Uptake, Localization, and Speciation of Iron and Boron in the Brown Algae Ectocarpus siliculosus

164 Pages·2015·3.69 MB·English

UC San Diego
UC San Diego Electronic Theses and Dissertations
Title
Uptake, Localization, and Speciation of Iron and Boron in the Brown Algae Ectocarpus
siliculosus and Macrocystis pyrifera
Permalink
https://escholarship.org/uc/item/3dz4729v
Author
Miller, Eric Paul
Publication Date
2015

Peer reviewed|Thesis/dissertation
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University of California
UNIVERSITY OF CALIFORNIA, SAN DIEGO
SAN DIEGO STATE UNIVERSITY

Uptake, Localization, and Speciation of Iron and Boron in the Brown Algae Ectocarpus
siliculosus and Macrocystis pyrifera

A dissertation submitted in partial satisfaction of the
requirements for the degree Doctor of Philosophy
in
Chemistry
by
Eric Paul Miller

Committee in charge:
University of California, San Diego
Professor Katja Lindenberg
Professor Arnold Rheingold

San Diego State University
Professor Carl Carrano, Chair
Professor Miriam Bennett
Professor Matthew Edwards

2015
Copyright
Eric Paul Miller, 2015
All rights reserved.
The Dissertation of Eric Paul Miller is approved and it is acceptable in quality and
form for publication on microfilm and electronically.

Chair

University of California, San Diego
San Diego State University
2015
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DEDICATION

