In: "Journey to Diverse Microbial Worlds: Adaptation to Exotic Environments" , ed. Joseph Seckbach; a volume which is part of the book series on Cellular Origin and Life in Extreme Habitats. Kluwer Academic Publishers, Dordrecht, The Netherlands. Chapter 27, pp. 387-398.
________________________________________________________________________________________
Terrestrial Microbes as Candidates
for Survival on Mars and Europa
Julian Chela-Flores
The Abdus Salam International Centre for Theoretical Physics, Miramare P.O.Box 586; 34136 Trieste, Italy, and
Instituto de Estudios Avanzados, (Universidad Simon Bolivar), Apartado 17606 Parque Central, Caracas 1015A,Venezuela.
1. Introduction
Mars and the ice-covered satellites of Jupiter are currently the most favourable sites for the search of extraterrestrial life. The motivation for the search for life in the Solar System is the evidence of liquid water in the early history of Mars and, at present, in the interior of at least two of the galilean satellites (Callisto and Europa). Hydrothermal vents on the Earth's sea floor have been found to sustain life forms that can live without direct solar energy. Similar possible geologic activity on Europa, caused by tidal heating and decay of radioactive elements, makes this Jovian moon the best target for identifying a separate evolutionary line. This search addresses the main problem remaining in astrobiology, namely, the distribution of life in the universe. We explore ideas related to Europa's likely degree of evolution, and discuss a possible experimental test. The total lack of understanding of the distribution of extraterrestrial life is particularly troublesome. Nevertheless, technical ability to search for extraterrestrial intelligence, by means of radioastronomy, has led to remarkable technological advances. In spite of this success, the theoretical bases for the distribution of life in the universe are still missing. The search for life in the Jovian satellites can provide a first step towards the still missing theoretical insight.
2. Eukaryogenesis as a cosmic imperative
We have suggested in our recent work (Chela-Flores, 1996; 1998a-c) that eukaryogenesis may be a universal phenomenon. We formulate the conjecture that the laws of physics and chemistry imply an 'imperative' appearance of eukaryogenesis during cosmic evolution [paraphrasing the well known sentence that "life is a cosmic imperative" (De Duve, 1995a)],
Life is not only a natural consequence of the laws of physics and chemistry, but once the living process has started, then the cellular plans, or blueprints, are also of universal validity: The simplest cellular blueprint (prokaryotic) will lead to a more complex cellular blueprint (eukaryotic). Eukaryogenesis will occur inexorably because of evolutionary pressures, driven by environmental changes in planets, or satellites, where conditions may be similar to the terrestrial ones.
In spite of the difficulty of identifying 'terrestrial-like environments', we have formulated a testable hypothesis related to the degree of extraterrestrial evolution on the pathway to intelligence. We emphasise that the conjecture has been formulated strictly at the cellular level; its relevance to the Drake Equation is discussed in Sec. 4.1.
3. A new approach to the distribution of life in the universe
Many hints suggest the conjecture of the universality of eukaryogenesis. One such hint concerns the combined action of natural selection, and the inevitable effect of symbiosis (Margulis, 1993). Besides, horizontal gene transfer (HGT) may also be a factor that drives prokaryotes into the more advanced eukaryotes (Smith et al., 1987).
However, an intriguing question is whether the transition prokaryote-eukaryote will occur in a suitable planetary, or satellite environment. We dwell on eukaryogenesis, since on Earth this was the first step towards multicellularity and, subsequently, intelligence. Insights derived form biogeochemical data suggest that the prokaryotic blueprint will make its first appearance on a planet, or satellite, in a relatively short geological time (Schildowski, 1988). There are even indications that sedimentary rocks older than 3.7 gigayears (Gyr) before the present (BP) from Isua contain reduced carbon of likely biogenic origin (Rosing, 1999). Additional support for an early onset of the prokaryotic blueprint was the result of analysis of banded iron formation from Isua and Akilia of some 3.8 Gyr BP (Mojzis et al., 1996). Once the period of heavy bombardment was over, some 3.9 to 4 Gyr BP, the appearance of prokaryotic life was almost instantaneous in the context of a geological time frame. We may assume that prokaryotes are bound to occur in environments where chemical and geological conditions may be similar to the terrestrial ones. This remark induces us to ask whether eukaryogenesis is a universal phenomenon, which is the bigger and deeper issue.
