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4 Limits of Life on Earth: Expansion of the Microbial World and Detection of Life The Task Group on Planetary Protection assessed past reports and current views on the range of environmental conditions believed to exist on Mars and reached the consensus that it is extremely unlikely that a terrestrial organism could grow on the surface of Mars, although survival for some time is possible. It is clear that the most extreme environments on Earth where organisms can replicate are still considerably less extreme in some parameters vital for life than are known to occur over most of the martian surface. Particularly important in this regard are the high levels of UV radiation, the thin atmosphere, the extremely low temperatures, and the absence of liquid water on the surface of Mars. This appraisal is based on our current understanding of the conditions on Earth that limit cell growth; however, the task group emphasizes that although it is extremely unlikely that terrestrial organisms could grow on the surface of Mars, this does not imply that life does not exist anywhere on Mars. There is far too little information to assess the possibility that life may exist in subsurface environments associated with hydrothermal activity or in selected microenvironments free from the harsh conditions already mentioned, or to conclude that organisms resembling terrestrial life forms did not evolve on Mars during the planet's early geological history. The primary residual concern of the task group is with forward contamination by intact cells or components of cells that could be detected by sophisticated molecular methods in future expeditions designed to look for evidence of extant or past life on Mars. The task group believes that this concern necessitates that those involved in the planning of present and future expeditions to Mars be appraised of new results obtained from 30

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studies of extreme environments as well as the inevitable extension of the limits of environments where growth and survival can take place. Underscoring all of these advances in the microbiology of extreme environments are parallel advances in the development of new methods and more accurate and sensitive instruments for detecting the presence of life and life-related molecules and for identifying their evolutionary relatedness. It is not a straightforward matter to define the ranges of physical and chemical conditions on Earth in which organisms can grow, replicate, or survive for extended periods. During the 13 years since the SSB's last report on planetary protection,1 bacteria have been detected or isolated from many hostile environments on Earth, including the dry, extremely cold surfaces and interstices of rocks in the dry valleys of the Antarctic, hot environments associated with submarine and terrestrial volcanoes and geothermal systems, and deep subsurface sediments and aquifers. These investigations are in their infancy, and we still know little about either most of the organisms inhabiting these environments or in many cases the geochemistry and geophysics of the environments. In the last decade or so, a variety of novel organisms have been isolated. They include hyperthermophiles capable of growing at 110°C, obligate barophiles capable of growing at the pressures found in the deepest ocean trenches, and anaerobes capable of using iron, manganese, or even uranium as electron acceptors. Similarly, a variety of strategies have been identified by which microorganisms can survive environmental conditions that do not allow growth, including low temperature and low nutrient conditions. Traditionally, endospore and cyst development were considered the principal mechanisms for long-term survival by microorganisms, but it is now clear that many microorganisms have mechanisms for long-term survival that do not involve spore or cyst formation. It is now recognized that the inability to culture many microorganisms is a widespread phenomenon apparent with environmental samples and that only a few percent (or less) of organisms detected by microscopic methods can usually be cultured. Examples of the manifestation of organismal survival mechanisms include both the miniaturization of cells and the attachment to surfaces. EXTREME THERMOPHILES AND VOLCANIC ENVIRONMENTS Important recent discoveries and hypotheses, published across a diversity of disciplines, have pointed to submarine hydrothermal vent systems, and specifically to their subsurface crustal environments, as the likely site of biochemical and even early biological evolution.2 The particular nature of organisms that might have evolved on Mars is unknown. As to organisms that may have been transported to Mars from 31

