Extremophiles are a unique group of microbes, mainly archea and bacteria, that thrive in harsh environments which were previously considered uninhabitable. Thermophiles are a category of extremophiles which are suited to temperatures greater than 45 degrees Celsius. Organisms that are heat resistant in temperatures of over 80 degrees C are referred to as hyperthermophiles(1). These microbes have managed to colonize a variety of extremely hot places such as hot springs, deep sea vents, and the deep subsurface. Their ecosystems and extraordinary survival skills are just beginning to be understood, but they already hold promise for major impacts on our society. Thermophiles and hyperthermophiles not only have great practical application, they are also considered as possible links to the origin of life and extraterrestrial life. THERMOPHILES OF HOT SPRINGS: The first hyperthermophiles, the archaean Sulfolobus acidocaldarius, were discovered only 30 years ago. The discovery was made by Thomas D. Brock of the University of Wisconsin-Madison in the hot springs of Yellowstone National Park. These microbes, found in a hot, acidic spring, exist in temperatures of up to 85 degrees C.(3) Brock collected hot spring specimens by immersing microscope glass slides in the spring for 7-10 days. When he retrieved them they were colonized by microbes. Some slides were so densely covered that the colonies were visible to the naked eye. Brock found other species of hyperthermophiles in Yellowstone pools of temperatures up to 95.5 degrees C. In New Zealand, microbes have been found in pools of temperatures up to 101 degrees C(4). Since these discoveries, subsequent explorations to various hot springs and deep-sea hydrothermal vents have led to the identification of more than 50 species of surface hyperthermophiles(5). THERMOPHILES OF DEEP SEA VENTS: Deep sea vents are hot springs in the ocean bed. These vents are often created along submarine rifts where two continental masses are separated by tectonic movement. The rifts allow the ocean water to be exposed to the geothermal heat within the crust, and the high pressure of the deep ocean allows the water to be liquid up to 350 degrees C(6). Pyrolobus fumarii, among the most heat resistant of surface hyperthermophiles, is found near deep sea vents. This microbe favors temperatures of 105 degrees C and has been found to reproduce in temperatures of up to 113 degrees C(8). The majority of the hyperthermophiles near deep sea vents are anaerobes, meaning they require no oxygen. This adaptation is necessary as there is little oxygen solubility in the hot water(9). THERMOPHILES OF THE DEEP SUBSURFACE: Other thermophiles live in remarkable depths in the earth's crust. Subsurface microbes have been found as far down as 2.8 kilometers below the surface. These specimens were collected in the Taylorsville Basin in an exploratory well drilled by the Texaco Oil Company. However, this record is likely to be broken soon. In South Africa, samples of rock from gold mines as deep as 3.5 kilometers show the existence of thermophiles in preliminary tests(10). Collection of subsurface microbes is a careful process in which scientists must take precautions not to contaminate the samples of crust with surface microbes. In explorations such as the Taylorsville Basin well, a coring tool was used to extract the subsurface rock. These samples were immediately placed in a sterile glove bag and the bags were filled with inert gases so that oxygen could not poison the microbes, which were suspected to be anaerobic. Sterile tools were then used to remove the outer portions of the samples, leaving only the inner portions that were least contaminated by the outside environment(11). The inner portions of the sample were then placed in a growth medium which replicated the temperatures, salinities, and acid levels of the deep environment. Months after the samples were placed in the growth media the scientists were rewarded by the appearance of several strains of thermophilic microbes(12). The microbes grown in the Taylorsville Basin samples proved to originate from the rock and not the outside environment because they were anaerobic. Surface microbes that existed in likely sample contaminants such as the drilling fluid of the coring tool required oxygen(13). Further studies of deep surface samples from other places yielded anaerobic microbes with physiological abilities specific to their unique environment. In the Columbia River Basin, for example, reactions between the basalt rock and water produced hydrogen gas and appropriately the microbes cultured from the sample utilized hydrogen(14). The specific abilities of these microbes which suited their unique environments indicated that they were not impostors from the surface. The basic requirements for a rock to harbor life are water, pores in the rock sufficiently big enough for the microbes to live in, and nutrients. Because the microbes are so small, they are virtually unaffected by the extremely high pressure of such