Bacteria in Ballast Water: the Shipping Industry’S
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BACTERIA IN BALLAST WATER: THE SHIPPING INDUSTRY’S CONTRIBUTIONS TO THE TRANSPORT AND DISTRIBUTION OF MICROBIAL SPECIES IN TEXAS A Thesis by ELIZABETH B. NEYLAND Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE August 2009 Major Subject: Biology BACTERIA IN BALLAST WATER: THE SHIPPING INDUSTRY’S CONTRIBUTIONS TO THE TRANSPORT AND DISTRIBUTION OF MICROBIAL SPECIES IN TEXAS A Thesis by ELIZABETH B. NEYLAND Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Approved by: Co-Chairs of Committee, Robin Brinkmeyer Susan Golden Committee Members, Michael Benedik Antonietta Quigg Head of Department, Uel J. McMahan August 2009 Major Subject: Biology iii ABSTRACT Bacteria in Ballast Water: The Shipping Industry’s Contributions to the Transport and Distribution of Microbial Species in Texas. (August 2009) Elizabeth B. Neyland, B.S., Texas A&M University Co-Chairs of Advisory Committee: Dr. Robin Brinkmeyer Dr. Susan Golden The transportation of organisms in the ballast water of cargo ships has been recognized as a source of invasive species despite current control measures. Pathogenic bacteria in the ballast tank have been studied but the total diversity of the ballast tank bacterial community has not been examined. This study is the first to characterize the total bacterial community within a ballast tank by constructing a clone library from a ballast water sample from a cargo ship in the Port of Houston, amplified ribosomal rDNA restriction analysis (ARDRA) and phylogenetic analysis. Bacterial communities in Texas ports and bays were also examined using denaturing gradient gel electrophoresis (DGGE), looking at both temporal and spatial variations for effects of deballasting activity. This ballast tank bacterial community had a high level of diversity (95%) with the clone library only representing 40% of the total community of the tank. Most probable originating habitats of the ballast bacteria were: marine pelagic (40%), estuarine (37%), coastal (6%), freshwater (3%) and other (14%), even though this ballast iv tank was exchanged with pelagic water. Predominate groups were alpha- and gammaproteobacteria, a few betaproteobacteria and bacteriodetes, and one each of verrucomicrobia, planctomycetes and actinobacteria, but no pathogens were detected. The data reveals a ballast tank that consists of half marine-pelagic, half port bacteria, revealing a low efficacy of exchange control methods and potentially invasive bacteria. The bacterial communities of five ships that exchanged ballast water in the Pacific Ocean shared on average 50% similarity. Two ships that exchanged ballast water in temperate latitudes were more similar than three other ships that exchanged in tropical latitudes, showing a correlation between location of exchange and community similarity. The bacterial communities of the Ports of Houston and Galveston exhibit stable, seasonal successions over one year. The port and bay systems of Texas exhibited spatial variations in bacterial communities related to salinity levels. Both experiments did not show evidence of community disruption by deballasting activities. This study shows that ballast water is a viable vector for invasive bacterial transport, although impact on Texas estuarine systems seems minimal. v DEDICATION To my dad, mom, and brother, Mathew: Thank you for your support, encouragement and love throughout this process. To Jeremiah: I couldn’t have made it through this without you! vi ACKNOWLEDGEMENTS I would like to thank my committee chairs, Dr. Robin Brinkmeyer and Dr. Susan Golden, and my committee members, Dr. Antonietta Quigg and Dr. Michael Benedik, for their guidance and support throughout the course of this research. Thanks also go to my colleagues in the Phytoplankton Dynamics Lab and the Coastal Health Lab for assistance, help and encouragement. Special thanks to Jamie Steichen, Federico Alvarez, Nicole Towers and Emily Kane for help with sample collection and processing, and to the undergraduate students who assisted with this project: Kaylyn Germ, Paul Kroesen, Melanie Britton and Autumn Patton. A very special thank you to the shipping agent in the Port of Houston who collected ballast water samples for us, and without whom none of the ballast work in the project could have been done. I also want to extend my gratitude to the Texas Advanced Research Program, which provided the funding for this study and to the Department of Biology at Texas A&M University for funding support. vii NOMENCLATURE BWE Ballast Water Exchange IMO International Maritime Organization PCR Polymerase Chain Reaction ARDRA Amplified Ribosomal rDNA Restriction Analysis DGGE Denaturing Gradient Gel Electrophoresis viii TABLE OF CONTENTS Page ABSTRACT.............................................................................................................. iii DEDICATION .......................................................................................................... v ACKNOWLEDGEMENTS ...................................................................................... vi NOMENCLATURE.................................................................................................. vii TABLE OF CONTENTS.......................................................................................... viii LIST OF FIGURES................................................................................................... x LIST OF TABLES .................................................................................................... xiii CHAPTER I INTRODUCTION................................................................................ 1 Introduction .......................................................................... 1 Research Objectives .............................................................. 15 Total Materials and Methods................................................. 20 II DIVERSITY OF BACTERIA IN THE BALLAST TANK OF A COMMERICAL CARGO SHIP IN THE PORT OF HOUSTON, TEXAS ................................................................................................. 35 Introduction .......................................................................... 35 Materials and Methods.......................................................... 39 Results ................................................................................... 45 Discussion ............................................................................. 60 III SPATIAL AND TEMPORAL COMMUNITY COMPARISONS IN BALLAST WATER, THE PORTS OF HOUSTON AND GALVESTON, AND TEXAS BAY AND PORT SYSTEMS ............ 75 ix CHAPTER Page Introduction .......................................................................... 75 Materials and Methods.......................................................... 85 Results ................................................................................... 92 Discussion ............................................................................. 128 IV CONCLUSIONS.................................................................................. 144 Conclusions .......................................................................... 144 REFERENCES.......................................................................................................... 159 VITA ......................................................................................................................... 171 x LIST OF FIGURES FIGURE Page I. 1. Ballast water uptake and discharge in cargo ships............................... 2 I. 2. Location in the water column of typical aquatic bacterial phyla ......... 6 I. 3. Locations of the Ports of Houston and Galveston................................ 9 I. 4. Texas river and coastal basins.............................................................. 12 I. 5. Texas ports and bays sampled during this study.................................. 21 I. 6. Locations of the Ports of Houston and Galveston................................ 23 I. 7. Location of ballast water exchange in ships sampled in this study...... 23 I. 8. Summary of methods used in this study............................................... 34 II. 1. Location of ballast water exchange by the ship examined in this study ..................................................................................................... 40 II. 2. Rarefaction curve for clone library AO06’57.3S008’15.3W............... 46 II. 3. Habitat characterization of ballast tank bacterial community members ............................................................................................... 47 II. 4. Habitat characterization of each major bacterial group: (a) α- proteobacteria, (b) γ-proteobacteria, (c) β-proteobacteria, and the (d) non-proteobacteria phyla...................................................................... 53 II. 5. Phylogenetic tree of alphaproteobacteria diversity in ballast water sample ‘06°57.3S, 008°15.3W’ and closest 16S rRNA relatives used to classify habitat.................................................................................. 56 II. 6. Phylogenetic tree of gammaproteobacteria diversity in ballast water sample ‘06°57.3S, 008°15.3W’ and closest 16S rRNA relatives used to classify habitat.................................................................................. 57 xi FIGURE Page II. 7. Phylogenetic tree of betaproteobacteria