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Uiopasdfghjklzxcvbnmqwertyui Ghjklzxcvbnmqwertyuiopasdfgh qwertyuiopasdfghjklzxcvbnmq wertyuiopasdfghjklzxcvbnmqw ertyuiopasdfghjklzxcvbnmqwer tyuiopasdfghjklzxcvbnmqwerty uiopasdfghjklzxcvbnmqwertyui Dissertation opasdfghjklzxcvbnmqwertyuiopDoan Thanh Loan asdfghjklzxcvbnmqwertyuiopas dfghjklzxcvbnmqwertyuiopasdf ghjklzxcvbnmqwertyuiopasdfgh jklzxcvbnmqwertyuiopasdfghjkl zxcvbnmqwertyuiopasdfghjklzx cvbnmqwertyuiopasdfghjklzxcv bnmqwertyuiopasdfghjklzxcvbn mqwertyuiopasghjklzxcvbnmq werty Page 0 of 208 uiopasdfghjklzxcvbnmqwertyui Microbial composition in biofloc-based shrimp aquaculture systems A dissertation by Doan Thanh Loan 2019 Page 1 of 208 STATEMENT OF ORIGINALITY I, Doan Thanh Loan with this certify that I am the sole author of this thesis and any assistance received is fully acknowledged. To the best of my knowledge, this thesis does not contain any material, which has been previously published or written by others. Any content of other work utilized in this thesis is cited following the standard referencing procedures. Date: ------------------------------------ Signed: ------------------------------------ Page 2 of 208 This thesis was conceived and written at the Leibniz Centre for Tropical Marine Ecology (ZMT) in Bremen and at the Laboratory of Aquaculture & Artemia Reference Centre (ARC) in Belgium between October 2014 and April 2019. This thesis was conducted under the supervision of Dr. Astrid Gärdes, Dr. Andreas Kunzmann, Prof. Dr. Matthias Wolff (ZMT) and Prof. Peter Bossier (ARC), and with non-panel additional supervision of Dr. Christiane Hassenrück (ZMT). Research for this thesis was funded by the Vietnamese Government, ARC, and ZMT. Thesis writing period was supported by Bremen Early Career Researcher Development BYRD (Universität Bremen). Chairperson: Prof. Dr. Michael Friedrich Co-examiners: Dr. Astrid Gärdes Prof. Dr. Matthias Wolff Prof. Dr. Patrick Sorgeloos Other non-voting members of the dissertation committee: Dini Adyasari (ZMT PhD candidate) Ramón Alejandro Plazas Gómez (ISATEC student) Date of Defense: 31/07/2019 Page 3 of 208 DEDICATION I dedicate this work to My parents, Doan Van Lai and Nguyen Thi Lien, My son, Nguyen Bao Kim, All the family members All my teachers For their endless love and unconditional support I would hereby like to thank the many people whose help has been invaluable during the completion of this work. “The Beauty of Science is to Make Things Simple” – Zymo research Page 4 of 208 ACKNOWLEDGEMENTS Writing this part has reminded me that I would never have completed this doctoral project without the help and support from my mentors, teachers, colleagues, advocates, family and friends. Many thanks to all! Specifically, I would first like to express my gratitude to my mentor, Dr Astrid Gärdes, for giving me an excellent chance to work on microbial composition in shrimp aquaculture with the application of biofloc technology at ZMT and ARC. If I had been in Berlin working on Yersinia and small animal diseases at FLI, it would not have been much benefit to the Fisheries Faculty, where I have been working on fish diseases for VNUA. Thanks so much, Astrid! In addition to my first mentor, I would like to express my deepest gratitude to Prof. Peter Bossier for his insightful comments and encouragement from the MSc thesis and from the moment I gave him the PhD literature review. He was such a patient and understanding advocate, giving me significant opportunities to learn. He listened to all my crazy ideas (e.g. about nitrobacteria, and PHB) whenever his office door was open after 6 pm, and he gave me direction. I will never forget the discussion I had with him in the Challenge Lab about my first findings on shrimp mortality, because he had to take off his shoes and wear the Challenge Lab boots. He slowly informed me that the key finding for my thesis would be TSS, with its role and relationship to operating systems, although I was arguing that it was pH. (I still have his handwritten note on how to calculate NH3 from pH and temperature). Thanks so much, Peter! I would also like to express my sincere gratitude to Prof. Matthias Wolff, Dr Andreas Kunzmann and Prof. Patrick Sorgeloos, who provided me ideas and comments over the last four years, beginning with key papers provided by Patrick to the end of this thesis with the meetings with panel members. Patrick and Mama Magda are always in my thoughts, such as when I attended a lovely dinner with all their family members, including 11 grandchildren. In addition, Andreas’ office door was always open to my questions and urgent help after 2 pm, and Andreas provided me with many critical papers in forming this thesis story. Thanks so much! Finally, thanks very much to Matthias for being my promotor at Bremen University. Special gratitude goes out to all the