depths. Instead, the limiting factor of their existence in the crust is based on the estimated upper limit of temperature that microbes can tolerate. In oceanic crust, the temperature rises 15 degrees C per kilometer of depth while in continental crust the temperature rises 25 degrees C per kilometer. Using these data and a 113 degrees C temperature limit, scientists have estimated that microbes can exist as far down as 7 kilometers below the surface of oceanic crust while under continental crust they can live as far down as 4 kilometers(15). Because deep subsurface life is limited by temperature, many of the very deepest organisms are likely to be hyperthermophiles. The Taylorsville Basin samples came from an environment of about 75 degrees C, making the organisms found there thermophilic(16). Subsurface microbes have been found in various types of sedimentary and igneous rock. Sedimentary rock is created from surface sand, silt and clay that are compressed over time. Plants and other organisms are also compressed and incorporated into the rock, making sedimentary rock rich in organic material. This organic material feeds the subsurface microbes. Sedimentary rock also provides nutrients through oxidized forms of sulfur, iron, and manganese. As sedimentary rock ages it continues to compress and the habitable space, or pores, within the rock become scarce and small. Consequently, the distribution of microbes within the rock tends to concentrate in small patches around pockets of nutrients(18). Igneous rock is formed from cooled molten magma. Unlike sedimentary rock, igneous rock contains little organic material for microbes to feed on. Many of the microbes that inhabit this environment are autotrophic and therefore produce organic compounds from inorganic substances. For example, some autotrophs are able to create organic carbon from carbon dioxidee. Others derive their energy from hydrogen gas that is produced through the oxygen-poor water and iron-bearing minerals within the rock. The organic compounds that autotrophic microbes produce are then ingested by heterotrophic, or organic consuming, microbes. A a self-sustaining community is created that is independent from the surface and the sun. The igneous environments in which such communities are found are often referred to as SLiME (subsurface lithoautotrophic microbial ecosystems)(19). Microbe communities in such deep environments may exist trapped within the crust for at least several million years, while some scientists theorize that some communities may have been isolated for up to 160 million years. Because igneous rock is originally molten and therefore sterile, microbes are introduced through the flow of groundwater after the rock cools. In some places, it has been deduced that subsurface groundwater has not been in contact with the surface for millions of years. Therefore, the communities that exist in these places must be at least as old as the last incoming groundwater (20). Other evidence for the extreme age of some subsurface microbe communities comes from the pore spaces of the rock. The pore spaces, extremely small, hollow pockets in the rock, are where the microbes reside. In the case of the Taylorsville Basin samples, the passages between pore spaces were at the largest only .03 microns wide. These passages were too small to allow microbes to pass through. Therefore, the microbes within the pore spaces were trapped and isolated from other microbes. The material which filled the passages between the pores was clay, including a substance called illite. With the help of a laser microprobe, the age of this illite was determined and it was concluded that the clay which filled the pore passages crystallized 80 million years ago. This could only mean that the microbes within the pore spaces had been trapped for at least 80 million years. After an analysis of the calcite and pyrite, microbial by-products, within the pores, it was concluded that the doubling time of these communities was of the order of thousands or even millions of years. This growth rate, 10^15 times slower than other microbes, illustrates that deep subsurface reproduction is extremely low and consequently, evolution of these microbes has been minimal. These microbes therefore, have been isolated with minimal change since the time of the dinosaurs, at least 80 million years ago(22). The extreme age of these communities is even more remarkable because of the scarcity of food and energy in the deep subsurface. In the cases of autotrophic microbes such as those in the SLiME described above, nutrients are created directly from the environment. In other communities the microbes have adapted to long periods of starvation. During a period of starvation, microbes undergo several changes. Cell membrane transport may be enhanced, DNA may change in both quantity and expression, and specific starvation proteins may be produced(23). These microbes have also been found to shrink to less than 1000th of their normal volume during extreme periods of