kinetics of iron uptake by the freshwater cyanobacterium
TRANSCRIPT
Kinetics of Iron Uptake by the
Freshwater Cyanobacterium
Microcystis aeruginosa
by
The Cuong Dang
A thesis submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
School of Civil and Environmental Engineering
The University of New South Wales
August, 2012
THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet
Surname or Family name: Dang
First name: The Cuong
Other name/s:
Abbreviation for degree as given in the University calendar: Ph.D.
School: Civil and Environmental Engineering
Faculty: Engineering
Title: Kinetics of Iron Uptake by the Freshwater Cyanobacterium Microcystis aeruginosa
Abstract
Kinetics of iron (Fe) uptake by the freshwater cyanobacterium Microcystis aeruginosa cultured under a variety of growth conditions are examined in this thesis. Visible light was observed to induce reductive dissociation of Fe(III) bound to ethylenediaminetetraacetic acid (EDTA) and to dramatically increase the short-term uptake rate of Fe by M. aeruginosa. A mathematical model based on photo-generated unchelated Fe(II) uptake by concentration gradient dependent passive diffusion of Fe(II) through outer-membrane channels adequately described the rate and extent of Fe uptake. Studies of the kinetics of Fe transport to periplasmic and cytoplasmic compartments of M. aeruginosa indicated that a Monod-type relationship exists between cytoplasmic Fe accumulation rates and steady-state concentrations of unchelated Fe in the periplasm and extracellular milieu, suggesting that translocation of Fe into the cytoplasm involves complexation of Fe by a limited number of Fe-binding sites in the periplasm followed by subsequent transport into the cytoplasm, possibly via energy-dependent plasma-membrane Fe transporters. Fe uptake kinetics were also examined in Fraquil
* medium containing a natural organic ligand, Suwannee River Fulvic Acid (SRFA). Reduced Fe
uptake rates in the presence of ferrozine and superoxide dismutase under both light and dark conditions indicated that approximately a quarter to a half of the total Fe uptake was accounted for by Fe(II) uptake likely produced via light-, SRFA- or superoxide-mediated reduction of Fe(III) bound to SRFA. To further investigate cellular characteristics under various levels of Fe stress, a chemostat system made of metal-free materials was developed and used to maintain Fe-limited cultures in nutrient-insufficient and replete Fraquil*. In the nutrient-insufficient case, Fe uptake rate was lower for cells grown under conditions of lower Fe availability, suggesting cells grown under severe Fe stress and other nutrients insufficiency are likely unable to synthesize sufficient resources required for Fe uptake. In contrast, reversion to the expected relationship between Fe uptake capacity and the degree of Fe-limitation was observed when cells were grown under nutrient-replete Fe limitation. A kinetic model describing Fe transformations and biological uptake was applied to determine the biologically available form of Fe in the continuous culture.
Declaration relating to disposition of project thesis/dissertation
I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only).
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THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS
The Cuong Dang 28 November 2012
ii
ORIGINALITY STATEMENT
‘I hereby declare that this submission is my own work and to the best of
my knowledge it contains no materials previously published or written
by another person, or substantial proportions of material which have
been accepted for the award of any other degree or diploma at UNSW or
any other educational institution, except where due acknowledgement is
made in the thesis. Any contribution made to the research by others,
with whom I have worked at UNSW or elsewhere, is explicitly
acknowledged in the thesis. I also declare that the intellectual content of
this thesis is the product of my own work, except to the extent that
assistance from others in the project's design and conception or in style,
presentation and linguistic expression is acknowledged.’
Signed ……………………………………………...........................
Date ......………………………………………….............................
The Cuong Dang
28 November 2012
iii
COPYRIGHT STATEMENT
‘I hereby grant the University of New South Wales or its agents the right
to archive and to make available my thesis or dissertation in whole or part
in the University libraries in all forms of media, now or here after known,
subject to the provisions of the Copyright Act 1968. I retain all proprietary
rights, such as patent rights. I also retain the right to use in future works
(such as articles or books) all or part of this thesis or dissertation.
I also authorise University Microfilms to use the 350 word abstract of my
thesis in Dissertation Abstract International (this is applicable to doctoral
theses only).
I have either used no substantial portions of copyright material in my
thesis or I have obtained permission to use copyright material; where
permission has not been granted I have applied/will apply for a partial
restriction of the digital copy of my thesis or dissertation.'
Signed ……………………………………………...........................
Date ......………………………………………….............................
AUTHENTICITY STATEMENT
‘I certify that the Library deposit digital copy is a direct equivalent of the
final officially approved version of my thesis. No emendation of content
has occurred and if there are any minor variations in formatting, they are
the result of the conversion to digital format.’
Signed ……………………………………………...........................
Date ......………………………………………….............................
The Cuong Dang
28 November 2012
The Cuong Dang
28 November 2012
iv
ACKNOWLEDGEMENTS
First and foremost I wish to express my deepest gratitude to my supervisor, Professor
David Waite, for his highly valuable supervision throughout my course of study. His
scientific spirit, knowledge, engagement and cheerfulness have extremely assisted me
in the completion of this thesis. Special thanks to my co-supervisor, Assistant
Professor Manabu Fujii, who encouraged and patiently guided me through the
dissertation process. I wish to express my sincerest appreciation and thanks to him for
his invaluable suggestions, discussions and help in the research work.
I gratefully acknowledge the support of the University of New South Wales through
the award of the University International Postgraduate Award.
Thanks to Dr. Gautam Chattopadhyay and Mr. Kelvin Ong for their assistance in my
laboratory work. Thanks also given to Ms. Pattie MacLaughlin and Mr. Patrick Vuong
for their administrative support.
I would like to take this opportunity to express my sincere appreciation and special
thanks to all my research group members. I just name a few here: Dr. Mark Bligh, Dr.
Andrew Kinsela, Dr. An Ninh Pham, Dr. Shikha Garg, Dr. Adele Johns, Dr. Chris
Miller, Ms. Anna Yeung, Mr. Daniel Boland, Mr. Di He, Ms. Tian Ma, Mr. Yongjia
Xin, Ms. Lam Ho, etc. I want to thank you all for your kind help, support, interest and
valuable hints.
I am very thankful to my friends: Mr. Khoa Vo, Mr. Thao Tran, Mr. Lam Dang, Mr.
Hoang Dao, Dr. Nhat Le and Ms. Trang Trinh for their support and encouragement.
Also, I am very grateful for the love, spiritual support and encouragement of my
parents, sisters and brothers throughout my study.
Finally, I feel a deep sense of gratitude to my wife, Ms. My Van La, for standing next
to me in life, understanding and encouraging me during the completion of this thesis
regardless of the 6,300 km between us.
v
ABSTRACT
Kinetics of iron (Fe) uptake by the freshwater cyanobacterium Microcystis aeruginosa
cultured under a variety of growth conditions are examined in this thesis. Visible light
was observed to induce reductive dissociation of Fe(III) bound to
ethylenediaminetetraacetic acid (EDTA) and to dramatically increase the short-term
uptake rate of Fe by M. aeruginosa. A mathematical model based on photo-generated
unchelated Fe(II) uptake by concentration gradient dependent passive diffusion of
Fe(II) through outer-membrane channels adequately described the rate and extent of Fe
uptake. Studies of the kinetics of Fe transport to periplasmic and cytoplasmic
compartments of M. aeruginosa indicated that a Monod-type relationship exists
between cytoplasmic Fe accumulation rates and steady-state concentrations of
unchelated Fe in the periplasm and extracellular environment, suggesting that
translocation of Fe into the cytoplasm involves complexation of Fe by a limited
number of Fe-binding sites in the periplasm followed by subsequent transport into the
cytoplasm, possibly via energy-dependent plasma-membrane Fe transporters. Fe
uptake kinetics were also examined in Fraquil* medium containing a natural organic
ligand, Suwannee River Fulvic Acid (SRFA). Reduced Fe uptake rates in the presence
of ferrozine and superoxide dismutase under both light and dark conditions indicated
that approximately a quarter to a half of the total Fe uptake was accounted for by Fe(II)
uptake likely produced via light-, SRFA- or superoxide-mediated reduction of Fe(III)
bound to SRFA. To further investigate cellular characteristics under various levels of
Fe stress, a chemostat system made of metal-free materials was developed and used to
maintain Fe-limited cultures in nutrient-insufficient and replete Fraquil*. In the
nutrient-insufficient case, Fe uptake rate was lower for cells grown under conditions of
lower Fe availability, suggesting cells grown under severe Fe stress and other nutrients
insufficiency are likely unable to synthesize sufficient resources required for Fe
uptake. In contrast, reversion to the expected relationship between Fe uptake capacity
and the degree of Fe-limitation was observed when cells were grown under nutrient-
replete Fe limitation. A kinetic model describing Fe transformations and biological
uptake was applied to determine the biologically available form of Fe in the
continuous culture.
vi
PUBLICATIONS
Journal papers
FUJII, M., DANG, T. C., ROSE, A. L., OMURA, T. & WAITE, T. D. 2011. Effect of
light on iron uptake by the freshwater cyanobacterium Microcystis aeruginosa.
Environmental Science & Technology, 45, 1391-1398
DANG, T. C., FUJII, M., ROSE, A. L., BLIGH, M. & WAITE, T. D. 2012.
Characteristics of the freshwater cyanobacterium Microcystis aeruginosa grown in
iron-limited continuous culture. Applied and Environmental Microbiology, 78, 1574-
1583.
FUJII, M., DANG, T. C., ROSE, A. L. & WAITE, T. D. Kinetics of extracellular iron
transport to periplasmic and cytoplasmic compartments of the freshwater
cyanobaterium Microcystis aeruginosa (under preparation for re-submission to Applied
and Environmental Microbiology).
FUJII, M., DANG, T. C., ROSE, A. L. & WAITE, T. D. Iron uptake kinetics by the
freshwater cyanobacterium Microcystis aeruginosa in the presence of Suwannee River
fulvic acid (under preparation for submission to Environmental Science &
Technology).
DANG, T. C., FUJII, M., ROSE, A. L., BLIGH, M. & WAITE, T. D. Characteristics
of the freshwater cyanobacterium Microcystis aeruginosa grown in iron-limited
continuous culture under nutrient-replete condition (under preparation for submission
to Applied and Environmental Microbiology).
Conference papers
DANG, T.C., FUJII, M., ROSE, A.L., BLIGH, M. & WAITE, T.D. 2012. Growth and
responses to iron stress of the freshwater cyanobacterium Microcystis aeruginosa in
both nutrient-insufficient and -replete continuous cultures. 2012 ASLO Aquatic
Sciences Meeting. July 8-13th
, 2012. Lake Biwa, Shiga, Japan.
vii
TABLE OF CONTENTS
Acknowledgements ...................................................................................................... iv
Abstract .......................................................................................................................... v
Publications .................................................................................................................. vi
Table of Contents ........................................................................................................ vii
List of Figures ............................................................................................................. xiii
List of Tables .......................................................................................................... xxviii
Chapter 1 ....................................................................................................................... 1
Introduction ................................................................................................................... 1
1.1. Background to the study ...................................................................................... 2
1.1.1. Importance of Iron in Natural Waters towards Cyanobateria ....................... 2
1.1.2. Transformations of Iron in Natural Waters ................................................... 3
1.1.3. Iron Uptake Models by Phytoplankton ......................................................... 4
1.1.4. Mode of Iron Acquisition by the Freshwater Cyanobacterium M.
Aeruginosa and Knowledge Gaps ........................................................................... 7
1.2. Objectives ............................................................................................................ 9
1.3. Layout of Thesis .................................................................................................. 9
Chapter 2 ..................................................................................................................... 12
General Methodology ................................................................................................. 12
2.1. Reagents ............................................................................................................. 13
2.2. Culturing Conditions .......................................................................................... 13
viii
2.2.1. Culture Medium .......................................................................................... 13
2.2.2. Long-term Culturing Conditions ................................................................. 14
2.2.3. Continuous Culturing Apparatus ................................................................ 15
2.3. Short-term Iron Uptake Experiment .................................................................. 17
2.4. Measurement of Iron .......................................................................................... 18
2.4.1. Measurement of FeIIFZ3 with Spectrophotometer ...................................... 18
2.4.2. Measurement of Radio-labeled 55
Fe with Scintillation Counter ................. 19
2.5. Measurement of Cellular Iron Quota ................................................................. 19
2.5.1. Acid Digestion Combined with Spectrophotometry Method ..................... 19
2.5.2. Radiometry Method .................................................................................... 20
2.6. Model Fitting ..................................................................................................... 20
2.7. Analytical Quality Control ................................................................................. 20
2.7.1. Procedural Blank ......................................................................................... 20
2.7.2. Replication .................................................................................................. 20
Chapter 3 ..................................................................................................................... 21
Effect of Light on Iron Uptake by the Freshwater Cyanobacterium Microcystis
aeruginosa .................................................................................................................... 21
3.1. Introduction ........................................................................................................ 22
3.2. Materials and Methods ....................................................................................... 23
3.2.1. Materials ..................................................................................................... 23
3.2.2. Light Conditions ......................................................................................... 24
3.2.3. Photochemical Experiments ........................................................................ 28
ix
3.2.4. Short-term 55
Fe Uptake Experiments .......................................................... 28
3.3. Results and Discussion ...................................................................................... 29
3.3.1. Effect of Light on Photoreductive Dissociation and Fe Uptake .................. 29
3.3.2. Effect of Light Wavelength ........................................................................ 33
3.3.3. Fe Substrate for Uptake .............................................................................. 37
3.3.4. Kinetic Model for Fe Species ..................................................................... 39
3.3.5. Fe Uptake Machinery .................................................................................. 43
3.4. Implication of Findings ...................................................................................... 46
Chapter 4 ..................................................................................................................... 48
Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic
Compartments of the Freshwater Cyanobacterium Microcystis aeruginosa ......... 48
4.1. Introduction ........................................................................................................ 49
4.2. Materials and Methods ....................................................................................... 51
4.2.1. Reagents ...................................................................................................... 51
4.2.2. 55
Fe Accumulation Experiments ................................................................. 52
4.2.3. Determination of Periplasmic Fe(II) ........................................................... 54
4.2.4. Determination of Steady-state Concentration of Extracellular Unchelated
Fe ........................................................................................................................... 54
4.2.5. Analysis of Genome Sequences .................................................................. 55
4.3. Results and Discussion ...................................................................................... 55
4.3.1. Accumulation of 55
Fe in the Periplasm and Cytoplasm .............................. 55
4.3.2. Fe Species Translocated from the External Environment to the Periplasm 60
x
4.3.3. Fe Species Translocated from the Periplasm to the Cytoplasm .................. 61
4.3.4. Model for Translocation of Fe from the External Environment ................. 62
4.3.5. Fe Redox Speciation in Periplasm .............................................................. 66
4.4. Conclusions ........................................................................................................ 70
Chapter 5 ..................................................................................................................... 71
Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa
in the Presence of Suwannee River Fulvic Acid ....................................................... 71
5.1. Introduction ........................................................................................................ 72
5.2. Materials and Methods ....................................................................................... 74
5.2.1. Reagents ...................................................................................................... 74
5.2.2. Culturing Media .......................................................................................... 75
5.2.3. Long-term Culturing Conditions ................................................................. 76
5.2.4. Light Condition ........................................................................................... 76
5.2.5. Short-term 55
Fe Uptake Experiments .......................................................... 77
5.2.6. Kinetic Model for Fe Transformation and Uptake ..................................... 78
5.3. Results and Discussion ...................................................................................... 79
5.3.1. 55
Fe Uptake as a Function of SRFA Concentration .................................... 79
5.3.2. Effect of Chemical Treatment on 55
Fe Uptake ........................................... 82
5.3.3. Mode of Dark Fe Uptake ............................................................................ 85
5.3.4. Mode of Light-mediated Fe Uptake ............................................................ 89
5.4. Implications of Findings .................................................................................... 91
Chapter 6 ..................................................................................................................... 93
xi
Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown
in Iron-limited Continuous Culture .......................................................................... 93
6.1. Introduction ........................................................................................................ 94
6.2. Materials and Methods ....................................................................................... 95
6.2.1. Materials ..................................................................................................... 95
6.2.2. Culturing Method ........................................................................................ 95
6.2.3. Chemostat Apparatus .................................................................................. 96
6.2.4. Cellular Fe Quota and External Fe Concentration ...................................... 96
6.2.5. Short-term 55
Fe and 14
C Uptake .................................................................. 97
6.2.6. Kinetic Model for Unchelated Fe(II) Calculation ....................................... 98
6.2.7. Modified Chemostat Theory ..................................................................... 101
6.3. Results and Discussion .................................................................................... 105
6.3.1. Growth Kinetics in Batch Culture ............................................................. 105
6.3.2. Performance of Chemostat System under Fe Limitation .......................... 108
6.3.3. Cellular Fe Quota ...................................................................................... 112
6.3.4. Fe Uptake Kinetics .................................................................................... 115
6.3.5. Cellular Response to Fe Limitation in Chemostat .................................... 120
6.3.6. Characteristics of Iron-limited Cultures of M. aeruginosa Grown
Continuously in Nutrient-replete Fraquil* Medium ............................................ 123
6.4. Conclusions ...................................................................................................... 140
Chapter 7 ................................................................................................................... 142
Conclusions and Recommendations ........................................................................ 142
xii
7.1. Conclusions ...................................................................................................... 143
7.2. Implications of the findings ............................................................................. 147
7.2.1. With Regard to Knowledge of Fe Transformation and Uptake Kinetics by
Freshwater Cyanobacteria in Natural Waters ..................................................... 147
7.2.2. With Regard to Application of the Continuous Culturing System for Study
of Trace Metal Interactions with Freshwater Phytoplankton .............................. 147
7.2.3. With Regard to Knowledge of the Composition of the Growth Medium for
Freshwater Phytoplankton .................................................................................. 148
7.3. Recommendations for Future Work ................................................................. 148
References .................................................................................................................. 150
Appendix 1 ................................................................................................................. 170
Appendix 2 ................................................................................................................. 193
Appendix 3 ................................................................................................................. 201
xiii
LIST OF FIGURES
Figure 1.1. Transformations between Fe(II) and Fe(III) species in oxygenated
natural waters (Rose and Waite, 2003c).
Figure 1.2. The Fe(II)s and FeL models of Fe acquisition by phytoplankton.
The most significant difference between the two models is that the FeL model
excludes the unchelated Fe(III) in the medium as an important source of Fe(II)
for phytoplankton uptake (adapted from Morel et al., 2008).
Figure 1.3. Kinetic model for iron uptake by C. marina with Fe(III) reduction
to Fe(II) occurring by either non-reductive dissociation (NRD) or superoxide-
mediated non-dissociative reduction (NDR) or dissociative reduction (DR). In
this model superoxide plays an important role in the reduction of Fe(III) into
the more soluble form Fe(II) for uptake by marine phytoplankton (adapted from
Garg et al., 2007).
Figure 1.4. Iron uptake model for the freshwater cyanobacterium M.
aeruginosa in Fraquil* medium buffered by the model ligand EDTA in the
absence of light (Fujii et al., 2010a)
Figure 2.1. The chemostat culturing system consisting of non-metal materials.
The system was operated at four different dilution rates in triplicate.
Figure 3.1. Irradiation spectra emitted from the cool-white fluorescent tube of
the culturing incubator in the (A) absence and (B-H) presence of light filter
treatments. The spectra were measured using an Ocean Optics USB 4000
spectrophotometer equipped with an optical fiber and cosine corrector lens
(CC-3-UV) calibrated against a DH-2000 VIS-light source (hydrogen lamp).
The measurement was performed using SpectraSuite software in absolute
irradiation mode. Various light spectra were obtained by placing the cut-off
light filter between the light source and the irradiance probe. The photon flux
4
5
6
8
16
25
xiv
densities calculated for each wavelength range are shown in Table 3.1
Figure 3.2. Absorbance spectra for plastic and glass vessels. (A) blank (no
materials), (B) 1 cm quartz spectrophotometer cuvette (Starna Pty Ltd,
Australia), (C) Scintillation glass vials (20 mL, Crown Scientific), (D) 1 cm
polystyrene spectrophotometer cuvette (Starna Pty Ltd, Australia), (E)
polycarbonate container (250 mL, Nalgene), (F) high-clarity polypropylene
tube (15 mL, BD Falcon), (G) polypropylene microtube (1.5 mL, Eppendorf),
(H) high-density polyethylene bottle (125 mL, Nalgene). The absorbance
spectra were measured using a Varian Cary 50 UV-Vis spectrophotometer
(Scan mode). During measurement, the containers were filled with ultrapure
water (Milli-Q water). For large materials, the sample holder was removed
from the instrument and the materials were placed between the light source and
detector.
Figure 3.3. Effect of light on Fe(II)' formation and 55
Fe uptake by M.
aeruginosa. Time-courses of (A) FeIIFZ3 formation ([Fe]T = 0, 1 or 10 µM,
[EDTA]T = 26 µM, [FZ]T = 1 mM) and (B) 55
Fe uptake ([55
Fe]T = 200 nM and
[EDTA]T = 26 µM). Effect of light wavelength on (C) 55
FeEDTA uptake
([55
Fe]T = 200 nM, [EDTA]T = 26 µM) and (D) FeIIFZ3 formation ([Fe]T = 10
µM, [EDTA]T = 26 µM, [FZ]T = 1 mM). The incubations were performed in
modified Fraquil* (pH 8) in the light or dark at 27
oC. In the light filter
treatments (panels C and D), filters were placed between the incubated samples
and the light source to allow transmission of wavelengths longer than 400, 450,
500, 550, 600, 650 or 700 nm. In the control treatment, no light filter was
inserted in front of the sample. Incubations were performed for 4 h for the
photo-reduction experiment and 2 h for the 55
Fe uptake experiment. Asterisks
indicate that light filter treatments were significantly different from the control
at the p < 0.05 level using a single-tailed heteroscedastic t-test. Symbols and
error bars represent average data ±standard deviation from duplicate (photo-
reduction) or triplicate (55
Fe uptake) experiments. Solid lines represent linear
regression.
27
31
xv
Figure 3.4. Relationships between (A) FeIIFZ3 formation rate and
55Fe uptake
and (B) total photon flux density and 55
Fe uptake rate. At each data point, the
parameters were obtained from the incubation experiments and measurements
of irradiation spectra using the same cutoff filter. Thus, the data for 55
Fe uptake
and FeIIFZ3 formation rate are the same as those shown in parts C and D of
Figure 3.3. Details of total photon flux density are listed in Table 3.1.
Figure 3.5. UV-VIS absorbance spectra for FeIII
EDTA complex (solid line,
[Fe(III)]T = 0.5 mM and [EDTA]T = 1.3 mM) and EDTA (dotted line, [EDTA]T
= 1.3 mM) at pH 8 buffered by 15 mM NaHCO3. Enlarged absorbance spectra
in the visible light range are also shown. The average molar absorptivity of
FeIII
EDTA complex in the wavelength range from 400 nm to 500 nm was
determined to be 37 M-1
cm-1
.
Figure 3.6. Effect of (A) ferrozine (FZ) and (B) excess EDTA on 55
Fe uptake
rate in the light. The 55
Fe uptake experiment was undertaken by incubating cells
in Fraquil* containing pre-equilibrated
55Fe
IIIEDTA complex and FZ or excess
EDTA ([Fe]T = 200 nM, [EDTA]T = 26-260 µM and [FZ]T = 1 mM). While
various cell densities (2.6 × 105 – 2.7 × 10
7 cell.mL-
1) were used in the FZ
experiment, the cell density was kept constant (3.5 × 106 cell.mL-
1) in the
excess EDTA experiment. One and two asterisks indicate that treatments with
FZ or excess EDTA were significantly different from the control ([Fe]T = 200
nM and [EDTA]T = 26 µM) at p < 0.05 and p < 0.01 levels, respectively, using
a single-tailed heteroscedastic t-test. Symbols and error bars are average data
and errors represent ±standard deviation from triplicate experiments.
Figure 3.7. Fe uptake model by M. aeruginosa in the presence of light.
Unchelated Fe(II) (i.e., Fe(II)') is formed from the photoreductive dissociation
of ferric EDTA complex (FeIII
EDTA). The photoproduced Fe(II) subsequently
passes through the nonspecific outer membrane channel (porins) by diffusion.
However, cellular Fe uptake competes with Fe(II)' complexation by
extracellular Fe-binding ligands such as ferrozine (FZ) and excess EDTA if
present at appropriate concentrations. Solid arrows represent major reactions
34
35
38
39
xvi
under conditions of the short-term 55
Fe uptake experiment, whereas dotted
arrows indicate relatively minor reactions. Rate constants depicted near the
arrows correspond to those listed in Table 3.3.
Figure 3.8. Effect of competitive ligand concentrations and cellular densities
on calculated Fe uptake rate using eq 3.2.
Figure 4.1. Time course of 55
Fe accumulation in (A) periplasm and (B)
cytoplasm for M. aeruginosa strains PCC7806 (filled symbols) and PCC7005
(open symbols) grown under moderate Fe limitation. Fe uptake assays were
performed for 9 h in Fraquil* at concentrations of 0.7 µM for
55Fe and 20 µM
for citrate. Symbols and error bars represent the mean and ± standard deviation
from triplicate experiments. Solid and dashed lines represent the calculated
values for PCC7806 and PCC7005, respectively, using (A) eq. 4.7 and (B) the
integrated form of eq. 4.5 with Fe uptake parameters listed in Table 4.1.
Detailed 55
Fe accumulation data are provided in Table A2.1 of Appendix 2.
Figure 4.2. Cytoplasmic accumulation rate of 55
Fe by Fe-limited M. aeruginosa
strains PCC7806 (filled symbols) and PCC7005 (open symbols) as a function
of (A) steady-state concentration of total periplasmic 55
Fe and (B) calculated
concentration of unchelated Fe in the extracellular environment and periplasm.
Data were obtained from the assay using Fraquil* at concentrations of 0.7 µM
for 55
Fe and 5-200 µM for citrate. Solid and dashed lines in panel A were
determined for PCC7806 and PCC7005, respectively, by linear regression
analysis (p<0.05, n=15). In panel B, the solid and dashed lines represent the
calculated values using eq. 4.6 for PCC7806 and PCC7005, respectively.
Detailed 55
Fe accumulation data are provided in Table A2.2 of Appendix 2.
Calculated values of unchelated Fe concentrations are provided in Table A2.3
of Appendix 2. Symbols and error bars represent the mean and ± standard
deviation from triplicate experiments.
Figure 4.3. Cellular 55
Fe accumulation in the various culturing media at pH 8
(plasmolysis solutions, Fraquil* and 2 mM NaHCO3). Plasmolysis solutions
44
56
57
58
xvii
were 0.5 M D-sorbitol, sucrose or NaCl (buffered by 10 mM Tris-HCl, 2 mM
NaHCO3 and 1 mM for Na2EDTA) and 0.5M D-sorbitol with high EDTA
concentration (10 mM). The incubation experiment was initiated by addition of
55FeEDTA to the culture media at final concentrations of 0.7 µM for
55Fe and 2
mM for EDTA. In case of the 0.5M D-sorbitol solution containing high EDTA,
the final concentration of EDTA was adjusted to 11 mM. All incubations were
performed for 30 or 60 min in the dark at pH 8 with Fe-limited Microcystis
aeruginosa (PCC7806).
Figure 4.4. Kinetic model for Fe transport from the extracellular environment
to the intracellular environment in cyanobacteria. In the extracellular
environment, unchelated Fe (i.e., Fe′) is formed due to the (thermal or
reductive) dissociation of chelated Fe. Unchelated Fe subsequently diffuses
through non-specific outer membrane channels (such as porins). Unchelated Fe
in the periplasm is then complexed by one or more periplasmic Fe-binding
ligands (FeXperi) followed by translocation of Fe into the cytoplasm (Fecyto) by
inner membrane Fe transporters. A possible mechanism of Fe(III) and Fe(II)
transformation in the periplasm is also illustrated. Solid arrows represent major
reactions considered in the model. Rate constants depicted near the arrows
correspond to those listed in Table 4.1. MCO: multi-copper oxidase, FeoB:
ferrous iron transporter, FutA: ferric iron transporter.
Figure 4.5. Effect of ascorbate and TTM on Fe(II) accumulation in the
periplasm of M. aeruginosa PCC7806; (A) oxidation kinetics of Fe(II) in the
periplasmic extract, and (B) percentage of Fe(II) extracted from the periplasm.
PCC7806 was incubated in Fraquil* (0.7 µM Fe and 100 µM citrate) in the
presence and absence of chemical treatments (1 mM ascorbate and 1 mM
ascorbate plus 100 µM TTM). The periplasm was extracted by the cold osmotic
shock method in cold Milli-Q water followed by measurement of Fe(II) in the
extract by the luminol chemiluminescence technique. The amount of
periplasmic Fe(II) was calculated by assuming that the observed maximum
value of the chemiluminescence signal corresponds to the amount of Fe(II) in
the periplasm. Error bars represent ±standard deviation from duplicate
63
67
xviii
experiments. A single-tailed heteroscedastic t-test indicated that the treatments
with ascorbate + TTM were different from the control at a p value of 0.14.
Figure 4.6. Effect of chemical treatments on cellular 55
Fe accumulation for M.
aeruginosa PCC7806. In the control, cells were incubated for 3 hr in Fraquil*
containing 55
Fe-citrate (total concentrations for Fe and citrate were 0.7 µM and
100 µM, respectively). In the chemical treatments, cells were incubated in the
additional presence of 100 µM TTM, 1 mM ascorbate and 1 mM ascorbate plus
100 µM TTM. Error bars represent ±standard deviation from duplicate
experiments. One asterisk indicates that chemical treatments were significantly
different from the control at a p value less than 0.05 using a single-tailed
heteroscedastic t-test.
Figure 5.1. Kinetic model for Fe chemical speciation and uptake by M.
aeruginosa.
Figure 5.2. 55
Fe uptake as a function of (A) SRFA and (B) model ligand
concentrations in the absence (black symbols and bars) and presence (white
symbols and bars) of light. The 55
Fe uptake assay was performed at
concentrations of 200 nM total Fe, 1-100 mg L-1
SRFA and 26-100 µM citrate
and EDTA. Solid and dotted lines indicate model fits to the data from the Bligh
and Rose model, respectively. Effect of (C) ferrozine (FZ) and (D) superoxide
dismutase (SOD) on 55
Fe uptake. In control treatments, Fe uptake assays were
undertaken under dark (black bar) and light (white bars) at concentrations of
200 nM for Fe and 1-25 mg L-1
for SRFA. In the chemical treatments, the
identical 55
Fe uptake assay was performed except for the additional presence of
either FZ or SOD under dark (gray bar) and light (shaded bar). (E) Effect of
reducing agents on 55
Fe uptake. The control treatments were undertaken under
dark (black bar) and light (white bars) (200 nM for Fe and 5 mg L-1
for SRFA).
Chemical treatments were performed, in addition, in the presence of FZ,
ascorbate (Asc), hydroxylamine hydrochloride (HH), xanthine/xanthine oxidase
(X/XO) or their combination. All short-term Fe uptake assays were performed
in Fraquil* for 2 h at cell density of ~2 × 10
6 cell mL
-1. Symbols and error bars
69
78
80
xix
represent averaged value and ±standard deviation from triplicate experiments.
In panels C-E, asterisks indicate that 55
Fe uptake rate in the presence of a
particular chemical treatment is significantly different from control at the levels
of p < 0.01 for **
and p < 0.05 for * using a single-tailed heteroscedastic t-test.
Figure 5.3. Simulated results for unchelated Fe concentrations (gray lines for
Fe(III) and black lines for Fe(II)) as a function of SRFA ligand concentration
by using the Rose (solid lines) and Bligh (dotted lines) models.
Figure 6.1. Model for Fe uptake by M. aeruginosa in the presence of light
(Adapted from Fujii et al. (2011a))
Figure 6.2. Growth curves in batch cultures of M. aeruginosa at different total
Fe concentrations in Fraquil*. Total Fe concentrations were varied from 10 nM
to 10 µM; all other media components were constant. Symbols represent the
mean and error bars represent the standard deviation from triplicate incubations
(filled diamonds = 10 nM [Fe]T, filled squares = 20 nM [Fe]T, filled triangles =
50 nM [Fe]T, open diamonds = 100 nM [Fe]T, open squares = 1 µM [Fe]T, and
crosses = 10 µM [Fe]T).
Figure 6.3. Relationship between specific growth rate µ (d-1
) and log
concentration of unchelated Fe(II)’ (where [Fe(II)’] is in molar (M) units) in
batch culture studies of M. aeruginosa. Non-linear regression analysis yielded a
half saturation constant for growth of 'S
K = 3.6 ± 0.32 fM (with respect to
Fe(II)’) and a maximum specific growth rate µmax = 0.80 ± 0.03 d-1
. Solid and
dotted lines represent the regression line and 95% confidential interval,
respectively. Symbols indicate data for experimentally determined growth rate
under different degrees of Fe limitation.
Figure 6.4. Predicted and measured steady-state cell density and substrate
concentration in continuous cultures of M. aeruginosa as a function of dilution
rate with different total Fe concentrations in the inflowing medium (50 nM and
20 nM). Symbols represent data for steady-state cell density in Fraquil*
89
99
106
107
109
xx
medium with total Fe of 50 nM (circles) and 20 nM (triangles). Dotted lines are
the theoretical values of steady-state cell density calculated from eq. 6.16 with
growth parameters estimated from batch culture studies (µmax = 0.80 ± 0.03 d-1
,
'S
K = 3.6 ± 0.32 fM with respect to Fe(II)’, TSK = 26 ± 2.3 nM with respect to
total Fe, and Y = 8.1 ± 0.21 × 1016
cell (mol Fe)-1
), while bold lines indicate the
theoretical steady-state cell density estimated with parameters obtained from
continuous culture studies ( 'S
K = 3.4 ± 0.82 fM, TSK = 25 ± 5.0 nM and Y =
1.1 ± 0.2 × 1017
cell mol-1
), except for µmax (0.80 ± 0.03 d-1
) which was
obtained from the batch studies. Dashed and chained lines indicate predicted
steady-state unchelated Fe(II)’ concentrations estimated using parameters from
batch and continuous culture studies, respectively.
Figure 6.5. Growth of M. aeruginosa in the continuous culture system at
different dilution rates with total Fe concentrations in the inflowing Fraquil*
medium. (A) [Fe]T = 50 nM, with dilution rates of 0.07 d-1
(diamonds), 0.15 d-1
(squares), 0.30 d-1
(triangles) and 0.45 d-1
(circles). (B) [Fe]T = 20 nM, with
dilution rates of 0.09 d-1
(diamonds), 0.14 d-1
(squares), 0.17 d-1
(triangles) and
0.25 d-1
(circles). Symbols represent the mean and error bars the standard
deviation from triplicate incubations. Dashed lines represent the 95%
confidence interval at steady-state.
Figure 6.6. Time-course of cellular Fe quotas for Fe-limited M. aeruginosa in
the chemostat with [Fe]T = 20 nM as radiolabelled 55
Fe in the inflowing
medium and dilution rates of 0.09 d-1
(diamonds), 0.14 d-1
(squares), 0.17 d-1
(triangles) and 0.25 d-1
(circles). Symbols represent the mean and error bars the
standard deviation from triplicate incubations.
Figure 6.7. Relationship between the cellular Fe quota (Q) and the (A) specific
uptake rate of Fe (µQ) or (B) specific growth rate (µ) for Fe-limited M.
aeruginosa under steady-state conditions in continuous cultures. The system
was operated at four different dilution rates (0.09, 0.14, 0.17 and 0.25 d-1
) and
fed with Fraquil* medium containing 20 nM radiolabeled
55Fe. In panel (A),
linear regression analysis (represented by the bold line) yielded the maximum
110
113
114
xxi
“impossible” growth rate µ’max = 0.37 ± 0.04 d-1
and minimum cellular quota
Qmin = 1.2 ± 0.2 × 103 zmol cell
-1. Symbols represent the mean and error bars
the standard deviation from triplicate incubations. In panel (B), the solid line
represents the theoretical curve calculated from the Droop equation using the
obtained estimated values of µ’max and Qmin. Symbols represent the mean from
triplicate incubations.
Figure 6.8. Time-course of 55
Fe uptake during batch short-term Fe uptake
assays using cells obtained at steady-state from the chemostat cultures grown
with [Fe]T = 20 nM in the inflowing medium and dilution rates of 0.09 d-1
(diamonds), 0.14 d-1
(squares), 0.17 d-1
(triangles) and 0.25 d-1
(circles). In the
short-term uptake assay, each culture was incubated in Fraquil* with either (A)
20 µM EDTA or (B) 200 µM EDTA and constant concentration of radiolabeled
55Fe (200 nM). Symbols represent the mean and error bars the standard
deviation from triplicate experiments. The continuous lines were obtained by
linear regression of data collected within 4 h (represented by closed symbols)
for each culture.
Figure 6.9. Eadie-Hofstee plots demonstrating the linear relationship between
the short-term 55
Fe uptake rate (ρFe) and the ratio ρFe/[Fe(II)’] (d-1
M-1
) for
cultures of M. aeruginosa. Linear regression analysis yielded comparable half-
saturation constants for Fe uptake (Kρ = 18 ± 1.9 fM, as Fe(II)’) but different
maximum specific uptake rates (ρmax of 270, 720, 950 and 1,010 zmol cell-1
hr-1
for cultures grown at dilution rates of 0.09 d-1
(diamonds), 0.14 d-1
(squares),
0.17 d-1
(triangles) and 0.25 d-1
(circles), respectively). Lines for 95%
confidential intervals were omitted for clarity.
Figure 6.10. Time-course of 14
C uptake during batch short-term uptake assays
using cells obtained at steady-state from the chemostat cultures grown with
[Fe]T = 20 nM in the inflowing medium and dilution rates of 0.09 d-1
(diamonds), 0.14 d-1
(squares), 0.17 d-1
(triangles) and 0.25 d-1
(circles).
Symbols represent the mean and error bars the standard deviation from
triplicate experiments.
116
118
122
xxii
Figure 6.11. Growth curves in batch cultures of M. aeruginosa at a constant
total Fe concentration in different modified Fraquil* growth media. Total Fe
concentration and its chelator EDTA were fixed at 10 and 26 µM while other
media components were varied as shown in Table 6.3. Symbols represent the
mean and error bars represent the standard deviation from duplicate incubations
(filled diamonds: control (i.e., Fraquil*); filled squares: Test 1; filled triangles:
Test 2; open diamonds: Test 3; open squares: Test 4 and open triangles: Test 5).
Figure 6.12. Growth curves in batch cultures of M. aeruginosa at various total
Fe concentrations in the nutrient-replete Fraquil* medium (i.e., Test 2 medium).
Total Fe concentration was varied from 0.05 to 10 µM while concentration of
EDTA was fixed at 26 µM. Symbols represent the mean and error bars
represent the standard deviation from duplicate incubations (filled diamonds:
[Fe]T = 0.05 µM; filled squares: [Fe]T = 0.1 µM, filled triangles: [Fe]T = 0.2
µM, filled circles: [Fe]T = 0.5 µM, open diamonds: [Fe]T = 1.0 µM, open
squares: [Fe]T = 2.0 µM, open triangles: [Fe]T = 5.0 µM, and open circles: [Fe]T
= 10 µM).
Figure 6.13. Relationship between specific growth rate µ (d-1
) and log
concentration of unchelated Fe(II)’ (M) in batch culture studies of M.
aeruginosa grown in nutrient-replete Fraquil* medium. Solid and dotted lines
represent the regression line and 95% confidential interval, respectively.
Symbols indicate data for experimentally determined growth rate under
different degrees of Fe limitation.
Figure 6.14. Growth of M. aeruginosa in the continuous culture system at
different dilution rates with total Fe concentrations in the inflowing nutrient-
replete Fraquil* medium [Fe]T = 100 nM, with dilution rates of 0.07 d
-1
(diamonds), 0.15 d-1
(squares), 0.30 d-1
(triangles) and 0.45 d-1
(circles).
Symbols represent the mean and error bars the standard deviation from
triplicate incubations. Dashed lines represent the 95% confidence interval at
steady-state.
125
128
130
132
xxiii
Figure 6.15. Predicted and measured steady-state cell density and substrate
concentration in continuous cultures of M. aeruginosa as a function of dilution
rate with different total Fe concentrations in the two inflowing media: Fraquil*
(20 nM and 50 nM) and nutrient-replete Fraquil* (100nM). Symbols represent
data for steady-state cell density in Fraquil* medium with total Fe of 20 nM
(triangles), 50 nM (squares) and 100 nM (diamonds). Dotted lines are the
theoretical values of steady-state cell density calculated from eq. 6.16 with
growth parameters estimated from batch culture studies in Fraquil* (µmax = 0.80
± 0.03 d-1
, 'S
K = 3.6 ± 0.32 fM with respect to Fe(II)’, TSK = 26 ± 2.3 nM with
respect to total Fe, and Y = 8.1 ± 0.21 × 1016
cell (mol Fe)-1
), while bold lines
indicate the theoretical steady-state cell density estimated with parameters
obtained from batch culture studies in nutrient-replete Fraquil* (µmax = 0.89 ±
0.03 d-1
, 'S
K = 3.1 ± 0.30 fM, TSK = 23 ± 2.2 nM, and Y = 2.7 ± 0.74 × 10
17
cell (mol Fe-1
)). Chained and dashed lines indicate predicted steady-state
unchelated Fe(II)’ concentrations estimated using parameters from batch
culture studies in Fraquil* and nutrient-replete Fraquil
*, respectively.
Figure 6.16. Relationship between the cellular Fe quota (Q) and the specific
growth rate (µ) for Fe-limited M. aeruginosa under steady-state conditions in
continuous cultures. The system was operated at four different dilution rates
(0.07, 0.15, 0.30 and 0.45 d-1
) and fed with nutrient-replete Fraquil* medium
containing 100 nM Fe. The solid line represents the theoretical curve calculated
from the Droop equation using the obtained estimated values of µ’max = 0.69 ±
0.05 d-1
and Qmin = 18 ± 2.6 amol cell-1
. Symbols represent the mean from
triplicate measurements.
Figure 6.17. Time-course of 55
Fe uptake during batch short-term Fe uptake
assays using cells obtained at steady-state from the chemostat cultures grown
with [Fe]T = 100 nM in the inflowing nutrient-replete Fraquil* medium and
dilution rates of 0.07 d-1
(diamonds), 0.15 d-1
(squares), 0.30 d-1
(triangles) and
0.45 d-1
(circles). In the short-term uptake assay, each culture was incubated in
nutrient-replete Fraquil* with 20 µM EDTA and 200 nM radiolabeled
55Fe.
133
135
137
xxiv
Symbols represent the mean and error bars represent the standard deviation
from triplicate experiments. The continuous lines were obtained by linear
regression of data collected within 4 h (represented by closed symbols) for each
culture.
Figure 6.18. Eadie-Hofstee plots demonstrating the linear relationship between
the short-term 55
Fe uptake rate (ρFe) and the ratio ρFe/[Fe(II)’] for cultures of M.
aeruginosa. Linear regression analysis yielded comparable half-saturation
constants for Fe uptake (Kρ = 45 ± 1.9 fM, as Fe(II)’) but different maximum
specific uptake rates (ρmax of 1.0 ± 0.046, 0.89 ± 0.079, 0.67 ± 0.087 and 0.48 ±
0.035 amol cell-1
hr-1
for cultures grown at dilution rates of 0.07 d-1
(diamonds), 0.15 d-1
(squares), 0.30 d-1
(triangles) and 0.45 d-1
(circles),
respectively). Lines for 95% confidential intervals were omitted for clarity.
Figure A1.1. Time-course of FeIIFZ3 formation from Fe
IIIEDTA in (A and C)
the light and (B and D) dark. For measurement of photo-reduction rate of
FeIII
EDTA, pre-equilibrated FeIII
EDTA complex and FZ were mixed in
Fraquil* at concentrations of 1-10 µM for Fe(III), 26 µM for EDTA and 1 mM
for FZ, followed by incubation for several hours at 27oC in the presence and
absence of the light (157 µmol quanta.m-2
.s-1
). Photo-reductive dissociation rate
constants were determined by applying eq. A1-5 to the measurements with (E)
1 µM and (F) 10 µM total Fe. Symbols and error bars indicate average data and
±standard deviation from triplicate experiments. Solid lines represent linear
regression lines.
Figure A1.2. Bioavailability of pre-photolyzed 55
FeEDTA complex in the dark.
The x-axis represents the time for which 55
FeEDTA complex was exposed to
the light (157 µmol photons.m-2
.s-1
) before the commencement of the 55
Fe
uptake experiment. Immediately after irradiation, the photolyzed 55
FeEDTA
complex was added at final concentrations of 200 nM Fe and 26 µM EDTA to
the Fe and EDTA-free Fraquil* containing M. aeruginosa cells at a density of 3
× 106 cell.mL
-1. Cells were then incubated for 2 h in the dark at 27
oC. Values
shown represent the average and ±standard deviation from triplicate
139
172
174
xxv
experiments.
Figure A1.3. Kinetic data for the dissociation of FeIIEDTA complex in Fraquil
*
(pH 8); (A) time-dependent formation of FeIIFZ3 complex over a range of
[EDTA]T and (B) plots of time versus ln[Fe(II)]T/([Fe(II)]T-[FeIIFZ3]). The
value of koverall[FZ]T3 was determined as the slope of the line in the panel B.
Figure A3.1. Time-course of 55
Fe uptake under the dark (closed symbol) and
light (open symbol) conditions. 55
Fe uptake was measured by incubating cells
(at density of 1.6 × 106 cell.mL
-1) in Fraquil
* containing pre-equilibrated
55Fe
IIISRFA complex at 27
oC. Concentrations of Fe and SRFA were 200 nM
and 1 mg.L-1
, respectively. Symbols represent experimental data. Solid and
dotted lines were yielded by applying a linear regression analysis to the data
collected within 2 h under the dark and light conditions, respectively.
Figure A3.2. Comparison of measured 55
Fe uptake rate to calculated Fe(III)
uptake for M. aeruginosa PCC7806. 55
Fe uptake rates were determined in the
short-term incubational assay under the dark in modified Fraquil* containing
200 nM for Fe, 1, 5 and 25 mg L-1
for SRFA and 1 mM for FZ. In the model
calculations, steady-state concentrations for Fe(III)' were determined at the
concentration identical to those employed in the short-term assay by using rate
constants for complexation and dissociation for FeIII
SRFA complex published
by Rose (square), Jones (diamond) and Bligh (triangle). Fe(III) uptake rates
were then calculated by use of Monod-type equation with parameters listed in
Table 5.1. Solid line represents linear line with 1:1 slope.
Figure A3.3. Comparison of measured 55
Fe(II) uptake rate to calculated Fe(II)
uptake for M. aeruginosa PCC7806. Measured Fe(II) uptake rates in this figure
were determined by subtracting 55
Fe uptake rate in the presence of FZ from that
measured in the absence of FZ. The short-term incubational assays were
performed in the absence and presence of FZ under the dark in modified
Fraquil* containing 200 nM for Fe, 1, 5 and 25 mg L
-1 for SRFA and 1 mM for
FZ. In the model calculations, steady-state concentrations for Fe(II)' were
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202
203
204
xxvi
determined at the concentration identical to those employed in the short-term
assay by using rate constants for complexation and dissociation for FeIISRFA
complex published by Rose (square) and Bligh (triangle). Fe(II) uptake rates
were then calculated by use of Monod-type equation with parameters listed in
Table 5.1. Solid line represents linear line with 1:1 slope. Error bar indicates
standard deviation from duplicate experiments.
Figure A3.4. Comparison of calculated steady-state concentration of Fe(II)’
under the dark and light conditions. Steady-state concentrations for Fe(II)' were
determined by using rate constants for complexation and dissociation for
FeIISRFA complex published by Rose (square) and Bligh (triangle), the
photochemical and non-photochemical reduction of FeIII
SRFA complex and
oxidation of FeIISRFA complex. Solid line represents linear line with 1:1 slope.
Figure A3.5. Effect of pH on the 55
Fe uptake rate for M. aeruginosa PCC7806
under (A) dark and (B) light. Effects of FZ (gray bar) and SOD (white bar) on
55Fe uptake were also examined compared to control treatment (black bar)
where addition of FZ or SOD was omitted. Error bar indicates standard
deviation from triplicate experiments. Asterisks indicate that 55
Fe uptake rate in
the presence of chemical treatment is significantly different from control (55
Fe
uptake rate in the absence of FZ or SOD) for each pH at the levels of p < 0.01
for ** and p < 0.05 for * using a single-tailed heteroscedastic t-test.