Our aim is to discuss experimental means for testing the conjecture of the universality of eukaryogenesis within the solar system; we also explore the implication of the conjecture in the context of the search for extraterrestrial intelligence (SETI).
4. Looking for sites where parallel evolution could occur within our Solar System
4.1. A NEW EQUATION FOR THE DRAKE PARAMETER fi
An early reference regarding the transition from prokaryotes to eukaryotes in relation with the Drake Equation was made by Carl Sagan. His remarks were in the context of a discussion of the SETI projects at a 1971 conference (Sagan, 1973). In order to make Sagan's general comment more specific, we consider the Drake Equation N = kfi , where N is the number of civilisations capable of interstellar communication, k is a constant of proportionality involving several factors that we need not discuss here; fi is the fraction of life-bearing planets or satellites where biological evolution produces an intelligent species (Drake and Sobel, 1992). We suggest that the Drake parameter fi is itself subject to the equation
fi = k1 fe fm , (1)
where k1 is a constant of proportionality, fe, fm denote, respectively, the fractions of planets or satellites where eukaryogenesis, or multicellularity may occur. Our conjecture motivates the search within our own solar system for a key factor (fe) in the distribution of life in the universe, including intelligent life. The presence of the parameter fm in our equation (1) can be understood, since once the eukaryotic level of evolution was reached on Earth multicellularity was bound to follow (De Duve, 1995b).
The extrapolation of the transition to multicellularity into an extraterrestrial environment is suggested by the selective advantage of organisms that go beyond the single-cell stage. Such organisms have the possibility of developing nervous systems and, eventually, brains and intelligence. If cell formation is possible in a short geological time frame, there are going to be evolutionary pressures on prokaryotes to evolve, due to symbiosis, HGT and natural selection (cf., Sec. 3. In fact, these evolutionary mechanisms are going to provide strong selective advantage to those cells that can improve gene expression by compartmentalisation of their genomes. (Larger genomes would be favoured, since organisms with such genetic endowment would have better capacity for survival, and hence better ability to pass their genes to their progeny.) Whether the pathway to eukaryogenesis in a Europan-like environment, or elsewhere in the cosmos, has been followed, is clearly still an open question.
4.2. CAN EXOGENOUS MICROORGANISMS EXIST?
Hydrothermal vents on the Earth's sea floor have been found to sustain life forms that can live without direct contact with solar energy. Similar possible volcanic activity on Europa, caused by its interaction with Jupiter and the other Galilean satellites, makes this Jovian moon the best target for a possible identification of a living micro-organism beyond our planet (Delaney et al., 1996). An open question is whether a habitable planet, or satellite, has to be in the "habitable zone" of its star; volcanism seems to be sufficient as a source of energy for driving chemical into biological evolution. Alternatively, another possibility is the decay of radioactive elements.
The present work aims at turning the question of distribution of life in the Universe, from the present realm of conjectures, into a well-defined scientific discipline that could be tested in the foreseeable future within our solar system. The current question of distribution of life in the universe will inevitably be faced with an 'armada' of space missions. Such efforts should be constrained to test only clearly formulated hypotheses (cf., Sec. 1), backed by the proposal of realistic, specific, and unambiguous experiments (cf., Sec. 5). We recall the recent proposal of a space mission called the Cryobot/Hydrobot, which in principle would be capable of investigating the possible existence of life in Europa (Horvath et al., 1997). Independently, in Japan there is a feasibility study of a space mission to explore the subsurface ocean of Europa and search for indicators of biological activity. Other projects will probably follow. In particular, the Japanese proposal includes instrumentation for in situ observation by means of a submersible of the type of the hydrobot (Raulin and Kobayashi, 1998).
