University of Dundee DOCTOR OF PHILOSOPHY Marine phytoplankton in a high CO2 world Crawfurd, Katharine Award date: 2010 Awarding institution: University of Dundee Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 16. Jun. 2016 DOCTOR OF PHILOSOPHY Marine phytoplankton in a high CO2 world Katharine Crawfurd 2010 University of Dundee Conditions for Use and Duplication Copyright of this work belongs to the author unless otherwise identified in the body of the thesis. It is permitted to use and duplicate this work only for personal and non-commercial research, study or criticism/review. You must obtain prior written consent from the author for any other use. Any quotation from this thesis must be acknowledged using the normal academic conventions. It is not permitted to supply the whole or part of this thesis to any other person or to post the same on any website or other online location without the prior written consent of the author. Contact the Discovery team ([email protected]) with any queries about the use or acknowledgement of this work. MARINE PHYTOPLANKTON IN A HIGH CO2 WORLD KATHARINE CRAWFURD A THESIS SUBMITTED IN PARTIAL FULFILLMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF DUNDEE COLLEGE OF LIFE SCIENCES IN COLLABORATION WITH PLYMOUTH MARINE LABORATORY MARCH 2010 AUTHORS DECLARATION I am the sole author of this thesis and have consulted all of the references cited either in full or abstract form. The work reported was all carried out by myself, with the exception of that performed by other members of the consortia in the Bergen mesocosm project and aboard the STS Lord Nelson, as acknowledged within the text. None of the work discussed in this thesis has previously been submitted for a higher degree. This programme of advanced study was financed with the aid of a studentship from the Natural Environment research Council. ACKNOWLEDGEMENTS I would like to express my sincere thanks to Dr. Ian Joint for his constant support and supervision of this project and to Prof. John Raven for his inspirational guidance. The success of the long term experiment would not have been possible without the aid of Emily Baxter. I would like to thank Glen Wheeler, Jack Gilbert, Mike Allen and Martin Mühling for their assistance in the art of molecular biology; Glen Tarran and Susan Kimmance for flow cytometry and fluorometry tuition. Many thanks to Declan Schroeder and George Sorensen-Pound for DGGE assistance, Paul Somerfield and Bob Clarke for help with statistical analyses. The work in Bergen and aboard the STS Lord Nelson was only possible with a dedicated team, my thanks to all those involved and also to the University of Plymouth electron microscopy unit. Thanks to Karen Weynberg and Nicole Bale for their encouragement and Helen Findlay for CO2 equilibration assistance. Finally I must thank Jez and Isabel for their unfailing patience, support and nourishment during this project. i LIST OF CONTENTS LIST OF FIGURES viii LIST OF TABLES xiv LIST OF ABREVIATIONS xviii ABSTRACT 1 Chapter 1. GENERAL INTRODUCTION 2 1.1 CARBON DIOXIDE AND THE OCEANS 3 1.1.1 Ocean acidification 3 1.1.2 Carbon emissions 3 1.1.3 Climate models 4 1.1.4 The ocean carbonate system 7 1.2 THE PHYSICAL, BIOLOGICAL AND CARBONATE PUMPS 9 1.3 MARINE PHYTOPLANKTON 10 1.4 PRIMARY PRODUCTION 12 1.4.1 Light 13 1.4.2 The light dependent reactions of photosystems I and II 13 1.4.3 The light independent reactions, Calvin-Benson cycle 14 1.4.4 RuBisCO, photorespiration and CCMs 15 1.5 THEORETICAL EFFECTS OF INCREASED CO2 ON PHYTOPLANKTON 19 1.6 REGULATION, ACCLIMATION AND ADAPTATION 21 1.7 THE EFFECTS OF INCREASED CO2 ON A RANGE OF PHYTOPLANKTON ATTRIBUTES 22 1.7.1 Specific growth rate (µ) 22 1.7.2 Stoichiometry and DIC uptake 25 1.7.3 Calcification 29 1.7.4 Photosynthetic parameters /Primary production 31 1.7.5 Enzyme activities and regulation 33 1.7.6 Community structure 34 1.8 GENERAL CONCLUSIONS 35 1.9 THESIS AIMS 35 ii Chapter 2. METHODS 39 2.1 CULTURE STRAINS 39 2.2 CULTURE GROWTH