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The Ascidian Styela Plicata As a Potential Bioremediator of Bacterial and Algal Contamination of Marine Estuarine Waters

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The Ascidian Styela Plicata As a Potential Bioremediator of Bacterial and Algal Contamination of Marine Estuarine Waters THE ASCIDIAN STYELA PLICATA AS A POTENTIAL BIOREMEDIATOR OF BACTERIAL AND ALGAL CONTAMINATION OF MARINE ESTUARINE WATERS by Lisa Denham Draughon A Dissertation Submitted to the Faculty of The Charles E. Schmidt College of Science In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Florida Atlantic University Boca Raton, Florida May, 2010 Copyright by Lisa Denham Draughon 2010 ii ACKNOWLEDGEMENTS I wish to express my sincerest gratitude to those who made this research possible; Ocean Restoration Corporation Associates (ORCA), the Link Foundation, and Florida Atlantic University (FAU) for their financial fellowships, and for the many hands that played a part in getting it all done. A special thank you goes to my committee members; Dr. James X. Hartmann (chairperson) and Dr. David Binninger of FAU, Dr. Ed Proffitt and Dr. John Scarpa of Harbor Branch Oceanographic Institute at FAU, and Dr. Ray Waldner of Palm Beach Atlantic University. You were all such an integral part of helping me achieve this goal. Special thanks also go to my friend Karen Halloway- Adkins who was always willing to help locate specimens and provide equipment when she had it. Mike Calinski of ORCA made trips from the Gulf Coast to deliver specimens to me. Sherry Reed of the Smithsonian Marine Station in Ft. Pierce was extremely helpful by keeping an eye out for the tunicates and at times gathering them for me. Dr. Patricia Keating was invaluable for her help in flow-cytometry analysis. Dr. Peter McCarthy of Harbor Branch Oceanographic Institute at FAU provided the bacteria for the filtration rate experiment. Dr. Esther Guzman and Tara Pitts, also from HBOI-FAU, were extremely helpful during the ATP assay. Ashley Callahan, your help in Panama is so much appreciated. And finally but definitely not least, I want to thank all the undergraduate students that assisted during the experiments, especially Catherine (Katie) Lord and Danielle (Dani) Nangle. I can’t thank you enough. iv PREFACE This dissertation, which focuses on the potential use of the tunicate Styela plicata for bioremediation, is organized into one general background chapter followed by individual chapters that have already been or will be submitted for publication. For this reason, there will be redundancy in most of the chapter introductions and discussions as different facets of the tunicate’s use are explored. Portions of Chapter Two have been compiled and reprinted with permission from two separate publications: Draughon, L., Scarpa, J., Keating, P., & Hartmann, J. (2008). Potential estuarine water quality improvement via marine invertebrate bioremediation. In M. Theophanides & T. Theophanides (Eds.), Environmental Awareness and Management (pp. 97-112). Athens: ATINER. and Draughon, L. D., Scarpa, J., & Hartmann, J. X. (2010). Are filtration rates for the rough tunicate Styela plicata independent of weight or size? Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 45(2), 168 - 176. During the summer of 2008, south Florida was hit with Tropical Storm Fay that resulted in massive rain totals along the Indian River Lagoon counties. Subsequently the salinities in the Lagoon decreased for a prolonged period of time resulting in the loss of v local Styela plicata populations. The same salinity decreases were found along the Gulf Coast of Florida, as well. For this reason, the tunicate Phallusia nigra was used in the last experiment (Chapter Six). These specimens were collected during the month of June, 2009 in Bocas del Toro, Panama. vi ABSTRACT Author: Lisa Draughon Title: The Ascidian Styela plicata as a Potential Bioremediator of Bacterial and Algal Contamination of Marine Estuarine Waters Institution: Florida Atlantic University Dissertation Advisor: Dr. James X. Hartmann Degree: Doctor of Philosophy Year: 2010 The tunicate Styela plicata (Ascidiacea) was investigated for its potential use in bioremediation of bacteria and microalgae blooms from estuarine waters. Salinity tolerances, filtration rate, substrate selection, and effects on digested bacteria and ichthyofaunal communities were examined. If acclimated slowly, Styela can be placed in salinities as low as 24 parts per thousand (ppt) before increased fatalities result. An immediate decrease in salinity from 32 ppt to 20 ppt was not detrimental at short term exposure (four days). One average-sized (~40g) Styela, exposed to 105 and 106 bacteria or microalgae mL-1, can filter as much as 4.7 L hr-1 and 3.3 L hr-1, respectively. Individual tunicate filtration rates varied from hour to hour, independent of organism size, indicating that filtration