If successful, these missions offer excellent possibilities for testing the eukaryogenesis conjecture (cf., Sec. 1). We have maintained that an appropriate experiment to test eukaryogenesis is feasible, in spite of the evident severe payload limitations that the nature of the mission is bound to impose on us.
4.3. IS THE EARTH ANALOGOUS TO EUROPA AND EARLY MARS?
Once we give up the chauvinistic point of view that has been forced upon us by the multicellular nature of Homo sapiens, the similarity between the environments of Europa, early Mars and the Earth becomes evident. We are beginning to realise that if life does exist on Europa it will be mainly deep, aquatic, cryophilic and most likely unicellular. Early Mars may have been analogous to the Earth as well.
On the other hand, it is worth underlining that deep, aquatic, low temperature environments for unicellular organisms are also predominant on Earth (Prieur et al., 1995; Gilichinsky, 1994). We need only recall that 70% of the Earth is covered by sea water, of which two-thirds have a temperature of around 2° C. Cryogenic conditions are widespread in our planet. More than 80% of the Earth biosphere, including the polar regions, is permanently cold (from the point of view of the mean annual temperature). The deep sea (> 1000 m) represents 88% of the Earth area covered by sea water and 75% of the total volume of the oceans; in other words, the deep sea represents 62% of the biosphere. Micro-organisms can be subject to extreme temperature fluctuations. In polar and tundra soil these fluctuations can have a lower bound of some -15° C. Finally, in taking advantage of the analogy between the Earth and Europa, we may make use of the wide experience with viable micro-organisms in permafrost, which may serve as the background against which to test conjectures. The possibility of detecting biomolecules in Europa, on the ice surface itself, rather than in the possible ocean underneath, was made recently (McKay, 1998). A possible mechanism for bringing biomolecules to Europa's surface was subsequently discussed (Chela-Flores, 1998d). Unfortunately, the Galileo Europa Mission is restricted to infrared and ultraviolet spectroscopy. Surface biogenic tests on Europa may have to wait for the further orbital mission to search for traces of putative Europan biochemistry, or signs of extant life.
4.4. CAN LIFE BEGIN IN THE TOTAL ABSENCE OF SUNLIGHT?
The answer to this question is important for the possible existence of life on Europa, or any of the other iced satellites of the Outer Solar System. Earth-bound eukaryotes depend on an oxygenic atmosphere, which was in turn produced by prokaryotic photosynthesis over billions of years. A possible scenario favouring the existence of Europan micro-organisms decouples hydrothermal-vent systems from surface photosynthesis. Indeed, experiments have already shown that chemical evolution leading to biological evolution is possible in conditions similar to those of hydrothermal vents (Huber and Wachtershauser, 1998). Further, the delivery of amino acids at hydrothermal vents is possible, either by cometary or by meteoritic delivery (Chyba, 1998). Rather than prebiotic evolution, the genesis of a primitive cell in the deep ocean independent of photosynthesis, is still a wider issue to be settled experimentally. The possibilities of primary deep-sea or, alternatively, deep-underground evolution, are at present open questions. We may recall some related evidence against hydrogen-based microbial eco-systems in basalt aquifers, namely ecosystems in rock formations containing water in recoverable quantiites (Anderson et al., 1998). The new experiment raises doubts on the specific mechanism proposed for life existing deep underground (Stevens and McKinley, 1995). However, the new evidence of Anderson and co-workers refers more to the specific Stevens-McKinley means of supporting microbial metabolism in the subsurface, rather than being an argument against the possible precedence of chemosynthesis before photosynthesis, which is really the wider and deeper issue to be settled. What remains to be shown in microbiology is that some barophilic and thermophilic micro-organism has a metabolism that can proceed in completely anoxic conditions, deprived from carbon and organic-nitrogen derived from surface photosynthesis. For example, such experiments probing the ability of a given micro-organism to survive in well-defined environments have already been performed; it was shown that Cyanidium, a primitive alga was able to thrive in a pure carbon-dioxide atmosphere (Seckbach et al., 1970). Thus, the case for life's origins, either through chemosynthesis first, or through a secondary reliance on photosynthesis at hydrothermal vents (by using oxygen dissolved in the sea-water), or deep underground, are still open questions. While this situation remains unsettled, plans for experiments have to be made by the space agencies, as the technological capability is consolidated for delivering equipment underneath the crust of satellites with iced surfaces, such as Europa, Callisto, Enceladus and Triton.
