UNRECOGNIZED DIVERSITY OF MICROBES LINKING METHANOTROPHY TO NITROGEN LOSS IN MARINE OXYGEN MINIMUM ZONES A Dissertation Presented to The Academic Faculty by Cory Cruz Padilla In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the School of Biological Sciences Georgia Institute of Technology December 2017 COPYRIGHT © 2017 BY CORY PADILLA UNRECOGNIZED DIVERSITY OF MICROBES LINKING METHANOTROPHY TO NITROGEN LOSS IN MARINE OXYGEN MINIMUM ZONES Approved by: Dr. Frank J Stewart, Advisor Dr. Jennifer B Glass School of Biological Sciences School of Earth and Atmospheric Georgia Institute of Technology Science Georgia Institute of Technology Dr. Thomas J DiChristina Dr. Bo Thamdrup School of Biological Sciences Department of Biology and Nordic Georgia Institute of Technology Center for Earth Evolution University of Southern Denmark Dr. Kostas T. Konstantinidis School of Biological Sciences Georgia Institute of Technology Date Approved: November 1, 2017 This work is dedicated to my Grandparents, Ofelio and Regina Padilla, and to my Uncle Charles Padilla. ACKNOWLEDGEMENTS I would like to thank Kate O’Neill for her constant love and support throughout the entire process from temporary lab positions to oceanographic cruises and finally the Ph.D. proceess, I look forward to our next steps in life. I would also like to thank my parents, James and Dwan Padilla, for their encouragement to follow my curiosities, wherever they may lead. I would also like to thank my advisor, Dr. Frank Stewart for his guidance and for offering me uncountable opportunities to get involved with amazing projects and people. Along those lines, I would like to express my sincerest gratitude toward the members of the Stewart Lab, past and present. It has been an honor working along side all of you. I am grateful to have such a large number of talented collaborators. In particular, I would like to recognize Laura Bristow and Bo Thamdrup, for their guidance and support both with generating exciting research and for offering much-needed comic relief and commiseration on cruises. Science is rarely a solo effort, and I have been more than fortunate work with and learn from all of you. Lastly, I would like to thank all my former labs, the Zehr lab, Repeta Lab, and Karl lab. The support from everyone in these labs helped to motivate me and gave me a vast background and experieance in oceanography, which allowed me to start graduate school running at full speed. iv TABLE OF CONTENTS Page ACKNOWLEDGEMENTS iv LIST OF TABLES viii LIST OF FIGURES ix LIST OF SYMBOLS AND ABBREVIATIONS xi SUMMARY xiv CHAPTER 1 INTRODUCTION 1 1.1 Microbial methane metabolisms 2 1.2 Oceanic methane cycling 8 1.3 Oxygen mimimum zones as sites of CH4 and N linkages 10 1.4 Objectives 13 1.5 References 16 2 STANDARD FILTRATION PRACTICES MAY SIGNIFICANTLY DISTORT MICROBIAL DIVERISTY ESTIMATES 22 2.1 Abstract 23 2.2 Introduction 23 2.3 Materials and Methods 26 2.4 Results 31 2.5 Discussion 39 2.6 Conclusion 45 2.7 References 47 v 3 NC10 BACTERIA IN MARINE OXYGEN MINIMUM ZONES 53 3.1 Abstract 54 3.2 Introduction 55 3.3 Materials and Methods 58 3.4 Results and Discussion 72 3.5 References 89 4 METAGENOMIC BINNING RECOVERS A TRANSCRIPTIONALLY ACTIVE GAMMAPROTEOBACTERIUM LINKING METHANOTROPHY TO PARTIAL DENITRICTION IN AN ANOXIC OXYGEN MINIMUM ZONE 95 4.1 Abstract 96 4.2 Introduction 