Thermophilic Bacteria of Yellowstone National Park
CEE:5154 Environmental Microbiology
Research Paper

University of Iowa Department of Civil and Environmental Engineering

December 14, 2015

Bruce McWilliams

Amid the vast, sparsely populated regions of Northwest Wyoming, lies one of the most diverse and extraordinary ecosystems in the world, Yellowstone National Park. Yellowstone is one of the world's foremost sites for the study and appreciation of the evolutionary history of the earth. The park has a globally unparalleled assemblage of surficial geothermal activity, thousands of hot springs, mudpots, fumaroles, and more than half of the world’s active geysers (NPS, 2013). Yellowstone is located on top of the Yellowstone Caldera, which is a volcanic hot spot where hot, molten rock from the earth’s mantle rises toward the surface. Volcanic activity from the Caldera produces geothermal activity on the park’s surface that has drawn more than 3 million visitors to the park since 2000 (NPS, 2015). Geysers, hot springs, and mudpots are extremely toxic due to high concentrations of sulfuric acid (sulfate concentrations measure up to 925 ppm near vents) and, temperatures measuring over 100oC. Many have recorded inhabitable pH levels ranging from 2 to 9.8 (Rowe/Founder/Morey, 1973). While these colorful and wondrous hot springs may appear stagnant and devoid of life to the common park visitor, they are actually a complex, intricate habitat teeming with a diverse array of microbial life. The existence of these organisms has puzzled and intrigued scientists for over 50 years, but continued research has allowed us to gain a better understanding of their existence. As more information has come to light about these complex organisms, the use of thermophilic microorganisms to curb the effects of environmental pollution has emerged as a realistic possibility. Bioremediation from thermophilic organisms could play a large role in the future of climate change. Yellowstone’s hydrothermal system is a direct expression of the immense caldera or “super volcano” located underneath the park. The tremendous amount of energy released from the underlying molten body of magma, heats groundwater and run off from melting snow and mountain streams that percolates through permeable rocks (NPS, 2014). As cooler surface water infiltrates the surface, it meets hot brine heated by the magma in the park’s subsurface. The surface water is heated well above the sea-level boiling point, but remains in a liquid state due to the great pressure of overlying water and rock. This creates super-heated water in the subsurface with temperatures exceeding 200oC (NPS, 2013). This superheated water is less dense than the cooler, heavier water that infiltrates and sinks around it, forming convection currents that allow the buoyant superheated water to migrate back toward the earth’s surface. As superheated water rises, it dissolves silica in the rhyolite and transports silica out of the subsurface (NPS, 2014). The water continues to move upward and begins to cool, causing the dissolved silica to precipitate out of the water. Precipitated silica is then deposited in cracks present in the overlying rock, creating an immense amount of pressure due and preventing superheated water from continuing its upward migration. Pressure builds in the subsurface until the silica deposits are overcome by the force of the rising water. This extreme amount of pressure causes mineral rich, superheated water to violently erupt through fissures in the rock and project out of the park’s surface (NPS, 2014). This geological phenomenon creates the thousands of geysers located throughout the natural wonder that is Yellowstone National Park. The upwelling of mineral rich superheated water is continually happening in the subsurface of the park, but it is not always as obvious and violent as the magnificent geysers. Hydrothermal features like hot springs, mudpots, fumaroles, and limestone terraces are also formed by the subsurface migration of thermal water. Hot springs are the most common hydrothermal features in the park, and are created by the convection of infiltrating precipitates and super-heated water similar to the geological process that forms geysers. Unlike in the case of geysers, however, convection currents constantly circulate water from the surface to the subsurface in these locations, preventing water from reaching a temperature that would trigger an eruption (NPS, 2013). Mudpots are formed much like hot springs, but occur in areas of the park where surface water cannot infiltrate the subsurface due to an impermeable depression of clay (NPS, 2013). Thermal water beneath this depression causes steam to rise and heat the surface water that is collected or absorbed by the impermeable clay lining. This causes the clay and water