5. On the ubiquity of eukaryotes in Antarctica
The relevance of the information to be retrieved from Lake Vostok can be made more evident by means of the following question:
What might be learned regarding eukaryogenesis from the possible study of viable diatoms from permafrost and deep ice?
Since the presence of biochemical traces on the Europan surface has been suggested , it becomes imperative to pursue analogous research in Lake Vostok, particularly concerning eukaryotes. Diatoms, discovered in 1702 by the microscopy pioneer Anton van Leeuwenhoek, are some of the most interesting micro-organisms to consider, given their ubiquity on Earth: In just one litre of sea water one may find as many as ten million diatoms, which may be considered the primary foodstuff of the sea. Marine species often form a brown coating on Arctic ice floes.
The ubiquity of diatoms may also be exemplified by what is known in the other Earth analogues of the Europan environment: Permanently frozen lakes in a series of dry valleys were discovered in 1905 by the British explorer Sir Robert Scott. From the point of view of geology and microbiology some of the best studied frozen lakes are in the Taylor Valley, namely Lake Fryxell and Lake Hoare; further north, Lake Vanda, in the Wright Valley, is also remarkable. Some species of diatoms (Pennales) are known to dwell under the permanently ice-covered lakes of the Antarctic dry valleys. Some further details of the Antarctic lakes are given elsewhere (Chela-Flores, 1997). Once the planetary protection protocols are duly taken into account, forthcoming knowledge of the micro flora that populates the substantial water volume of Lake Vostok will be of great value for anticipating, and testing the instrumentation requirements that might be needed. Amongst the micro-organisms that are permanently living in the Antarctic lakes there are examples of eukaryotes, a few of which are illustrated in our previous work (Chela-Flores, 1997).We have emphasised the presence of diatoms before mentioning other examples of algae, because diatoms comprise the largest number of algae in the benthic mats of these singular biotopes; our main motivation, however, is to underline the significance of eukaryogenesis in astrobiological research. In the Antarctic biotope, eukaryotes have demonstrated to thrive in Europa-like conditions. By the mechanism explained by Wharton and co-workers (Wharton et al., 1983; Parker et al., 1981, 1983), vertical transport of diatoms and other micro-organisms is possible in the permanently frozen lakes. Hence, it is hardly surprising that diatoms have been found recently in the permafrost and deep ice of the Lake Vostok region. It should be kept in mind, however, that the mechanism of vertical transport that applies in the dry valley lakes, where the ice covering the lakes measures a few meters, may not apply in the case of Lake Vostok, where the depth of the ice covering the lake is measured in kilometres. The Cryobot/Hydrobot (CH) mission (Horvath et al., 1997) would benefit form the experience that has been gained in the dry valley lakes of Antarctica.
6. On the search for extraterrestrial eukaryotes
6.1. POSSIBLE EXPERIMENTS ON OR BELOW ICED SATELLITE SURFACES
To settle the question whether the iced satellites are potential sources of parallel evolution for micro-organisms, in this section and the next one we shall discuss possible experiments that may be carried out on and below the ice surface, which could be implemented by means of a landing craft, of the type of the cryobot; indeed, it seems feasible to search for extant life by means of the either of the two subsurface-oriented missions (cf., Sec. 4.2).
We have argued above that a factor in the lack of uniformity in surface brightness and colour of the Europan surface may be the presence of micro-organisms, or their biomolecules. In other words, the search for extraterrestrial biochemistry, or biology on the surface of Europa ought to be a possible straightforward and evident aspect of the Europa campaign. The biogenic hypothesis can be tested, for instance, by spectroscopic search of the Europan surface ice. On the other hand, it seems reasonable to test directly for surface biochemistry, or organisms.