Figure A3.6. Primary kinetic data of FeIIFZ3 formation in the (A) light and (B)
dark conditions. The time-dependent FeIIFZ3 formation in Fraquil
* (pH 8) was
spectrophotometrically monitored for 4 h at concentrations of 1 µM for Fe(III),
1 mM for FZ, 1 mg.L-1
for SRFA, 26 µM for EDTA and 100 µM for citrate.
Symbols and error bars indicate average data and ±standard deviation from
triplicate experiments.
Figure A3.7. Determination of rate constants for photo-reduction of Fe(III)-
ligand complexes in Fraquil* (pH 8). The experimental conditions, symbols and
error bars are identical to those in Figure A3.6, except that the data measured
205
206
209
212
xxvii
under the light were only shown. The solid lines represent linear regression
lines in each ligand system.
Figure A3.8. Effect of pH on reduction of FeIII
SRFA under the light and dark.
213
xxviii
LIST OF TABLES
Table 3.1. Photon flux density at each wavelength range.
Table 3.2. Parameters used for the determination of quantum yield.
Table 3.3. Kinetic model for Fe transformation and uptake under the light.
Table 3.4 Measured and calculated Fe uptake parameters for various culture
conditions.
Table 4.1. Kinetic parameters used for the calculation of intracellular Fe
transport and extracellular Fe transformation.
Table 5.1. Kinetic model and rate constants used in this study.
Table 6.1. Kinetic model for Fe transformation and uptake in the presence of
light by M. aeruginosa (adapted from Fujii et al. (2011a) and therein).
Table 6.2. Measured and calculated Fe uptake parameters under the conditions
of short-term 55
Fe uptake experiments for four different steady-state cultures of
M. aeruginosa.
Table 6.3. Compositions of the modified nutrient-replete Fraquil* media
examined in this study.
Table 6.4. Summary of the growth constants in the batch cultures and the
behaviors of the Fe-limited chemostat cultures at different dilution rates of M.
aeruginosa grown in both Fraquil* and nutrient-replete Fraquil
*.
Table A1.1. Formation rate constant of FeIIEDTA complex (kf-EDTA) in Fraquil
*
(pH 8).
26
36
41
42
59
83
100
119
126
141
177
xxix
Table A1.2. Dissociation rate constant of FeIIEDTA complex (kd-EDTA) in
Fraquil* (pH 8).
Table A1.3. Published values of porin properties for various Gram-negative
bacteria.
Table A1.4. Range of uptake rate constant (kup) calculated using published
parameters (a, porin radius; l, channel length; Nporin, porin density; D, diffusion
coefficient of metal ions; As, surface area of Microcystis aeruginosa PCC7806).
Table A2.1. Measured and modelled values for the time course of 55
Fe
accumulation in the periplasmic and cytoplasmic fractions.
Table A2.2. Measured and modelled values for the steady-state periplasmic 55
Fe
concentration and accumulation rate of cytoplasmic 55
Fe over a range of
Fe:citrate ratios.
Table A2.3. Calculated values of unchelated Fe concentrations in the
extracellular environment and periplasm.
Table A3.1. Reduction rate constants for organically complexed Fe(III) in
Fraquil* (pH 8).
Table A3.2. Reduction rate constants for organically complexed Fe(III) in
Fraquil* (pH 6-9).
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188
198
199
200
214
214
1
CHAPTER 1
INTRODUCTION
Chapter 1. Introduction
2
1.1. BACKGROUND TO THE STUDY
1.1.1. Importance of Iron in Natural Waters towards Cyanobateria
Iron (Fe) is one of the most essential micronutrients for almost all living organisms
because of its critical roles in various metabolic processes (Crichton, 2009).
Cyanobacteria in particular have a relatively high Fe requirement since Fe is needed
for the processes of photosynthetic and respiratory electron transfer and, in some cases,
nitrogen fixation (Straus, 1994). Therefore, growth of cyanobacteria is influenced
strongly by Fe availability (Wilhelm, 1995). In surface waters at circumneutral pH,
concentrations of ferrous iron (Fe[II]) and ferric iron (Fe[III]) in biologically available
unchelated inorganic forms are typically low due to rapid oxidation of Fe(II) (Rose and
Waite, 2003a) and strong complexation of Fe(III) (Kuma et al., 1996, Liu and Millero,
2002) by a range of naturally occurring ligands (Fujii et al., 2008a). When Fe is a
growth-limiting nutrient, photochemically and biologically mediated reduction of
Fe(III) to more soluble Fe(II) may become critical steps in increasing Fe availability
(Sunda, 2001, Salmon et al., 2006, Fujii et al., 2010a).
Occurrence of the bloom-forming freshwater cyanobacterium Microcystis aeruginosa
in lakes, reservoirs and slowly-flowing rivers poses serious social and ecological
concerns with excessive growth typically deteriorating water quality and jeopardizing
human and ecological health (DECC, 2005). Evidence exists that growth of this
organism can be limited by supply of the trace nutrient Fe (Nagai et al., 2006).
Additionally, Fe nutrition alters basal metabolic functions of the organism including
photosynthesis, respiration and nutrient uptake (Imai et al., 1999, Kosakowska et al.,
2007, Xing et al., 2007) as well as potentially inducing the biosynthesis of secondary
metabolites such as the potent hepatotoxin (microcystin), possibly to prevent cellular
damage from reactive oxygen species that are generated by oxidative stress (Alexova
et al., 2011). Recent studies suggested that iron limitation can induce an increase in
toxin generation of M. aeruginosa (Sevilla et al., 2008, Alexova et al., 2011). In fact,
Microcystis species can produce a wide range of secondary metabolites including
microcystins, aeruginosins, microginins, anabaenopeptins, cyanopeptolins,
microviridins and cyclamides (Welker and von Dohren, 2006) which can affect both
Chapter 1. Introduction
3
animals and human beings (Carmichael and Falconer, 1993). Microcystins, with a
molecular weight between 900-1100 daltons, are of particular concern as they are
potent hepatotoxins and are the most widespread of cyanotoxins in brackish and
freshwater environments (Sivonen and Jones, 1999, Welker and von Dohren, 2006).
In order to understand ecological functioning and adaptation of M. aeruginosa and to
provide insight into the management of aquatic environments to reduce both the
occurrence of the blooms and the toxicity potentially associated with these blooms, it
is necessary to thoroughly understand the Fe uptake mechanism of this organism.
1.1.2. Transformations of Iron in Natural Waters
In natural waters Fe occurs in five major forms including unchelated inorganic
complexes of Fe(II) and Fe(III), complexes with natural organic ligands (FeIIL and
FeIII
L) and Fe(oxy)hydroxides which may (or may not) be associated with other
(inorganic and organic) particles (Bruland and Rue, 2001). The major transformations
that occur between various iron species in natural waters are summarised in Figure 1.1
(Rose and Waite, 2003c). Ferric iron species are thermodynamically favourable in oxic
waters because of rapid oxidation of ferrous iron, particularly with dissolved oxygen at
circumneutral pH (King et al., 1995). Ferric irons tend to undergo rapid polymerisation
at low concentrations (reaction 2, Figure 1.1) to generate insoluble forms. However, in
the presence of natural organic matter (NOM) both Fe(II) and Fe(III) may additionally
form complexes with NOM, also maintaining the iron in dissolved form (reactions 4
and 6, Figure 1.1). Ferrous iron, either as inorganic ferrous species or bound to NOM,
can be returned to the ferric oxidation state by reaction with oxygen or by hydrogen
peroxide (reactions 1 and 3, Figure 1.1). In general, ferrous forms are more soluble
than Fe(III) due to the lower stability of complexes of Fe (II) with organic chelators.
Thus, the reduction of Fe(III) to Fe(II), which can be mediated by photochemical
(reaction 8, Figure 1.1) and biological processes, increases the concentration of labile
form of Fe and subsequent biological uptake.
Chapter 1. Introduction
4
Figure 1.1. Transformations between Fe(II) and Fe(III) species in oxygenated natural
waters (Rose and Waite, 2003c).
1.1.3. Iron Uptake Models by Phytoplankton
There have been various strategies to solubilise and acquire Fe by phytoplankton. The
siderophore-mediated iron uptake mechanism represents one of the most studied of
iron acquisitions. The ability of phytoplankton to synthesize siderophores during iron
limitation and actively transport them into cells has been known for some time (Estep
et al., 1975, Murphy et al., 1976, Simpson and Neilands, 1976, Wilhelm and Trick,
1994, Neilands, 1995, Martinez et al., 2001). Under iron-deplete conditions some
microorganisms have been shown to release siderophores (Greek: siderous = iron,
phorus = bearer) - low molecular weight, specific iron-binding compounds
(ferrisiderophore complex) - and to subsequently acquire iron specifically from these
strong ferric complexes. The presence of siderophores is expected to affect the cycling
of iron in the systems potentially increasing the pool of biologically available iron by
the amount of iron complexed to the siderophores.
Chapter 1. Introduction
5
Recently, numerous studies have focused on the extracellular reductive strategy of Fe
acquisition by phytoplankton. Following the Fe’ model (Hudson and Morel, 1990) in
which unchelated Fe(III) (Fe(III)’), Fe(OH)2+ , Fe(OH)3, and Fe(OH)4
- is considered as
the main substrate for uptake by marine phytoplankter, some studies reported that iron
bound in strong complexes can be taken up by some species and that both chelated
(FeIII
L) and unchelated Fe(III) (Fe(III)’) must be reduced prior to internalization
(Soriadengg and Horstmann, 1995, Maldonado and Price, 2001, Rose et al., 2005).
More recent models of Fe acquisition by marine phytoplankton have focused on the
role of Fe(II) in uptake including two well-known reduction mechanisms: the Fe(II)s
model (part A of Figure 1.2) described by Shaked et al. (2005) and the FeL model (part
B of Figure 1.2.) proposed by Salmon et al. (2006).
Figure 1.2. The Fe(II)s and FeL models of Fe acquisition by phytoplankton. The most
significant difference between the two models is that the FeL model excludes the
unchelated Fe(III) in the medium as an important source of Fe(II) for phytoplankton
uptake (adapted from Morel et al., 2008).
In the Fe(II)s model, both the chelated and unchelated Fe(III) serve as sources of
Fe(II)s at the cell surface for uptake where the parameter Fe(II)s represents both
chelated and unchelated Fe(II) which are formed by cell surface reductase at the cell
surface. Meanwhile, in the FeL model, the chelated Fe(III) serve as a sole source of
either unchelated or chelated Fe(II), for uptake. Generally, current models for Fe
acquisition suggest that unchelated Fe(II) and Fe(III) can be taken up by marine
microalgae and that reduction of both chelated and unchelated Fe(III) by either
Chapter 1. Introduction
6
photochemical and/or biological processes in the external medium or near the cell
surface prior to uptake is critical to the iron uptake process. The precise mode of
reduction may be organism specific with reduction occurring either in bulk solution
and driven by light or reduced extracellular metabolites or at the cell surface by
mediated by membrane-bound enzymes. It has also been suggested by Rose et al.
(2005) that membrane bound oxido-reductase enzymes may reduce oxygen to
superoxide which, in turn, could reduce Fe(III) to Fe(II). Garg et al. (2007)
investigated this mechanism further for the prolific superoxide producer Chattonella
marina and showed that the precise mode of Fe(III) reduction was dependent upon the
strength of any Fe(III)L complex present with both dissociative and non-dissociative
modes of superoxide-mediated Fe(III) reduction possible (Figure 1.3).
Figure 1.3. Kinetic model for iron uptake by C. marina with Fe(III) reduction to Fe(II)
occurring by either non-reductive dissociation (NRD) or superoxide-mediated non-
dissociative reduction (NDR) or dissociative reduction (DR). In this model superoxide
plays an important role in the reduction of Fe(III) into the more soluble form Fe(II) for
uptake by marine phytoplankton (adapted from Garg et al., 2007).
In conclusion, studies to date on iron uptake models for phytoplankton have shown
that not only the form of iron in the external medium but also the kinetics of
transformation between different species of iron, often mediated by the microorganism
Fe(II)'
Transporter
Fe(III)'
Transporter
kup2
kup1
kd2 kf2 kf1 kd1
FeIIIL
, kox2
, kox1
, kr1 FeIIL
FeIII FeII
−•2O
2O
2O
−•2O
Oxido-reductase
−•2O
2O
, kr2
NDR
DR
NRD
Chapter 1. Introduction
7
itself or by photochemical processes, are critical to bioavailability of iron and its rate
of acquisition by the microorganism.
1.1.4. Mode of Iron Acquisition by the Freshwater Cyanobacterium
M. aeruginosa and Knowledge Gaps
Although the modes of Fe acquisition by marine phytoplankton have been extensively
studied, little is known of the mode of Fe acquisition by freshwater phytoplankton. In
terms of the freshwater cyanobaterium M. aeruginosa, in contrast to many
cyanobacteria which can produce siderophores to facilitate Fe uptake (Murphy et al.,
1976, Simpson and Neilands, 1976, Kerry et al., 1988, Wilhelm and Trick, 1994),
excretion of siderophores to assist in acquiring Fe is not believed to be used by this
organism (Fujii et al., 2011b, Schleiff et al., 2008). A siderophore-independent iron
acquisition mechanism was also observed in Fe-limited cells of siderophore-forming
freshwater cyanobacterium Anabaena flos-aquae (Wirtz et al., 2010). Recently, a
kinetic model incorporating uptake of both unchelated Fe(II) and Fe(III) for this
organism under darkness (Figure 1.4) has been proposed (Fujii et al., 2010a). This
model suggested that in the presence of strong Fe chelators such as
ethylenediaminetetraacetate (EDTA), superoxide-mediated reductive dissociation of
organically-complexed Fe(III) with subsequent uptake of unchelated Fe(II) was a
significant pathway for Fe uptake. In contrast, assimilation of unchelated Fe(III) was
favoured in the presence of the weak Fe-binding ligand citrate (≤ 100 µM).
Chapter 1. Introduction
8
Figure 1.4. Iron uptake model for the freshwater cyanobacterium M. aeruginosa in
Fraquil* medium buffered by the model ligand EDTA in the absence of light (Fujii et
al., 2010a)
While the mode of Fe acquisition in batch cultures of M. aeruginosa in Fraquil*
medium buffered by the model ligand EDTA in the absence of light has apparently
been described satisfactorily, a variety of important questions relating to iron uptake
by M. aeruginosa remain including:
(i) Does light, which is recognized to induce a net increase in the more soluble
and bioavailable form – extracellular unchelated Fe(II) - facilitate Fe uptake
by M. aeruginosa?
(ii) How is Fe transported from the extracellular medium into the periplasmic
and cytoplasmic compartments of M. aeruginosa?
(iii) Is the Fe uptake kinetics by M. aeruginosa based on the studies using model
ligand, for example (EDTA, citrate) consistent with the mode of Fe uptake
occurring in natural waters where Fe is generally buffered by chemically
heterogeneous natural organic matter (NOM)?
Fe(III)' Fe(II)'
Fe(III)L Fe(II)L
kd1
kred1 ,
kox2 ,
kred2 ,
kf1kd2 kf2
kox1 ,
Ks
ρFeOxido-reductase
Fe-binding site
Outer membrane
; kox3 ,
O2
O2
O2 O2
•−O2•−
O2•−O2•−
O2•−O2•−
O2•−O2•−
Chapter 1. Introduction
9
(iv) What are the growth characteristics of Fe-limited continuous cultures of M.
aeruginosa and can a model of Fe uptake for this organism developed from
batch culture studies be used to describe Fe uptake by a continuous culture
of M. aeruginosa?
1.2. OBJECTIVES
The overall aim of this study is to investigate the Fe uptake kinetics of the bloom-
forming freshwater cyanobacterium M. aeruginosa under Fe limitation with particular
attention given to:
(i) Effect of light on the Fe uptake by M. aeruginosa in a chemically well-
defined culture medium (Fraquil*) in the presence of a single metal
chelator, ethylenediaminetetraacetic acid (EDTA);
(ii) Intracellular Fe transport processes of M. aeruginosa in Fraquil* medium
buffered by EDTA;
(iii) Fe uptake kinetics by M. aeruginosa in the presence of a natural organic
ligand, Suwannee River fulvic acid (SRFA); and
(iv) Characteristics of M. aeruginosa grown in iron-limited continuous culture.
1.3. LAYOUT OF THESIS
In order to achieve these objectives, the thesis is divided into seven chapters as
described below:
Chapter 1: Background information on the significance of Fe towards cyanobacteria,
literature review on iron transformations in natural waters and mechanisms of Fe
acquisition by phytoplankton as well as the objectives of the study are presented in this
chapter.
Chapter 2: Information on the experimental and computational methods used as well
as a description of analytical quality control measures used in this thesis is presented in
Chapter 1. Introduction
10
this chapter. Particular attention is given to description of the culturing conditions of
M. aeruginosa in batch and continuous incubations, the methods used to determine Fe
concentrations, the short-term radio-labeled 55
Fe uptake experiments and the methods
used to quantify cellular Fe quota.
Chapter 3: The effect of light on Fe transformation and uptake kinetics by M.
aeruginosa PCC7806 in Fraquil* (pH 8) with the free iron activity buffered by the
ligand EDTA is described in this chapter. A kinetic model for Fe acquisition by M.
aeruginosa based on photo-generation and subsequent uptake of unchelated Fe(II) is
presented.
Chapter 4: In this chapter, insight into kinetics of extracellular Fe transport to
periplasmic and cytoplasmic compartments for strains PCC7806 and 7005 of M.
aeruginosa in Fraquil* (pH 8) buffered by EDTA is obtained. Specifically, short-term
Fe accumulation in periplasmic and cytoplasmic compartments of Fe-limited M.
aeruginosa is examined using radiolabeled 55
Fe combined with the cold osmotic shock
method to quantify the amount of Fe in each compartment.
Chapter 5: Fe uptake by M. aeruginosa grown in batch culture in pH 8 Fraquil*
containing the natural organic compound Suwannee River fulvic acid (SRFA) using
short-term radiolabeled 55
Fe uptake assays is assessed in this chapter. A kinetic model
that describes extracellular Fe transformations is developed and used to elucidate the
key processes involved in cellular Fe uptake by M. aeruginosa.
Chapter 6: In this chapter, the development of a continuous culturing system
(chemostat) made of metal-free materials, used to maintain cultures of M. aeruginosa
PCC7806 in both nutrient-insufficient and nutrient-replete Fraquil* media at
nanomolar iron (Fe) concentrations (20-100 nM total Fe) in which Fe availability
limited growth is described. A modified chemostat theory for Fe-limited
phytoplankton growth is developed and applied to describe the behaviour (including
steady state cell density, Fe cell quota and Fe uptake kinetics) of M. aeruginosa strain
PCC7806 grown continuously in Fraquil* and nutrient-replete Fraquil
* media with Fe
activity buffered by the organic ligand EDTA.
Chapter 1. Introduction
11
Chapter 7: In this final chapter, the major findings of this research are summarised
and recommendations for future studies provided.
12
CHAPTER 2
GENERAL METHODOLOGY
Chapter 2. General Methodology
13
2.1. REAGENTS
All reagents were prepared in ultrapure water (18 MΩ·cm resistivity Milli-Q water,
MQ) and all solutions stored in the dark at 4oC when not in use, unless stated
otherwise. All chemicals used were of high purity (at least analytical grade). All pH
measurements were made using a pH meter (pH/ION 340i, WTW, Germany)
calibrated by pH 4.01 and pH 6.88 buffers on the free hydrogen ion activity scale.
Adjustment of pH was performed using 1 and 5 M HCl and NaOH solutions, which
were prepared from highly purified 30% w/v HCl (Fluka) and NaOH (Riedel-deHaën,
Germany). The plastic-ware used was soaked in 0.1 M HCl for at least a day, rinsed
with MQ and then dried prior to use.
2.2. CULTURING CONDITIONS
2.2.1. Culture Medium
A modified Fraquil medium (referred to as Fraquil*) designed for the study of trace
metal interactions with freshwater phytoplankton was used throughout and prepared as
previously described (Andersen, 2005). Briefly, all salt and trace metal stocks were
made up in MQ individually rather than as a mixture. Then, the stocks were mixed in
~1 L MQ, except for Fe and EDTA. The 1.0 × 10-3
M stock of ferric chloride (FeCl3,
Ajax Finechem, Australia) in 0.1 M HCl was mixed with a 1.3 × 10-2
M solution of
disodium ethylenediaminetetraacetic acid (Na2EDTA, Sigma) prior to mixing with the
other stock solutions in order to prevent precipitation of Fe(III). The pH of the medium
was then adjusted to 8, as in previous work (Fujii et al., 2010a). The medium was
sterilized using a 700 W microwave oven for 10 min in intervals of 3, 2, 3 and 2
minutes. After cooling to room temperature, filter-sterilized vitamin solution was
added. Final concentrations of the major salts, trace metals and vitamins in Fraquil*
were 2.6 × 10-4
M for CaCl2, 1.5 × 10-4
M for MgSO4, 1.0 × 10-3
M for NaNO3, 1.0 ×
10-5
M for K2HPO4, 5.0 × 10-4
M for NaHCO3, 1.0 × 10-3
M for HEPES, 1.6 × 10-7
M
for CuSO4, 5.0 × 10-8
M for CoCl2, 6.0 × 10-7
M for MnCl2, 1.2 × 10-6
M for ZnSO4,
1.0 × 10-8
M for Na2SeO3, 1.0 × 10-8
M for Na2MoO24, 3.0 × 10-7
M for thiamine HCl
Chapter 2. General Methodology
14
(Vitamin B1, Sigma), 2.1 × 10-9
M for biotin (Vitamin H, Sigma) and 3.6 × 10-10
M for
cyanocobalamin (Vitamin B12, Sigma). For the Fe uptake and photochemical
experiments, culturing media were prepared with nutrient concentrations identical to
Fraquil* except that Fe and EDTA was omitted.
To avoid Fe contamination, the culture media were prepared inside a trace-metal clean
room supplied with HEPA-filtered air. Based on the Fe concentration in the reagent
grade salts using the manufacturers’ specifications, Fe contamination was calculated to
be far less than 20 nM Fe (which is the lowest concentration used in this work); thus
no additional cleaning procedures (e.g., Chelex treatment; (Andersen, 2005)) were
undertaken. In addition to this calculation, Fe contamination was measured to be below
detection limit (~1 nM) by using a colorimetric method combined with an Ocean
Optics 1 m spectrophotometry system. The apparatus and configuration of the
spectrophotometry system and detailed procedure used to measure total Fe
concentration can be found elsewhere (Fujii et al., 2008b).
2.2.2. Long-term Culturing Conditions
Toxic and non-toxic strains of M. aeruginosa (PCC7806 and PCC 7005, respectively)
were generously provided by Dr. Brett Neilan (Cyanobacteria and Astrobiology
Research Laboratory, University of New South Wales). Batch axenic cultures of M.
aeruginosa cells were maintained in Fraquil* under conditions where cellular growth
was moderately limited by Fe availability as documented earlier (Fujii et al., 2010a).
Briefly, cells were incubated at 27oC under a 14 h:10 h light:dark cycle. Light was
supplied by three cool-white fluorescent tubes (36W, 28 mm diameter, 1.2 m length,
Philips) with total radiation intensity of 157 µmol.m-2
.s-1
. In the long-term culturing
medium, total concentrations for Fe and EDTA were 0.1 µM and 26 µM, respectively.
Cells were regularly subcultured into fresh media when cultures reached stationary
growth phase (generally ~2 weeks after inoculation with initial cell density of ~104 cell
mL-1
). Cells in the cultures were enumerated using a Neubauer hemocytometer (0.1
mm depth) under an optical microscope (Nikon, Japan). All experiments were
performed using cells harvested by filtering during the light phase of the light:dark
cycle in exponential growth phase.
Chapter 2. General Methodology
15
2.2.3. Continuous Culturing Apparatus
Continuous culturing systems (chemostats) have been used by several workers (Novick
and Szilard, 1950, Herbert et al., 1956, Tempest, 1969, Gerhardt and Drew, 1994,
Hoskisson and Hobbs, 2005). In this work, a chemostat system made of metal-free
materials was successfully developed and used to maintain cultures of M. aeruginosa
PCC7806 in both Fraquil* and nutrient-replete Fraquil
* media at nanomolar Fe
concentrations (20-100 nM total Fe) in which Fe availability limited growth.
Composition of the nutrient-replete Fraquil* medium will be discussed in details in
Chapter 6.
The metal-free sterile chemostat system was developed for four different flow-rates
with three replicates (Figure 2.1). The culture medium reservoirs consisted of two 2 L
polycarbonate bottles containing sterile Fraquil* medium with screw top caps vented
by a 0.22 µm air filter. Sterile fresh medium was distributed at four different flow-rates
into the 12 cultures in 250 mL polycarbonate culturing vessels using a high precision
24-channel peristaltic medium pump (Ismatec). Inline 0.22 µm-filtered air was
supplied by a 4-channel aquarium style diaphragm air pump (Aqua-one) to create the
positive pressure required in the head-space of the culture vessels. The air gap created
between the culture and waste vessels flushed out the excess culture over the elevated
weir into a 10 L polycarbonate waste vessel via an overflow vent. The volume of the
culture in each vessel was therefore maintained constant at approximately 200 mL. To
avoid sedimentation of cells, the culturing vessels were continuously gently shaken
using a Benchtop digital shaker (Thermoline Scientific, Australia) at a rotation rate of
135 ± 5 rpm. A sampling port was equipped with a sterile one-way sampling valve that
allowed sampling of the culture without bacterial or metal contamination.
The system was placed in an incubator (Thermoline Scientific, Australia) to control the
temperature at 27oC under a 14 hr:10 hr light:dark cycle with light intensity of 157
µmol photons m-2
s-1
. Prior to use, all chemostat apparatus was sterilized by
autoclaving. During this treatment, all materials were protected from bulk trace metal
contamination from metal leaching inside the autoclave by placement in a double-layer
plastic bag.
Chapter 2. General Methodology
16
Figure 2.1. The chemostat culturing system consisting of non-metal materials. The
system was operated at four different dilution rates in triplicate.
Chapter 2. General Methodology
17
2.3. SHORT-TERM IRON UPTAKE EXPERIMENT
The short-term uptake of radio-labelled 55
Fe by M. aeruginosa was measured by
incubating cells in Fraquil* containing
55Fe-ligand complex in the presence or absence
of light. Briefly, cells of the original batch or continuous cultures were harvested onto
a 25 mm diameter, 0.65 µm PVDF membrane (Millipore) during daytime in
exponential growth phase for the batch cultures and in steady state phase for the
continuous cultures of M. aeruginosa. To remove any adsorbed Fe from the cell
surface, washing solutions containing 50 mM Na2EDTA and 100 mM Na2oxalate at
pH 7 (hereafter referred to as “EDTA/oxalate”) and 2 mM NaHCO3 at pH 8 were used
(Tang and Morel, 2006). The filtered cells were washed gently at 1 mL.min-1
with
EDTA/oxalate solution (pH 7) and subsequently rinsed with 2 mM bicarbonate buffer
(pH 8) by continuously passing 5 mL of each solution through the harvested cells for
~10 min. Washed cells were re-suspended into Fe and EDTA-free Fraquil* medium at
densities of interest. The Fe uptake experiment was initiated by adding different pre-
equilibrated 55
FeIII
-ligand stock solutions with different Fe:ligand ratios into the
cultures to obtain expected concentrations of 55
Fe(III) and ligand. The solutions of
55Fe(III) complexed by ligand were made by mixing radiolabeled
55FeCl3 solution with
the ligand stock (i.e., EDTA, citrate or SRFA) in the bottom of a polypropylene
microtube, followed by addition of 2mM bicarbonate buffer (pH 8) to the mixture to
maintain pH 8 and let the Fe(III)-ligand stock stored for 24 h in the dark at ambient
temperature to equilibrate.
In some experiments, the culturing medium was prepared with addition of either a
single reagent such as: ascorbate (Asc), ferrozine (FZ), superoxide dismutase (SOD),
hydroxylamine hydrochloride (HH), xanthine/xanthine oxidase (X/XO), etc. or a
combination of these reagents depending on the purposes of the particular experiment
of interest. Cells were incubated at 27oC for 0-12 h in the presence and absence of light
and cut-off filters.
After incubation, cells were vacuum-filtered onto the 0.65 µm PVDF membrane filters,
and then rinsed three times with 1 mL of EDTA/oxalate solution and three times with
1 mL of 2 mM NaHCO3 solution (total rinsing time was ~10 min). The filtered cells
were then placed in glass scintillation vials with 5 mL of scintillation cocktail
Chapter 2. General Methodology
18
(Beckman ReadyScint) for further measurement of radioactivity by using a Packard
TriCarb Liquid Scintillation Counter (see Section 2.4.2).
2.4. MEASUREMENT OF IRON
2.4.1. Measurement of FeIIFZ3 with Spectrophotometer
A Cary 50 Bio UV-Visible spectrophotometer (with a detection limit of ~0.1 µM) was
used to determine micromolar concentrations of the FeIIFZ3 by measurement of the
absorbance of the complex at 562 nm. Further details of the FZ method are provided
by Stookey (1970) and Viollier et al. (2000).
When determination of nanomolar concentrations of iron was required, a 1 m path-
length “waveguide” (LWCC Type II, World Precision Instruments) combined with an
Ocean Optics spectrophotometer was employed. The spectrophotometer configuration
consisted of a deuterium tungsten halogen light source (DH-2000), a UV-VIS
spectrophotometer (USB 2000 UV-VIS), two optical fibers (P400 UV-VIS) and an
Ocean Optics 1 m pathlength spectrophotometry system. In order to undertake the
analyses, Fe-free Fraquil* was initially introduced into the 1 m flow cell by pushing the
solution with a peristaltic pump and the absorbance at 562 nm was zeroed. FZ and
sample solutions were mixed and incubated under experimental conditions of interest
prior to being introduced into the 1 m cell, followed by monitoring absorbance at 562
nm for several minutes with the OOIBase 32 computer program provided by Ocean
Optics. The absorbance at 562 nm was baseline-corrected using the absorbance at 750
nm as a reference to obtain a stable absorbance.
A stock solution of Fe(II) was made by dissolving ammonium ferrous sulfate (Sigma)
in 1.0 mM HCl at a final concentration of 4 mM. Calibration of FeIIFZ3 concentration
was performed by adding known amounts of the Fe(II) stock to FZ solutions, yielding
molar absorptivity of ε562= ~28,000 M-1
.cm-1
.
Chapter 2. General Methodology
19
2.4.2. Measurement of Radio-labeled 55
Fe with Scintillation Counter
Radioactivity of radio-labelled 55
Fe was measured using a Packard TriCarb Liquid
Scintillation Counter with quench correlation. Scintillation counts (counts per minute)
of the samples were converted to moles of Fe by using concurrent counts of 1-50 µL of
55Fe-ligand stock in 5 mL scintillation cocktail.
2.5. MEASUREMENT OF CELLULAR IRON QUOTA
In this study the cellular Fe quota of M. aeruginosa was quantified using either acid
digestion combined with spectrometry or the radiometry (radiolabeled 55
Fe) method as
described below.
2.5.1. Acid Digestion Combined with Spectrophotometry Method
In order to determine the intracellular Fe content of M. aeruginosa cells, 5 mL of the
culture grown in growth medium containing non-radiolabeled Fe was filtered on to a
0.65 µm PVDF membrane filter (Millipore) and gently rinsed three times with 1 mL of
EDTA/oxalate solution and three times with 1 mL of 2 mM NaHCO3 solution (total
rinsing time was ~10 min) in order to eliminate non-specifically adsorbed Fe from the
cell surface. The filter was then immersed in 2 mL of 50% HNO3 and heated at ~100
0C for 4 h. The digest solution was brought up to a final volume of 10 mL by MΩ
water and centrifuged (3000 rpm for 10 min) to separate the residue from the acid
soluble fraction. The supernatant was complexed with FZ by addition of 1 mM
hydroxylamine hydrochloride in the presence of 1 mM FZ (allowed to react overnight
to reduce all Fe to Fe(II)) followed by addition of 10 M ammonium acetate buffer (pH
9.5) to bring the pH of the final solution to a value of approximately 6 where FeIIFZ3 is
significantly formed (Tang and Morel, 2006, Fujii et al., 2010a). The total Fe
concentration was determined by quantifying the absorbance of the FeIIFZ3 complex at
562 nm using a 1 m Ocean Optics long pathlength spectrophotometry system as
described previously in Section 2.4.1.
Chapter 2. General Methodology
20
2.5.2. Radiometry Method
M. aeruginosa cells were grown in growth medium containing radiolabeled 55
Fe
instead of non-radiolabeled Fe. The amount of 55
Fe incorporated within cells was
measured by filtering 1 mL of the culture on to a 25 mm diameter 0.65 µm PVDF
membrane (Millipore). The filtered cells were then gently rinsed with EDTA/oxalate
solution and 2 mM NaHCO3 solution (total rinsing time was ~10 min). Subsequently,
the washed cells were placed in glass scintillation vials filled with 5 mL of scintillation
cocktail. The activity of radioisotope 55
Fe in the washed cells was measured in a
Packard TriCarb Liquid Scintillation Counter as previously described in Section 2.4.2.
2.6. MODEL FITTING
In this thesis, the best fit of the model to the experimental data was determined by
using a least-squares method in which the mean square error between the model value
and the average of the experimental data was minimized. For the model fit, Microsoft
Excel was used.
2.7. ANALYTICAL QUALITY CONTROL
2.7.1. Procedural Blank
Procedural blanks were prepared and analyzed exactly like, and along with, the
samples. The procedural blanks provided an indication of the response of the
measurement system to a sample with zero concentration of analyte. In addition, the
procedural blanks provided an indication of analyte contamination that may occur
during sample preparation and analysis. The procedural blank responses could also be
used to estimate the detection limit of the measurement system. If the analyte
concentration of the procedural blank was less than the detection limit, no corrective
action was necessary.
2.7.2. Replication
Sample analysis was performed in triplicate, unless otherwise stated.
21
CHAPTER 3
EFFECT OF LIGHT ON IRON UPTAKE
BY THE FRESHWATER
CYANOBACTERIUM MICROCYSTIS
AERUGINOSA
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobacterium Microcystis aeruginosa
22
3.1. INTRODUCTION
Iron (Fe) is an essential element for primary producers because it plays critical roles in
various metabolic processes including photosynthesis, respiration and nitrogen fixation
(Crichton, 2009), and limits phytoplankton growth in some open ocean waters because
of its low availability (e.g., Martin and Fitzwater (1988)). Although Fe is not generally
considered a major growth-limiting nutrient for freshwater phytoplankton (Hassler et
al., 2009), Fe nutrition still affects biosynthesis of primary and secondary metabolites
and cells must amend their physiological functions depending on Fe availability
(Alexova et al., 2011); for example, Fe availability may influence toxin gene
expression and toxin generation under low Fe conditions (Sevilla et al., 2008, Alexova
et al., 2011).
Fe(III) is thermodynamically favored in air-saturated surface waters because of rapid
oxidation of Fe(II) by dissolved oxygen at circumneutral pH (Pham and Waite, 2008a).
Nevertheless, substantial evidence exists that reduction of Fe(III) to Fe(II) is promoted
by photochemical and biochemical processes in surface waters (Fan (2008) and
references therein). Consequently, analytically measureable amounts of Fe(II), which
is more available than insoluble Fe(III) for biological uptake (Shaked et al., 2005, Rose
et al., 2005), are formed in surface waters (e.g., Waite et al. (1995)). Light-mediated Fe
redox reactions such as ligand-to-metal charge transfer (LMCT) and reactions with
secondarily produced organic and inorganic radicals may play critical roles in the
formation of Fe(II) during the daytime (Fan, 2008, Rose and Waite, 2006, Waite and
Morel, 1984). During LMCT, irradiation of Fe(III) complexes with aminocarboxylate,
carboxylate and catecholate ligands (among others) leads to the oxidation or
decarboxylation of the ligands at certain sites, and induces Fe(III) reduction by
electron transfer to the metal center (Barbeau et al., 2001, Sunda and Huntsman, 2003).
Kinetic modeling of LMCT Fe transformations indicates that the photoreductively
mediated dissociation of chelated Fe(III) and subsequent oxidation of unchelated Fe(II)
(Fe(II)') to unchelated Fe(III) (Fe(III)') markedly increases the concentration of
unchelated Fe (Fe') in sunlit surface waters (Sunda, 2001). Laboratory and shipboard
culturing studies have consistently shown that exposure to natural sunlight enhances
the availability of Fe when bound to various organic ligands (in situ chelators,
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobacterium Microcystis aeruginosa
23
aminocarboxylates and siderophores) for uptake by in situ and laboratory strains of
marine phytoplankta by a factor of 2-15 (Barbeau et al., 2001, Maldonado et al., 2005,
Anderson and Morel, 1982) though exceptions have been reported (Hassler and Twiss,
2006).
Given the underlying concepts of classical and current Fe uptake models (Hudson and
Morel, 1990, Morel et al., 2008), where Fe' is considered the most assimilable pool of
Fe for phytoplankton, enhanced biological Fe uptake may be linked to increased Fe'
concentration in the light. However, these Fe uptake models have been essentially
developed from biological Fe assimilation assays undertaken in the absence of light.
Therefore, the mode of biological Fe uptake in the presence of light has yet to be
established despite the relatively well-defined phototransformations for certain Fe
complexes. In this work, we mechanistically examine the effect of light on Fe
transformation and uptake by a common bloom-forming freshwater cyanobacterium,
Microcystis aeruginosa, in Fraquil* (modified Fraquil) medium with the free iron
activity buffered by ethylenediaminetetraacetic acid (EDTA).
3.2. MATERIALS AND METHODS
3.2.1. Materials
Unless otherwise stated, chemicals were purchased, prepared and stored as described
in Section 2.1, Chapter 2. pH (values reported on the free hydrogen ion activity scale)
was measured using a pH/ION 340i pH meter (WTW, Germany). Only plasticware
was used for solution preparation, storage and sample incubation to prevent Fe
contamination. Plasticware was acid-cleaned by soaking in 0.1 M hydrochloric acid for
at least a day and rinsed with ultrapure Milli-Q water (MQ; Millipore, 18 MΩ.cm
resistivity) before use.
Long-term cell culturing was performed in Fraquil* medium at pH 8, prepared as
described (Andersen, 2005) excepting modified concentrations of 100 nM Fe and 26
µM EDTA (see Section 2.2.1, Chapter 2 for details). To avoid Fe contamination, the
culture medium was prepared inside a trace metal clean room supplied with HEPA-
filtered air. Fe contamination in the medium has been previously determined to be < ~1
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobacterium Microcystis aeruginosa
24
nM (Fujii et al., 2010a), much less than the lowest Fe concentration used in this work.
For photochemical and Fe uptake experiments, Fraquil* media were prepared with the
omission of Fe or Fe and EDTA, but concentrations of all nutrients other than Fe and
EDTA were unchanged. Then an appropriate volume of pre-equilibrated FeIII
EDTA
stock was added shortly before the experiment. The FeIII
EDTA stock was prepared by
mixing either non-radiolabeled 1-10 mM FeCl3 (in 0.1 M HCl, Ajax Finechem,
Australia) or radiolabeled 8.3 mM 55
FeCl3 (in 0.5 M HCl, 185 MBq, Perkin-Elmer,
Australia) with an appropriate volume of 1-100 mM EDTA solution (pH 8, Sigma) in a
polypropylene microtube, followed by addition of 2-50 mM bicarbonate buffer (pH 8,
Sigma) to maintain pH 8. The FeIII
EDTA stocks were then stored for at least 2 h in the
dark at ambient temperature to equilibrate.
For use in Fe uptake and photo-reductive dissociation experiments, stock solutions of
ferrozine (FZ; 3-(2-pyridyl)-5,6-diphenyl-1,2, 4-triazine-4’,4’’-disulfonic acid sodium
salt, Sigma) were prepared in MQ at concentrations of 0.1 M. The pH of the solutions
was adjusted to 8.0 to avoid a significant pH change when added to the culture
medium.
3.2.2. Light Conditions
Most experiments and cell culturing were performed in a light- and temperature-
controlled incubator at 27oC (Thermoline Scientific). Light was vertically supplied by
three cool-white fluorescent tubes (36W, 28 mm diameter, 1.2 m length, Philips).
Samples were consistently incubated 10 cm from the fluorescent tubes, at which
distance total radiation intensity was determined to be 157 µmol quanta.m-2
.s-1
(part A
of Figure 3.1 for the emission spectrum) using an Ocean Optics USB 4000
spectrophotometer equipped with an optical fiber and cosine converter (CC-3-UV) that
was calibrated against a DH-2000 VIS light source. Samples were incubated in either
polystyrene spectrophotometer cuvettes (Starna) or polycarbonate vessels (Nalgene)
that minimally interfere with visible light transmission (see Figure 3.2 for absorbance
spectra). During dark incubations, samples were covered with aluminum foil.
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
Figure 3.1. Irradiation spectra emitted from the cool-white fluorescent tube of the culturing incubator in the (A) absence and (B
spectra were measured using an Ocean Optics USB 4000 spectrophotometer equipped with an optical fiber and cosine corrector le
VIS-light source (hydrogen lamp). The measurement was per
the cut-off light filter between the light source and the irradiance probe. The photon flux densities calculated for each wavelength
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
white fluorescent tube of the culturing incubator in the (A) absence and (B-H) presence
spectra were measured using an Ocean Optics USB 4000 spectrophotometer equipped with an optical fiber and cosine corrector lens (CC
light source (hydrogen lamp). The measurement was performed using SpectraSuite software in absolute irradiation mode. Various light spectra were obtained by placing
off light filter between the light source and the irradiance probe. The photon flux densities calculated for each wavelength range are s
25
H) presence of light filter treatments. The
ns (CC-3-UV) calibrated against a DH-2000
formed using SpectraSuite software in absolute irradiation mode. Various light spectra were obtained by placing
range are shown in Table 3.1.
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
26
Table 3.1. Photon flux density at each wavelength range.
Wavelength range
Photon flux density (µmol photons.m-2
.s-1
)
No filter Light filter treatments
400nm 450nm 500nm 550nm 600nm 650nm 700nm
400nm-450nm 29.1 25.4 13.5 1.4 1.5 1.0 0.5 0.4
450nm-500nm 23.9 22.0 22.1 11.1 4.2 0.8 0.5 0.4
500nm-550nm 38.1 36.3 37.4 36.6 25.5 1.0 0.8 0.6
550nm-600nm 25.4 24.2 24.9 24.7 22.1 9.1 4.8 0.4
600nm-650nm 33.8 32.2 33.1 33.0 31.8 29.8 19.1 0.6
650nm-700nm 6.2 5.7 5.6 5.7 5.4 5.2 4.7 2.2
Total 156.5 145.8 136.7 112.7 90.6 47.0 30.4 4.6
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis. aeruginosa
Experiments examining the effect of
biological uptake employed visible long
Corporation), which permit the transmission of radiation at wavelengths that are
nominally longer than 400, 450, 500, 550, 60
cut-off is defined as the wavelength where ~50% of peak transmission occurs, light
transmission was typically completely blocked at a wavelength 25 nm less than this
nominal value (e.g., 475 nm for the 500 nm filter)
3.1). Experiments were conducted by placing samples in a cardboard box covered on
five sides with aluminum foil, with the front of the box (facing the light source) fitted
with a light filter to allow passage of specific wav
A control sample was incubated without placing any filters in the front of the box.
Figure 3.2. Absorbance spectra for plastic and glass vessels. (A) blank (no materials),
(B) 1 cm quartz spectrophotometer cuvette (
Scintillation glass vials (20 mL, Crown Scientific), (D) 1 cm polystyrene
spectrophotometer cuvette (Starna Pty Ltd, Australia), (E) polycarbonate container
Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis. aeruginosa
Experiments examining the effect of irradiation wavelength on Fe photochemistry and
biological uptake employed visible long-wave-pass edge filters (50 mm sq., Andover
Corporation), which permit the transmission of radiation at wavelengths that are
nominally longer than 400, 450, 500, 550, 600, 650 or 700 nm. Although the nominal
off is defined as the wavelength where ~50% of peak transmission occurs, light
transmission was typically completely blocked at a wavelength 25 nm less than this
nominal value (e.g., 475 nm for the 500 nm filter) (parts B-H Figure 3.1 and Table
Experiments were conducted by placing samples in a cardboard box covered on
five sides with aluminum foil, with the front of the box (facing the light source) fitted
with a light filter to allow passage of specific wavelengths of light as described above.
A control sample was incubated without placing any filters in the front of the box.
Absorbance spectra for plastic and glass vessels. (A) blank (no materials),
(B) 1 cm quartz spectrophotometer cuvette (Starna Pty Ltd, Australia), (C)
Scintillation glass vials (20 mL, Crown Scientific), (D) 1 cm polystyrene
spectrophotometer cuvette (Starna Pty Ltd, Australia), (E) polycarbonate container
Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis. aeruginosa
27
irradiation wavelength on Fe photochemistry and
pass edge filters (50 mm sq., Andover
Corporation), which permit the transmission of radiation at wavelengths that are
0, 650 or 700 nm. Although the nominal
off is defined as the wavelength where ~50% of peak transmission occurs, light
transmission was typically completely blocked at a wavelength 25 nm less than this
H Figure 3.1 and Table
Experiments were conducted by placing samples in a cardboard box covered on
five sides with aluminum foil, with the front of the box (facing the light source) fitted
elengths of light as described above.
A control sample was incubated without placing any filters in the front of the box.
Absorbance spectra for plastic and glass vessels. (A) blank (no materials),
Starna Pty Ltd, Australia), (C)
Scintillation glass vials (20 mL, Crown Scientific), (D) 1 cm polystyrene
spectrophotometer cuvette (Starna Pty Ltd, Australia), (E) polycarbonate container
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis. aeruginosa
28
(250 mL, Nalgene), (F) high-clarity polypropylene tube (15 mL, BD Falcon), (G)
polypropylene microtube (1.5 mL, Eppendorf), (H) high-density polyethylene bottle
(125 mL, Nalgene). The absorbance spectra were measured using a Varian Cary 50
UV-Vis spectrophotometer (Scan mode). During measurement, the containers were
filled with ultrapure water (Milli-Q water). For large materials, the sample holder was
removed from the instrument and the materials were placed between the light source
and detector.
3.2.3. Photochemical Experiments
The rate of unchelated Fe(II) formation during FeIII
EDTA photolysis was
spectrophotometrically determined using ferrozine (FZ), a strong Fe(II) complexing
agent. Experiments were initiated by spiking appropriate volumes of the pre-
equilibrated FeIII
EDTA and 0.1 M FZ (pH 8, Sigma) stock solutions into Fe and
EDTA-free Fraquil* at final concentrations of 1-10 µM for Fe, 26 µM for EDTA and 1
mM for FZ, and samples incubated under light or dark conditions in the presence and
absence of light filters. At various incubation times (0-10 h) after adding FeIII
EDTA
and FZ, the FeIIFZ3 concentration of the sample was measured by monitoring the
absorbance at 562 nm where FeIIFZ3 absorbs most strongly (Thompsen and Mottola,
1984). Absorbance was measured using an Ocean Optics spectrophotometry system
with a 1 m long path length flow cell for the 1 µM Fe system and a Varian Cary 50
UV-VIS spectrophotometer with 1 cm path length cuvette for the 10 µM Fe systems
(see Section 2.4.1. for details). The effect of Fe contamination in reagents on the Fe(II)
formation rate was examined by repeating the experiments without adding Fe(III) to
the sample.
3.2.4. Short-term 55
Fe Uptake Experiments
Cells of the original cultures of M. aeruginosa PCC7806 were harvested onto a 25 mm
diameter, 0.65 µm PVDF membrane (Millipore) during daytime in exponential growth
phase at densities of 1-2 × 106
cell.mL-1
. To remove any adsorbed Fe, the filtered cells
were washed gently at 1 mL.min-1
with EDTA/oxalate solution (pH 7) and
subsequently rinsed with 2 mM bicarbonate buffer (pH 8) by continuously passing 5
mL of each solution through the harvested cells for ~10 min. Washed cells were re-
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis. aeruginosa
29
suspended into Fe and EDTA-free Fraquil* medium at densities of 2.6 × 10
5 to 2.7 ×
107 cell.mL
-1. The Fe uptake experiment was initiated by adding the pre-equilibrated
55Fe
IIIEDTA stock at final concentrations of 200 nM for
55Fe and 26-260 µM for
EDTA. In some experiments, the culturing medium was prepared with addition of 1
mM FZ. In all cases, cells were incubated for 2 h in the presence and absence of light
and cutoff filters. All Fe uptake experiments were performed under nonsaturating
conditions (i.e., measured uptake rate was less than the maximum uptake rate of 2.0
amol.cell-1
.hr-1
(Fujii et al., 2010a) at least by a factor of 5). Under the Fe
concentration and cell densities examined here, the amount of 55
Fe taken up by cells
during the short-term incubation were calculated to be only 3% of total 55
Fe in culture
medium at a maximum.
