adaptation of a gas chromatograph seems more feasible. Gas chromatographs can be small, and they are extremely sensitive and capable of good resolution, provided a volatile phase can be generated. Finally, there exist sensitive techniques capable of giving yes or no answers about the presence of organic compounds; for example, spectro-acoustical techniques capable of detecting C—H bonds in picomole quantities now exist,31 and they can possibly be adapted to penetrators. In summary, penetrators have the advantages that they can be directed to numerous areas that are deemed of high biological promise* and that they would provide subsurface sampling. They have the disadvantages that only the impacted sites are sampled, that the vertical range of subsurface sampling is restricted, that the instrumentation is far more restricted than on a soft lander, and that there is far less potential for adaptivity in the conduct of the experi- ments. The major unknown is their specific instrumental capability. Specific flight-configuration instruments for collecting biologically critical data do not yet exist. D. Further Considerations Concerning the Strategy of Exploration To repeat, from the biological viewpoint, the first phase of post-Viking explo- ration of Mars should be to acquire subsurface samples from areas likely to contain sediments and ice-regolith interfaces, and to characterize these sam- ples with respect to organic content and carbon and sulfur isotope ratios, the abundance and state of water and water-soluble electrolytes, the abundance and types of occluded gases, and the geochemistry and morphology of the sediments. It is not clear whether the necessary characterization can be achieved by a hard lander or a soft lander alone, or whether it will require both, probably in sequence. The reason it is not clear is that there are at least two major areas of ignorance: (1) A soft lander can unquestionably carry the necessary instrumentation to characterize subsurface samples, but there are serious questions as to whether it can reach the areas that are prime candidates for sampling; (2) hard landers can probably reach the areas that are prime candidates for sampling, but there are unresolved questions as to whether *It should be noted that the error ellipse for the point of impact of penetiators is com- parable in size to that for soft landers; hence, penetrators should not be considered capable of hitting small targets. Moreover, although penetrators can carry the variety of instruments listed above, limitations in space and demands from other experiments make it unlikely that each could carry the full complement of biological instruments. 18

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they can carry the instrumentation necessary to characterize the samples satisfactorily. It is our strong recommendation that NA SA carefully address the matter of mobility of soft landers and the matter of instrument payload on hard landers before deciding on subsequent missions to the surface of Mars* At- tention has been devoted to the achievement of mobility on a soft lander. But to our knowledge, little attention has been given as to whether the lander should conduct a random walk or whether it should be directed to some target preselected from orbit. We opt for the latter. If the latter, then, one must estimate how much mobility is required and whether the required mo- bility is feasible. The decision may be in favor of the random walk approach, but, if so, it should be as a consequence of deliberation and not by default. With respect to penetrators, there is an urgent need to determine whether the considerable potential of these devices can be translated into specific instruments that will adequately characterize samples of Martian subsurface. Whether the samples are characterized by a soft lander or hard lander, the extent to which they possess reduced carbon, water, soluble electrolytes, gas disequilibria, and 13 Cand 3*S depletion should determine the priority ac- corded to the initiation of a subsequent phase in the biological exploration of Mars, namely, a detailed examination of samples for direct evidence of current or former life. E. Direct Examination of Samples for Evidence of Current or Former Life There are two options for conducting the detailed examination: One would be to experiment remotely on the Martian surface; the other is to return samples to Earth and experiment on them here. We believe the arguments strongly favor sample return. 1. Evidence for current life. One second-generation instrument capable of chemically characterizing samples and searching remotely for existing Martian life is currently under development26"28—the so-called "Unified Biology Exper- iment" (now termed the "Integrated Chemistry and Biology Instrument"). The instrument is essentially a chemistry laboratory preloaded with a variety of reagents that can be added to Martian samples in desired sequences or amounts. It uses a mass spectrometer to monitor the gaseous products re- sulting from metabolism or chemical reaction. As indicated on p. 15, the instrument as presently envisaged would be capable of determining the presence of organic compounds and perhaps characterizing them to some extent, and of measuring gas disequilibria and carbon and sulfur isotope *In emphasizing these contrasting problems, we are not of course intending to minimize the necessity for intensive study of soft lander payloads. 19

