BIOLOGY OF ACID-SULFATE-CHLORIDE SPRINGS IN YELLOWSTONE NATIONAL PARK, WYOMING, UNITED STATES OF AMERICA by Eric Stephen Boyd A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology MONTANA STATE UNIVERSITY Bozeman, Montana July 2007 © COPYRIGHT by Eric Stephen Boyd 2007 All Rights Reserved ii APPROVAL of a dissertation submitted by Eric Stephen Boyd This dissertation has been read by each member of the dissertation committee and has been found to be satisfactory regarding content, English usage, format, citations, bibliographic style and consistency, and is ready for submission to the Division of Graduate Education. Dr. Gill G. Geesey Approved for the Department of Microbiology Dr. Timothy E. Ford Approved for the Division of Graduate Education Dr. Carl A. Fox iii STATEMENT OF PERMISSION TO USE In presenting this dissertation in partial fulfillment of the requirements for a doctoral degree at Montana State University, I agree that the Library shall make it available to borrowers under rules of the Library. I further agree that copying of this dissertation is allowable only for scholarly purposes, consistent with “fair use” as prescribed in the U.S. Copyright Law. Requests for extensive copying or reproduction of this dissertation should be referred to ProQuest Information and Learning, 300 North Zeeb Road, Ann Arbor, Michigan 48106, to whom I have granted “the exclusive right to reproduce and distribute my dissertation in and from microform along with the non- exclusive right to reproduce and distribute my abstract in any format in whole or in part.” Eric Stephen Boyd July 2007 iv ACKNOWLEDGEMENTS Firstly, I would like to extend my gratitude to Dr. Gill Geesey who for the past four busy years has always had time to discuss project ideas and to edit manuscripts. Equally, I would like to thank Marisa for always believing in me and for all she has done over the past two and half years to making my life more enjoyable. I would also like to thank my friends, both close and afar, and of course my loving family for their steadfast support. I would also like to acknowledge my best friend Abbey who, irregardless of my mood or degree of frustration, greeting me each evening with a doggie smile. I am grateful to all of those who I have worked with and learned from in the Geesey Lab: Katie, Wil, Bob, Trevor, Andy, and Gem. I am grateful to Dr. Timothy McDermott for his passioned enthusiasm for researching geothermal biology and to Dr. William Inskeep for teaching me the benefits of studying biology using a systems approach. I would also like to thank the members of my committee: Dr. Mensur Dlakic for helpful discussions and Dr. James Becker for not only representing the graduate college, but also showing genuine interest in my research. I also wish to thank the Inland Northwest Research Alliance for funding two years of my graduate education and the Montana Water Center and the Ferguson Scholars Program for financial aid during the final year of my graduate education v TABLE OF CONTENTS 1. SCOPE OF THESIS ......................................................................................................1 2. BIOLOGY OF ACID-SULFATE-CHLORIDE GEOTHERMAL SPRINGS LITERATURE REVIEW.............................................................................4 Introduction to ASC Geothermal Springs......................................................................4 Sulfur Zone....................................................................................................................7 Introduction..............................................................................................................7 Microorganisms Involved In Sulfur Reduction .......................................................9 Thermocladium-like sp. .....................................................................................9 Caldisphaera-like sp........................................................................................10 Caldococcus-like sp.........................................................................................11 Thermofilum-like sp.........................................................................................12 Stygiolobus-like sp...........................................................................................13 Desulfurella-like sp..........................................................................................13 Microorganisms Involved In Hydrogen Sulfide Oxidation ...................................15 Hydrogenobaculum-like sp..............................................................................15 Sulfur Zone Synthesis............................................................................................16 Iron/Arsenic Zone........................................................................................................19 Introduction............................................................................................................19 Microorganisms Involved In Fe(II) Oxidation ........................................................22 Acidimicrobium-like sp....................................................................................22 Sulfobacillus-like sp.........................................................................................23 Metallosphaera-like sp. ...................................................................................25 Microorganisms Involved In As(III) Oxidation .......................................................26 Hydrogenobaculum-like sp..............................................................................26 Microorganisms Involved In As(III) and Fe(II) Oxidation .......................................27 Thiomonas-like sp............................................................................................27 Microorganisms Involved In Heterotrophy ...........................................................29 Alicyclobacillus-like sp....................................................................................29 Meoithermus-like sp.........................................................................................29 Marinithermus-like sp......................................................................................30 Iron/Arsenic Zone Synthesis..................................................................................31 Phototrophic Zone........................................................................................................33 Introduction............................................................................................................33 Thermophilic Phototrophs ....................................................................................34 Cyanidioschyzon-like sp. .................................................................................34 Galdieria-like sp. .............................................................................................36 Non-Thermophilic Phototrophs .............................................................................38 Zygogonium-like sp..........................................................................................38 vi TABLE OF CONTENTS CONTINUED Chlamydomonas-like sp...................................................................................38 Chlorella-like sp. .............................................................................................39 Phototrophic Zone Synthesis .................................................................................40 Food Webs ...................................................................................................................42 Introduction............................................................................................................42 Invertebrates Involved in ASC Food Webs ...........................................................44 Ephydra thermophila .......................................................................................44 Bezzia setulosa.................................................................................................44 Food web synthesis ..............................................................................................45 References ....................................................................................................................47 3. ISOLATION, CHARACTERIZATION, AND ECOLOGY OF SULFUR-RESPIRING CRENARCHAEA INHABITING ACID-SULFATE-CHLORIDE GEOTHERMAL SPRINGS IN YELLOWSTONE NATIONAL PARK .....................................................................64 Abstract........................................................................................................................64 Introduction..................................................................................................................65 Materials and Methods.................................................................................................67 Sample Collection and Enrichment and Isolation of Spring Microorganisms ...............................................................................................67 DNA Extraction and Denaturing Gradient Gel Electrophoresis............................69 Characterization of Physicochemical Properties of the Isolates............................71 Carbon Source and Electron Donors......................................................................71 Alternative Electron Acceptors..............................................................................72