To my family

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TABLE OF CONTENTS
SIGNATURE PAGE ......................................................................................................... iii
DEDICATION ................................................................................................................... iv
TABLE OF CONTENTS .....................................................................................................v
LIST OF FIGURES .............................................................................................................x
LIST OF TABLES ......................................................................................................... xviii
ACKNOWLEDGEMENTS ...............................................................................................xx
VITA ............................................................................................................................... xxii
ABSTRACT OF THE DISSERTATION ...................................................................... xxiii
I. Cell Surface Binding, Uptake, and Storage of Iron in Brown Algae ...............................1
I.1. Introduction .................................................................................................................. 1
I.2. Cell Surface Binding, Uptake, and Storage of Iron in Ectocarpus siliculosus ............. 7
I.2.1. Cell Surface Binding .......................................................................................... 7
I.2.1.1. Results ......................................................................................................... 7
I.2.1.1.1. Kinetics and thermodynamics of surface-bound iron ............................7
I.2.1.1.2. Localization of iron on the surface ......................................................11
I.2.1.1.3. Nature of the surface bound iron .........................................................13
I.2.1.2. Discussion .................................................................................................. 18
I.2.1.2. Methods ..................................................................................................... 22
I.2.1.2.1. Algal culture .........................................................................................22
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I.2.1.2.2. Time-independent cellular iron ............................................................22
I.2.1.2.3. Cell surface-EDTA binding competition .............................................23
I.2.1.2.4. Calculation of the cell surface effective binding constant, K’ ..........24
eff
I.2.1.2.5. 55Fe pulse/pulse-chase ..........................................................................30
I.2.1.2.6. Ferrocyanide-diaminobenzidine (Perls-DAB) staining .......................31
I.2.1.2.7. Energy-dispersive X-ray spectroscopy (EDS) .....................................32
I.2.1.2.8. Transmission Mössbauer Spectroscopy ...............................................33
I.2.1.2.9. Extended X-ray absorption fine structure (EXAFS) ............................34
I.2.2. Iron Uptake in Ectocarpus siliculosus ............................................................. 35
I.2.2.1. Results and Discussion .............................................................................. 35
I.2.2.2. Discussion .................................................................................................. 40
I.2.2.3. Methods ..................................................................................................... 43
I.2.2.3.1. Algal culture .........................................................................................43
I.2.2.3.2. Time-dependent cell-associated iron ...................................................44
I.2.2.3.3. Concentration-dependent iron uptake ..................................................45
I.2.2.3.4. Cell surface reductase activity .............................................................46
I.2.2.3.5. Metabolic and reductive inhibition ......................................................47
I.2.3. Iron Storage in Ectocarpus siliculosus ............................................................. 48
I.2.3.1. Results ....................................................................................................... 48
vi
I.2.3.2. Discussion .................................................................................................. 56
I.2.3.3. Methods ..................................................................................................... 58
I.2.3.3.1. Ferrocyanide-diaminobenzidine (Perls-DAB) staining .......................58
I.2.3.3.2. Transmission Mössbauer spectroscopy (TMS) ....................................59
I.2.3.3.3. Extended X-ray absorption fine structure (EXAFS) ............................60
I.3. Cell Surface-Binding and Iron Storage in Macrocystis pyrifera ................................ 61
I.3.1. Cell Surface-binding of Iron in Macrocystis pyrifera ...................................... 61
I.3.1.1. Results ....................................................................................................... 61
I.3.1.1.1. Kinetics and thermodynamics of surface-bound iron ..........................61
I.3.1.1.2. Localization of iron on the surface ......................................................69
I.3.1.1.3. Nature of the surface bound iron. ........................................................74
I.3.1.2. Discussion .................................................................................................. 76
I.3.1.3. Methods ..................................................................................................... 79
I.3.1.3.1. Algal culture ........................................................................................... 79
I.3.1.3.2. Time-dependent and age-dependent surface binding studies ..............80
I.3.1.3.3. Ferrocyanide-diaminiobenzidine (Perls-DAB) staining ......................81
I.3.1.3.4. 7-(4-methylpiperazin-1-yl)-4-nitrobenzo-2-oxa-1,3-diazole . .............81
I.3.1.3.5. Energy-dispersive X-ray spectroscopy (EDS) .....................................84
I.3.1.3.6. Enzymatic digestion of alginate in Macrocystis blade ........................85
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I.3.1.3.7. EDTA-cell surface binding competition ..............................................86
I.3.1.3.8. Calculation of the cell surface effective binding constant, K’ ..........87
eff
I.3.1.3.9. Transmission Mössbauer spectroscopy (TMS) ....................................92
I.3.2. Iron Storage in Macrocystis pyrifera ............................................................... 93
I.3.2.1. Results ....................................................................................................... 93
I.3.2.2. Discussion .................................................................................................. 96
I.3.2.3. Methods ..................................................................................................... 97
I.3.2.3.1. 7-(4-methylpiperazin-1-yl)-4-nitrobenzo-2-oxa-1,3-diazole . .............97
I.3.2.3.2. Transmission Mössbauer spectroscopy (TMS) ....................................98
I.4. Appendix .................................................................................................................... 99
Titanium(III)-citrate-EDTA reagent ...................................................................... 99
I.5. Acknowledgements .................................................................................................... 99
II. Boron Uptake, Localization, and Speciation in Ectocarpus siliculosus and Macrocystis
pyrifera .............................................................................................................................101
II.1. Introduction ............................................................................................................. 101
II.2. Results ..................................................................................................................... 103
II.2.1. Boron Uptake ................................................................................................ 103
II.2.2. Boron Localization ........................................................................................ 107
II.2.3. Boron Speciation ........................................................................................... 117
II.3. Discussion ............................................................................................................... 120
viii
II.4. Methods ................................................................................................................... 124
II.4.1. Algal Culture ................................................................................................. 124
II.4.2. Time-dependent boron uptake ...................................................................... 125
II.4.3. Concentration-dependent boron uptake ........................................................ 126
II.4.4. B-FITC histochemistry ................................................................................. 127
II.4.5. Energy-dispersive X-ray spectroscopy (EDS) .............................................. 127
II.4.5. Raman microspectroscopy ............................................................................ 128
II.4.6. 11B Nuclear magnetic resonance (NMR) ...................................................... 128
II.4. Acknowledgements ................................................................................................. 129
III. References ..................................................................................................................130

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