FLOW CYTOMETRIC INVESTIGATION OF THE SIZE SPECTRUM OF NORTH SEA PHYTOPLANKTON COMMUNITIES Katy R. Owen A thesis submitted to the School of Environmental Sciences, at the University of East Anglia, for the degree of Doctor of Philosophy, May 2014. © This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. 2 ABSTRACT Marine biogeochemical processes are closely linked to phytoplankton community assemblages. Cell abundance and biomass are a measure of the successful conversion of inorganic to organic carbon. Carbon estimates are therefore often used to analyse metabolism and energy transfers within marine environments, and carbon is frequently the main parameter used in ecosystem models. Phytoplankton can be divided into functional types based on cell size: microplankton (<200 µm), nanoplankton (2-20 µm) and picoplankton (≤ 3 µm). Differences in cell volume govern variations in carbon content, nutrient uptake and influence cell fate. Reduced diameters equate to lower sedimentation rates and promote participation within the microbial loop and recycling of carbon within surface waters. Larger diameters can increase settling rates, resulting in the loss of carbon from surface waters. Current North Sea monitoring and research programmes typically only consider larger micro- and nanoplankton cells, or the bulk phytoplankton community as a whole: there is little separation by functional type. Inclusion of picoplankton and the delineation of biomass contribution by cell size are required for accurate depictions of phytoplankton productivity within this region, but this is not feasible with current water sampling protocols. Flow cytometry is a new multiparametric analysis technique offering high-speed enumeration and assessment of particles. Phytoplankton cells from 2-200 µm can be easily distinguished from debris and reproducible data on cell size and pigment content is supplied within minutes. This research uses flow cytometry to provide detailed assessments of phytoplankton community structure at a range of spatial and temporal scales. Distribution patterns are related to environmental parameters and observed patterns are used to test existing paradigm and advance current ecological theory. 3 4 LIST OF CONTENTS List of contents………………………………………………………………………….5 List of figures…………………………………………………………………………..11 List of tables……………………………………………………………………………19 List of abbreviations……………………………………………………………………23 Dedications……………………………………………………………………………. 27 Acknowledgements…………………………………………………………………….29 Chapter 1 Introduction .......................................................................................................... 31 1.1. What are plankton? ................................................................................................ 31 1.2. Ecological and biogeochemical relevance of phytoplankton ................................ 32 1.2.1. Photosynthesis and the global carbon cycle ....................................................... 33 1.3. Phytoplankton diversity ......................................................................................... 36 1.3.1. Diatoms (Bacillariophyta) .................................................................................. 39 1.3.2. Dinoflagellates (Dinophyta) ............................................................................... 40 1.3.3. Golden brown flagellates (Haptophyta, Chrysophyta) ....................................... 41 1.3.4. Green algae (Chlorophyceae, Prasinophyceae) .................................................. 42 1.3.5. Cyanobacteria ..................................................................................................... 43 1.4. Phytoplankton Functional Types (PFT) ................................................................ 44 1.4.1. PFT distribution .................................................................................................. 46 1.4.2. Picophytoplankton .............................................................................................. 50 1.5. The microbial loop ................................................................................................ 51 1.6. Mathematical ecosystem models ........................................................................... 54 1.7. Phytoplankton analysis techniques ........................................................................ 55 1.7.1. Microscopy ......................................................................................................... 55 1.7.2. Chlorophyll a (chl a) and accessory pigments ................................................... 55 1.7.3. Coulter counters ................................................................................................. 57 1.7.4. Flow cytometry ................................................................................................... 57 5 1.7.5. Molecular analysis techniques ............................................................................ 61 1.7.6. Remote sensing ................................................................................................... 63 1.8. Study area: the North Sea ...................................................................................... 64 1.9. Project rationale and primary research objectives ................................................. 66 Chapter 2 General methodology .......................................................................................... 71 2.1. Phytoplankton culturing techniques ...................................................................... 71 2.1.1. Media preparation ............................................................................................... 72 2.1.1.1. f/2 + Si medium ............................................................................................... 72 2.1.1.2. L1 medium ....................................................................................................... 73 2.1.1.3. K medium ........................................................................................................ 74 2.1.1.4. SN medium ...................................................................................................... 75 2.2. Culture sampling.................................................................................................... 76 2.3. Environmental sampling ........................................................................................ 76 2.3.1. Chlorophyll determination .................................................................................. 78 2.3.2. HPLC .................................................................................................................. 80 2.3.3. Nutrient analysis ................................................................................................. 81 2.3.4. Salinity determination......................................................................................... 82 2.4. Phytoplankton analysis by flow cytometry ........................................................... 82 2.4.1. Development of flow cytometry ......................................................................... 83 2.4.2. Operational principles ......................................................................................... 84 2.4.2.1. Fluidics ............................................................................................................ 84 2.4.2.2. Optics ............................................................................................................... 86 2.4.2.3. Electronics ....................................................................................................... 89 2.4.3. CytoSense flow cytometer .................................................................................. 91 2.4.4. Environmental phytoplankton analysis .............................................................. 94 2.4.5. Treatment of data ................................................................................................ 95 2.4.6. Machine monitoring and maintenance ............................................................... 97 2.4.6.1. Volume calibration .......................................................................................... 98 2.4.6.2. Scatter calibration .......................................................................................... 100 6 2.4.6.3. Laser and PMT calibration ............................................................................ 101 2.4. Statistical analyses ............................................................................................... 103 Chapter 3 Analysis of North Sea phytoplankton biomass by size based PFT during late summer (2010) ............................................................................................................... 105 3.1 Introduction .......................................................................................................... 106 3.2. Materials and methods ......................................................................................... 109 3.2.1. Data collection and processing ......................................................................... 109 3.2.2. Statistical analyses ...........................................................................................