famine. The metabolism rate of the microbes is also drastically lowered to allow survival. Metabolism may slow down to the point where the microbes undergo cell division less than once a century(24). The starvation resistance changes can also make the microbes more resistant to disinfectants and toxins(25). The population density of deep subsurface communities is considerably lower than the population density of surface microbes. For example, the density of microbes grown from the Taylorsville Basin samples was only 1-10 microbes per gram of rock. In comparison, there may be as many as 10^9 microbes in one gram of surface rock or soil(27). However, the density of subsurface communities is often uncertain. Because the metabolism of the microbes is often is often so slow, it is sometimes difficult for scientists to determine if the microbes are actually alive. Dead cells in the subsurface decompose slowly due to the lack of movement of nutrients and water, so they can easily be mistaken for live cells. Also, it is difficult to sufficiently reproduce the subsurface environment in a laboratory to encourage the microbial growth that will accurately indicate living cells(28). Thomas Brock, a pioneer in the study of extremophiles, stated only 21 years ago, "It is unlikely that deep subsurface microbial activity occurs, due to the lack of O2.(29) However, as of October of last year more than 9000 strains of subsurface microbes have been catalogued. Thomas Gold of Cornell University has calculated that the weight of all subterranean microbes could equal the weight of all surface organisms(30). The world of subsurface microbes has been discovered only recently, but it is already recognized as a significant part of our biosphere. PRACTICAL APPLICATIONS OF THERMOPHILES: Thermophiles hold great practical interest for our society. Because of their unique environments, hyperthermophiles produce tough enzymes which can operate in high temperatures. These unique enzymes have several practical applications. For example, enzymes from the hyperthermophile Pyrococcus furiosus of deep sea vents are used by forensic scientists in DNA testing. These enzymes are especially stable and can replicate tiny amounts of DNA into huge quantities so that it can be identified. The heat resistance of hyperthermophile enzymes could also be put to use in industry for products such as high-temperature detergents. Enzymes from deep sea hyperthermophiles may also lead to a future of hydrogen energy that is environmentally clean and unlimited. The use of hydrogen energy has been hampered in the past because large quantities of hydrogen gas have been relatively difficult to obtain. However, enzymes from hyperthermophiles may allow hydrogen gas to be created from agricultural wastes that would normally be buried or burned. This would require a two step process in which the cellulose of the plants is first broken down into glucose and then the glucose is broken down to produce hydrogen. The first step of this process can be completed with an enzyme from Pyrococcus furiosus, and the second from another deep sea hyperthermophile called Thermoplasma(31). Hyperthermophile enzymes are also being studied for use in cancer and AIDS treatments(32). Subsurface microbes could play a role in deep groundwater pollution clean-up and underground nuclear waste storage. They could also be used to consume toxic chemicals in industrial wastewater that is too hot for normal microbes to survive in.(33) THERMOPHILES AND THE ORIGIN OF LIFE: Hyperthermophiles are likely to be our closest link to the very first organisms. Upon the beginning of life, the earth was a hotter planet due to the increased greenhouse effect of a carbon dioxide rich atmosphere. Therefore, it is logical to say that the first organisms were accustomed to high temperatures and adapted to cooler temperatures as the earth cooled(34). This early atmosphere also did not contain oxygen until 2 billion years ago. Because life started at least 3.5 billion years ago, life was exclusively anaerobic for at least 1.5 billion years(35). As stated above, many subsurface and deep sea thermophiles are anaerobic as well. It appears that the unique heat resistance and anaerobic nature of many hyperthermophiles could be traits of the earliest organisms. Deep sea microbes are one type of hyperthermophile which may be involved in the origin of life. Deep sea vents release iron nickel sulfides which catalyze the production of methyl thioacetate. Methyl thioacetate is significant because it resembles acetyl-coenzyme A which is necessary for the production of various important biomolecules. Therefore, early biomolecules could have originated near deep sea vents starting with the iron nickel sulfides that are released there. These biomolecules may have led to the first microbes which could resemble the hyperthermophiles found there today. The unique environment of the underground makes subsurface microbes another possible link to the beginning of life on our planet. Meteorites and