IDENTIFICATION, ENUMERATION AND DIVERSITY OF AMMONIA-OXIDIZING ARCHAEA IN THE LAURENTIAN GREAT LAKES Maitreyee Mukherjee A Dissertation Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY August 2013 Committee: Dr. George S. Bullerjahn, Advisor Dr. William H. O’ Brien Graduate Faculty Representative Dr. Robert M. McKay Dr. Scott O. Rogers Dr. Zhaohui Xu © 2013 Maitreyee Mukherjee All Rights Reserved iii ABSTRACT George Bullerjahn, Advisor Nitrification is a major process of the global nitrogen cycle in which ammonia is oxidized to nitrate. Previously it was assumed that bacteria (e.g., Nitrosomonas and Nitrobacter) are responsible for this process. Over the past decade, researchers have discovered different groups of nitrifying archaea, specifically Thaumarchaeota Groups I.1a, and I.1b, in both marine and freshwater environments. The physiological characteristics of these organisms are best suited to the levels of ammonia found in aquatic systems. Since 1900, Lake Superior has seen a steady increase in nitrate, yielding a severe stoichiometric imbalance of nitrogen to phosphorus. Stable isotopic analyses indicate that nitrate increases result from in-lake biological processes. By contrast, mesotrophic Lake Erie is prone to transient hypoxia, yielding nitrogen losses to the atmosphere via denitrification and anammox. In this study, I examined these unique nitrifiers contributing to nitrification in both lakes. Phylogenetics of amoA sequences from lake seston reveals that ammonia oxidizing archaea (AOA) dominate the Superior pelagic microbial community, although AOA diversity in Lake Superior was observed to be low, indicating only a few key ecotypes can survive the extreme oligotrophic conditions prevalent in the lake. CARD- FISH probes specific for AOA, ammonia oxidizing bacteria (AOB) and nitrite oxidizers (NOB) allow their enumeration in samples obtained from 2010-12. During the stratified period in Superior, AOA and NOB are detectable in the hypolimnion and oxic sediments (up to 7 x 104 mL-1), and absent in the epilimnion. AOB are not detectable (<101 mL-1) in Superior, but dominate in Erie samples. For summer 2010, AOA abundance reflects parallel assays of nitrification rates. This study concludes that Thaumarchaeota (AOA) dominate the ammonia- iv oxidizing microbial population in Lake Superior, whereas, the ammonia oxidizing bacterial population is negligible in the lake. In contrast, Thaumarchaeota are almost absent or negligible in Lake Erie, whereas, the ammonia oxidizing bacterial are the dominant nitrifiers in the lake, indicating towards a possible association of the AOA in environments with lower ammonium concentrations, whereas that of the AOBs with environments with higher ammonium concentrations. Thaumarchaeota (AOA), in spite of being so abundant in Lake Superior, tend to be less diverse in the lake, indicating that only few species of these ammonia-oxidizers can survive the extreme oligotrophic conditions of the lake. All the ammonia oxidizers, irrespective of whether they are bacteria or archaea, tend to be absent from surface waters, whereas their abundance increases with depth of water column in the lakes. v Dedicated to my dad, the teacher, philosopher, and guide of my life; to my mom, who made every possible sacrifice to see this day of my life; to the memory of my grandfather, who would have been proud; to my grandmother, for always believing in me; and last but not the least, to my husband, without whose immense support, this work wouldn’t be completed. | েতামায় িদলাম | vi ACKNOWLEDGMENTS First of all, I want to express my gratitude and thanks to my advisor Dr. George Bullerjahn, without whose immense support, guidance, and valuable advice, this work would not be possible. I am grateful to him for taking me in his lab in the first place, making science so much fun, and giving me enough independence and freedom to do this research. In addition to being the best scientist and guide I’ve come across, I particularly admire his character of being able to provide a considerable sense of freedom to his students; to allow them to do their work in their own time and space. It was an absolute honor being his PhD advisee, and his student in the past five years, and I cannot thank him enough for making my scientific journey at Bowling Green State University such amazing and memorable. I would like to extend my special thanks to my co-advisor Dr. Robert Michael McKay for all his invaluable advice throughout this work. I also want to thank all the members of my PhD committee Dr. Scott Orland Rogers, Dr. Zhaohui Xu and Dr. William O’Brien for all their suggestions and advice towards this work. I also want to take this opportunity to thank all the members of the Bullerjahn-Mckay lab group for their helpful advices, trainings, and the wonderful science that we did together in the group. I want to thank all the staff of the Department of Biological Sciences and the BGSU Graduate College for accepting me in their graduate program, and for making my PhD journey so wonderful and memorable. Finally, I want to thank my family: my parents and my grandparents, for making every sacrifice to see this day of my life, for believing in me and taking immense pride in me forever. I want to thank all my great friends in Bowling Green and in India for supporting my work throughout these five years. And finally, I want to thank my husband without whose immense support and sacrifices, I wouldn’t have seen this day of my life. Thank you all. vii TABLE OF CONTENTS Page CHAPTER I. INTRODUCTION .......................................................................................... 1 1.The Nitrogen cycle........................................................................................ 1 2.Nitrification....................................................................................................3 3.Denitrification................................................................................................5 4.Chemolithotrophy..........................................................................................6 5.The Nitrifiers................................................................................................... 7 6.The anaerobic Ammonia-oxidizers: Anammox Organisms............................. 11 7.The Ammonia Oxidizing Archaea (AOA) or the Thaumarchaota.................13 7.1. Habitat Diversity of the AOA............................................................... 18 7.2. Cell structure, Physiology, Growth of the AOA................................... 21 7.3. Stoichiometry and Kinetics of ammonia oxidation of AOA................. 23 7.4. The biochemistry of ammonia oxidation and Nitrous oxide production by the AOA......................................................................... 25 7.5. Autotrophic, Mixotrophic and Heterotrophic growth in AOA.............. 29 8.Nitrification in the Laurentian Great Lakes: two lakes, two different stories....................................................................................... 31 8.1. Structure and characteristics of Lake Superior vs. Lake Erie............... 32 8.2. Nitrification and trends in nitrate levels in Lake Superior vs. Lake Erie.......................................................................................... 33 9. Objective and significance of the current study............................................. 36 REFERENCES.............................................................................................. 37 CHAPTER II. MATERIALS AND METHODS .................................................................. 52 viii 1. Sampling ........................................................................................................ 52 2. DNA Extraction Procedures.......................................................................... 57 3. Catalyzed Reporter Deposition Fluorescence in-situ Hybridization (CARD-FISH)............................................................................................... 58 4. PCR Methods................................................................................................. 71 5. Bioinformatics Tools...................................................................................... 75 REFERENCES .............................................................................................. 76 CHAPTER III. RESULTS .................................................................................................... 79 1. Sampling site characteristics .......................................................................... 79 2. Temperature Profiles...................................................................................... 84 3. Diversity of ammonia oxidizing archaea (AOA) in Lake Superior and Lake Erie................................................................................................. 85 3.1. PCR analysis of 16S rDNA of environmental samples in Lake Superior........................................................................................... 86 3.2. Phylogenetic Analysis of 16S rDNA sequences from Lake Superior.................................................................................. 88 3.3. PCR analysis of amoA sequences from environmental samples from Lake Superior and Lake Erie........................................................... 91 3.4. Phylogenetic analysis of amoA sequences from Lake Superior and Lake Erie..........................................................................................