Heterotrophic Microbial Colonization of the Interior of Shocked Rocks from the Haughton Impact Structure, Devon Island, Nunavut, Canadian High Arctic David Andrew Fike Churchill College Thesis submitted in partial fulfillment of the requirements for the degree of Master of Philosophy in Polar Studies. Scott Polar Research Institute University of Cambridge June 2002 12..?~9? ~TJ1,) Declaration In accordance with the University of Cambridge regulations, I do hereby declare that: This thesis represents my own original work and conforms to accepted standards of citation in those instances in which I have availed myself of the work of others. This thesis is not now being submitted, nor has been submitted in the past, for any other degree, diploma or similar qualification at any university or similar institution. This thesis does not exceed the maximum allowable length of 20,000 words, excluding footnotes, tables, appendices, and references. ~// David A. Fike Abstract The polar desert is one of the most extreme environments on Earth. In these regions, microorganisms have had to develop novel strategies and adaptations in order to survive. One of the most effective such strategies has been developed by mkroorganisms, known as endoliths, which live in the interior of rocks, escaping or mitigating the hazards of the polar desert and fully utilizing the resources available in the rock environment. The most studied groups of polar endoliths are near-surface phototrophic communities inhabiting porous sedimentary rocks in Antarctica. Here we examine a novel environment for endolithic communities: crystalline rocks that have undergone shock metamorphosis as a result of a comet or asteroid impact. Specifically, we present a characterization of the heterotrophic endolithic community and its environment in the interior of impact-shocked gneiss and breccia samples from Haughton Impact structure on Devon Island, Nunavut, in the Canadian High Arctic. The high-latitude and arid, polar climate at Haughton preclude significant populations of higher-order organisms, naturally restricting the impact structure ecosystem to microbial communities. As such, it provides a unique opportunity to examine, in a natural setting, the microbiological colonization of impact-shocked rocks. This colonization is facilitated primarily by the creation of interconnected fissures and vesicles throughout the sample, which serve as microbial habitats. Twenty-seven heterotrophic bacteria have been isolated from the samples of shocked rocks: fourteen from shocked gneiss and thirteen from breccia. Genes encoding the 16S rRNA of the isolates were sequenced to identify the isolates and characterize the community inhabiting the shocked rocks. The bacteria inhabiting the shocked gneiss and the breccia show great similarity to each other, and also to other heterotrophic communities isolated from polar environments. Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis were used together to document the in situ growth of these microbes, either in small groups or in large biofilms, in the interior of the samples, where they take advantage of impact-induced inhomogeneities in surface composition and inhabit cavities created by the impact-induced shock. The interiors of shocked crystalline rocks are observed to provide abundant habitats for heterotrophic bacteria, particularly as compared to unshocked samples, demonstrating, through habitat generation, the beneficial role that impact events can play in microbial ecosystems. The discovery of these heterotrophic communities within impact-shocked crystalline rocks extends our knowledge of the habitable biosphere on Earth. The colonization of the interiors of these samples has significant astrobiological applications both for considering terrestrial, microbiological contamination of meteorites from the Antarctic ice sheet and for investigating possible habitats for microbial organisms on the early Earth, and more speculatively, on Mars. 11 Table of Contents Declaration Abstract Table of Contents List of Tables and Figures List of Terms List of Abbreviations Ac know ledgments Chapter 1: Introduction Polar Desert Advantages of an Endolithic Existence in a Polar Desert Endolithic Community Structure Mechanisms of Bacterial Deposition into Rocks A Comparison of Arctic and Antarctic Environments Containing Endoliths Rocks Hosting Endoliths Meteorites Description of Present Study Chapter 2: Materials and Methods Study Site Sample Collection Physical Characterization of Samples Inductively Coupled Plasma-Atomic Emission Spectroscopy Biological Characterization of Samples Isolation of Bacteria 16S rRNA Sequencing of Isolates Microscopic/Microanalytic Characterization of Samples Scanning Electron Microscopy Energy Dispersive X-ray Spectroscopy Chapter 3: Results Physical Characterization of Samples Inductively Coupled Plasma-Atomic Emission Spectroscopy Biological Characterization of Samples Isolated Heterotrophic Bacteria Microscopic/Microanalytic Characterization of Samples Scanning Electron Microscopy Energy Dispersive X-ray Spectroscopy Chapter 4: Discussions Geological Interpretations Biological Interpretations Comparison with Known Polar Heterotrophic Communities Origin of Isolates iii II 111 V VI Vil Vlll 1 8 10 13 15 16 16 18 19 24 24 24 26 26 26 29 30 30 32 32 35 35 42 42 47 51 52 52 54 Assessment of SEM/EDX Technique for Identifying Endolithic Microorganisms Relevance to Meteorites Life on Mars Chapter 5: Summary Conclusions Future Work References IV 55 55 56 57 58 59 - --- List of Tables 1. Nomenclature for Organisms in Low-Temperature and Low-Nutrient 6 Environments 2. Nomenclature for Lithophytic Organisms 7 3. Bacterial Metabolisms 11 4. Mineralogy of Gneiss and Breccia from the Haughton Impact Structure 21 5. Physical Parameters of Unshocked and Shocked Gneiss from Haughton 22 Impact Structure 6. Mineralogy of Unshocked Gneiss, Shocked Gneiss, and Breccia 33 7. Trace Element Concentrations of Unshocked Gneiss, Shocked Gneiss, and Breccia 34 8. Phylogenetic Identification of Isolates from Shocked Gneiss, Breccia, 37 and Antarctic Soil. 9. Known Habitats of Identified Bacteria. 39 10. Summary of Physiological and Growth Characteristics for Isolated Bacteria. 