rates for this species would be better reported on a population basis rather than on an individual weight or size basis. Bacterial viability in tunicate feces was assessed by ATP analysis. Bacteria were found to be non-viable after vii passage through the tunicate digestive tract. Filtration of eggs or larvae of recreationally or commercially important fish is of concern. The smallest eggs (~0.6mm) reported in the Indian River Lagoon belong to Cynoscion nebulosus (spotted seatrout) and Bairdiella chrysoura (silver perch). Over the course of two hours, 72.5% of 0.5 mm glass beads (simulated fish eggs) were initially retained, but many were later expelled by the tunicates. For 1.0 mm beads, 49.4% were initially retained and for 2.0 mm beads, 43.5% were initially retained. Neither the size of the oral siphon nor the size of the overall animal was correlated to bead retention. Substrate selection was investigated using the tunicate Phallusia nigra. Newly hatched larvae preferred settling on wood (53.7%), as compared to polyethylene (30.9%), high-density polyethylene (13.0%), and polyvinyl chloride (2.4%). Overall, results of this study indicate S. plicata is very effective at removing bacteria and microalgae from estuarine waters. However, further testing is warranted regarding the unwanted removal of fish eggs and larvae before utilization of S. plicata for bioremedial purposes. viii DEDICATION I dedicate this work and this book to my wonderful and supportive family who encouraged me all along the way, especially to my wonderful husband, Randy. I couldn’t have done this without him. Most times, he worked multiple jobs to help make ends meet, never once complaining. He ate late dinners, leftovers, and sometimes put up with “fend-for-yourself” night. He is truly as perfect of a man as I have ever met. He’s not the only one who had to eat late meals at times. I also dedicate this book to Sammie, my daughter, whose homework often times stumps me. I hope I was there for her enough when she needed me. She is such a blessing in our lives. My Dad passed away on Feb. 2, 2003 before he even saw me complete my undergraduate degree. He would have been very proud to know I achieved this. Still, I think he does know. Daddy, this is also for you. Many, like my husband, Mom, and daughters Kelly, Kasey, and Sammie prayed a lot for me. Thank you to all of you. Most important, I thank God, who in a very confusing way brought me to this place. I would have never believed it but He works in strange ways. For those who know me, you know the story. For those of you who don’t, I’d love to share it with you some day. TABLE OF CONTENTS List of Tables ................................................................................................................... xiv List of Figures................................................................................................................... xv CHAPTER ONE: General Introduction.............................................................................. 1 Problems Facing the Indian River Lagoon ................................................................... 1 Styela plicata History.................................................................................................... 5 Species Populations ...................................................................................................... 6 Styela plicata Filtration Advantages............................................................................. 7 CHAPTER TWO: Potential Estuarine Water Quality Improvement Via Filtration Benefits of Tunicate Styela plicata (LeSueur 1823)........................................................... 9 Abstract......................................................................................................................... 9 Introduction................................................................................................................. 10 Materials and Methods................................................................................................ 13 Collection and Acclimation of Organisms............................................................ 13 Experimental Design and Statistical Analysis ...................................................... 14 Algal Filtration Rate ............................................................................................. 14 Establishment of Bacterial Filtration Rate............................................................ 16 Establishment of bacterial region for flow cytometry analysis. ..................... 16 Bacterial filtration rate. ................................................................................... 18 Preparation of bacterial water samples for flow cytometry............................ 19 x Calculation of Filtration Rate...............................................................................
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