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Earth during Archaean bombardments, there is strong phylogenetic evidence, based on both 16S rRNA sequence comparisons of a large number of organisms and geological evidence, that the earliest groups of microorganisms to inhabit Earth were anaerobic hyperthermophiles (growth at 90°C or higher).3,4 These organisms, which utilize carbon and energy sources found in hydrothermal and geothermal systems and possess unusual mechanisms for growth at temperatures exceeding 100°C, constitute a distinct phylogenetic group of organisms that share some characteristics with other bacteria and eucaryotes as well. Originally classified as a distinct kingdom, the archaebacteria are now classified in the domain Archaea and are more closely related to the domain of the Eucarya (formerly eucaryota) than to the Bacteria (formerly eubacteria).5 Hyperthermophiles are significant to a discussion of planetary protection issues because of evidence already presented that active volcanism may occur on Mars today. Implied is that hydrothermal activity would accompany volcanism because of water entrapped in the martian crust. Although the chance that a hyperthermophilic Archaea from Earth would contaminate Mars at a location that would allow growth is extremely remote, these organisms could be more significant with respect to back contamination, and as Earth analogues to past martian life if life ever existed on Mars. Among the many unusual properties of hyperthermophilic Archaea, those properties important to concerns about planetary protection include the probability of survival and growth under any of the ranges of physical and chemical conditions that exist on Mars. Unfortunately, we know considerably less about the survival of hyperthermophilic Archaea than we know about Bacteria, spores, fungi, and viruses. Only recently have extremely thermostable enzymes from vent hyperthermophiles been purified and characterized. For example, an amylase from Pyrococcus furiosus—a heterotroph capable of growing at temperatures up to 103°C— has a half-life of 2 hours in an autoclave at 120°C and is active at 140°C.6 A purified -glucosidase, with a half-life of 48 hours at 98°C, reaches optimal activity in the temperature range of 105 to 115°C.7 The other extremely thermal stable enzymes studied from hyperthermophiles include ferredoxin, hydrogenase, serine protease, glyceraldehyde-3-phosphate dehydrogenase, and a never-before-described tungsten-iron-sulfur enzyme from P. furiosus that catalyzes a dehydrogenase-like reaction of very low potential at 100°C.8 Besides proteins, other macromolecules from hyperthermophiles, including DNA and membrane lipids, must also have some unusual properties. Recently, the presence of a reverse gyrase, which catalyzes positive supercoiling of circular DNA, was discovered in all hyperthermophiles tested.9 It was suggested that supercoiling of DNA imparts thermostability. Questions regarding thermal stability of Archaea cells and their macromolecules and synthetic systems have only recently been addressed. Preliminary results, however, point to unique structures and mechanisms 32

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for growth and survival under some of the more extreme conditions on Earth, although these conditions are not nearly so severe as surface conditions on Mars. LIFE IN EXTREME ENVIRONMENTS Dormant Forms of Life Endospores from the gram-positive bacteria are ubiquitous and perhaps the most resistant and survivable form of life on Earth. They are known to survive for thousands of years and are resistant to freezing, desiccation, and vacuum and are highly resistant to many disinfectants. Bacterial spores are also moderately resistant to heat and to UV and ionizing radiation. Some spores germinate whenever there is free water, ranging in temperature from subfreezing to superboiling. Recognition of the ability of spores to survive such harsh conditions has led previous committees to focus on bacterial endospores as a major concern in planetary protection. Recent studies utilizing the NASA Long Duration Exposure Facility (LDEF) have shown good survival of multilayers of bacterial spores, which had been fortified with buffer and nutrients, after 6 years of exposure to space vacuum.10 When spores were not shielded from solar radiation, their survival was reduced to 10-2 to 10-4 percent. These results suggest that the vacuum and cold conditions of space pose no particular barriers for spore survival, but in the absence of shielding from UV radiation, there is little chance for the survival of dormant spores transported through the space environment. Other studies have focused on comparisons of the survival rates of spores exposed to UV irradiation under atmospheric and vacuum conditions, and at a variety of