41 List of Figures 1. Lithophytic Organisms and Their Relationship with the Rock Surface 7 2. Geographical Location 20 3. Unshocked and Shocked Gneiss 23 4. Isolation of Bacteria 27 5. Select Bacterial Isolates 36 6. SEM Images of Unshocked Gneiss, Shocked Gneiss, and Breccia 43 7. SEM Images of Bacteria on Shocked Gneiss 44 8. EDX Spectra 48 9. EDX Transects across Surfaces of Unshocked and Shocked Gneiss 50 V List of Terms Aerobe: Organism that can grow and metabolize in the presence of oxygen. Autotroph: Organism whose cellular carbon is derived from inorganic carbon (C02). Chemotroph: Organism whose energy is derived from the oxidation of chemical compounds. Chemotrophs can be divided into two categories: chemoorganotrophs (energy derived from the oxidation of organic chemicals); chemolithotrophs (energy derived from the oxidation of inorganic chemicals). Endolith: Organism living in the interior of a rock. Facultative anaerobe: Aerobic organism that can also grow and metabolize in the absence of oxygen. Gneiss: Metamorphic rock that has typically been subjected to deep burial, and exhibits a segregation of minerals into separate bands of different composition. Heterotroph: Organism whose cellular carbon is derived from organic carbon compounds. Meteorite: A solid mass of mineral or rock matter, derived from a comet or asteroid, but which did not completely vaporize in the Earth's atmosphere, nor upon impact with the ground. Obligate aerobe: Organism that can only grow and metabolize in the presence of oxygen. Obligate oligotroph: Organism adapted to low-nutrient environments. Optimal growth exhibited in low-nutrient conditions. Oligotroph: Organism inhabiting low-nutrient environments. Optimal growth exhibited in abundant nutrient conditions. Omithogenic: Derived from birds (refers to soils in Antarctica). Phototroph: Organism whose energy is derived from photochemical reactions. Psychrophile: Organism adapted to cold environments. Optimal growth exhibited below 15°C. Psychrotroph: Organism inhabiting cold environments. Optimal growth exhibited above 20°C. 16S rRNA: A conserved portion of the ribosomal RNA. It is used to identify organisms and determine evolutionary relationships. 16S rDNA: DNA genes encoding the 16S rRNA. VI -~- List of Abbreviations BAS: British Antarctic Survey (National Environmental Research Council), Cambridge, UK. EDX: Energy Dispersive X-ray Spectroscopy (also known as EDS). GenBank: Genetic database containing 16S rDNA sequences of organisms. 0Pa: Giga-Pascal = 109 Pascal= 109 N/m2 (measure of pressure). ICP-AES: Inductively Coupled Plasma - Atomic Absorption Spectroscopy. PAR: Photosynthetically Available Radiation (usable by organisms for photosynthesis). PCR: Polymerase Chain Reaction (process to amplify DNA prior to sequencing). rDNA: ribosomal DNA (deoxyribonucleic acid). rRNA: ribosomal RNA (ribonucleic acid). SEM: Scanning Electron Microscopy. SPRI: Scott Polar Research Institute, University of Cambridge, Cambridge, UK. TSA: Tryptone Soy Agar. UV: Ultraviolet radiation (wavelengths between 200 - 400 nm). UVB: most dangerous category of UV radiation for organisms on the surface (280 - 320 nm). VII Acknowledgments I would like to acknowledge the support and assistance that made this research possible. Foremost, I would like to thank my supervisor Dr. Charles Cockell (British Antarctic Survey), under whose guidance this study took shape and from whom I have learned much over the past year. I would also like to thank Dr. David Pearce (British Antarctic Survey) for his work with the PCR and sequencing of the isolated bacteria, Dr. Jon Ward (British Antarctic Survey) for access to the SEM and EDX machines, and Jacqui Duffett and Dr. Sarah James for the use of the ICP-AES machine and sample preparation facilities at Royal Holloway, University of London. I am grateful to the Scott Polar Research Institute (SPRI) for providing me with support, friendship, and the opportunity to conduct this research. Finally, I would like to acknowledge the funding of my research by the Winston Churchill Foundation, and funding for the presentation of my research at the 2002 Astrobiology Science Conference at NASA Ames Research Center by the Scott Polar Research Institute and Churchill College. viii Polar Desert Chapter 1 Introdu~tion The polar desert is one of the least hospitable biomes on the Earth. The principle challenges faced by organisms attempting to survive in such regions are extremely low temperatures and limited nutrient availability (Wynn-William, 2000). These challenges are inescapable for organisms living in this environment. Organisms can react in two ways when faced with such environmental stresses: they can either yield to the stress conditions and make suitable provisions for survival, or attempt to resist the stress (Franks et al., 1990). The organisms surviving in a polar desert have either evolved a mechanism to protect themselves from these extremes or adopted a strategy to allow for survival in spite of them (see Table 1). The primary challenge facing organisms in a polar desert is to survive and metabolize under extremely low temperatures. Temperature extremes to which polar organisms are exposed can reach -41.2°C at Mount Fleming in Antarctica (Wynn-Williams, 2000) or -26.l°C at Haughton impact structure, Devon Island in the Arctic (Cockell et al., 2002), and these temperatures can be sustained for several months during the polar winter. However, in spite of these temperatures, surviving bacterial communities have been found at Mount Fleming, where at the rock surface the annual mean temperature was -24.2°C and the minimum temperature was -41.2°C (Wynn- Williams, 2000). The organisms that can survive in these low temperature environments may be divided into psychrotrophs and psychrophiles. Psychrotrophs have not evolved specific adaptations to the extremely low temperatures of the polar desert, and merely tolerate cold environments, where their growth is sub-optimal (Morita, 2000a). Although growth can occur around 4 °C, optimal growth temperatures are between 15°C and 35°C (Bowman' et al., 1997). The majority of heterotrophic bacteria that have been isolated in the Arctic and Antarctica are psychrotrophs (Franzmann, 1996). These organisms have optimal growth temperatures significantly higher than the usual temperature of their environment, suggesting poor adaptation to their environment, because for optimal fitness, the maximal growth rate should occur within the environmental range of the habitat (Franzmann, 1996). 1 On the other hand, psychrophilic organisms thrive in and actually require cold environments (Morita, 1975). They have minimal growth temperatures less than 0°C, optimal growth temperatures below 15°C, and show no growth above 20°C (Morita, 2000a; Bowman et al., 1997; Morita, 1975). Alternately, Franzmann et al. (1990) used the operational definition of psychrophiles as those strains that grow at I0°C, but not at 25°C. Psychrophiles have evolved specific mechanisms to cope with the thermal extremes of the polar desert (Morita, 2000a). As an example, Kappen and Friedmann (1983) found that metabolic activity among psychrophiles occurred at temperatures down to -I0°C. However, at temperatures much below -I0°C, the metabolism of (known) psychrophiles ceases and these microorganisms enter cryobiosis, a state of anabiosis (dormancy) resulting when ambient temperatures decrease below the minimal growth temperature (Morita, 2000a). When temperatures rise above the minimal growth temperature, these microbes resume normal function (Morita, 2000a; Nienow & Friedmann, 1993). Examples of particular adaptations these organisms have evolved to survive and increase their productivity in these environments include: the development of enzymes capable of functioning at low temperatures; and the ability to manipulate cell membrane composition (Feller et al., 1996; Nichols et al., 1995; Rotert et al., 1993). In order for growth in polar environments, enzymes need to possess the ability to catalyze reactions at a sufficient rate despite extremely low temperatures (Nienow & Friedmann, 1993). Psychrophilic organisms have evolved the ability to produce enzymes adapted to function at low temperatures (Feller et