After incubation, cells were vacuum filtered on to 0.65 µm filters, rinsed three times
with 1 mL of EDTA/oxalate solution then three times with 1 mL of 2 mM NaHCO3
(total rinsing time was ~10 min). The filtered cells were placed in glass scintillation
vials with 5 mL of scintillation cocktail (Beckman ReadyScint) and their activity
measured in a Packard TriCarb Liquid Scintillation Counter, with scintillation counts
(counts per minute) of the samples converted to moles of Fe using concurrent counts of
1-5 µL of 55
FeEDTA stock in 5 mL scintillation cocktail. Process blanks were
determined by performing the procedure in the absence of cells.
3.3. RESULTS AND DISCUSSION
3.3.1. Effect of Light on Photoreductive Dissociation and Fe Uptake
Photoreductive dissociation of FeIII
EDTA in Fraquil* was examined by measuring
time-dependent Fe(II) formation in the presence of FZ. During irradiation (by the
fluorescent tubes in the culture cabinet), the concentration of FeIIFZ3 complex
increased linearly with time (R2 > 0.99). Time-dependent increases were negligible or
very small in the dark and in the absence of Fe. A concentration of 1 mM FZ was
chosen as Fe(II)' complexation by FZ at this concentration is rapid and should
outcompete other reactions involving Fe(II)' such as oxidation by dioxygen and
recomplexation by intact EDTA. Under these conditions therefore, assuming that the
rate of FeIIFZ3 formation is equal to the rate of Fe(II)' production would appear
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis. aeruginosa
30
reasonable. Because FeIIFZ3 formation followed a first-order relationship with respect
to FeIII
EDTA concentration, the rate of photochemical Fe(II)' generation in our system
can be written as:
d[FeIIFZ3]
dt=
d[Fe(II)']
dt= khv[Fe
IIIEDTA] (3.1)
where khv is a first-order rate constant for photoreductive dissociation of FeIII
EDTA
under the conditions examined. Approximating III II
T 3[Fe EDTA] [Fe] - [Fe FZ ]≈ and
[FZ] ≈ [FZ]
T (where subscript T indicates total concentration) followed by integration
of the resulting expression yields a relationship between [FeIIFZ3] and time (see part
A1.1 and Figure A1.1 of Appendix 1 for details). As shown in part A of Figure 3.3, khv
was determined to be 6.5 (± 0.25) × 10-6
s-1
for 1 µM total Fe and 6.2 (± 0.20) × 10-6
s-1
for 10 µM total Fe by linear regression analysis. Effect of total Fe concentration on khv
was statistically insignificant. In addition, the reaction proceeded in a first-order
manner with respect to total Fe concentration. These results suggest that secondary
radicals such as reactive oxygen species, which could be generated to a larger extent at
higher Fe concentrations or accumulate with time, play a minor role in Fe(II)'
formation. The minor effect of secondary radicals is probably due to either the
presence of radical scavengers in the medium (e.g., copper) or insignificant
participation of secondary radicals in the Fe(II)'-producing photoredox processes.
The effect of light on 55
Fe uptake rate was examined under conditions identical to
those used in the photochemical experiments except for the presence of M. aeruginosa
cells (exponential growth phase, ≤ 3.5 × 106
cell.mL-1 ) and the use of different
concentrations of EDTA and radiolabeled 55
Fe(III) (as a replacement for
nonradiolabeled Fe). In the absence and presence of light, 55
Fe accumulated in cells
linearly with time over the 2 h incubations (part B of Figure 3.3). At 26 µM EDTA and
200 nM Fe, the 55
Fe uptake rate (0.33 ± 0.065 amol.cell-1
.hr-1
, n=13) under the light
was two orders of magnitude greater than that under darkness (0.003 ± 0.006 amol.cell-
1.hr
-1, n=3), indicating a significant role of light in Fe uptake.
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microc
Figure 3.3. Effect of light on Fe(II)' formation and
[EDTA]T = 26 µM, [FZ]T = 1 mM) and (B) 55
Fe uptake
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
formation and 55
Fe uptake by M. aeruginosa. Time-courses of (A) FeIIFZ3 formation ([Fe]
Fe uptake ([55
Fe]T = 200 nM and [EDTA]T = 26 µM). Effect of light wavelength on (C)
31
formation ([Fe]T = 0, 1 or 10 µM,
. Effect of light wavelength on (C) 55
FeEDTA
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
32
uptake ([55
Fe]T = 200 nM, [EDTA]T = 26 µM) and (D) FeIIFZ3 formation ([Fe]T = 10 µM, [EDTA]T = 26 µM, [FZ]T = 1 mM). The incubations
were performed in modified Fraquil* (pH 8) in the light or dark at 27
oC. In the light filter treatments (panels C and D), filters were placed
between the incubated samples and the light source to allow transmission of wavelengths longer than 400, 450, 500, 550, 600, 650 or 700 nm. In
the control treatment, no light filter was inserted in front of the sample. Incubations were performed for 4 h for the photo-reduction experiment
and 2 h for the 55
Fe uptake experiment. Asterisks indicate that light filter treatments were significantly different from the control at the p < 0.05
level using a single-tailed heteroscedastic t-test. Symbols and error bars represent average data ±standard deviation from duplicate (photo-
reduction) or triplicate (55
Fe uptake) experiments. Solid lines represent linear regression.
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
33
3.3.2. Effect of Light Wavelength
Transport of Fe across the (cyto)plasmic membrane, is likely energy-dependent
process (Andrews et al., 2003). Thus, it is possible that the Fe uptake system in the
presence of photosynthetically active radiation (PAR) can be activated by immediate
use of the energy (e.g., ATP) produced during photosynthesis. To investigate this, we
determined which wavelengths (λ) of light particularly affected Fe uptake and
photoreduction. Use of cutoff filters to manipulate the wavelengths of light reaching
the culture indicated that only light from λ = 400-500 nm significantly contributed to
55Fe uptake by M. aeruginosa, with the uptake rate at these wavelengths comparable to
that in the control treatment (no light filter) (part C of Figure 3.3). Irradiation with
light at λ = 500-700 nm, which accounts for the majority (66%) of the total photon flux
(Table 3.1) and which has a substantial impact on cyanobaterial photosynthesis (Kirk,
1994), had almost no effect on cellular Fe uptake (part C of Figure 3.3). Similarly,
significant FeIIFZ3 formation only occurred when the sample was exposed to light in
the range λ = 400-500 nm (part D of Figure 3.3). In contrast to the total photon flux
density, the FeIIFZ3 formation rate was a linear function of
55Fe uptake rate (Figure
3.4), supporting the contention that specific light wavelength rather than total intensity
is important for the short-term 55
Fe uptake and photoreduction. These results strongly
suggest that photoinduced abiotic Fe transformations rather than biological factors are
more important for cellular Fe uptake. Such an interpretation is consistent with
previous studies (Anderson and Morel, 1982, Hudson and Morel, 1990) showing that
Fe uptake by coastal phytoplankton (the coccolithophorid Pleurochrysis carterae and
diatom Thalassiosira weissflogii) grown under Fe limitation was affected negligibly or
only slightly (by 0-37% relative to dark) by any light-mediated increase in cellular
metabolism. Although the absorbance of FeIII
EDTA rapidly decreases in the visible
light region (Figure 3.5), this complex is still capable of capturing some light at 400-
500 nm. Over this wavelength range, the average quantum yield for FeIII
EDTA (ϕ =
[FeIIFZ3 formation rate]/[number of photons absorbed]) was 0.010 (Table 3.2),
comparable to the published value at similar pH and irradiation wavelength (ϕ =
0.011, when quantum yield data ranging from ϕ = 0.005 at 500 nm to ϕ = 0.02 at 400
nm are averaged (Kari et al., 1995)).
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
34
Figure 3.4. Relationships between (A) FeIIFZ3 formation rate and
55Fe uptake and (B)
total photon flux density and 55
Fe uptake rate. At each data point, the parameters were
obtained from the incubation experiments and measurements of irradiation spectra
using the same cutoff filter. Thus, the data for 55
Fe uptake and FeIIFZ3 formation rate
are the same as those shown in parts C and D of Figure 3.3. Details of total photon flux
density are listed in Table 3.1.
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
Figure 3.5. UV-VIS absorbance spectra for Fe
= 0.5 mM and [EDTA]
8 buffered by 15 mM NaHCO
are also shown. The average molar
wavelength range from 400 nm to 500 nm was determined to be 37 M
Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
VIS absorbance spectra for FeIII
EDTA complex (solid line, [Fe(III)]
= 0.5 mM and [EDTA]T = 1.3 mM) and EDTA (dotted line, [EDTA]
8 buffered by 15 mM NaHCO3. Enlarged absorbance spectra in the visible light range
are also shown. The average molar absorptivity of FeIII
EDTA complex in the
om 400 nm to 500 nm was determined to be 37 M
Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
35
EDTA complex (solid line, [Fe(III)]T
= 1.3 mM) and EDTA (dotted line, [EDTA]T = 1.3 mM) at pH
. Enlarged absorbance spectra in the visible light range
EDTA complex in the
om 400 nm to 500 nm was determined to be 37 M-1
cm-1
.
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
36
Table 3.2. Parameters used for the determination of quantum yield.
Parameters Symbol Value Unit
FeIII
EDTA concentration c 10 µM
Average molar absorptivity at 400-500nma)
ε 37.3 M-1
.cm-1
Length of light pathb)
l 1.0 cm
Absorbance A =εcl 2.0 × 10-4
cm-1
Incident light at 400-500 nmc)
I0 53.0 µmol.m-2
.s-1
Light absorbed Ia =I0(1-10-εcl
) 4.6 pmol.cm-3
.s-1
FeIIFZ formation rate
d) d[Fe
IIFZ3]/dt 0.046 pmol.cm
-3.s
-1
Quantum yield φ 0.010
a) The average molar absorptivity was determined from the Fe
IIIEDTA spectrum shown in Figure 3.5.
b) A 1 cm polystyrene spectrophotometer cuvette was used to incubate samples in the Fe
IIIEDTA photoreduction experiment at 10 µM Fe.
c) Values were calculated from Table 3.1.
d) Data from the photoreduction experiment at 10 µM Fe.
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
37
3.3.3. Fe Substrate for Uptake
A plausible explanation for the mechanism of light-facilitated uptake is that Fe
availability increased due to an increase in the concentration of photo-produced Fe(II)'
and, potentially, Fe(III)'. The presence of 1 mM FZ significantly decreased Fe uptake
by 27-70% (p < 0.05, part A of Figure 3.6), supporting the hypothesis that
photoproduced Fe(II)' aided Fe uptake since, in this event, complexation by
membrane-impermeable FZ would be expected to inhibit cellular uptake. The lesser
effect of FZ at higher cell densities suggests that M. aeruginosa cells can effectively
compete with high concentrations of FZ for Fe(II). Such a high affinity of the cell for
Fe(II) may explain the previously reported absence of effect of FZ on Fe uptake by the
macrophytic cyanobacterium Lyngbya majuscula (Rose et al., 2005). A significant
decrease in rate of Fe uptake was also observed with increasing EDTA concentration
(part B of Figure 3.6), indicating that complexation by EDTA at concentrations
examined here effectively competes with cellular uptake, as direct FeIII
EDTA
acquisition is unlikely. The limited availability of 55
Fe-EDTA for dark uptake, even
after preliminary photolysis, consistently indicates that light-facilitated Fe uptake is
tightly coupled with the availability of photo-produced Fe. This Fe is available for
uptake during illumination but readily transforms to an unavailable form once
illumination ceases, presumably via oxidation of photoproduced Fe(II) following
complexation (part A1.2 and Figure A1.2 of Appendix 1).
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis ae
Figure 3.6. Effect of (A) ferrozine (FZ) and (B) excess EDTA on
the light. The 55
Fe uptake experiment was undertaken by incubating cells in Fraquil
containing pre-equilibrated
200 nM, [EDTA]T = 26
× 105 – 2.7 × 10
7 cell.mL
constant (3.5 × 106 cell.mL
indicate that treatments with FZ or excess EDTA were significantly different from the
control ([Fe]T = 200 nM and
respectively, using a single
average data and errors represent ±standard deviation from triplicate experiments.
Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis ae
Effect of (A) ferrozine (FZ) and (B) excess EDTA on
Fe uptake experiment was undertaken by incubating cells in Fraquil
equilibrated 55
FeIII
EDTA complex and FZ or excess
26-260 µM and [FZ]T = 1 mM). While various cell densities (2.6
cell.mL-1) were used in the FZ experiment, the cell density was kept
cell.mL-1) in the excess EDTA experiment. One and two asterisks
indicate that treatments with FZ or excess EDTA were significantly different from the
200 nM and [EDTA]T = 26 µM) at p < 0.05 and
respectively, using a single-tailed heteroscedastic t-test. Symbols and error bars are
errors represent ±standard deviation from triplicate experiments.
Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
38
Effect of (A) ferrozine (FZ) and (B) excess EDTA on 55
Fe uptake rate in
Fe uptake experiment was undertaken by incubating cells in Fraquil*
FZ or excess EDTA ([Fe]T =
. While various cell densities (2.6
) were used in the FZ experiment, the cell density was kept
excess EDTA experiment. One and two asterisks
indicate that treatments with FZ or excess EDTA were significantly different from the
< 0.05 and p < 0.01 levels,
test. Symbols and error bars are
errors represent ±standard deviation from triplicate experiments.
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis ae
3.3.4. Kinetic Model for
Transformation of Fe species under the various conditions employed in the Fe uptake
incubations was examined using a kinetic model based on the chemical reactions
shown in Table 3.3 and presented schematically in Figure 3.7.
Figure 3.7. Fe uptake model
Fe(II) (i.e., Fe(II)') is formed from the photoreductive dissociation of ferric EDTA
complex (FeIII
EDTA). The photoproduced Fe(II) subsequently passes through the
nonspecific outer membrane channel (por
uptake competes with Fe(II)
ferrozine (FZ) and excess EDTA if present at appropriate concentrations. Solid arrows
represent major reactions under conditions o
whereas dotted arrows indicate relatively minor reactions. Rate constants depicted near
the arrows correspond to those listed in Table 3.3.
Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis ae
3.3.4. Kinetic Model for Fe Species
Transformation of Fe species under the various conditions employed in the Fe uptake
incubations was examined using a kinetic model based on the chemical reactions
Table 3.3 and presented schematically in Figure 3.7.
Fe uptake model by M. aeruginosa in the presence of light. Unchelated
) is formed from the photoreductive dissociation of ferric EDTA
EDTA). The photoproduced Fe(II) subsequently passes through the
nonspecific outer membrane channel (porins) by diffusion. However, cellular Fe
uptake competes with Fe(II)' complexation by extracellular Fe-binding ligands such as
ferrozine (FZ) and excess EDTA if present at appropriate concentrations. Solid arrows
represent major reactions under conditions of the short-term 55
Fe uptake experiment,
whereas dotted arrows indicate relatively minor reactions. Rate constants depicted near
the arrows correspond to those listed in Table 3.3.
Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
39
Transformation of Fe species under the various conditions employed in the Fe uptake
incubations was examined using a kinetic model based on the chemical reactions
in the presence of light. Unchelated
) is formed from the photoreductive dissociation of ferric EDTA
EDTA). The photoproduced Fe(II) subsequently passes through the
ins) by diffusion. However, cellular Fe
binding ligands such as
ferrozine (FZ) and excess EDTA if present at appropriate concentrations. Solid arrows
Fe uptake experiment,
whereas dotted arrows indicate relatively minor reactions. Rate constants depicted near
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
40
Key processes in this model are photoreductive dissociation of FeIII
EDTA to Fe(II)',
oxygenation of Fe(II)' to Fe(III)', and formation and dissociation of FeIIEDTA. This
model is similar to that used by Sunda and Huntsman (2003) in describing the
photochemical transformation of FeEDTA complexes in seawater except that rate
constants differed as different media and light intensities were used, and
recomplexation of Fe(II)' followed by oxidation of the complex was also considered
here. Because inorganic Fe complexes are nonphotoreactive at circumneutral pH (King
et al., 1993), photo-reduction of Fe(III)' was ignored.
The rate constants for photo-reductive dissociation of FeIII
EDTA determined in this
work were reasonably similar to the values reported in seawater by Sunda and
Huntsman (2003) (4.4 × 10-6
s-1
at light intensity of 500 µmol-quanta.m-2
.s-1
) and
Anderson and Morel (1982) (1.7 × 10-6
s-1
at 95 µmol-quanta.m-2
.s-1
). However, in
contrast to the Sunda and Huntsman model (Sunda and Huntsman, 2003), Fe(III)'
formation via oxidation of photoproduced Fe(II)' is negligible in the freshwater model
presented here because complexation of Fe(II)' by EDTA or FZ occurs much faster
than Fe(II)' oxygenation (by a factor of at least 104 under the experimental conditions
described here, Table 3.4). Although rates of oxidation and photoproduction of Fe(II)'
are similar in both seawater and freshwater systems, recomplexation of Fe(II)' by
EDTA was ignored in the Sunda model as complexation of Fe' by EDTA in seawater is
very slow (23 M-1
.s-1
) due to substantial precomplexation of EDTA by Ca at the high
Ca concentrations in seawater, a subject that has been extensively discussed elsewhere
(Fujii et al., 2010a).
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
41
Table 3.3. Kinetic model for Fe transformation and uptake under the light.
Chemical reactions / diffusion parameters Rate constants/Parameter values
(a) Chemical reactions
FeIII
EDTA + hv → Fe(II)' + EDTAox khv 6.4 × 10-6 a)
s-1
Fe(II)' + EDTA → FeIIEDTA kf-EDTA 2.1 × 10
6 b) M
-1.s
-1
Fe(II)' + 3FZ → FeIIFZ3 kf-FZ 3.1 × 10
11 c) M
-3.s
-1
FeIIEDTA→Fe(II)' + EDTA kd-EDTA 1.2 × 10
-3 b) s
-1
FeIIFZ3→Fe(II)' + 3FZ kd-FZ 4.3 × 10
-5 c) s
-1
Fe(II)' + O2 → Fe(III)' + O2- kox 8.8
d) M
-1.s
-1
FeIIEDTA + O2 → Fe
IIIEDTA + O2
- kox-EDTA 31
e) M
-1.s
-1
Fe(II)' → uptake kup 3.9 × 10-9
L.cell-1
.s-1
(b) Diffusion parameters f)
Diffusion coefficient D 0.5-1 × 10–9
m2.s
-1
Radius of porin a 0.5-1.5 × 10–9
M
Length of porin channel l 2-7.5 × 10–9
M
Density of porin Nporin 0.79-3.3 × 1016
m-2
Surface area per cell As 1.1 × 10-10
m2
a) Average value for khv determined in the 1 µM and 10 µM total Fe systems. EDTAox represents photo-oxidized EDTA formed in the ligand-to-metal charge
transfer (LMCT) process. b)
kf-EDTA and kd-EDTA were determined in this work. See part A1.3 of Appendix 1 for detailed discussion of methods and results.
c) Thompsen and Mottola (1984).
d) Pham and Waite (2008a).
e) Fujii et al. (Fujii et al., 2010a).
f) Parameters for diffusion and bacterial outer-membrane porin
properties are carefully examined in part A1.5 of Appendix 1 (See also Tables A1.3 and A1.4 of Appendix 1).
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis
Table 3.4. Measured and calculated Fe uptake parameters for various culture conditions.
a) The first-order oxygenation rate constant for Fe(II)' at pH 8 was calculated by multiplying the second
saturated dissolved oxygen concentration of 0.24 mM at 27
experiments. All experiments were performed under nonsaturating conditions.
eq. 3.2. Average and ±standard deviations for all
calculate the steady-state concentrations for Fe(II)' and Fe
were performed on different dates. Although the
were slightly different possibly due to the different preconditioning of cells used.
Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
Measured and calculated Fe uptake parameters for various culture conditions.
order oxygenation rate constant for Fe(II)' at pH 8 was calculated by multiplying the second-order rate constant of 8.8 M
saturated dissolved oxygen concentration of 0.24 mM at 27oC.
b) Averaged
55Fe uptake rates and ±standard de
experiments. All experiments were performed under nonsaturating conditions. c)
kup value for each experimental condition was determined using
verage and ±standard deviations for all kup were also calculated. d)
See part A1.4 of Appendix 1 for detailed methods employed to
state concentrations for Fe(II)' and FeIIEDTA and time-averaged concentrations for Fe
IIFZ
were performed on different dates. Although the 55
Fe:EDTA ratio and cellular density were similar in these two experiments, the uptake rates
were slightly different possibly due to the different preconditioning of cells used.
42
order rate constant of 8.8 M-1
.s-1
by a
Fe uptake rates and ±standard deviations from triplicate
value for each experimental condition was determined using
A1.4 of Appendix 1 for detailed methods employed to
FZ3. e)
The 55
Fe uptake experiments
tio and cellular density were similar in these two experiments, the uptake rates
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
43
3.3.5. Fe Uptake Machinery
Under nonsaturating conditions, the rate of uptake of a particular substrate is
reasonably assumed to be proportional to the substrate concentration. Provided that the
steady-state concentration of Fe(II)' ([Fe(II)']ss) is controlled by photo-reductive
dissociation of FeIII
EDTA, complexation of photo-produced Fe(II)' by EDTA and FZ
(if present), dissociation of FeIIEDTA and Fe
IIFZ (if present), oxidation of Fe(II)' and
cellular uptake, then Fe uptake rate (ρFe mol.cell-1
.s-1
) can be described by:
ρFe
= kup
[Fe(II)']ss
=k
upk
hv[FeIIIEDTA] + k
d-EDTA[FeIIEDTA] + k
d-FZ[FeIIFZ
3]( )
kf-EDTA
[EDTA] + kf-FZ
[FZ]3 + k
up[cell] + k
ox[O
2]
(3.2)
where [cell] is the cell density (cell.L-1
) and relevant reaction details are listed in Table
3.3. As explained in part A1.4 of Appendix 1, the thermal dissociation of Fe(II)
complexes with EDTA or FZ significantly influences [Fe(II)']ss while Fe(II)' oxidation
has a negligible effect on this parameter. The uptake rate constant kup was estimated by
substituting 55
Fe uptake rates measured under various conditions together with other
known kinetic parameters into eq. 3.2 (see part A1.4 of Appendix 1 for details),
yielding an average value (± one standard deviation) of 3.9 (±1.9) × 10-9
L.cell-1
.s-1
(Table 3.4). Using this value, Fe uptake rates for a range of cell densities and
competitive ligand concentrations were calculated (Figure 3.8). This model
demonstrates that higher cell densities result in a decrease in [Fe(II)']ss and thus a
decrease in 55
Fe uptake rate (Figure 3.6). This situation will arise when the rate of
cellular uptake is comparable to or higher than the rate of formation of Fe(II)
complexes with EDTA and/or FZ (i.e., k
up[cell] ≥ k
f-EDTA[EDTA] + k
f-FZ[FZ]3 ).
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
Figure 3.8. Effect of competitive ligand concentrations and cellular densities on calculated Fe uptake rate using eq
Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
concentrations and cellular densities on calculated Fe uptake rate using eq
44
concentrations and cellular densities on calculated Fe uptake rate using eq. 3.2
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
45
The apparent high affinity of the cell for Fe(II) was further examined by considering
the possibility of passive diffusion of photo-produced Fe(II)' to the intracellular space.
In cyanobacteria, nutrients must pass through the outer-membrane into the periplasmic
space prior to active translocation across the inner-membrane to the cytosol. Some
specific forms of Fe (e.g., ferric siderophore complexes) are recognized by outer-
membrane receptors and actively transported into the intracellular compartments,
however for most small-sized hydrophilic nutrients (including metal ions) transport
from the external environment into the periplasmic space most likely occurs via
movement through non-selective transmembrane channels called porins, which are
ubiquitous in almost all Gram-negative bacteria investigated so far including
cyanobacteria (Nikaido, 2003). Given the concentration gradient across the outer-
membrane, a relationship between uptake rate and diffusional flux of a substrate in a
nonreactive cylindrical channel (Jporin mol.porin-1
.s-1
) may be formulated as follows:
ρFe
=Jporin
Nporin
AS = − Dπa2 1,000∆[Fe']
l
N
porinA
S (3.3)
where D is the diffusion coefficient (m2.s
-1), a is the radius of a porin (m), l is the
length of the channel (m), ∆[Fe'] (= in out[Fe'] - [Fe'] ) is the difference between Fe'
concentrations inside and outside the outer-membrane (M), Nporin is the number of
porins per square meter and AS is the surface area of a cell (m2). The diffusion layer
thickness of metal complexes in aqueous solution is generally on the order of tens of
micrometers (Buffle et al., 2009) such that the metal flux in proximity of the cell
surface would not be influenced by physical diffusion in the case of small
phytoplankton such as M. aeruginosa (cellular radius ~4 µm). Assuming that
unchelated Fe which enters the periplasm is rapidly captured by periplasmic Fe
transporters under the nonsaturating conditions investigated here (i.e., [Fe']in ≈ 0), then
out[Fe'] ~ -[Fe']∆ = [Fe(II)']ss, leading to an alternative expression for the uptake rate
constant kup:
k
up=
1,000Dπa2Nporin
AS
l (3.4)
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
46
Calculation using literature values for the diffusion coefficient of metal ions and
reported values for the properties of porins from several Gram-negative bacterial
species (Table 3.3) indicates that diffusion of Fe through such channels is faster than
an active transport process by a factor of 101-10
3. The upper limit for calculated kup
(0.06-4.1 × 10-9
L.cell-1
.s-1
, Table A1.4 of Appendix 1) is in accordance with the
measured value (i.e., 3.9 × 10-9
L.cell-1
.s-1
), regardless of the fact that all parameters
used were determined for bacterial species other than M. aeruginosa. Any
underestimation of the calculated uptake rate using this model may be because the
permeability of the outer-membrane of M. aeroginosa (determined by porin diameter,
length and density and Fe concentration gradient across the membrane) is greater than
that estimated here based on reported porin characteristics for other bacteria. The
diffusional model developed here for cyanobaterial Fe uptake contrasts with the Fe(II)s
uptake model for eukaryotic phytoplankta in the sense that the latter model considers
assimilation of extracellular Fe(II) as a process mediated by active membrane
transporters (Shaked et al., 2005).
3.4. IMPLICATION OF FINDINGS
The rate of uptake of Fe by M. aeruginosa in EDTA-buffered medium increased
substantially in the presence of light due to the photochemical transformation, at
wavelengths <500 nm, of FeIII
EDTA to a more labile form of Fe suitable for cellular
uptake. The inhibitory effect of the presence of both FZ and excess EDTA on 55
Fe
uptake rates, combined with prediction of the Fe species present using kinetic
modelling, consistently suggests that photochemically formed Fe(II)' is the major
substrate for uptake. Negligibly small uptake rates of Fe in the dark indicate that Fe
acquisition dominantly occurs during the daytime. Therefore, Fe influx during the day
is likely adequate for the biological functioning of the organism, which also occur in
the dark (e.g., respiration and dark reaction of photosynthesis). Consistent with this
notion, the steady-state Fe uptake rate (0.25-0.32 amol.cell-1
.hr-1
) calculated by
multiplying specific growth rate (µ=0.56-0.74 day-1
) by Fe quota (QFe = 7.2 amol.cell-
1) for 100 nM Fe and 26 µM EDTA culture medium (Fujii et al., 2010a) is comparable
to the short-term 55
Fe uptake rate measured at 200 nM Fe and 26 µM EDTA (0.18-0.23
amol.cell-1
.hr-1
, averaged over a 14 h:10 h light/dark cycle).
Chapter 3. Effect of Light on Iron Uptake by the Freshwater Cyanobaterium Microcystis aeruginosa
47
Although the uptake model developed using the well-characterized ligand EDTA
should be useful in ascertaining likely Fe availability to phytoplankton in laboratory
studies and natural environments, caution should be exercised before extending the
model to other microorganisms or aquatic systems. In contrast to previous observations
for Fe-stressed M. aeruginosa PCC7806 during short-term 55
Fe uptake incubations
(Fujii et al., 2010a), some cyanobacteria (e.g. Anabaena and Synechococcus) have
been shown to upregulate a siderophore-mediated Fe uptake system under Fe stress
(Ito and Butler, 2005), which presumably dominates over Fe influx by passive
diffusion. The biological response to external environment conditions and its effect on
Fe chemistry must be carefully evaluated in such cases. Additionally, the thermal- and
photolability of any Fe complexes present are important determinants of biological
uptake as are specific variables that depend on the nature of the ligand. For example,
using identical methods and conditions to those employed in this work, khv values for
the weaker ligands citrate and Suwannee River fulvic acid were determined to be
around one order of magnitude higher than that for EDTA (khv = 3.2 × 10-5
s-1
for
citrate and 1.7 × 10-5
s-1
for SRFA, unpublished data) suggesting that the rate of
bioavailable Fe formation will also be an order of magnitude greater. Under these
conditions the uptake rate reaches saturation (i.e. ~1-3 amol.cell-1
.hr-1
, unpublished
data), when ligand concentrations similar to those employed here or in natural
environments (e.g., <~10 mg.L-1
for SRFA) are used. When Fe transporters resident in
the periplasm or inner-membrane are saturated with Fe, the assumption that
out[Fe'] ~ -[Fe']∆ will not be valid and the Fe influx through the outer-membrane will
be influenced by the free Fe concentration in the periplasm. Such effects should be
investigated in future work by undertaking studies over a wider range of available Fe
concentrations. Regardless of the limitations of the model, this study provides
compelling evidence of the connection between Fe photolysis and biological uptake
and, as such, furthers understanding of Fe uptake by phytoplankton in natural waters.
48
CHAPTER 4
KINETICS OF EXTRACELLULAR IRON
TRANSPORT TO PERIPLASMIC AND
CYTOPLASMIC COMPARTMENTS OF
THE FRESHWATER CYANOBACTERIUM
MICROCYSTIS AERUGINOSA
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
49
4.1. INTRODUCTION
Iron (Fe) is of great importance for the growth of phytoplankton including
cyanobacteria as a cofactor of a range of proteins responsible for primary metabolic
processes including photosynthesis, chlorophyll synthesis, nitrogen fixation and
respiration as well as regulation of incidental oxidative stress (Crichton, 2009). Fe is
recognized as one of the critical nutrients limiting the growth of phytoplankton in one-
third of the world’s open ocean (Boyd et al., 2007) and some specific areas of coastal
and fresh waters where Fe availability is low (Hutchins and Bruland, 1998, Nagai et
al., 2006). Fe nutrition has also been found to be highly relevant to the biosynthesis of
secondary metabolite cyanotoxins, although the physiological functioning of these
mysterious molecules is still under debate (Alexova et al., 2011).
Over the last three decades, the kinetics and mechanisms of Fe acquisition by
phytoplankton have been studied with particular attention given to eukaryotes
(Anderson and Morel, 1982, Hudson and Morel, 1990, Shaked et al., 2005, Morel et
al., 2008) partially due to the relative abundance of diatoms in coastal and marine
ecosystems (Boyd et al., 2007). Although a number of recent studies have been
undertaken with a view to clarifying the mode of Fe uptake by cyanobacteria (Fujii et
al., 2010a, Fujii et al., 2011a, Salmon et al., 2006), existing kinetic models for Fe
uptake assume that only a single (plasma-)membrane acts as a major “permeability
barrier” in the selective transport of extracellular Fe into the cytoplasm. Cyanobacteria
are taxonomically classified as gram-negative bacteria, which possess two structurally
different phospholipid membranes surrounding the cellular body (i.e., the outer and
inner membranes) in addition to a thylakoid membrane, which is noncontiguous with
the inner plasma-membrane in the case of phototorophs (Spence et al., 2003).
Therefore, to transport nutrients from the external environment to the thylakoid or
cytoplasm where most nutrient-dependent biochemical metabolic reactions take place,
the nutrients must sequentially cross these membranes as well as the periplasmic space
between the outer and inner membranes.
The permeability of the lipid bilayer outer membrane depends on the type of transport
system involved (i.e., specific or non-specific transport) and the physicochemical
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
50
properties of substrates including size, polarity and charge as well as specificity to
outer membrane receptors. In some cases, uptake of specific substrates such as ferric
siderophore complexes and vitamin B12 is facilitated by specific recognition of these
substrates by outer membrane receptors (e.g., FepA and FhuA for enterobactin and
ferrichrome respectively) and subsequent active transport to intracellular
compartments via what is known as the Ton system, involving a giant protein complex
spanning from the inner to outer membrane (Faraldo-Gomez and Sansom, 2003,
Andrews et al., 2003). In contrast to such a high-affinity transport system, most small
hydrophilic nutrients (including ionic metals) are passively transported to the
periplasm via an alternative relatively lower-affinity pathway through water-filled
transmembrane channels embedded in the outer membrane, which is otherwise
impermeable to hydrophilic molecules (Hoiczyk and Hansel, 2000, Nikaido, 2003,
Jones and Niederweis, 2010). Pore-forming protein channels characterized by a β-
barrel structure (referred to as porins) are the major type of protein that is ubiquitously
found in the outer membrane of almost all gram-negative bacteria investigated so far,
including cyanobacteria (Hoiczyk and Hansel, 2000, Nikaido, 2003). Previous work
has indicated that this type of membrane channel in proteobacteria allows nonselective
permeation of hydrophilic molecules with size less than ~550-650 Da (Nikaido, 1976,
Nikaido, 1979) with the movement of nutrient in the porin most likely driven by
molecular diffusion or in some cases by slight interaction with low-affinity binding
sites (Nikaido, 2003, Pages et al., 2008). Although such information for cyanobacteria
is limited, recent studies using Fe-limited freshwater cyanobacteria Microcystis
aeruginosa PCC7806 and Anabaena flos-aquae UTEX1444 have provided indirect
evidence that only unchelated Fe is capable of permeating the outer membrane during
a short-term incubational assay with this process being independent of the siderophore-
mediated system (Fujii et al., 2010a, Wirtz et al., 2010).
Mechanisms involved in intracellular Fe transport may be quite different for the outer
membrane, periplasm and inner plasma-membrane. In the periplasm, putative
periplasmic Fe binding proteins (FutA) have been identified for cyanobacteria such as
Microcystis (Alexova et al., 2011) and Synechocystis (Katoh et al., 2001, Waldron et
al., 2007, Badarau et al., 2008), implying the selective transport or storage of Fe in this
compartment. Fe translocation across the inner membrane of proteo- and cyanobacteria
is most likely an energy-dependent process mediated by ATP-binding cassette (ABC)
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
51
type transporters for ferrous (FeoB) (Velayudhan et al., 2000, Andrews et al., 2003)
and ferric iron (FutB and FutC) (Katoh et al., 2001) in contrast to the diffusive
permeation of nutrients through the outer membrane.
Given the presence of an additional compartment enclosed by the outer and inner lipid
bilayers in cyanobacterial cells, the Fe uptake machinery for this type of phytoplankton
must couple the process of Fe entering the outer compartment with energy-dependent
Fe transport to the inner compartment. To our knowledge, there are no detailed studies
of Fe transport by cyanobacteria taking such intracellular compartments into account.
In this work we investigate the kinetics of transport of extracellular Fe to periplasmic
and cytoplasmic spaces in the freshwater cyanobacterium Microcystis aeruginosa by
measuring the accumulation kinetics of radio-labeled 55
Fe in chemically well-defined
media. Periplasmic and cytoplasmic 55
Fe concentrations were monitored using the cold
osmotic shock technique that has previously been used for the extraction of
periplasmic substances in a range of gram-negative bacterial species including
cyanobacteria (Neu and Heppel, 1965, Fulda et al., 1999). A recently developed
kinetic model (Fujii et al., 2010a) is extended to accommodate the Fe uptake data
obtained here.
4.2. MATERIALS AND METHODS
4.2.1. Reagents
Unless otherwise stated, chemicals were purchased, prepared and stored as described
in Section 2.1, Chapter 2. For cell culturing, Fraquil* medium was prepared at pH 8
(see Section 2.2.1, Chapter 2 for the detailed preparation of Fraquil* medium) where
the transformation kinetics of extracellular Fe are well understood (Fujii et al., 2010a).
To avoid Fe contamination, the culturing medium was prepared in a trace metal clean
room supplied with HEPA-filtered air using reagent grade chemicals, resulting in Fe
contamination of less than 1 nM, as described elsewhere (Fujii et al., 2010a). Solutions
of 100 mM Na2EDTA (disodium ethylenediaminetetraacetate, Sigma) and 2.6-26 mM
Na3citrate (Sigma) were prepared at pH 8 as 55
Fe-binding ligand stock solutions.
Solutions of radiolabelled Fe complexes were then made by mixing 55
FeCl3 solution
(in 0.5 M HCl, 185 MBq, Perkin-Elmer, Australia) either with the Na2EDTA or
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
52
Na3citrate solutions in 1.5 mL polypropylene tubes followed by addition of 2 mM
NaHCO3 (Sigma, pH 8) to maintain circumneutral pH, providing final concentrations
of 24 µM 55
Fe, 34 mM EDTA, 0.17-6.9 mM citrate and 1-1.3 mM NaHCO3. The 55
Fe-
labelled complex stock solutions were equilibrated for 24 h prior to use.
A pH 7 solution containing 50 mM Na2EDTA and 100 mM Na2oxalate (Sigma)
(hereafter referred to as “EDTA/oxalate”) was used to remove Fe that was
nonspecifically retained on the cell surface and filter (Tang and Morel, 2006). A
solution of 2 mM NaHCO3 (pH 8) was also used to rinse the cells after the
EDTA/oxalate wash. Plasmolysis solutions at pH 8 were prepared with final
concentrations of 10 mM Tris-HCl (Sigma), 2 mM NaHCO3, 1 mM Na2EDTA and
either 0.5 M D-sorbitol, sucrose or NaCl (Sigma). All plasticware and glassware used
for cell culturing and analysis were acid cleaned using 0.1 M HCl.
4.2.2. 55
Fe Accumulation Experiments
The short-term accumulation rate of 55
Fe by M. aeruginosa strains PCC7806 and PCC
7005 was measured by incubating cells in Fraquil* containing
55Fe complexed by
either citrate or EDTA (see Section 2.3, Chapter 2 for the detailed description of the
short-term 55
Fe uptake experiment). Prior to the Fe accumulation assay, the cultured
cells were filtered on to a 25 mm diameter, 0.65 µm pore size PVDF membrane
(Millipore) and then rinsed firstly by passing 5 mL of EDTA/oxalate solution three
times and subsequently 10 mL of 2 mM NaHCO3 three times through the filter (total
rinsing time was ~10 min). The washed cells were then re-suspended into Fe- and
ligand free Fraquil* medium to provide cell densities of ~3 × 10
6 cell.mL
-1. Fe
accumulation experiments were initiated by adding a solution containing the 55
Fe-
ligand complex to the cultures at concentrations of 0.7 µM Fe, 2-11 mM EDTA and 5-
200 µM citrate. To examine the effect of Fe(II) and Fe(III) transformation on the rate
and extent of 55
Fe uptake, the assay was also undertaken in the presence of 1 mM
ascorbate and 100 µM ammonium tetrathiomolybdate (TTM). All Fe accumulation
experiments were conducted at 27oC under dark conditions to avoid complexities
associated with photochemical transformation of Fe complexes with EDTA and citrate.
After incubation, samples were harvested by vacuum-filtering on to 0.65 µm PVDF
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
53
membrane filters then rinsed three times with 1 mL of EDTA/oxalate solution and
once again with 1 mL of 2 mM NaHCO3 (total rinsing time was ~10 min).
To determine the amounts of 55
Fe accumulated in periplasmic and cytoplasmic spaces,
the harvested cells were subjected to cold osmotic shock in order to extract periplasmic
substances as a cold water fraction. In this process, filtered cells were incubated in 5
mL of 0.5 M D-sorbitol for 10 min. After vacuum-filtering to remove the solution, the
cells were then exposed to cold ultrapure water (5 mL, Milli-Q) for a further 10 min.
The cold water extract (containing the periplasmic 55
Fe) and the remaining cellular
mass on the filter (containing cytoplasmic 55
Fe as well as any Fe tightly bound to the
inner membrane) were each placed in glass scintillation vials. After the addition of 5
mL of scintillation cocktail (Beckman ReadyScint) to the fractionated samples, the
activity was measured in a Packard TriCarb Liquid Scintillation Counter. Scintillation
counts (counts per minute) of the samples were converted to moles of Fe by using
concurrent counts of 5-50 µL of 55
Fe-ligand stock in 5 mL of scintillation cocktail.
Process blanks were determined by performing the entire procedure in the absence of
cells.
The permeability of the outer membrane to Fe species was examined using the
plasmolysis solutions and bicarbonate buffer as the assay medium instead of Fraquil*.
Three types of plasmolysis solutions (D-sorbitol, sucrose and NaCl) were used to
examine the effect of solute molecular size on periplasmic 55
Fe accumulation. After
cells were resuspended in the assay medium, short-term 55
Fe accumulation assays were
initiated by adding 55
Fe-EDTA solution to the cultures at final concentrations of 0.7
µM 55
Fe and 2 mM EDTA. In the plasmolysis solution, the bacterial periplasmic space
can be enlarged to ~40-50% of total cell volume due to the high osmotic pressure
(Nikaido, 1979, Rose, 1982). In addition, in this assay, the presence of excess EDTA
ensured that 55
Fe was present almost exclusively in complexed form with minimal
transport of unchelated Fe into the cytoplasm. The assay was also undertaken at a
particularly high concentration of EDTA (11 mM) by using the plasmolysis solution
with D-sorbitol, which was prepared with 10 mM Na2EDTA. After incubating for 0.5
and 1 h at 27oC in the dark, cells were harvested by filtration and rinsed with the assay
medium. Total 55
Fe in the cells was then determined by collecting the filtered cells and
measuring radioactivity with the liquid scintillation counter.
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
54
4.2.3. Determination of Periplasmic Fe(II)
To determine the concentration of periplasmic Fe(II), the well-developed FeLume
chemiluminescence technique that has now been widely used for determination of
subnanomolar Fe(II) (King et al., 1995) was employed. In a manner identical to that
described for the 55
Fe accumulation experiment, M. aeruginosa PCC7806 cells were
harvested from the long-term incubation by filtration. The cells were then resuspended
in fresh Fraquil* which contains non-radiolabelled Fe and citrate at concentrations of
0.7 µM and 100 µM, respectively. Incubation was performed under dark condition
overnight. The incubation was also undertaken in the presence of 1 mM ascorbate and
100 µM TTM in order to examine the effect of Fe(II) and Fe(III) transformation on
Fe(II) accumulation in the periplasmic space. After the incubation, the cells were
filtered, washed with the EDTA/oxalate and bicarbonate solution, and subjected to the
cold osmotic shock protocol using the 0.5 M sorbitol solution. The cold water extract
(pH of 7.0-7.1) was immediately introduced to the flow cell of the FeLume system
where it was mixed with 0.5 mM luminol (5-amino-2,3-dihydro-1,4-phthalazinedione,
Sigma) chemiluminescence reagent prepared in 1 M ammonia (pH 10.3). The emitted
light arising from the reaction of the luminol reagent and Fe(II) present in the extract
was quantified by photomultiplier tube. The preparation of reagents, instrument
settings, measurement conditions and system calibration were all performed in a
manner identical to that described elsewhere (Fujii et al., 2010b). The total amount of
periplasmic Fe present was also determined by the procedure described above
involving the use of radiolabelled Fe.
4.2.4. Determination of Steady-state Concentration of Extracellular
Unchelated Fe
Speciation of extracellular Fe for the various conditions in the 55
Fe accumulation
experiments was calculated by use of the kinetic model described in Table 4.1A
(details of the calculation are provided in parts A2.1 and A2.2 of Appendix 2). As
discussed in Appendix A2.1, the effect of diffusional influx in the calculation of
steady-state concentration of extracellular unchelated Fe (Fe′) is negligible due to the
small concentration gradient created by extracellular and periplasmic Fe′.
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
55
4.2.5. Analysis of Genome Sequences
The genome sequences of M. aeruginosa strains PCC7806 and NIES843 (whose
genomes have been completely decoded (Kaneko et al., 2007, Frangeul et al., 2008)
were examined by use of the National Center for Biotechnology Information database.
4.3. RESULTS AND DISCUSSION
4.3.1. Accumulation of 55
Fe in the Periplasm and Cytoplasm
In the Fe(III)-dominant system buffered by citrate (concentrations of Fe and citrate
were 0.7 µM and 20 µM, respectively, yielding an extracellular pFe′ of 9.3), 55
Fe
concentrations in the periplasmic and cytoplasmic fractions increased monotonically
and approached steady-state after several hours (Figure 4.1). The concentration of
periplasmic 55
Fe (< ~0.2 amol.cell-1
) was an order of magnitude less than the
cytoplasmic 55
Fe concentration (< ~5 amol.cell-1
) over the duration of the experiments
described here. Accumulation of periplasmic and cytoplasmic 55
Fe was also examined
over a range of Fe:citrate ratios yielding pFe′ from 8.6 to 10.8. The rate of cytoplasmic
55Fe accumulation was linearly correlated with the steady-state concentration of
periplasmic 55
Fe measured in 9 h incubations (R2 > 0.97 for both strains, part A of
Figure 4.2). The cytoplasmic accumulation rate also increased with increasing
extracellular Fe' concentration (part B of Figure 4.2).
In common culturing media with low osmotic pressure, the periplasmic space of gram-
negative bacteria has been determined to constitute approximately 5-40% of the total
cellular volume, depending on the type of microorganism and method of analysis
employed (Nikaido, 1979, Rose, 1982). If we assume that the concentration of 55
Fe in
the periplasm is comparable to that in the cytoplasm at saturation (e.g., after 5 h),
measured 55
Fe concentrations in each compartment under these conditions (Figure 4.1)
indicate that the periplasmic space has a volume of only 2.5-3% that of the cytoplasm
for both strains of M. aeruginosa. In the 1 h incubations,
the amount of 55
Fe
accumulated in the plasmolysed cells where the periplasmic space is enlarged (< 0.05
amol.cell-1
, pFe′ = 14.0, Figure 4.3) was even less than the periplasmic 55
Fe that was
extracted from the non-plasmolysed cells using cold osmotic shock (0.07-0.12
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
56
amol.cell-1
in 1h incubation with Fraquil*, part A of Figure 4.1). Addition of EDTA at
5.5-fold higher concentration (11 mM EDTA yielding pFe′ of 14.7) or use of media
with low osmotic pressure (Fraquil* and 2 mM bicarbonate buffer) yielded no
substantial changes in periplasmic 55
Fe accumulation.
Figure 4.1. Time course of 55
Fe accumulation in (A) periplasm and (B) cytoplasm for
M. aeruginosa strains PCC7806 (filled symbols) and PCC7005 (open symbols) grown
under moderate Fe limitation. Fe uptake assays were performed for 9 h in Fraquil* at
concentrations of 0.7 µM for 55
Fe and 20 µM for citrate. Symbols and error bars
represent the mean and ± standard deviation from triplicate experiments. Solid and
dashed lines represent the calculated values for PCC7806 and PCC7005, respectively,
using (A) eq. 4.7 and (B) the integrated form of eq. 4.5 with Fe uptake parameters
listed in Table 4.1. Detailed 55
Fe accumulation data are provided in Table A2.1 of
Appendix 2.
Cy
top
lasm
ic 5
5F
e
(am
ol.
cell
-1)
Pe
rip
lasm
ic 5
5F
e
(am
ol.
cell
-1)
0
1
2
3
4
5
6
7
0 2 4 6 8 10
B.
0
0.05
0.1
0.15
0.2
0.25
0 2 4 6 8 10
A.