temperatures.11,12 Conditions simulating interstellar space or those of the surface of Mars inactivate spores rather quickly, suggesting that any long- term survival of unshielded spores on Mars would be impossible. It has not been possible to specify the mechanism of spore inactivation, although it appears that spore photoproducts, such as thymine dimers, are not responsible.13 On the basis of such laboratory experiments, it has been proposed that with proper shielding, bacterial spores might survive UV irradiation for very long periods, perhaps millions of years.14 Deep-Subsurface Microbes Experiments since 1984 supported by the Department of Energy (DOE) have allowed the recovery of viable bacteria from subsurface sediments 500 to 700 meters below Earth's surface.15 In these experiments, there is clear evidence that the organisms recovered from the deep 33

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subsurface are not contaminants from the surface, from more superficial sedimentary horizons, or from the drilling fluids. These organisms are found in Middendorf Cretaceous sediments, which are 100 million years old and form an aquifer in which the time for ground water recharge is at least 15,000 to 20,000. The ground water from the bore holes contains traces of refractory carbon and about 2 milligrams of oxygen per liter. For the oxygen to be maintained in the face of a living microbiological community of 106 microbes per cubic centimeter, the growth rate in terms of the doubling time of these organisms must be between 10,000 and 20,000 years.16 Based on analysis of total 16 S rRNA sequences from these organisms, it is known that they constitute primarily a subset of unique Pseudomonas and Arthrobacter species. They clearly have the capacity to exist in a viable but dormant state for very long periods of time. These organisms appear to have a highly developed capacity to repair their DNA as evidenced by their very high resistance to UV radiation. The ability of Earth microbes with a full complement of enzymes to exist in relatively suspended animation for extended periods, yet to be ready for instant growth, has direct implications for planetary protection requirements related to forward contamination as well as sample return. The ability to maintain efficient DNA repair in the absence of cell division (which these organisms are apparently able to do) is a property that may be of great advantage for long-term survival in space and on Mars. Extreme Halophiles A preliminary (and as yet unpublished) report involving halophilic bacteria was presented to the task group and deserves at least a passing mention here. Microorganisms embedded in crystals of salt have recently been identified at the DOE Waste Isolation Pilot Project high-intensity storage site in New Mexico. The salt deposits have been dated as being approximately 200 million years old. Extremely halophilic bacteria, of the domain Archaea, have been cultured from the interior of the salt crystals, which suggests the possibility that bacteria have remained viable within the salt crystals. Such a potential for extended survival and the capacity of these organisms for growth in brines, which may be present in the martian subsurface, make this group of extreme halophiles very interesting with regard to possible types of organisms to look for in the search for extant or past life on Mars. With regard to forward contamination, the possibility that such an organism could reach a suitable environment, even if it survived the trip through space, seems vanishingly small. Cryptoendoliths Some microorganisms in the Antarctic have adapted to extremes of low temperature, high winds, and lack of water by forming communities 34

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within sandstone. Inside the rock, an increased relative humidity provides adequate water for growth, and light penetration is adequate for photosynthesis for very short periods that occur no more often than 2 to 5 days per year.17 For example, in the Dry Valley region of Antarctica, a microbial ecosystem exists in the interstices of porous sandstone, complete with primary producers (lichen algae, green algae, and cyanobacteria) and primary microbial consumers (decomposers such as yeast, bacteria, and filamentous fungi), but lacking higher trophic-level consumers.18,19 This microbial community has apparently adapted to life in these rocks to avoid the harsh external conditions, which include (1) high-velocity winds, (2) low temperatures (the rocks warm to above-freezing temperatures due to their low albedo and high thermal inertia), (3) low moisture (the rocks retain water from snow melt), and (4) high UV flux on the surface. Conditions of light, temperature, and water that permit slow metabolic rates occur only rarely, perhaps