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Characterization of Aerobic Respiration in Great Basin Hot Springs UNLV Theses, Dissertations, Professional Papers, and Capstones 2009 Characterization of aerobic respiration in Great Basin Hot Springs Caitlin N. Murphy University of Nevada Las Vegas Follow this and additional works at: https://digitalscholarship.unlv.edu/thesesdissertations Part of the Environmental Microbiology and Microbial Ecology Commons Repository Citation Murphy, Caitlin N., "Characterization of aerobic respiration in Great Basin Hot Springs" (2009). UNLV Theses, Dissertations, Professional Papers, and Capstones. 70. http://dx.doi.org/10.34870/1374011 This Thesis is protected by copyright and/or related rights. It has been brought to you by Digital Scholarship@UNLV with permission from the rights-holder(s). You are free to use this Thesis in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Thesis has been accepted for inclusion in UNLV Theses, Dissertations, Professional Papers, and Capstones by an authorized administrator of Digital Scholarship@UNLV. For more information, please contact [email protected]. CHARACTERIZATION OF AEROBIC RESPIRATION IN GREAT BASIN HOT SPRINGS by Caitlin N Murphy Bachelor of Science in Microbiology University of Michigan 2007 A thesis submitted in partial fulfillment of the requirements for the Master of Science Degree in Biological Sciences School of Life Sciences College of Sciences Graduate College University of Nevada, Las Vegas August 2009 ABSTRACT Characterization of Aerobic Respiration in Great Basin Hot Springs by Caitlin N Murphy Dr. Brian Hedlund, Committee Chair Professor of Biology University of Nevada, Las Vegas Despite a wide diversity of possible electron donors available to fuel chemolithotrophy, it has been proposed that hydrogen is the single most important electron donor in geothermal ecosystems. To directly test this hypothesis, a simple system was devised to determine whether microorganisms in hot spring water and sediment are capable of using hydrogen and other electron donors for aerobic respiration using microrespirometry. The protocol for these experiments was developed using pure cultures of Thermocrinis ruber to determine the effect of growth conditions on the rate of oxygen consumption following the addition of electron donors. For field experiments, samples were collected without introduction of oxygen or change in temperature and measurements of oxygen consumption were initiated within ten minutes of collection. After a baseline oxygen consumption rate was established, possible electron donors were added and oxygen consumption was measured over a twenty-minute window. This 2- system was used to evaluate the impact of electron donor addition (1 mM each of S 2O3 , formate/lactate/acetate/propionate mixture, yeast extract/peptone, NH 3 and 10 µM each iii H2 and CH 4) on oxygen consumption in source water and surface sediment of two near- neutral pH hot springs in the Great Basin, Great Boiling Spring (GBS, 80°C, pH 7) and Sandy’s Spring West (SSW, 83°C, pH 7). The composition of the microbial community in the corresponding environments was also evaluated using a combination of terminal restriction fragment length polymorphism (T-RFLP) and fluorescent in situ hybridization (FISH). The resulting data showed that both the simple water-borne microbial communities (~2-4 species) and the more complex sediment microbial communities (~20-35 species) have diversified to take advantage of a variety of electron donors. These data do not support the hypothesis that hydrogen is the most important electron donor in the source water and sediments of the springs that were examined because hydrogen only stimulated an increased rate of oxygen consumption in two of the four environments tested. This study provides an important step in providing a more complete understanding of the energy budgets of high-temperature ecosystems and provides a template to study other habitats. iv TABLE OF CONTENTS ABSTRACT ....................................................................................................................... iii LIST OF FIGURES ........................................................................................................... vi LIST OF TABLES ............................................................................................................ vii ACKNOWLEDGEMENTS ............................................................................................ viii CHAPTER 1 INTRODUCTION .....................................................................................1 Hydrogen and Hyperthermophiles ..................................................................................1 Thermodynamic Predictions .........................................................................................10 Testing the “H 2 hypothesis” ..........................................................................................13 Great Basin Hot Springs ...............................................................................................18 Microbial Community of GBS and SSW......................................................................20 References .....................................................................................................................30 CHAPTER 2 MATERIALS AND METHODS ............................................................36 Microrespirometry ........................................................................................................36 Terminal Restriction Fragment Length Polymorphism ................................................42 Fluorescent In Situ Hybridization .................................................................................46 Reverse Transcriptase Polymerase Chain Reaction ......................................................49 References .....................................................................................................................52 CHAPTER 3 RESULTS & DISCUSSION ...................................................................55 Thermocrinis rube r Pure Culture Studies .....................................................................55 Microrespiration Studies in GBS and SSW ..................................................................59 Enrichments ..................................................................................................................72 Assessing the Microbial Community in GBS and SSW ...............................................75 RNA Extraction and RT-PCR .......................................................................................89 References .....................................................................................................................90 CONCLUSION ..................................................................................................................94 VITA ..................................................................................................................................96 v LIST OF FIGURES Figure 1.1 The versatility of molecular hydrogen as an electron donor .........................4 Figure 1.2 Thermodynamic modeling in Yellowstone National Park (YNP) ................7 Figure 1.3 The pathway of hydrogen oxidation ..............................................................9 Figure 1.4 Thermodynamic modeling in the Great Basin and YNP .............................12 Figure 1.5 Anticipated rate of O 2 consumption in respirometry experiments .............16 Figure 1.6 Map of the Great Basin................................................................................19 Figure 1.7 Great Boiling Spring and Sandy’s Spring West ..........................................20 Figure 1.8 Phylogenetic tree of the bacterial phylotypes in GBS and SSW .................24 Figure 1.9 Phylogenetic tree of the archaeal phylotypes in GBS and SSW .................28 Figure 2.1 Schematic representation of the O 2 microsensor experimental set-up ........38 Figure 2.2 Interpretation of microrespirometry with liquid electron donors ................49 Figure 2.3 Interpretation of microrespirometry with gaseous electron donors .............41 Figure 2.4 Schematic representation of T-RFLP procedure .........................................43 Figure 3.1 Rate of O 2 consumption after H 2 addition (Minutes 1-10) ..........................56 Figure 3.2 Rate of O 2 consumption after H 2 addition (Minutes 21-30) ........................57 2- Figure 3.3 Rate of O 2 consumption after S 2O3 addition (Minutes 1-10) ....................58 Figure 3.4 Unamended rate of O 2 consumption in GBS and SSW ...............................60 Figure 3.5 Rate of O 2 consumption in GBS and SSW water ........................................63 Figure 3.6 Rate of O 2 consumption in GBS and SSW sediment ..................................69 Figure 3.7 Epifluorescence microscopic images of T. ruber ........................................78 Figure 3.8 Epifluorescence microscopic images of GBS bulk water (Arc v. Bac) ......79 Figure 3.9 Epifluorescence microscopic images of GBS bulk water (Cren v. Eury) ...80 Figure 3.10 Epifluorescence microscopic images of SSW bulk water (Arc v. Bac) ......82 Figure 3.11 Epifluorescence microscopic images of common cell type seen in SSW ...83 Figure 3.12 Epifluorescence microscopic mixed images of SSW water (Arc
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