Time (hr)
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
57
Figure 4.2. Cytoplasmic accumulation rate of 55
Fe by Fe-limited M. aeruginosa strains
PCC7806 (filled symbols) and PCC7005 (open symbols) as a function of (A) steady-
state concentration of total periplasmic 55
Fe and (B) calculated concentration of
unchelated Fe in the extracellular environment and periplasm. Data were obtained
from the assay using Fraquil* at concentrations of 0.7 µM for
55Fe and 5-200 µM for
citrate. Solid and dashed lines in panel A were determined for PCC7806 and
PCC7005, respectively, by linear regression analysis (p<0.05, n=15). In panel B, the
solid and dashed lines represent the calculated values using eq. 4.6 for PCC7806 and
PCC7005, respectively. Detailed 55
Fe accumulation data are provided in Table A2.2 of
Appendix 2. Calculated values of unchelated Fe concentrations are provided in Table
-20
-19.5
-19
-18.5
-18
-17.5
-17
-12 -11 -10 -9 -8
B.
Acc
um
ula
tio
n r
ate
of
cyto
pla
sm
ic 5
5F
e (
am
ol.
ce
ll-1
.hr-
1)
log [Fe'] (≈ log [Fe'peri] ) (M)
y = 10.0x - 0.22R² = 0.96
y = 6.9x - 0.14R² = 0.94
-0.5
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.1 0.2 0.3 0.4
A.
Steady-state periplasmic 55Fe (amol.cell-1)
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
58
A2.3 of Appendix 2. Symbols and error bars represent the mean and ± standard
deviation from triplicate experiments.
Figure 4.3. Cellular 55
Fe accumulation in the various culturing media at pH 8
(plasmolysis solutions, Fraquil* and 2 mM NaHCO3). Plasmolysis solutions were 0.5
M D-sorbitol, sucrose or NaCl (buffered by 10 mM Tris-HCl, 2 mM NaHCO3 and 1
mM for Na2EDTA) and 0.5M D-sorbitol with high EDTA concentration (10 mM). The
incubation experiment was initiated by addition of 55
FeEDTA to the culture media at
final concentrations of 0.7 µM for 55
Fe and 2 mM for EDTA. In case of the 0.5M D-
sorbitol solution containing high EDTA, the final concentration of EDTA was adjusted
to 11 mM. All incubations were performed for 30 or 60 min in the dark at pH 8 with
Fe-limited Microcystis aeruginosa (PCC7806).
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.1
30 min 60 min
D-sorbitol
Sucrose
Sodium Chloride
High EDTA
Fraquil*
Bicarbonate
Incubation period
Ce
llu
lar
55F
e a
ccu
mu
lati
on
(am
ol
cell
-1)
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
59
Table 4.1. Kinetic parameters used for the calculation of intracellular Fe transport and
extracellular Fe transformationa.
Parameters Unit
Microcystis strains
PCC7806 PCC7005
A. Rate constants for calculation of unchelated Fe concentration in extracellular
environment
kf-Cit b
M
-1.s
-1 2.1 × 10
5
kd-Cit c
s
-1 2.7× 10
-4 – 2.8 × 10
-3
kf-EDTA d
M
-1.s
-1 3.5 × 10
5
kd-EDTA e
s
-1 1.0× 10
-5
B. Parameters for calculation of Fe uptake and intracellular Fe transport
max
Fe'(peri)ρ f amol.cell
-1.h
-1 2.1 (±0.38)
*** 2.9 (±0.54)
***
Fe'(peri)K
f
pM 360 (±97)**
420 (±110)*
[Xperi]T g
amol.cell-1
0.30 0.29
k+1 h M
-1.s
-1 1.2 × 10
9 2.5 × 10
8
k-1 i s
-1 4.3 × 10
-1 1.0 × 10
-1
k2 j s
-1 1.9 × 10
-3 2.8 × 10
-3
kdif k
L.cell-1
.s-1
3.9 × 10-9
a Detailed procedure for determination of parameters was described in part A2.1 of
Appendix 2.
b kf-Cit: complexation rate constant of ferric citrate complex (Fujii et al., 2010a)
(reaction, f-Cit IIIFe(III)+Cit Fe Citk→ ).
c kd-Cit: dissociation rate constant for ferric citrate (Fujii et al., 2010a) (reaction,
d-CitIIIFe Cit Fe(III)+Citk→ ), which is a function of citrate concentration ([Cit]) due
to the formation of two mononuclear ferric citrate complexes (FeIII
Cit and FeIII
Cit2).
d kf-EDTA: complexation rate constant for ferric EDTA complex (Fujii et al., 2010a)
(reaction, f-EDTA IIIFe(III)+EDTA Fe EDTAk→ )
e kd-EDTA: dissociation rate constant for ferric EDTA complex (Fujii et al., 2010a)
(reaction, d-EDTAIIIFe EDTA Fe(III)+EDTAk→ ).
f To determine
max
Fe'(peri)ρ and Fe'(peri)K , the log-transformed eq 4.6 (eq. A2-1 of Appendix
2) was fitted to the data shown in part B of Figure 4.2 by nonlinear regression analyses
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
60
using R version 2.13.0 (free software for statistical computation). Asterisks represent
statistically significant levels as follows: ***, P < 0.001; **, P < 0.01; *, P < 0.05.
g [Xperi]T was determined from max
Fe'(peri)ρ = k2[Xperi]T.
h k+1 was determined from by fitting eq. 4.7 to the data shown in part A of Figure 4.1.
i k-1 was calculated from
Fe'(peri)K = (k-1+k2)/k+1.
j k2 was determined by linear regression analysis (eq. 4.5) to the data shown in part A
of Figure 4.2.
k Diffusional constant for unchelated Fe (Fujii et al., 2011a).
4.3.2. Fe Species Translocated from the External Environment to the
Periplasm
The apparent dependency of cytoplasmic Fe accumulation rates on the unchelated Fe
concentration (part B of Figure 4.2) suggests that Fe uptake is initiated by influx of
extracellular unchelated Fe into the periplasmic space. However, there is some
evidence that proteobacterial porin channels allow permeation of hydrophilic
molecules with molecular weight less than 650 Da (Nikaido, 1976, Nikaido, 1979),
potentially including chelated Fe as used in this work. Since it is difficult to identify
the Fe species resident in the periplasmic space when the periplasmic 55
Fe
concentration is substantially smaller than the total cellular 55
Fe concentration, 55
Fe
accumulation assays were performed in plasmolysis solutions where the periplasmic
space can be enlarged to approximately half of the total cell volume. As the
plasmolysis solutions contain excess EDTA, Fe transport to the cytoplasm is negligibly
slow under these conditions due to low Fe′ concentration (pFe′ > 14), and almost all
detectable 55
Fe in the medium and inside cells (if present) is expected to be present as
FeEDTA. We estimated that ~1.6-2.5 amol.cell-1
would be accumulated in the enlarged
periplasmic space if FeEDTA were to pass through the outer membrane. However,
55Fe accumulation in the plasmolysed cells was very small in 1 h incubations (< 0.05
amol.cell-1
; Figure 4.3). Therefore, the results support the notion that Fe bound to
relatively small metal-buffering organic ligands is unable to cross the outer membrane
of Microcystis, consistent with the previous finding that only unchelated Fe is capable
of crossing the outer membrane of the bacterium Micobacterium smegmai in citrate-
buffered medium (Jones and Niederweis, 2010).
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
61
The relatively low size-exclusion limit (less than ~340 Da) for porins in Microcystis
cells seems reasonable given the lower single channel conductance (i.e., size-
exclusion) of cyanobacterial porins (e.g., SomA) compared to typical proteobacterial
porins (e.g., OmpF and OmpC) (Hoiczyk and Hansel, 2000). It also has been reported
that while the complexation of polyamines to typical bacterial porin channels inhibits
substrate transport by modulating the channel proteins to a closed state, such treatment
does not significantly affect cyanobacterial Fe uptake (Sonier et al., 2011), suggesting
that types of porin other than OmpF and OmpC may be responsible for cyanobacterial
outer membrane nutrient transport (Hoiczyk and Hansel, 2000). This argument is
further supported by the absence of genes encoding for typical porins in proteobacteria
in the genome sequences of M. aeruginosa strains PCC7806 and NIES843.
Although one of the protein homologues involved in the siderophore-mediated system
(TonB) has been identified in cyanobacterial genomes, including those for the strains
of M. aeruginosa used in this work and in Anabaena sp. (Nicolaisen et al., 2008),
recent studies on cyanobacterial Fe uptake consistently suggest that, in contrast to
proteobacteria (Andrews et al., 2003), at least short-term Fe acquisition proceeds in a
manner independent of cellular exudates such as siderophores even under Fe-limitation
(Wirtz et al., 2010, Fujii et al., 2010a). Furthermore, the direct uptake of ferric citrate
by the specific receptor and transporters (FecA-E) used by proteobacteria (Andrews et
al., 2003) appears unlikely for Microcystis due to the dependency of 55
Fe uptake on the
unchelated Fe concentration rather than total ferric citrate concentration (part B of
Figure 4.2).
4.3.3. Fe Species Translocated from the Periplasm to the Cytoplasm
Once extracellular Fe enters the periplasm, specific forms of Fe (e.g., Fe bound to
periplasmic or transporter proteins) are eventually translocated to the cytoplasm. For
some proteobacteria and cyanobacteria such as Synechosystis PCC6803, free or
membrane-anchored periplasmic Fe-binding proteins (FutA) and membrane
transporters (FutB, FutC and FeoB) play significant roles in ferrous and ferric iron
transport into the cytoplasmic space (Velayudhan et al., 2000, Katoh et al., 2001,
Andrews et al., 2003, Waldron et al., 2007). Examination of gene sequences from M.
aeruginosa strains PCC7806 and NIES843 indicates that these strains also have a
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
62
homologue of the GTP-dependent plasma-membrane Fe(II) transporter (FeoB).
Although no information on the synthesis of FutA proteins was retrieved from a
similar genome analysis, a recent study on M. aeruginosa PCC7806 has suggested the
presence of FutA2 for ferric iron transport that is expressed in the periphery of cells
(Alexova et al., 2011). Since FutA2 is generally fractionated in the extract of the cold
osmotic shock method (Waldron et al., 2007) as water-soluble membrane-free
proteins, Fe bound to this protein is also expected to be found in the periplasmic Fe
fraction of M. aeruginosa. In contrast to FutA2, FutA1 (which is a water-insoluble
membrane-associated protein generally copurified with the photosystem) has been
identified recently as a homologue of an Fe deficiency-induced protein (IdiA) for the
protection of photosynthesis (Tolle et al., 2002).
4.3.4. Model for Translocation of Fe from the External Environment
Given the evidence that (i) small hydrophilic molecules such as unchelated metals are
capable of diffusing into outer membrane porin channels, and (ii) M. aeruginosa
generally possesses periplasmic Fe-binding proteins and active Fe transporters across
the inner membrane, a simple kinetic model to describe Fe transport from the
extracellular medium to the intracellular environment is proposed in Figure 4.4. The
model presented is based on the following assumptions:
(i) Unchelated Fe in the extracellular environment is capable of crossing outer
membrane channels into the periplasmic space in a diffusive manner. This
process is reversible, such that unchelated Fe in the periplasm also diffuses out to
the extracellular environment.
(ii) Unchelated Fe in the periplasm forms complexes (FeXperi) with periplasmic Fe-
binding ligands (Xperi) followed by irreversible translocation of Fe into the
thylakoid membrane or cytoplasm (Fe) by inner membrane transporters, with the
latter process occurring in a first order manner. Although we assign FeXperi as a
single major substrate for the transport of iron to the cytoplasm for simplicity,
FeXperi can be considered to represent any form of periplasmic ferric and ferrous
iron transported into the thylakoid membrane or cytoplasm.
(iii) FeXperi accounts for the majority of periplasmic Fe (Feperi) (i.e., [Feperi] =
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
63
[FeXperi] + [Fe′peri] ≈ [FeXperi]). Given the average cellular volume of M.
aeruginosa (~270 µm3) (Wiedner et al., 2003) and a periplasmic volume of ~3%
relative to total cell volume, the concentration of total periplasmic Fe is
calculated to be ~10-5
M, which is much higher than [Fe′] (< 10-9
M). As
described below, the steady-state concentration of Fe′ in the periplasm (Fe′peri)
was determined to be approximately equal to the concentration of extracellular
unchelated Fe (i.e., [Fe′peri] ≈ [Fe′]).
Figure 4.4. Kinetic model for Fe transport from the extracellular environment to the
intracellular environment in cyanobacteria. In the extracellular environment,
unchelated Fe (i.e., Fe′) is formed due to the (thermal or reductive) dissociation of
chelated Fe. Unchelated Fe subsequently diffuses through non-specific outer
membrane channels (such as porins). Unchelated Fe in the periplasm is then
complexed by one or more periplasmic Fe-binding ligands (FeXperi) followed by
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
64
translocation of Fe into the cytoplasm (Fecyto) by inner membrane Fe transporters. A
possible mechanism of Fe(III) and Fe(II) transformation in the periplasm is also
illustrated. Solid arrows represent major reactions considered in the model. Rate
constants depicted near the arrows correspond to those listed in Table 4.1. MCO:
multi-copper oxidase, FeoB: ferrous iron transporter, FutA: ferric iron transporter.
The chemical reactions that describe Fe transport to the intracellular environment can
be described as follows:
' '
periFe FeDiffusion→←
(4.1)
1
1
'
peri peri periFe + X FeX k
k
+
−
→← (4.2)
2
peri cytoFeX Fek→ (4.3)
where k+1, k-1, and k2 represent rate constants for formation and dissociation of the
periplasmic Fe complex and translocation of periplasmic Fe to the cytoplasm,
respectively. According to this model, the time-dependent changes of periplasmic and
cytoplasmic Fe concentrations can be described as follows:
'
peri peri peri
peri '
1 peri peri 1 2 peri
[Fe ] ([Fe ] [FeX ])
[FeX ][Fe ][X ] ( )[FeX ]
d d
dt dt
dk k k
dt+ −
+=
≈ = − +
(4.4)
cyto
2 peri
[Fe ][FeX ]
dk
dt= (4.5)
Making a quasi-steady-state approximation for FeXperi, the rate of Fe accumulation in
the cytoplasm ( Fe'ρ ) can be described, in accord with previous findings (Fujii et al.,
2010a), by Monod-type kinetics in terms of the periplasmic Fe′ concentration as
follows:
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
65
max '
Fe'(peri) peri
Fe' 2 peri SS '
Fe'(peri) peri
[Fe ][FeX ]
[Fe ]k
K
ρρ = =
+
(4.6)
where [FeXperi]SS indicates steady-state concentration of the periplasmic Fe complex,
max
Fe'(peri)ρ (= k2[Xperi]T where [Xperi]T is the total concentration of the periplasmic Fe-
binding ligand) is the maximum uptake rate and Fe'(peri)K (= (k-1+k2)/k+1) is the half
saturation constant under the conditions of the experiment. Integration of eq. 4.4 yields
the concentration of periplasmic Fe as a function of time as follows (see part A2.3 of
Appendix 2 for detailed derivation):
'
peri peri T '
peri Fe'(peri) peri 1'
Fe'(peri) peri
[Fe ][X ][FeX ] 1 exp ( +[Fe ])
+[Fe ]K k t
K+
= − − ⋅ (4.7)
At steady-state, the translocation rate of periplasmic Fe to the cytoplasm (i.e., Fe'ρ ) is
equivalent to the net rate of diffusional Fe influx that occurs across the outer
membrane (JFe′ mol.cell-1
.s-1
), yielding the following relationship between average
concentrations of Fe′ in the periplasm ([Fe′peri]) and bulk external environment ([Fe′]):
Fe' Fe'J ρ=
max '
Fe'(peri) peri'
dif '
Fe'(peri) peri
' max ' max 2 2 '
dif Fe'(peri) dif Fe'(peri) dif Fe'(peri) dif Fe'(peri) dif Fe'(peri)'
peri
dif
[Fe ][Fe ]
[Fe ]
( [Fe ] ) ( [Fe ] ) 4 [Fe ][Fe ]
2
kK
k K k k K k k K
k
ρ
ρ ρ
− ∆ =+
− − + + − + +=
(4.8)
where kdif is the diffusional constant for unchelated Fe (L.cell-1
.s-1
), and ∆[Fe′] is the
difference between unchelated Fe concentrations in extracellular bulk medium and
periplasmic space (∆[Fe′]=[Fe′peri]-[Fe′]).
By fitting eqs. 4.5-4.7 to the data in Figure 4.1 and part B of Figure 4.2 under the
assumption of [Fe′peri] ≈ [Fe′], parameters for Fe uptake and intracellular transport
were obtained as listed in part B of Table 4.1. Values for [Fe′peri] and [Fe′] were then
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
66
calculated using eq. 4.8 and the parameters in part B of Table 4.1, suggesting that
[Fe′peri] is comparable to [Fe′] across a range of [Fe′] concentrations from 1 fM to 10
nM ([Fe′peri]/[Fe′] > 0.999). Therefore, the approximation [Fe′peri] ≈ [Fe′] was justified
in this work (see part A2.2 of Appendix 2 for details). Overall, this model (with
relevant parameters) described the time- and concentration-dependent kinetics of Fe
transport reasonably well (Figures 4.1 and 4.2).
An intriguing finding is that the diffusional flux of Fe across the outer membrane of M.
aeruginosa (reported to be 3.9 × 10-9
L.cell-1
.s-1
; see kup in eq. 4.2 shown in reference
(Fujii et al., 2011a) is three orders of magnitude greater than k+1[Xperi]T in this work
(~10-12
L.cell-1
.s-1
). This result suggests that the diffusion of extracellular Fe′ occurs at
much faster rate than complexation by the periplasmic Fe-binding ligand and is a
particularly important factor controlling Fe′ concentration in the periplasm. Another
interesting result is that a higher transport rate of periplasmic Fe by the membrane
transporter (k2) was seen in the non-toxic strain. This finding is consistent with the
previously reported upregulation of Fe transporter proteins for the non-toxic strain
under Fe stress (Alexova et al., 2011).
4.3.5. Fe Redox Speciation in Periplasm
Although we do not consider, for simplicity, the redox state of Fe in the model
presented here, Fe redox reactions inside cells or near the cell surface are
acknowledged to be important in Fe acquisition by phytoplankton (Shaked et al., 2005,
Salmon et al., 2006). Therefore, to further investigate the effect of chemical speciation
on periplasmic and inner-membrane Fe transport, the redox state of Fe present in
periplasm was examined. For the case where Microcystis cells were incubated in the
Fe(III)-dominant system (the control in Figure 4.5), a significant amount of Fe(II)
(33% of total periplasmic Fe) was detected in the periplasmic extract. Periplasmic
Fe(II) was also detected in the Fe(II)-dominant system where ascorbate was added to
the culture (in order to reduce all extracellular and possibly some periplasmic Fe(III) to
Fe(II)). Interestingly, the amount of extracted Fe(II) present in the ascorbate treated
system was comparable to that determined in the control, suggesting that the redox
state of periplasmic Fe was negligibly influenced by the presence of extracellular
reducing agents which might also enter the periplasmic space.
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
67
Figure 4.5. Effect of ascorbate and TTM on Fe(II) accumulation in the periplasm of
M. aeruginosa PCC7806; (A) oxidation kinetics of Fe(II) in the periplasmic extract,
and (B) percentage of Fe(II) extracted from the periplasm. PCC7806 was incubated in
Fraquil* (0.7 µM Fe and 100 µM citrate) in the presence and absence of chemical
treatments (1 mM ascorbate and 1 mM ascorbate plus 100 µM TTM). The periplasm
was extracted by the cold osmotic shock method in cold Milli-Q water followed by
measurement of Fe(II) in the extract by the luminol chemiluminescence technique. The
amount of periplasmic Fe(II) was calculated by assuming that the observed maximum
value of the chemiluminescence signal corresponds to the amount of Fe(II) in the
periplasm. Error bars represent ±standard deviation from duplicate experiments. A
0
500
1000
1500
2000
2500
3000
3500
0 100 200 300 400
Sig
na
l
Time (s)
Control
Ascorbate
Ascorbate + TTM
A
0
20
40
60
80
100
120
control Ascorbate Ascorbate + TTM
Pro
po
rtio
n o
f p
erip
lasm
ic F
e(I
I)
rela
tive
to
tal p
erip
lasm
ic F
e (
%)
B
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
68
single-tailed heteroscedastic t-test indicated that the treatments with ascorbate + TTM
were different from the control at a p value of 0.14.
The comparable concentrations of periplasmic Fe(II) in both Fe(III)- and Fe(II)-
dominated systems suggests that extracellular Fe present as either Fe(III) or Fe(II) can
be reduced or oxidized in the periplasm. Thus, it is likely that the redox state of Fe is
tightly regulated in the periplasm and is likely to be substantially different from the
redox state in the external environment. Although the identity of such Fe redox
regulators in the periplasm remains largely unknown, redox reactions of extracellular
Fe with plasma-membrane reductases and multi-copper oxidases (MCO) have been
suggested to be important processes in Fe acquisition for some eukaryotic
phytoplankta (Maldonado et al., 2006). Since MCO (a periplasmic laccase) is also
identified in the genome of M. aeruginosa (PCC 7806 and NIES 843), we examined
the effect of TTM (a compound that inhibits MCO activity) on Fe(II) accumulation in
the periplasm. As shown in Figure 4.5, the presence of TTM and ascorbate in the
culture medium resulted in an increase in accumulation of periplasmic Fe(II), implying
that this enzyme plays a role in controlling the redox state of periplasmic Fe for M.
aeruginosa PCC 7806.
The effect of chemical treatments on 55
Fe accumulation in the whole of cell was also
examined (Figure 4.6). The presence of ascorbate increased the cellular 55
Fe
accumulation by 1.3-fold indicating that ascorbate-mediated reduction of Fe(III) to
Fe(II) increased unchelated Fe concentration and uptake for the Fe:Cit ratio of 0.007
(resulting in pFe′ = 10.3) where Fe(III) uptake was not saturated. The single addition
of TTM resulted in an insignificant change in the Fe accumulation compared to the
control system. In contrast, when ascorbate and TTM were simultaneously present in
the assay culture, the 55
Fe accumulation increased by 3.6-fold, which is in contrast to
the observation by Maldonado et al. (2006) that the presence of TTM in the culture
medium resulted in inhibition of Fe uptake by marine diatoms. The facilitated
periplasmic Fe(II) accumulation and cellular 55
Fe(II) uptake (but not Fe(III) uptake) in
the presence of TTM plus ascorbate for M. aeruginosa suggests that MCO-mediated
oxidation of periplasmic Fe(II) retards the transport of Fe to the cytoplasm. The
increased Fe accumulation in the absence of MCO-mediated Fe(II) oxidation suggests
that Fe(II) in the periplasm is preferably transported to inner compartments by a
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
69
plasma-membrane transporter (e.g., FeoB) at a faster rate than is the case for Fe(III)
(e.g., through FutA) (Figure 4.4).
Figure 4.6. Effect of chemical treatments on cellular 55
Fe accumulation for M.
aeruginosa PCC7806. In the control, cells were incubated for 3 hr in Fraquil*
containing 55
Fe-citrate (total concentrations for Fe and citrate were 0.7 µM and 100
µM, respectively). In the chemical treatments, cells were incubated in the additional
presence of 100 µM TTM, 1 mM ascorbate and 1 mM ascorbate plus 100 µM TTM.
Error bars represent ±standard deviation from duplicate experiments. One asterisk
indicates that chemical treatments were significantly different from the control at a p
value less than 0.05 using a single-tailed heteroscedastic t-test.
A negligible effect of TTM on 55
Fe accumulation in the Fe(III)-dominant system was
observed when an exogenetic reductant was absent (Figure 4.6). This lack of effect is
surprising as it was expected that the presence of TTM (which inhibits oxidation of
Fe(II) through shut-down of the MCO) would lead to a relative increase in the rate of
Fe(III) reduction. The non-observable effect of TTM in the Fe(III)-dominant system
could result from secondary effects of TTM such as inhibition of the activity of other
enzymes responsible for the reduction of periplasmic Fe(III) to Fe(II) though the
molecules responsible and detailed mechanism involved remain unclear. In addition to
oxidases and oxidoreductases such as MCO and flavin enzymes respectively,
secondary produced reactive oxygen species (ROS) are also candidates for the redox
55F
e u
pta
ke
ra
te
(re
lative
to
co
ntr
ol)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Control TTM Ascorbate Ascorbate +TTM
*
*
Cellu
lar
55F
e a
ccum
ula
tion
(rela
tive to
contr
ol)
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic and Cytoplasmic Compartments of
the Freshwater Cyanobacterium Microcystis aeruginosa
70
regulation of periplasmic Fe, given that Fe redox cycling is potentially relevant to
generation of free radical intermediates. Superoxide, for example, can be formed in the
periplasm of Escherichia coli possibly as a result of the adventitious autooxidation of
menaquinone in the cytoplasmic membrane (Korshunov and Imlay, 2006), eventually
resulting in diffusion through the outer membrane to the extracellular environment. M.
aeruginosa also produces superoxide though the mechanism of production is unknown
(Fujii et al., 2010a). We believe that considerable scope exists for further investigation
of such processes with regard to their role in cyanobacterial Fe uptake.
4.4. CONCLUSIONS
In the present work, a previously developed model for Fe uptake by phytoplankton has
been extended to accommodate the processes involved in Fe transport from the
extracellular environment to the cytoplasm. The model incorporates the conclusion
from the current study that only Fe′ crosses the outer membrane by diffusion into the
periplasm with subsequent conversion into a form suitable for transport into the
cytoplasm. This is in contrast to the existing model in which direct internalization of
extracellular Fe′ by plasma-membrane transporters is assumed. Our model provides
consistency not only with previous kinetic studies indicating that the rate of
cyanobacterial Fe uptake follows Monod-type kinetics with respect to the
concentration of Fe′ but also with studies identifying the nature of intracellular
molecules responsible for Fe transport (Andrews et al., 2003). However, further
studies are clearly required to properly account for the effect of periplasmic redox
processes on intracellular Fe transport.
71
CHAPTER 5
IRON UPTAKE KINETICS BY THE
FRESHWATER CYANOBACTERIUM
MICROCYSTIS AERUGINOSA IN THE
PRESENCE OF SUWANNEE RIVER
FULVIC ACID
Chapter 5. Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa in the
Presence of Suwannee River Fulvic Acid
72
5.1. INTRODUCTION
Iron (Fe) is one of the micronutrients essential for growth of almost all organisms. Of
the microorganisms, cyanobacteria have a particularly high requirement for Fe due to
its critical roles in metabolisms including photosynthesis, respiration, nitrogen fixation
and regulation of reactive oxygen species (Crichton, 2009). Because of the low
solubility of inorganic Fe(III) in circumneutral pH waters (e.g., 10-11
M at pH 7.5-9
(Liu and Millero, 1999)), a majority of dissolved Fe(III) in natural waters is present as
complexes with natural organic matter (NOM) including siderophores (Vraspir and
Butler, 2009), humic substances (Liu and Millero, 2002, Tipping, 2002) and possibly
polysaccharides (Hassler et al., 2011). In air-saturated surface waters at circumneutral
pH, ferrous iron (Fe[II]) is rapidly oxidized to ferric iron (Fe[III]) by dissolved oxygen
and secondarily produced organic and inorganic radicals including reactive oxygen
species with a half-life time of several minutes (Millero and Sotolongo, 1989, Rose
and Waite, 2002, Pham and Waite, 2008a). As such, Fe(III) is recognized to be the
thermodynamically favoured redox state in surface waters. As a result of extensive
investigations of Fe redox chemistry over the last decade, however, it is now evident
that Fe(II), an important substrate for Fe uptake by phytoplankton, can be generated at
appreciable rates in euphotic waters via biological, photochemical and thermal
reduction of Fe(III) species in many cases mediated by cellular membrane reductase
(Maldonado and Price, 2001, Shaked et al., 2005), superoxide (Rose and Waite, 2005,
Fan, 2008, Rose, 2012), ligand-to-metal charge transfer (LMCT) (Faust and Zepp,
1993, Waite et al., 1995) and humic substances (Pullin and Cabaniss, 2003).
The Fe uptake machinery in both freshwater and marine phytoplankton has been
investigated by many researchers in recent decades. One of the important consensuses
from the previous studies is that organically-complexed Fe, including the complexes
with metal buffering ligands used for algal culturing medium, are too large or
hydrophilic to directly permeate plasma-membrane (lipid-bilayer) of eukaryotic and
prokaryotic phytoplankta. Thus, Fe availability for uptake by phytoplankton is
described as a function of unchelated Fe concentration rather than total or chelated Fe
(Maldonado and Price, 2001, Shaked et al., 2005, Fujii et al., 2011a). Although Fe
bound to some specific molecules such as siderophores and citrate may be recognized
Chapter 5. Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa in the
Presence of Suwannee River Fulvic Acid
73
by bacterial outer-membrane receptor followed by active transport to intracellular
compartments via energy-dependent processes, recent studies have indicated that, even
under Fe-limited conditions, the siderophore-independent uptake (i.e., uptake of
unchelated Fe) is dominant for freshwater cyanobacteria such as Microcystis (Fujii et
al., 2010a) and Anabaena sp. (Wirtz et al., 2010). The lack of siderophore-associated
genes in marine phytoplankton consistently suggests that Fe uptake independent of
siderophore may be prevalent among the prokaryotic phytoplankton (Hopkinson and
Barbeau, 2012). There is also evidence that the reduction of organically complexed
Fe(III) to Fe(II) via photochemical and biological processes is a critical step in
increasing Fe bioavailability under Fe-limited environments (Maldonado et al., 2005,
Fujii et al., 2011a, Kranzler et al., 2011).
Previous studies have undoubtedly provided significant insights toward understanding
the mode of Fe uptake by phytoplankton in natural and culturing systems. However,
the underlying experimental and theoretical findings were basically provided by
incubational assays using model Fe-binding ligands such as ethylenediaminetetraacetic
acid (EDTA), desferrioxamine B (DFB) and citric acid (Maldonado and Price, 2001,
Rose et al., 2005, Shaked et al., 2005, Garg et al., 2007, Fujii et al., 2010a, Fujii et al.,
2011a), as transformation kinetics for extracellular Fe bound to these ligands are well
defined. Therefore, one of the important issues remaining to be addressed is whether
the current consensus on the Fe uptake kinetics based on the studies using model
ligands is indeed consistent with the mode of Fe uptake occurring in natural waters
where Fe is generally buffered by structurally and chemically heterogeneous NOM.
Transformation kinetics of Fe bound to NOM have been extensively studied over the
last decade, particularly for one of the most commonly used standard humic
substances: Suwannee River fulvic acid (SRFA). Using Fe complexed with this
heterogeneous molecule, chemical reactions potentially occurring in natural surface
waters including formation and dissociation of FeSRFA complexes and redox
reactions involving Fe and SRFA have been carefully examined. Consequently, a set
of published rate constants is currently available for predicting the transformation of
the FeSRFA complex under particular solution conditions (e.g., at pH~8, Table 5.1)
with these rate constants potentially useful in elucidating and/or describing the key
processes involved in Fe uptake by phytoplankton.
Chapter 5. Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa in the
Presence of Suwannee River Fulvic Acid
74
In this work, we have investigated the mechanism of Fe uptake mechanism by the
freshwater cyanobacterium Microcystis aeruginosa in the presence of SRFA with
particular attention given to examining the effect of non-photochemical and
photochemical transformations of Fe on Fe uptake. Although there are only a few
reports on the growth limitation of freshwater phytoplankton due to low Fe availability
(Nagai et al., 2006) compared to marine systems, it is recognized that the Fe nutritional
status influences the synthesis of primary and secondary metabolites including
cyanotoxins even under relatively higher Fe availability where optimal growth rates
are observed (Voelker et al., 2010, Alexova et al., 2011). Therefore, insight into the
mode of Fe uptake is considered to be of great importance in understanding the
environmental and nutritional factors influencing growth and cellular metabolism of
this organism. Proper understanding of ecological function and adaptation of
cyanobacteria is considered to be particularly important for the occurrence and
management of toxic phytoplankton blooms in water reservoirs and bodies used for
drinking water supplies.
5.2. MATERIALS AND METHODS
5.2.1. Reagents
Detailed information on the grade of chemicals used, the mode of storage of stock
solutions, pH measurement and approach to cleaning of containers has been described
previously (Section 2.1, Chapter 2).
Stock solutions of Suwannee River fulvic acid (SRFA), ethylenediaminetetraacetic
acid (EDTA) and citric acid were prepared at concentrations of 1-100 g L-1
, 1-100 mM
and 1-100 mM by dissolving SRFA (International Humic Substance Society),
Na2EDTA (Sigma) and Na3citrate (Sigma) to MQ, respectively. A 0.1 M ferrozine
(FZ; 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4’, 4’’-disulfonic acid sodium salt,
Sigma) solution was prepared in MQ. pH of these ligand stocks were all adjusted to
8.0 to avoid a significant pH change when added to the culture medium (Fraquil* pH
8).
Chapter 5. Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa in the
Presence of Suwannee River Fulvic Acid
75
Stock solutions of non-radiolabeled Fe(III) (FeIII
Cl3, Ajax Finechem, Australia) were
made in 0.01 M HCl at concentrations of 1-10 mM. For the Fe uptake experiments, a
23 mM stock of radiolabeled ferric chloride (55
FeIII
Cl3 in 0.5 M HCl, 185 MBq,
Perkin-Elmer, Australia) was diluted with the 1 mM non-radiolabeled FeCl3 by 24-fold
to produce a final Fe concentration of 1.9 mM. A 2 mM bicarbonate buffered solution
at pH 8 was made by dissolving sodium hydrogen carbonate (NaHCO3, Sigma) in MQ.
Solutions of organically complexed Fe(III) (FeIII
L) were then made by placing the 1.9
mM Fe(III) solution in the bottom of a 1.5 mL polypropylene container followed by
addition of an appropriate volume of SRFA, EDTA and citrate stocks. After the
bicarbonate buffer was pipetted into the mixture, the solution pH was adjusted to 8.
Before use, the solution was stored for 24 hr under dark conditions at 25oC to reach
equilibrium.
A stock solution of 6000 unit mL-1
superoxide dismutase (SOD) was prepared by
dissolution of 2519 unit mg-1
SOD (Sigma) in MQ and frozen in 100 µL aliquots at -
80oC when not in use. Denatured SOD (d-SOD) was also prepared by heating SOD
solution in boiling water for 10 min. As Fe reducing reagents, stock solutions of 100
mM ascorbate (sodium L-ascorbate, Sigma) and 100 mM hydroxylamine
hydrochloride (Sigma) were prepared in MQ followed by pH adjustment to 8.0. In the
experiments where superoxide was artificially generated, a stock solution of 12.5 mM
xanthine (Sigma) was prepared in 0.01 M NaOH solution and the pH of the solution
adjusted to 9.8 with HCl. A 1 kU.L-1
stock solution of xanthine oxidase (XO, Sigma)
was prepared in MQ water and 1 mL aliquots were individually frozen at -86 oC until
use. To remove metals non-specifically adsorbed to cell surfaces, cells were washed by
using a chelate solution containing 50 mM Na2EDTA (Sigma) and 100 mM Na2oxalate
(Sigma) (hereafter referred to as “EDTA/oxalate”) (Tang and Morel, 2006).
5.2.2. Culturing Media
The detailed preparation procedure and nutrient composition of a modified Fraquil
medium (Fraquil*) were described previously (Section 2.2.1, Chapter 2). Briefly, for
long-term incubation, Fraquil* buffered by EDTA was prepared using at least reagent
grade salts inside a trace clean room supplied with HEPA-filtered air to provide final
concentrations of 0.1 µM for Fe and 26 µM for EDTA and pH 8. For short-term 55
Fe
Chapter 5. Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa in the
Presence of Suwannee River Fulvic Acid
76
uptake studies, Fe- and ligand-free Fraquil* was prepared with the procedure identical
to those described for the long-term culturing medium, except that addition of Fe and
ligand was omitted and final pH of the medium was adjusted to 6-9.
5.2.3. Long-term Culturing Conditions
Long-term culturing conditions are described in detail in Section 2.2.2, Chapter 2.
Briefly, a batch culture of the unicellular cyanobacterium Microcystis aeruginosa
PCC7806 was incubated under sterile conditions in a temperature- and light-controlled
incubator (Thermoline Scientific) at 27oC. Cells were regularly subcultured into fresh
media when cultures reached stationary growth phase. The subculturing was
performed approximately two-weeks after the commencement of incubation with
cellular concentration of ~104 cell mL
-1. Cell numbers in the cultures were counted on
a Neubauer hemocytometer (0.1 mm depth) under an optical microscope (Nikon,
Japan). A specific growth rate in the long-term incubation was determined to be ~0.7
d-1
.
5.2.4. Light Condition
For all incubation and experiments undertaken in this work, light was vertically
supplied by three cool-white fluorescent tubes (36W, 28mm diameter, 1.2 m length,
Philips) on a 14:10 light:dark cycle. Cell culture and other abiotic samples were
consistently incubated at a distance of 10 cm from the fluorescent tubes. At this
distance, total radiation intensity was determined to be 157 µmol-quanta.m-2
.s-1
(see
part A of Figure 3.1, Chapter 3 for the emission spectrum) using an Ocean Optics USB
4000 spectrophotometer equipped with an optical fiber and cosine converter (CC-3-
UV) that was calibrated against a DH-2000 VIS light source. All incubations and
experiments were performed either in 1 cm polystyrene spectrophotometer cuvettes
(Starna Pty Ltd, Australia) or polycarbonate vessels (Nalgene) that minimally interfere
with visible light transmission between 400-800 nm (Figure 3.2, Chapter 3). For the
dark incubations, the vessels were covered with aluminium foil to prevent any light
penetration into the solution.
Chapter 5. Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa in the
Presence of Suwannee River Fulvic Acid
77
5.2.5. Short-term 55
Fe Uptake Experiments
Prior to short-term Fe uptake assays, Microcystis cells in the long-term culture were
harvested onto a 25 mm diameter, 0.65 µm PVDF membrane (Millipore) during the
daytime of late-exponential growth phase (at densities ~3 × 106
cell mL-1
). The filtered
cells were washed gently with a chelate solution (50 mM Na2EDTA and 100 mM
Na2oxalate at pH 7, hereafter referred to as “EDTA/oxalate”) and subsequently rinsed
with 2 mM NaHCO3 in order to remove metals non-specifically adsorbed onto cell
surfaces. The washing treatment was achieved by passing ~30 mL of the solutions
through the filter for ~10 minutes. The washed cells were re-suspended into the Fe-
and ligand-free Fraquil* medium to provide cell densities of ~2 × 10
6 cell mL
-1. In
experiments where the effect of chemical treatment on 55
Fe uptake were examined, the
Fe- and ligand-free culture was prepared in the additional presence of either 1 mM FZ,
60 kU.L-1
superoxide dismutase (SOD), 1 mM ascorbate, 1 mM hydroxylamine
hydrochloride, 100 µM xanthine plus 1 kU.L-1
xanthine oxidase (XO) or 1 mM FZ
plus the reducing agent (ascorbate or hydroxylamine hydrochloride) by appropriately
supplementing chemical stock solutions described in Section 5.1.
A solution of Fe(III) complexed by SRFA was prepared by mixing radiolabeled ferric
chloride (55
FeCl3) stock with SRFA stock (International Humic Substance Society).
After dilution of a 23 mM radiolabeled ferric chloride stock (55
FeCl3 in 0.5 M HCl,
185 MBq, Perkin-Elmer, Australia) with 1 mM non-radiolabeled FeCl3 in 0.01 M HCl
at total Fe concentration of 1.9 mM, the 55
Fe solution was mixed with an appropriate
volume of 10 g.L-1
SRFA in the bottom of a polypropylene microtube. A 2 mM
NaHCO3 was then added to the mixture to maintain circumneutral pH followed by
acid-base titration to adjust the pH of the mixture to 6-9 depending on the purpose of
the experiments. The 55
FeIII
SRFA solution was equilibrated for 24 h under dark
conditions and ambient temperature before use.
The 55
Fe uptake assay was initiated by adding pre-equilibrated 55
FeIII
SRFA at final
concentrations of 0.2 µM in 55
Fe and 1-100 mg.L-1
SRFA to cell suspensions. In all
cases, except for the experiments where time course of 55
Fe uptake was examined,
cells were incubated at 27oC for 2 h (based on the linearity of
55Fe uptake) in the
absence and presence of light. For comparison, 55
Fe uptake experiments were also
Chapter 5. Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa in the
Presence of Suwannee River Fulvic Acid
78
undertaken in the identical manner described above except that EDTA or citrate was
used as Fe-binding ligand instead of SRFA.
After the incubation, cells were vacuum-filtered onto 0.65 µm PVDF membrane filters,
then rinsed three times with 1 mL EDTA/oxalate solution and twice with 1 mL of 2
mM NaHCO3 (total rinsing time was ~10 min). The filtered cells were then placed in
glass scintillation vial with 5 mL of scintillation cocktail (Beckman ReadyScint). The
activity was measured in a Packard TriCarb Liquid Scintillation Counter, with
scintillation counts (counts per minute) of the samples converted to moles of Fe using
concurrent counts of 5-50 µL of 55
Fe-ligand stock in 5 mL scintillation cocktail.
Process blanks were determined by performing the procedure in the absence of cells.
5.2.6. Kinetic Model for Fe Transformation and Uptake
The kinetic model used for the calculation of Fe chemical species and uptake by M.
aeruginosa is shown in Table 5.1 and illustrated in Figure 5.1.
Figure 5.1. Kinetic model for Fe chemical speciation and uptake by M. aeruginosa.
FeIIISRFA
Fe(III)'
kup
FeIIFZ3
KFe'
FeIISRFA
Fe(II)'
Outer membranePorin
Fe'
Fe(III)
fk
Fe(III)
d1k
Fe(II)
fk
Fe(III)
d2k
and Fe(II)
dk
Fe(II)
ox1k Fe(II)
ox4kto
Fe(III)
redk
IIFe L
ox1k to
IIFe L
ox4k
IIIFe L
redk ,
IIIFe L
red-darkk and
IIIFe L
red-lightk
Chapter 5. Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa in the
Presence of Suwannee River Fulvic Acid
79
In this work, we assume that only unchelated Fe (Fe') is available for Fe uptake.
Steady-state concentration of unchelated Fe ([Fe']SS) was calculated over a range of
conditions appropriate to the 55
Fe uptake assay experiments by summing steady-state
concentrations of Fe(III)' and Fe(II)' as described in Section 5.3. Monod-type
saturation theory was then used to describe the rate of Fe uptake (ρs, amol.cell-1
.hr-1
) as
follows:
[S]
[S]
S
max
SS
+=
K
ρρ (5.1)
where [S] indicates the steady-state concentration of the biologically available portion
of Fe in the extracellular environment (i.e., [Fe'] = [Fe(III)'] + [Fe(II)']). KS and max
Sρ
represent the half saturation constant and the maximum uptake rate under the
conditions examined, respectively. In order to reduce unknown complexity in
competitive uptake between Fe(II) and Fe(III), cellular affinity and uptake capacity for
the two redox states of Fe were assumed to be equal. This assumption is consistent
with the outer-membrane structure of cyanobacteria, where only small-size nutrients
are capable of passing through the outer-membrane transport channel by a
concentration-dependent diffusive process (Fujii et al., 2011a). Although the short-
term Fe uptake can be regulated by the cellular nutritional status during the
preconditioning stage, the uptake parameters for M. aeruginosa PCC7806 acclimated
under the conditions identical to those employed in this work have been previously
published (Fujii et al., 2010a, Fujii et al., 2011a) (Table 5.1).
5.3. RESULTS AND DISCUSSION
5.3.1. 55
Fe Uptake as a Function of SRFA Concentration
55Fe uptake in the absence and presence of irradiation by fluorescent tubes (hereafter
referred to as dark and light uptake) indicate that the amount of 55
Fe accumulated in
cells increases with time over several hours (Figure A3.1 of Appendix 3). Linear
regression analysis was applied to the data collected within 2 h, yielding relatively
good linearity with correlation coefficients (R2) of 0.87 for dark uptake and 0.94 for
Chapter 5. Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa in the
Presence of Suwannee River Fulvic Acid
80
light uptake. The non-linear accumulation after a few hours’ incubation may suggest
that either (i) the concentration of Fe available for uptake varied in a time-dependent
manner due to the heterogeneity of Fe-binding strength or redox properties of SRFA,
(ii) cellular uptake became saturated during this period, and/or (iii) cellular growth or
activity varied significantly. Due to uncertainties regarding the processes responsible
for the non-linear uptake, further 55
Fe uptake assays were undertaken with only 2 hr
incubations. The results of the 2 hr 55
Fe uptake assays for a range of SRFA
concentrations (while total Fe concentration was kept constant) indicated that both
dark and light 55
Fe uptake rates decrease with increasing SRFA concentration (part A
of Figure 5.2).
Figure 5.2. 55
Fe uptake as a function of (A) SRFA and (B) model ligand
concentrations in the absence (black symbols and bars) and presence (white symbols
0
0.5
1
1.5
2
2.5
3
3.5
1 5 25
C*
****
****
0
0.5
1
1.5
2
2.5
3
3.5
1 5 25
D
**
**
SRFA concentration (mg.L-1) SRFA concentration (mg.L-1)
SRFA concentration (mg.L-1)
55F
e u
pta
ke
ra
te (
am
ol.
cell
-1.h
r-1)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
5 mg/L 25 mg/L 26 μM 100 μM 26 μM 100 μM
SRFA Citrate EDTA
B
Ligand concentrations
0
0.5
1
1.5
2
2.5
3
3.5
0 20 40 60 80 100 120
A
0
0.2
0.4
0.6
0.8
1
1.2
Control FZ Asc Asc+FZ HH HH+FZ X/XO X/XO+FZ
No
t m
ea
sure
d
No
t m
ea
sure
d
E**
** ****
*
**
*
**
Chapter 5. Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa in the
Presence of Suwannee River Fulvic Acid
81
and bars) of light. The 55
Fe uptake assay was performed at concentrations of 200 nM
total Fe, 1-100 mg L-1
SRFA and 26-100 µM citrate and EDTA. Solid and dotted lines
indicate model fits to the data from the Bligh and Rose model, respectively. Effect of
(C) ferrozine (FZ) and (D) superoxide dismutase (SOD) on 55
Fe uptake. In control
treatments, Fe uptake assays were undertaken under dark (black bar) and light (white
bars) at concentrations of 200 nM for Fe and 1-25 mg L-1
for SRFA. In the chemical
treatments, the identical 55
Fe uptake assay was performed except for the additional
presence of either FZ or SOD under dark (gray bar) and light (shaded bar). (E) Effect
of reducing agents on 55
Fe uptake. The control treatments were undertaken under dark
(black bar) and light (white bars) (200 nM for Fe and 5 mg L-1
for SRFA). Chemical
treatments were performed, in addition, in the presence of FZ, ascorbate (Asc),
hydroxylamine hydrochloride (HH), xanthine/xanthine oxidase (X/XO) or their
combination. All short-term Fe uptake assays were performed in Fraquil* for 2 h at cell
density of ~2 × 106 cell mL
-1. Symbols and error bars represent averaged value and
±standard deviation from triplicate experiments. In panels C-E, asterisks indicate that
55Fe uptake rate in the presence of a particular chemical treatment is significantly
different from control at the levels of p < 0.01 for **
and p < 0.05 for * using a single-
tailed heteroscedastic t-test.
It is an intriguing result that the light 55
Fe uptake were generally similar to the dark
uptake particularly in the higher [SRFA] cases (e.g., uptake increased to only 1.1-2.2
folds under the light at 5 mg L-1
SRFA or greater) where 55
Fe uptake rates were
substantially lower than the maximum uptake rate. This result is in contrast to those
observed by using model ligands including EDTA and citrate where 55
Fe uptake in
these systems increased significantly on illumination (e.g., to 40-51-fold in EDTA
system and 1.8-6.2-fold in citrate system, part B of Figure 5.2) under similar or slightly
lower Fe(III)' availability. Since all cells used in this work were preconditioned in an
identical manner, the different effects of the light on 55
Fe uptake between the homo-
and heterogeneous Fe-binding ligands are expected to be associated with abiotic
processes rather than cellular activities (e.g., Fe uptake affinities). In addition, previous
finding indicated that biological factors that may be activated during light irradiation is
unlikely to facilitate the short-term 55
Fe uptake in the Fraquil* system (Fujii et al.,
2011a). Under identical preconditioning conditions, the light 55
Fe uptake is most likely
Chapter 5. Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa in the
Presence of Suwannee River Fulvic Acid
82
determined by abiotic factors (i.e., Fe transformation due to photochemical reduction)
rather than biological factors.