for about 100 to 200 hours per year, and rates of cellular metabolism and growth in these communities are perhaps the lowest found on Earth. (For example, the carbon turnover, a reflection of metabolic rate, has been estimated to be on the order of 10,000 to over 20,000 years.20 All the inorganic nutrients needed for growth come from the minerals in the rock matrix and thus are not limiting to the community.21 The community can carry out photosynthetic metabolism at temperatures as low as -8°C. Cryptoendolithic lipids, which can stay fluid to -20°C, may be important for the organisms to metabolize in such cold conditions.22 Clearly these microbes have adapted to harsh environmental conditions, and these communities may provide reasonable models for survival strategies that might be adopted by microbes as conditions change from above-freezing temperatures and flowing water to temperatures below 0°C and limited free water. Barophiles If liquid water is present at kilometer depths on Mars, microbial life in that environment may face environmental conditions similar to those experienced by the barophilic bacteria isolated from the deep sea on Earth. Pressure is a significant factor in the growth of the barophilic bacteria, with the optimal pressure for growth being similar to the pressures found in the deep-sea regions from which the bacteria were isolated.23 Studies of these bacteria will help to establish the limits of pressure that can be tolerated by life on Earth and will guide future life- detection experiments conducted beneath the surface of Mars. For instance, is it possible that liquid water exists at depth (due to geothermal heating) and supports a community of barophilic organisms similar to those described for Earth organisms? 35

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Radiation-Resistant Bacteria There is a wide range of microbial sensitivity to radiation stress induced by either UV or ionizing radiation. Although the two types of radiation are physically quite different, they are usually considered together, as the site of damage for both is the genetic material (DNA), and the modes of coping with the ensuing radiation damage are fundamentally similar.24 Although microorganisms are generally resistant to the radiolytic effects of low-level25 and chronic irradiation, only a few notable species are known to survive high levels of irradiation even remotely similar to those that would be faced on Mars, or in interstellar space. Vegetative cells of Deinococcus (formerly Micrococcus) radiodurans isolates can typically withstand doses of UV light and gamma radiation characteristically withstood by bacterial spores. The dose-response curves show large shoulders that extrapolate to 500 Jm-2 for UV radiation and 700 Krad for gamma radiation. Some isolates have been found to survive single doses of 104 ergs cm-2, or 106 rads.26-28 In addition, within a core of the Three Mile Island reactor and other commercial reactors, microorganisms have been isolated after exposure to very high levels of radiation. Probably all vegetative cells, even those of the extremely resistant forms, possess similar mechanisms for coping with radiation stress.29 These mechanisms have been studied intensively in Escherichia coli and other well-characterized bacteria. The alterations that occur and that allow organisms to tolerate levels of radiation flux higher by orders of magnitude than those tolerated by E. coli are not yet well characterized. In general, both prokaryotes and eukaryotes show greater rates of mutation when subjected to increased UV fluxes, and these mutations are thought to be induced during the processes that repair radiation-induced lesions. Although it seems clear that increased mutagenesis is associated with rapid DNA repair, the repair mechanisms are not well understood for populations subject to UV stress for extended periods of time. It should also be mentioned that the studies of these responses discussed here have focused primarily on vegetative cells; as discussed elsewhere, the situation for the highly resistant bacterial spores might be quite different. LIFE DETECTION FOR PLANETARY PROTECTION (INCLUDING BIOBURDEN DETERMINATION) Techniques for assessing the existence of microorganisms have advanced dramatically since pre-Viking days, and these advances will strongly affect bioburden assessment procedures as well as future life- detection experiments. Until the mid 1970s, the major methodology used for detecting microbes was the counting of viable colony-forming units (CFUs) on various defined media. NASA procedures carefully outlined the swabbing procedures and media to be used to assess "cleanliness."30,31 36

