Yellowstone Hot Springs
Yellowstone Hot Springs
What are hot springs?
Hot springs are geothermal springs that are substantially higher in temperature than the air temperature of the surrounding region.1
Creation of Hot Springs
Where is Yellowstone?
Yellowstone is a U.S. National Park located in Wyoming, Montana, and Idaho. It is also America's first national park; and is a home to a large variety of wildlife including grizzly bears, wolves, bison, and elk. Preserved within Yellowstone National Park are Old Faithful and a collection of the world's most extraordinary geysers and hot springs, and the Grand Canyon of the Yellowstone.2
Creation of Yellowstone Hot Springs
What Microbes Live in Yellowstone Hot Springs?
The varieties of microbes found in Yellowstone National Park hot springs are thermophilic archaea and bacteria. Their classification “thermophile,” translates literally to “heat loving”; these organisms can tolerate or even thrive in temperatures that many organisms are not well adapted to. The temperature range found at Yellowstone is approximately 30º to 100º C with a variable pH range and low concentration of organic matter.
Due to the unique nature of their environment, these thermophiles have adapted a number of different features to help them survive in extreme conditions. Among the advantages that come with increased temperature are higher reaction rates, higher solubility of most chemicals, and increased fluidity and diffusion rates. Conversely, increased temperature could also result in protein denaturation and could prove detrimental to cellular processes. To compensate for the harmful effects of higher temperature, thermophilic microbes have unique features that allow them to thrive in their environment. They tend to have a higher melting temperature due to the high content of C and G nucleotides. Also, archaea that live in high-sulfur environments can gain energy be reducing sulfur anearobically. The majority of thermophillic archaea are actually anaerobes due to the low solubility of oxygen at high temperature. Other common features that allow archaea to live in extreme environments include cell wall components that include pseudomurein, special proteins and polysaccharides. Their membrane lipids consist of glycerol and isopranyl ethers as opposed to the acid esters of bacteria. Thermophilic bacteria can generally survive in maximum temperatures lower than thermophilic archaea. The survival mechanisms of bacterial thermophiles could involve modification of their cell wall (greater charged amino acids), lipids, and protein compositions. They also have modified cellular processes. For example, in the thermus species, the electron transport chain, when compared to mesophiles, shows a lower molar growth yield for glucose, possibly explained by the higher membrane permeability of thermophiles.
Temperature/Microbial Zones of Hot Springs
Pigmentation of thermophiles exposed to sunlight in the hot springs is a good indicator of the changes in temperature. We can divide the temperature ranges into four zones characterized by different microbes that are dominant in each zone.
- High Temperature Zone (>73ºC)
- The high temperature zone hosts heterotrophic and lithotrophic bacteria and archaea that are nonphotosynthetic.
- Synechococcus-Chloroflexus Zone (73-60ºC)
- Synechococcus-Chloroflexus zone is characterized by the presence of abundant amount of Synechococcus and Chloroflexus microbes that make up the yellow, orange, and greenish bacterial biofilms on water surfaces and line the bottom of hot springs where temperature is below 73º C. Synechococcus are cyanophytes and are observed to make yellow and green biofilms on surfaces in contact with water temperature ranging from 67 to 73º C. The mats provide a natural system in which the organic matter such as photoexcreted glycolate formed by Synechococcus could be utilized by heterotroph organisms. Where Synechococcus makes yellow and green biofilms, the photosynthetic filamentous bacterium Chloroflexus auraniacus makes green and orange laminated mats on water surfaces ranging from 60 to 68º C. Thus, no bacterial mats in High temperature zones is observed where water is higher than 73 º C, but as the water cools down around these zones, an outline of yellow and green is present, and as temperature cools down further away, orange mats indicates the presence of Chloroflexus bacterium. Synechococcus and Chloroflexus also line the bottom of hot springs where water temperature is lower than 73º C, where Chloroflexus can metabolize organic acids produced by the fermentation of organic matter.
- Phormidium zone (60-30ºC)
- In the Phormidium zone, where water temperature ranges from 30 to 60ºC, the dominant species of filamentous cyanobacteria Phormidium forms extensive bacterial mats of varying morphologies with eukaryotic algae and fungi. These morphologies include:
- o Active streams: longitudinal bacterial streamers
- o Flat rubbery bacterial sheets: complex bacterial community, including :Mastigocladus laminosus, which can tolerate freezing and dessication
- o Terrace pools: smalls subaqueous conical stromatolites and flat-topped stromatolites, both are varieties of Phormidium
- o Terrace fronts: thick rubbery mats
- In addition to these bacteria, Synechococcus and Chloroflexus are also present in this zone.