al., 1996). These enzymes are structurally different than their counterpart enzymes in mesophilic or thermophilic organisms; enzymes isolated from psychrophilic organisms are characterized by increased structural flexibility as .a result of weakened intramolecular interactions and increased enzyme- solvent interactions (Feller et al., 1996). The result is that enzymes isolated from psychrophiles can catalyze reactions three times faster at temperatures aroun_d 25°C, six times faster at I0°C, and approximately ten times faster around 4°C (Feller et al., 1996). The cost for these low temperature adaptations, however, is a decrease in the thermostability of these enzymes, which characteristically undergo rapid inactivation at temperatures above 30°C (Feller et al., 1996). A functional, fluid cell wall is another requirement for cell survival and growth (Rotert et al., 1993). Low temperatures increase the crystalline phase of cell membranes, which is detrimental to cell funct ion (Nichols et al., 1995). Optimal growth rates can be maintained as 2 long as 50% of the membrane remains in a fluid state; a cell with a membrane that is less than 50% fluid has sub-optimal growth, which can be sustained until membrane fluidity decreases to 5 _ 10% (Nichols et al., 1995). To prevent cell membranes from entering the crystalline phase, many bacteria regulate their membrane phospholipids in response to temperature. This is achieved by manipulation of cellular fatty acid composition, increasing the proportion of lower melting-point fatty acids in response to decreased ambient temperature (Rotert et al., 1993). This can involve three approaches: increasing the degree of unsaturation of individual fatty acids, and/or the overall production of unsaturated fatty acids ; shortening the fatty acid chain length; and/or increasing the proportion of methyl-branched-chain fatty acids (Nichols et al., 1995). While such changes can be observed phenotypically, the capacity to perform them must have evolved in the genome. The production of polyunsaturated fatty acids (PUFAs) appears to be a key physiological adaptation, which bacteria from the Antarctic appear to have evolved (Nichols et al., 1995). While Kappen & Friedmann (1983) observed growth to cease at temperatures below -10°C, experiments have shown that a PUFA membrane can remain sufficiently fluid to support growth to temperatures below -20°C (Nichols et al., 1995), indicating that there are perhaps 'extreme' psychrophiles, as yet undetected, with similar abilities in nature. It is logical to believe that in a polar desert there should only be psychrophiles, since under the prevailing environmental conditions they would experience optimal growth, while psychrotrophs would endure sub-optimal growth conditions. Harder & Veldkamp (1971) demonstrated in laboratory experiments that psychrophiles out-compete psychrotrophs at low temperatures, because their growth rate is higher at these temperatures. Psychrotrophs are able to overcome this disadvantage by adopting other competitive strategies. Examples of such strategies include increasing substrate affinities, particularly in nutrient-poor environments, so that growth becomes dependent on the concentration of growth-rate-limiting substrate, and not on temperature; or developing broad tolerance for survival in fluctuating extremes, such as temperature, salinity, and relative humidity (Franzmann, 1996). The abundance of psychrotrophs in polar deserts may be explained in part by ambient temperatures that can fluctuate greatly between polar summers and winters, which specialized psychrophiles may not be able to tolerate (Franzmann, 1996). Furthermore, the low abundance of psychrophiles (and the resulting prevalence of psychrotrophs) may be due to the fact that organisms inhabiting these environments may not 3 have had sufficient time for their genomes to evolve to suit the polar environment (i.e., to become 'psychrophiles'). The evolution of psychrophiles has paralleled the development of permanently cold regions on Earth, those with temperatures less than 5°C, which have only existed on Earth for the last 36 - 38 million years (Gazdzicki et al., 1992, cited in Franzmann, 1996; Kennet & Shackleton, 1976). The divergence of genes from ribosomal RNA (rRNA) is observed to proceed at a rate of 1 % every 25 - 50 million years (Moran et al., 1993; Ochman & Wilson, 1987). Although it is not known how much of the genome must be changed to shift the minimal and optimal growth temperatures of organisms, it is presumably a significant amount and Franzmann (1996) estimates that considerable shifts in these temperatures may take millions of years. Therefore, it is ljkely that the observed abundance of psychrotrophs, in environments apparently more suited to psychrotrophs, may be partly due to the slow pace of evolution, as these organisms adapt to these relatively 'new' permanently cold environments. As in all biological systems, there is no sharp discontinuity between psychophiles and psychrotrophs. In practice, there is a continuum of organisms that ranges between 'extreme' psychrophiles, those with optimal growth temperatures below 7°C, to moderate psychrophiles, to psychrotrophs, to mesophiles, and to thermophiles (Nichols et al., 1995). For example, psychrotrophic Antarctic species do generally show reduced optimal growth temperatures (on the order of 10° - 20°C) when compared with their nearest non-Antarctic taxonomic counterparts (Franzmann, 1996). Although these temperatures are still higher than those for organisms deemed 'psychtophilic', this supports the suggestion that psychrotrophs are in the process of evolving to their environment, and, with time, would likely have temperature characteristics more similar to psychrophiles. Further complications arise when using these terms, because the label 'psychrophile' or 'psychrotroph' is customarily applied based upon observations of an organism under particular environmental conditions; however, the optimal, maximal, and minimal growth temperatures for organisms are observed to vary with experimental conditions (Nichols et al., 1995). For · example, a decrease in water activity results in an increase to the minimum growth temperature (Nichols et al., 1995). Further, salinity has been found to affect the maximum growth temperature of several psychrophilic and psychrotrophic organisms. In one case, the obligate psychrophile Vibrio marinus MP-I, was able to grow at 21.2°C in 3.5% salinity, but had a 4 maximum growth temperature of 10.5°C when the salinity was decreased to 0.7% (Nichols, et al., 1995). The second maJor hazard to which organisms m a polar desert are exposed is the oligotrophic (low nutrient) environment. Organisms that have successfully dealt with this challenge can be grouped into two categories: oligotrophs and obligate oligotrophs. Oligotrophs are capable of surviving and reproducing under low nutrient conditions, although these organisms have not evolved any particular adaptations to increase their fitness in the low nutrient environment (Morita, 2000b). As a result, their growth under these conditions is sub-optimal (Nienow & Friedmann, 1993). On the other hand, obligate oligotrophs have evolved mechanisms to cope with