97 4.3 Materials and Methods 103 4.4 Results and Discussion 111 4.5 Conclusion 127 4.6 References 130 5 CONCLUSION AND SUGGESTIONS 139 5.1 OMZs as niche for CH4 oxidation coupled to N loss 139 5.2 Suggestions and further questions 141 5.3 Final remarks 146 APPENDIX A: CRYPIC OXTGEN CYCLING IN ANOXIC MARINE ZONES 147 APPENDIX B: SUPPLEMENTARY MATERIAL FOR CHAPTER 2: STANDARD FILTRATION PRACTICES MAY SIGNIFICANTLY DISTORT MICROBIAL DIVERISTY ESTIMATES 176 APPENDIX C: SUPPLEMENTARY MATERIAL FOR CHAPTER 3: NC10 BACTERIA IN MARINE OXYGEN MINIMUM ZONES 182 vi APPENDIX D: SUPPLEMENTARY MATERIAL FOR CHAPTER 4: METAGENOMIC BINNING RECOVERS A TRANSCRIPTIONALLY ACTIVE GAMMAPROTEOBACTERIUM LINKING METHANOTROPHY TO PARTIAL DENITRICTION IN AN ANOXIC OXYGEN MINIMUM ZONE 188 APPENDIX E: ATTEMPTS AT ENRICHING DENITRIFICATION-DEPENDENT METHANOTROPHS FROM THE EASTERN TROPICAL NORTH PACIFIC 194 VITA 206 vii LIST OF TABLES Page Table 3.1: Process rates*, as measured during 10-day anoxic incubations. 83 Table 4.1: Representation of methane oxidation and denitrification genes present (+/-) in GD_7 (bin 010) and abundance in the coupled 90 m metatranscriptome. 122 Table A.1: Depth-integrated oxygen production and carbon fixation rates. 168 Table B.1: Bacterial 16S rRNA gene copies per mL in sample water from experiments 1 and 2. 176 Table B.2: Percentages variation (R2) in weighted UniFrac distances explained by filtered water volume differences, based on adonis tests in QIIME. 176 Table B.3: Abundances of microbial orders in experiment 1, expressed as a percent of toal 16S rRNA gene amplicons. 177 Table B.4: Abundances of microbial orders in experiment 2, expressed as a percent of toal 16S rRNA gene amplicons. 179 Table E.1: Enrichment treatment conditions. 197 Table E.2: Sampling information for the CC samples over a 1-month period. 198 viii LIST OF FIGURES Page Figure 2.1: Total DNA yield (A) and filtration time (B) as a function of filtered water volume. 33 Figure 2.2: Microbial community relatedness (A,C) and taxon abundances (B,D) in experiment 1 prefilter (>1.6 mm; A,B) and Sterivex (0.2-1.6 mm; C,D) samples 35 Figure 2.3: Microbial community relatedness (A,C) and taxon abundances (B,D) in experiment 2 prefilter (>1.6 mm; A,B) and Sterivex (0.2-1.6 mm; C,D) samples 37 Figure 2.4: Chao1 estimates of operational taxonomic unit (97% similarity cluster) richness in prefilter (>1.6 mm; A,B) and Sterivex (0.2-1.6 mm; C,D) samples in experiments 1 and 2 38 Figure 3.1: Water column chemistry and microbial biomass in the ETNP OMZ in May 2014 (a-e) and GD OMZ in January 2015 (f-j). 74 Figure 3.2: Particulate methane monooxygenase subunit A (PmoA) gene phylogeny. 76 Figure 3.3: Figure 3.3 Transcription of denitrification-dependent AOM genes in the ETNP (a,b) and GD OMZs (c,d). 79 Figure 4.1: Methanotroph 16S rRNA gene phylogeny. 102 Figure 4.2: Water column chemistry and Methylococcales OTU abundances in the GD water column. 113 Figure 4.3: Concatenated gene phylogeny of methanotrophic genomes and description of N utilizing genes. 116 Figure 4.4: Nitrate reductase (narG) and nitrite reductase (nirK) phylogenies. 119 Figure 4.5: Transcripts mapping to GD_7. 