- Calothrix zone (<30ºC)
- The Calothrix zone, with water temperature lower than 30ºC, is characterized by grayish-brown flat and postular mats of filamentous Cyanobacteria Calothrix. This zone tends to be shallow (less than five cm deep) and has closely packed vertical and subvertical bacterial filaments that are, like Phormidium, coated with silica. These filaments form small microspicular branching shrublike masses that are denser and more resistant to crushing than the bacterial mats formed by Phormidium.
Yellowstone Hot Spring Regions
Lower Geyser Basin
Lower Geyser Basin is the largest geyser basin of Yellowstone National Park, locating between Old Faithful area and Madison Junction. After the Yellowstone Caldera erupted about 600,000 years ago and the floor of the giant crater dropped downward along a ring-shaped fault, molten rock, hot water, and steam move through conduits at various levels beneath the caldera to shape up what is now the Lower Geyser Basin [ ]. Within the 12-square-mile geyser lays a variety of thermal features such as mud pots, geysers, pools, fumaroles and the most- studied hot springs, Octopus Spring and Mushroom Spring.
- Mushroom Springs
- Mushroom Spring is silicious and moderately alkaline with pH around 8 and has a temperature range of 60ºC’s. The spring contains very low sulfate concentration with reduced activity of sulfur cycle with depths due to both sulfate and carbon limitation. However, its microbial mat sustains a highly active sulfur cycle by sulfate-respiring prokaryotes (SRP) [RA5].The highly active population of SRP mediates both oxidizing and reducing pathways of the sulfur cycle even with low concentration of sulfate. There is also a measurable amount of glycolate which governs the relationship of many glycolate-excreting bacteria and glycolate-incorporating bacteria such as Synochococcus lividus and filamentous heterotrophs. This relationship shows that many bacteria in Mushroom Spring as well as in other Yellowstone hot springs operate closely in communities.
- Synechococcus lividus and filamentous heterotrophs share a cross-feeding relationship. The photosynthesis by S. lividus promotes glycolate photoexcretion while filamentous heterotrophs incorporate the excreted glycolate [RA21]. Glycolate is a major photoexcretion product whose production is light-dependent and increased in the presence of O2. However, the oxidation of glycolate to CO2 is not completely light-dependent, suggesting that there is a working relationship between both photoheterotrophic and chemoheterotrophic organisms to metabolize glycosylated. These two types of bacteria work in adjacent to each other to synthesize and utilize glycolate.
- Sulfate-respiring Prokaryotes (SRP) are obligate anaerobes that can reduce sulfate with the highest rates after the depletion of oxygen chemocline at dusk or in immediate response to shading. The reoxidation of sulfide occurs via either aerobic oxidation or anoxic photosynthesis, but is inhibited when both oxygenic and anoxygenic photosynthesis is attenuated by shading. [RA5]. Sulfate respiration is an important process in thermophilic microbial mats despite fluctuating oxygen levels and very low sulfate concentration of the spring.
- Octopus Springs
- Octopus Spring is warmed by the heat generating from the magma below the surface. The hot water dissolves the silica of volcanic rock and precipitates it, giving the silicate deposits, or sinter, a grayish white color [ ]. The spring has a temperature range of 95ºC near the source to 60ºC. There is also a lack of hydrogen sulfide in the water, making it alkaline with a pH around 8. The spring has a high concentration of hydrogen gas due to the bacteria that utilize it. There is a diverse collection of bacteria thriving in the hot spring mats including photoautotrophic, photoheterotrophic, chemoautotrophic, and heterotrophic organisms [RA3].