the low nutrient conditions, in which they exhibit optimal growth; however, they are unable to grow and metabolize efficiently in the presence of abundant nutrients (Hirsch et al., 1988; Siebert & Hirsch, 1988; Johnston & Vestal, 1986). While organisms have traditionally been categorized separately based upon their preferred temperature and nutrient ranges, there is evidence that these ranges are coupled (Wiebe et al., 1992). In most studies of psychrophiles and cold-tolerant bacteria, substrate concentrations have been in the grams-per-liter range, and most often the temperatures used have been from 2 - 5°C (Wiebe et al., 1992). However, the question of prime interest to environmental scientists is not at what temperatures or nutrient conditions can these organisms be grown in a laboratory, but what is their growth in the ranges characteristic of their natural environment (Baross & Morita, 1978). Given that, the most interesting question is whether psychrotrophic or psychrophilic organisms dominate under the actual environmental conditions. Bacteria isolated on low-nutrient media at low-temperatures have been found to be more nutritionally versatile, although they are most often neither psychrophiles nor obligate oligotrophs, because they often grow at higher temperatures and nutrient concentrations (Wiebe et al. 1992; Horowitz et al., 1983). Wiebe et al. (1992) examined growth rates over a range of substrate concentrations, where it was noted that, at 15° - 20°C, growth rates of bacteria isolated from temperatures below 0°C were independent of changes to substrate concentrations on the order of 104. However, in every case where the temperature was lowered, an increased substrate concentration was required for optimal growth, with the amount of additional substrate required increasing as temperatures decreased, until a maximum was reached around 4°C (Wiebe et al. 1992). These results demonstrate that extrapolation of temperature or nutrient preference from 5 experimental conditions to in situ conditions is, at best, problematic, and that designations of psychrophile vs. psychrotroph and obligate oligotroph vs. oligotroph have no meaning without the context of the environment in which they were observed (Wiebe et al. 1992). Term Definition Psychrophile Organism adapted to cold environments Optimal growth exhibited below 15°C. Psychrotroph Organism inhabiting cold environments. Optimal growth exhibited above 20°C. Oligotroph Organism inhabiting low-nutrient environments. Optimal growth exhibited in Abundant nutrient conditions. Obligate Organism adapted to low-nutrient environments. Optimal growth exhibited in Oligotroph Low-nutrient conditions. Table 1: Nomenclature for Organisms in Low-Temperature and Low-Nutrient Environments. While there are organisms, such as psychrophiles and obligate oligotrophs, which have evolved mechanisms for overcoming the particular challenges of low temperatures and limjted nutrient availability, organisms in a polar desert must cope with additional environmental difficulties including frequent freeze-thaw cycles, extreme aridity, seasonally high UVB stress, abrasive winds, and a short growing season (Wynn-Williams, 2000; Nichols et al., 1995). In the face of these remaining hazards, many polar microorganisms have developed a strategy that either eliminates or significantly mitigates the danger posed by these environmental conditions. One such adaptation that is widespread in the polar desert is the ability of microorganisms to occupy and exploit lithic (rock) habitats (Nienow & Friedmann, 1993). Microorganisms that use rock as the substratum for their growth are known as lithophytes (see Figure 1; Table 2) and can be divided into three distinct categories: epilithic, hypolithic (or sublithic), or endolithfo. organisms (Nienow & Friedmann, 1993). Epilithic organisms live on exposed surfaces of rocks, whereas hypolithic organisms live underneath small translucent rocks that transmit sufficient light to sustain photosynthesis, albeit at reduced levels (Smith et al. 2000; Nienow & Friedmann, 1993). Studies of these communities reveal that both are composed primarily of cyanobacteria and lichens, although heterotrophic bacteria are also present (Smith et al. 2000; Wynn-Williams et al. 2000; Nienow & Friedmann, 1993; Vincent, 1988). Organisms in both of these groups, while utilizing some of the protective elements of the rocks, remain on the rock's exterior, and an in-depth discussion of their characteristics is beyond the scope of this study. 6 Endolithic organisms inhabit the interior spaces of rocks, where they can fully utilize the protection and resources available beneath the rock surface (Vincent 1988; Nienow & Friedmann, 1993; Wynn-Williams 2000). There are two main forms of endolithic organisms: cryptoendoliths and chasmoendoliths (Nienow & Friedmann, 1993). Cryptoendoliths colonize the interior pore space of rocks, whereas chasmoendoliths inhabit surface fractures in rocks. Although, in principle, these are distinct categories, in practice it is often difficult to make a clear distinction between these two forms of endolithic growth (Nienow & Friedmann, 1993), and the term endolith shall be used throughout this study. Endoliths \ Figure 1: Lithophytic Organisms and Their Relationship with the Rock Surface. Term Definition Epilith Organism that lives on the surface of a rock Sublithic Organism that lives under a translucent rock Endolith Organism that lives in the interior of a rock Cryptoendo lith Organism that lives within the interior pore space of a rock Chasmoendo lith Organism that lives within cracks and crevices of a rock Table 2: Nomenclature for Lithophytic Organisms. · 7 Advantages of an Endolithic Existence in the Polar Desert Rocks serve two primary functions for the endolithic organisms that inhabit them. First, they provide protection from many of the environmental extremes that threaten microbial existence in these surroundings. Secondly, the rocks act as a reservoir for water, nutrients, and heat, giving endoliths an advantage over organisms trying to survive outside the lithic environment. The interior of a rock offers protection from several environmental hazards that otherwise severely limit microbial growth on rock surfaces, including frequent freeze-thaw cycles, seasonally high UVB stress, and abrasive winds (Wynn-Williams, 2000). Temperatures near freezing pose a real danger to organisms in the polar desert as cell walls can rupture (despite their increased flexibility) and/or the cells can be lysed by the formation of internal ice crystals (Feller et al., 1996; Nienow & Friedmann, 1993). As such, rapid, usually wind-driven, temperature fluctuations can be deadly for organisms which otherwise might survive the extreme cold (Wynn-Williams, 2000; Friedmann, 1982). The interior of a rock provides protection from these fluctuations because temperatures slightly below the rock surface vary significantly less than those at the surface, affording endoliths increased protection from freeze-thaw cycles. For example, within a 42-minute period during midsummer on Linnaeus Terrace, Antarctica, the surface temperature of a rock was observed to fluctuate between -l.8°C and 5.9°C, crossing the 0°C freezing point 14 times, whereas over the same interval the temperature 3 mm beneath the rock surface remained positive, fluctuating between 1. 