5.3.2. Effect of Chemical Treatment on 55
Fe Uptake
To examine the contribution of Fe redox transformations on net 55
Fe uptake, the 55
Fe
uptake assay was undertaken in the presence of the strong Fe(II) chelator, FZ (Rose et
al., 2005, Garg et al., 2007, Fujii et al., 2010a, Fujii et al., 2011a) (part C of Figure
5.2). The presence of this membrane non-permeable chemical resulted in the reduction
of rate of Fe uptake by 22-63% and 39-79% under dark and light conditions (at pH 8),
respectively, depending on SRFA concentration. Although there is a concern that use
of high concentrations of FZ (≥ 400 µM) might actively reduce Fe(III)' and result in
artificial inhibition of Fe uptake by simply lowering [Fe(III)'] (Shaked et al., 2004), use
of FZ at a concentration of 1 mM in previous work resulted in a negligible inhibition
of Fe(III) uptake in citrate-buffered Fraquil* (where Fe(III)' uptake dominates) (Fujii et
al., 2010a). Therefore, the reduced 55
Fe uptake in the FZ-treated system suggests that
the reduction of Fe(III) to Fe(II) is a significant process in Fe uptake under both dark
and light conditions.
It is now acknowledged that the photochemical reduction of chelated Fe(III) is
mediated by several mechanisms including ligand-to-metal charge transfer (LMCT),
superoxide-mediated Fe(III) reduction (SMIR) and Fe(III) reduction by photo-
generated organic radicals (e.g., semiquinone-type radicals). Recent studies have
proposed that while LMCT is important at acidic pHs, SMIR becomes a more
significant pathway in the photo-reduction of FeIII
SRFA at higher pH. Therefore, the
55Fe uptake assay was also undertaken at pH 8 in the presence of SOD to examine the
effect of superoxide on reductive Fe uptake in our system (part D of Figure 5.2).
Indeed, addition of SOD resulted in significant inhibition of the light-mediated 55
Fe
uptake (49-72%), while small effects were seen for the dark 55
Fe uptake (-3-4%). The
result that 55
Fe uptake decreased only in the light system is consistent with previous
findings that SMIR is a major pathway of FeIII
SRFA reduction in sunlit natural waters
at circumneutral pH (Garg et al., 2012). Under dark conditions, other mechanisms are
responsible for Fe(III) reduction as discussed below.
Chapter 5. Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa in the
Presence of Suwannee River Fulvic Acid
83
To examine if reducing entities other than light-mediated reduction influence
unsaturated 55
Fe uptake, the 55
Fe uptake assay was performed in the presence of
exegetic Fe-reducing reagents ascorbate and hydroxylamine hydrochloride (part E of
Figure 5.2). Similar to the light system, the exegetic Fe reductants, if solely present,
had negligible impact on both dark and light 55
Fe uptake. However, when FZ was
added to these systems, 55
Fe uptake was strongly inhibited. In the experiment where
superoxide was artificially generated by oxidation of xanthine with XO, 55
Fe uptake
was also substantially reduced when FZ was present. These results suggest that sole
reduction of Fe by reducing entities does not increase unsaturated 55
Fe uptake, possibly
as a result of the rapid reoxidation of FeIISRFA prior to the complex dissociation (as
discussed below), whilst sequestration of reduced Fe by FZ substantially decreases
55Fe uptake.
Table 5.1. Kinetic model and rate constants used in this study.
No. Reaction Rate constant Reference
1. Rate constants for organically complexed Fe
1 FeIII
L → Fe(II)' + L IIIFe L
red-darkk 1.3 × 10-6
s-1
This study
2 FeIII
L + hv → Fe(II)' + L
IIIFe L
red-lightk 1.6 × 10-5
s-1
This study
3 FeIII
L + O2- →Fe
IIL + O2
IIIFe L
redk 2.8 × 105 M
-1 s
-1 Rose and Waite (2005)
4 FeIIL + O2 →Fe
IIIL + O2
•-
IIFe L
ox1k 1.5 × 102 M
-1 s
-1 Miller et al. (2009)
5 FeIIL +
1O2 →Fe
IIIL + O2
•-
IIFe L
ox2k ~1010
M-1
s-1
Garg et al. (2012)
6 FeIIL + O2
- →Fe
IIIL + H2O2
IIFe L
ox3k 1.2 × 106 M
-1 s
-1 Fujii et al. (2010b)
7 FeIIL + H2O2 →Fe
IIIL + OH
-
IIFe L
ox4k 3.2 × 105 M
-1 s
-1 Miller et al. (2009)
8 Fe(III)' + L →FeIII
L Fe(III)
fk 8.7 × 10
5 –
7.9 × 106
M-1
s-1
Rose and Waite
(2003b), Jones et al.
(2009), Bligh and
Waite (2010)
Chapter 5. Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa in the
Presence of Suwannee River Fulvic Acid
84
9 FeIII
L1→ Fe(III)' + L1 Fe(III)
d1k 2.0 × 10
-6 –
3.2 × 10-4
s
-1
Rose and Waite
(2003b), Jones et al.
(2009), Bligh and
Waite (2010)
10 FeIII
L2→ Fe(III)' + L2 Fe(III)
d2k 3.2 × 10
-4 –
3.8 × 10-3
s
-1
Rose and Waite
(2003b), Jones et al.
(2009)
11 Fe(II)' + L →FeIIL
Fe(II)
fk 2.5 × 10
4 -
4.5 × 104
M-1
s-1
Rose and Waite
(2003b), Bligh and
Waite (2010)
12 FeIIL→ Fe(II)' + L
Fe(II)
dk 7.9 × 10
-4 -
4.5 × 10-1
s
-1
Rose and Waite
(2003b), Bligh and
Waite (2010)
13 % Fe bound to strong ligand
class R 61 - 100 %
Rose and Waite
(2003b), Jones et al.
(2009)
14 Fe binding capacity of SRFA CFe 260 µmol.g-1
Rose and Waite
(2003b)
2. Rate constants for unchelated Fe
15 Fe(III)' + O2- →Fe(II)' + O2
Fe(III)
redk 1.5 × 108 M
-1 s
-1
Rush and Bielski
(1985)
16 Fe(II)' + O2 →Fe(III)' + O2•-
Fe(II)
ox1k 8.8 M-1
s-1
Pham and Waite
(2008a)
17 Fe(II)' + O2- →Fe(III)' + H2O2
Fe(II)
ox2k 1.0 × 107 M
-1 s
-1
Rush and Bielski
(1985)
18 Fe(II)' + H2O2 →Fe(III)' + OH•
Fe(II)
ox3k 5.0 × 104 M
-1 s
-1
Millero and Sotolongo
(1989)
19 Fe(II)' + OH• → Fe(III)' + OH
-
Fe(II)
ox4k 5.0 × 108 M
-1 s
-1 Zuo and Hoigne (1992)
3. Rate constants for superoxide
20 O2- +
O2
-→ H2O2 + O2 kdisp 5.0 × 10
4 M
-1 s
-1 Bielski et al. (1985)
Chapter 5. Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa in the
Presence of Suwannee River Fulvic Acid
85
a A is the redox-active organic moiety which catalytically disproportionates superoxide
in the dark, but whose catalytic activity is inhibited during irradiation due to reaction
of the reduced form of A with singlet oxygen. Total concentration for A ([AT]) is 20
µmol (g-SRFA)-1
.
b Uptake parameters were determined for M. aeruginosa PCC7806 grown in Fraquil
*
under the conditions identical to those employed in this work (e.g., exponential growth
phase, light condition and nutrient compositions including Fe availability).
5.3.3. Mode of Dark Fe Uptake
To investigate the mechanism of dark Fe uptake in further detail, we examined the
chemical speciation of Fe at the Fe:SRFA ratios used in this work. The significant
cellular 55
Fe accumulation even in the presence of FZ (part C of Figure 5.2) suggests
that a certain amount of Fe was taken from the Fe(III) pool most likely via the thermal
dissociation of FeIII
SRFA. To quantitatively describe the processes involved in dark
Fe(III) uptake, the steady-state concentration of Fe(III)' present in the culture medium
([Fe(III)']SS) was calculated from the balance of FeIII
SRFA formation and dissociation
processes. The calculation of [Fe(III)']SS requires knowledge of the various Fe-binding
sites present in fulvic acid with different affinities for Fe that are classically
characterized by continuous or discrete ligand models (Tipping, 2002). While
recognised to be a simplification, this trait of fulvic acid was considered by assigning
21 Microcystis cells → O2- kprod 1.2 × 10
-18
mol cell-1
hr-1
Fujii et al. (2010a)
22 O2- +
A → A
- + O2, kSRFA
(3.3 × 10-2
)
/[AT] a
M-1
s-1
Garg et al. (2011)
4. Parameters for Fe uptake b
23
Fe' → uptake:
max
Fe'Fe'
Fe
[Fe ]
[Fe ]K
ρρ
′
′=
+ ′
KFe' 3.3 × 10-11
M Fujii et al. (2010a)
max
Fe'ρ 3.3 × 10-18
mol cell
-1
hr-1
Fujii et al. (2010a)
Chapter 5. Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa in the
Presence of Suwannee River Fulvic Acid
86
different rate constants for two representative ligand classes: i.e., strong and weak
ligand classes (L1 and L2, Table 5.1). The dissociation reactions can be described as:
( )
Fe(III)d1III
1 1Fe L Fe III ' Lk
→ + (5.2)
( )
Fe(III)d2III
2 2Fe L Fe III ' Lk
→ + (5.3)
Formation of a metal–ligand complex is generally controlled by the rate of water-loss
from the metal center of the outer-sphere complex (Margerum et al., 1978). Formation
rates of Fe complexes with each ligand class have previously been assumed to be
identical with this assumption successfully describing the kinetic data of FeIII
SRFA
complexation (Rose and Waite, 2003b). Thus, in this work, the formation reaction is
described as;
( )
Fe(III)f IIIFe III ' L Fe L
k+ → (5.4)
Due to the predominance of FeIII
SRFA relative to other Fe species, FeIII
SRFA
concentration can be approximated to be equal to the initial Fe total concentration in
the system (i.e., [FeT]≈[FeIII
L1]+[FeIII
L2]). Thus, [Fe(III)']SS was calculated using the
following equation:
Fe(III) III Fe(III) III
d1 1 d2 2SS Fe(III)
f
Fe(III) Fe(III)
d1 T d2 T
Fe(III)
f T T
[Fe L ] [Fe L ] [Fe(III)'] =
[L]
[Fe ]×R [Fe ]×(1-R)
([L ]-[Fe ])
k k
k
k k
k
+
+=
(5.5)
where R represents the proportion of Fe(III) bound to strong-binding sites. [L] and [LT]
indicate the concentrations of free Fe-binding ligand and total ligand, respectively.
[LT] is calculated from the product of SRFA concentration (mg.L-1
) and Fe binding
capacity of SRFA (CFe µmol.g-1
).
For calculation, we considered three combinations of R and rate constants for
dissociation and complexation of FeIII
SRFA published so far at pH~8 (reactions 8, 9,
Chapter 5. Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa in the
Presence of Suwannee River Fulvic Acid
87
10, 13 and 14 in Table 5.1, hereafter referred to as Rose, Bligh and Jones Fe(III)
models) (Rose and Waite, 2003b, Bligh and Waite, 2010, Jones et al., 2009). The
reported rate constants vary by up to three orders of magnitude due presumably to
different contributions of other competing reactions depending on the methods and
media employed. The competing reactions may include Fe precipitation and adjunctive
processes associated with competitive ligands that will occur at similar time-scales to
FeIII
SRFA complexation and dissociation but which are not fully considered in the
previous kinetic models, resulting in the different estimates of rate constants and Fe-
binding capacity. Comparison of calculated Fe(III) uptake (by substituting [Fe(III)']SS
into eq. 5.1) to the measured 55
Fe uptake in the presence of FZ (where a majority of
55Fe is most likely assimilated from Fe(III) pool) indicated that the measured uptake
rates fall within the range of predicted uptake rate using the three different models with
the Rose and Bligh models providing reasonable agreements (Figure A3.2 of Appendix
3). Although caution should be exercised in use of the published parameters,
particularly where unaccounted differences exist, the reasonable fit of the two models
is generally supportive of the notion that concentration of Fe(III) available for uptake
is determined solely by complexation and dissociation of FeIII
SRFA, which is
consistent with previous work using model ligands EDTA and citrate (Fujii et al.,
2010a). In this context, the decreased 55
Fe uptake with increasing concentration of
SRFA (part A of Figure 5.2) can be explained by the lower availability of unchelated
Fe at higher SRFA concentration.
With regard to Fe(II) uptake, the result that dark 55
Fe uptake was significantly reduced
in the FZ treatment (part C of Figure 5.2) suggests that cellular Fe intake is
accompanied by Fe(II) generation via non-photochemical reductive process(es). It has
been acknowledged that (i) thermal reduction by SRFA (the redox-active moieties
including hydroquinones and semiquinone-type radicals which are typically present
intrinsically in SRFA) (Pullin and Cabaniss, 2003) and (ii) SMIR (Rose, 2012)
facilitates non-photochemical Fe(II) formation. Given the negligible effect of SOD on
the dark 55
Fe uptake at pH 8 (part D of Figure 5.2), however, the latter process is
unlikely to be responsible for dark Fe(II) uptake (though the effect of SOD on 55
Fe
uptake appears to be significant at lower pH 6 as can be seen from Figure A3.5 of
Appendix 3). Thus, to examine the importance of thermal Fe(III) reduction by SRFA
at pH 8, a first-order rate constant for the SRFA-mediated Fe(III) reduction (kdark) was
Chapter 5. Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa in the
Presence of Suwannee River Fulvic Acid
88
determined in this work (which is consistent with the reported value (Pullin and
Cabaniss, 2003) as described in part A3.2 of Appendix 3). Subsequently, the steady-
state concentration of Fe(II)' ([Fe(II)']SS) was calculated by considering this effect as
well as other competing reactions for Fe(II)' (i.e., oxygenation to Fe(III)' and
recomplexation to form FeIISRFA with second order rate constants of Fe(II)
ox1k and Fe(II)
fk ,
respectively), as follows:
III IIIFe L III Fe L
red-dark red-dark TSS Fe(II) Fe(II) Fe(II) Fe(II)
ox1 2 f ox1 2 f T T
[Fe L] [Fe ][Fe(II)'] =
[O ]+ [L] [O ]+ ([L ]-[Fe ])
k k
k k k k≈ (5.6)
[Fe(II)']SS was determined by using two sets of rate constants available for FeIISRFA
complexation and dissociation (namely the Rose and Bligh Fe(II) models, reactions 11
and 12 in Table 5.1) by assuming the presence of a single ligand class (rather than the
two ligand class) with the use of this model being justified for FeIISRFA complexation
kinetics in the previous work (Rose and Waite, 2003b, Bligh and Waite, 2010). The
Fe(II) uptake rates were then calculated by substituting the two substrate
concentrations of [Fe'] and [Fe(III)'] into eq. 5.1 followed by taking a subtraction for
these two uptake rates (i.e., Fe' Fe(III)'ρ ρ− ), as experimentally determined Fe(II) uptake
represents the difference of 55
Fe uptake rates in the absence and presence of FZ
treatment. Comparison of calculated values with measurement indicate that both the
Rose and Bligh models reasonably account for the measured 55
Fe(II) uptake (Figure
A3.3 of Appendix 3) with this result supporting the conclusion that SRFA-mediated Fe
reduction facilitates Fe uptake. As found for Fe(III) uptake, the decrease in Fe(II)
uptake rate with increase in SRFA concentration is reasonably explained by the
associated decline of concentration of Fe(II)' available for uptake.
By using the Rose and Bligh models, concentrations of unchelated Fe for the two
redox states were calculated as a function of ligand concentration (Figure 5.3) resulting
in the prediction that Fe(II)' is generated at concentrations comparable to Fe(III)' at [L]
> ~10-7
M (corresponding to free SRFA concentration of 0.38 mg.L-1
). At [L] < ~10-7
M, [Fe(II)'] was calculated to be almost independent of [L] whilst [Fe(III)'] increased
with decreasing [L] over the ligand concentrations used. This prediction suggests that
Chapter 5. Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa in the
Presence of Suwannee River Fulvic Acid
89
the thermal reduction of Fe(III) by SRFA is the rate limiting step in Fe(II)' formation at
lower ligand concentrations.
Figure 5.3. Simulated results for unchelated Fe concentrations (gray lines for Fe(III)
and black lines for Fe(II)) as a function of SRFA ligand concentration by using the
Rose (solid lines) and Bligh (dotted lines) models.
5.3.4. Mode of Light-mediated Fe Uptake
The lack of a marked effect of light on 55
Fe uptake in the SRFA system suggests that
the concentration of Fe available for uptake in the light is comparable to that in the
dark. However, the photochemical reduction of Fe(III)-fulvic acid complex is
acknowledged to facilitate Fe(II) formation at circumneutral pH. Consistent with this
evidence, we also found that the visible light irradiation used in this work is capable of
reducing FeIII
SRFA to Fe(II) at a one order of magnitude greater rate than the dark
reduction by using the FZ-trapping method where Fe(II) formed in the system of
interest is determined by measuring time-course of FeIIFZ3 concentration (see part
A3.2 of Appendix 3 for details). Therefore, one might expected that the steady-state
concentration of Fe(II) species in the light is much higher than that in the dark. One of
the plausible explanations for the apparent contradiction here is that the photo-
generated Fe(II) species involve not only unchelated Fe(II) but also chelated Fe(II) that
-14
-13
-12
-11
-10
-9
-8
-10 -9 -8 -7 -6 -5 -4
Logarithm of ligand concentration (M)
Log
ari
thm
of
Fe
(III
)' o
r
Fe
(II)
' co
nce
ntr
ati
on
(M
)
Chapter 5. Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa in the
Presence of Suwannee River Fulvic Acid
90
is readily accessible to FZ (at a similar rate as that of unchelated Fe(II)) but not
available for uptake.
The comparable Fe availability under light and dark is likely accounted for by the
relatively similar availability of Fe(II) in the dark and light due to the rapid reoxidation
of photo-generated Fe(II) prior to dissociation of the Fe(II)SRFA species formed. The
dark Fe(II) oxygenation in the SRFA solution is recognized to be faster (150 M-1
.s-1
)
than the inorganic Fe(II) oxidation (8.8 M-1
.s-1
) and other synthetic ligands EDTA (31
M-1
.s-1
) and citrate (2.9 M-1
.s-1
). Furthermore, Fe(II) oxidation is facilitated during
photolysis due to the participation of photo-produced singlet oxygen (1O2) and
(possibly) other inorganic and organic radicals such as superoxide (O2-) and
semiquinones. According to Garg et al. (2012), the steady-state concentration of Fe(II)
species ([Fe(II)]SS) in photolyzed SRFA solution is well accounted for by the balance
of photo-reduction of FeIII
SRFA (IIIFe L
red-lightk ) and oxidation of FeIISRFA by dissolved
oxygen (IIFe L
ox1k ) and singlet oxygen (IIFe L
ox2k ) with the assumption that light-mediated Fe
reduction primarily generates chelated Fe(II), as follows:
III III
II II II II
Fe L III Fe L
red-light red-light T
SS Fe L Fe L 1 Fe L Fe L 1
ox1 2 ox2 2 ox1 2 ox2 2
[Fe L] [Fe ][Fe(II)] =
[O ]+ [ O ] [O ]+ [ O ]
k k
k k k k≈ (5.7)
Assuming an apparent 1O2 concentration of [
1O2]app = 3.5 pM in the photolyzed NOM
solution and a diffusion-controlled rate for the 1O2-mediated Fe(II) oxidation (
IIFe L
ox2k
~1010
M-1
.s-1
) (Garg et al., 2012), the steady-state concentration of photo-generated
Fe(II) was calculated to be 45 pM. Comparison of calculated [Fe(II)']SS under the dark
and light conditions provides relatively similar Fe(II)' concentrations when the Rose
model is used (Figure A3.4 of Appendix 3).
The photochemical FeIIFZ3 formation and
55Fe uptake were also measured in weakly
acidic to alkaline pH where large shifts in the redox potential of Fe complexed by
SRFA have been suggested (Pullin and Cabaniss, 2003) (Figure A3.5 of Appendix 3).
The time-dependent measurement of FeIIFZ3 concentration indicates that
photochemical reduction of FeIII
SRFA is substantially more significant at pH 6-7
compared to pH 8-9. In contrast, only a slight reduction of unsaturated 55
Fe uptake was
Chapter 5. Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa in the
Presence of Suwannee River Fulvic Acid
91
observed at the lower pH, suggesting that the formation of Fe(II) available for uptake
negligibly increased on light irradiation even though photochemical FeIIFZ3 formation
proceed relatively rapidly. These findings are consistent with the notion that
photochemical reduction of FeIII
SRFA does not facilitate Fe uptake. The relatively
large inhibitory effect of FZ on 55
Fe uptake at the lower pH and in the presence of light
(parts C and E of Figure 5.1, and Figure A3.5 of Appendix 3) is well accounted for by
the facilitated reduction of FeIII
SRFA followed by formation of biologically
unavailable FeIIFZ3. The formation of Fe
IIFZ3 results in the decrease in concentration
of the Fe(III) pool (basically both FeIII
SRFA and Fe(III)') and, as such, reduces not
only Fe(II) but also Fe(III) availability. In the scenario discussed above, it is important
to recognise that FZ sequesters not only unchelated Fe(II) but also photo-generated
FeIISRFA directly at an appreciable rate. Although previous reports are mixed, the
ability of FZ and other strong Fe-chelator (such as DFB) to associate adjunctively with
Fe bound to weak Fe-binding ligands is now recognised (Pullin and Cabaniss, 2003,
Pham and Waite, 2008b, Ito et al., 2011).
5.4. IMPLICATIONS OF FINDINGS
Regardless of the inherent complexity of metal binding by natural organic matter, we
have attempted to elucidate the mechanism of Fe uptake by Microcystis aeruginosa in
the presence of the natural organic SRFA to the best of our knowledge with results of
Fe uptake kinetics obtained under both dark and light conditions. The negligible
impact of visible light on 55
Fe uptake by M. aeruginosa is intriguing and in contrast to
the Fe uptake in the EDTA and citrate systems. The findings of the present study are
consistent with the notion that Fe' generated by thermal and reductive dissociation of
the Fe-fulvic acid complex is a primary substrate for uptake by M. aeruginosa..
The rate of Fe uptake at fulvic acid concentrations typical of freshwaters has been
found to be relatively high at pH 8 (Figure 5.3). If ligand concentrations are
standardized by their molecular weights (~2,000 Da for SRFA) (Her et al., 2002), Fe
uptake in the presence of SRFA is comparable to that observed in the presence of
EDTA in the light but a little lower than found for uptake in the presence of citrate in
both the dark and light. Only when the fulvic acid concentration is very high (e.g., >25
mg.L-1
), is the growth rate likely to be limited by Fe availability. The 55
Fe uptake
Chapter 5. Iron Uptake Kinetics by the Freshwater Cyanobacterium Microcystis aeruginosa in the
Presence of Suwannee River Fulvic Acid
92
measured for SRFA concentrations <25 mg.L-1
(0.14-2.3 amol.cell-1
.hr-1
) and the
cellular Fe quota under Fe-replete and limited conditions (~1-3 amol.cell-1
) (Dang et
al., 2012) suggests that Fe uptake during daytime is sufficient to sustain the optimal
rate of growth (>~0.76 day-1
).
The difference in light quality between the incubator fluorescent light used in this
work and natural sunlight is recognised. Natural sunlight for example includes
radiation in the UV range which will reduce Fe(III) bound to NOM at a much faster
rate than will visible light. In addition, the light intensity of 157 µmol m-2
s-1
used in
this work is much less than natural sunlight during the day (e.g., ~2 mmol m-2
s-1
).
Thus, under the incubational conditions used in the studies reported here, the rate and
extent of Fe(III) photoreduction is expected to be substantially lower than would be the
case in natural surface waters. Despite this, the finding that the intrinsic Fe(III)
reducing ability of NOM (at least as exemplified by SRFA in the studies described
here) is sufficient to maintain growth of M. aeruginosa at maximum rates is of
considerable significance.
93
CHAPTER 6
CHARACTERISTICS OF THE
FRESHWATER CYANOBACTERIUM
MICROCYSTIS AERUGINOSA GROWN IN
IRON-LIMITED CONTINUOUS
CULTURE
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
94
6.1. INTRODUCTION
Most previous investigations of the cellular phenotype expressed under nutrient
limitation employed batch culture incubations. However, temporal changes to physico-
chemical properties of the medium occur during incubation including pH, nutrient
concentrations and metabolic products (Hoskisson and Hobbs, 2005), such that the
batch method suffers severe limitations with regard to accurately assessing the effect
of growth conditions on cellular response. In addition, the response of cultured
microorganisms varies throughout the growth cycle, which typically consists of a lag
phase, exponential growth, stationary phase and death phase in batch cultures
(Tempest, 1969). In contrast, the growth of microorganisms in continuous culture is
maintained at steady-state throughout the incubation, with metabolic processes and
resultant growth occurring at a constant rate in a relatively stable environment (Herbert
et al., 1956).
The growth response of phytoplankton has been widely investigated in chemostats
operating under nutrient limitation by not only macro-nutrients including nitrogen
(Caperon and Meyer, 1972a, Caperon and Meyer, 1972b, Gotham and Rhee, 1981a)
and phosphorus (Fuhs, 1969, Burmaster, 1979, Gotham and Rhee, 1981b) but also
trace metals (particularly Fe) (Wilhelm and Trick, 1995, Xue et al., 1998, Weger,
1999, Weger and Espie, 2000, Collins et al., 2001, Middlemiss et al., 2001, Weger et
al., 2002, Gress et al., 2004, Weger et al., 2006, Weger et al., 2009, Sonier and Weger,
2010, Wirtz et al., 2010). However, a mathematical theory of trace-metal-limited
continuous culture is lacking. In fact, the chemostat theory used for describing cellular
growth has been developed and subsequently reviewed thoroughly by several authors
(Monod, 1950, Novick and Szilard, 1950, Herbert et al., 1956, Gerhardt and Drew,
1994, Hoskisson and Hobbs, 2005, Andersen, 2005), and shown to be applicable to
macro-nutrients studies where the concentration of limiting substrate is considered as
the total concentration of the macro-nutrient. In contrast, chemostat behavior and
theory under trace metal limited conditions is significantly different to that under
macro-nutrient limited conditions because the limiting substrate is buffered by excess
organic ligands and also because some trace metals (including Fe and Cu) are photo-
reactive. Indeed, under Fe limiting conditions, the “traditional” chemostat theory is no
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
95
longer suitable for description of the growth and behavior of microorganisms since,
under these conditions, Fe availability is a function of unchelated Fe concentration and
not total Fe concentration with the kinetics of both light-mediated reduction of Fe(III)
species and the oxidation of Fe(II) species critical determinants of steady state
concentration of unchelated Fe. In this work, a modified chemostat theory for Fe-
limited phytoplankton growth is developed and applied to description of the behavior
(including steady state cell density, Fe cell quota and Fe uptake kinetics) of M.
aeruginosa strain PCC7806 grown continuously in Fraquil* medium with Fe activity
buffered by the organic ligand EDTA. The modified chemostat theory developed here
and used to describe the results obtained in this study is presented in full in Section
6.2.7.
6.2. MATERIALS AND METHODS
6.2.1. Materials
The grade, preparation and storage of all reagents, pH measurements, cleaning
procedure of all lab-ware are described in Section 2.1, Chapter 2.
6.2.2. Culturing Method
Cells of the toxic strain PCC7806 of M. aeruginosa were cultured in Fraquil* medium
in which the speciation of trace metals present could be precisely defined. The detailed
preparation of Fraquil* medium is described in Section 2.2.1, Chapter 2. Briefly, the
medium is buffered by single metal chelator, EDTA, and contains 0.26 mM CaCl2,
0.15 mM MgSO4, 0.5 mM NaHCO3, 0.1 mM NaNO3, 0.01 mM K2HPO4, 1 mM
HEPES, 160 nM CuSO4, 50 nM CoCl2, 600 nM MnCl2, 1.2 µM ZnSO4, 10 nM
Na2SeO3, 10 nM Na2MoO4, 300 nM thiamine HCl, 2.1 nM biotin and 0.41 nM
cyanocobalamin. In this work, the EDTA concentration was maintained constant at 26
µM, while total Fe concentration varied from 10 nM to 10 µM. All salt, trace metal and
vitamin stock solutions were made up in MQ individually rather than as a mixture.
Then, the stocks were mixed in ~1 L MQ, except for Fe and EDTA. The 1 mM stock
of ferric chloride (FeIII
Cl3, Ajax Finechem, Australia) in 0.1 M HCl was mixed with a
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
96
26 mM solution of EDTA (Na2EDTA, Sigma) prior to mixing with the other stock
solutions in order to prevent precipitation of Fe(III). After mixing all nutrient stocks,
the pH of the medium was adjusted to 8.0 ± 0.05 using concentrated NaOH. The
medium was then sterilized using a 700 W microwave oven for 10 minutes in intervals
of 3, 2, 3 and 2 minutes. After cooling to room temperature, the filter-sterilized vitamin
solutions were added into the medium. Fraquil* with radiolabeled Fe was also prepared
by an identical procedure except for use of radiolabeled 23 mM 55
FeIII
Cl3 (in 0.5 M
HCl, 185 MBq, PerkinElmer, Australia) instead of non-radiolabeled 1.0 mM FeIII
Cl3.
All cultures of M. aeruginosa PCC7806 in Fraquil* were grown in a temperature- and
light-controlled incubator (Thermoline Scientific, Australia) at 27oC under a 14hr:10hr
light:dark cycle with light intensity of 157 µmol photons m-2
s-1
vertically supplied by
cool-white fluorescent tubes. In the original culture used for long-term batch
cultivation, total Fe and EDTA concentrations of 100 nM and 26 µM respectively were
used. Cells were regularly sub-cultured into fresh media when cultures reached
stationary growth phase. Cell density in the culture was counted on a Neubauer
hemacytometer (0.1 mm depth) under an optical microscope (Nikon, Japan). Cellular
size was determined using a Mastersizer 2000 particle size analyzer (Malvern).
6.2.3. Chemostat Apparatus
A metal-free sterile chemostat system was developed for four different flow-rates with
three replicates (see Section 2.2.3, Chapter 2 for the detailed description of the
chemostat system used in this study).
6.2.4. Cellular Fe Quota and External Fe Concentration
In order to quantify steady-state cellular Fe quotas and extracellular Fe concentrations,
the chemostat system was operated at an inflowing 55
Fe concentration of 20 nM with
four dilution rates (0.09, 0.14, 0.17 and 0.25 d-1
). For this purpose, cells were
previously grown batch-wise in 50 nM non-radiolabeled Fe Fraquil* and harvested
during late exponential growth phase by filtration. The filtered M. aeruginosa cells
were then resuspended in 200 mL of Fraquil* medium containing 20 nM radiolabeled
55Fe for each dilution rate in triplicate at a cellular density of ~2.5 × 10
8 cell L
-1. The
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
97
continuous system was maintained by introducing fresh Fraquil* medium prepared
with 20 nM radiolabeled 55
Fe. The amount of 55
Fe incorporated within cells was then
monitored in triplicate every 2 d until the system approached steady-state using the
following procedure: (i) sampling 1 mL of the cultures, (ii) filtering through a 25 mm
diameter 0.65 µm PVDF membrane (Millipore), (iii) gently washing the filtered cells
at 1 mL min-1
with a solution containing 50 mM Na2EDTA (Sigma) and 100 mM
Na2oxalate (Sigma) adjusted to pH 7 (hereafter referred to as EDTA/oxalate solution)
for 15 minutes in order to eliminate non-specifically adsorbed Fe from the cell surface
(Tovar-Sanchez et al., 2004), (iv) subsequent rinsing with 2 mM sodium bicarbonate
buffer (pH 8) and (v) placing the washed cells in glass scintillation vials with 5 mL of
scintillation cocktail. When the chemostat system reached steady-state, in addition to
the cellular Fe quota, steady-state Fe concentrations were also determined by
collecting the filtrates from the filtration step and setting aside for radioactivity
measurement. The activity (counts per minute) of radioisotope 55
Fe in the washed cells
and the filtrates was measured in a Packard TriCarb Liquid Scintillation Counter and
converted to moles of Fe by performing concurrent counts of 1-5 µL of 55
FeEDTA
stock in 5 mL scintillation cocktail. Procedural blanks were measured by repeating the
identical procedure but with cells absent.
6.2.5. Short-term 55
Fe and 14
C Uptake
To prepare steady-state Fe-limited cells used for the short-term uptake experiments,
the chemostat system was operated with 20 nM non-radiolabeled Fe at four different
dilution rates (0.09, 0.14, 0.17 and 0.25 d-1
). 200 mL of batch culture acclimated in
Fraquil* containing 50 nM non-radiolabeled Fe was removed in late exponential
growth phase (cellular density was ~1.5 × 109 cell L
-1) and transferred to the
continuous culture apparatus. The cell density of the cultures was then monitored
regularly every 2 d for a period of ~1 mo. When steady-state conditions were achieved,
cells were harvested onto PVDF membrane filters and rinsed with 5 mL of 2 mM
NaHCO3 for 5 min. The washed cells were then re-suspended into Fe- and EDTA-free
Fraquil* medium at cell densities of (5-7) × 10
8 cell L
-1. Pre-equilibrated
55Fe
IIIEDTA
stock solutions with different Fe:EDTA ratios were added into the cultures to obtain
concentrations of 200 nM 55
Fe and 20-200 µM EDTA. Cells were incubated at 27oC
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
98
for 1-12 h under light with intensity of 157 µmol photons m-2
s-1
. After the incubation,
cells were again vacuum-filtered onto PVDF membrane filters then rinsed three times
with 1 mL EDTA/oxalate solution and twice with 1 mL of 2 mM NaHCO3 (total
rinsing time was about 10 min). The filtered cells were then collected in scintillation
vials. The radioactivity was measured as described in the procedure above for
determination of cellular Fe quota. Processing steps in the experiment examining
short-term 14
C uptake by M. aeruginosa were identical to those described in the short-
term 55
Fe uptake experiments, except that cells were incubated in Fraquil* medium
([Fe]T = 20 nM and [EDTA]T = 26 µM) containing 0.5 mM 14
C prepared by
substituting the non-radiolabeled NaHCO3 stock with radiolabeled NaH14
CO3
(PerkinElmer, Australia).
6.2.6. Kinetic Model for Unchelated Fe(II) Calculation
In the presence of light, photo-produced unchelated ferrous iron (i.e., Fe(II)’) rather
than total Fe becomes the main substrate for uptake by M. aeruginosa in Fraquil*
medium, as described in detail elsewhere (Fujii et al., 2011a). In a manner similar to
that described in the previous work, the steady-state Fe(II)’ concentration ([Fe(II)’]ss)
was calculated using a kinetic model of Fe transformations that accounts for a variety
of processes including photo-reductive dissociation of FeIII
EDTA into Fe(II)’,
complexation of photo-produced Fe(II)’ by EDTA, dissociation of FeIIEDTA and
oxidation of generated Fe(II)’ to Fe(III)’ by oxygen. The [Fe(II)’]ss was calculated
from the total Fe concentration (≈ [FeIII
EDTA]) and kinetic constants using the
following expression:
[ ] [ ]
' ss
ss2
( )
III II
d Eh DTA
f E A
v
DT ox
Fe EDTA Fe Ek k
k k
DTAFe II
EDTA O
−
−
+ =
+
(6.1)
where the unknown [FeIIEDTA]ss represents the steady-state Fe
IIEDTA concentration
and can be determined from knowledge of the rate of complexation of Fe(II)’ by
EDTA and rates of dissociation and oxidation of FeIIEDTA, i.e.:
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
99
[ ]
[ ]
'
2
( )f EDTAII ss
ssd EDTA ox EDTA
k Fe IIEDTFe DTA
k
AE
Ok
−
− −
+= (6.2)
Figure 6.1. Model for Fe uptake by M. aeruginosa in the presence of light (Adapted
from Fujii et al. (2011a))
Rate constants reported by Fujii et al. (2011a) were assumed appropriate for use in eqs.
6.1 and 6.2 given that very similar experimental conditions were employed in both
studies (Table 6.1 and Figure 6.1). Assuming that dissolved oxygen is saturated (i.e.,
[O2]~0.25 mM at 25oC) and that [Fe
IIIEDTA] ≈ [Fe]T and [EDTA] ≈ [EDTA]T when
EDTA is in considerable excess of Fe, where the subscript T denotes total
concentration, the two unknown parameters [Fe(II)']ss and [FeIIEDTA]ss were
calculated from eqs. 6.1 and 6.2 using an iterative trial and error method (i.e., by
assuming an initial value of [Fe(II)']ss and calculating [FeIIEDTA]ss from eq. 6.2 then
substituting the calculated [FeIIEDTA]ss into eq. 6.1 to obtain a new value of [Fe(II)']ss;
repeating the process using the new estimate for [Fe(II)']ss and continuing until the
calculated [Fe(II)']ss was equal to the assumed value of [Fe(II)']ss whereupon the
solution had converged). Under the conditions examined here, the calculated steady-
state [Fe(II)’] was approximately proportional to [Fe]T (≈ [FeIII
EDTA]) (see eq. 6.1).
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-limited Continuous Culture
100
Table 6.1. Kinetic model for Fe transformation and uptake in the presence of light by M. aeruginosa (adapted from Fujii et al. (2011a) and
therein).
Reaction Rate constant/parameter Value Unit
FeIII
EDTA + hυ → Fe(II)' + EDTAox khυ 6.4 × 10-6
s-1
Fe(II)' + EDTA → FeIIEDTA kf-EDTA 2.1 × 10
-6 M
-1 s
-1
FeIIEDTA → Fe(II)' + EDTA kd-EDTA 1.2 × 10
-3 s
-1
Fe(II)' + O2 → Fe(III)' + O2- kox 8.8 M
-1 s
-1
FeIIEDTA + O2 → Fe
IIIEDTA + O2
- kox-EDTA 31 M
-1 s
-1
Fe(II)' → uptake ρmax or Kρ
'
max '
( )
( )
ssFe
ss
Fe II
Fe IIKρ
ρ ρ
= +
a mol cell
-1 hr
-1 or M
-1
a Fe uptake parameters (ρmax and Kρ) were determined as described in text.
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
101
6.2.7. Modified Chemostat Theory
The theory of continuous culturing was first described by Monod (1950) and Novick
and Szilard (1950) independently and reviewed subsequently by a number of authors
(Herbert et al., 1956, Gerhardt and Drew, 1994, Hoskisson and Hobbs, 2005,
Andersen, 2005). Growth kinetics in a chemostat system are characterized by key
parameters including the specific growth rate µ (d-1
), the growth rate constant µmax (d-1
)
(which is equal to the maximum value of µ at saturation levels of the limiting substrate
S (M)), the half-saturation constant Ks (M) (which represents the concentration of the
growth limiting substrate that yields a growth rate of 0.5µmax) and the yield constant Y
(cell mol-1
of limiting substrate). Such growth parameters are typically estimated from
a preliminary batch culture experiment prior to commencement of a chemostat
incubation.
The specific growth rate during the exponential growth phase in a batch system is
calculated using the following equation (Monod, 1950, Herbert et al., 1956):
dxx
dtµ= (6.3)
where x (cell L-1
) represents the population size and t (d) is the incubation period. A
straight line can be fitted to a semi-log plot of the ratio of the population size at an
arbitrary time to the initial population size (i.e.,0
lnx
x) versus time interval t. The
specific growth rate during the exponential phase µ then corresponds to the slope of
the fitted line. Both the growth rate constant and half-saturation constant can be
obtained by observing specific growth rates over a range of limiting substrate
concentrations and applying the Monod equation:
max
s
S
K Sµ µ
=
+ (6.4)
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
102
The yield constant Y is defined as the reciprocal of cellular nutrient quota and
quantified using growth rate and rate of utilization of the limiting nutrient as follows:
0
0
Number of organism formed
Weight of limiting substrate used S S
dxx xdtY
dSdt
−= − = =
− (6.5)
where x0 (cell L-1
) is the density of inoculated organism and S0 (M) is the initial total
concentration of limiting substrate in the medium under conditions where the total
substrate is available for cellular uptake. During stationary growth phase in a batch
culture, it is reasonable to assume that all of the limiting substrate present in the
medium has been converted to cellular material. In this situation, the yield constant in
eq. 6.5 may be estimated using the following equation:
0
0
–ssx xY
S= (6.6)
where xss (cell L-1
) is the cell density in stationary growth phase. The estimated values
of µmax, Ks and Y together with eqs. 6.3 to 6.5 provide both a quantitative description of
the growth cycle of algal cells in batch cultures and preliminary data for predicting the
growth behavior of cells in continuous cultures.
In continuous cultures operating under perfect mixing conditions, net rates of change
in organism density dx
dt
and substrate concentration dS
dt
can be expressed as
follows:
( ) max s
dx Sx D x D
dt K Sµ µ
= − = −
+ (6.7)
( ) ( )
maxI I
s
xdS x SD S S D S S
dt Y Y K S
µµ = − − = − −
+ (6.8)
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
103
where D (d-1
) is the dilution rate (defined as the ratio of the inflow rate of the feed
medium F (L d-1
) and the culture volume V (L)) and SI is the concentration of substrate
in the feed medium. When the continuous system reaches steady-state (i.e.,
0dx dS
dt dt= = ) with constant SI and D, the steady-state concentrations of substrate ( %S )
and cells ( %x ) are uniquely defined as follows:
%S = Ks
D
µmax
− D
(6.9)
%x = Y SI
− %S( ) = Y SI
− Ks
D
µmax
− D
(6.10)
Under steady-state conditions, D is equal to the specific growth rate
µ = µmax
%S
Ks+ %S
. D has a maximum value, generally referred to as the critical dilution rate (Dc), which
is equal to the highest value of µ obtained when S has its highest value (i.e., SI), i.e.
Ic max
s I
SD
K Sµ
=
+ . If D > Dc, any cells present in the culture vessel will be washed
out completely.
Eqs. 6.7 to 6.10 describe completely the dependency of growth of microorganisms in
continuous cultures on the total concentration of limiting substrate. However, these
equations are not applicable to continuous cultures with trace metals as the limiting
substrate since the trace metal is generally buffered by excess organic ligands in the
culturing medium, with only a small portion of the total metal present available for
uptake at any given time. While eq. 6.7 describes growth kinetics as a function of the
concentration of limiting substrate available for uptake, eq. 6.8 is derived from a total
mass balance of the substrate in the reactor. Provided the concentration of buffered
trace metal is approximately proportional to the total metal concentration, the
following relationships between total and available portions of limiting trace metals
can be introduced:
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
104
' T
S mS= (6.11)
' TS S
K mK= (6.12)
where ST and S’ (M) represent the total and available concentrations for the limiting
substrate in the reactor respectively, TS
K and 'S
K (M) are the half-saturation constants
obtained when ST and S’ are considered as substrate concentration, and m is a constant.
Using these relationships, eqs. 6.7 and 6.8 can be re-written as follows:
( ) m '
'
ax
S
dx Sx D x D
dt K Sµ µ
′
= − = −
+ (6.13)
( )I T
dS xD S S
dt Y
µ= − − (6.14)
At steady-state, where 0dx dS
dt dt= = and µ = D, eqs. 6.9 and 6.10 become:
%S' = K
S '
D
µmax
− D
(6.15)
%x = Y SI− %S
T( ) = Y SI
− KS
T
D
µmax
− D
(6.16)
where
%ST
= m %S' = mK
S '
D
µmax
− D
= K
ST
D
µmax
− D
When µmax, 'S
K , TS
K and Y for a given organism and growth medium are known, the
behavior of a continuous culture at steady-state under limitation of a buffered trace
metal can be completely defined by eqs. 6.15 and 6.16, where the steady-state
concentrations of substrate and cells depend solely on the values of the total substrate
concentration in the inflowing medium SI and the dilution rate D. With a fixed
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
105
inflowing substrate concentration, variation of dilution rate leads to variation of the
steady-state cell density and substrate concentration in the continuous system. At D =
0, for example, cell density becomes maximum and the limiting substrate
concentration approaches zero, approximately corresponding to the latter stages of a
batch culture. As the dilution rate increases towards Dc, steady-state cell density and
substrate concentration approach zero and SI, respectively. With a fixed dilution rate 0
< D < Dc, the substrate concentration in the culture must reach a level that is
independent of SI so that the specific growth rate µ is equal to the dilution rate D,
whilst cell density increases with increasing SI.
6.3. RESULTS AND DISCUSSION
6.3.1. Growth Kinetics in Batch Culture
As discussed above (Section 6.2.7), if the values of four growth constants: maximum
specific growth rate µmax (d-1
), yield constant Y (cell mol-1
) and half-saturation
constants TS
K and 'S
K (M) obtained when total and available concentrations (ST and
S’ (M), respectively) of the limiting substrate are considered as substrate concentration
are known, the behavior of a continuous culture at steady-state under limitation of a
buffered trace metal can be completely defined by eqs. 6.15 and 6.16.
To predict the behavior of Fe-limited chemostat cultures of M. aeruginosa PCC7806, a
preliminary study of the growth kinetics of this organism in batch cultures was
conducted under incubation conditions identical to those used in the chemostat study
with regard to the culture volume and vessels, growth medium, temperature and light
intensity. Only [Fe]T was modified in order to investigate the growth kinetics under
various degrees of Fe limitation. The parent culture in exponential growth phase was
sub-cultured and grown in triplicate in Fraquil* media with [Fe]T ranging from 0.01 to
10 µM. Application of the exponential growth equation (eq. 6.3) to the initial linear
section of a semi-log plot provided specific growth rates of M. aeruginosa PCC7806 in
the batch culture ranging from 0.23 ± 0.012 to 0.82 ± 0.049 d-1
(Figure 6.2). [Fe]T > 1
µM was found to be sufficient to support optimal growth of M. aeruginosa, whilst at
[Fe]T ≤ 0.1 µM the growth rate of M. aeruginosa declined due to the depletion of Fe
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
106
available for uptake. These growth rates were consistent with previously reported
values for the specific growth rates of M. aeruginosa PCC7806 in batch Fraquil*
culture at somewhat higher [Fe]T of 0.1-10 µM (Fujii et al., 2010a, Fujii et al., 2011b).
Figure 6.2. Growth curves in batch cultures of M. aeruginosa at different total Fe
concentrations in Fraquil*. Total Fe concentrations were varied from 10 nM to 10 µM;
all other media components were constant. Symbols represent the mean and error bars
represent the standard deviation from triplicate incubations (filled diamonds = 10 nM
[Fe]T, filled squares = 20 nM [Fe]T, filled triangles = 50 nM [Fe]T, open diamonds =
100 nM [Fe]T, open squares = 1 µM [Fe]T, and crosses = 10 µM [Fe]T).
Maximum growth rate and half-saturation constants were estimated via non-linear
regression of the data using the Monod equation (i.e.,
max
s
S
K Sµ µ
=
+ , Figure 6.3).
The regression analysis was performed for cases where both total Fe and calculated
steady-state Fe(II)’ were treated as the appropriate substrate concentration. Since the
7.0
7.5
8.0
8.5
9.0
9.5
0 2 4 6 8 10 12 14 16
Logari
thm
of
cell
den
sity
(ce
ll L
-1)
Time (d)
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
107
steady-state [Fe(II)’] is essentially proportional to the total [Fe] (≈[FeIII
EDTA]), in
each case the theoretical specific growth rates as a function of Fe concentration fitted
well the measured growth rates of M. aeruginosa, yielding the same value for the
growth constant (µmax = 0.80 ± 0.03 d-1
). In contrast, a much lower value of the half-
saturation constant 'S
K = 3.6 ± 0.32 fM with respect to Fe(II)’ was deduced compared
with TS
K = 26 ± 2.3 nM for total Fe. Although response of growth rate to Fe limitation
is typically expressed in terms of total Fe, expression of 'S
K in terms of Fe(II)’ is more
appropriate given that the bioavailable form of Fe in our system is unchelated Fe(II) as
a result of the photoreductive dissociation of organically complexed Fe.
Figure 6.3. Relationship between specific growth rate µ (d-1
) and log concentration of
unchelated Fe(II)’ (where [Fe(II)’] is in molar (M) units) in batch culture studies of M.
aeruginosa. Non-linear regression analysis yielded a half saturation constant for
growth of 'S
K = 3.6 ± 0.32 fM (with respect to Fe(II)’) and a maximum specific
growth rate µmax = 0.80 ± 0.03 d-1
. Solid and dotted lines represent the regression line
and 95% confidential interval, respectively. Symbols indicate data for experimentally
determined growth rate under different degrees of Fe limitation.
