ANAMMOX AND DENITRIFICATION IN THE CAPE FEAR RIVER ESTUARY: ANAMMOX BACTERIAL DIVERSITY AND SIGNIFICANCE IN SEDIMENTARY NITROGEN REMOVAL Matthew D. Hirsch A Thesis Submitted to the University of North Carolina at Wilmington in Partial Fulfillment of the Requirements for the Degree of Master of Science Center for Marine Science University of North Carolina Wilmington 2010 Approved by Advisory Committee Lawrence B. Cahoon Craig R. Tobias Bongkeun Song Chair Accepted by ______________________________ Dean, Graduate School JOURNAL PAGE Chapter 2 is in the format for Microbial Ecology Chapter 3 is in the format for Limnology and Oceanography ii TABLE OF CONTENTS ABSTRACT.....................................................................................................................................v ACKNOWLEDGEMENTS.......................................................................................................... vii LIST OF TABLES......................................................................................................................... ix LIST OF FIGURES .........................................................................................................................x CHAPTER 1. INTRODUCTION ....................................................................................................1 Literature review..................................................................................................................2 Hypotheses...........................................................................................................................7 CHAPTER 2. ANAMMOX BACTERIAL DIVERSITY IN VARIOUS AQUATIC ECOSYSTEMS BASED ON THE DETECTION OF HYDRAZINE OXIDASE GENES (hzoA/hzoB)......................................................................................................................................8 Abstract................................................................................................................................9 Introduction........................................................................................................................10 Materials and methods .......................................................................................................13 Results and discussion.......................................................................................................17 Conclusion .........................................................................................................................30 Figure Legends...................................................................................................................31 Tables.................................................................................................................................32 Figures................................................................................................................................36 CHAPTER 3. ANAMMOX AND DENITRIFICATION RATES TO ESTIMATE N REMOVAL POTENTIAL IN THE CAPE FEAR RIVER ESTUARY, NC, USA ...........................................41 Abstract..............................................................................................................................42 Introduction........................................................................................................................43 iii Methods..............................................................................................................................46 Results................................................................................................................................49 Discussion..........................................................................................................................52 Figure legends....................................................................................................................58 Tables.................................................................................................................................60 Figures................................................................................................................................67 References......................................................................................................................................76 Conclusion .....................................................................................................................................84 iv ABSTRACT + Anaerobic ammonium oxidation (anammox) is the microbial N removal pathway of NH4 - oxidation coupled to NO2 reduction, and is mediated by bacteria belonging to the phylum Planctomycetes. A comparative analysis of 16S rRNA and hydrazine oxidase genes (hzoA/hzoB) was conducted to reveal anammox bacterial diversity and distribution in various aquatic environments, focusing on the Cape Fear River Estuary (CFRE). Phylogenetic analyses of 16S rRNA and hzoA/hzoB genes showed the dominance of Scalindua organisms in marine ecosystems, but there was no congruence of 16S rRNA and hzoA/hzoB gene phylogenies among the freshwater anammox bacteria associated with Brocadia sp., Jettenia sp., and Anammoxoglobus sp. Much higher diversity of anammox bacteria was revealed based on hzoA/hzoB genes than 16S rRNA genes in the examined environments. Thus, molecular detection and resulting phylogeny of the hzoA/hzoB gene generated a better understanding of anammox bacterial diversity and their ecological distribution in various aquatic ecosystems. The spatiotemporal variability of anammox and denitrification in the CFRE were assessed using sediment slurry incubation experiments with 15N isotope. Samples were collected at 15 stations along the estuarine gradient of the CFRE on the banks and in the shipping channel during the spring and fall of 2009. Potential N2 production rates were slightly higher but not -1 -1 statistically different in the spring than in the fall (6.42 ± 4.42 and 5.85 ± 3.87 nmol N2 g h , respectively), no clear pattern was seen along the estuarine gradient. Total N2 production rates were higher, on average, in bank sediments than in the channel. Denitrification was the dominant N-removal process in all sediments, accounting for 81 to 99% of total N2 production. Of the physical and chemical parameters that were measured, mean sediment grain size was most 2 significantly correlated (R = 0.153) to the N2 production rates in the spring sampling. The v correlation between denitrification and anammox rates in the spring (R2 = 0.7015) and fall (R2 = 0.5469) explained more variance than that of any of the environmental parameters. vi ACKNOWLEDGEMENTS Funding for this research was provided by NC Sea Grant, and NSF (OCE-0851435). I am extremely grateful for generous funding from the Ahuja Water Quality Fellowship. I acknowledge T. Brian Shirey, Jennifer Bagwell, Jessica Lisa, Daniel Tozour, Yossi Shirazi, Cassie Martin, D. Alex Vance, Jessica Lisa, Andrew Long, Chris Swanson, Ryan Young and Rena Haltom, David Ring, Sam Perkins, Ann Arfken, Vinca Puri, Lisa Nguyen and Matt Lettrich for assistance in the laboratory and field as well as Dr. Steve Carini and Dr. Patrick Erwin and Tse-Lynn Loh for editorial discussions and Yvonne Marsan for creating a map of the sampling stations, Kimberly Duernberger for creating contour plots, and Dr. Zachary Long for assitance with statistical analysis in Chapter 2. I also would like to acknowledge Dr. Michael Mallin, Mr. Matthew McIver and the cruise members of the Lower Cape Fear River Monitoring Program for providing sediment samples and measurements of environmental parameters. I appreciate Dr. Kartik Chandran and Dr. Costantino Vetriani for providing activated sludge and hydrothermal vent sediment, respectively and thank Dr. Martin Palmer and Dr. Rachel Mill at the University of Southampton and the members of 2007 JC18 Cruise for the Montserrat samples as well as the Gillings Family Foundation for travel support. I would like to thank my parents and the rest of my family for supporting me throughout my educational career and life in general. I thank Dr. Cahoon for teaching in his unique discussion-based, chalkboard-using and Socratic manner and for highlighting the role of microbes in biological oceanography. I thank Dr. Tobias for helping young molecular biologists like myself to understand the utility and mechanisms of stable isotope biogeochemistry in his tenure at UNCW. Lastly, I must thank Dr. Song for being demanding yet understanding, for vii putting up with me, and for the many one-on-one discussions which have helped solidify my growing knowledge of molecular microbial ecology. viii LIST OF TABLES Table Page 1. Summary of sampling sites, detected genes and DOTUR analyses ..................................41 2. PCR primers for hzoA/hzoB gene detection.......................................................................42 3. Environmental parameters measured at different sampling sites ......................................43 4. hzoA/hzoB gene amplifications..........................................................................................44 5. Summary of rate data from spring and fall seasons...........................................................72 6. Average values for physical and chemical parameters......................................................73 7. Rate data and relative anammox importance.....................................................................74 8. Environmental parameter data from spring sampling period ............................................75