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Microorganisms Associated with Ulva Grown in Abalone Effluent Water: Implications for Biosecurity

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Microorganisms Associated with Ulva Grown in Abalone Effluent Water: Implications for Biosecurity MICROORGANISMS ASSOCIATED WITH ULVA GROWN IN ABALONE EFFLUENT WATER: IMPLICATIONS FOR BIOSECURITY DJGKRI001 KRISTIN DE JAGER Dissertation presented in the partial fulfilment of the requirements for the degree of Master of Science (BIO 5006W Applied Marine Science) Department of Biological Science University of Cape Town February 2019 Supervisor: Prof John J. Bolton (University of Cape Town) Co-supervisor: Dr Brett M. Macey University(Department of Agriculture, of Forestry Cape and Fisheries) Town The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town P a g e | 1 DECLARATION I know the meaning of plagiarism and declare that all of the work in the dissertation (or thesis), other than that which was carried out by the University of the Western Cape Next Generation Sequencing Facility and that which is properly acknowledged, is my own work as assisted by my supervisors. This thesis has not been submitted in this or any form to another university. Experimental work discussed in this thesis was carried out under the supervision of Prof. J. J. Bolton of the Department of Botany, University of Cape Town and Dr. B.M. Macey of the Department of Agriculture, Forestry and Fisheries. Mr Kristin de Jager Date: P a g e | 2 DEDICATIONS I dedicate this thesis to all things green, as without words they strive to answer questions we have never asked. P a g e | 3 ABBREVIATIONS ANOVA Analysis of variance ASW Autoclaved seawater AEW Abalone effluent wastewater CFU Colony forming units DAFF Department of Agriculture, Forestry and Fisheries ™ dH2O Distilled water (Millipore) DNA Deoxyribonucleic acid dNTP Deoxyribonucleotide triphosphate EDTA Ethylene-diamenetetra-acetic acid ES Effluent system FS Fertilised system FSW Fertilised seawater FTS Flow through system IMTA Integrated multitrophic aquaculture LEfSe Linear discriminate analysis effect size ML Maximum likelihood MRA Marine Research Aquarium NGS Next generation sequencing NMDS Non-metric multidimensional scaling analysis OUT Operational Taxonomic Unit PCoA Principal co-ordinate analysis rbcL Ribulose-1,5-bisphosphate carboxylase RAS Recirculating aquaculture system RNA Ribonucleic acid TCBS Thiosulfate Citrate Bile Sucrose Agar TSA Tryptone Soy Agar P a g e | 4 TABLE OF CONTENTS DECLARATION .................................................................................................................................... 1 DEDICATIONS ..................................................................................................................................... 2 ABBREVIATIONS ................................................................................................................................ 3 TABLE OF CONTENTS ...................................................................................................................... 4 PREFACE .............................................................................................................................................. 7 ACKNOWLEDGEMENTS ................................................................................................................... 8 ABSTRACT ........................................................................................................................................... 9 SECTION 1. GENERAL INTRODUCTION .................................................................................... 11 1.1 Brief history of the cultivation of abalone in South Africa ......................................................... 11 1.2. The global seaweed industry: species, production and markets ............................................... 14 1.3 General overview of Ulva cultivated in South Africa .................................................................. 15 1.3.1 Taxonomy....................................................................................................... 15 1.3.2 Morphology and reproduction ......................................................................... 16 1.3.3 Ulva cultivars maintained on South African Abalone farms ............................. 17 1.4 Uses and benefits of Ulva grown in integrated aquaculture systems ........................................ 18 1.4.1 Ulva as a feed additive ................................................................................... 18 1.4.2 Ulva and Ulva extracts for improving larval settlement .................................... 19 1.4.3 Use of Ulva in IMTA systems and for bioremediation of farm effluent water.... 21 1.5 Role of bacteria in integrated abalone seaweed systems and the beneficial associations between bacteria, Ulva and abalone ................................................................................................... 23 1.5.1 Host-bacterial interactions .............................................................................. 23 1.5.2 Role of bacteria in morphogenesis and reproduction of Ulva .......................... 25 1.5.3 Impacts of bacteria on algal sporulation and settlement.................................. 26 1.5.4 The role of algal associated bacteria on abalone larval and adult digestive processes, growth and health .................................................................................... 27 1.6 Diseases in aquaculture and the biosecurity concerns associated with the use of effluent grown Ulva as a feed or supplementary feed .................................................................................................. 29 1.7 Methods for the detection, quantification and assessment of microbial community structure in complex ecosystems ......................................................................................................................... 32 1.8 Concluding remarks .................................................................................................................... 33 1.9 Objectives of this study .............................................................................................................. 34 SECTION 2. MATERIALS AND METHODS .................................................................................. 36 2.1 Collection of water and Ulva samples ........................................................................................ 36 P a g e | 5 2.1.1 Study site ....................................................................................................... 36 2.1.2 Ulva collection ................................................................................................ 37 2.1.3 Water collection .............................................................................................. 37 2.1.4 Description of tanks and integrated raceway systems sampled ...................... 37 2.2 Molecular analysis of Ulva .......................................................................................................... 41 2.2.1 DNA extraction and purification ...................................................................... 41 2.2.2 Polymerase chain reaction (PCR) amplification and DNA sequencing ............ 42 2.2.3 Sequence assembly and phylogenetic analysis .............................................. 42 2.3 Enumeration of culturable bacteria in seawater and on Ulva (Selective Plating) ...................... 44 2.3.1 Media preparation ........................................................................................... 44 2.3.2 Enumeration of bacteria in seawater ............................................................... 45 2.3.3 Enumeration of bacteria on Ulva ..................................................................... 45 2.3.4 Isolation, identification and storage of culturable bacteria ............................... 45 2.3.5 Statistical analyses for colony forming unit (CFU) data ................................... 47 2.4 Bacterial community profiling by 16S rRNA Illumina MiSeq sequencing ................................... 47 2.4.1 DNA Extraction and library preparation ........................................................... 47 2.4.2 Raw data processing, data filtration and normalisation. .................................. 48 2.4.3 Overview of sequencing and microbial diversity at a higher level .................... 49 2.4.4 Comparison of bacterial community structure among Ulva and water libraries. 50 2.4.5 Comparison of bacterial community structure among sample types ............... 50 SECTION 3. RESULTS ..................................................................................................................... 52 3.1 Molecular analysis of Ulva .......................................................................................................... 52 3.2 Enumeration of culturable bacteria in seawater and on Ulva ................................................... 54 3.3 Identification of culturable bacteria ........................................................................................... 55 3.4 Bacterial community profiling by 16S rRNA Illumina MiSeq sequencing ................................... 57 3.4.1 Overview of OTU sequence clustering and richness estimation ...................... 57 3.4.2 Comparison of bacterial community structure among Ulva and water libraries. 61 3.4.3 Comparison of bacterial community
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