- Synechococcus is a thermophilic cyannobacterium which has the highest temperature tolerance of the cyanobacteria in this ecosystem, up to 71ºC [ ]. The bacteria have evolved to temporally separate photosynthesis and N2 fixation to maximize the use of organic sources in Octopus Spring. There are critical regulatory switches that control many metabolic processes within the bacteria as well as in other organisms in the microbial mat. The bacteria have nif genes that are expressed when the mat becomes anoxic under declining photon irradiance in the late afternoon. The gene is correspondingly related to the increase in nitrogenase activity which fixes N2 during the night (RA3). Fixing N2 requires a lot of energy; therefore, the bacteria rely on fermentation process to acquire energy to power N2 fixation at night time [ ]. Overall, Synechococcus rely on photosynthesis and respiration during the day when O2 level is high, but switch to fermentation and N2 fixation at night.
- Methanobacterium thermoautotrophicus is a strictly anaerobic, autotrophic extremophile that is irregularly shaped as curved rods which often formed long filaments [RA 16]. M. thermoautotrophicus have an optimum temperature for growth and methanogenesis of 65ºC to 70ºC. The bacteria use ammonia as nitrogen source, sulfide as sulfur source and hydrogen- carbon dioxide as main energy and carbon source. Their ability to use hydrogen for reducing power and carbon dioxide classifies them as hydrogen-oxidizing autotroph. The ability of the bacteria’ ribosomes to undergo a 20% increase in hyperchromicity and endure high heat plays an important role in governing the maximal growth temperature of the bacteria depending on the areas [RA16]. Overall, M. thermoautotrophicus seem to optimally adapt to the temperatures at which they are found [RA10].
Norris Geyser Basin
Norris Geyser Basin (Norris Basin) is located north from the Yellowstone caldera and between the Hebgen Lake and Mammoth Springs. This location in Yellowstone can be broken down into three main areas called the Porcelain Basin, Back Basin, and One Hundred Springs Plain. Norris Basin is better known for the geysers it is home to, which includes Steamboat Geyser, the largest and highest shooting active geyser in the world, and Echinus Geyser. To many scientists, researchers, and tourists that visit Yellowstone National Park, Norris Geyser Basin is considered one of the most interesting and diverse regions. This is attributed to the fact that the Basin contains a variety of hot spots and thermal activity in the form of hot springs, geysers, and mud volcanoes, and is also one of the few regions in Yellowstone that undergoes drastic changes in thermal activity and landscape. One of the noteworthy changes observed is when a hot spring transforms into a geyser or a geyser changes into a hot spring. (L1)
Besides the abundance of geysers, Norris Basin also consists of hot springs that are inhabited by various microbes. The groups of hot springs that exist in this region are mostly acidic (between pH 2 and 3) with the temperature of each individual hot spring varying across the region. Most hot springs in this regions are considered acid-sulfate-chloride (ACS) springs due to the high acid and sulfate content. The microbes that inhabit acidic environments (including the hot springs here) and have a mode of survival in low pH are collectively called acidophiles. Some of the hot springs that are home to these microbes which have been characterized include Green Dragon Springs and Beowulf Springs. The best characterized microbe known to be abundant in Norris Basin is the thermophile Sulfolobus known for oxidizing hydrogen sulfide to sulfuric acid at high temperature and acidic pH (L1). Recent studies on acid-sulfate-chloride geothermal springs have shown that chemical gradients of energy resources (i.e. Hydrogen Gas) create a distinct community dependent on the distance from the geothermal discharge source of the spring with distinct resources being more concentrated or less concentrated going away from the source such as arsenite oxidation being inhibited in the high-sulfide zone of the spring (L2, L3, L4).
- Green Dragon Springs
- This hot spring is located in the One Hundred Springs Plain of Norris Geyser Basin. Dragon Springs has been well characterized as a near boiling acid-sulfate-chloride spring due to its high chemical composition of sulfate and chloride. The spring’s pH is about 3 with the temperature ranging from 66 to 73 degrees celsius. Other components of the hot spring include various organic carbon sources, hydrogen sulfide (H2S), Iron (II), As (III), and an abundance of elemental sulfur. Due to the temperature, pH, and available energy sources, microbes living in Dragon Springs (as well as other hot springs with similar conditions) have acquired the ability to utilize available elements or ions for energy. Some of the microbes recently identified include two Crenarchaeas (phylum within the domain Archaea), chemo-organotrophes that uses elemental sulfur for energy, and an new class of chemolithotrophic microbes that oxidize As(III) to As(V) for energy.