mixture in the depression to boil from the release of hydrogen sulfide gas out of the clay; this phenomenon gives the park its notorious rotten egg smell (NPS, 2014). Hydrogen sulfide gas is oxidized at the surface of mudpot, creating an environment rich in elemental sulfur and sulfuric acid which drastically lowers the pH of the local environment (Rowe/Grogan/Morey, 1973). Fumaroles, also known as steam vents, are hydrothermal features that lack a significant amount of water to produce geysers, hot springs, or mudpots. This is because the small amount of water in the hydrothermal vent system boils away before it reaches the surface. Steam and other gases are released from surface vents into the atmosphere through these structures (NPS, 2014). The intense levels of heat (ranging from 50oC to over 200oC), concentrations of toxic compounds, lack of organic substrates, and violent geological tendencies of these hydrothermal features make the possibility of life in these locations seem preposterous. When biological research began in Yellowstone during the 1960’s, the presence of thermophiles was not suspected. The upper limit of life was thought to be around 73oC for phototrophic microbes like cyanobacteria (Brock, 1994). At the time, thermophilic bacteria were even considered to prefer lower temperatures around 55oC (Grogan, 2010). This hypothesis was based on the known effects of temperature on important biological structures including DNA. Enzymes were known to degrade in high heat and are destroyed through boiling. Many concluded that life would be impossible in the thermal features of the park. Field observations in Yellowstone proved otherwise, however, as evidence of biological activity appeared in certain springs. The microbes in these springs were found to have enzymes that are very tolerant of heat and are active in the boiling waters of Yellowstone’s famous geysers (Brock, 1994). Microscopic life in the hydrothermal features of Yellowstone exists in the form of Archaea, Eukarya, Bacteria, and even several types viruses. This diverse array of life has made Yellowstone an oasis for biological and microbial research due to the unique characteristics of this historically unaltered environment. Thermophilic organisms are classified as k-strategists due to their ability to thrive in harsh conditions with limited resources. Archaea exist under the most extreme conditions in the park, relative to their bacterial and eukaryotic neighbors, and have been discovered at temperatures greater than 90oC (Grogan, 2010). Sulfolobus is a genus of Archaea most often isolated in Yellowstone from the hot, highly sulfuric mudpots in the Norris Geyser Basin. This genus of Archaea prefers an environment with a pH range of 0-4 and has an optimal temperature range from 40oC to 55oC. The Sulfolobus community in the Norris Basin is providing new research directions for scientists because it has recently been found to harbor previously unknown viral strains (NPS, 2014). Eukaryotes can also adapt to extreme conditions, and millions of unseen, microscopic members of this kingdom exist throughout Yellowstone. Many are phototrophic algae or fungi that form symbiotic relationships through microbial mats to increase survival in toxic environments. The most common thermophilic eukaryotes in the park are red and green algae, or Cyanidioschyzon and Zygogonium. Both of these photosynthetic eukaryotes prefer an environment with a pH range of 0-4, and can exist in temperatures as high as 55oC (NPS, 2014). Thermophilic viruses exist in every type of hydrothermal feature in the park, and can be found in almost every region. This is not only because these microorganisms are highly resistant to toxic conditions, but because viruses are not considered to be alive (Grogan, 2010). Since most viruses have no semipermeable lipid membrane or recognizable organelle, only a protein “envelope” that encloses their genetic material, they depend on the existence of other microorganisms to reproduce. Sulfolobus Turreted Icosahedral Virus (STIV) is most understood virus in the park and was only recently discovered among the archaeal Sulfolobus. STIV injects Sulfolobus with its genetic information for reproduction through viral replication (NPS, 2014). Most thermophilic viruses in Yellowstone are unnamed, and exist in an optimal pH range of 2-3 and temperatures of 70oC - 75oC (Grogan, 2010). The most abundant thermophiles found in Yellowstone are bacteria, and some are considered to be similar or even direct descendants of the first life forms capable of photosynthesis (NPS, 2014). Historically, cyanobacteria are believed to be first organisms to reduce carbon dioxide. The reduction of CO2 ultimately formed glucose and produced oxygen as a byproduct. Cumulatively this changed the overall anaerobic environment