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ratios. It would provide some measure of the amount of water present, but little information on the phases present at subzero temperatures. Like the existing Viking instruments, it would use metabolic activity as the chief criterion of life detection. But in our view there is an inherent dif- ficulty in demonstrating that data from metabolic experiments which are consonant with biological activity are in fact uniquely ascribable to biological activity. The basic premise of metabolic experiments is that organisms modify substrates and other components of the environment by heat-labile catalysts. While this is true, the modification of substrates by heat-labile catalysts is not unique to living forms. Furthermore, of necessity, most metabolic experi- ments tend to be highly geocentric. Thus two of the three current Viking experiments are in fact terrestrial microbial experiments in which soil samples are exposed to terrestrially oriented organic substrates under terrestrial con- ditions of temperature and water. Proposed second-generation experiments are of necessity also geocentric (e.g., challenge soils with various substrates and metabolic poisons). There are, however, other attributes which are generally considered to be characteristic of living systems, for example: motility in the absence of ex- ternal vectorial forces; increase in size and number, with descendants similar in chemical composition, form, function, and behavior to the parents; con- version of low-molecular-weight organic compounds into high-molecular- weight compounds, or in general a reduction in entropy within a delineated compartment; and objects predominantly composed of specific optical isomers. The detection and assessment of all these characteristics require sufficient "biomass" for direct observation or direct chemical analysis. Direct detection requires (a) high sensitivity and versatility in analytical procedures, (b) fractionation and concentration of samples, or (c) prior biological amplifi- cation of the sample (i.e., growth) sufficient to permit detection. All three are far more readily achievable on samples returned to Earth than on samples examined remotely on Mars. 2. Evidence for fossil life. The question of whether life ever evolved on Mars is of the same order of importance as the question of whether it now exists. Only a returned sample seems capable of permitting the application of the full armamentarium of paleontological and geochemical techniques: detailed microscopic examination of morphology; the detailed determination of isotope ratios of carbon and sulfur; the absolute dating of the samples; the detailed mineralogical composition; and the localization and relative abun- dances of carbonates, sulfates, sulfides, phosphates, and nitrates. Remote experimentation also would preclude the powerful approach of simultaneously correlating micromorphology with chemical analysis by such methods as com- binations of scanning electron microscopy and electron microprobe analysis. 20

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3. Adaptivity. While Viking is remarkably adaptive in comparison with prior missions, its adaptive capabilities pale in comparison with those of hu- man hands and the human brain on Earth. Adaptiveness is especially impor- tant in that a search for current or past life is a search for items about which we will know little in advance. 4. Instrument sophistication and obsolescence. The instrumentation that can be brought to bear in repetitive experiments on samples returned to Earth will always represent a far greater array of far greater sophistication than can ever be launched to Mars. Less obvious is the fact, illustrated by Viking, that any instrument that lands on Mars is of necessity some 10 years out of date by the time it arrives. This obsolescence arises, of course, from the need to freeze instrument design far in advance of the flight. The chief (and perhaps only) scientific argument against a returned sample (apart from back contamination issues) is the danger of alterations in the sam- ple during the return flight. This potential problem will require extensive study. Perhaps it could be obviated by cryogenic storage. Since Martian samples will have been exposed daily to temperatures below 220°K and will have been exposed annually to temperatures as low as 149°K, it is likely that they could be cooled to and maintained at <120°K without significant alteration, tem- peratures which preclude nearly all thermally driven chemical reactions. These conclusions about the high scientific potential of returning unsteri- lized Martian samples to Earth reaffirm existing Space Science Board pol- icy1' p'19 and are consistent with the conclusions of several publications, internal reports, and workshops.32"35 The matter of the physical contain- ment of returned unsterilized samples has also been analyzed by these same and other groups. Containment is considered necessary to protect the Martian samples by providing them with a controlled nonterrestrial environment, and to isolate the Earth from possible Martian organisms until scientific evidence accumulates to show that the risk from interaction is nil or vanishingly small. The reports consider such containment technically feasible. F. Phases of Biological Exploration versus Missions Our recommended strategy for the post-Viking exploration of Mars consists of, first, an initial phase of exploration to characterize Martian samples with respect to the possession of several attributes of paramount biological im- portance; second, a decision as to whether the characteristics of the samples are sufficiently promising to warrant the initiation of a succeeding phase of biological exploration; and, if so, the third step would be this succeeding phase of exploration that would examine Martian samples in detail for evi- dence of current or past life. There might or might not be a one-to-one correspondence between the 21

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two phases of exploration and the number of missions flown. One could con- ceive of a single mission that would combine both phases of exploration, but such a combined mission would preclude the intermediate decision step that we are recommending. ,4 minimum of one mission then would have to be devoted to the first characterization phase. If the decision based on that first phase were positive, our strategy would dictate that the next mission initiate the detailed biological study, and that it be a Mars Sample Return (MSR).* This MSR would have to include aspects of the first phase as well, for clearly it will not be able to acquire samples from precisely the same sites and depths as did the preceding mission or missions. *The current acronym for this mission is MSSR (Mars Surface Sample Return). We re- name it MSR because we recommend subsurface samples. 22