7°C and 6.1 °C (Friedmann, 1982). Endolithic organisms at or below this depth within a rock would be protected from freeze-thaw cycles. Thus, organisms that have adapted to survive within the rocks are buffered from sudden changes in temperature, drastically reducing the number of freeze-thaw cycles they undergo, which has been identified by Friedmann (1982) as one of the primary restrictions on population growth in the polar desert. Additionally, the rocks provide endoliths with protection from excessive doses of UVB radiation and wind abrasion, two of the primary dangers that organisms on the surface must face (Cockell et al., 2002; Wynn-Williams, 2000; Nienow & Friedmann, 1993). While UVB exposure is a hazard for all organisms living at the surface, its danger increases dramatically for those living in polar areas, where, during the summer months, the surface organisms are exposed to UVB continually for up to six months. Furthermore, the presence of polar ozone holes in the 8 atmosphere, which significantly increase the level of UVB reaching the surface, compounds the danger from UVB in polar environments. For endolithic organisms, however, the overlying rock attenuates the UVB and organisms that are greater than 1 mm below the surface of a rock exist in an essentially UVB-free environment (the exact depth associated with this UVB protection depends on the density and translucence of the particular rock and may vary from less than 0.5 mm up to 3 mm) (Cockell et al., 2002). Endolithic organisms living 0.5 mm below the rock surface receive over the course of an Arctic summer the equivalent UVB radiation dose of organisms exposed on the surface for a single day (Cockell et al., 2002). Furthermore, endolithic organisms are protected from the abrasive winds that frequently scour polar deserts, particularly in the Antarctic, where katabatic winds from the continental interior, produced by the flow of cold dense air down a slope, sweep across surfaces at high speeds, essentially precluding epilithic (surface-dwelling) colonization of rocks exposed to the wind. Again, the retreat into the interior of rocks provides endolithic organisms a means of escaping from this hazard. In addition to serving as protection from environment extremes, rocks increase the productivity of the endolithic communities they host by providing water and heat reservoirs as well as a source and reservoir for nutrients. While water is often a scarce commodity on the surface of the polar desert, the interstices and fractures of rocks serve as reservoirs to retain water. For several days following precipitation, the relative humidity in the pore spaces of a rock remains significantly higher than that of the ambient atmosphere, allowing microorganisms within the rock to metabolize and grow for longer periods of time (Nienow & Friedmann, 1993; Kappen et al. 1981). Kappen et al. (1981) report that after a snowfall on Linnaeus Terrace, Antarctica, the relative humidity within the interstitial spaces of a rock remained above 80% (sufficient to allow metabolic activity) for five days, whereas it repeatedly dropped below 20% in the ambient atmosphere over the same length of time. The water retained within rocks after precipitation can dramatically extend the growing season of encloliths, as compared to surface organisms. Rocks provide polar endolithic organisms with a heat reservoir during the months when the sun is above the horizon. Due to their high heat capacity, rocks can reach temperatures that are significantly higher than ambient during these times. As a result of solar heating during the summer months, the surface temperature of rocks has been observed to be as much as 20°C 9 higher than the ambient temperature (Cockell et al., 2002; Nienow & Friedmann, 1993; Vincent 1988). This extra reserve of heat allows endolithic organisms to remain active for significantly longer periods than those living on the surface (Nienow & Friedmann, 1993). Additionally, thermal radiation from the rock can melt nearby snow or ice, increasing the supply of water (and dissolved nutrients) that endoliths may access from within the rock. A further benefit to endolithic organisms is the reserve of nutrients that exists within rocks. Nutrient availability derived from the lithic structure itself varies with rock-type and age, but Hirsch et al. (1988) suggest that it can be sufficient to meet the needs of an endolithic community that lacks primary producers. In addition to its nutrients content, a rock serves as a reservoir for nutrients obtained from the environment. It is estimated that nitrate precipitation from the atmosphere, approximately 21 mg N m-2 yf 1 (R. L. Mancinelli and E. I. Friedman, unpublished, cited in Nienow & Friedmann, 1993), is sufficient to ensure that endolithic communities are not nitrate limited. Furthermore, Johnston and Vestal (1986) have demonstrated no significant increase in primary productivity when a selection of Antarctic endolithic communities were exposed to increased concentrations of nitrate, ammonia, phosphate, manganese, or iron, suggesting that the concentration of these nutrients were not limiting factors on the growth in these communities. In fact, in all cases where phosphate was added to samples, the productivity decreased (Johnston & Vestal, 1986), indicating perhaps that the communities are composed of obligate oligotrophs. Endolithic Community Structure Within an endolithic community there are a variety of different niches filled by microorganisms with different metabolic pathways (Nienow & Friedmann, 1993; Hirsch et al. , 1988; Siebert & Hirsch, 1988). Two useful ways to characterize microorganisms are by the energy and carbon sources that they utilize (Egli, 2000). Microbes have developed two different systems for extracting energy from their environments: phototrophic organisms utilize light, whereas chemotrophs utilize chemicals as their energy source (Yoon et al., 2000; Moat & Foster, 1995). Chemotrophs are further divided into two different categories: chemolithotrophs derive their energy from reduced inorganic chemicals; chemoorganotrophs use reduced organic chemicals as their energy source (Staley, 2000; Moat & Foster, 1995). Microbes can also be distinguished by two different systems for incorporating carbon into their cells: autotrophic 10 organisms are capable of converting inorganic carbon (C02) into organic molecules (Yoon et al., 2000); heterotrophic organisms are unable to utilize C02 and must rely upon exterior sources of organic carbon (e.g., glucose) for their survival (Staley, 2000). The two primary constituents of endolithic communities are photoautotrophs and chemoorganoheterotrophs (Hirsch et al., 1998; Siebert & Hirsch, 1988; Nienow & Friedmann, 1993), although chemolithoautotrophs may play an important role in communities lacking photoautotrophs (Hirsch et al., 1988). Note that since photoheterotrophic organisms are confined to three specialized bacterial groups (green gliding bacteria, Gram-positive bacteria, and photosynthetic Proteobacteria), chemoorganoheterotrophs are customarily referred to