ISOLATION AND CHARACTERIZATION OF IRON-OXIDIZING BACTERIA FROM BOILING SPRINGS LAKE AND THE POTENTIAL ROLE OF FERROUS IRON IN CARBON AND SULFUR CYCLING By Francine Arroyo A Thesis Presented to The Faculty of Humboldt State University In Partial Fulfillment of the Requirements for the Degree Master of Science in Biology Committee Membership Patricia L. Siering, Ph.D., Committee Chair Mark S. Wilson, Ph.D. Matthew Hurst, Ph.D. Kristine Brenneman, Ph.D. Michael Mesler, Ph.D., Graduate Coordinator December, 2012 ABSTRACT ISOLATION AND CHARACTERIZATION OF IRON-OXIDIZING BACTERIA FROM BOILING SPRINGS LAKE AND THE POTENTIAL ROLE OF FERROUS IRON IN CARBON AND SULFUR CYCLING Francine Arroyo Boiling Springs Lake (BSL) is a 52˚C, pH 2, iron and sulfur-rich thermal feature in Lassen National Volcanic Park (California, USA). Previous community composition studies of small subunit rRNA and RuBisCo genes revealed an abundance of phylotypes closely related to an Acidimicrobium strain isolated from Yellowstone National Park. As an ideal candidate to examine its contribution to primary production in BSL, we attempted to isolate Acidimicrobium and related iron-oxidizing Bacteria from BSL. We obtained 23 isolates that shared 99% rRNA gene identity with their closest cultured relative: 16 were identified as Sulfobacillus acidophilus, four as Alicyclobacillus sp., and three isolates had nearly identical rRNA gene sequences to the previously identified Acidimicrobium clones. We characterized most isolates for pH and temperature growth range and optima, and we assessed their abilities to oxidize iron, pyrite, sulfur and tetrathionate. Morphology of Acidimicrobium isolates was analyzed with transmission and scanning electron microscopy. The Acidimicrobium isolates were Gram positive, non-endospore-forming rods with a complex cellular envelope. Optimal growth ii temperature and pH for many isolates correlated with conditions at BSL. Acidimicrobium and Sulfobacillus isolates oxidized 10 mM iron when amended with 0.01% yeast extract. Acidimicrobium isolates were inhibited at iron concentrations ≥25 mM. Acidimicrobium and some Sulfobacillus isolates were able to oxidize pyrite when amended with yeast extract. We found no evidence for sulfur or tetrathionate oxidation by any of the isolates. These results will further our understanding of the potential role of Acidimicrobium and related iron-oxidizing Bacteria in the iron and carbon cycles in BSL. iii ACKNOWLEDGEMENTS I would first like to thank my advisor, Patricia Siering, for her unyielding support and time dedicated to making sure that I complete my thesis in a timely manner. I cannot accurately express my gratitude for her efforts these past three and a half years. She is a role model for hard work, passion for teaching, and all-around joyful person to work with. I can only hope to maintain my head above water in the wake that she leaves behind. Many thanks to my committee members for their continued support and time dedicated for reviewing this endless thesis. Mark was always available to answer any questions that I would have, from assistance with sequence alignments to recipes for posole soup. Many thanks to Matt for all his help in setting up the iron oxidation assays and allowing me access to the Chemistry department’s diode array spectrophotometer. To Dr. B for showing me how much fun it is to monitor the Secchi depth. My work utilizing electron microscopy could not have been done without the patience and guidance of Casey Lu. I would also like to thank Marty Reed and Lewis McCrigler for helping me through the technical difficulties with the microscopes. Dr. Barrie Johnson from the University of Wales (Bangor, U.K.) provided me with insight on successful growth maintenance of the finicky A. ferrooxidans ICPT. Many members of the H.E.A.T. lab contributed to my work. Jennifer Hampton’s initial attempts of isolating Acidimicrobium provided me with the framework of methods and media to use in my isolation attempts. Jon Schultz and Clay Carey assisted me with iv clone library construction. Connor Fitzhugh eagerly helped me analyze my experimental data using AICc under the guidance of Dr. Rob Van Kirk. I would like to thank my partner, Max Cannon, for moving to Arcata with me and personally supporting me through my emotional vicissitudes. Thanks to the helpful and optimistic staff and faculty of the Department of Biology. It has been a pleasure. Funding was provided by the National Science Foundation (Patricia Siering, PI, grant no. MCB-0702018). v TABLE OF CONTENTS ABSTRACT .......................................................................................................... ………..ii ACKNOWLEDGEMENTS ................................................................................... ………iv TABLE OF CONTENTS……………………………………………………………..