intense ultra-violet rays created a hostile surface environment in the earth's early history. Early meteor bombardments could have caused impacts large enough to flash-heat the surface of the earth, vaporizing the oceans in the process. Based on a study of the moon's craters, scientists calculated the end of the meteor bombardments here on earth at 3.8 billion years ago. This time was set as a limit to the origin of surface life because it is unlikely that any organism could have survived the sterilizing impacts of the meteors. However, rocks from this time period have been found to contain evidence of highly evolved microbes which lived in colonies and performed photosynthesis. These microbes must have evolved from simpler life forms. If life began 3.8 billion years ago on the surface and rocks from this era already indicate highly evolved microbes, early evolution must have been extremely fast. A more likely possibility is that life began before 3.8 billion years ago in the only place it could, the subsurface. At this time it is likely that the subsurface was the most habitable environment as it offered protection from the surface-sterilizing impacts of meteorites. Ultraviolet rays would also have been much stronger back then due to a lack of ozone, and the subsurface would have provided necessary protection from the radiation. The subsurface would also have had the most stable temperature. Taking all of these factors into account, the first habitable environment on the planet would have most likely been the subsurface in the area between the intense geothermal heat of the earth below and the meteorite impacts above(36). Based on this evidence, it is not unreasonable to theorize that the origins of life began with subsurface, thermophilic, anaerobic microbes similar to those being discovered today. SUBSURFACE THERMOPHILES AND EXTRA-TERRESTRIAL LIFE: Subsurface microbes may be similar to extraterrestrial life as well. Some autotrophic communities, as described earlier, produce their own nutrients and are independent from the sun. Life, therefore, does not need to exist at a certain distance or "habitable zone" from a star. It seems to be limited mainly by liquid water, and it has been estimated that 5-10% of planets have liquid water. This figure results in a huge number of inhabitable planets, and multiple possibilities within our own solar system. As Todd O. Stevens of Battelle Pacific Northwest Laboratory states, "If primary production can occur underground, where it is disconnected from photosynthesis, then there is no reason that (subsurface) life couldn't exist on several planets in the solar system."(37) The nearest candidate for extraterrestrial life is Mars, and the closest link to possible microbes there are subsurface extremophiles. Mars is considerably cooler than earth with high temperatures of 20 degrees C and lows of at least -125 degrees C on the surface. These temperatures do not allow liquid water to exist on the surface, though water still exists in limited quantities as ice. Temperature changes within a Martian day may exceed 100 degrees C. This fluctuation in temperature is due to Mars' thin atmosphere which is only 1/150 as dense as Earth's. This atmosphere also lacks enough ozone to provide a shield from ultraviolet light(38). The lack of liquid water, unstable temperatures, and strong ultraviolet radiation on the surface of Mars lead many scientists to believe that Mars is a sterile planet. However, many of these drawbacks are diminished in the subsurface. Though Mars does not have frequent geological activity, volcanoes such as Olympus Mons attest to geothermal heat under the surface. It is therefore likely that the Martian subsurface may provide a warmer environment that also offers protection from ultraviolet light. Because the heat originates from the interior of the planet rather than the sun, temperatures would also be more stable. The liquid water that once existed on the surface of Mars might still exist in the warmer Martian interior(39). Therefore, if organisms exist on Mars it is likely that they reside in the deep subsurface and our closest link to these extraterrestrials would be subsurface microbes on our own planet. By studying the emissions of subsurface microbes, such as biochemical products, here on earth, scientists may know what compounds to look for in the search for Martian extremophiles(40). SUMMARY: From hot, acidic springs to miles within the earth's crust, thermophiles have shown us a few of the ingenious ways in which life can adapt to survive in almost all possible places. Their unique environments and abilities may one day reveal to us secrets from our ancient history in the origin of life. Or perhaps they will lead us to an exciting future through hydrogen powered cars and the discovery of extra-terrestrial life. Hyperthermophiles have been quietly existing unnoticed for billions of years, but now that their world is beginning to open up they show promise to impact our society in great ways. 1 Michael Madigan and Barry Marrs, "Extremophiles", Scientific American, (April 1997), pp. 83. 