of the earth to an aerobic one, beginning around 2.3 billion years ago (NPS, 2014). This is known as the Great Oxygenation Event, a landmark development caused by bacteria similar to those found in the harsh environments of the park. During the period of the Great Oxygenation Event, conditions on the earth’s surface were similar to that of Yellowstone’s hydrothermal features, leading biologists to believe that cyanobacteria in the park have undergone minimal evolution from their primitive ancestors (NPS, 2014). Cyanobacteria are not the only bacteria to be found in the Yellowstone. Sulfur oxidizing bacteria like Green Sulfur Chlorobium and Green non-sulfur Chloroflexus also exist symbiotically with cyanobacteria. These bacteria utilize sulfur for primary metabolism and are prevalent in the Mammoth Hot Springs and Calcite Springs portion of Yellowstone (NPS, 2014). Chlorobium are photolithotrophs that use light energy to oxidize hydrogen sulfide to sulfate and elemental sulfur in hot springs and mudpots. They are considered to be one of the main biological underlying the persistent rotten egg smell in Yellowstone due to their use of hydrogen sulfide as an electron acceptor for metabolism (Gorgan, 2010). Chloroflexus, despite their name (non-sulfur), also uses reduced forms of elemental sulfur or hydrogen sulfide as a source of electrons for metabolism. Chloroflexus bacteria is a photolithotroph like Chlorobium and uses light energy to drive photosynthesis (Gorgan, 2010). Chemolothitrophs like Aquifex Hydrogenobaculum are found in the park as well. Hydrogenobaculum uses carbon dioxide and hydrogen sulfide as an energy source and resides in the Norris Geyser Basin and Amphitheater Springs. Despite the existence of these prokaryotes, there is today no other bacteria more important to Yellowstone National Park than the thermophile Thermus aquaticus. In 1966, Thermus aquaticus, was the first thermophilic bacterium discovered in Yellowstone by Thomas D. Brock and Hudson Freeze of Indiana University (NPS, 2014).Thermus aquaticus, a gram negative bacterium, was found in springs located in the Great Foundation portions of the Lower Geyser Basin, and has since been found in similar thermal conditions around the globe (Gorgan, 2010). The microbe was found to prefer temperatures around 70oC, but has been isolated from springs ranging 50oC to 80oC. Thermus aquaticus is a cylindrical, rod shaped chemolithotroph that undergoes chemosynthesis during metabolism (Brock, 1994). It is also known to symbiotically exist with cyanobacteria, and use energy from their photosynthesis for growth in environments that can support both microbes (Brock, 1994). Cyanobacteria and T. aquaticus form microbial mats at the bottom of many hot springs in Yellowstone, giving them their iconic orange-yellow color (NPS, 2014). The discovery of Thermus aquaticus has had a large impact on the microbial community because of the unique proteins it expresses. The bacterium’s ability to thrive in extreme environments was unlike anything that had been discovered. Subsequent laboratory research was conducted on T. aquaticus, allowing biologists the opportunity to isolate a key enzyme contained in the organism. Due to its high tolerance of heat, this enzyme proved useful for DNA amplification via the polymerase chain reaction (PCR). The enzyme Taq DNA polymerase, named after Thermus aquaticus, does not degrade during the thermal cycling stage of PCR when the temperature is raised to denature the DNA being amplified (Kondratas, 1992). T. aquaticus persists in the environment at temperatures from 70-80oC, and 72oC was determined to be the optimal activity temperature for Taq DNA polymerase. During PCR, Taq DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand. DNA is synthesized by condensing dNTPs and extending DNA in the 5’ to 3’ direction. At an optimal temperature, there are few practical limits to the DNA sequences that the Taq enzyme can amplify (neglecting limited substrates or reagents), making Taq DNA polymerase a powerful tool in modern molecular biology (Kondratas, 1992). The isolation of Taq DNA Polymerase was a breakthrough, and the enzyme became the basis of a multimillion dollar industry. The enzyme has revolutionized the medical diagnosis of the acquired immune deficiency syndrome (AIDS), forensics (DNA fingerprinting), and bio remediation of toxic waste. After Dr. Brock studied T. aquaticus, samples of the microbe were deposited in a public repository, the American Type Culture Collection (ATCC) (Kondratas, 1992). This nonprofit organization allowed other scientists to access the unique microbe. As the commercial potential of Taq DNA polymerase became apparent in the 1990’s, Dr. Kary Mullis used Taq to develop PCR as we know it today. Dr. Mullis’ research and incorporation of the heat stable DNA