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Microbial Diversity in the Thermal Springs Within Hot Springs National Park E Journal of the Arkansas Academy of Science Volume 72 Article 9 2018 Microbial diversity in the thermal springs within Hot Springs National Park E. Taylor Stone Hendrix College, [email protected] Richard Murray Hendrix College, [email protected] Matthew .D Moran Hendrix College, [email protected] Follow this and additional works at: https://scholarworks.uark.edu/jaas Part of the Biodiversity Commons, and the Environmental Microbiology and Microbial Ecology Commons Recommended Citation Stone, E. Taylor; Murray, Richard; and Moran, Matthew D. (2018) "Microbial diversity in the thermal springs within Hot Springs National Park," Journal of the Arkansas Academy of Science: Vol. 72 , Article 9. Available at: https://scholarworks.uark.edu/jaas/vol72/iss1/9 This article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able to read, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. This Article is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academy of Science by an authorized editor of ScholarWorks@UARK. For more information, please contact [email protected], [email protected]. Microbial diversity in the thermal springs within Hot Springs National Park Cover Page Footnote We wish to thank Hot Spring National Park staff for their assistance in collecting the samples and accessing decommissioned bathhouse facilities. The eH ndrix College Odyssey Program provided generously provided funds for this research. This article is available in Journal of the Arkansas Academy of Science: https://scholarworks.uark.edu/jaas/vol72/iss1/9 Journal of the Arkansas Academy of Science, Vol. 72 [], Art. 9 Microbial Diversity in the Thermal Springs within Hot Springs National Park E.T. Stone1, R. Murray, and M.D. Moran Department of Biology, Hendrix College, 1600 Washington Ave., Conway, AR 72032 1Corresponding author The authors of this manuscript declare no conflicts of interest. Running Title: Microbial Diversity in Arkansas Hot Springs Abstract work in thermophilic bacteria have yielded discoveries with far-reaching implications, ranging from The thermal water systems of Hot Springs National biogeochemistry to biotechnology (Leis et al. 2015; Park (HSNP) in Hot Springs, Arkansas exist in relative Shrestha et al. 2018; Undsworth and Koutsopoulos isolation from other North American thermal systems. 2007). Evolving in relative isolation from other The HSNP waters could therefore serve as a unique extremophiles provides opportunities for unique center of thermophilic microbial biodiversity. However, communities to develop, resulting in community these springs remain largely unexplored using culture- structures that vary tremendously, even among similar independent next generation sequencing techniques to study sites (Amin et al. 2017). Investigation of classify species of thermophilic organisms. individual microbes in extreme environments has Additionally, HSNP has been the focus of yielded the discovery of thermostable enzymes critical anthropogenic development, capping and diverting the to biotechnological advances such as PCR (Brock springs for use in recreational bathhouse facilities. 1969), treatment of industrial waste (Shrestha et al. Human modification of these springs may have 2018), and industrial chemistry (Leis et al.2015). impacted the structure of these bacterial communities The in-depth characterization these communities compared to springs left in a relative natural state. The warrant cannot be accomplished with culture-dependent goal of this study was to compare the community methods alone, as it has been hypothesized that less than structure in two capped springs and two uncapped 1% of environmental bacteria are likely to be cultured springs in HSNP, as well as broadly survey the in laboratory settings (Staley and Konopka 1985). Given microbial diversity of the springs. We used Illumina 16S the stringent growth requirements for extremophiles, rRNA sequencing of water samples from each spring, and the observation that some thermophiles appear to be the QIIME workflow for sequence analysis, and co-culture dependent (Stewart 2012), the proportion of generated measures of genera and phyla richness, culturable thermophiles is likely smaller. The diversity, and evenness. In total, over 700 genera were limitations of culture-dependent characterization render detected and most individual samples had more than 100 these methods insufficient for improving understanding genera. There were also several uncharacterized of these microbial communities. However, with the sequences that could not be placed in known taxa, increasing accessibility of