simply as heterotrophs (Cavicchioli & Thomas, 2000; Staley, 2000), which will be adopted throughout this study. Term Definition Autotrophy Metabolism in which cellular carbon is derived from inorganic carbon (C02) Metabolism in which cellular carbon is derived from organic carbon Heterotroph y compounds Phototrophy Metabolism in which energy is derived from photochemical reactions Metabolism in which energy is derived from the oxidation of chemical Chemotrophy compounds Metabolism in which energy is derived from the oxidation of organic Chemoorganotrophy chemicals Metabolism in which energy is derived from the oxidation of inorganic Chemolithotrophy chemicals Table 3: Bacterial Metabolisms. The majority of research to-date has focused on the photoautotrophs, such as lichen and cyanobacteria, which are the near-surface, photosynthetic 'primary producers' of the endolithic world (Vincent, 1988; Nienow & Friedmann, 1993). These organisms are easily detected and identified due to their large size and the distinctive pigmentation necessary for the photosynthetic process. Often these organisms appear to have an epilithic origin and have subsequently migrated into the interior of the rock, trading increased protection from temperature fluctuations, UV radiation, and desiccation for decreased photosynthetically available radiation (PAR). This has been observed, for example, in lichen that has abandoned its · characteristic thallus morphology to live within the protected pore spaces of rocks (Nienow & Friedmann, 1993). The photoautotrophic community structure is necessarily limited by the depth to which PAR can penetrate in sufficient quantities to support photosynthesis. PAR decreases by approximately 11 70 - 95% for each mm below the rock surface, although factors such as the presence of pigmented microorganisms and the degree of water saturation can decrease and increase, respectively, this figure by up to an order of magnitude (Nienow & Friedmann, 1993). When the PAR falls below 0.005% of its intensity at the rock surface - at a typical distance of 3 - 5 mm, photoautotrophs can no longer survive (Nienow & Friedmann, 1993). Although Nienow & Friedmann (1993) conclude that endolithic colonization is limited to this few-millimeter-deep zone below the rock surface, they have not taken into account the possibility that heterotrophic endolithic communities could exist at greater depths. This oversight may be due to the envisioned difficulty for heterotrophic organisms to survive in the interior without the photosynthetic 'primary producers' to provide them with the necessary sources of organic carbon. However, as Hirsch et al. (1988) noted, organics within the rock structure itself may be enough to support endoliths in the absence of primary producers. Furthermore, precipitation and snowmelt penetrating into the interior of the samples would allow the accumulation of organics from the phototrophic outer layers. Endolithic organisms in the near-surface environment are not limited to photoautotrophs; a variety of heterotrophic bacteria and fungi are found in this region as well (Wynn-Williams, 2000; Nienow & Friedmann, 1993; Hirsch et al. 1988). In fact, Parker et al. (1977) noted that heterotrophs were by far the most numerous organisms observed in their samples from Dufek Massif, Antarctica. Wynn-Williams (2000) concludes that a large, diverse population of heterotrophic bacteria can survive in this region, either by utilizing extracellular organic chemicals produced by photoautotrophs (or possibly by chemo(litho)autotrophs) or preymg directly upon them. However, as Siebert & Hirsch (1988) note, "little is known so far about heterotrophic bacteria and their part in endolithic microbial ecosystems." This paucity of knowledge is primarily due to their relatively small size and lack of distinctive pigmentation, which causes difficulties in identifying heterotrophic species, and even genera. However, the increased use and practicality of sequencing the genes encoding for 16S rRNA (16S rDNA) eliminates these constraints by allowing the detailed investigation and identification of these organisms, often to the species level, even when present in extremely low concentrations (Vincent, 2000). Molecular techniques are particularly valuable in polar environments, where the psychrophilic nature of many bacteria makes culturing them problematic (Morita 2000a) and the novelty of many organisms provides no reliable comparison against which to identify them 12 (Franzmann, 1996). Indeed, since the first application of phylogenetic techniques to polar endolithic communities (Colwell et al., 1989), "all Antarctic strains sequenced to date have represented new species" (Franzmann, 1996). This underscores how little is understood about polar endolithic communities and their origins. Mechanisms of Bacterial Deposition into Rocks Despite inhabiting the interior of a rock, endolithic organisms are not completely isolated from the world outside their lithic habitat. In fact, interactions with the exterior environment are required for an endolithic community to develop, both as a means of introducing would-be endoliths into the rock interior and as a source of colonizing microorganisms. Wind, precipitation, and snowmelt serve as mechanisms that may inoculate the interior of rocks with organisms from the immediate environment (Wynn-Williams, 2000). Wind (or windblown snow) can deposit microorganisms into cracks and crevices in a rock surface, where, if motile, they may move further into the rock interior, and if nonmotile, they can be carried deeper into the rock by water or snowmelt, which deposits them while percolating through the interior pore spaces of a rock (Wynn-Williams, 2000). Soil, ice, and water from the vicinity of a given rock are the typical sources for these organisms. However, in many cases, the ultimate origin of the endolithic organisms may not be from the immediate environment of the rocks that they eventually colonize. In fact, many polar endolithic species are believed to have undergone long- distance transport prior to their entry into an endolithic community. Various dispersal mechanisms, including atmospheric circulation, snowmelt, ocean currents, birds, fish, marine mammals, and human vectors, have been identified to transport bacteria across vast distances to the environment immediately surrounding the rock to be colonized (Vincent, 2000; Franzmann, 1996). Due to the efficacy of these dispersal mechanisms, Franzmann (1996) believes that the concept of spatial isolation is not tenable to microbes, that as a result of these dispersal mechanisms, the majority of microbes have a global distribution. Similarly, Nichols et al. (1995) conclude that, given the ubiquitous presence of microorganisms, the ease with which they are dispersed, and their remarkable, albeit poorly understood, survival characteristics, it is expected to find species present at widely separated geographical locations. Of these mechanisms, atmospheric circulation is likely to be the most significant because it has the power to entrain microbes and subsequently deposit them on a global level (Vincent, 13 2000; Burckle & Delaney, 1999; Franzmann, 1996; Burckle & Wasell, 1995; Burckle 1995a, 