6.2. ON POSSIBLE BIOLOGY EXPERIMENTS
Missions to the Galilean satellites would benefit form the experience that has been gained in the dry valley lakes of Antarctica. In the biology experiment presumably the maximum size of the Cryobot would be some 10 to 15 cm diameter and 1.5 - 2 m long (its equivalent in an alternative mission would presumably be subject to similar constraints). Within the restricted space available there would be an "in-situ chemistry laboratory". The submersible instrumentation would aim to determine whether the ocean exhibited one set of requirements for "life we would recognise" (Horvath et al., 1997). We believe that the detection of life and its evolutionary stage should inevitably be one of the primary goals of any exploration program aiming at the exploration of the iced satellites in the outer solar system. We wish to define in some detail the minimum equipment that is needed in the biology experiment proposed earlier (Chela-Flores, 1998b). The optical system that would be proposed for the submersible depends on the chemical composition of the putative micro-organisms. The technique relies on the material being able to induce luminescence by the application of various dyes. Fluorescent dyes are detected with a fluorescent microscope. If microscopic fluorescence is used to probe for life, some advantages and some challenges are immediately evident.
First, in contrast to the Martian search for life, the typical resolution needed for fluorescence micrographs of chromosomes is 10 mm (Lodish et al., 1995). Such resolution is well within the scope of a light microscope. Martian research is linked to the electron microscope, since the nodules in the Allan Hills meteorite, which are currently under discussion, have been suggested to be nanobacteria, 50-75 nm long and 10-20 nm in diameter (Benoit and Tauton, 1997). The difficulties of going beyond a light microscope in the Hydrobot are evident. Hence, it is feasible to think in terms of a robotic biologist'. It would be contained within the Hydrobot and consist of simple optics and sampling arms. At later stages in the cell cycle this structure serves as the basis for further folding, ending up at the highest degree of folding observed at the metaphase chromosome. This is an extremely fortunate feature from the experimental point of view. We only need to recall that the ultimate aim of the biology experiment is to develop tests that are compatible with the reduced dimensions available. Indeed, chromosomes stain easily, in a well-defined manner. The biochemical basis for the difference between heterochromatin (the more compact structure of chromatin) and euchromatin (its less compact form), remains unknown. Heterochromatin is not only a clear hallmark of eukaryogenesis (Chela-Flores, 1998a), but it is also a unique indicator of eukaryoticity, which is amenable to the tasks that the 'mechanical biologist' will have to perform. We confine our attention to the clearest hallmark for eukaryogenesis: heterochromatic genomes that respond in an unambiguous manner to well-defined dyes, the result of which could be recorded with video equipment for later analysis, after relaying the results to an Earth-bound laboratory.
6.3. WHEN CHEMICAL DETAIL OF THE GENOME IS UNCERTAIN
Quinacrine fluorescent dye inserts itself between base pairs in the DNA helix producing the so-called Q-bands, which for the planned mission would probably suffice. We suggested earlier (Chela-Flores, 1998b) the more involved use of Giemsa stain to produce the more permanent R-bands (Alberts et al., 1994). This is probably an unnecessary complication. Adjacent areas stain differently. The bands give a clear indication of slightly different modes of DNA packaging. It is the tightness of the genomic material that would be an indicator of a higher degree of evolution. The question is not so much what is the chemical detail of the genome, but what is the degree to which it has been packaged. It may be argued that gene activity is correlated with light-staining bands. (For instance, genes that are transcriptionally active are light-staining (Watson et al., 1987). This aspect of the experiment is its relevant, since it does not force upon us the requirement of previous detailed knowledge of the putative Europan biochemistry. The main scope of the experiment is to expose eukaryoticity at the level of gene expression, whose most characteristic indicator is heterochromaticity .