polymerase in PCR earned him the 1993 Nobel Prize in Chemistry (Kondratas, 1992). Cetus, a commercial biotechnology firm, patented PCR and Taq and eventually sold those patents to the Swiss health-care company Hoffmann-La Roche for $330 million, from which they have received an estimated $2 billion in royalties (Kondratas, 1992). The National Park Service labeled this development as the “Great Taq Rip-off.” Now researchers working in National Parks are required to sign a series of “benefits sharing” agreements that send a portion of profits from private revenue on federal land to the Park Service (DOI, 2014). Thermophiles have proven to be beneficial for humanity, and research continues to find ways we can to utilize their unique characteristics. Thermophiles have been applied in the detoxification of effluents generated from industry and remediation of hydrocarbon contamination from subsurface petroleum storage through stripping and steam injection (Turner/Mamo/Karlsson, 2007). This technology enhances the degradation of compounds like benzene, toluene, ethylbenzene, and xylene as well as chlorinated solvent contamination from industrial activity (Das, 2014). Optimal temperature and nutrient gradients are major challenges facing bioremediation via thermophiles, because these microbes thrive in nutrient depleted habitats (Das, 2014). Deep sites below layers of bedrock prove difficult for the implementation of thermophilic bacteria due to the impacts of changing pressure and temperature in the subsurface. These conditions exert a synergistic negative impact on the bacteria, despite the fact that thermophiles in their native environment are augmented by elevated temperatures (Turner/Mamo/Karlsson, 2007). Regardless, there is a bright future for the application of thermophiles, and as more research is conducted, thermophilic bioremediation will become more prevalent in limiting the effect of environmental pollutants. Thermophilic bacteria, similar those discovered in Yellowstone, have been found in hydrothermal features located in Iceland, Italy, Japan and New Zealand (Brock, 1994). These locations have been affected by geothermal power developments and health spas which have destroyed the natural thermal features. Human influence has affected microbes negatively in these regions and many organisms have died as a result (Brock, 1994). Since it has undergone limited influence by humanity, microbial research in Yellowstone is so unique and crucial. However, Yellowstone has seen some adverse effects on microbial life, most notably in Morning Glory Pool. Known for its vibrant colors and diverse microbial community, Morning Glory has begun to lose its iconic color due to the ignorance of a park visitors. People have thrown trash and other foreign objects into the pool, clogging the entryway of the spring, preventing the upwelling of superheated water that is required to maintain a temperature which supports thermophilic bacteria (NPS, 2013). This example indicates how fragile this particular ecosystem is and the importance of protecting these unique features. Thermophiles have also been found in ocean vents located in the Abyssopelagic zone. These organisms share similar metabolic characteristics with their terrestrial relatives, but have proven challenging to research. The intense amount of pressure present at the ocean floor is difficult to duplicate in a laboratory setting, and these fragile organisms will die without the level of pressure that they have adapted to on the ocean floor (Brock, 1994). As more research is conducted and technology advances, oceanic thermophiles might prove to be just as useful as their counterparts in Yellowstone. Yellowstone National Park is a unique wonderland of unparalleled natural beauty that is home to one of the last, nearly intact ecosystems in Earth’s temperate zone. This mountain wilderness contains grizzly bears, wolves, elk, and some of the last undomesticated herds of bison in the American West. These magnificent animals and untamed wilderness are what draw millions of visitors to this mountainous oasis every year. Most of these visitors are oblivious to the life below the surface of the park. Throughout the boiling waters of Old Faithful and its neighbors, in each bubbling mudpot, and in the brilliant waters of The Grand Prismatic Spring exists an ecosystem of life exponentially more diverse than what lives on the surface. Yellowstone and its resident microorganisms are the origin of breakthroughs in molecular biology that have provided substantial advancements in the fields of medicine, forensics, and remediation. The protection of this natural beauty is crucial for the survival of these unique organisms for the next breakthrough could be lying just below the surface.

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