1995b). Global transport of microorganisms has been supported by several studies describing bacteria that have been detected in environments that appear to be radically different from their indigenous environments, as suggested by 16S rRNA genetic analysis. Examples include an Uncultured Antarctic Ice bacterium found in hot springs at Angel Terrace, Yellowstone National Park, U. S. A. (B. Fouke, unpublished); and various thermophilic bacteria isolated on the Antarctic ice sheet (Vincent, 2000; Atlas & Bartha, 1993). Due to the temperature difference between their respective optimal temperatures and that of the environment in which they were found, it is unlikely that any of these bacteria were actually active in the environments from which they were isolated. Additional mechanisms, such as meltwater flow and migration by birds and sea mammals are also believed to play a significant role in the transport of bacteria to, from, and among polar environments. For example, studies have demonstrated the efficacy of these mechanisms by observing the impact of Antarctic omithogenic (derived from birds) soils on seawater bacterial microflora (Delille, 1990, 1987). A distinct difference has also been observed to exist between heterotrophic communities inhabiting sea ice and the underlying water column, between which Delille (1993) concluded, based on both physiological and taxonomic data, there exists no direct relationship. In addition to divergence resulting simply from the different natures of their respective physical environments, likely sources of divergence between these communities include the introduction of bacteria derived from avian and mammalian visitors, the run-off of glacial meltwater, and direct deposition from the atmosphere onto sea ice. Examinations of microfossils and microorganisms within rocks exposed in Antarctica support long-distance transport of bacteria. A study of microfossils within four meteorites from the Allan Hills and Queen Alexandra Range, Antarctica has revealed the presence of modem freshwater and saltwater diatoms, as well as specimens representing extinct species (Burckle & Delaney, 1999). This indicates that both continental erosion and marine evaporation played a role in entraining microorganisms and microfossils. Opal phytoliths, microscopic particles produced by plants and released during their incineration, were also found in each meteorite (Burckle & Delaney, 1999). These data support the work of Kellogg & Kellogg (1996), which found that diatoms and opal phytoliths were transported to the Antarctic ice surface by atmospheric circulation. Freshwater, marine, and terrestrial species of diatoms have been 14 observed to occur from Maud Land, the McMurdo Dry Valleys, and Marie Byrd Land, Antarctica (Burckle, 1995b). From the variety of diatoms and the identification of several species present in all locations, Burckle (1995b) concluded that atmospheric processes are most likely responsible for their deposition. Another study involving igneous rocks from James Ross Island, Antarctica revealed the presence of marine, lagoonal, freshwater, and terrestrial diatoms, as well as plant fragments, pollen, and spores, within cracks and crevices in the rock surface, which were concluded to have been deposited after atmospheric transport (Burckle & Wasell, 1995). A companson of contamination between igneous (Marie Byrd Land, Antarctica) and sedimentary rocks (Beacon Supergroup, Antarctica) showed that both contained identical species, and that the igneous rocks had a much higher amount of micro fossil deposition (Burckle, 1995a). This suggests that the surface characteristics of igneous rocks may make them more suited than sedimentary rocks for the accumulation of microfossils (and microorganisms) transported through the atmosphere. The high degree of exogenous organic matter and organisms present in Antarctic samples, despite the relative isolation of Antarctica from global patterns of atmospheric circulation (Vincent, 2000), suggests that other terrestrial environments, including the Arctic, would be exposed to more substantial nutrient and organism deposition. A Comparison of Arctic and Antarctic Environments Containing Endoliths Endolithic communities have been identified in terrestrial regions of both poles (Arctic: Cockell et al., 2001; Cockell & Lee, 2000; Antarctic: Wynn-Williams, 2000; Nienow & Friedmann, 1993; Friedmann et al., 1988; Vincent, 1988; Friedmann, 1982; Friedmann, 1977; Friedmann & Ocampo, 1976). While these communities share a polar desert climate, there are several differences in their environments. The endolithic environments from Antarctica are, for the most part, characterized by lower temperatures, higher winds, decreased precipitation, and decreased entrained nutrient deposition relative to their cqunterparts in Arctic endolithic communities (Nienow & Friedmann, 1993; Cockell et al. 2002). As such, Antarctic endoliths are subjected to a more 'extreme' environment and must be correspondingly better adapted to their environment. Although a comparative study has yet to be conducted, it is likely that the percentage of psychrophiles among Antarctic endolithic communities will be significantly higher 15 than amongst the Arctic endoliths, where a larger percentage may be composed of psychrotrophic organisms. Rocks Hosting Endoliths Since the notion of polar endoliths was first put forward (Friedmann & Ocampo, 1976), porous sedimentary rocks, such as the Beacon sandstone formation in Antarctica, have been identified as the most important for endolithic communities, because their high porosity favors colonization by endolithic bacteria (Nienow & Friedmann, 1993). While crystalline rocks, such as gneiss and granite, have also been studied in Antarctica, their typically low porosities make them poor hosts for endolithic bacteria. However, the Arctic contains a unique environment in which endolithic organisms can readily colonize crystalline rocks, specifically impact-shocked gneisses and breccias from Haughton impact structure on Devon Island in the Canadian High Arctic. Crystalline rocks, such as gneiss, can host endolithic organisms if they have undergone impact-induced shock metamorphosis, one of the many effects of an asteroid or comet impact (Cockell et al., 2002; Cockell et al., 2001; Dressler & Reimold, 2001; Cockell & Lee, 2000). Such impact-induced shocking can heavily fracture a rock and partially volatilize its constituent minerals, generating habitats suitable for endolithic colonization. Meteorites Impact-induced shock metamorphism affects the impacting body (comet or asteroid), as well as the rocks in the vicinity of the impact. Melosh (1989) showed that the shock is greatest at the point immediately below the impact point and within the impacting object itself, where pressures can exceed 100 0Pa. The majority of an impacting body can be vaporized during the impact process. In cases where a sizable portion of the original impactor remains as a meteorite, it is highly shocked throughout (Dressler & Reimold, 2001; Melosh, 1989). In many cases, meteorites are covered by a fusion crust, formed during reentry or in the heat of the impact (Dressler & Reimold, 2001 ; Steele et al., 2000). This