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Beginning with epifluorescence microscopy, new methods with greater sensitivity and specificity have rapidly appeared. The task group strongly recommends that efforts be made to explore current analytical methods for use in bioburden assessment and inventory procedures. New procedures for bioburden assessment must be established before the spacecraft that are designed to detect life are assembled and launched. In addition to the epifluorescent microscopic techniques developed for counting viable cells, many other new methods have been developed that involve the detection of specific biomarkers that are components of cells. Such biomarkers may provide a sensitive means for detection without the necessity for release of attached microbes from the substratum (needed for most microscopic counting) or the efficient growth of each propagule. Circumventing the requirement for cultivation is crucial, since it is estimated that fewer than 10 percent of the microorganisms present in most environmental samples have been cultivated. Thus there is a risk that techniques that rely on cultivation will not detect the majority of the microbial population in a given sample. In addition to obviating the need for cultivation, these techniques are appealing because of their extreme sensitivity. In some cases, single cells can be detected and identified. However, due to this sensitivity, life- detection experiments using these techniques may be compromised if the bioload of the spacecraft is not also monitored using the same technologies. Viable But Nonculturable Organisms A recently recognized problem is that some organisms are fully functional even though they are not culturable with the usual microbiological techniques. This has been shown most clearly with the cholera-causing pathogen Vibrio cholerae. This organism, when attached to chitin substrata under starvation conditions, is not culturable in any of the standard media, but it is fully infectious if given to a suitable host animal .32 It is now recognized that nonculturability is a widespread phenomenon in environmental samples. In surface soils, direct microscopic counts of stained bacteria show that less than 1 percent of the organisms seen by epifluorescence microscopy can subsequently be recovered by direct plating and grown to form colonies. Clearly, the previously used procedures for counting viable organisms are insufficient to help assess potential contamination by organisms that could possibly reproduce on another planet or on spacecraft components. 37

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Epifluorescence Microscopy In the early 1970s epifluorescence microscopy began to be used for the detection of potentially viable (i.e., nucleic acid-containing) microbes. The method involves using fluorescent dyes, such as acridine orange, that bind to DNA and RNA and then directly examining and counting fluorescent particles under UV illumination. Such procedures showed that viable count methodology (e.g., the methods used in bioload assessment for the Viking mission) drastically underestimates the actual microbial population. Although obtaining acridine dye epifluorescent counts cannot give information as to species composition, coupling fluorescent microscopy to other approaches can do so. Specific oligonucleotide probes labeled with fluorescent dyes can be used to identify and quantitate individual taxonomic groups.33,34 Such technology may have great importance in the identification and quantitation of targeted groups of microbes during bioburden assessment. Lipids as Biomarkers One technique that has recently been widely used to detect and identify microbes is based on the extraction of membrane lipids.35 Lipids provide two advantages as biomarkers. The extraction of lipids from cells in their environmental matrix is quantitative and allows both a simple purification as well as a concentration step. Extraction has classically been performed with a one-phase chloroform-methanol-water system that requires the use of potentially toxic solvents as well as a prolonged period of exposure to the extraction solutions. Recent use of supercritical fluid carbon dioxide with suitable polar modifiers has made rapid and semi- automatable extraction techniques possible. Detailed analysis of the extracted lipid biomarkers provides quantitative evidence for the presence of viable components of the microbial community. The polar lipid fraction of the extract is polar by virtue of the presence of primarily phosphate esters. These polar lipid phosphate esters are metabolically labile. During growth or after death, the polar lipids show a relatively rapid turnover by phospholipases inside or external to the cells so that the polar lipid content rapidly disappears from nonliving cells. The dephosphorolated neutral lipid molecular components of the original polar lipids are then readily detected. Consequently, the detection of specific polar lipids provides a quantitative definition of the viable or potentially viable cellular biomass and requires no growth or recovery of intact microbes. Because different groups of microbes contain identifiable specific patterns of lipid components, detailed examination of the structure of the lipid allows definition of the community structure of the microbial 38