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
-15.0 -14.5 -14.0 -13.5 -13.0 -12.5 -12.0 -11.5
Sp
eci
fic
gro
wth
ra
te µ
(d
-1)
log([Fe(II)'] (M))
R 2 = 0.9689
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
108
In a batch culture where growth rate is controlled solely by the concentration of a
single limiting nutrient, it would be reasonable to assume that the limiting substrate in
the growth medium has been completely consumed upon reaching stationary growth
phase. Hence, assuming that the concentration of limiting nutrient is approximately
zero at this point, the yield constant Y of M. aeruginosa under Fe limitation ([Fe]T of
0.01-0.1µM) was determined to be 8.1 ± 0.21 × 1016
cell (mol Fe)-1
by use of eq. 6.6.
6.3.2. Performance of Chemostat System under Fe Limitation
Total Fe concentrations of less than 50 nM in the Fraquil* growth medium were used
in continuous cultures in this study to ensure that cultures were maintained under Fe-
limited conditions. Using the growth parameters for M. aeruginosa PCC7806 obtained
from the Fraquil* batch culture studies, expected values of both the steady-state
concentrations of M. aeruginosa cells and unchelated photo-reductively produced
Fe(II)’ concentrations were calculated as a function of dilution rate, as illustrated in
Figure 6.4. Critical dilution rates were determined to be 0.34 d-1
for [Fe]T = 20 nM and
0.52 d-1
for [Fe]T = 50 nM. The continuous cultures were then maintained in Fraquil*
medium at dilution rates < Dc with 50 nM Fe (dilution rates of 0.07, 0.15, 0.30 and
0.45 d-1
) and 20 nM Fe (dilution rates of 0.09, 0.14, 0.17 and 0.25 d-1
) for a period of 4
wk (Figure 6.5).
In the system with [Fe]T = 50 nM (part A of Figure 6.5) there was a 4-day lag before
cells began to grow, suggesting that the cells took some time to adjust to the change in
medium conditions. At lower dilution rates (0.07, 0.15 and 0.30 d-1
), cell density
increased with time after day 4. In contrast, at the highest dilution rate (0.45 d-1
), the
cell number declined significantly from day 4 to day 20 implying that the wash-out
rate was initially higher than the net growth rate under these conditions. The
continuous cultures appeared to be at steady-state after ~20 d when the variation of the
cell density with time was less than 5% of the average cell density. In the system with
20 nM radiolabeled 55
Fe, a lag of 2 d after inoculation was also observed, which was
then followed by stable increase of cells from day 2 to day 12, when each system
achieved almost maximum cell yields. The cell concentrations then slightly decreased
to reach steady-state growth at around day 20 (part B of Figure 6.5). In both systems,
as expected, the steady state cell density declined with decreasing degree of iron
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
109
limitation (i.e. increasing dilution rate) which is consistent with data reported in other
Fe-limited chemostat studies (Weger, 1999, Weger et al., 2002).
Figure 6.4. Predicted and measured steady-state cell density and substrate
concentration in continuous cultures of M. aeruginosa as a function of dilution rate
with different total Fe concentrations in the inflowing medium (50 nM and 20 nM).
Symbols represent data for steady-state cell density in Fraquil* medium with total Fe
of 50 nM (circles) and 20 nM (triangles). Dotted lines are the theoretical values of
steady-state cell density calculated from eq. 6.16 with growth parameters estimated
from batch culture studies (µmax = 0.80 ± 0.03 d-1
, 'S
K = 3.6 ± 0.32 fM with respect to
Fe(II)’, TS
K = 26 ± 2.3 nM with respect to total Fe, and Y = 8.1 ± 0.21 × 1016
cell (mol
Fe)-1
), while bold lines indicate the theoretical steady-state cell density estimated with
parameters obtained from continuous culture studies ( 'S
K = 3.4 ± 0.82 fM, TS
K = 25 ±
5.0 nM and Y = 1.1 ± 0.2 × 1017
cell mol-1), except for µmax (0.80 ± 0.03 d
-1) which was
obtained from the batch studies. Dashed and chained lines indicate predicted steady-
state unchelated Fe(II)’ concentrations estimated using parameters from batch and
continuous culture studies, respectively.
0
10
20
30
40
50
60
0 0.1 0.2 0.3 0.4 0.5 0.6
[Fe(
II)'
]ss
(fM
)
or
[Cel
l]ss
10
8(c
ell
L-1
)
Dilution rate (d-1)
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in
limited Continuous Culture
Figure 6.5. Growth of
dilution rates with total Fe concentrations in the inflowing Fraquil
= 50 nM, with dilution rates of 0.07 d
(triangles) and 0.45 d-
(diamonds), 0.14 d-1
(squares), 0.17 d
represent the mean and error bars the standard deviation from triplicate incubations.
Dashed lines represent
8.8
8.9
9.0
9.1
9.2
9.3
9.4
9.5
9.6
9.7
9.8
0 5
Logari
thm
of
cell
den
sity
(ce
ll L
-1)
8.2
8.4
8.6
8.8
9.0
9.2
9.4
9.6
0 5Logari
thm
of
cell
den
sity
(ce
ll L
-1)
Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in
Growth of M. aeruginosa in the continuous culture system at different
dilution rates with total Fe concentrations in the inflowing Fraquil*
= 50 nM, with dilution rates of 0.07 d-1
(diamonds), 0.15 d-1
-1 (circles). (B) [Fe]T = 20 nM, with dilution rates of 0.09 d
(squares), 0.17 d-1
(triangles) and 0.25 d-1
represent the mean and error bars the standard deviation from triplicate incubations.
the 95% confidence interval at steady-state.
10 15 20
Time (d)
10 15 20
Time (d)
Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
110
in the continuous culture system at different
medium. (A) [Fe]T
(squares), 0.30 d-1
= 20 nM, with dilution rates of 0.09 d-1
(circles). Symbols
represent the mean and error bars the standard deviation from triplicate incubations.
25 30
25 30
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
111
When the chemostat system supplied with 20 nM 55
Fe reached steady-state, cell
density and the total concentration of Fe in each reactor was determined. The steady-
state cell density () and steady-state total Fe () were subsequently used to re-
calculate the half saturation constant and the yield constant using the following
equations derived from eqs. 6.15 and 6.16 (Herbert et al., 1956):
KS
T
= %ST
µmax
− D
D
(6.17)
Y =%x
SI
− %ST
(6.18)
This produced a value of Y = 1.1 ± 0.2 × 1017
cell (mol Fe)-1
, which is comparable to
the value of 8.1 × 1016
cell (mol Fe)-1
derived in the batch culture studies. Although we
assumed that all Fe was completely consumed by cells at the stationary phase, a slight
underestimation of Y from the batch cultures suggests that during these incubations,
some portion of Fe present in the medium is transformed to a non-available form of Fe,
possibly due to precipitation of Fe (as a ferric oxyhydroxide) or loss of Fe by
adsorption to vessel surfaces. Similarly, eq. 6.17 produced a value of TS
K = 25 ± 5.0
nM in the continuous culture studies, with eq. 6.12 providing 'S
K = 3.4 ± 0.82 fM.
These values are also consistent with those determined in batch cultures.
The values for 'S
K , TS
K and Y obtained from the continuous culture studies and the
estimated value of µmax in the batch culture studies were used to re-calculate the
theoretical steady-state cell and Fe concentrations. For comparative purposes,
measured steady-state cell densities at different dilution rates are plotted on the same
graph as the theoretical data (Figure 6.4). The theoretically calculated values are in
reasonable agreement with the experimentally determined data, indicating that the cell
density decreases in accordance with the increase in dilution rates, while increasing
[Fe]T from 20 to 50 nM leads to an increase in the cell number.
Finally, to verify the hydraulic performance of the chemostat system, the water level
inside the culture vessels was monitored every 2 d and the medium inflow rates were
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
112
measured before and after the system had been operated continuously for 4 wk. No
significant change was observed in either parameter over this period. Thus the
chemostat system developed for the study of M. aeruginosa in this work is able to
operate continuously for a period of at least one month without any evidence of
contamination or other problems and, as such, cultures of this microorganism could be
maintained at steady-state over a range of Fe nutritional conditions.
6.3.3. Cellular Fe Quota
The amount of 55
Fe internalized by cells was measured every 2 d for a month in the
cultures supplied with 20 nM Fe (Figure 6.6). During the non-steady-state phase,
relatively large fluctuations in cellular Fe quotas were observed at each dilution rate.
After day 20, when the system approached steady-state growth, the cellular Fe quotas
at each dilution rate became constant with time. Cellular Fe quotas (Q) under steady-
state conditions increased as dilution rates (i.e. specific growth rates) increased,
consistent with Droop theory (Droop, 1973). According to this theory, specific growth
rates are predicted to hyperbolically increase with increasing steady-state Fe quota
(part A of Figure 6.7) as follows:
' minmax
Q Q
Qµ µ
−=
(6.19)
or rearranging:
' '
max max minQ Q Qµ µ µ= − (6.20)
where µ’max (d-1
) is the maximum (“impossible”) specific growth rate achieved at
infinite cellular Fe quota, Qmin (zmol cell-1
) is the minimum subsistence quota and µQ
(zmol cell-1
d-1
) represents the specific Fe uptake rate or the long-term uptake rate for
growth ρµ (µQ). The cellular Fe quota parameter µ’max in eqs. 6.19 and 6.20 (which
relates to internal substrate concentration) is unrelated to the growth rate constant µmax
(0.80 d-1
) in eq. 6.4 (which relates to external substrate concentration).
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
113
Figure 6.6. Time-course of cellular Fe quotas for Fe-limited M. aeruginosa in the
chemostat with [Fe]T = 20 nM as radiolabelled 55
Fe in the inflowing medium and
dilution rates of 0.09 d-1
(diamonds), 0.14 d-1
(squares), 0.17 d-1
(triangles) and 0.25 d-1
(circles). Symbols represent the mean and error bars the standard deviation from
triplicate incubations.
Plotting against Q produces a linear transformation of the Droop equation with a
slope of µ’max and an intercept of µ’maxQmin, as shown in part A of Figure 6.7 for long-
term Fe uptake by M. aeruginosa. This plot yields values of Qmin= 1.2 ± 0.24 amol
cell-1
and µ’max = 0.37 ± 0.04 d-1
. Assuming that cell diameter of M. aeruginosa
PCC7806 is about 4.0 µm, i.e., cell volume of ∼33.5 µm3, this results in Qmin = 3.58 ×
10-5
mol Fe per liter-cell which was comparable to 2.1 ± 0.05 × 10-5
mol Fe per liter-
cell of Thalassiosira weissflogii (Anderson and Morel, 1982) or lower than those of
other species reported previously such as 4.8-33.3 × 10-5
mol Fe per liter-cell of
Dunaliella tertiolecta (Davies, 1970); 6.9-15.9 × 10-5
mol Fe per liter-cell of Pavlova
lutherii (Droop, 1973) and 9.3-18.6 × 10-5
mol Fe per liter-cell of T. weissflogii
(Harrison and Morel, 1986). The lower value of Fe quotas of M. aeruginosa observed
in this study relative to those of other microorganisms in previous studies may partly
due to removal of extracellular iron during washing step by EDTA/oxalate solution.
0
500
1000
1500
2000
2500
3000
3500
4000
0 5 10 15 20 25 30
Cel
lula
r F
e q
uota
(zm
ol
cell
-1)
Time (d)
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
114
Figure 6.7. Relationship between the cellular Fe quota (Q) and the (A) specific uptake
rate of Fe (µQ) or (B) specific growth rate (µ) for Fe-limited M. aeruginosa under
steady-state conditions in continuous cultures. The system was operated at four
different dilution rates (0.09, 0.14, 0.17 and 0.25 d-1
) and fed with Fraquil* medium
containing 20 nM radiolabeled 55
Fe. In panel (A), linear regression analysis
(represented by the bold line) yielded the maximum “impossible” growth rate µ’max =
0.37 ± 0.04 d-1
and minimum cellular quota Qmin = 1.2 ± 0.2 × 103 zmol cell
-1. Symbols
represent the mean and error bars the standard deviation from triplicate incubations. In
Ay = 0.373x - 460
R² = 0.95
0
200
400
600
800
1000
1000 1500 2000 2500 3000 3500 4000
Sp
ecif
ic u
pta
ke
rate
µQ
(zm
ol
cell
-1d
-1)
Cellular Fe quota Q (zmol cell-1)
B
0.0
0.1
0.2
0.3
0.4
0 1000 2000 3000 4000 5000 6000 7000
Sp
ecif
ic g
row
th r
ate
µ (
d-1
)
Cellular Fe quota Q (zmol cell-1)
Critical dilution rate Dc = 0.35
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
115
panel (B), the solid line represents the theoretical curve calculated from the Droop
equation using the obtained estimated values of µ’max and Qmin. Symbols represent the
mean from triplicate incubations.
As expected, the calculated µ’max was comparable to the critical dilution rate Dc = 0.35
d-1
for the system supplied with 20 nM Fe. The hyperbolic relationship between the
cellular Fe quotas and the specific growth rates in this work (part B of Figure 6.7) is
consistent with other observations for cyanobacteria such as Anabaena sp. and
Microcystis sp. under nitrogen or phosphorous limitation (Gotham and Rhee, 1981b,
Ahlgren, 1985, Olsen, 1989) and eukaryotic phytoplankta such as green algae
Chlamydomonas sp. and diatom Thalassiosira sp. under Fe, Mn, vitamin B12 or
phosphorus limitation (Goldman and Mccarthy, 1978, Sunda and Huntsman, 1985,
Sunda and Huntsman, 1986, Harrison and Morel, 1986).
6.3.4. Fe Uptake Kinetics
Short-term 55
Fe uptake assays were undertaken using cells collected from the
chemostat supplied with 20 nM non-radiolabelled Fe. In this assay, four batch
experiments were prepared by filtering cells from the steady-state chemostat cultures
at each of the dilution rates examined and subsequently resuspending them in Fe- and
ligand-free Fraquil*. The short-term uptake assay was then initiated by adding pre-
equilibrated 55
FeIII
EDTA to each batch experiment. Intracellular 55
Fe accumulated
during incubation in the light was then measured every 1-2 h for 12 h. Incubations
were conducted in the light based on previous findings that 55
Fe uptake rates by M.
aeruginosa in EDTA-buffered Fraquil* medium are substantially higher under light
irradiation than in the dark due to the higher concentration of bioavailable substrate
resulting from photoreductive dissociation of the FeIII
EDTA complex present in the
medium (Fujii et al., 2011a). The Fe uptake rate of each steady-state culture of M.
aeruginosa was investigated over a range of unchelated photo-produced Fe(II)’
concentrations by varying the EDTA concentrations from 20 to 200 µM, while 55
Fe
concentration was maintained constant (200 nM). Intracellular 55
Fe accumulated in a
linear manner with respect to time for at least 4 h, followed by rapid decline in uptake
rate after 6 h, indicating that uptake became saturated in the latter stages of the
experiment (Figure 6.8). A Fe mass balance in these experiments indicated that
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
116
depletion of extracellular Fe available for uptake was unlikely to have occurred over
the duration of the short-term assays.
Figure 6.8. Time-course of 55
Fe uptake during batch short-term Fe uptake assays using
cells obtained at steady-state from the chemostat cultures grown with [Fe]T = 20 nM in
the inflowing medium and dilution rates of 0.09 d-1
(diamonds), 0.14 d-1
(squares),
0.17 d-1
(triangles) and 0.25 d-1
(circles). In the short-term uptake assay, each culture
was incubated in Fraquil* with either (A) 20 µM EDTA or (B) 200 µM EDTA and
R² = 0.9979
R² = 0.9932
R² = 0.9775
R² = 0.9925
0
1000
2000
3000
4000
5000
6000
7000
0 2 4 6 8 10 12 14
Acc
um
ula
ted
5
5F
e (z
mol
cell
-1)
Time (hr)
A. [EDTA] = 20 µM
R² = 0.9986
R² = 0.9974
R² = 0.9969
R² = 0.9844
0
200
400
600
800
1000
1200
1400
1600
0 2 4 6 8 10 12 14
Acc
um
ula
ted
5
5F
e (z
mol
cell
-1)
Time (hr)
B. [EDTA] = 200 µM
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
117
constant concentration of radiolabeled 55
Fe (200 nM). Symbols represent the mean and
error bars the standard deviation from triplicate experiments. The continuous lines
were obtained by linear regression of data collected within 4 h (represented by closed
symbols) for each culture.
Assuming that the short-term uptake rate for M. aeruginosa follows classical
Michaelis-Menten kinetics, the Fe uptake rate can be described as follows:
'
max '
( )
( )
ssFe
ss
Fe II
Fe IIKρ
ρ ρ
= +
(6.21)
where ρmax (mol cell-1
s-1
) is the maximum uptake rate and Kρ (M) is the half-saturation
constant for short-term Fe uptake. Steady-state concentrations of Fe(II)’ were
calculated from eqs. 6.1 and 6.2 using a trial and error method (Table 6.2). To
determine the uptake parameters, an Eadie-Hofstee linear transformation was applied
to the measured 55
Fe uptake rates in each culture as shown in Figure 6.9. Linear
regression analysis gave comparable half-saturation constants (Kρ = 18 ± 2.2 fM as
Fe(II)’) but significantly different maximum uptake rates (ρmax of 0.27 ± 0.030, 0.72 ±
0.060, 0.95 ± 0.080 and 1.0 ± 0.090 amol Fe cell-1
hr-1
) for the cultures grown at
different dilution rates, suggesting that the cells in the continuous cultures adjusted to
different degrees of Fe-limitation by varying their maximum uptake rate ρmax rather
than the half-saturation constant Kρ. This ability to regulate short-term uptake has
previously been suggested in studies of algal growth under limitation by macro-
nutrients such as N, P and Si (Tilman and Kilham, 1976, Goldman and Mccarthy,
1978, Mccarthy, 1981) as well as by Fe (Harrison and Morel, 1986). In comparison to
other studies, the maximum uptake rates ρmax obtained in this study (assuming a cell
volume of M. aeruginosa of ∼33.5 µm3) ranged from 0.8 to 2.9 × 10
-5 mol Fe liter-cell
-
1 hr
-1 and was one order lower than those of Thalassiosira weissflogii reported in
Anderson and Morel (1982) (ρmax = 1.1 – 2.1 × 10-4
mol Fe liter-cell-1
hr-1
), Harrison
and Morel (1986) (ρmax = 2.4 ± 0.02 × 10-4
mol Fe liter-cell-1
hr-1
) and Morel (1987)
(ρmax = 3.2 × 10-4
mol Fe liter-cell-1
hr-1
). This is likely because lower Fe availability in
marine systems may lead to higher uptake capacity of marine microorganisms relative
to those of freshwater microorganisms.
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
118
Figure 6.9. Eadie-Hofstee plots demonstrating the linear relationship between the
short-term 55
Fe uptake rate (ρFe) and the ratio ρFe/[Fe(II)’] (d-1
M-1
) for cultures of M.
aeruginosa. Linear regression analysis yielded comparable half-saturation constants
for Fe uptake (Kρ = 18 ± 1.9 fM, as Fe(II)’) but different maximum specific uptake
rates (ρmax of 270, 720, 950 and 1,010 zmol cell-1
hr-1
for cultures grown at dilution
rates of 0.09 d-1
(diamonds), 0.14 d-1
(squares), 0.17 d-1
(triangles) and 0.25 d-1
(circles), respectively). Lines for 95% confidential intervals were omitted for clarity.
y = -15.4x + 273
R² = 0.94
y = -18.7x + 718
R² = 0.99
y = -20.7x + 951
R² = 0.99
y = -18.4x + 1014
R² = 0.95
0
100
200
300
400
500
600
700
800
0 10 20 30 40 50 60
55F
e u
pta
ke
rate
(zm
ol
cell
-1h
r-1)
55Fe uptake rate/[Fe(II)']ss (µmol cell-1 hr-1 M-1)
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-limited Continuous Culture
119
Table 6.2. Measured and calculated Fe uptake parameters under the conditions of short-term 55
Fe uptake experiments for four different steady-
state cultures of M. aeruginosa.
Dilution rate Initial concentrations Calculated steady-state concentrations Measured and calculated Fe uptake rates
[FeIII
EDTA]initial [EDTA]initial [Fe(II)']ssa
[FeII
EDTA]ssa
ρFe-measured ρFe-calculatedb
d-1
M M M M zmol cell-1
hr-1
zmol cell-1
hr-1
0.09
2.0 × 10-7
2.0 × 10-5
3.54 × 10-14
1.72 × 10-10
180 ± 22 180
2.0 × 10-7
5.0 × 10-5
1.42 × 10-14
1.72 × 10-10
118 ± 12 119
2.0 × 10-7
1.0 × 10-4
7.08 × 10-15
1.72 × 10-10
85 ± 11 76
2.0 × 10-7
2.0 × 10-4
3.54 × 10-15
1.72 × 10-10
44 ± 2 44
0.14
2.0 × 10-7
2.0 × 10-5
3.54 × 10-14
1.72 × 10-10
478 ± 24 474
2.0 × 10-7
5.0 × 10-5
1.42 × 10-14
1.72 × 10-10
301 ± 18 313
2.0 × 10-7
1.0 × 10-4
7.08 × 10-15
1.72 × 10-10
198 ± 22 201
2.0 × 10-7
2.0 × 10-4
3.54 × 10-15
1.72 × 10-10
115 ± 11 117
0.17
2.0 × 10-7
2.0 × 10-5
3.54 × 10-14
1.72 × 10-10
592 ± 33 627
2.0 × 10-7
5.0 × 10-5
1.42 × 10-14
1.72 × 10-10
395 ± 21 415
2.0 × 10-7
1.0 × 10-4
7.08 × 10-15
1.72 × 10-10
246 ± 14 265
2.0 × 10-7
2.0 × 10-4
3.54 × 10-15
1.72 × 10-10
135 ± 19 154
0.25
2.0 × 10-7
2.0 × 10-5
3.54 × 10-14
1.72 × 10-10
886 ± 52 669
2.0 × 10-7
5.0 × 10-5
1.42 × 10-14
1.72 × 10-10
540 ± 25 443
2.0 × 10-7
1.0 × 10-4
7.08 × 10-15
1.72 × 10-10
350 ± 24 283
2.0 × 10-7
2.0 × 10-4
3.54 × 10-15
1.72 × 10-10
220 ± 13 164
a The two unknown concentrations [Fe(II)’]ss and [Fe
IIEDTA]ss calculated from eqs. 6.1 and 6.2 using trial and error method where ρFe is a measured
55Fe uptake rate.
b The Fe
uptake rate estimated from eq. 6.21 using the constant half-saturation constant (Kρ =18 ± 1.9 × fM as Fe(II)’) and different maximum uptake rates (ρmax of 270, 720, 950 and
1,010 zmol cell-1
hr-1
for the cultures grown at dilution rates of 0.09 d-1
, 0.14 d-1
, 0.17 d-1
and 0.25 d-1
, respectively) where Fe(II)’ is considered as the limiting substrate.
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
120
6.3.5. Cellular Response to Fe Limitation in Chemostat
The half-saturation constant with respect to Fe uptake (Kρ) was determined to be
higher than that for the growth rate (KS’) by ~5-fold, suggesting that the decline in Fe
uptake due to low Fe availability does not necessarily result in a decline in capacity for
cellular growth. Rather, an optimal growth rate is maintained until Fe concentrations
are imposed that are substantially lower than those at which a decline in Fe uptake
begins to occur, after which growth rate starts to decrease. This is consistent with
previous observations that cells of the marine cyanobacterium Synechococcus exhibit
symptoms of Fe stress under low Fe availability (e.g., declining photosynthetic
activity) well before growth rate is affected, implying that cellular division is accorded
a higher priority than general metabolic functioning even in low nutrient environments
(Henley and Yin, 1998). Different preconditioning of cells in the Fe-limited chemostat
resulted in altered responses of cells with regard to Fe uptake. The Fe uptake capacity
increased as the degree of Fe-limitation decreased from the most-starved condition to
the least starved. A similar trend was also found in iron-limited chemostat cultures of
green algae Chlamydomonas reinhardtii (Weger, 1999), Chlorococcum
macrostigmatum and Stichococcus bacillaris (Weger et al., 2002) where an increase in
dilution rate (or decrease in degree of Fe limitation) led to an increase in plasma
membrane ferric chelate reductase (FC-R) activity, hence, likely an increase in Fe(II)
uptake capacity. However, this trend is the reverse of the expected relationship
between cellular Fe quota and uptake rate where cells with a higher degree of nutrient
limitation generally exhibit higher uptake rates (Gotham and Rhee, 1981a, Gotham and
Rhee, 1981b, Olsen, 1989).
Provided that Fe uptake is mediated by concentration gradient dependent passive
diffusion through non-specific trans-membrane channels (porins) as suggested by Fujii
et al. (2011a) for M. aeruginosa and by Jones and Niederweis (2010) for
Mycobacterium smegmatis, Fe uptake rate is likely proportional to cell surface area. A
slight increase (by a factor of ~1.5) in average cell volume (and hence cell surface
area) was found in P-limited continuous cultures of Monochrysis lutheri with increase
in dilution rate from 0.1 d-1
to 1.0 d-1
(Burmaster, 1979). A similar trend was found in
N-limited continuous cultures of Chlorella pyrenoidosa (Williams, 1965). Thus, an
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
121
increase in dilution rate may result in an increase in cell surface area and hence in cell
normalized Fe uptake rate. However, there was no substantial change in the size of M.
aeruginosa cells with increasing dilution rates in this work (mean diameters of 4.1 ±
0.07, 3.8 ± 0.01, 3.8 ± 0.05 and 3.8 ± 0.01 µm in cultures grown at dilution rates of
0.09, 0.14, 0.17 and 0.25 d-1
, respectively), consistent with the observed invariant Kρ
values among the four cultures. Therefore, change in cellular size could not account for
the ~5-fold relative increase in 55
Fe uptake rate observed in the culture grown at the
highest dilution rate versus that grown at the lowest dilution rate.
Another possible explanation is that the starved cells grown under extreme Fe stress
require time to recover Fe uptake machinery before functioning optimally, whilst less
starved cells can immediately acquire Fe at optimal rates. As a result, within the
recovery period, the less starved cells would exhibit a higher Fe uptake rate (i.e. the Fe
uptake trend observed in this work), but beyond this recovery period, more starved
cells would exhibit greater Fe uptake rates, corresponding to the expected trend
suggested by others (Morel, 1987). However, the decrease in accumulated cellular Fe
with increasing dilution rate during both the linear Fe uptake period (0-4 h) and the
non-linear Fe uptake period (4-12 h) shown in Figure 6.8 implies that such an
explanation is unlikely.
Since the transport of Fe across the (cyto)plasmic membrane of cyanobacteria is
generally mediated by ATP-binding cassette (ABC) transporters (Andrews et al.,
2003), a more plausible reason is that decline in either the efficiency of energy-
dependent processes or of energy (i.e., ATP) production during photosynthesis causes
decreasing Fe uptake under extreme Fe stress. For serverely Fe-limited cells (those
grown at lower dilution rates in this study), it is likely that the photosynthetic capacity
and subsequent ATP production from the cyclic electron transport are minimal due to
very low Fe availability, resulting in these cells being unable to drive high Fe uptake
rates. This possibility is supported by results from short-term 14
C uptake assays using
the same steady-state cultures as those used for the short-term Fe uptake experiments.
As shown in Figure 6.10, the accumulation rate of radio-labeled carbonate in cells
decreased with decreasing dilution rate during an incubation period of 12 h, similarly
to the trend observed in the short-term 55
Fe accumulation studies. Therefore, a shortage
of resources necessary for Fe uptake such as an internal transporter or ATP may
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
122
account for the short-term Fe uptake trend in this study. Additionally, a physiological
trade-off may occur under Fe stress between the affinity for Fe at the cell surface (i.e.
the number of surface uptake sites or Fe channels) and the maximum rate at which Fe
can be assimilated (i.e. the number of internal transporters which assimilate Fe once it
is encounted) (Smith et al., 2009). When grown under Fe stress for a long period, cells
may acclimate by maintaining the number of surface uptake sites while decreasing the
number of internal enzymes available for Fe uptake. In the presence of any pulse of Fe,
cells with fewer internal transporters will likely exhibit lower Fe uptake rates relative
to non-stressed cells. This physiological acclimation strategy has been used to explain
the observed pattern of nitrate uptake by phytoplankton in the ocean (Smith et al.,
2009).
Figure 6.10. Time-course of 14
C uptake during batch short-term uptake assays using
cells obtained at steady-state from the chemostat cultures grown with [Fe]T = 20 nM in
the inflowing medium and dilution rates of 0.09 d-1
(diamonds), 0.14 d-1
(squares),
0.17 d-1
(triangles) and 0.25 d-1
(circles). Symbols represent the mean and error bars
the standard deviation from triplicate experiments.
0
20
40
60
80
100
120
140
0 2 4 6 8 10 12 14
Acc
um
ula
ted
14C
(fm
ol
cell
-1)
Time (hr)
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
123
6.3.6. Characteristics of Iron-limited Cultures of M. aeruginosa Grown
Continuously in Nutrient-replete Fraquil* Medium
The observation that Fe uptake rates were lower in the more Fe deficient cultures and
the supposition that this effect may be related to the overall health of the organism is
highly speculative and requires further investigation. It is likely that the insufficiency
of nutrients other than Fe in the culture medium (i.e., Fraquil*) may also contribute to
the weakening strength of M. aeruginosa cells. It is worth noting that, due to the
variable composition of freshwaters compared to a remarkably constant composition of
major ions in seawater, freshwater phytoplankton are usually adapted to their ambient
chemistry. As a result, one freshwater medium may support good growth of a
microorganism but not others and even minor changes of a medium composition such
as the [Na+]/[K
+] ratio may inhibit the growth of some freshwater microorganism
species (Andersen, 2005). In fact, although the concentration of trace metal ions in
Fraquil* designed to support optimal algal growth in freshwater is comparable to those
in the synthetic seawater Aquil* medium and about an order of magnitude higher than
those in the original Fraquil medium, the concentration of most major ions and trace
components in this medium are still significantly lower than that in the growth medium
BG-11 which is widely used for the culturing of freshwater, soil, thermal, and marine
cyanobacteria (Allen and Stanier, 1968, Morel et al., 1975, Morel et al., 1979, Price et
al., 1988, Andersen, 2005). For example, the concentrations of phosphate, magnesium,
manganese, copper and cobalt in BG-11 are about 17.5, 2, 15, 2 and 3 times,
respectively, higher than in Fraquil* and, particularly, nitrate and molybdenum in BG-
11 are about 2 orders of magnitude greater than in Fraquil* (~176 and 161 times,
respectively) (see Table 6.3). Thus, in this study, the composition of Fraquil* used in
our previous chemostat study were revised to find appropriate concentrations of the
culture medium composition that likely support optimal growth of M. aeruginosa.
Following the medium selection, the chosen-modified Fraquil* medium (referred to as
nutrient-replete Fraquil*) was used to revisit the behavior (including steady state cell
density, Fe cell quota and Fe uptake kinetics) of M. aeruginosa strain PCC7806 grown
continuously in the chemostat system. All the experimental methods used in this work
were identical to those used in our previous chemostat study, except nutrient-replete
Fraquil* was employed as the culture medium instead of Fraquil
*.
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
124
6.3.6.1. Selection of the Nutrient-replete Fraquil*
Medium
Fraquil* medium has been widely used for examining the effects of metals on growth,
nutrient uptake, photosynthetic activity, morphology, etc. of freshwater phytoplankton
(Rueter and Ades, 1987, Rueter, 1988, Gensemer, 1990, Gensemer et al., 1993, Fujii et
al., 2010a, Fujii et al., 2011a, Alexova et al., 2011) due to its chemically well-defined
composition which can facilitate studies of trace metal interactions with freshwater
phytoplankton. Therefore, the Fraquil* recipe was chosen as the basis of the new
culture medium (i.e., nutrient-replete Fraquil*) used in this batch-wise medium
selection study as well as our further chemostat studies on behavior of Fe-limited
cultures of M. aeruginosa grown continuously under nutrient-replete conditions.
However, the concentrations of certain nutrients in the original Fraquil* was adjusted
to levels which provide improved growth of M. aeruginosa compared to that in the
original Fraquil*.
In this medium selection study, the growth of M. aeruginosa in batch incubation
cultures was examined for various nutrient concentrations of Fraquil*, with particular
attention given to the variation of nitrate and molybdenum due to the exceptionally
high concentrations of these two nutrients in BG-11 relative to Fraquil* (~176 and 161
times higher, respectively), while the concentration of bio-available Fe was maintained
at a level sufficient to prevent the adverse effect of Fe deficiency on the growth of M.
aeruginosa. The batch-wise growth of M. aeruginosa in the control medium (i.e.,
Fraquil*) was compared to those in five different nutrient-replete Fraquil
* media,
defined here as Test 1, Test 2, Test 3, Test 4 and Test 5. These nutrient-replete Fraquil*
media were prepared by simultaneously increasing the concentrations of both nitrate
and molybdenum in Fraquil* by a factor of 10 (Test 1), 20 (Test 2), 40 (Test 3), 80
(Test 4), and 176 (for nitrate) and 161 (for molybdenum) (i.e., equivalent to those in
BG-11, Test 5). Meanwhile, the concentration of the other major and trace nutrients,
except for iron, was increased to the concentration of the corresponding nutrients in
BG-11 or held at the same concentration as in Fraquil* if the concentration of that
nutrient in BG-11 was lower than that in the original Fraquil* medium. The recipes of
these culture media are presented in details in Table 6.3. The concentration of Fe and
its synthetic organic chelator EDTA in all the studied culture media was fixed at 10
and 26 µM, respectively, to ensure a sufficient level of Fe available for growth of M.
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
125
aeruginosa as previously reported in Section 6.3.1 and elsewhere (Fujii et al., 2010a,
Dang et al., 2012).
Figure 6.11. Growth curves in batch cultures of M. aeruginosa at a constant total Fe
concentration in different modified Fraquil* growth media. Total Fe concentration and
its chelator EDTA were fixed at 10 and 26 µM while other media components were
varied as shown in Table 6.3. Symbols represent the mean and error bars represent the
standard deviation from duplicate incubations (filled diamonds: control (i.e., Fraquil*);
filled squares: Test 1; filled triangles: Test 2; open diamonds: Test 3; open squares:
Test 4 and open triangles: Test 5).
0
2
4
6
8
10
12
14
16
18
0 2 4 6 8 10 12 14 16 18 20 22
Cel
l d
ensi
ty (
10
9×
cell
L-1
)
Time (d)
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-limited Continuous Culture
126
Table 6.3. Compositions of the modified nutrient-replete Fraquil* media examined in this study.
Composition Ratio of
[nutrient]original BG-11/[nutrient]original Fraquil*
Ratio of [nutrient]examined Fraquil*/[nutrient]original Fraquil*
Control Test 1 Test 2 Test 3 Test 4 Test 5
Salt solutions
CaCl2.2H2O 0.9 1.0 1.0 1.0 1.0 1.0 1.0
MgSO4.7H2O 2.0 1.0 2.0 2.0 2.0 2.0 2.0
NaHCO3 0.4a 1.0 1.0 1.0 1.0 1.0 1.0
NaNO3 176 1.0 10 20 40 80 176
K2HPO4 17.5 1.0 17.5 17.5 17.5 17.5 17.5
HEPES N.A.b 1.0 1.0 1.0 1.0 1.0 1.0
Trace metal solutions
CuSO4.5H2O 2.0 1.0 2.0 2.0 2.0 2.0 2.0
CoCl2.6H2O 3.4 1.0 3.4 3.4 3.4 3.4 3.4
MnCl2.4H20 15.2 1.0 15.2 15.2 15.2 15.2 15.2
ZnSO4.7H2O 0.6 1.0 1.0 1.0 1.0 1.0 1.0
Na2SeO3 N.A. 1.0 1.0 1.0 1.0 1.0 1.0
Na2MoO4.2H20 161 1.0 10 20 40 80 161
Fe(III)-ligand solutions
Na2EDTA.2H2O N.A. 1.0 1.0 1.0 1.0 1.0 1.0
FeCl3.6H2O N.A. 1.0 1.0 1.0 1.0 1.0 1.0
Vitamin solutions
Thiamine.HCl N.A. 1.0 1.0 1.0 1.0 1.0 1.0
Biotin N.A. 1.0 1.0 1.0 1.0 1.0 1.0
Cyanocobalamin N.A. 1.0 1.0 1.0 1.0 1.0 1.0
a This value represents ratio of the concentration of Na2CO3 in BG-11 and the concentration of NaHCO3 in Fraquil
*.
b N.A means “not applicable”
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
127
As seen in Figure 6.11, the growth of M. aeruginosa significantly increased when
additional nutrients were added. The lowest specific growth rate of M. aeruginosa
(0.81 ± 0.02 d-1
) was found in the control medium (i.e., Fraquil*) while the growth
rates in the other culture media used were comparable and averaged 0.90 ± 0.01 d-1
.
The cell density of M. aeruginosa grown in the control medium reached the maximum
value after 14 incubation days while the M. aeruginosa cell density in the modified
Fraquil* media continued to increase over the 20-d incubation period. These results
clearly show the obvious deficiency of nutrients for optimal growth of M. aeruginosa
in the original Fraquil* medium used in our previous studies. The lower cell densities
after day 10 in Test 4 and Test 5 media compared to those in the other modified
Fraquil* media (i.e., Test 1-3) suggest that the excessive concentrations of nitrate and
molybdenum in these two media adversely affected the growth of M. aeruginosa.
Overall, among the culture medium examined here, Test 1, Test 2 and Test 3 culture
media apparently provided the best growth conditions for M. aeruginosa with growth
of this organism in these culture media relatively similar. Thus, either Test 1, Test 2 or
Test 3 culture media could be chosen as the nutrient-replete medium yielding optimal
growth of M. aeruginosa. In this study, the Test 2 medium (hereafter referred to as
nutrient-replete Fraquil*) was chosen as the nutrient-replete culture medium for further
studies of M. aeruginosa growth.
6.3.6.2. Growth Kinetics in Batch Culture
In order to predict the behaviour of the Fe-limited chemostat cultures of M. aeruginosa
PCC7806 grown in nutrient-replete Fraquil* and thereby to decide on the chemosat
operating parameters (i.e., total Fe in the inflowing nutrient-replete Fraquil* medium
and the critical dilution rate, etc.) for the continuous culturing system, batch-wise
growth of this organism in nutrient-replete Fraquil* was investigated over a range of Fe
concentrations ([Fe]T = 0.05-10 µM).
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-limited Continuous Culture
128
Figure 6.12. Growth curves in batch cultures of M. aeruginosa at various total Fe concentrations in the nutrient-replete Fraquil* medium (i.e., Test 2 medium).
Total Fe concentration was varied from 0.05 to 10 µM while concentration of EDTA was fixed at 26 µM. Symbols represent the mean and error bars represent
the standard deviation from duplicate incubations (filled diamonds: [Fe]T = 0.05 µM; filled squares: [Fe]T = 0.1 µM, filled triangles: [Fe]T = 0.2 µM, filled
circles: [Fe]T = 0.5 µM, open diamonds: [Fe]T = 1.0 µM, open squares: [Fe]T = 2.0 µM, open triangles: [Fe]T = 5.0 µM, and open circles: [Fe]T = 10 µM).
0
2
4
6
8
10
12
14
0 2 4 6 8 10 12 14 16 18 20 22
Cel
l d
ensi
ty (
10
9×
cell
L-1
)
Time (d)
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
129
Although specific growth rate of M. aeruginosa under various Fe conditions was
relatively comparable (with an average value of 0.19 ± 0.011 d-1
) during the late
exponential growth phase (i.e., 8-14 d), it increased significantly with increasing Fe
concentration in the early exponential phase (i.e., 2-6 d), therefore, resulting in a
substantial increase in cell density in batch cultures of M. aeruginosa under high Fe
conditions (Figure 6.12). The specific growth rates in the early exponential phase for
cultures grown in the ≤ 2 µM Fe nutrient-replete Fraquil* growth media range from
0.61 ± 0.011 to 0.85 ± 0.007 d-1
and are significantly (p < 0.0002 using a single-tailed
heteroscedastic t-test) lower than the comparable growth rates of cultures grown in 5
and 10 µM Fe nutrient-replete Fraquil* (0.90 ± 0.05 and 0.92 ± 0.013 d
-1, respectively).
Thus, ≤ 2 µM of initial Fe induced growth inhibition while 5-10 µM Fe ensured
optimal growth of M. aeruginosa in nutrient-replete Fraquil*. As expected, when
grown in the same Fe concentration under identical incubation conditions, the batch
incubation cultures of M. aeruginosa in nutrient-replete Fraquil* consistently exhibited
higher specific growth rates (0.61 ± 0.011; 0.71 ± 0.022; 0.84 ± 0.011; and 0.92 ±
0.013 d-1
for [Fe]T = 0.05; 0.1; 1 and 10 µM, respectively) compared to those (0.57 ±
0.022; 0.65 ± 0.005; 0.77 ± 0.05; and 0.79 ± 0.006 d-1
for [Fe]T = 0.05; 0.1; 1 and 10
µM, respectively) in Fraquil* (Dang et al., 2012). These results are consistent with our
previous finding that the low concentration of nutrients in Fraquil* was not enough to
support optimal growth of M. aeruginosa (see Section 6.3.6.1).
The values of four growth constants of M. aeruginosa in the batch culture grown in
nutrient-replete Fraquil*: maximum specific growth rate µmax, half-saturation constants
TSK (for total Fe) and '
SK (for Fe(II)’) and yield constant Y were estimated using
identical calculation methods employed for the batch cultures of M. aeruginosa grown
in Fraquil* as discussed previously in Section 6.3.1. As such, the maximum growth
rate and the half-saturation constant were estimated via nonlinear regression of the data
using the Monod equation (eq. 6.4) for cases where both total Fe and calculated
steady-state Fe(II)’ were treated as the appropriate substrate for growth, yielding two
half-saturation constant 'S
K of 3.11 ± 0.30 fM with respect to Fe(II)’ and
TSK of 23 ±
2.2 nM for total Fe, and the same value for the growth constant (µmax = 0.89 ± 0.09 d-
1). The theoretical data of specific growth rate calculated using eq. 6.4 based on these
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
130
growth values fitted the observed growth rates of M. aeruginosa very well with R2 of
0.93 (Figure 6.13). Meanwhile, the average yield constant Y of M. aeruginosa under
Fe limitation condition ([Fe]T of 0.01 and 0.02 µM) was determined to be 27 ± 0.74 ×
1016
cell (mol Fe)-1
by using eq. 6.6. Compared to the growth constants of M.
aeruginsoa obtained from the batch studies in Fraquil*, the values of the half-
saturation constants 'S
K and
TSK and the growth constant µmax of this organism under
nutrient replete condiitons were comparable while the yield constant Y was about 3-
fold higher which accounts for the substantial increase in cell density when nutrient-
replete Fraquil* was used as the growth medium of M. aeruginosa instead of Fraquil
*.
Figure 6.13. Relationship between specific growth rate µ (d-1
) and log concentration of
unchelated Fe(II)’ (M) in batch culture studies of M. aeruginosa grown in nutrient-
replete Fraquil* medium. Solid and dotted lines represent the regression line and 95%
confidential interval, respectively. Symbols indicate data for experimentally
determined growth rate under different degrees of Fe limitation.
R2 = 0.9276
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
-14.5 -14 -13.5 -13 -12.5 -12 -11.5
Sp
ecif
ic g
row
th r
ate
µ (
d-1
)
log([Fe(II)'] (M))
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
131
6.3.6.4. Comparison between the Behavior of M. aeruginosa Grown Continuously in
Nutrient-replete Fraquil* and in Original Fraquil
*
A summary of the growth constants in the batch cultures and the behaviour of the
chemostat cultures of M. aeruginosa grown in both Fraquil* and nutrient-replete
Fraquil* is presented in Table 6.4. Detailed description of the behaviour (including
steady state cell density, Fe cell quota, cell size and Fe uptake kinetics) of M.
aeruginosa strain PCC7806 grown continuously in the nutrient-replete Fraquil*
medium with Fe activity buffered by the organic ligand EDTA is given below.
a. Performance of chemostat system under Fe limitation
Since the ≤ 2 µM Fe cultures were inhibited by depletion of Fe available for growth, a
total Fe concentration of 100 nM in the nutrient-replete Fraquil* growth medium was
used in this study to ensure that steady state chemostat cultures of M aeruginosa were
maintained under Fe limitation. At this initial Fe concentration (i.e., 100 nM Fe) in the
inflowing growth medium, the corresponding critical dilution rate for complete wash-
out (Dc) was determined to be 0.72 d-1
. Hence, the continuous cultures were
maintained in 100 nM Fe nutrient-replete Fraquil* at dilution rates of 0.07, 0.15, 0.30
and 0.45 d-1
which were less than the critical dilution rate Dc for a period of 40 d
(Figure 6.14). M. aeruginosa cells grew continuously in 100 nM nutrient-replete
Fraquil* in a similar growth pattern as seen in the 20 nM Fe Fraquil
* chemostat
cultures (part B of Figure 6.5), i.e. after 2-d lag phase, cells density of the four dilution
rates increased exponentially and appeared to reach steady state concentrations after
day 25.
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
132
Figure 6.14. Growth of M. aeruginosa in the continuous culture system at different
dilution rates with total Fe concentrations in the inflowing nutrient-replete Fraquil*
medium [Fe]T = 100 nM, with dilution rates of 0.07 d-1
(diamonds), 0.15 d-1
(squares),
0.30 d-1
(triangles) and 0.45 d-1
(circles). Symbols represent the mean and error bars
the standard deviation from triplicate incubations. Dashed lines represent the 95%
confidence interval at steady-state.
Similar to the previous chemostat studies which used Fraquil* as growth medium, the
theoretical steady state cell and Fe concentrations in the nutrient-replete chemostat
cultures as a function of dilution rate were estimated by substituting the values for the
growth constants: 'S
K , TS
K , µmax and Y into eqs 6.15 and 6.15. These data are plotted
in Figure 6.15 together with observed steady state cell densities at different dilution
rates. For comparative purposes, measured and theoretical steady state cell densities
obtained previously from the batch studies in Fraquil* are also presented in this graph.
In all cases, the observed steady state cell densities at the lowest dilution rate were
always greater than the corresponding theoretical values. This is probably because at
the lowest dilution rate (i.e., the lowest inflow rate) the evaporation rate of water inside
the culture vessel was likely comparable to the flow-rate of the inflowing medium
which leads to accumulation of nutrients in the culture vessel, hence, increase in the
8.8
9.0
9.2
9.4
9.6
9.8
10.0
10.2
10.4
10.6
10.8
0 5 10 15 20 25 30 35 40 45
Loga
rith
m o
f ce
ll d
en
sity
(ce
ll L
-1)
Time (d)
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
133
cell density. Overall, the theoretical steady state cell densities as function of dilution
rate fitted well the observed steady state cell densities of M. aeruginsoa, indicating that
the modified chemostat theory proposed in Section 6.2.7 (Dang et al., 2012) was
applicable to predicting the steady state cell densities in the Fe-limited chemostate
cultures of M. aeruginosa grown in both Fraquil* and nutrient-replete Fraquil
* media.