crust, however, is frequently fractured, cracked, and weathered and serves to trap dust particles (including microfossils) that have been transported into the proximity of the meteorites either within the ice, or more likely, on the ice surface (Burckle & Delaney, 1999; Steele et al., 2000). In addition to microfossils and microorganisms found on meteorite surfaces (Burckle & Delaney, 1999; Burckle & Wasell , 16 1995; Burckle, 1995a, 1995b), detailed microscopic investigations of the interior of the meteorite Allan Hills 84001 suggest that it may also be contaminated by endoliths (Steele et al., 2000). Burckle & Delaney (1999) suggest that entrapment of micrometer-scale life forms by meteorites may be a ubiquitous process in Antarctica. Meteorites are important because they provide physical samples of extraterrestrial materials from the solar system, yielding information concerning Solar System origin, the geochemical evolution of primitive parent objects, and the irradiation histories of material in space (Lipschutz, 1995). Of particular interest to planetary scientists are the Martian meteorites, which constitute the only direct source of knowledge of Martian geology, chemistry, atmospheric composition, and biology that scientists may access. The interest in these Meteorites focuses on understanding the early history of Mars, which is believed to have been similar to that of Earth, to better understand how Mars came to have its present climate, and how this can be applied to the climate stability of Earth (McKay, 1997). In recent years, much excitement has been raised by the announcement of possible relic biological activity inside of the Martian meteorite Allan Hills 84001 (McKay et al., 1996). The possible origin and/or existence of life on Mars are topics of extreme scientific (and philosophical) interest. However, when investigating these questions, care must be taken to ensure the absence of terrestrial biological contamination. Several studies have shown terrestrial surface contamination in meteorites obtained from the Antarctic ice sheet (from which Allan Hills 84001 was obtained). Burckle & Delaney (1999) conclude that terrestrial contamination, via atmospheric transport, of Antarctic meteorites is ubiquitous. Entrainment of terrestrial microfossils is believed to be the natural result of interactions between meteorites and the Antarctic (Burckle & Delaney, 1999). Microfossils and viable microorganisms have been found in several meteorites, including Allan Hills 84001 (Burckle & Delaney, 1999; Steele et al., 2000). Given the high degree of impact-induced shock that these meteorites underwent, it is likely that terrestrial contamination is not limited to the surface, and that heterotrophic microbial communities could exist within the interior of these meteorites. The presence of these communities would alter the biogeochemical environment of the meteorites, possibly removing or altering any traces of relic biological activity from Mars. 17 Description of Present Study To date, no work has been published identifying and characterizing the heterotrophic endolithic communities that exist significantly below the region of photosynthesis. Support for the existence of these communities has come from the work of Hirsch et al. (1988), who have identified near-surface, endolithic communities that appear to lack photosynthetic primary producers. A decrease in heterotrophic abundance with depth beneath the surface is expected in environments below the phototrophic zone, due to the decreased penetration of nutrients from the surface and decreased availability of nutrients associated with the lack of primary producers. However, it is also likely that, given the lack of photoautotrophs within the interior of rocks (and the resulting absence of competition), the abundance of chemoautotrophs in this environment may increase significantly. If this is the case, then the concentration of nutrients in the interior may not decrease as much as expected for rocks whose interiors are inhabited by chemoautotrophic endoliths. The aim of the present study is to investigate possible colonization by heterotrophic bacteria of the interior of samples of impact-shocked gneiss and breccia from the -23 Myr-old Haughton impact structure on Devon Island in the Canadian High Arctic. In particular, this study examines the origin of the possible colonizing bacteria by isolating and sequencing microorganisms present within the rock interior and comparing them with a microbial database to determine the habitat from which the isolate likely originated. Furthermore, this study aims to determine, through a combination of biological, microscopic, and spectroscopic techniques, the extent to which heterotrophic endolithic communities are present in rock interiors significantly below the photic zone. In the process, the combination of scanning electron microscopy (SEM) and X-ray energy dispersive spectroscopy will be assessed as a tool for the identification of microorganisms inhabiting impact-shocke~ crystalline rocks. 18 Study Site Chapter 2 Materials and Methodology Haughton Impact structure is located at 75°22'N, 89°41 'W in the northwestern portion of Devon Island, Nunavut in the Canadian High Arctic (Figure 2). The impact structure is approximately 24 km in diameter (Grieve, 1988) and was created by the impact of a comet or asteroid 23.4 ± 1.0 Myr ago during the early Miocene Period (Jessberger, 1988). Furthermore, Haughton impact structure is located in a polar desert, where the paucity of organics derived from higher-order organisms makes it an ideal location for the study of microbial colonization of impact-shocked rocks and microbial interactions with the post-impact environment of a crater. At the time of the impact, PreCambrian gneiss formed the crystalline basement on Devon Island and was overlain by 1750 m of Paleozoic sedimentary rocks (Frisch & Thorsteinsson, 1978; Robertson & Sweeney, 1983; Metzler et al., 1988). These sedimentary layers were composed predominantly of dolomite and limestone, interbedded with gypsum, shale, and quartzose sandstone (Frisch & Thorsteinsson, 1978). The presence of significant amounts of gneiss, either incorporated into impact-melt breccias or as individual, impact-shocked rocks, throughout the surface of the impact structure indicates that the excavation depth for the impact exceeded 1750 m (Dressler & Reimold, 2001; Metzler et al., 1988). A survey of crystalline basement rocks by Metzler et al. (1988) has identified thirteen different rock types which can be grouped into eight categories: (1) sillimanite- and garnet- bearing gneiss; (2) alkali feldspar-rich aplitic or biotite-hornblende-bearing gneiss; (3) biotite and hornblende gneiss; (4) apatite-rich biotite and biotite-hornblende gneiss; (5) calcite-diopside gneiss; (6) amphibolite; (7) tonalitic orthogneiss; and (8) basalts (See Table 4) . Samples of post- impact crystalline rocks have been observed to display shock metamorphism to varying degrees, ranging from pressures less than 5 GPa up to 60 0Pa, with the following distribution (in 0Pa): 0- 5: 4.5%; 10- 25: 9%; 25- 35: 33%; 35-45: 29%; 45-55: 18%; 'and 55- 60: 6.5% (Metzler et al. 1988). 19 --~~ retie Ocean f "\ 20 GPa) Low-Shock Rock (