- Beowulf Springs
- This spring is located not too far from Green Dragon Springs in the One Hundred Springs Plain of Norris Geyser Basin, YNP. The specific conditions for Beowulf Springs is also similar to Green Dragon Springs and consists of a temperature near 70°C and pH of 3.2. Beowulf Springs is also considered and acid-sulfate-chloride geothermal spring like many other springs in Norris Geyser Basin. While Green Dragon Springs is best characterized for its elemental sulfur, the Fe(III) oxide (Hydrous Ferric Oxide) microbial mats that exist in this spring is best characterized. The Fe(III) oxide mats have been observed to have thermophilic crenarchaea which have the capability of oxidizing iron. Besides iron metabolism, like many other hot springs in Norris Geyser Basin, other sources like sulfate are used as an energy source. (Kozubal et al.)
Hot Springs of Other Countries
One country known for having hot springs is Chile. The water found in the hot springs originates from volcanoes found throughout the country. Most of the hot springs are surrounded by lush vegetation and can be found near an inlet of water. A majority of the hot spring’s temperature ranges from 125˚F to 185˚F (52˚C to 85˚C). Some hot springs such as the Puyuhuapi have a pH of 6.3 while others such as Huife have a slightly more basic pH of 8.8. The hot springs are rich in minerals such as magnesium, chlorides, bromides, calcium, sodium, zinc, sulfur, etc. These hot springs have therapeutic benefits as well since in some hot springs the water can be ingested as tonic water or can be used to relieve rheumatism, arthritis, bronchitis, fatigue and skin conditions. [N1]
Situated on the fault between the North American and European plates, Iceland is volcanically and geologically active with numerous geothermal features, such as hot springs, mud pots, geysers, and fumaroles. Geothermal hot springs in Iceland are divided into high temperature fields and low temperature fields. High temperature areas, which are only found on the active volcanic rift zones, have temperatures of at least 150°C with a heat source of magma chamber. The low temperature fields, found in the vicinity of Reykjavik, have temperatures of less than 150°C at a depth of one kilometer [C1]. These varying features provide habitats for different groups of thermophilic life. Some famous hot springs in Iceland include the one in Grindavik, and Europe’s highest flow rate hot spring Deildartunguhver, which has a flow rate of 180 liters/second emerging at 97°C. Some of the water is used for heating as energy sources [C2].
A volcanically active island country, Japan has approximately 150 hot springs (onsen) with 14000 individual springs. The hot springs are broken down by different temperature ranges: less than 25°C, 25-34°C, 34-42°C, and higher than 42°C, and are called rai sen, bi on sen, on sen, and kou on sen, respectively. pH value of hot springs differ from place to place. The natural acidic hot springs have pH values of less than 5. For most of the onsen, pH ranges from 5 to 8 [C3]. In contrast with Yellowstone hot springs, Japanese hot springs are mainly places to relax and are considered to have various medical effects. Different minerals or chemicals in onsen’s water have different therapeutic uses. For example, the carbonate springs, which haves less than 1 gram of radical carbon and other mineral elements in each liter, are good for heart disease, blood circulation disorders, neurological disorders, and female disorders. Simple springs, which have water temperature higher than 25°C, are thought to be good for neuralgia, rheumatism and long term rehabilitations [C4]. Some of the popular springs in Japan are Kusatsu Onsen, Hakone, Kamuiwakka Falls, and Beppu.