Figure 6.15. Predicted and measured steady-state cell density and substrate
concentration in continuous cultures of M. aeruginosa as a function of dilution rate
with different total Fe concentrations in the two inflowing media: Fraquil* (20 nM and
50 nM) and nutrient-replete Fraquil* (100nM). Symbols represent data for steady-state
cell density in Fraquil* medium with total Fe of 20 nM (triangles), 50 nM (squares)
and 100 nM (diamonds). Dotted lines are the theoretical values of steady-state cell
density calculated from eq. 6.16 with growth parameters estimated from batch culture
studies in Fraquil* (µmax = 0.80 ± 0.03 d
-1, '
SK = 3.6 ± 0.32 fM with respect to Fe(II)’,
TSK = 26 ± 2.3 nM with respect to total Fe, and Y = 8.1 ± 0.21 × 10
16 cell (mol Fe)
-1),
while bold lines indicate the theoretical steady-state cell density estimated with
parameters obtained from batch culture studies in nutrient-replete Fraquil* (µmax = 0.89
± 0.03 d-1
, 'S
K = 3.1 ± 0.30 fM, TS
K = 23 ± 2.2 nM, and Y = 2.7 ± 0.74 × 1017
cell
0
50
100
150
200
250
300
350
400
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
[Fe(
II)'
]ss
(fM
)
or
[Cel
l]ss
(10
8ce
ll L
-1)
Dilution rate (d-1)
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
134
(mol Fe-1
)). Chained and dashed lines indicate predicted steady-state unchelated Fe(II)’
concentrations estimated using parameters from batch culture studies in Fraquil* and
nutrient-replete Fraquil*, respectively.
b. Cellular Fe quota and cell size under iron limitation
The intracellular Fe content of cells in the steady state chemostat cultures grown in 100
nM Fe nutrient-replete Fraquil* at different dilution rates was examined by using acid
digestion combined with spectrophotometry as described in Section 2.5.1. The steady-
state cellular Fe quotas at dilution rates of 0.07, 0.15, 0.30 and 0.45 d-1
were
determined to be 17.5 ± 2.1; 26.5 ± 0.7; 33.7 ± 2.4 and 48 ± 1.4 amol cell-1
,
respectively which are about one-order of magnitude higher than those observed in
Fraquil*. Similar to the data for the chemostat cultures of M. aeruginosa grown in
Fraquil*, the specific growth rates (i.e., the dilution rates) of this organism in nutrient-
replete Fraquil* increased hyperbolically with increasing steady state Fe quota (Figure
6.16) which is in accordance with the empirical Droop equation (eq. 6.19). These
results are consistent with other observations for cyanobacteria (Gotham and Rhee,
1981b, Ahlgren, 1985, Olsen, 1989) and eukaryotic phytoplankta (Goldman and
Mccarthy, 1978, Sunda and Huntsman, 1985, Sunda and Huntsman, 1986, Harrison
and Morel, 1986).
In terms of cell size, there was no significant change in the cell diameter with
increasing dilution rates observed in this study. However, M. aeruginosa cells were
slightly larger when grown continuously in nutrient-replete Fraquil* with an average
diameter of 4.01 ± 0.09 µm than in Fraquil* with an average diameter of 3.87 ± 0.15
µm. The higher nutrient levels (including Fe) in nutrient-replete Fraquil* compared to
those in Fraquil* possibly account for the increase in both the Fe quotas and the cell
size of the nutrient-replete chemostat cultures in this work.
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
135
Figure 6.16. Relationship between the cellular Fe quota (Q) and the specific growth
rate (µ) for Fe-limited M. aeruginosa under steady-state conditions in continuous
cultures. The system was operated at four different dilution rates (0.07, 0.15, 0.30 and
0.45 d-1
) and fed with nutrient-replete Fraquil* medium containing 100 nM Fe. The
solid line represents the theoretical curve calculated from the Droop equation using the
obtained estimated values of µ’max = 0.69 ± 0.05 d-1
and Qmin = 18 ± 2.6 amol cell-1
.
Symbols represent the mean from triplicate measurements.
c. Fe uptake kinetics and cellular response to Fe limitation in chemostat
Short-term 55
Fe uptake assays in the presence of light were undertaken using Fe-
limited cells from the steady-state chemostat cultures of M. aeruginosa grown in
100nM Fe nutrient-replete Fraquil* at dilution rates of 0.07; 0.15; 0.30 and 0.45 d
-1.
Processing steps in these short-term Fe uptake experiments were identical to those
described in Section 6.2.5 in which the examined cells were incubated in Fraquil*
medium (Dang et al., 2012). Since the photo-produced unchelated ferrous iron (i.e.,
Fe(II)’) has previously been shown to be the major form of Fe acquired by M.
aeruginosa in Fraquil* buffered by the synthetic organic ligand EDTA (Fujii et al.,
2011a, Dang et al., 2012), the Fe uptake rate of each steady-state culture of M.
aeruginosa was investigated over a range of photoproduced unchelated Fe(II)’
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 20 40 60 80 100
Sp
eci
fic
gro
wth
ra
te µ
(d
-1)
Cellular Fe quota (amol cell-1)
Critical dilution rate Dc = 0.72 d-1
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
136
concentrations by varying the EDTA concentration from 5 to 200 µM while
maintaining 55
Fe concentration at a constant value of 200 nM. As seen in Figure 6.17,
intracellular 55
Fe accumulated linearly (R2 = 0.991 ÷ 0.998) over the first 4 h during
incubation in the light followed by non-linear increase of the intracellular 55
Fe after 4-
8 h incubation, indicating that uptake reached saturated levels during the later stages of
the experiment.
Insight into Fe uptake kinetics was undertaken by applying Eadie-Hofstee linear
transformation to the measured 55
Fe uptake rates in each culture as shown in Figure
6.18, provided that the short-term uptake rates for M. aeruginosa followed classical
Michaelis-Menten kinetics (see eq. 6.21). Linear regression analysis (R2 = 0.91-0.99)
yielded comparable half-saturation constants for uptake with an average value of Kρ =
45 ± 1.9 fM but significantly different maximum uptake rates (ρmax of 1005 ± 46; 887
± 79; 684 ± 87 and 483 ± 35 zmol Fe cell-1
h-1
) for the chemostat cultures grown at
dilutions rates of 0.07; 0.15; 0.30 and 0.45 d-1
, respectively). The invariant nature of
the half-saturation constant Kρ at different dilution rates and the significant
dependence of the maximum uptake rate on the degree of Fe stress of cells are
consistent with previous classical findings where Kρ was generally observed to be
constant while ρmax was observed to increase with the degree of nutrient starvation of
cells (Gotham and Rhee, 1981a, Gotham and Rhee, 1981b, Olsen, 1989, Harrison and
Morel, 1986, Morel, 1987).
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
137
Figure 6.17. Time-course of 55
Fe uptake during batch short-term Fe uptake assays
using cells obtained at steady-state from the chemostat cultures grown with [Fe]T =
100 nM in the inflowing nutrient-replete Fraquil* medium and dilution rates of 0.07 d
-1
(diamonds), 0.15 d-1
(squares), 0.30 d-1
(triangles) and 0.45 d-1
(circles). In the short-
term uptake assay, each culture was incubated in nutrient-replete Fraquil* with 20 µM
EDTA and 200 nM radiolabeled 55
Fe. Symbols represent the mean and error bars
represent the standard deviation from triplicate experiments. The continuous lines were
obtained by linear regression of data collected within 4 h (represented by closed
symbols) for each culture.
The short-term Fe uptake data of M. aeruginsoa grown under nutrient replete
conditions were compared with those reported previously for Fe-limited chemostat
cultures of this organism grown in Fraquil* (Dang et al., 2012). As observed
previously, the half saturation-constant for uptake, Kρ, was again found to be
independent of the degree of Fe stress. However, an obvious difference between the
two studies is that the value of Kρ for the Fe-limited chemostat cultures grown under
nutrient-replete conditions (i.e., in nutrient-replete Fraquil*) was ~2.5 times higher than
that obtained in nutrient-deplete conditions (i.e., in Fraquil*), suggesting that when
R² = 0.9982
R² = 0.996
R² = 0.9971
R² = 0.9912
0
500
1000
1500
2000
2500
3000
3500
4000
0 2 4 6 8 10
Acc
um
ula
ted
55F
e (z
mol
cell
-1)
Time (hr)
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
138
grown under greater nutrient deficiency (including Fe), Fe-limited M. aeruginosa cells
become more competitive for photoproduced unchelated Fe(II)’. The most significant
difference in responses of cells with regard to Fe uptake between this study and the
previous chemostat study in which Fraquil* was used as growth medium was the Fe
uptake trend over the degree of Fe stress. In terms of Fe-limited cells grown in
nutrient-replete Fraquil*, Fe-starved cells exhibited much higher maximum uptake
rates as expected. In contrast, for Fe-limited cells grown in the nutrient-insufficient
growth medium (i.e., Fraquil*), the maximum short-term uptake was also dependent on
the degree of Fe stress but surprisingly decreased with increase in extent of Fe
deficiency.
Overall, when grown under nutrient-replete conditions, the kinetics of steady state
growth and short-term Fe uptake by Fe-limited cells in chemostat cultures of M.
aeruginosa strictly followed the classical hyperbolic expressions for steady state
growth (i.e., the Droop equation) and uptake (Michaelis-Menten equation), implying
that the nutrient-replete Fraquil* medium provided sufficient nutrient conditions for
optimal growth of M. aeruginosa cells. There were no abnormal responses of cells in
Fe-limited chemostat with regard to Fe uptake rate when nutrient-replete Fraquil* was
used as the inflowing growth medium, meaning that the M. aeruginosa cells and their
Fe uptake machinery functioned properly under these conditions. In comparison, under
nutrient deficient conditions (i.e., Fraquil*), although the steady state long-term uptake
kinetics of M. aeruginosa cells were not affected (i.e., the empirical Droop equation
was still followed), the short-term Fe uptake was found to surprisingly increase with
decreasing degree of Fe-limitation. It would appear that this difference in kinetics of
steady state growth and short-term Fe uptake between the cells in the Fe-limited
chemostat cultures grown in Fraquil* and those in nutrient-replete Fraquil
* stemmed
from the difference in the nutrient levels of the two culture media used.
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
139
Figure 6.18. Eadie-Hofstee plots demonstrating the linear relationship between the
short-term 55
Fe uptake rate (ρFe) and the ratio ρFe/[Fe(II)’] for cultures of M.
aeruginosa. Linear regression analysis yielded comparable half-saturation constants
for Fe uptake (Kρ = 45 ± 1.9 fM, as Fe(II)’) but different maximum specific uptake
rates (ρmax of 1.0 ± 0.046, 0.89 ± 0.079, 0.67 ± 0.087 and 0.48 ± 0.035 amol cell-1
hr-1
for cultures grown at dilution rates of 0.07 d-1
(diamonds), 0.15 d-1
(squares), 0.30 d-1
(triangles) and 0.45 d-1
(circles), respectively). Lines for 95% confidential intervals
were omitted for clarity.
The results from both the batch culture and chemostat culture studies in this work
suggest that the Fraquil* medium often used for study of trace metal interactions with
freshwater phytoplankton by several workers (Rueter and Ades, 1987, Rueter, 1988,
Gensemer, 1990, Gensemer et al., 1993, Fujii et al., 2010a, Fujii et al., 2011a, Alexova
et al., 2011, Dang et al., 2012) was insufficient to provide optimal growth of M.
aeruginosa. It would seem reasonable to conclude that the “unhealthy” organisms
present under nutrient deficient conditions were more prone to Fe deficiency and were
increasingly incapable of acquiring Fe as the extent of Fe deficiency increased. In
y = -42.7x + 1,005
R² = 0.99
y = -45.3x + 887
R² = 0.96
y = -47.2x + 684
R² = 0.91
y = -46.4x + 483
R² = 0.97
0
100
200
300
400
500
600
700
800
900
0 5 10 15 20 25
55Fe
up
tak
e r
ate
(zm
ol
cell
-1h
r-1)
55Fe uptake rate/[Fe(II)']ss (µmol cell-1 hr-1 M-1)
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-
limited Continuous Culture
140
comparison, organisms grown with sufficient major nutrients appear able to respond to
increasing Fe deficiency by increasing their short term Fe uptake rate.
6.4. CONCLUSIONS
In this chapter we have shown that a continuous culturing system made of metal-free
material provides a valuable tool to investigate the cellular responses of Fe-limited M.
aeruginosa PCC7806 in both nutrient-insufficient and nutrient-replete Fraquil* media.
The system was successfully operated to produce steady-state cultures with different
cell densities and different cellular properties. In both nutrient-insufficient and
nutrient-replete cases, the cellular response to steady-state Fe limitation in the
chemostat system followed the Droop equation, i.e. cellular Fe quotas increased with
increasing Fe availability. Under Fe stress, cells of steady-state cultures of M.
aeruginosa regulated their short-term Fe uptake by varying their uptake capacity ρmax,
but not their affinity for Fe (i.e. the half saturation constant Kρ). Under nutrient-
insufficient case, Fe uptake data from this study show that Fe-limited M. aeruginosa
cells grown under severe Fe stress (i.e., lower dilution rates) are likely unable to
synthesize sufficient resources (such as internal transporters and/or ATP) required for
Fe uptake, and therefore exhibit lower Fe uptake ability compared to cells grown under
conditions in which Fe is more available (i.e., at higher dilution rates). In contrast, the
relationship between Fe uptake capacity and the degree of Fe-limitation reverted to
that expected with the short-term Fe uptake rate increasing with the degree of Fe stress
when Fe-limited M. aeruginosa cells are grown under nutrient-replete conditions
where M. aeruginosa cells are “healthy” and functioning normally.
Chapter 6. Characteristics of the Freshwater Cyanobacterium Microcystis aeruginosa Grown in Iron-limited Continuous Culture
141
Table 6.4. Summary of the growth constants in the batch cultures and the behaviors of the Fe-limited chemostat cultures at different dilution
rates of M. aeruginosa grown in both Fraquil* and nutrient-replete Fraquil
*
Parameters Growth medium
Unit Fraquil
* Nutrient-replete Fraquil
*
A. Batch cultures
(i) Fe limitation condition ≤ 0.1 ≤ 2.0 µM
(ii) Growth constants
- µmax 0.80 ± 0.09 0.89 ± 0.03 d-1
- TS
K 26 ± 2.3 23 ± 2.2 nM
- 'S
K 3.6 ± 0.32 3.1 ± 0.30 fM
- Y 8.1 ± 0.21 × 1016
27 ± 0.74 × 1016
cell (mol Fe)-1
B. Continuous cultures
(i) Operating parameters
- [Fe]initial 20 100 nM
- Critical dilution rate 0.34 0.72 d-1
- Operating dilution rate 0.09; 0.14; 0.17 and 0.25 0.07; 0.15; 0.30; 0.45 d-1
(ii) Cellular Fe quota Follow Droop equation (expected) Follow Droop equation (expected)
- Qmin 1.2 ± 0.24 18 ± 2.6 amol cell-1
- µ'max 0.37 ± 0.04 0.69 ± 0.05 d
-1
(iii) Cell diameter 3.87 ± 0.15 4.01 ± 0.09 µm
(v) Fe uptake kinetics Maximum uptake rate increases with
increasing dilution rates (unexpected)
Maximum uptake rate decreases with
increasing dilution rates (expected)
- ρmax 0.27 ± 0.030, 0.72 ± 0.060, 0.95 ± 0.080
and 1.0 ± 0.090 (varied)
1.0 ± 0.046; 0.89 ± 0.079; 0.67 ± 0.087
and 0.48 ± 0.035 (varied)
amol Fe cell-1
hr-1
- Kρ ~18 ± 2.2 (constant) ~45 ± 1.9 (constant) fM
142
CHAPTER 7
CONCLUSIONS AND
RECOMMENDATIONS
Chapter 7. Conclusions and Recommendations
143
7.1. CONCLUSIONS
The results of studies described in this thesis provide new insights into the Fe uptake
kinetics of the bloom-forming freshwater cyanobacterium M. aeruginosa, particularly
in regard to: (i) effect of light on iron uptake by M. aeruginosa in the presence of a
single metal-chelator, ethylenediaminetetraacetic acid (EDTA); (ii) intracellular Fe
transport processes of M. aeruginosa in a chemically well-defined culture medium
(Fraquil*) buffered by EDTA; (iii) iron uptake kinetics by M. aeruginosa in the
presence of the natural organic ligand, Suwannee River Fulvic Acid (SRFA); and (iv)
characteristics of M. aeruginosa cells grown in iron-limited continuous culture. The
main conclusions obtained from this research are summarized below.
Chapter 3. Effect of Light on Iron Uptake by the Freshwater
Cyanobacterium Microcystis aeruginosa
The aim of the studies described in this chapter was to elucidate the effect of light on the
iron uptake by M. aeruginosa in a chemically well-defined culture medium (Fraquil*)
in the presence of a single metal chelator, ethylenediaminetetraacetic acid (EDTA).
Major findings of this aspect of the study are provided below.
(i) Visible light was observed to induce reductive dissociation of organically
complexed Fe and dramatically increase the short-term uptake rate of
radiolabeled Fe by M. aeruginosa PCC7806 in Fraquil* medium at pH 8 buffered
by EDTA. Only wavelengths < 500 nm activated Fe uptake indicating that Fe
photochemistry rather than biological factors is responsible for the facilitated
uptake.
(ii) The measured rate of photochemical Fe(II) production combined with a
significant decrease in 55
Fe uptake rate in the presence of ferrozine (a strong
ferrous chelator) confirmed that photo-generated unchelated Fe(II) was the major
form of Fe acquired by M. aeruginosa under the conditions examined.
(iii) Mathematical modeling based on unchelated Fe(II) uptake by concentration
Chapter 7. Conclusions and Recommendations
144
gradient dependent passive diffusion of Fe(II) through the non-specific
transmembrane channels (porins) could account for the magnitude of Fe uptake
and a variety of other observed effects.
(iv) Steady-state uptake rates indicated that M. aeruginosa acquires Fe
predominantly during the light cycle. This study confirms that Fe photochemistry
has a dominant impact on Fe acquisition and growth by M. aeruginosa in EDTA-
buffered culture medium.
Chapter 4. Kinetics of Extracellular Iron Transport to Periplasmic
and Cytoplasmic compartments of the Freshwater Cyanobaterium
Microcystis aeruginosa
The aim of the studies described in this chapter was to gain insight into the kinetics of
extracellular Fe transport to periplasmic and cytoplasmic compartments for strains
PCC7806 and 7005 of M. aeruginosa. Major findings of this aspect of the study are
provided below.
(i) A negligibly small amount of chelated 55
Fe accumulated in the periplasm of
plasmolysed cells, confirming that only unchelated Fe is capable of crossing the
outer membrane, most likely by a diffusive process.
(ii) The observed Monod-type relationship between cytoplasmic 55
Fe accumulation
rates and steady-state concentrations of unchelated Fe in the periplasm and
extracellular environment suggests that translocation of Fe into the cytoplasm
involves complexation of Fe by a limited number of Fe-binding sites in the
periplasm followed by subsequent transport into the cytoplasm, possibly via
energy-dependent plasma-membrane Fe transporters.
(iii) Further experimental evidences suggest that redox state of intracellular Fe is
relevant to the transport of periplasmic Fe into the cytoplasm with Fe redox state
possibly regulated by oxidoreductase enzymes such as multi-copper oxidase
resident in the periplasm.
Chapter 7. Conclusions and Recommendations
145
Chapter 5. Iron Uptake Kinetics by a Freshwater Cyanobacterium
Microcystis aeruginosa in the Presence of Suwannee River Fulvic Acid
The aim of the studies described in this chapter was to determine the effect of the
presence of the natural organic compound, Suwannee River fulvic acid (SRFA), on
iron uptake kinetics by M. aeruginosa. Major findings of this aspect of the study are
provided below.
(i) A kinetic model was developed which satisfactorily described the results
obtained and which provided insight into the significant reactions and
processes involved in cellular Fe uptake in the presence of SRFA.
(ii) During incubations under dark and visible light, 55
Fe uptake rates similarly
decreased in an exponential manner as SRFA concentration increased, even
though photochemical reduction rate of Fe(III) bound to SRFA (FeIII
SRFA)
was one order of magnitude greater than non-photochemical reduction. The
similarity of 55
Fe uptake rate under the dark and light was accounted for by
relatively rapid oxidation of photo-generated Fe(II) prior to dissociation of
the complex forming unchelated Fe(II), resulting in the comparable Fe(II)
and Fe(III) availability in the dark and light.
(iii) Model prediction suggested that the decreased trend of 55
Fe uptake at
higher SRFA concentrations is due to limited availability of unchelated Fe
for cellular uptake.
(iv) The inhibitory effect of strong Fe(II) chelator (ferrozine) on 55
Fe uptake
indicates that approximately a half of total Fe uptake was accounted for by
uptake of unchelated Fe(II) likely produced via reductive dissociation of
FeIII
SRFA.
(v) The findings in this study suggest that Fe uptake is generally near
saturation for the fulvic acid concentrations typically encountered in natural
waters (e.g., < ~10 mg.L-1
).
Chapter 7. Conclusions and Recommendations
146
Chapter 6. Characteristics of the Freshwater Cyanobacterium
Microcystis aeruginosa Grown in Iron-limited Continuous Culture
The aim of studies described in this chapter was to determine the characteristics of M.
aeruginosa grown in iron-limited continuous culture. Major findings of this aspect of
the study are provided below.
(i) A kinetic model describing Fe transformations and biological uptake was
developed and applied to determination of the biologically available form of Fe
(i.e., unchelated ferrous iron) that is produced by photoreductive dissociation of
the ferric EDTA complex.
(ii) Prediction by chemostat theory modified to account for the light-mediated
formation of bioavailable Fe was in good agreement with growth characteristics
of M. aeruginosa under Fe limitation.
(iii) In both nutrient-insufficient and nutrient-replete cases, cellular Fe quota
increased with increasing dilution rate in a manner consistent with Droop theory.
Short-term Fe uptake assays using cells maintained at steady-state indicated that
M. aeruginosa cells vary their maximum Fe uptake rate (ρmax) depending on the
degree of Fe stress.
(iv) Under nutrient-insufficient conditions, the rate of Fe uptake was lower for cells
grown under conditions of lower Fe availability (i.e., lower dilution rate)
suggesting that cells in the continuous cultures adjusted to Fe-limitation by
decreasing ρmax whilst maintaining a constant affinity for Fe. This result implies
that Fe-limited M. aeruginosa cells grown under severe Fe stress and other
nutrients insufficiency are likely unable to synthesize sufficient resources
required for Fe uptake.
(v) In contrast, under nutrient-replete conditions M. aeruginosa cells are “healthy”
and functioning normally. As such, the relationship between Fe uptake capacity
and the degree of Fe-limitation reverted to that expected with the short-term Fe
uptake rate increasing with the degree of Fe stress.
Chapter 7. Conclusions and Recommendations
147
7.2. IMPLICATIONS OF THE FINDINGS
7.2.1. With Regard to Knowledge of Fe Transformation and Uptake
Kinetics by Freshwater Cyanobacteria in Natural Waters
This work provides compelling evidence of the connection between the thermal and
photochemical transformation of Fe species and biological uptake where unchelated
Fe(II)’ formed by photochemical and thermal reduction of Fe(III) species is an
important substrate for growth and represents one of the important growth factors
controlling Fe uptake by phytoplankton. Also, insights gained into the kinetics of
transport of extracellular Fe to periplasmic and cytoplasmic spaces in the freshwater
cyanobacterium Microcystis aeruginosa should be applicable to other cyanobateria
given their similarity in cell structure.
7.2.2. With Regard to Application of the Continuous Culturing
System for Study of Trace Metal Interactions with Freshwater
Phytoplankton
In this thesis, the chemostat system was successfully developed for maintaining
steady-state continuous cultures of M. aeruginosa at different dilution rates under Fe
limited conditions. The performance of the system was checked and the results
indicate that this system was effectively used to grow the Fe-limited chemosat cultures
under steady state conditions for more than one month (at least 40 d) without any
biological and trace metal contamination. In addition, the compactivity of the system
makes it possible to be incorporated into an incubator which allows experimenters to
easily control not only the chemical growth conditions such as pH, oxygen,
concentrations of nutrients, etc. but also the physical conditions such as light intensity
and temperature. These advantages of this continuous culture system make it a suitable
apparatus for investigating growth and behaviours of microorganism in steady-state
chemosat cultures under micro-nutrients limitation.
Also, the modified chemostat theory for Fe-limited phytoplankton growth proposed in
this thesis described well the observed steady state cell density of M. aeruginosa strain
Chapter 7. Conclusions and Recommendations
148
PCC7806 grown continuously in growth medium (Fraquil* or nutrient-replete Fraquil
*)
with Fe activity buffered by the organic ligand EDTA. It can be applicable to trace
metal-limited chemostat studies where the concentration of limiting nutrient is
considered as the concentration of the bio-available forms for uptake (i.e., photo-
produced unchelated Fe(II)’ in this thesis) rather than the total concentration of
nutrient used in macronutrient-limited chemostat studies. Hence, it can be adopted as a
new model of chemostat functioning under trace metal-limited condition and used to
predict the steady-state concentrations of microorganism being cultured and limiting
substrate in the culture vessel for any value of the dilution rate and the concentration of
the inflowing limiting substrate.
7.2.3. With Regard to Knowledge of the Composition of the Growth
Medium for Freshwater Phytoplankton
The results obtained from both batch and continuous culture studies in this thesis
indicate that Fraquil* contained insufficient nutrients to support the optimal growth of
the freshwater cyanobacterium M. aeruginosa.. In comparison, the nutrient-replete
Fraquil* medium used in the studies described in this thesis was able to sustain optimal
growth of M. aeruginosa and can be recommended as a suitable medium for future
studies on the effects of metals on growth, nutrient uptakes, photosynthetic activity, or
morphology, etc. of this organism. While testing would be required to ensure that
nutrient concentrations are adequate, this modified Fraquil* medium could also be used
for the study of trace metal interactions with other freshwater phytoplankton.
7.3. RECOMMENDATIONS FOR FUTURE WORK
In order to improve our understanding of Fe uptake kinetics by M. aeruginosa in
natural waters, additional studies are required. Specific recommendations for future
work are noted below.
(i) It should be noted that all the M. aeruginosa cells in both batch and continuous
cultures used in this research was grown in the growth medium (Fraquil* or
nutrient-replete Fraquil*) with the free iron activity buffered by the well-
characterized model ligand EDTA before being harvested for further various
Chapter 7. Conclusions and Recommendations
149
experiments of interest. In order to reflect more closely the natural aquatic
systems, study of the kinetics of Fe acquisition using Fe-limited M. aeruginosa
cells grown batch-wise or continuously in the presence of iron either complexed
to natural organic matter (NOM) rather than the model organic ligand EDTA or
present as amorphous ferric oxyhydroxide is considered necessary.
(ii) As mentioned previously in Section 5.4, Chapter 5, the nature of the light
provided by the incubator fluorescent light used in this research and natural
sunlight is acknowledged to be substantially different (e.g., light intensity of 157
µmol m-2
s-1
compared to 2,000 µmol m-2
s-1
), leading to significant differences
in photo-reduction rate of Fe(III) to Fe(II) in laboratory compared to natural
systems. As such, there is justification for further studies to investigate the Fe
uptake kinetics of M. aeruginosa using a solar simulator.
(iii) Fe redox reactions inside cells or near the cell surface are known to be important
in Fe acquisition by phytoplankton. However, for simplicity, the redox state of Fe
in the periplasmic and cytoplasmic spaces of M. aeruginosa cells has not been
considered in the model for translocation of Fe from the external medium to the
intracellular compartments developed here. Therefore, further investigation on
the effect of Fe speciation in the periplasm and cytoplasm of M. aeruginosa cells
on intracellular Fe transport is required.
References
150
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APPENDIX 1
SUPPLEMENTAL MATERIAL FOR
CHAPTER 3 - EFFECT OF LIGHT ON
IRON UPTAKE BY THE FRESHWATER
CYANOBACTERIUM MICROCYSTIS
AERUGINOSA
Appendix 1. Supplemental Material for Chapter 3
171
A1.1. FEII
FZ3 FORMATION FROM FEIII
EDTA IN THE
LIGHT AND DARK
Primary kinetic data for FeIIFZ3 formation measured in the light and dark are shown in
parts A-D of Figure A1.1. FeIIFZ3 concentration substantially increased with time in
the light, while the time-dependent increase in FeIIFZ3 concentration was small or
negligible in the dark. Although FeIIFZ3 formation was unnoticeable at low Fe
concentration (1 µM), the linear increase in FeIIFZ3 concentration with time (R
2 =
0.99) was observed at a high Fe concentration (10 µM) possibly due to the active
reduction of Fe(III) species by FZ (Shaked et al., 2004) . In the high Fe concentration
system, therefore, the photo-produced FeIIFZ3 concentration was correctly calculated
by subtracting the FeIIFZ3 concentration produced in the dark from that produced in
the light at each incubation time.
In the FeIII
EDTA system, the ligand-to-metal charge transfer (LMCT) reaction will
ultimately produce unchelated Fe(II) (Fe(II)') and photo-oxidized EDTA (EDTAox). In
the presence of a high concentration of FZ, this strong Fe(II) complexing agent would
be expected to outcompete EDTA for complexation of liberated Fe(II)' and would also
be expected to prevent the oxygenation of Fe(II)' with subsequent formation of
FeIII
EDTA. This assumption was verified by kinetic calculations using relevant rate
constants as discussed below. Thus, the system can be simply described by the
following two reactions:
FeIIIEDTA + hν
khv*
→ Fe(II)' + EDTAox
(A1-1)
3
IIFZFe 3FZ Fe(II)' FZf →+ −k (A1-2)
where k*hv and kf-FZ are rate constants for photo-reductive dissociation of Fe
IIIEDTA
and for Fe(II) complexation by FZ, respectively. The value of kf-FZ has been previously
determined to be 3.1 × 1011
M-3
s-1
in 0.1 M NaClO4 (Thompsen and Mottola, 1984)
and 2.0 × 1011
M-3
s-1
in seawater (Lin and Kester, 1992). The effect of solution pH on
kf-FZ is insignificant in the range of pH 3 to 8. Since the ionic strength was closer to
Appendix 1. Supplemental Material for Chapter 3
172
that of this work, the value of 3.1 × 1011
M-3
s-1
was chosen. At the FZ concentration
employed in this work (1 mM), the complexation reaction of Fe(II)' by FZ is very fast
(kf-FZ[FZ]3 = 310 s
-1) and outcompetes any other competing reactions (i.e., oxidation by
oxygen, kox[O2] = 0.0021 s-1
, and re-complexation of Fe(II)' by EDTA, kf-EDTA[EDTA]
= 52 s-1
, Table 3.4). The FeIIFZ3 complex dissociates with a first-order rate constant of
4.3 × 10-5
s-1
(Thompsen and Mottola, 1984). However, the effect of FeIIFZ3
dissociation on the photo-formation rate of FeIIFZ3 is negligible, as any Fe(II)'
produced is predominantly recaptured by FZ under the conditions of photochemical
experiment. Therefore, the FeIIFZ3 formation rate was assumed to be equal to the
photo-production rate of Fe(II)'. The rate law for the photo-reductive dissociation of
FeIII
EDTA can be written as:
d[FeIIFZ3]
dt=
d[Fe(II)']
dt= k
hv
* [hν][FeIIIEDTA] (A1-3)
where [hv] represents light intensity. Under constant illumination, k*hv[hv] is also
invariant, i.e.:
d[Fe(II)']
dt= k
hv
* [hν][FeIIIEDTA] = khv
[FeIIIEDTA] (A1-4)
where khv indicates a first-order rate constant for photo-reductive dissociation of
FeIII
EDTA under the constant irradiation intensity examined. Approximating
[FeIII
EDTA] ≈ [Fe]Total – [FeIIFZ3] (due to the low [Fe(II)'], e.g., [Fe(II)'] = 2.0×10
-
8[Fe
IIIEDTA] + 1.4×10
-7[Fe
IIFZ3]) and [FZ] ≈ [FZ]Total (due to the high [FZ]Total, e.g.,
[FZ]Total = 1 mM >> [Fe]Total = 0.01-0.001 mM) followed by the integration gives the
relationship between FeIIFZ3 concentration and time, as follows:
ln[Fe]
T
[Fe]T
− [FeIIFZ
3]
= k
hv⋅ t (A1-5)
The value of khv was determined as the slope from linear regression of plots of time
versus ln([Fe]T/([Fe]T-[FeIIFZ3])) (parts E-F of Figure A1.1).
Appendix 1. Supplemental Material for Chapter 3
Figure A1.1. Time-course of
and (B and D) dark. For
equilibrated FeIII
EDTA
10 µM for Fe(III), 26
several hours at 27oC in the presence and absence of the light (
1). Photo-reductive dissociati
to the measurements with (E)
indicate average data and
represent linear regression lines.
A1.2. AVAILABILITY O
IN THE DARK
To constrain the chemical
examined the availability of
purpose, a photolyzed
Material for Chapter 3
course of FeIIFZ3 formation from Fe
IIIEDTA in
and (B and D) dark. For measurement of photo-reduction rate of Fe
EDTA complex and FZ were mixed in Fraquil* at concentrations
26 µM for EDTA and 1 mM for FZ, followed by incubation for
in the presence and absence of the light (157
reductive dissociation rate constants were determined by
s with (E) 1 µM and (F) 10 µM total Fe. Symbols and error bars
indicate average data and ±standard deviation from triplicate experiments
linear regression lines.
A1.2. AVAILABILITY OF PRE-PHOTOLYZED FE
chemical form of Fe available for uptake by M.
examined the availability of pre-photolyzed 55
FeEDTA for dark uptake
ed 55
FeEDTA solution was made by exposing the
173
(A and C) the light
reduction rate of FeIII
EDTA, pre-
concentrations of 1-
for FZ, followed by incubation for
µmol quanta.m-2
.s-
on rate constants were determined by applying eq. A1-5
Symbols and error bars
plicate experiments. Solid lines
PHOTOLYZED FE-EDTA
M. aeruginosa, we
FeEDTA for dark uptake. For this
the pre-equilibrated
Appendix 1. Supplemental Material for Chapter 3
174
55Fe
IIIEDTA stock to light supplied from the fluorescent tube for 0.5-48 h. The treated
solution was used for the 55
Fe uptake experiment immediately after the irradiation
treatment. The 55
Fe uptake experiment was undertaken following a procedure identical
to that described in Section 3.2.4, except that (i) the pre-photolyzed 55
FeEDTA
solution instead of pre-equilibrated 55
FeIII
EDTA stock was added to the culture at
concentrations of 200 nM 55
Fe and 26 µM EDTA and (ii) the culture was incubated in
the dark for 2 h.
As shown in Figure A1.2, measured 55
Fe uptake rates were independent of the duration
of pre-exposure to the light (0.5 to 48 hrs, p > 0.05), and no significant changes in
uptake rate were seen in the experiments with and without pre-photolysis treatment.
Importantly, the observed rates were substantially smaller than the light uptake rate
measured at identical concentrations of 55
Fe and EDTA by a factor of ~102. Such a low
availability of the pre-photolyzed Fe-EDTA indicates that the Fe uptake facilitated in
the light is tightly coupled with the availability of photo-produced Fe, but Fe is not
taken up by M. aeruginosa in the dark.
Assuming that photo-oxidized EDTA (EDTAox) is produced at a rate identical to that
of Fe(II)' via LMCT, the EDTAox concentration resulting from pre-photolysis was
computed to account for only 1% relative to total EDTA even after 48 h exposure. This
calculation suggests that intact EDTA still largely exists after the irradiation treatment.
Even though Fe uptake experiments were commenced immediately (~5 min) after the
photolysis treatment, such a short period would be sufficient to form a biologically
unavailable Fe complex, as the complexation of photo-produced Fe(II)' by the intact
EDTA and subsequent oxygenation of the complex formed are rapid and significantly
occur within this time scale.
In this regard, the photochemical cycle of Fe in our EDTA-buffered Fraquil* system
appears to be different from the system of aquachelin, a suite of siderophores produced
by the marine bacterium Halomonas aquamarina strain DS40M3. Barbeau et al.
(2001) found that Fe-aquachelin C complex undergoes photolysis in natural sunlight,
resulting in oxidative cleavage of the ligand at the site of the β-hydroxyasparate
residue. Although the photo-oxidized aquachelin still retains Fe(III)-binding capacity,
its binding strength for Fe(III)' was significantly lower than that of intact aquachelin
Appendix 1. Supplemental Material for Chapter 3
175
(conditional stability constants log cond
Fe'FeL,K measured by CLE-ACSV were 12.5 ± 0.3 M-
1 for intact aquachelin and 11.6 ± 0.2 M
-1 for photo-oxidized aquachelin).
Consequently, the availability of the photolyzed 59
Fe-aquachelin complex for uptake
by a natural phytoplankton assemblage collected in the oligotrophic North Atlantic
Ocean (under 1-2% ambient light level) was significantly higher than that of the intact
complex.
Figure A1.2. Bioavailability of pre-photolyzed 55
FeEDTA complex in the dark. The x-
axis represents the time for which 55
FeEDTA complex was exposed to the light (157
µmol photons.m-2
.s-1
) before the commencement of the 55
Fe uptake experiment.
Immediately after irradiation, the photolyzed 55
FeEDTA complex was added at final
concentrations of 200 nM Fe and 26 µM EDTA to the Fe and EDTA-free Fraquil*
containing M. aeruginosa cells at a density of 3 × 106 cell.mL
-1. Cells were then
incubated for 2 h in the dark at 27oC. Values shown represent the average and
±standard deviation from triplicate experiments.
0
0.002
0.004
0.006
0.008
0.01
0.012
0 0.5 1 2 48
Pre-exposure time to light (hr)
55F
e u
pta
ke
rate
(am
ol cell-1
hr-1
)
Appendix 1. Supplemental Material for Chapter 3
176
A1.3. DETERMINATION OF FORMATION AND
DISSOCIATION RATE CONSTANTS FOR FEII
EDTA
COMPLEX
A1.3.1 Formation Rate of FeIIEDTA Complex
The rate of Fe(II) complexation by EDTA was determined using the FZ competition
method. In this method, the concentration of FeIIFZ3 complex was
spectrophotometrically determined at a wavelength of 562 nm shortly after addition of
inorganic Fe(II) solution into Fraquil* containing EDTA and FZ. At particular
concentrations of EDTA and FZ, the concentration of FeIIFZ3 complex formed is a
function of the two rate constants for the competing reactions of Fe(II) complexation
(eq. A1-8) as stated below.
A1.3.1 .1 Methods
Sample solutions were prepared by pipetting appropriate volumes of FZ and EDTA
stocks into the Fe and EDTA-free Fraquil* to create ~2 mL of solution. While the final
concentration of FZ was kept constant at 1 mM, EDTA concentration was varied from
50 to 500 µM. In order to undertake the analyses, the solution was transferred to a 1
cm polystyrene cuvette and placed in the sample holder of Cary 1E UV–Visible
spectrophotometer. The absorbance of the solution was initially zeroed. The solution in
the cuvette was then spiked with Fe(II) stock to a final concentration of 5 µM or 10
µM and mixed by shaking. Immediately, the concentration of FeIIFZ3 complex formed
was spectrophotometrically monitored. The time lag between the addition of Fe(II)
stock (t = 0) and the measurement of initial data was approximately 3-5 s. The pH
change of the solution after addition of the chemicals was previously determined to be
less than 0.1 pH units. Calibration was performed by addition of Fe(II) stock to
Fraquil* containing 1 mM FZ (in the absence of EDTA). Correlation coefficients of the
linear calibration curves of r2 > 0.99 and a molar absorptivity of ε562 = ~28,000 M
-1
cm-1
were obtained. Measurements were made at 25oC.
Appendix 1. Supplemental Material for Chapter 3
177
A1.3.1.2 Results and Discussion
When Fe(II) is added to a solution containing the strong complexing ligand FZ, Fe(II)'
rapidly reacts with FZ to form the FeIIFZ3 complex. If the solution additionally
contains EDTA, EDTA effectively competes with FZ for Fe(II)' complexation. As
Fe(II)' oxidation is negligible, the system can be simply described by two competing
reactions:
3
IIFZFe 3FZ Fe(II)' FZf →+ −k (A1-6)
Fe(II)' + EDTA k
f −EDTA → Fe IIEDTA (A1-7)
where kf-EDTA is a rate constant for Fe(II)' complexation by EDTA. Under the
conditions examined, Fe(II)' complexation by FZ is rapid. For example, calculation
using the rate law equation for reaction eq. A1-6 indicates that 99.9% of Fe(II) binds
with FZ in < 0.03 s, such that the competitive complexation reactions by FZ and
EDTA are completed within a few seconds after the addition of Fe(II) stock. In
contrast, dissociation of the complexes is relatively slow and the concentration of Fe
complexes on this timescale can be described by the two formation reactions only (eqs.
A1-6 and A1-7).
Approximating [FZ] = [FZ]T - [FeIIFZ3] ≈ [FZ]T, [EDTA] = [EDTA]T - [Fe
IIEDTA] ≈
[EDTA]T, [Fe(II)]T = [FeIIFZ3] + [Fe
IIEDTA] (where subscript T indicates the total
concentration) and considering that the formation reactions are complete (t > 0.03 s), a
simple first order differential equation derived from eqs. A1-6 and A1-7 can be solved
using an identical procedure to that employed by Fujii et al. (2008). This analysis
generates the following relationship between kf-EDTA and the concentration of the
FeIIFZ3 complex detected (see Electronic Annex in Fujii et al. (2008) for a complete
derivation):
kf-EDTA
= k
f-FZ[FZ]
T
3
[EDTA]T
[Fe(II)]T
[FeIIFZ
3]
− 1
(A1-8)
Appendix 1. Supplemental Material for Chapter 3
178
The rate constant kf-EDTA was calculated to be 2.1 (± 0.2) × 106 M
-1.s
-1 (Table A1.1) by
substituting the total concentrations of Fe(II), FZ and EDTA, the value of kf-FZ, and the
spectrophotometrically determined [FeIIFZ3] into eq. A1-8. The relatively small
variation of the determined rate constants with varying EDTA and Fe(II)
concentrations indicates that eq. A1-8 is applicable over the range of EDTA
concentrations used in this work.
Table A1.1. Formation rate constant of FeIIEDTA complex (kf-EDTA) in Fraquil
* (pH
8).
[EDTA]T [Fe(II)]T [FZ]T [FeIIFZ3]
a) kf-EDTA Average kf-EDTA
µM µM µM µM M-1
.s-1
M-1
.s-1
50 5 1000 3.85 1.9 × 106
2.1(±0.2)×106
50 10 1000 7.33 2.3 × 106
100 5 1000 3.14 1.8 × 106
100 10 1000 6.06 2.0 × 106
250 5 1000 1.86 2.1 × 106
250 10 1000 3.64 2.2 × 106
500 5 1000 1.10 2.2 × 106
500 10 1000 1.98 2.5 × 106
a) Spectrophotometrically measured data.
Appendix 1. Supplemental Material for Chapter 3
179
A1.3.2. Dissociation of FeIIEDTA Complex
The dissociation rate of the FeIIEDTA complex was also determined in the presence of
FZ. For this measurement, a pre-equilibrated FeIIEDTA complex was initially prepared
and thermal dissociation of the complex was measured by spectrophotometrically
monitoring the time-dependent increase in FeIIFZ3 concentration. Ascorbate was used
to prevent significant oxidation of Fe(II) during the measurement.
A1.3.2.1. Methods
Pre-equilibrated FeIIEDTA solutions were made by mixing appropriate volumes of
EDTA, Fe(II) and ascorbate stocks, followed by addition of the mixture to ~5 mL of
Fraquil*. The solution was left for 1 h to reach equilibrium, then FZ stock was added to
the solution. The final concentration of total EDTA in the solution was varied from 4
µM to 400 µM, whereas concentrations of other chemicals were kept constant at 1 mM
for FZ, 1 mM for ascorbate and 4 µM for Fe(II). The absorbance at 562 nm was zeroed
immediately after FZ stock was added. The dissociation of the FeIIEDTA complex was
then monitored by measuring absorbance at 562 nm in the dark for 30 min using a
Cary 1E UV–Visible spectrophotometer. The pH change of the solution (pH 8) after
addition of the stocks was previously determined to be less than 0.1 pH units. The
effect of ascorbate on Fe(II) complexation by FZ was also determined to be
insignificant by examining time-dependent formation of FeIIFZ3 complex in the
presence and absence of 1 mM ascorbate at pH 5. For this purpose, Fraquil* adjusted to
pH 5 using HCl was prepared. The solution pH 5 instead of pH 8 was employed, since
Fe(II) oxidation at this pH is very slow even in the absence of ascorbate and as such is
negligible over the duration of the experiment. Since pKa for ascorbate are 4.2 and
11.6, we would expect that protonation does not substantially influence to the
complexation of Fe(II) by ascorbate in the pH range from 5 to 8. Measurements were
made at 25oC.
A1.3.2.2. Results and Discussion
When the FZ stock is added to Fraquil* containing Fe
IIEDTA, the ligand-exchange
reaction will proceed as follows:
Appendix 1. Supplemental Material for Chapter 3
180
Fe
IIEDTA + 3FZ
koverall → Fe
IIFZ
3 (A1-9)
where, koverall indicates a fourth-order rate constant for overall ligand-exchange
reaction. Then, the rate law for FeIIFZ3 formation can be written as:
d[FeIIFZ3]
dt= k
overall[FeIIEDTA][FZ]3 (A1-10)
Approximations [FeIIEDTA] ≈ [Fe]T – [Fe
IIFZ3] (due to the relatively low [Fe(II)'])
and [FZ] = [FZ]T – [FeIIFZ3] ≈ [FZ]T (due to the relatively low [Fe
IIFZ3]) followed by
the integration give relationship between FeIIFZ3 concentration and time, as follows:
ln[Fe(II)]
T
[Fe(II)]T
− [FeIIFZ
3]
= k
overall[FZ]
T
3 ⋅ t (A1-11)
The value of koverall[FZ]T was determined from linear regression analysis of plots of
time versus ln[Fe(II)]T/([Fe(II)]T-[FeIIFZ3]) (Figure A1.3, Table A1.2).
Assuming a disjunctive mechanism, the ligand exchange reaction between FeIIEDTA
and FZ can be described by thermal dissociation of FeIIEDTA and subsequent
complexation of liberated Fe(II)′ by FZ, as follows:
FeIIEDTA
kd-EDTA
kf-EDTA
→← Fe(II)' + EDTA (A1-12)
Fe(II)' + 3FZ
kf −FZ → Fe
IIFZ
3 (A1-13)
where kd-EDTA represents the rate constant for the dissociation of FeIIEDTA.
Appendix 1. Supplemental Material for Chapter 3
Figure A1.3. Kinetic data for the dissociation of Fe
8); (A) time-dependent formation of Fe
plots of time versus ln
determined as the slope of
Material for Chapter 3
inetic data for the dissociation of FeIIEDTA complex
dependent formation of FeIIFZ3 complex over a range of [EDTA]
ln[Fe(II)]T/([Fe(II)]T-[FeIIFZ3]). The value of
determined as the slope of the line in the panel B.
181
EDTA complex in Fraquil* (pH
a range of [EDTA]T and (B)
The value of koverall[FZ]T3 was
Appendix 1. Supplemental Material for Chapter 3
182
Table A1.2. Dissociation rate constant of FeIIEDTA complex (kd-EDTA) in Fraquil
* (pH 8).
[EDTA]T [Free EDTA]Initiala)
[Fe(II)]T [FZ]T koverall[FZ]T3
kd-EDTA Average kd-EDTA
µM µM µM mM ×10-3
s-1
×10-3
s-1
×10-3
s-1
4 0 4 1 1.0 1.0
1.2 (±0.23)
5 1 4 1 1.0 1.0
6 2 4 1 1.1 1.1
8 4 4 1 1.0 1.0
14 10 4 1 1.0 1.1
44 40 4 1 0.89 1.1
104 100 4 1 0.82 1.4
100 96 4 1 0.55 0.9
200 196 4 1 0.59 1.4
200 196 4 1 0.73 1.7
200 196 4 1 0.36 0.8
400 396 4 1 0.33 1.2
400 396 4 1 0.33 1.2
a) Initial concentration of EDTA that is not bound to Fe.
Appendix 1. Supplemental Material for Chapter 3
183
Due to the rapid complexation of Fe(II)' by EDTA and FZ at pH 8, we assume that the
intermediate Fe(II)' is highly reactive and does not accumulate to a significant extent.
Therefore, assuming steady-state for this intermediate, the FeIIFZ3 formation rate can
be written as follows:
d[FeIIFZ3]
dt= k
f-FZ[FZ]3[Fe(II)'] = k
f-FZ[FZ]3
kd-EDTA
[FeIIEDTA]
kf-EDTA
[EDTA] + kf-FZ
[FZ]3
(A1-14)
Comparison with eq. A1-10 yields:
kd-EDTA
=k
overall(k
f-EDTA[EDTA] + k
f-FZ[FZ]3)
kf-FZ
(A1-15)
If the term kf-EDTA[EDTA] is much smaller than kf-FZ[FZ]3 due to the low concentration
of free EDTA (e.g., [EDTA]T is less than 10 µM), reformation of FeIIEDTA complex
can be ignored and kd-EDTA is simply described as:
k
d-EDTA= k
overall[FZ]3 (A1-16)
The value of kd-EDTA was determined to be 1.2 (±0.23) × 10-3
s-1
by using eqs. A1-15
and A1-16 under a range of [EDTA]T (Table A1.2). Under the condition of [EDTA]T >
10 µM where the term for Fe(II)' re-formation by EDTA in eq. A1-15 becomes
significant, the kd-EDTA values were reasonably consistent with those determined in the
low [EDTA]T system, confirming that the kf-EDTA value reported in the previous section
was also reasonable.