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community. The changes in the lipid component structures also correlate with the nutritional status of the microbiota and with recent exposures to some toxic stresses. Thus the lipid analysis can provide direct evidence of lipid synthetic gene activity as well as the viable biomass, community structure, and nutritional status of the community. Since this technique could provide a means to detect the presence of extant or fossil life on Mars, it is important to prevent potential contamination of spacecraft by specific microbial lipids. Nucleic Acids as Biomarkers Nucleic acids are the second group of biomarkers that have received considerable attention in the past several years. Both RNA and DNA provide suitable markers for identifying and quantitating groups of organisms or individual strains of microorganisms. Much of this research in microbial ecology has focused on the use of ribosomal RNAs (rRNAs), both for the identification of microorganisms and for the production of unique nucleic acid probes used for quantitation and for in situ hybridizations. One obvious advantage to using nucleic acids for microbial identification is that limiting amounts of nucleic acids can readily be amplified up to a millionfold, permitting the analysis of only a few molecules of nucleic acid. Amplifications are currently performed either with the polymerase chain reaction, in which a thermostable DNA polymerase is used to amplify a template following denaturation of the template DNA, annealing of suitable primers, and extension of the primed template; or with self-sustained sequence replication, an isothermal mode of amplification modeled after viral replication. Following the acquisition of nucleic acids, the targeted gene sequences (usually from an rRNA-encoding gene) are analyzed and used to determine the identity of microorganisms in the sample. This information can also be used to design nucleic acid probes for use in future studies and for monitoring the relative abundances of microorganisms in a sample. Since this technology is currently available, life-detection experiments that may use these techniques need to be conducted in an environment that is not contaminated by nucleic acids. Detection of Spore-forming Bacteria The swab-and-culture technique used to detect spore-forming microbes as sterilization-resistant contaminants can be improved by biomarker recovery, which obviates the requirement that organisms be cultured for detection. Recovery of 2-4 diamino pimelic acid and/or a signature rRNA sequence could provide a quantitative biomarker for gram-positive sporeforming bacteria. 39

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Detection of Chirality as an Indicator of Bioprocesses It is particularly important to apply stringent bioload-reduction technology to those missions anticipated to involve the detection of past or present life. One of the most sensitive detection methods will involve the determination of a significantly greater than expected chirality in components of polymers such as peptides. This is one of the most characteristic features of life on Earth. Recent advances in the use of chiral derivatizing agents or stationary phases in column chromatography coupled with the detection of specific analytes on cooled germanium disks (which allow matrix-assisted microscopic Fourier transform infrared spectroscopy) provide ultrasensitive methodology that could be adapted to systems for detecting microbial contamination of spacecraft. It should be mentioned, however, that such techniques require significant amounts of material in comparison to molecular amplification methods such as the polymerase chain reaction. REFERENCES 1. Space Science Board, National Research Council. 1978. Recommendations on Quarantine Policy for Mars, Jupiter, Saturn, Uranus, Neptune, and Titan. Committee on Planetary Biology and Chemical Evolution. National Academy of Sciences, Washington, D.C. 2. Waldrop, M.M. 1990. "Goodbye to the Warm Little Pond?" Science 250:10781080. 3. Achenbach-Richter, L., R. Gupta, K. Stetter, and C. Woese. 1987a. "Were the Original Eubacteria Thermophiles?" Syst. Appl. Microbiol. 9:34-39. 4. Achenbach-Richter, L., K. Setter, and C.R. Woese. 1987b. "A Possible Missing Link Among Archaebacteria." Nature 327:348-349. See also Achenbach-Richter, L., et al., 1987a. 5. Woese, C.R. 1987. "Bacterial Evolution." Microbiol. Rev. 51:221-271. 