New Zealand is very popular for having many hot springs throughout the country. In the 19th century, there were about 220 erupting hot springs, but now there are approximately 58, most of which have been turned into spas and resorts. The water found in the hot springs comes from 3 geological features: younger erupting volcanoes, older cooler volcanoes and water near fault lines. The temperature at the center of the volcano of the hot springs can be even greater than 300˚C, but most have been cooled to approximately 30˚ to 45˚C to be used in spas. The hot springs are rich in minerals such sodium, calcium, sulfur, potassium, magnesium, and chlorides. These hot springs are often used for therapeutic effects, such as relieving arthritis pains, bronchial infections and for cleaning the skin. [N2], [N3], [N4]
There are some microbes and microbe products that were found in some of the New Zealand hot springs. A thermophilic anaerobic spirochete was found to grow in optimum temperature of 45˚C to 50˚C and pH of 7.0-7.5. Also oncoids, which are coated grains that are produced by microbes such as cyanobacteria were found in the hot springs of New Zealand. [N5], [N6]
One of the microbes that was discovered by bacteriologist Thomas Brock in the 1960’s was Thermus aquaticus. This microbe was a major discovery because it could not only survive in temperatures near 100C and was one of the first microbes that can be considered an archaea, but also provided an enzyme that was essential for the PCR reaction. TAQ polymerase, which is the enzyme produced by the thermus aquaticus is a thermostable DNA polymerase. It is also used for 3”A-tailing of blunt ends, primer extensions and DNA sequencing. PCR is a technique that allows researchers to produce millions of copies of specific DNA sequences and amplify DNA is a short amount of time. [N7], [N8], [N9]
(1) Mycobacterium parascrofulaceum in Acidic Hot Springs in Yellowstone National Park
- Mycobacteria have a high resistance to chlorine and biocides in water and are able to form biofilm. Their tolerance to extreme temperatures and starvation survival allow them to persist in extreme environments. Samples of Mycobacterium parascrofulaceum were collected from two thermal springs and their respective runoffs in the One Hundred Spring Plain. This system of two springs is very acidic (pH 3.0) with temperature ranging from 56.1°C to 40.0°C. To confirm the temperature resistance of M. parascrofulaceum isolates, the strains were incubated at 42°C and 55°C for 60 days. Samples were extracted and tested by PCR targeting of part of the 16S-23S spacer region, and PCR primers. Fluorescence microscopy was used to assess the presence of mycobacteria. All samples were positive by both assays for the entire duration of the assay. The experiment performed the first isolation of mycobacteria and confirmed that M. parascrofulaceum could grow at this temperature. (C1)
(2) High Rates of Sulfate Reduction in a Low-Sulfate Hot Spring Microbial Mat are Driven by a Low Level of Diversity of Sulfate-Respiring Microorganisms
- Cyanobacterial mats found in the Mushroom Spring in Yellowstone National Park depend heavily on sulfur respiration. The results demonstrated that this bacterial community was able to sustain an active sulfur cycle despite the very low sulfate concentration. It was found that the highest rates of sulfate respiration were measured close to the surface of the mate when photosynthetic oxygen production stopped. This study helped the understanding of the geobiochemistry of the early Earth, in which oxidized sulfur species were less abundant that today. It also revealed a highly active sulfate-reducing community composed of dsrAB genotypes, especially active during the switch from net photosynthetic to net respiratory conditions. (C2)
(3) NASA Research Focuses on Yellowstone’s Hot Springs and Compares Findings to Rocks from Mars
- The unanswered fundamental questions of where life originated and how it evolved are being answered at the Yellowstone Hot Springs. The microorganisms that inhibit these hot waters can help form a picture of the history of life and climate changes on Earth. Scientists are studying thermophiles and the fossils of thermophiles to see the evolutionary process that has taken place. The scientists are also hoping to be able to compare the fossils of the thermophiles to rocks found on Mars to determine if life did exist or if life can exist in Mars’ conditions. [N10]
(4) Microbes in Hot Springs Test Notion of Global Travel
- It has been a long standing notion that “Everything is everywhere” when applied to microorganisms. Two teams of scientists have recently discovered that this saying may not be true. They have discovered that the microbes that live in Yellowstone differ from those that live in New Zealand or anywhere else. It has been noted that the further the distance from each hot spring, the more genetically different the microbes are. This new discovery is now going to be tested on other bacteria to see if they are also genetically isolated like the thermophiles which live in hot springs throughout the world. [N11]
L1 White, Donald E., Hutchinson, Roderick A., and Keith, Terry E.C., 1988, The geology and remarkable thermal activity of Norris Geyser Basin, Yellowstone National Park, Wyoming: U.S. Geological Survey Professional Paper 1456, 84 p., 1 plate, scale 1:2,400.
L2 MACUR, R. E., LANGNER, H. W., KOCAR, B. D. AND INSKEEP W. P. Linking geochemical processes with microbial community analysis: successional dynamics in an arsenic-rich, acid-sulphate-chloride geothermal spring. Geobiology, 2004 2:163-177.