modern high-throughput indicating the sampled springs contain undescribed sequencing techniques, researchers have improved bacteria. There was great variation both between sites understanding of high-temperature microbial and within samples, so no significant differences were community structure, allowing the development of detected in community structure between sites. Our metagenomic libraries specific to thermophiles (Mirete results suggest that these springs, regardless of their et al. 2016). In spite of these advances, many isolated human modification, contain a considerable amount of regions of thermophilic activity remain unexplored, biodiversity, some of it potentially unique to the study limiting knowledge of prospective biodiversity. site. Hot Springs National Park is an isolated thermophilic environment located in the Ouachita Key Words: Hot Springs National Park, Microbial Mountains of Arkansas, and is the only hot water Diversity, Next Generation Sequencing, Thermophiles thermal spring complex with average water temperatures above 50°C in the central U.S. (NOAA Introduction NCEI 2018). Given its geographic isolation, opportunities for biological exchange with other North Constraints governing the biochemical processes at American thermophile communities are likely very Journal of the Arkansas Academy of Science, Vol. 72, 2018 31 Published by Arkansas Academy of Science, 31 Journal of the Arkansas Academy of Science, Vol. 72 [], Art. 9 E.T. Stone, R. Murray, and M.D. Moran limited. While the hot water springs have been used for individual flows and previously used for recreation and human recreation for thousands of years, intense tourism. The two capped sites, the Fordyce Bath House development of the area began in the 1830s (Hanor Spring (F) and the Hale Bath House Spring (H), have 1980). Modern development has included heavy been decommissioned for public use. The remaining landscaping and the capping and diversion of the springs sites, the Lamar Display Spring (L) and the Tunnel to recreational facilities, such as bath houses. Today, Display Spring (T), are open-air springs on display to only ten spring complexes are uncapped and open to the the public. environment (Yeatts 2006). In the open springs, a presumably more complex ecological community exists Sample Collection and Preparation with thermophilic algae (Smith 2010) and crustaceans Duplicate water samples from each site were (Meg O’Connor, personal correspondence). By contrast, collected aseptically at the surface level using sterile 50 the capped springs, which lack a light source, are likely mL conical tubes and stored immediately on ice in to contain a simpler community consisting mostly of transit to the laboratory for further processing. Samples chemotrophs (probably chemolithotrophs). Our goal in were processed within four hours of collection for DNA this study was to characterize the microbial extraction. All water samples were filtered via a communities in both capped and uncapped hot springs Millipore® filtration apparatus containing a filter with a within Hot Springs National Park. We hypothesized that pore size of 0.45 μm. DNA extraction was performed the microbial diversity would be lower and the using the Zymo Research® ZR Fecal DNA Miniprep community structure distinctive in capped springs Kit (Catalog No: D6010) according to manufacturer compared to the uncapped springs. While some novel specifications. Purified product was quantified by thermophiles have been identified in this system via spectrophotometry and stored at -80°C until preparation traditional laboratory culture and 16S rRNA sequencing for Illumina Sequencing. at specific sites within the park (Marks et al. 2012), the community structures of these different springs have not Sequencing and Analysis been broadly compared with culture-independent PCR-amplification targeted variable regions V3 & methods. Therefore, our study may provide insight into V4 of the 16S ribosomal subunit and amplified from the biodiversity in this isolated system, detect human 515bp to 806 bp within the gene using the Illumina impacts on the communities, and assess the 515FB/806RB primer pair (Caporaso et al. 2011; Parada conservation value of the capped and uncapped springs. et al. 2016) for a total of 291bp. This PCR- amplification, as well as barcoding and high-throughput Materials and Methods sequencing on an Illumina platform, was performed by Wright Labs (Huntingdon, PA 16652) using primers Sampling Sites utilized in previous studies. All sequence data was All sampling sites were located within Hot Springs analyzed with access to computing cluster located at National Park (HSNP)
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