6. Koch, R., P. Zablowski, A. Spreinat, and G. Antranikian. 1990. "Extremely Thermophilic Amylolytic Enzyme from the Archaebacterium Pyrococcus furiosus." FEBS Microbiol. Lett. 71:21-26. 7. Constantino, H.R., S.H. Brown, and R.M. Kelly. 1990. "Purification and Characterization of an a-glucosidase from a Hyperthermophilic Archaebacterium, Pyrococcus furiosus, Exhibiting a Temperature of 105 to 115°C." J. Bacteriol. 172:3654-3660. 8. Mukund, S., and M. Adams. 1990. "Characterization of a Tungsten- Iron-Sulfur Protein Exhibiting Novel Spectroscopic and Redox Properties from a Hyperthermophilic Archaebacterium Pyrococcus furiosus." J. Biol. Chem. 265:11508-11517. 40

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9. Bauthier de la Tour, C., C. Portemer, M. Nadel, K. Stetter, P. Forterre, and M. Duguet. 1990. "Reverse Gyrase, a Hallmark of the Hyperthermophilic Archaebacteria." J. Bacteriol. 172:6803-6808. 10. Horneck, G., H. Buecker, and G. Reitz. 1991. "Long-term Exposure of Bacterial Spores to Space." Nature (London) (in press). 11. Weber, P., and J.M. Greenberg. 1985. "Can Spores Survive in Interstellar Space?" Nature 316:403-407. 12. Lindberg, C., and G. Horneck. 1991. "Action Spectra for Survival and Spore Photoproduct Formation of Bacillus subtilis Irradiated with Short-wavelength (200-300 nm) UV at Atmospheric Pressure and in Vacuo." J. Photochem. Photobiol. B:Biol. 11:69-80. 13. See Lindberg and Horneck, 1991. 14. See Weber and Greenberg, 1985. 15. Fliermans, C., and D. Balkwill. 1989. "Microbial Life in Deep Terrestrial Subsurfaces." BioScience 39:370-377. 16. Phelps, T.J., E. Murphy, S. Pfiffner, and D.C. White. 1992. "Comparison of Geochemical and Biological Estimates of Subsurface Microbial Activity." Appl. Environ. Microbiol. 58 (in press). 17. Johnston, D., and J. Vestal. 1991. "Photosynthetic Carbon Incorporation and Turnover in Antarctic Cryptoendolithic Microbial Communities: Are They the Slowest Growing Communities on Earth?" Appl. Environ. Microbiol. 57:23082311. 18. Vestal, J.R. 1988. "Primary Production of the Cryptoendolithic Microbiota from the Antarctic Desert." Polarforschung 58:193- 198. 19. Friedmann, E.I. 1980. "Endolithic Microbial Life in Hot and Cold Deserts." Origins Life 10:223-235. 20. Vestal, J.R. 1988. "Carbon Metabolism of the Cryptoendolithic Microbiota from the Antarctic Cold Desert." Appl. Environ. Microbiol. 54:960-965. See also Johnston and Vestal, 1991. 21. Johnston, C.G., and 1.R. Vestal. 1989. "Distribution of Inorganic Species in Two Cryptoendolithic Microbial Communities." Geomicrobiol. J. 7:137-153. See also Vestal, J.R., 1988. 22. Finegold, L., M. Sincer, T. Federele, and J. Vestal. 1990. "Composition and Thermal Properties of Membrane Lipids in Cryptoendolithic Lichen Microbiotat from Antarctica." Appl. Environ. Microbiol. 56:1191-1194. 23. Yayanos, A. 1986. "Evolutional and Ecological Implications of the Properties of Deep-sea Barophilic Bacteria." Proc. Natl. Acad. Sci. USA 83:9542-9546. 24. Bridges, B.A. 1976. "Survival of Bacteria Following Exposure to Ultraviolet and Ionizing Radiations." Symp. Soc. Gen. Microbiol. 26:183-208. 25. Frances, A.J. 1985. "Low-level Radioactive Wastes in Subsoils." In Soil Reclamation Processes: Microbiological Analyses and 41

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Applications. R.L. Tate and D. Klein (eds.). Marcel Dekker, Inc., New York. 26. See Bridges, B.A., 1976. 27. McCabe, A. 1990. "The Potential Significance of Microbial Activity in Radioactive Waste Disposal." Experientia 46:779-787. 28. Nasim, A. 1978. "Life Under Conditions of High Irradiation." In Microbial Life in Extreme Environments. D.J. Kushner (ed.). Academic Press, London. 29. See Bridges, B.A., 1976. 30. National Aeronautics and Space Administration (NASA). 1980. NASA Standard Procedures for the Microbiological Examination of Space Hardware. NHB 53401B. NASA, Washington, D.C. 31. National Aeronautics and Space Administration (NASA). 1990. Lessons Learned from the Viking Planetary Quarantine and Contamination Control Experience. NASA Contract Document No. NASW-4355. NASA, Washington, D.C. 32. Colwell, R.R., P.A. West, D. Maneval, E.F. Remmers, E.L. Elliott, and N.E. Carlson. 1984. "Ecology of Pathogenic Vibrios in Chesapeake Bay." Pp. 367387 in Vibrios in the Environment. R.R. Colwell (ed.). John Wiley and Sons, New York. 33. Sayler, G.S., and A.C. Layton. 1990. "Environmental Application of Nucleic Acid Hybridization." Ann. Rev. Microbiol. 44:625-648. 34. DeLong, E.F., G.S. Wickham, and N.R. Pace. 1989. "Phylogenetic Stains of Ribosomal RNA-based Probes for the Identification of Single Cells." Science 245:1360-1363. 35. Wilkinson, S.G., and C. Ratledge. 1988. Microbial Lipids. Volume 1. Academic Press, New York, 750 pp. 42