Iron Acquisition in Cynobacteria Synechococcus sp. PCC 7002 Culture Ayten Pehlivan Environmental Toxicology and Chemistry Submission date: May 2018 Supervisor: Murat Van Ardelan, IKJ Co-supervisor: Martin.F Hohmann-Marriott, IKJ Norwegian University of Science and Technology Department of Chemistry Abstract Iron is an essential micronutrient for the growth and health of phytoplankton and low overall iron concentrations in the High Nutrient Low Chlorophyll (HNLC) zones in the ocean have been reported. Cyanobacteria is one of the most important organism involved in primary productivity and iron-limitation could indirectly affect the global carbon cycle and CO2 sequestration. This project aims to study the influence of the type of iron mineral and iron acquisition mechanism efficiency in Cyanobacteria. Two incubation experiments were carried out using a tropical strain of Synechococcus sp. PCC 7002 at room temperature (25 °C). Two different forms of Fe were used in cultures with different solubilities; one culture was added with 50 nM FeCl3 (74.4 g/100 mL) and the other culture was added with 50 nM Goethite (FeO(OH)) (HCl soluble), respectively. Biological parameters such as growth and chlorophyll a concentration as well as particulate and intracellular Fe were analysed. Growth of Synechococcus sp. PCC 7002 was significantly slow in both cultures regardless of Fe concentrations which was predicted to be due to cells already being in stationary phase during the sampling period. High particulate and intracellular Fe were detected in FeCl3 culture and no physiological stress response was observed. On the other hand, cells grown in FeO(OH), had less particulate and intracellular Fe however, the days where intracellular Fe was recorded, the Fe concentration was high. Overall, this study suggests that FeCl3 is more available to cells than the less soluble goethite FeO(OH) and the cells grown in FeO(OH) culture are able to utilise the particulate Fe via PilA1 mechanism. However, future work must be carried out in order to conclude the experiment. iii iv Acknowledgements I would like to thank my supervisor, Professor Murat, for the opportunity to work on this project. I would also like to thank Annie Vera Hunnestad for being very helpful and supportive throughout this project, and for teaching me a lot of useful skills. This project would not be completed without the hard work of Maria Villegas, I would like to thank her for all the time and work she had put in this project with me. A big thank you to Cafe-Sito at Realfagbygget for providing delicious cinnamon buns on Wednesdays and fuelling us up on long lab days! v vi Table of Contents Abstract ................................................................................................................................. iii Acknowledgements ................................................................................................................ v List of Figures ....................................................................................................................... xi List of Tables ....................................................................................................................... xiii List of Equations .................................................................................................................. xv List of Appendix Tables ..................................................................................................... xvii Abbreviations ...................................................................................................................... xix 1 INTRODUCTION ............................................................................................................ 1 2 HYPOTHESIS AND OBJECTIVES .............................................................................. 3 3 BACKGROUND AND THEORY ................................................................................... 5 3.1 Sources of iron ........................................................................................................... 5 3.2 Iron in the oceans ....................................................................................................... 7 3.2.1 Biogeochemistry and speciation of iron in the oceans ........................................... 7 3.2.2 Oceanic iron cycling .............................................................................................. 9 3.3 Iron as a nutrient ....................................................................................................... 11 3.3.1 Iron limitation and fertilization ............................................................................ 14 3.4 Phytoplankton in the ocean ...................................................................................... 16 3.5 Iron uptake mechanisms in Cyanobacteria .............................................................. 18 3.5.1 Siderophores ......................................................................................................... 18 3.5.2 Reductive iron uptake ........................................................................................... 19 3.5.3 Particulate bound iron via PilA1 ......................................................................... 21 3.6 Iron starvation in Cyanobacteria .............................................................................. 22 4 MATERIALS AND METHODS................................................................................... 25 4.1 Clean work and acid washing ................................................................................... 25 4.1.1 Acid cleaning procedure ...................................................................................... 25 4.2 Synthetic ocean water preparation ........................................................................... 27 4.3 Cleaning of Aquil with Chelex-100 ......................................................................... 31 4.4 Microwave sterilisation ............................................................................................ 33 4.5 Experimental setup ................................................................................................... 34 4.6 Important biological indicators ................................................................................ 35 4.6.1 Extracted chlorophyll a ........................................................................................ 35 vii 4.6.2 Optical density (OD730nm) ................................................................................. 36 4.6.3 Particulate and intracellular iron ........................................................................ 37 4.6.4 Particulate Organic Carbon (POC)/ Particulate Organic Nitrogen (PON) ....... 39 4.6.5 Temperature and pH ............................................................................................ 39 5 RESULTS ........................................................................................................................ 41 5.1 Blanks and limit of detection ................................................................................... 41 5.2 Growth ...................................................................................................................... 42 5.2.1 FeCl3 ..................................................................................................................... 42 5.2.2 FeO(OH) .............................................................................................................. 44 5.2.3 FeCl3 vs FeO(OH) ................................................................................................ 46 5.3 Temperature and pH ................................................................................................. 47 5.3.1 FeCl3 ..................................................................................................................... 47 5.3.2 FeO(OH) .............................................................................................................. 49 5.4 Chlorophyll a ............................................................................................................ 52 5.4.1 FeCl3 ..................................................................................................................... 53 5.4.2 FeO(OH) .............................................................................................................. 55 5.5 Intracellular and particulate iron .............................................................................. 57 5.5.1 FeCl3 ..................................................................................................................... 59 5.5.2 FeO(OH) .............................................................................................................. 60 6 DISCUSSION ................................................................................................................. 63 6.1 Growth ...................................................................................................................... 63 6.2 Temperature and pH ................................................................................................. 64 6.3 Chlorophyll a ............................................................................................................ 65 6.4 Intracellular and particulate iron .............................................................................. 67 6.4.1 FeCl3 ....................................................................................................................