7. Discussion
7.1. EXCLUSION OF REFUGES AGAINST EVOLUTION
There seems to be a strong case for the exclusion of refuges against evolution (Little et al., 1997). Cambrian fauna, such as lamp shells (inarticulate brachiopods) and primitive molluscs (Monoplacophora), were maintained during Silurian times by micro-organisms that lived in hydrothermal vents. In the current Cenozoic Era these hot environments demonstrate that such fauna no longer inhabits these environments and hence has been unable to escape evolutionary pressures. Hence, this remark rules out the possibility that these deep-sea environments are refuges against evolutionary pressures. In other words, the evidence so far does not support the idea that there might be environments, where ecosystems might escape biological evolution, not even at the very bottom of deep oceans. It is then reasonable to assume that any micro-organism, in whatever environment on Earth, or elsewhere, would be inexorably subject to evolutionary pressures. On this planet eukaryotes seem to have been the consequence of over 2 Gyr of evolutionary pressures acting on the prokaryotic blueprint. The first appearance in the fossil record of eukaryotes occurred during the Proterozoic Eon, after prokaryotic communities (stromatolites) were well established in the previous Archean Eon, some 3.5 Gyr BP (Schopf, 1993). It still remains to be confirmed, or rejected, whether the Europan environment may have had liquid water in a geological time frame. In such a favourable environment a primordial archaea community would have had sufficient time for evolutionary pressures have modelled a primordial archaea community. If these conditions occurred on Europa, then, according to our conjecture (cf., Sec. 2), eukaryogenesis would have been inevitable. Recent observational evidence does suggest the presence of an ocean (Carr et al., 1998; Khurana et al., 1998).
7.2. THE POSSIBILITY OF EUKARYOGENESIS ON EUROPA AND MARS
There are several reasons why Mars may have experienced a more rapid environmental evolution towards an atmosphere rich in oxygen (McKay, 1996; 1998). This may have favoured a restricted period a rapid pace of evolution of the background population of prokaryotic cells towards eukaryogenesis. Such an oxic environment is favourable to the first appearance of the eukaryotic blueprint. On Earth eukaryogenesis occurred as far back as 2 Gyr BP, according to the micropalaentological data. Clearly such a Martian "Eden" may not have lasted for long on a geologic time scale, although we should recall that we are continually improving our understanding of the geologic history of Mars, as the evidence of crater statistics for recent volcanism demonstrates (Hartmann et al., 1999). Some work of "exo-palaentology" clearly remains to be done (Farmer, 1997), which could be facilitated by new technical support currently being discussed, such as the JPL aerobot, or a Martian aeroplane.
On the other hand, the eukaryotic transition may be a general consequence of geological changes on an Earth-like planet, or satellite coupled to the effect of natural selection. We have argued that evolution may have occurred in Europa and that the experimental test of this conjecture is feasible through a space mission. We have seen that difficult instrumentation issues are involved. The preparation of a package to search for life either on Mars or Europa is a formidable task. The method elaborated for the exploration of Mars (Kobayashi et al., 1998), which is based on fluorescence microscopy, would still require further miniaturisation before it could be adapted to the case of Europa. In relation with the possibility of eukaryogenesis having occurred on Mars, it has been pointed out (McKay, 1996; 1998) that since there are several factors that may have accelerated oxygenation on Mars (no tectonic recycling of organic sediments, less volcanic activity, smaller oceans during the first billion years), then before the environment deteriorated, Mars may have experienced eukaryogenesis earlier than when it occurred on Earth.
7.3. OUTLOOK
Unlike the situation concerning the distribution of life in the universe, two aspects of astrobiology have already sound scientific approaches. Firstly, the study of the origin of life, the first aspect of astrobiology, is based on the theory of chemical evolution, which is a time-honoured scientific discipline. The second aspect of astrobiology, the evolution of life, has scientific bases provided by the two well-established insights of Darwin: "the theory of common descent", and "natural selection" as a mechanism for biological evolution.In the present work we have defended the thesis that if the conjecture in Sec. 1 were to be tested successfully within our own solar system by the biological experiment discussed in Sec. 5, it would not only show the non vanishing of the all-important parameter fi [(cf., Eqn. (1)], one of the most controversial parameters of the Drake Equation, but at the same time it would bridge a remaining gap in astrobiology, namely the distribution of life in the universe. In other words, a preliminary test of the conjecture in Sec. 1 on the iced satellites would serve as a firm scientific basis on which to develop eventually the science of the distribution of life in the universe.
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