CONDITIONS 39 2.2.1 Media, F/2 (+Si) and F/50 39 2.2.2 Axenic cultures 40 2.3 EXPERIMENTAL PROCEDURES 42 2.3.1 Acclimation 42 2.3.2 Bubbling of cultures 42 2.3.3 Reservoirs of pre-equilibrated media 42 2.3.4 Buffers 42 2.3.5 Semi-continuous cultures 43 2.3.6 Continuous cultures 43 2.3.7 Evaluation experiments 44 2.3.7 .a 900ml bottle experiment 45 2.3.7 b 1.5ml plate experiment 46 2.4 MEASUREMENTS 46 2.4.1. Coulter Counter 46 2.4.2 Analytical flow cytometry (AFC) 47 2.4.2.a Side scatter and forward scatter 48 2.4.2.b Auto-fluorescence 49 2.4.2.c Separating different groups of phytoplankton 49 2.4.3 Satlantic FIRe Fluorescence Induction and Relaxation system 50 2.4.4 C:N 52 2.4.5 Total Chlorophyll 52 2.4.6 Scanning electron microscopy (SEM) 54 2.4.7 pH 55 2.4.8 Salinity and Temperature 56 2.4.9 Measuring primary production and photosynthetic ability 56 2.4.10 pCO2 and total alkalinity 57 2.4.11 Nutrients 57 2.4.12 Pigments 57 2.4.13 Phytoplankton identification 57 iii 2.4.14 Gene expression as determined by quantitative RT PCR 58 2.4.14a Introduction 58 2.4.14b Quantitative RT- PCR 58 2.4.14c Assay design 60 2.4.14d Primer design 60 2.4.14e Genes of interest 60 2.4.14f Housekeeping genes 61 2.4.14g Harvesting cells 62 2.4.14h RNA extraction 62 2.4.14i RNA Quantification 63 2.4.14j Reverse transcription 64 2.4.14k Gene quantification 65 2.4.14l Primer optimization 67 2.4.14m Standard curves 67 2.4.14n Validation of housekeeping genes 67 2.4.14o Data analysis 68 2.4.14p Statistical analysis 69 2.4.15 DGGE 70 2.4.15 a. Introduction 70 2.4.15 b. Sampling 70 2.4.15 c. DNA extraction 71 2.4.15 d. PCR 72 2.4.15 e. Denaturing gradient gel electrophoresis (DGGE) 73 2.4.15 f. DGGE data Analysis 74 2.5 DATA ANALYSIS 74 2.5.1 Transformation of data 75 2.5.2 Matrix generation 76 2.5.3 Permutation 76 2.5.4 Univariate tests, ANOSIM 76 2.5.5 Multivariate tests, PERMANOVA 78 2.5.6 Multivariate techniques 79 iv Chapter 3. INVESTIGATION OF THE EFFECT OF INCREASED CO2 ON LABORATORY CULTURES OF TWO PHYTOPLANKTON SPECIES 80 3.1 INTRODUCTION 80 3.1.1 Carbonate system control 80 3.1.2 Phytoplankton species 82 3.2 BATCH CULTURE OF THALASSIOSIRA PSEUDONANA AT PRESENT DAY AND HIGH CO2 84 3.3 THALASSIOSIRA PSEUDONANA CCMP1335 AND EMILIANIA HUXLEYI CCMP 1516 AT INCREASED CO2; USING LOW CELL DENSITY SEMI-CONTINUOUS CULTURE TO MAINTAIN PH 84 3.4 EFFECTS OF INCREASED CO2 ON CALCIFYING EMILIANIA HUXLEYI CCMP 371 USING SEMI-CONTINUOUS CULTURE TO MAINTAIN PH. 89 3.5 LONG TERM CONTINUOUS CULTURE OF THALASSIOSIRA PSEUDONANA MAINTAINED AT INCREASED CO2 FOR THREE MONTHS, APPROXIMATELY 100 GENERATIONS. 92 3.5.1 pH control 92 3.5.2 Measurements of cell physiology 94 3.5.3 Carbon and nitrogen content of the cultures 94 3.5.4 Photophysiology 96 3.5.5 Fluorescence and light scatter by flow cytometry 96 3.5.6 Gene expression as determined by quantitative PCR 97 3.6 EVIDENCE FOR ACCLIMATION OF THALASSIOSIRA PSEUDONANA TO HIGH CO2 AFTER APPROXIMATELY 100 GENERATIONS. 99 3.6.1. Large volume (900ml) experiment 99 3.6.1a pH 99 3.6.1b Specific growth rates 100 3.6.1c Carbon and nitrogen content 102 3.6.1d Photophysiology 102 3.6.1e Flow cytometry 103 3.6.1f Gene expression 104 3.6.2 High replication experiment to evaluate of the effects growth of Thalassiosira pseudonana at 760 ppm CO2 108 3.7 DISCUSSION 109 v Chapter. 4 EXAMINATION OF THE EFFECTS OF INCREASED CO2 ON A NATURAL PHYTOPLANKTON ASSEMBLAGE FROM A NORWEGIAN FJORD 116 4.1 INTRODUCTION 116 4.2 BACKGROUND TO FACILITY 117 4.2.1 The Raunefjorden and its phytoplankton community 117 4.2.2 Bergen mesocosm system 118 4.2.3 Experimental design 120 4.3 RESULTS 122 4.3.1 Phytoplankton growth and carbon drawdown 123 4.3.1 a Chlorophyll 126 4.3.1b 14C to directly measure primary production 129 4.3.1c Particulate Organic carbon (POC) 129 4.3.1d C:N ratio 130 4.3.1e Calculated carbon drawdown, Δ[DIC*] and calcification by the alkalinity anomaly method 130 4.3.2 Flow cytometry 134 4.3.3 Microscopy analysis 141 4.3.4 Coccolithophores 142 4.3.4a Growth of E.