UNIVERSITY OF CALIFORNIA, SAN DIEGO Biological Alteration of Basaltic Glass: Laboratory and Field Studies on Basalt Hosted Microbial Ecosystems A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Earth Sciences by Brad E. Bailey Committee in charge: Hubert Staudigel, Chair Bradley Tebo, Co-Chair Lihini Aluwihare Joris Gieskes Alexis Templeton Mark Thiemens 2014 ii The Dissertation of Brad E. Bailey is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Co-Chair Chair University of California, San Diego 2014 iii DEDICATION For my Mom, Who always reminded me to “Feed my ‘Friends’” iv EPIGRAPH The most exciting phrase to hear in science, the one that heralds the most discoveries, is not "Eureka!" (I found it!) but "That's funny..." Isaac Asimov A geobiologist talks geology to the biologists and biology to the geologists. Then he meets another geobiologist and they just talk about sports. Author unknown v TABLE OF CONTENTS Signature Page ........................................................................................................... iii Dedication ................................................................................................................. iv Epigraph .................................................................................................................... v Table of Contents ...................................................................................................... vi List of Figures ........................................................................................................... ix List of Tables ............................................................................................................. x Acknowledgements ................................................................................................... xi Vita ............................................................................................................................ xiv Abstract of the Dissertation ....................................................................................... xvi Chapter 1. Introduction ............................................................................................. 1 1.1. Preface ................................................................................................... 1 1.2. Biological Alteration of Basaltic Glass ................................................. 2 1.3. The Deep Oceanic Biosphere ................................................................ 4 1.4. Iron Biogeochemistry and Related Microorganisms ............................. 6 1.5. Phylogenetic Characterization of Microbial Communities on Loihi and In the Basaltic Deep Biosphere ............................................................. 8 1.6. Outline of the Thesis ............................................................................. 11 1.7. References ............................................................................................. 12 Chapter 2. A New Laboratory DNA Extraction Protocol for Spores and Metal-Rich Low Biomass Samples ........................................................................... 17 2.1. Introduction ........................................................................................... 18 2.2. Materials and Methods .......................................................................... 19 2.2.1. Samples .................................................................................. 19 2.2.2. Sample Preparation ................................................................ 20 2.2.3. Extraction Protocols ............................................................... 20 2.3. Results and Discussion .......................................................................... 22 2.4. Acknowledgements ............................................................................... 26 2.5. References ............................................................................................. 27 Chapter 3. Fe Moessbauer Spectroscopy as a Tool in Astrobiology ........................ 28 3.1. Introduction ........................................................................................... 29 3.2. Moessbauer Spectroscopy ..................................................................... 31 vi 3.2.1. Moessbauer Effect .................................................................. 32 3.2.2. A Simple Moessbauer Spectrometer ...................................... 33 3.2.3. Moessbauer Parameters .......................................................... 34 3.2.3.1. Isomer Shift .............................................................. 35 3.2.3.2. Quadrupole Splitting ................................................. 36 3.2.3.3. Magnetic Hyperfine Splitting ................................... 37 3.2.3.4. Relative Areas ........................................................... 39 3.2.4. The Miniaturized Moessbauer Spectrometer MIMOS II ....... 39 3.3. Mars ....................................................................................................... 40 3.3.1. Gusev Crater ........................................................................... 41 3.3.2. Meridiani Planum ................................................................... 43 3.4. Loihi Deep Seamount ............................................................................ 46 3.5. Conclusions ........................................................................................... 52 3.6. Acknowledgements ............................................................................... 54 3.7. References ............................................................................................. 55 Chapter 4. Utilization of substrate components during basaltic glass colonization by Pseudomonas and Shewanella isolates ............................................. 67 4.1. Introduction ........................................................................................... 68 4.2. Materials and Methods .......................................................................... 71 4.2.1. Basalt Substrates .................................................................... 71 4.2.2. Bacterial Consortia, Isolation and Media ............................... 72 4.2.3. Experiments ............................................................................ 73 4.3. Results ................................................................................................... 76 4.4. Discussion ............................................................................................. 80 4.4.1. Iron Cycling in Auto-and Heterotrophic Growth ................... 84 4.4.2. Nutrient Leaching from Apatite Infused Basaltic Glass ........ 86 4.5. Conclusions ........................................................................................... 87 4.6. Acknowledgements ............................................................................... 88 4.7. References ............................................................................................. 89 Chapter 5. An Interlaboratory Comparison of DNA-based Methods for Assessing Microbial Diversity of Seafloor Basalts .................................................. 92 5.1. Introduction ........................................................................................... 93 5.2. Materials and Methods .......................................................................... 95 5.2.1. Sample Collection .................................................................. 95 5.2.2. T-RFLP Analysis .................................................................... 97 5.2.3. 16S rRNA Gene Clone Library Construction and in silico T-RFLP Analysis .................................................................... 98 5.2.4. Calculation of Diversity Indices ............................................. 98 5.3. Results ................................................................................................... 99 5.4. Discussion ............................................................................................. 107 5.5. Acknowledgements ............................................................................... 111 5.6. References ............................................................................................. 112 vii Chapter 6. A First-Order Assessment of Microbial Diversity at Loihi Seamount Using Terminal Restriction Fragment Length Polymorphism ................ 115 6.1. Introduction ........................................................................................... 116 6.2. Sample Sites .......................................................................................... 118 6.3. Materials and Methods .......................................................................... 124 6.3.1. Sample Collection .................................................................. 124 6.3.2. DNA Extraction ...................................................................... 124 6.3.3. T-RFLP ................................................................................... 125 6.3.4. Clone Libraries and in-silico T-RF Generation ...................... 126 6.4. Results ................................................................................................... 127 6.5. Discussion ............................................................................................. 131 6.5.1. Basalt Hosted Microbial Communities .................................