people at Vietnam International Education Development (VIED), ARC, ZMT and BYRD for helping and providing the funding for the work. ZMT is my second home at the moment, where I can learn important knowledge to bring to my university. Page 5 of 208 These benefits should open more opportunities for those at the university, and my future students may be able to come work at ZMT on new projects. There are several people at ZMT whose help during my stay and study I can never forget, namely, Achim, Mercè, Sonja, Constanze, Stefanie, Christina, Kai, Agostino, Jailson, Jennifer, Inken, Febrina, Nan, Dini, Agustin, and Akuila. As a special mention, Christiane introduced me to R language so that I could understand her guidance in bioinformatics and data analysis. Thank you very much, Christiane! Another special mention goes to Gérman and Anna, who shared their time and unconditional support with me. Mainly, I would like to thank Dr Janine Reinhard, ZMT’s board of directors and members working for ZMT for establishing this second home for many international people, who can benefit from this environment through learning and cooperation. I thank my lab mates for the stimulating discussions, the sleepless nights we spent working together until 3 am and all the fun we had in the ARC laboratory during the experiments. Special mentions are owed to Tom B, Anita, Brigitte, Caroline, Barbara, Peter S, Jorg, Alex, Geert and Marc, who helped me in this research. It was fantastic to have the opportunity to carry out most of my thesis work in the ARC laboratory. It was an excellent place to work on Litopenaeus vannamei disease and bioflocs, and I gained knowledge and friendships there. My sincere gratitude is also extended to VNUA’s college, who have been working on my job’s responsibilities. They helped me to have time to stay in ZMT to complete this PhD. Thanks very much! I am also grateful to the members of my thesis examination committee for their invaluable remarks and suggestions for the improvement of my PhD thesis. I am also thankful for the hard questions which motivated me to widen my research from various perspectives. Finally, I would like to thank my family members for their prayers and trust, encouraging me to study to the highest level and taking care of my son. Mom, it was hard for me when you broke your wrist while I was away! Many thanks to my son for his understanding of my absence in his important moments at school, for his smiles and laughter, for his kisses and hugs; I love him so much. Thanks to Liem for being with me during the whole time of my studying in Bremen and visiting me in Ghent with jokes and smiles, surprise presents and flowers … Thanks, Liem. Thank you all for your encouragement! Page 6 of 208 ABBREVIATIONS A-Floc/Afloc Autotrophic biofloc system AHL N-acylhomoserine lactone AHPND Acute hepatopancreatic necrosis disease AIC Akaike information criterion BFT Bioflocs Technology C/N carbon/nitrogen CFU Colony forming units DNA Deoxyribonucleic acid DO Dissolved oxygen EMS Early Mortality Syndrome FAO Food and agriculture organization FCR Food conversion ratio FT Flow-through aquaculture system GLMMs General linear mixed models H-Floc/ Hfloc Heterotrophic biofloc system NH3-N un-ionized ammonia + NH4 -N ammonia-N/ ammonium NMDS Non-metric multidimensional scaling - NO2 -N nitrite-N - NO3 -N nitrate-N OTU Operational taxonomic unit PCA Principal component analysis PCR Polymerase chain reaction PHB Poly-β-hydroxybutyrate pVpAHPND potentially AHPND-causing strain of Vibrio parahaemolyticus RAS Recirculating aquaculture system RDA Redundancy analysis SS Setting solids suc+Vibrio spp. Vibrio bacteria have yellow colour colony suc-Vibrio spp. Vibrio bacteria have green colour colony TAN Total Ammonia Nitrogen TCBS Thisulfate citrate bile salt sucrose agar TIN Total Inorganic Nitrogen TSS Total suspended solids VSS Volatile suspended solids WGR Weight gain rate Page 7 of 208 TABLE OF CONTENTS ABSTRACT -----------------------------------------------------------------------------------------------------------------------------------------11 CHAPTER 1 -----------------------------------------------------------------------------------------------------------------------------------------18 INTRODUCTION ----------------------------------------------------------------------------------------------------------------------------------18 1.1 MAKING A DIFFERENCE THROUGH RESPONSIBLE AQUACULTURE -------------------------------------------------------------------------19 1.2 MICROBIOTA AND DISEASE IN AQUACULTURE: ISSUES RELATED TO THE VIBRIO GENUS -------------------------------------------------21 1.3 STABLE MICROFLORA SYSTEMS -------------------------------------------------------------------------------------------------------------22 1.4 MICROBIAL
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