124 Figure A.1: Maps with sampled stations and main characteristics of the upper part of the (A–F) ETNP AMZ and (G–L) ETSP AMZ. 159 Figure A.2: Oxygen production and carbon fixation during incubations of samples from the SCM off Mexico (A - C) and Peru (D - F) 161 ix Figure A.3: Water column dissolved oxygen (O2), chorophyll concentrations (Chl), and microbial transcript abundances at station T6 in the ETNP in 2013 (A- D) and 2014 (E-H) 166 Figure B.1: Total bacterial 16S rRNA gene counts as function of filtered water volume. 181 Figure C.1: Sampling sites in the ETNP and GD OMZs off Mexico and Costa Rica, respectively. 182 Figure C.2: Figure C.2 PmoA gene phylogeny. 183 Figure C.3: Abundance of rRNA transcripts matching NC10 bacteria. 184 Figure C.4: Transcription of n-damo genes in the ETNP (a,b) and GD (c,d) identified by LCA analysis in MEGAN5. 185 Figure C.5: Phylogeny and motif structure of NC10-like qNor transcripts in the ETNP OMZ. 186 Figure C.6: Methanogen abundance in the ETNP OMZ. 187 Figure D.1: Sample site (star) in Golfo Dulce, Costa Rica. 188 Figure D.2: Gene organization of bin 010 (GD_7). 189 Figure D.3: Transcription of partial denitrification, methane oxidation, RuMP, and partial serine pathways in the GD_7 bin. 190 Figure D.4: Recruitment of transcriptome to the GD_7 bin. 191 Figure D.5: Rank abundance plot of the top transcribed GD_7 open reading frames (ORFs) represented in the 90 m metatranscriptome. 192 Figure D.6: Taxonomic classification and abundance of Methylococcales mRNA. 193 Figure E.1: Taxon abundance at the Family level presented as a proportion of total 16S sequences. 202 Figure E.2 Methanotroph and methylotroph OTUs and N dynamics in CC - 10 µM enrichments. 203 x LIST OF SYMBOLS AND ABBREVIATIONS CH4 Methane CO2 Carbon dioxide DO Dissolved Oxygen EDTA Ethylene diamine tetraacetic acid H2S Hydrogen sulfide N Nitrogen N2 Dinitrogen gas N2O Nitrous oxide NED N-(1-Naphthyl)ethylenediamine + NH4 /NH3 Ammonium NO Nitric oxide - NO2 Nitrite - NO3 Nitrate O2 Oxygen gas pH Hydrogen ion concentration SDS Sodium Dodecyl Sulfate 2- SO4 Sulfate Tris-HCl 2-Amino-2-hydroxymethyl1-1,3-propanediol hydrochloride AOA Ammonia oxidizing archaea AOB Ammonia oxidizing bacteria NOB Nitrite oxidizing bacteria OPU Operationial PMO unit amo Ammonia monooxygenase fdh Fomate dehydrogenase hao Hydroxyalamine oxidoreductase HATO High-affinity terminal oxidases LATO Low-affinity terminal oxidases mdh Methanol dehydrogenase MMO Methane monooxygenase nap Periplasmic nitrate/nitrite oxidoreductase nar Nitrate reductase nas Assimilatory nitrate reductase nir Nitrite reductase nod Nitric oxide dismutase nor Nitric oxide reductase nos Nitrous oxide reductase nxr Nitrite oxidoreductase pmo Particulate methane monooxygenase qnor Quinol-dependent nitric oxide reductase xox Methanol dehydrogenase - rare earth element xi Ek Light intensities kDA Kilodalton Km Michaelis-Menten constant L Litre m Meter nM Nanomolar µM Micromolar µm Micrometer BLAST Basic Local Alignment Search Tool CTD Conductivity, Temperature, and Depth HMM Hidden Markov Model KAAS KEGG Automatic Annotation Server LCA Lowest Common Ancestor PCR Polymerase chain reaction PF Prefilter qPCR Quantitative polymerase chain reaction STOX Switchable trace amount oxygen sensor cDNA Complementary DNA DNA Deoxyribonucleic Acid dsDNA Double stranded DNA gDNA Genomic DNA KEGG Kyoto Encylopedia of Genes and Genomes mRNA Messenger RNA ORF Open Reading Frame OTU Operational taxonomic unit RNA Ribonucleic Acid rRNA Ribosomal RNA Ca.