I Sustainable aquaculture effluent treatment systems utilizing biodegradable plastics through microbial remediation A Thesis Presented to The Faculty of the College of Arts and Sciences Florida Gulf Coast University In Partial Fulfillment of the Requirement for the Degree of Master of Science By Cristina Rose Lopardo 2018 II APPROVAL SHEET This thesis is submitted in partial fulfillment of the requirements for the degree of Master of Science Cristina Rose Lopardo Approved: 2018 Hidetoshi Urakawa, Ph.D. Committee Chair / Advisor William J. Mitsch, Ph.D. Li Zhang, Ph.D. Jong-Yeop Kim, Ph.D. The final copy of this thesis has been examined by the signatories, and we find that both the content and the form meet acceptable presentation standards of scholarly work in the above mentioned discipline. III Acknowledgements I would like to thank my mentor Dr. Hidetoshi Urakawa for his support and guidance throughout my years of study at Florida Gulf Coast University. You helped me to further my knowledge in the field of microbiology and to reach my academic goals through being a member of your laboratory and as a mentee. Thank you very much for helping me to contribute to the scientific field through publication of my research, something I did not think possible before working with you. I thank Dr. William J. Mitsch as a member of my committee and co-collaborator for his insight and expertise in the field of wetland ecology which helped to bring a different insight into my studies which I otherwise would not have. I thank Dr. Li Zhang as a member of my committee and co-collaborator for her guidance throughout our time working together and for your feedback of my thesis. I thank Dr. Jong-Yeop Kim for being a member of my committee and for providing feedback and constructive criticism of my thesis. Special thanks to the Everglades Wetland Research Park team under the guidance of Dr. William Mitsch and Dr. Li Zhang for their help and support in field and laboratory work. Also special thanks to Megan Feeney with laboratory work and Matthew Gamel and Nicolas Culligan for help with my aquarium setup and care of my pinfish, in addition to the rest of our lab team whom I had the privilege to know and work with over the past 2 ½ years who provided feedback and emotional support. I thank Haruka Urakawa for her guidance and care, your assistance contributed to my successes both professionally and personally at FGCU. You are much appreciated for all that you do. A special thanks to my parents, without your support I would literally not be where I am today. Your guidance and teachings have shaped me to be the person I became and helped me to reach my goals in life. I thank Stefanie, you are not only my sister but my best friend. You always help to remind me to not be so serious all the time and to live life with childish enthusiasm. Thanks IV to all my family and friends for your emotional support and guidance throughout my program and to always keep me grounded, you are too many to list but are always in my heart. Last but certainly not least, I would like to thank God. Without Him my dreams would not have happened and am grateful for all the opportunities I have been given and people I have met along the way. I thank the following scholarship foundations for their monetary support allowing me to complete my masters program at FGCU; the Marco Island Shell Club, the Blair Foundation, and the Dorothy M. Rygh Fellowship Foundation. I also thank Joe Veradino at JoeFish Aquatics for providing aquariums and Dr. Timothy Hovanec at Dr Tim’s Aquatics for providing polyhydroxyalkanoate plastic beads. I thank our funding agencies for allowing us to complete our research, FGCU Office of Research and Graduate Studies internal grant program and the Florida Sea Grant college program with support from the National Oceanic and Atmospheric Administration (NOAA), Office of Sea Grant, U.S. Department of Commerce, Grant (PD-15-2). V Abstract Demand of marine aquaculture has risen worldwide with human population growth and need for reliance on a steady protein supply. Direct discharges from on-land marine aquaculture systems are exceptionally high in nitrogen and phosphorous, which have negative impacts to downstream waters leading to eutrophication. Bioremediation methods using biodegradable plastic applications were explored through this thesis; a closed marine recirculating aquarium system (RAS) and using two different biodegradable plastic media in a bioreactor system and a marine aquaculture effluent from pinfish (Lagodon rhomboides) and wetlands using a vertical-flow (VFTW) and floating treatment (FTW) construction. The objectives explored were to; apply ecological engineering techniques for the treatment of nutrient rich marine aquaculture effluent, determine the nitrogen and phosphorous removal rates from these systems, and to determine the impact of biodegradable plastics on microbial community composition. In the marine RAS study, the TN removal efficiency was 92.1-98.5% with the use of biodegradable plastic, polycaprolactone (PCL) and polyhydroxyalkanoate (PHA), and an ASN medium and 61.5-62% TN removal efficiency with pinfish effluent. TN removal efficiency was shown to be greater with biodegradable plastic than a nonbiodegradable plastic control for both conditions. Alternatively, use of a biodegradable plastic (PCL) as an external carbon input to a treatment wetland showed minimal increases in TN retention in a FTW system with greater efficiency effects shown between wetland construction, the VFTW system showed an increased TN, 87-91%, and TP, 74-81%, nutrient retention compared to the FTW system. The use of biodegradable plastic for treatment of high nutrient marine aquaculture effluent was shown to be effective and this technology can be used on a larger scale application for sustainable wastewater treatment practices. VI Table of Contents Acknowledgements ........................................................................................................................III Abstract ........................................................................................................................................... V Table of Contents .......................................................................................................................... VI List of Tables ............................................................................................................................... VII List of Figures ............................................................................................................................. VIII Chapter 1: Introduction - Plastic waste in marine environments .............................................2 Objectives of Thesis .........................................................................................................................8 Figures............................................................................................................................................13 References ......................................................................................................................................14 Chapter 2: Performance and microbial diversity of a closed recirculating aquaculture effluent treatment system using polycaprolactone and polyhydroxyalkanoate as carbon source and biofilm carrier ...........................................................................................................21 Abstract ..........................................................................................................................................22 Introduction ....................................................................................................................................23 Materials and Methods ...................................................................................................................24 Results & Discussion .....................................................................................................................30 Conclusion .....................................................................................................................................37 Figures & Tables ............................................................................................................................39 References ......................................................................................................................................49 Chapter 3: Comparison of nutrient retention efficiency between vertical-flow and floating treatment wetland mesocosms with and without biodegradable plastic ................................57 Abstract ..........................................................................................................................................58 Introduction ....................................................................................................................................59 Materials and Methods ...................................................................................................................61 Results & Discussion .....................................................................................................................66 Conclusion .....................................................................................................................................74 Figures & Tables ............................................................................................................................75 References