2 Source unknown 3 Michael Madigan and Barry Marrs, "Extremophiles", Scientific American, (April 1997), pp. 83. 4 Thomas Brock, Thermophilic Microorganisms and Life at High Temperatures , (New York: Springer-Verlag, 1978) pp. 44, 46, 313. 5 Michael Madigan and Barry Marrs, "Extremophiles", Scientific American, (April 1997), pp. 83. 6 John Postgate, The Outer Reaches of Life, (Cambridge: Cambridge University Press, 1994), p. 14. 7 Source unknown 8 Michael Madigan and Barry Marrs, "Extremophiles", Scientific American, (April 1997), pp. 84. 9 John Postgate, The Outer Reaches of Life, (Cambridge: Cambridge University Press, 1994), p. 15. 10 Richard Monastersky, "Deep Dwellers," Science News, v. 151(March 1997) , pp. 192-193., 11 "The Microbiology of Deep Subsurface Environments." Online. World Wide Web. 5 October 1997. Available http://geo.princeton.edu/geomicrobio/smilodonTCO.html 12 "The Microbiology of Deep Subsurface Environments." Online. World Wide Web. 5 October 1997. Available http://geo.princeton.edu/geomicrobio/smilodonTCO.html 13 Richard Monastersky, "Deep Dwellers," Science News, v. 151(March 1997) , pp. 192-193. 14 Todd Stevens, 'Subsurface Microbiology and the Evolution of the Biosphere,' in Penny Amy and Dana Haldeman, ed., The Microbiology of the Terrestrial Deep Subsurface (Boca Raton: CRC Lewis Publishers, 1994) pp. 215-219. 15 James Fredrickson and Tullis Onstott, "Microbes Deep inside the Earth, " Scientific American, v. 275 (October 1996), pp. 71. 16 Richard Monastersky, "Deep Dwellers," Science News, v. 151(March 1997) , pp. 192-193., 17 James Fredrickson and Tullis Onstott, "Microbes Deep inside the Earth, " Scientific American, v. 275 (October 1996), pp. 68. 18 James Fredrickson and Tullis Onstott, "Microbes Deep inside the Earth, " Scientific American, v. 275 (October 1996), pp. 71. 19 James Fredrickson and Tullis Onstott, "Microbes Deep inside the Earth, " Scientific American, v. 275 (October 1996), pp. 72. 20 James Fredrickson and Tullis Onstott, "Microbes Deep inside the Earth, " Scientific American, v. 275 (October 1996), pp. 72. 21 James Fredrickson and Tullis Onstott, "Microbes Deep inside the Earth, " Scientific American, v. 275 (October 1996), pp. 71. 22 "The Microbiology of Deep Subsurface Environments." Online. World Wide Web. 5 October 1997. Available http://geo.princeton.edu/geomicrobio/smilodonTCO.html 23 Todd Stevens, 'Subsurface Microbiology and the Evolution of the Biosphere,' in Penny Amy and Dana Haldeman, ed., The Microbiology of the Terrestrial Deep Subsurface (Boca Raton: CRC Lewis Publishers, 1994) pp. 215-219. 24 James Fredrickson and Tullis Onstott, "Microbes Deep inside the Earth, " Scientific American, v. 275 (October 1996), pp. 73. 25 Todd Stevens, 'Subsurface Microbiology and the Evolution of the Biosphere,' in Penny Amy and Dana Haldeman, ed., The Microbiology of the Terrestrial Deep Subsurface (Boca Raton: CRC Lewis Publishers, 1994) pp. 215-219. 26 Todd Stevens, 'Subsurface Microbiology and the Evolution of the Biosphere,' in Penny Amy and Dana Haldeman, ed., The Microbiology of the Terrestrial Deep Subsurface (Boca Raton: CRC Lewis Publishers, 1994) pp. 215-219. 27 "The Microbiology of Deep Subsurface Environments." Online. World Wide Web. 5 October 1997. Available http://geo.princeton.edu/geomicrobio/smilodonTCO.html 28 James Fredrickson and Tullis Onstott, "Microbes Deep inside the Earth, " Scientific American, v. 275 (October 1996), pp. 73. 29 Thomas Brock, Thermophilic Microorganisms and Life at High Temperature s, (New York: Springer-Verlag, 1978)p. 36. 30 Richard Monastersky, "Deep Dwellers," Science News, v. 151(March 1997) , pp. 192-193., 31 John Newell, "Earth's Oldest, Toughest Organisms," World Press Review v. 43 (October 1996), pp. 48-49. 32 "Researchers Seek Origin of Deep Subsurface Bacteria." Online. World Wide Web. 19 October 1997. Available http://www.agu.org/sci_soc/deepbact.html. 33 Richard Monastersky, "Deep Dwellers," Science News, v. 151(March 1997) , pp. 192-193., 34 John Postgate, The Outer Reaches of Life, (Cambridge: Cambridge University Press, 1994), pp. 15, 18-19. 35 Howard Gest, The World of Microbes (Madison: Science Tech Publishers, Inc., 1987), p 209. 36 Todd Stevens, 'Subsurface Microbiology and the Evolution of the Biosphere,' in Penny Amy and Dana Haldeman, ed., The Microbiology of the Terrestrial Deep Subsurface (Boca Raton: CRC Lewis Publishers, 1994) pp. 215-219. 37 Richard Monastersky, "Deep Dwellers," Science News, v. 151(March 1997) , pp. 192-193., 38 Donald Goldsmith and Tobias Owen, The Search for Life in the Universe, (Reading: Addison-Wesley Publishing Company, 1992), pp. 304-305. 39 Richard Monastersky, "Deep Dwellers," Science News, v. 151(March 1997) , pp. 192-193., 40 Todd Stevens, 'Subsurface Microbiology and the Evolution of the Biosphere,' in Penny Amy and Dana Haldeman, ed., The Microbiology of the Terrestrial Deep Subsurface (Boca Raton: CRC Lewis Publishers, 1994) pp. 215-219.