…..vi LIST OF TABLES…………………………………………………………………….…..x LIST OF FIGURES…………………………………………………………………....…xi LIST OF APPENDICES………………………………………………………...……...xiii CHAPTER 1: INTRODUCTION ....................................................................................... 1 Project Overview……………………………………………………………….....1 Literature Review: Physiology of Acidophiles……………………………………2 Coping mechanisms for an acidophilic lifestyle…………………………..2 Thermoacidophily…………………………...…………………………….4 Metabolic diversity of acidophiles………………………………………...7 Iron and sulfur oxidation…………………………………………….…….9 Background on Boiling Springs Lake…………………...……………………….13 Site description……………………………………………………..….…13 Microbiology of BSL………………………………………………….…16 Production in BSL……………………………………………………….17 Comparison with acid mine drainage sites………………………………18 CHAPTER 2: MATERIALS AND METHODS .............................................................. 19 Media Preparation………………………………………………………….…….19 vi Isolation of Sulfobacillus and Alicyclobacillus Strains…………………….…….22 Sample collection……………………………..………………………….22 Winogradsky column enrichments…………………………………...….23 Selection and purification of isolates…………………………………….24 Isolation of Acidimicrobium sp. from BSL……………….……………….……..26 Sample collection………………………………………………….……..26 Isolation and purification…………………………………………...……26 Identification of Isolates by 16S rRNA Gene Sequencing………………………29 Acidimicrobium-specific 16S rRNA primer design……………………...29 SSU rRNA gene amplification by PCR………………………………….30 Clone library construction and sequence analysis……………………….31 Morphology of Acidimicrobium by Electron Microscopy…………………….…34 Transmission electron microscopy (TEM)……………………………....34 Scanning electron microscopy (SEM)……………………………...……35 Physiological Characterization of Isolates……………………………………….35 Preparation of inocula and growth assessment…………………………..35 Determination of pH and temperature range and optima for growth…….37 Growth experiments………………………………………...……37 Linear regression models and Akaike information criterion (AICc)……………………………………………………….……37 Iron and pyrite as energy sources…………………………………….…..38 Iron and pyrite oxidation via ferrozine assay…………………………….39 vii Sulfur and tetrathionate oxidation………………………………………..40 CHAPTER 3: RESULTS .................................................................................................. 41 Isolation, Identification and Naming of Alicyclobacillus and Sulfobacillus Isolates…………………………………………………………………………...41 Isolation, Identification and Naming of Acidimicrobium Isolates…...…………..43 Electron Microscopy of Acidimicrobium…………………………………...……50 Physiological Characterization of Isolates……………………………………….54 Determination of temperature and pH range and optima………….……..54 Growth experiments……………………………………….…..…54 Linear regression modeling of pH and temperature data………...64 Evaluation of iron and pyrite as potential energy sources……………….69 Measurements of iron oxidation……………………………………...….74 Pyrite oxidation…………………………………………………………..75 Sulfur and tetrathionate oxidation………………………………………..80 CHAPTER 4: DISCUSSION ............................................................................................ 86 Attempts to Isolate Acidimicrobium from BSL……………………………….…86 Isolation and physiological characterization of Acidimicrobium…....…..89 Cell morphology of Acidimicrobium…………………………………….97 Caveats associated with growth measurements…………………..…….100 Isolation and physiological characterization of Sulfobacillus…..…..….100 Using AIC analysis to differentiate between strains of Sulfobacillus and between species of Sulfobacillus and Acidimicrobium…….…....…103 viii Isolation and physiological characterization of Alicyclobacillus….……103 Speculation on Role of Acidophilic Isolates in BSL…………….…………..…106 Summary………………………………………………………………..………109 Future Work…………………………………………………………………….111 APPENDICES…………………………………………………………………….……115 REFERENCES ............................................................................................................... 119 ix LIST OF TABLES Table Page 1 16S rRNA primers utilized……………..……………………………………..…33 2 16S rRNA gene sequence analysis, isolation conditions, and morphology of isolates…………………………………………………………………..…….…46 3 Summary of temperature and pH ranges and optima for Acidimicrobium, Sulfobacillus, and Alicyclobacillus isolates………………………….……..……63 4 AICc of the top linear regression models of division rate (generations/time) according to pH, temperature, and strain of Sulfobacillus isolates.……….……..66 5 Sulfobacillus isolate sub-groups organized by common temperature and pH ranges/optima………………………………………………………………….....67 6 AICc of the top linear regression models of division rate (generations/time) according to pH, temperature, strain,