L4 Jackson C R; Langner H W; Donahoe-Christiansen J; Inskeep W P; McDermott T R Molecular analysis of microbial community structure in an arsenite-oxidizing acidic thermal spring. Environmental microbiology 2001;3(8):532-42
L5 Boyd, E. S., Jackson, R. A., Encarnacion, G., Zahn, J.A., Beard, T., Leavitt, W. D., Pi, Y., Zhang, C. L., Pearson, A., and Geesey G. G. Isolation. Characterization, and Ecology of Sulfur-Respiring Crenarchaea Inhabiting Acid-Sulfate-Chloride-Containing Geothermal Springs in Yellowstone National Park. Appl. Environmental Microbiol. October 15, 2007; 73(20): 6669 - 6677
L6 D'Imperio, S., Lehr, C. R., Breary, M., McDermott, T. R. Autecology of an Arsenite Chemolithotroph: Sulfide Constraints on Function and Distribution in a Geothermal Spring Appl. Environ. Microbiol. 2007 73: 7067-7074
L7 Kozubal, M., Macur, R. E., Korf, S., Taylor, W. P., Ackerman, G. G., Nagy, A., Inskeep, W. P. Isolation and Distribution of a Novel Iron-Oxidizing Crenarchaeon from Acidic Geothermal Springs in Yellowstone National Park. Appl. Environ. Microbiol. 2007 0: AEM.01200-07
L8 White, D. E., Hutchinson, R. A. & Keith, T. E. C. The geology and remarkable thermal activity of Norris Geyser Basin, Yellowstone National Park, Wyoming. US Geol. Surv. Prof. Pap. 1456, 1–84 (1988)
[N1] Hot Springs Guide. 2000. <http://www.gochile.cl/eng/guide/chiletermas/Chile-Termas.asp>
[N2] Stewart, Carol. 'Hot Springs, Mud Pools and Geysers', Te Ara - the Encyclopedia of New Zealand, 5-Nov-2007 <http://www.TeAra.govt.nz/EarthSeaAndSky/HotSpringsAndGeothermalEnergy/HotSpringsMudPoolsAndGeysers/en>
[N3] Hot Mineral Pools. 2005. <http://www.taupohotsprings.com/?PK_CATEGORY_ID=2>
[N4] The Pools. 27-August-2008 <http://hanmersprings.co.nz/thermal/pools/>
[N5] Patel, B.K.C., H.W. Morgan & R.M. Daniel. 'Thermophilic Anaerobic Spirochetes in New Zealand Hot Springs.' 2 October 1984. <http://www3.interscience.wiley.com/cgi-bin/fulltext/119517857/PDFSTART>
[N6] Renaut, Robin W., Brian Jones, Michael R. Rosen. 'Primary Silica Oncoids from Orakeikorako Hot Springs, North Island, New Zealand.' Palaios, v.11, p.446-458, 1996 <http://www.jstor.org/sici?sici=0883-1351(199610)11%3A5%3C446%3APSOFOH%3E2.0.CO%3B2-X>
[N7] 'Forecast: Hot and Humid.' University of Wisconsin, Board of Regents, 1999 <http://whyfiles.org/022critters/hot_bact.html>
[N8] TAQ Polymerase. Genscript Corporation, 2007. <http://taqpolymerase.net/>
[N9] Polymerase Chain Reaction. Gene Almanac. <http://www.dnalc.org/ddnalc/resources/pcr.html>
[N10] Yellowstone Park Foundation. 'NASA Research Focuses on Yellowstone’s Hot Springs and Compares Findings to Rocks from Mars.' 10-March-2004. <http://www.astrobiology.com/news/viewpr.html?pid=13809>
[N11] Yoon, Carol Kaesuk. 'Microbes in Hot Springs Test Notion of Global Travel.' New York Times, 26-August-2003. <http://query.nytimes.com/gst/fullpage.html?res=9B03E1DA1239F935A1575BC0A9659C8B63>
C1 Geothermal Resources in Iceland. 1995. <http://www.energy.rochester.edu/is/reyk/resources.htm>
C2 Deildartunguhver. <http://www.husafell.is/ensku_sidurnar/e_nagrenni/e_deildartunguhver/e_deildartunguhver.htm>
C3 Japanese Onsen Spa Guide. Japan Travel. 2008. <http://gojapan.about.com/cs/traditionculture/a/hotsprings.htm>
C4 Hot Spring in Japan. East Creative Inc., 2004. <http://www.east.co.jp/oyu/english/>
Edited by [Yu-Chen Chiu, Ngoc Dinh, Jenny Lee, Christina Pham, Lucas Puttock, Naon Shin], students of Rachel Larsen