Appendix 1. Supplemental Material for Chapter 3
184
A1.4. EFFECT OF FE(II) OXIDATION AND
DISSOCIATION ON CALCULATION OF STEADY-
STATE FE(II) CONCENTRATION IN THE 55
FE UPTAKE
EXPERIMENT
A1.4.1. Fe(II)' Oxidation
First-order rate constants for Fe(II)' oxidation and complexation by ligands under the
conditions of 55
Fe uptake experiments were calculated as follows:
kox[O2] = 8.8 × 0.24 × 10-3
= 2.1 × 10-3
(s-1
) (A1-17)
kf-EDTA[EDTA] = 2.1×106 × 2.6-26 × 10
-5 = 55-550 (s
-1) (A1-18)
kf-FZ[FZ]3 = 3.1 × 10
11 × (1 × 10
-3)3 = 310 (s
-1) (A1-19)
By comparing calculated values, the Fe(II)' oxidation rate was found to be 4-5 orders
of magnitude less than the rates of Fe(II)' complexation by EDTA or FZ. Therefore,
the effect of the oxidation reaction on the steady-state Fe(II)' concentration is
negligibly small.
A1.4.2. Dissociation of FeIIFZ3 and Fe
IIEDTA Complexes
The effect of thermal dissociation of FeIIEDTA and Fe
IIFZ3 on steady-state Fe(II)'
concentration was also examined under the conditions employed in 55
Fe uptake
experiments.
The dissociation rate of FeIIFZ3 accumulated during the uptake experiment was
calculated by multiplying the dissociation rate constant (kd-FZ) by the average FeIIFZ3
concentration during the experiment. Assuming that Fe(II)' formation is equal to
FeIIFZ3 formation at high FZ concentration, then the Fe
IIFZ3 formed at any given point
in time in the 55
Fe uptake experiment is calculated as follows:
Appendix 1. Supplemental Material for Chapter 3
185
[FeIIFZ
3]
t= [FeIIIEDTA]
Initial1− exp −k
hvt( ) (A1-20)
At the end of the 55
Fe uptake experiment (i.e., after 2 hr, [FeIII
EDTA]Initial = 200 nM
and khv = 6.4 × 10-6
s-1
), the final FeIIFZ3 concentration is estimated to be 9.0 nM.
Since FeIIFZ3 concentration increases in a linear manner with respect to time (Figure
A1.1), the time-averaged concentration for FeIIFZ3 ([Fe
IIFZ3]ave) over the duration of
experiment is half of this value (i.e., 4.5 nM). The average dissociation rate is then
calculated as follows:
kd-FZ[FeIIFZ3]ave = 4.3 × 10
-5 × 4.5 × 10
-9 = 1.9 × 10
-13 (M.s
-1) (A1-21)
For the EDTA complex, we assume that the concentration of FeIIEDTA is determined
by formation, dissociation and oxidation rates of the FeIIEDTA complex, i.e.:
d[Fe IIEDTA]
dt= k
f-EDTA[EDTA][Fe(II)'] − k
d-EDTA[Fe
IIEDTA] − k
ox-EDTA[O
2][Fe
IIEDTA]
(A1-22)
The steady-state FeIIEDTA concentration ([Fe
IIEDTA]ss) can be calculated as follows:
[FeIIEDTA]
SS=
kf-EDTA
[EDTA][Fe(II)']SS
kd-EDTA
+ kox-EDTA
[O2]
(A1-23)
where [Fe(II)']ss represents steady-state Fe(II)' concentration. The [Fe(II)']ss is
determined by rates of cellular uptake, complexation by ligands, dissociation of
formed complexes and oxidation, i.e.:
[Fe(II)']ss
=k
hv[FeIIIEDTA]
Initial+ k
d-EDTA[FeIIEDTA]
SS+ k
d-FZ[FeIIFZ
3]
ave
kf-EDTA
[EDTA] + kf-FZ
[FZ]3 + kup
[cell] + kox
[O2]
(A1-24)
The three unknown parameters [Fe(II)']SS, [FeIIEDTA]ss and kup were then calculated
from the three equations A1-23, A1-24 and ρ
Fe= k
up[Fe(II)']
ss using the trial and error
method (where ρ
Fe is the measured
55Fe uptake rate). Calculated data are shown in
Appendix 1. Supplemental Material for Chapter 3
186
Table 3.4. Using the computed [FeIIEDTA]ss, the thermal dissociation rate of
FeIIEDTA is calculated as follows:
kd-EDTA[FeIIEDTA]SS = 1.2 × 10
-3 × 0.20-1.7 × 10
-10 = 0.24-2.0 × 10
-13 (M.s
-1) (A1-25)
We can also calculate the rate of photo-dissociation of FeIII
EDTA as follows:
khv[FeIII
EDTA] = 6.4 × 10-6
× 2 × 10-7
= 1.3 × 10-12
(M.s-1
) (A1-26)
Therefore, dissociation of FeIIEDTA and Fe
IIFZ3 complexes will account for up to
23% of net Fe(II)' formation.
A1.5. OUTER-MEMBRANE PERMEABILITY AND
REPORTED PARAMETERS FOR PORIN PROPERTIES
Gram-negative bacteria have two structurally different phospholipid membranes; i.e.,
outer- and inner-membranes. The space between these two membranes is referred to as
the periplasm. To assimilate nutrients, this class of microorganisms must allow
molecules or ions to pass through the outer-membrane. Permeability of the outer-
membrane depends on the type of transport system involved (e.g., specific or non-
specific transport) and physicochemical properties of substrates such as size, polarity,
charge and specificity to outer-membrane receptors. Hydrophobic molecules have
generally lower permeability, as lipopolysaccharide (LPS) anchored in outer leaflet of
the asymmetric phospholipid bilayer act as a permeability barrier (Nikaido, 2003).
Once LPS synthesis is genetically knocked out, however, the permeability of
hydrophobic solutes increases (Nikaido, 1976). In contrast, almost all small
hydrophilic nutrients (generally less than 600 Da) including metal ions can passively
diffuse into the periplasmic space in a concentration gradient manner (Nikaido and
Rosenberg, 1981). This type of transport is mediated by the water-filled
transmembrane channel known as porins. The porin is one of the most abundant outer-
membrane proteins, in contrast to other minor transporter proteins for uptake of
specific nutrients such as vitamin B12 and ferric siderophore complexes (Hall and
Silhavy, 1981). Porins have been found for almost all Gram-negative bacteria
investigated so far including cyanobactria (Nikaido, 2003). Among these, porins of E.
Appendix 1. Supplemental Material for Chapter 3
187
coli. may be the best studied. Three different classes of proteins designated as OmpF,
OmpC and PhoE, have been found in this organism and each protein has molecular
mass of 36,000-38,000 Da (Nikaido, 2003). Diameters of the transmembrane channels
surrounded by the 16 β-barrel structure have been reported to be 1.0 nm to 1.2 nm
based on the crystal structure and the Stokes radius of nutrients which are capable of
passing through the channels (hereafter reported parameters for porin properties and
associated references are listed in Table A1.3, unless otherwise stated). The length of
porins has been reported to be around 2.8-7.5 nm, which is equal to or less than the
thickness of the outer-membrane bilayer. The density of porins in the outer-membrane
have been reported in the range of 7.9 × 1015
to 3.3 × 1016
porins per square meters.
These parameters for E. coli porins are somewhat different from porins of other
microorganisms (Table A1.3). Although studies on cyanobacterial porins are limited,
the molecular weight of pore-forming outer-menbrane proteins for cyanobacteria such
as Synechococcus sp. (52,000 Da (Hansel and Tadros, 1998, Hansel et al., 1994) and
Anabaena variabilis (40,000-80,000 Da, (Benz and Bohme, 1985)) seems to be larger
than the other bacterial porins. A relatively large pore diameter of 1.7 nm has been
reported for Anabaena variabilis. The single channel conductance determined in 1 M
KCl ranges from 0.4 nS to 5.5 nS (Benz and Bohme, 1985, Hansel et al., 1994, Hansel
and Tadros, 1998), which is more variable than the 2-3 nS reported for E. Coli
(Nikaido, 2003), suggesting that the permeability of the outer-membrane for
cyanobacteria varies over a relatively wide range.
Appendix 1. Supplemental Material for Chapter 3
188
Table A1.3. Published values of porin properties for various Gram-negative bacteria.
Microorganism Class of pore- Pore diameter Channel length Porins Surface area Porin density Reference forming protein 2a (nm) l (nm) per cell of cell (m2) Nporin (m
-2)
Escherichia coli B Pore-forming protein 1.2 4–7.5a)
1.0×105 3.0×10
-12 3.3×10
16 Nikaido and Rosenberg (1981)
Escherichia coli ML308 Pore-forming protein 1.16 4–7.5 1.1×105 1.4×10
-11 7.9×10
15 West and Page (1984)
Escherichia coli K12 OmpF 1.13–1.16 6 Hancock (1987)
Escherichia coli K12 OmpC 1.02–1.13 6 Hancock (1987)
Escherichia coli K12 PhoE 1.06–1.13 6 Hancock (1987)
Escherichia coli K12 PhoE 1.0 a)
2.8–4.5 Jap et al. (1991)
Rhodopseudomonas capsulata
ATCC 23782 Outer-membrane porin 1.6 4–7.5a)
Flammann and Weckesser (1984)
Rhodobacter capsulatus 37b4 Outer-membrane porin 1.0 a)
2–4 Weiss et al. (1991)
Pseudomonas aeruginosa Protein F 1.63–2.9 6 Hancock (1987)
Anabaena variabilis
ATCC29423 Pore-forming protein 1.7 7.5 Benz and Bohme (1985)
Microcystis aeruginosa
PCC7806 1.1×10-10
Fujii et al. (2010)
a) Values were assumed in this work; pore diameter = ~1 nm and channel length = ~4–7.5 nm.
Appendix 1. Supplemental Material for Chapter 3
189
Table A1.4. Range of uptake rate constant (kup) calculated using published parameters (a, porin radius; l, channel length; Nporin, porin density; D,
diffusion coefficient of metal ions; As, surface area of Microcystis aeruginosa PCC7806).
Microorganism Reference πa2/l (×10
-9 m)
a) kup = 1,000Dπa2NporinAS/l (×10
-9 L.cell
-1.s
-1)b) Lower limit Average Upper limit Lower limit Average Upper limit
Escherichia coli B Nikaido and Rosenberg (1981) 0.15 0.22 0.28 0.07 0.57 1.1
Escherichia coli ML308 West and Page (1984) 0.14 0.20 0.26 0.06 0.53 1.0
Escherichia coli K12 (OmpF) Hancock (1987) 0.17 0.17 0.18 0.07 0.37 0.7
Escherichia coli K12 (OmpC) Hancock (1987) 0.14 0.15 0.17 0.06 0.35 0.6
Escherichia coli K12 (PhoE) Hancock (1987) 0.15 0.16 0.18 0.07 0.36 0.7
Escherichia coli K12 (PhoE) Jap et al. (1991) 0.17 0.23 0.28 0.08 0.57 1.1
Rhodopseudomonas capsulata
ATCC 23782 Flammann and Weckesser (1984) 0.27 0.39 0.50 0.12 1.0 1.9
Rhodobacter capsulatus 37b4 Weiss et al. (1991) 0.20 0.29 0.39 0.09 0.78 1.5
Pseudomonas aeruginosa Hancock (1987) 0.35 0.72 1.10 0.16 2.2 4.1
Anabaena variabilis ATCC29423 Benz and Bohme (1985) 0.30 0.52 a)
For πa2/l, upper and lower limits and average value were calculated using the range of published parameters for microorganisms listed. The
values used are shown in Table A1.3.
Appendix 1. Supplemental Material for Chapter 3
190
b) For the calculation of kup, porin radius (a), channel length (l) and porin density (Nporin) listed in Table A1.3 were used. Reported parameters for
diffusion coefficient of metal ions D = 0.5-1×10-9
m2.s
-1 (Buffle et al., 2009) and surface area of Microcystis aeruginosa PCC7806 AS = 113 µm
2
(Fujii et al., 2010) were also used. The average represents the mean value of lower and upper limits.
Appendix 1. Supplemental Material for Chapter 3
191
REFERENCES
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cycling of iron in the surface ocean mediated by microbial iron(III)-binding
ligands. Nature, 413, 409-413.
BENZ, R. & BOHME, H. 1985. Pore formation by an outer-membrane protein of the
cyanobacterium Anabaena variabilis. Biochimica et Biophysica Acta, 812, 286-
292.
BUFFLE, J., WILKINSON, K. J. & VAN LEEUWEN, H. P. 2009. Chemodynamics
and bioavailability in natural waters. Environmental Science & Technology, 43,
7170-7174.
FLAMMANN, H. T. & WECKESSER, J. 1984. Porin isolated from the cell-envelope
of Rhodopseudomonas capsulata. Journal of Bacteriology, 159, 410-412.
FUJII, M., ROSE, A. L., OMURA, T. & WAITE, T. D. 2010. Effect of Fe(II) and
Fe(III) transformation kinetics on iron acquisition by a toxic strain of
Microcystis aeruginosa. Environmental Science & Technology, 44, 1980-1986.
FUJII, M., ROSE, A. L., WAITE, T. D. & OMURA, T. 2008. Effect of divalent
cations on the kinetics of Fe(III) complexation by organic ligands in natural
waters. Geochimica et Cosmochimica Acta, 72, 1335-1349.
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HANCOCK, R. E. W. 1987. Role of porins in outer-membrane permeability. Journal
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characterization of porin from the outer-membrane of Synechococcus
PCC6301. Archives of Microbiology, 161, 163-167.
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isolated from the outer membrane of Synechococcus PCC 6301. Current
Microbiology, 36, 321-326.
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membrane channel as determined by electron crystallography. Nature, 350,
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NIKAIDO, H. 1976. Outer membrane of Salmonella typhimurium. Transmembrane
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NIKAIDO, H. 2003. Molecular basis of bacterial outer membrane permeability
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NIKAIDO, H. & ROSENBERG, E. Y. 1981. Effect of solute size on diffusion rates
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193
APPENDIX 2
SUPPLEMENTAL MATERIAL FOR
CHAPTER 4 - KINETICS OF
EXTRACELLULAR IRON TRANSPORT
TO PERIPLASMIC AND CYTOPLASMIC
COMPARTMENTS OF THE
FRESHWATER CYANOBACTERIUM
MICROCYSTIS AERUGINOSA
Appendix 2. Supplemental Material for Chapter 4
194
A2.1. DETERMINATION OF PARAMETERS RELEVANT
TO FE UPTAKE AND INTRACELLULAR TRANSPORT
Assuming that the Fe′ concentration in the periplasm ([Fe′peri]) is comparable to that in
the bulk ([Fe′]) (this assumption is justified in part A2.2 of Appendix 2), log-
transformation of the Monod-type expression for cytoplasmic 55
Fe accumulation can
be described as follows:
max ' max '
cyto Fe'(peri) peri Fe'(peri)
' '
Fe'(peri) peri Fe'(peri)
[Fe ] [Fe ] [Fe ]log log log
[Fe ] [Fe ]
d
dt K K
ρ ρ = ≈ + +
(A2-1)
The steady-state concentration of extracellular Fe′ was firstly calculated under the
particular conditions of the 55
Fe accumulation experiments undertaken in this study
from the balance of formation and dissociation of the extracellular Fe-ligand
complexes by using the kinetic model in part A of Table 4.1. The calculation of the
steady-state concentration of extracellular unchelated Fe was justified as follows.
Since concentrations of extracellular Fe′ for particular Fe:Cit ratios can be calculated
from the balance between dissociation and recomplexation rates of ferric citrate (k-d-
L[FeL] and kf-L[L][Fe′], respectively) and total diffusional flux of unchelated Fe into
the periplasm (JFe′[cell] mol.s-1
), the time-dependent change of unchelated Fe
concentration can be described as follows:
''
d-L f-L Fe'
' '
d-L f-L dif
' ' '
d-L f-L dif peri
'
d-L f-L
'
d-L T f-L T T
[Fe ][FeL] [L][Fe ] [cell]
[FeL] [L][Fe ] [Fe ][cell]
[FeL] [L][Fe ] ([Fe ] [Fe ])[cell]
[FeL] [L][Fe ]
[Fe] ([L] [Fe] )[Fe ]
dk k J
dt
k k k
k k k
k k
k k
= − +
= − + ∆
= − + −
≈ −
= − −
(A2-2)
where [cell] is cell density (cell.L-1
), and the effect of diffusional influx in the
calculation of steady-state concentration of extracellular Fe′ is negligibly small due to
the assumption that [Fe′peri] ≈ [Fe′]. In the calculation of [Fe′], [FeL] and [L] were
Appendix 2. Supplemental Material for Chapter 4
195
approximated to be equal to total Fe and ligand concentrations in the system ([Fe]T and
[L]T), respectively, as Fe′ concentration is substantially lower than chelated Fe
concentration under the conditions examined in this work. Thus, at steady-state, the
following expression can be used to calculate the extracellular Fe′ concentration
([Fe′]SS):
' d-L TSS
f-L T T
[Fe][Fe ]
([L] [Fe] )
k
k=
− (A2-3)
The Fe uptake parameters max
Fe'(peri)ρ and Fe'(peri)K were then determined by fitting eq. A2-
1 to the data shown in part B of Figure 4.2. In the fitting process, nonlinear regression
analysis was performed using R version 2.13.0 (free software for statistical
computation). Subsequently, the rate constant for translocation of periplasmic Fe to the
cytoplasm (k2) was determined by fitting eq. 4.5 to the data in part A of Figure 4.2.
The best fit of the model to the experimental data was obtained by Microsoft Excel
using a least-squares method in which the sum of the mean square error using the
average values of the experimental data was minimized. k+1 was determined by fitting
eq. 4.7 to the data shown in part A of Figure 4.1 then k-1 was calculated from Fe'(peri)K =
(k-1+k2)/k+1. Finally, the value for [Xperi]T was determined from max
Fe'(peri)ρ = k2[Xperi]T.
A2.2. CALCULATION OF STEADY-STATE
CONCENTRATION OF PERIPLASMIC UNCHELATED
FE
The Fe′ concentration in the periplasm was calculated by assuming that, under steady-
state conditions, the flux of periplasmic Fe transported to the cytoplasm (ρFe′) is equal
to the diffusional flux of Fe across the outer membrane (JFe′ mol.cell-1
.s-1
). This
assumption yields the following relationship between Fe′ concentrations in the
periplasm ([Fe′peri]) and bulk ([Fe′]):
Appendix 2. Supplemental Material for Chapter 4
196
Fe' Fe'
max '
Fe'(peri) peri'
dif '
Fe'(peri) peri
' ' ' max '
dif peri peri Fe'(peri) Fe'(peri) peri
' 2 ' ' ' '
dif peri peri Fe'(peri) peri Fe'(peri)
[Fe ][Fe ]
[Fe ]
([Fe ] [Fe ])([Fe ] ) [Fe ]
([Fe ] [Fe ] [Fe ][Fe ] [Fe ] )
J
kK
k K
k K K
ρ
ρ
ρ
ρ
=
− ∆ =+
− − + =
− + − − = max '
Fe'(peri) peri
' 2 ' max ' '
dif peri dif Fe'(peri) dif Fe'(peri) peri dif Fe'(peri)
' max ' max 2
dif Fe'(peri) dif Fe'(peri) dif Fe'(peri) dif Fe'(peri)'
peri
[Fe ]
[Fe ] ( [Fe ] )[Fe ] [Fe ] 0
( [Fe ] ) ( [Fe ] )[Fe ]
k k K k k K
k K k k K k
ρ
ρ ρ
+ − + − =
− − + + − +=
2 '
dif Fe'(peri)
dif
4 [Fe ]
2
k K
k
+
(A2-4)
where kdif is the diffusion constant for unchelated Fe (L.cell-1
.s-1
), and ∆[Fe′] is the
difference between unchelated Fe concentrations in the extracellular bulk medium and
periplasmic space. By using eq. A2-4 and relevant parameters determined in part A2.1
of Appendix 2 (Table 4.1), [Fe′peri] was calculated over a range of [Fe′] concentrations
from 1 fM to 10 nM. Calculations suggested that [Fe′peri] was similar to [Fe′]
([Fe′peri]/[Fe′] was greater than 0.999). Therefore, we assumed that [Fe′peri] ≈ [Fe′] in
our work.
A2.3. EQUATION FOR TIME-DEPENDENT CHANGE OF
PERIPLASMIC FE
According to eq. 4.4 in the text, the time-dependent change of periplasmic Fe
concentration is described as follows:
peri peri
'
1 peri peri 1 2 peri
[Fe ] [FeX ]
[Fe ][X ] ( )[FeX ]
d d
dt dt
k k k+ −
≈
= − +
(A2-5)
Substitution of the mass balance equation for Fe-binding sites (i.e., [Xperi]T = [FeXperi]
+ [Xperi]) and Fe'(peri)K = (k-1+k2)/k+1 yields the following relationship:
Appendix 2. Supplemental Material for Chapter 4
197
peri ' '
1 peri peri T 1 2 1 peri peri
' '1 2peri peri T Fe'(peri) peri peri
Fe'(peri)
[FeX ][Fe ][X ] ( + [Fe ])[FeX ]
[Fe ][X ] ( +[Fe ])[FeX ]
dk k k k
dt
k kK
K
+ − +
−
= − +
+= −
(A2-6)
Then, integration and rearrangement give the following:
peri ' 1 2Fe'(peri) peri'
peri peri T Fe'(peri)
peri'
Fe'(peri) peri
' '
peri peri T Fe'(peri) peri 1 2
peri'
Fe'(peri) peri Fe'(peri)
[FeX ]( +[Fe ])
[Fe ][X ][FeX ]
+[Fe ]
[Fe ][X ] ( +[Fe ])( )ln [FeX ]
+[Fe ]
d k kK dt
K
K
K k k
K K
−
−
+=
−
+− − = ⋅
∫ ∫
t C+
(A2-7)
where C is an integration constant. The condition that [FeXperi] = 0 at time zero gives:
'
peri T
'
Fe peri
[Fe ][X]ln
+[Fe ]C
K
= −
(A2-8)
By substituting C in eq. A2-7,
' '
peri peri T peri peri T'
peri Fe'(peri) peri 1' '
Fe'(peri) peri Fe'(peri) peri
[Fe ][X ] [Fe ][X ]ln [FeX ] ( +[Fe ]) ln
+[Fe ] +[Fe ]K k t
K K+
− − = ⋅ −
(A2-9)
'
peri peri T '
peri Fe'(peri) peri 1'
Fe'(peri) peri
[Fe ][X ][FeX ] 1 exp ( +[Fe ])
+[Fe ]K k t
K+
= − − ⋅ (A2-10)
Appendix 2. Supplemental Material for Chapter 4
198
Table A2.1. Measured and modelled values for the time course of 55
Fe accumulation in the periplasmic and cytoplasmic fractions.
No.
55
Fe concentration (amol.cell-1
) PCC7806 strain PCC7005 strain
Incubation Periplasm Cytoplasm Periplasm Cytoplasm
time (hr) experiment model experiment model experiment model experiment model
1 0 0.03 0.0 0.14 0.0 0.01 0.0 0.13 0.0
2 0 0.01 0.0 0.18 0.0 0.01 0.0 0.18 0.0
3 0 0.01 0.0 0.16 0.0 0.004 0.0 0.16 0.0
4 1 0.12 0.16 1.54 1.1 0.08 0.12 1.44 1.2
5 1 0.13 0.16 2.17 1.1 0.06 0.12 1.45 1.2
6 1 0.09 0.16 1.38 1.1 0.07 0.12 1.42 1.2
7 3 0.18 0.16 3.66 3.3 0.07 0.12 4.28 3.7
8 3 0.22 0.16 2.82 3.3 0.08 0.12 3.05 3.7
9 3 0.15 0.16 2.84 3.3 0.07 0.12 3.14 3.7
10 5 0.09 0.16 4.25 5.5 0.09 0.12 5.21 6.2
11 5 0.09 0.16 4.43 5.5 0.10 0.12 5.97 6.2
12 5 0.12 0.16 4.71 5.5 0.10 0.12 4.49 6.2
13 7 0.16 0.16 3.84 -a 0.14 0.12 4.49 -
a
14 7 0.21 0.16 3.86 -a 0.12 0.12 5.09 -
a
15 7 0.13 0.16 5.27 -a 0.15 0.12 5.67 -
a
16 9 0.20 0.16 4.11 -a 0.12 0.12 4.65 -
a
17 9 0.16 0.16 3.89 -a 0.13 0.12 4.10 -
a
18 9 0.16 0.16 4.42 -a 0.12 0.12 5.73 -
a
a Value not calculated.
Appendix 2. Supplemental Material for Chapter 4
199
Table A2.2. Measured and modelled values for the steady-state periplasmic 55
Fe concentration and accumulation rate of cytoplasmic 55
Fe over a
range of Fe:citrate ratiosa.
55Fe in periplasm (amol.cell
-1) Accumulation rate of cytoplasmic
55Fe (amol.cell
-1.hr
-1)
No. Sample name PCC7806 PCC7005 PCC7806 PCC7005 experiment model experiment model experiment model experiment model
1 FeCit-5µM 0.36 0.29 0.33 0.26 2.1 1.9 3.0 2.4
2 FeCit-5µM 0.34 0.29 0.30 0.26 2.7 1.9 3.4 2.4
3 FeCit-5µM 0.31 0.29 0.38 0.26 2.2 1.9 3.3 2.4
4 FeCit-20µM 0.20 0.16 0.12 0.13 0.82 0.96 0.93 1.1
5 FeCit-20µM 0.16 0.16 0.13 0.13 0.78 0.96 0.82 1.1
6 FeCit-20µM 0.16 0.16 0.12 0.13 0.88 0.96 1.1 1.1
7 FeCit-50µM 0.15 0.080 0.09 0.070 0.71 0.42 0.90 0.48
8 FeCit-50µM 0.13 0.080 0.13 0.070 0.64 0.42 0.90 0.48
9 FeCit-50µM 0.10 0.080 0.11 0.070 0.58 0.42 0.93 0.48
10 FeCit-100µM 0.053 0.053 0.061 0.048 0.29 0.23 0.31 0.26
11 FeCit-100µM 0.070 0.053 0.085 0.048 0.31 0.23 0.40 0.26
12 FeCit-100µM 0.059 0.053 0.076 0.048 0.33 0.23 0.39 0.26
13 FeCit-200µM 0.016 0.031 0.020 0.030 0.086 0.079 0.11 0.077
14 FeCit-200µM 0.013 0.031 0.025 0.030 0.17 0.079 0.14 0.077
15 FeCit-200µM 0.020 0.031 0.024 0.030 0.059 0.079 0.093 0.077
a The Fe:citrate ratios were identical to those listed in Table A2.3.
Appendix 2. Supplemental Material for Chapter 4
200
Table A2.3. Calculated values of unchelated Fe concentrations in the extracellular milieu and periplasm.
PCC7806 strain PCC7005 strain
No. Sample name [Fe]T [Cit]T kf-cit kd-cit [Fe'peri] [Fe'] pFe' Ratio of [Fe'peri] [Fe'] pFe' Ratio of
(nM) (µM)
(x 105 M
-1s
-
1) (x 10
-3 s
-1) (pM) (pM) (M) [Fe'peri]/[Fe'] (pM) (pM) (M) [Fe'peri]/[Fe']
1 FeCit-5µM 700 5 2.1 3.5 2691 2691 8.6 0.999949 2691 2691 8.6 0.999948
2 FeCit-20µM 700 20 2.1 2.9 507 507 9.3 0.999845 507 507 9.3 0.999840
3 FeCit-50µM 700 50 2.1 2.2 152 152 9.8 0.999768 152 152 9.8 0.999766
4 FeCit-100µM 700 100 2.1 1.6 54 54 10.3 0.999732 54 54 10.3 0.999734
5 FeCit-200µM 700 200 2.1 1.0 17 17 10.8 0.999715 17 17 10.8 0.999722
201
APPENDIX 3
SUPPLEMENTAL MATERIAL FOR
CHAPTER 5 - IRON UPTAKE KINETICS
BY THE FRESHWATER
CYANOBACTERIUM MICROCYSTIS
AERUGINOSA IN THE PRESENCE OF
SUWANNEE RIVER FULVIC ACID
Appendix 3. Supplemental Material for Chapter 5
202
A3.1. SUPPLEMENTAL FIGURES
Figure A3.1. Time-course of 55
Fe uptake under the dark (closed symbol) and light
(open symbol) conditions. 55
Fe uptake was measured by incubating cells (at density of
1.6 × 106 cell.mL
-1) in Fraquil
* containing pre-equilibrated
55Fe
IIISRFA complex at
27oC. Concentrations of Fe and SRFA were 200 nM and 1 mg.L
-1, respectively.
Symbols represent experimental data. Solid and dotted lines were yielded by applying
a linear regression analysis to the data collected within 2 h under the dark and light
conditions, respectively.
0
2
4
6
8
10
12
14
0 2 4 6 8 10 12 14
Ce
llu
lar
55Fe
(a
mo
l.ce
ll-1
)
Time (h)
y = 1.95x + 0.44
R² = 0.94y = 1.28x + 0.46
R² = 0.87
Appendix 3. Supplemental Material for Chapter 5
203
Figure A3.2. Comparison of measured 55
Fe uptake rate to calculated Fe(III) uptake for
M. aeruginosa PCC7806. 55
Fe uptake rates were determined in the short-term
incubational assay under the dark in modified Fraquil* containing 200 nM for Fe, 1, 5
and 25 mg L-1
for SRFA and 1 mM for FZ. In the model calculations, steady-state
concentrations for Fe(III)' were determined at the concentration identical to those
employed in the short-term assay by using rate constants for complexation and
dissociation for FeIII
SRFA complex published by Rose (square), Jones (diamond) and
Bligh (triangle). Fe(III) uptake rates were then calculated by use of Monod-type
equation with parameters listed in Table 5.1. Solid line represents linear line with 1:1
slope.
-20
-19.5
-19
-18.5
-18
-17.5
-17
-20 -19.5 -19 -18.5 -18 -17.5 -17
Rose model
Jones model
Bligh model
1:1
0
0.5
1
1.5
2
2.5
3
3.5
0 0.5 1 1.5 2 2.5 3 3.5
Rose model
Jones model
Bligh model
1:1
Logarithm of calculated Fe(III) uptake rate (mol.cell-1.hr-1)
Me
asu
red
55Fe
(III
) u
pta
ke r
ate
(am
ol.
cell-1
.hr-1
)
Calculated Fe(III) uptake rate (amol.cell-1.hr-1)
Loga
rith
m o
f m
ea
sure
d 5
5Fe
(III
)
up
take
rat
e (
amo
l.ce
ll-1.h
r-1)
Appendix 3. Supplemental Material for Chapter 5
204
Figure A3.3. Comparison of measured 55
Fe(II) uptake rate to calculated Fe(II) uptake
for M. aeruginosa PCC7806. Measured Fe(II) uptake rates in this figure were
determined by subtracting 55
Fe uptake rate in the presence of FZ from that measured in
the absence of FZ. The short-term incubational assays were performed in the absence
and presence of FZ under the dark in modified Fraquil* containing 200 nM for Fe, 1, 5
and 25 mg L-1
for SRFA and 1 mM for FZ. In the model calculations, steady-state
concentrations for Fe(II)' were determined at the concentration identical to those
employed in the short-term assay by using rate constants for complexation and
dissociation for FeIISRFA complex published by Rose (square) and Bligh (triangle).
Fe(II) uptake rates were then calculated by use of Monod-type equation with
0
0.2
0.4
0.6
0.8
1
0 0.2 0.4 0.6 0.8 1
Rose model
Bligh model 1:1
Me
asu
red
55F
e(I
I) u
pta
ke
ra
te
(am
ol.
cell
-1.h
r-1)
Calculated Fe(II) uptake rate (amol.cell-1.hr-1)
-20
-19.5
-19
-18.5
-18
-17.5
-20 -19.5 -19 -18.5 -18 -17.5
Rose model
Bligh model 1:1
Log
ari
thm
of
me
asu
red
55F
e(I
I)
up
tak
e r
ate
(a
mo
l.ce
ll-1
.hr-1
)
Logarithm of calculated Fe(II) uptake rate (mol.cell-1.hr-1)
Appendix 3. Supplemental Material for Chapter 5
205
parameters listed in Table 5.1. Solid line represents linear line with 1:1 slope. Error bar
indicates standard deviation from duplicate experiments.
Figure A3.4. Comparison of calculated steady-state concentration of Fe(II)’ under the
dark and light conditions. Steady-state concentrations for Fe(II)' were determined by
using rate constants for complexation and dissociation for FeIISRFA complex
published by Rose (square) and Bligh (triangle), the photochemical and non-
photochemical reduction of FeIII
SRFA complex and oxidation of FeIISRFA complex.
Solid line represents linear line with 1:1 slope.
-14
-12
-10
-8
-6
-14 -12 -10 -8 -6
Log
ari
thm
of
calc
ula
ted
[Fe
(II)
']S
Su
nd
er
da
rk (
M)
Logarithm of calculated
[Fe(II)']SS under light (M)
Appendix 3. Supplemental Material for Chapter 5
206
Figure A3.5. Effect of pH on the 55
Fe uptake rate for M. aeruginosa PCC7806 under
(A) dark and (B) light. Effects of FZ (gray bar) and SOD (white bar) on 55
Fe uptake
were also examined compared to control treatment (black bar) where addition of FZ or
SOD was omitted. Error bar indicates standard deviation from triplicate experiments.
Asterisks indicate that 55
Fe uptake rate in the presence of chemical treatment is
significantly different from control (55
Fe uptake rate in the absence of FZ or SOD) for
each pH at the levels of p < 0.01 for ** and p < 0.05 for * using a single-tailed
heteroscedastic t-test.
0
0.2
0.4
0.6
0.8
1
1.2
6 7 8 9
Dark
Dark-FZ
Dark-SOD
A.
* *
*
*
0
0.2
0.4
0.6
0.8
1
1.2
6 7 8 9
Light
Light-FZ
Light-SOD
B.
*
*
*
***
****
55F
e u
pta
ke
ra
te (
am
ol.
cell
-1.h
r-1)
pH
Appendix 3. Supplemental Material for Chapter 5
207
A3.2. PHOTOCHEMICAL AND NON-PHOTOCHEMICAL
FE(III) REDUCTION EXPERIMENT
A3.2.1. Methods
A3.2.1.1. Fe(III) reduction experiment
Photo- and thermal reduction rates of FeIII
L were spectrophotometrically determined
by measurement of the absorbance of ferrous-ferrozine complex (FeIIFZ3) at a
wavelength of 562 nm, where the FeIIFZ3 absorbs most strongly. During the
measurement, the absorbance at 562 nm (i.e., FeIIFZ3 concentration) increased over
time after mixing the pre-equilibrated FeIII
L solution and FZ stocks in Fraquil*, as
FeIII
L is reduced to Fe(II) followed by Fe(II) complexation by FZ. All stock solutions
and vessels used in the experiments were prepared as described in Section 5.1, Chapter
5.
The solutions of FeIII
L and FZ (prepared at pH 6-9 depending on the pH of Fraquil*
interest) were spiked into an appropriate volume of the Fe- and ligand-free Fraquil*
(pH 6-9) in order to provide a total volume of 2-100 mL in an acid-washed 1 cm path
length polystyrene spectrophotometer cuvette or polycarbonate bottles (depending on
the total Fe concentration: the cuvette for 10 µM Fe and the polycarbonate bottle for 1
µM Fe) at final concentrations of 1-10 µM for Fe and 1 mM for FZ. The final
concentrations of the Fe-binding ligands were 1-250 mg L-1
for SRFA, 26 µM for
EDTA and 100 µM for citrate. The samples were then incubated either under light or
dark condition at 27oC. The photo- and thermal reduction rates were determined under
the condition identical to the Fe uptake experiment by using the same light source and
distance between the sample and the fluorescent light tube. At various times from 1
min to 36 h after mixing the FeIII
L and FZ stocks, FeIIFZ3 concentration in the sample
was measured by a Varian Cary 50 UV-VIS spectrophotometer for samples with 10
µM Fe or an Ocean Optics 1 m pathlength spectrophotometry system for samples with
1 µM Fe. In the latter measurement, the Ocean Optics spectrophotometry instruments
were used in order to determine concentrations of the FeIIFZ3 at nanomolar level (see
Section 2.4.1, Chapter 2 for the detailed description).
Appendix 3. Supplemental Material for Chapter 5
208
To examine the effect of Fe contamination in reagents such as the fulvic acid stock on
the FeIIFZ3 formation rate, the Fe(II) formation experiment was also performed
without addition of Fe(III) stock to the sample. The FeIIFZ3 formation so measured
was negligibly small, suggesting that Fe contamination in the sample was negligible.
Although natural organic matters such as fulvic acid significantly absorb visible light,
the absorbance of the solution at 562 nm was measured to be insignificant.
A3.2.1.2. Model fitting
The best fit of the model to the experimental data was determined by using a least-
squares method in which the mean square error between the model value and the
average of the experimental data was minimized.
A3.2.2. RESULTS AND DISCUSSION
A3.2.2.1. Rate of photochemical and non-photochemical reduction
Reduction rates for Fe(III) complexed by various ligands including SRFA, EDTA and
citrate was examined in Fraquil* by measuring Fe
IIFZ3 formed in the absence and
presence of light with primary kinetic data shown in Figure A3.6. Under both light and
dark conditions FeIIFZ3 accounting for ~10% of total Fe was rapidly formed within 1
min’s incubation after FZ was added to Fraquil* containing Fe complexed by SRFA.
Followed by the initial jump, the FeIIFZ3 concentration linearly increased with respect
to time over 4 h. The initial increase of FeIIFZ3 is likely due to rapid reaction of Fe(II)
with FZ, suggesting that Fe(II) may be formed significantly in the pre-equilibrated
FeIII
SRFA stock during the dark storage (Pullin and Cabaniss, 2003). Concentration of
FeIIFZ3 increased with time at substantially higher rate under the light compared to that
in the dark condition for the three ligands. However, only for SRFA, the dark FeIIFZ3
formation was not negligible (part B of Figure A3.6).
Appendix 3. Supplemental Material for Chapter 5
209
Figure A3.6. Primary kinetic data of FeIIFZ3 formation in the (A) light and (B) dark
conditions. The time-dependent FeIIFZ3 formation in Fraquil
* (pH 8) was
spectrophotometrically monitored for 4 h at concentrations of 1 µM for Fe(III), 1 mM
for FZ, 1 mg.L-1
for SRFA, 26 µM for EDTA and 100 µM for citrate. Symbols and
error bars indicate average data and ±standard deviation from triplicate experiments.
To investigate factor(s) influencing the time-dependent FeIIFZ3 formation in the dark
SRFA system, we repeated the Fe(III) reduction experiment under identical conditions
except that SRFA was replaced by other synthetic Fe chelators such as citrate and
EDTA. Since the dark FeIIFZ3 production was not discernible in such cases, it is
unlikely that light leakage into the sample or any chemicals present in Fraquil* are
0
100
200
300
400
500
0 50 100 150 200 250 300
A.
-50
0
50
100
150
200
250
300
0 50 100 150 200 250 300
Time (minutes)
EDTA
Citrate
SRFA
B.[Fe
(II)
FZ
3] (n
M)
Appendix 3. Supplemental Material for Chapter 5
210
responsible for the dark FeIIFZ3 formation. Although presence of FZ has been reported
to promote the Fe(III) reduction under some conditions via direct complexation of
Fe(III)' by FZ (Shaked et al., 2004), this phenomenon is unlikely to account for the
observed FeIIFZ3 formation, as negligible Fe
IIFZ3 formation was seen in the EDTA and
citrate systems which cover a range of steady-state Fe(III)' concentration from pFe' =
11.96 for EDTA to pFe' = 10.10 for citrate (part B of Figure A3.6). The dark FeIIFZ3
formation in the SRFA system, therefore, rather indicates that Fe(II) is continuously
formed by the thermal reduction of some portion of FeIII
SRFA, probably due to the
presence of redox-active moieties in fulvic acid (e.g., hydroquinones), as reported
previously (Garg et al., 2012, Pham et al., 2012).
The experiments were undertaken in excess of FZ in which any other competing
reactions relevant to Fe(II) such as complexation of Fe(II)' and re-oxygenation to
Fe(III) are likely to be ignored. For example, the SRFA-mediated Fe(II)' formation
may be followed by either complexation by FZ or recomplexation by SRFA
(represented by L in eq. A3-2). The two competing reactions can be simply described
as follows:
Fe(II)f FZ II
3Fe(II)' 3FZ Fe FZk −+ → (A3-1)
Fe(II)f-L II
Fe(II)' L Fe Lk
+ → (A3-2)
where Fe(II)
f-FZk and Fe(II)
f-Lk are bimolecular rate constants for the Fe(II) complexation by
FZ and SRFA respectively. The Fe(II)
f-FZk has been previously determined to be 3.1 × 1011
M-3
.s-1
in 0.1 M NaClO4 (Thompsen and Mottola, 1984) and 2.0 × 1011
M-3
.s-1
in
seawater (Lin and Kester, 1992). The effect of solution pH on Fe(II)
f-FZk is small in the
range of pH 3 to 8. The value of 3.1 × 1011
M-3
s-1
was used in this work as the ionic
strength was closer to that of this work (assuming that Fe(II)
f-FZk value is not influenced by
the presence of light). In the presence of 1 mM FZ, the calculation using the rate law
equation for the reaction shown in eq. A3-2 indicates that 99.9% of inorganic Fe(II)
can be complexed by FZ within < 0.03 s. In addition, the complexation rate for FeIIFZ3
( Fe(II)
f-FZk [FZ]3 = 310 s
-1) is calculated to be much larger than the complexation and
Appendix 3. Supplemental Material for Chapter 5
211
oxygenation rates for FeIII
SRFA ( Fe(II)
f-Lk [L] = > ~2 s-1
and kox[O2] = 0.036 s-1
) under the
condition examined. The dissociation of the FeIIFZ3 complex is relatively slow with a
first-order rate constant of 4.3 × 10-5
s-1
(Thompsen and Mottola, 1984). Given that FZ
possess such a high affinity to Fe(II), change of FeIIFZ3 concentration due to the
complex dissociation is negligible on a timescale examined in this work.
Under this condition, therefore, it is reasonable to assume that FeIIFZ3 formation rate is
equal to the Fe(II) formation rate. In addition, observed FeIIFZ3 concentration
increased in a first-order manner with respect to time regardless of the absence and
presence of light. The Fe(II) formation rates for dark and light conditions, respectively,
can be described as follows;
IIIII3
dark
[Fe FZ ] [Fe(II)][Fe L]
d dk
dt dt= = (A3-3)
IIIII3
light
[Fe FZ ] [Fe(II)][Fe L]
d dk
dt dt= = (A3-4)
where kdark and klight are first-order rate constants for the SRFA-mediated thermal
reduction of FeIII
SRFA and photochemical reduction rate of FeIII
SRFA, respectively.
Approximations [FeIII
L] = [FeT] – [FeIIFZ3] (due to the low concentration of Fe'
compared to total Fe [FeT]) and [FZ] ≈ [FZT] (due to the relatively high total FZ
concentration [FZT]) followed by the integration gives relationship between [FeIIFZ3]
and time, as follows;
Tdark/lightII
T 3
[Fe ]ln
[Fe ] [Fe FZ ]k t
= ⋅
− (A3-5)
where kdark/light represent either kdark or klight. The first-order rate constant for the thermal
reduction (kdark) for SRFA was then determined to be 1.3 (±0.0) ×10-6
s-1
from a slope
of linear regression line in plots of time versus ln([Fe]T/([Fe]T-[Fe(II)FZ3])). Similarly,
the photoreduction rate constant (klight) was determined to be 1.6 (±0.02) ×10-5
s-1
for
SRFA, 6.5 (±0.25) ×10-6
for EDTA and 3.2 (±0.03) ×10-5
for citrate (Figure A3.7) by
using the corrected FeIIFZ3 concentration by subtracting the Fe
IIFZ3 concentration
Appendix 3. Supplemental Material for Chapter 5
212
measured under dark. The determined rate constants were listed in Table A3.1. The
effect of pH on Fe(III) reduction was also examined for SRFA system by using the
identical experimental and analytical procedures except that pH of solutions such as
Frqauil* and Fe
IIISRFA was adjusted to 6-8 and higher total Fe (10 µM) and SRFA
concentrations (50 mg.L-1
) with Varian Cary 50UV-Vis spectrophotometer (1 cm
cuvette) were employed. As shown in Figure A3.8, the FeIIFZ3 formation rate
increased with decrease in pH in both dark and light conditions, indicating that Fe(III)
reduction is facilitated in acidic pH. The rate constants determined by using eq. A3-5
were listed in Table A3.2.
Figure A3.7. Determination of rate constants for photo-reduction of Fe(III)-ligand
complexes in Fraquil* (pH 8). The experimental conditions, symbols and error bars are
identical to those in Figure A3.6, except that the data measured under the light were
only shown. The solid lines represent linear regression lines in each ligand system.
y = 0.00039x - 0.0041
R2 = 0.99
y = 0.0019x + 0.012
R2 = 0.99
y = 0.0010x + 0.037
R2 = 0.95
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 50 100 150 200 250 300
Time (minutes)
EDTA
Citrate
SRFA
ln([
Fe] T
/([F
e] T
-[F
eF
Z3])
Appendix 3. Supplemental Material for Chapter 5
213
Figure A3.8. Effect of pH on reduction of FeIII
SRFA under the light and dark.
0
1
2
3
4
5
6
7
8
9
10
0 2 4 6 8
Fe
(II)
FZ
3fo
rma
tio
n (
μM
)
Time (hr)
(A) pH 6, [SRFA] = 50 mg/L
0
1
2
3
4
5
6
7
8
9
10
0 2 4 6 8
Fe
(II)
FZ
3fo
rma
tio
n (
μM
)
Time (hr)
(B) pH 7, [SRFA] = 50 mg/L
0
0.5
1
1.5
2
2.5
3
0 2 4 6 8
Fe
(II)
FZ
3fo
rma
tio
n (
μM
)
Time (hr)
(D) pH 9, [SRFA] = 50 mg/L
0
1
2
3
4
5
6
7
8
9
10
0 2 4 6 8
Fe
(II)
FZ
3fo
rma
tio
n (
μM
)
Time (hr)
(E) pH 6, [SRFA] = 50 mg/L
0
1
2
3
4
5
6
7
8
9
10
0 2 4 6 8
Fe
(II)
FZ
3fo
rma
tio
n (
μM
)
Time (hr)
(F) pH 7, [SRFA] = 50 mg/L
0
1
2
3
4
5
6
7
8
9
10
0 2 4 6 8
Fe
(II)
FZ
3fo
rma
tio
n (
μM
)
Time (hr)
(H) pH 9, [SRFA] = 50 mg/L
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 2 4 6 8 10
Fe
(II)
FZ
3fo
rma
tio
n (
μM
)
Time (hr)
(C) pH 8, [SRFA] = 50 mg/L
0
0.5
1
1.5
2
2.5
3
0 2 4 6 8 10
Fe
(II)
FZ
3fo
rma
tio
n (
μM
)
Time (hr)
(G) pH 8, [SRFA] = 50 mg/L
Appendix 3. Supplemental Material for Chapter 5
214
Table A3.1. Reduction rate constants for organically complexed Fe(III) in Fraquil*
(pH 8).
Ligand Thermal reduction rate constant Photoreduction rate constant
kdark (s-1
) klight (s-1
)
SRFA 1.3 (±0.0) ×10-6
1.6 (±0.02) ×10-5
EDTA N.Da)
6.5 (±0.25) ×10-6
Citrate N.Da)
3.2 (±0.03) ×10-5
a) Not determined.
Table A3.2. Reduction rate constants for organically complexed Fe(III) in Fraquil*
(pH 6-9)a)
.
Ligand pH Thermal reduction rate constant Photoreduction rate constant
kdark (s-1
) klight (s-1
)
SRFA 6 3.6 ×10-5
1.3 ×10-4
SRFA 7 3.2 ×10-5
8.3 ×10-5
SRFA 8 5.5 ×10-6
1.4 ×10-6
SRFA 9 7.8 ×10-6
2.5 ×10-5
a) The Fe reduction experiment was performed at concentrations of 10 µM for Fe, 50
mg.L-1
for SRFA and 1mM for FZ.
Appendix 3. Supplemental Material for Chapter 5
215
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LIN, J. & KESTER, D. R. 1992. The kinetics of Fe(II) complexation by ferrozine in
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PHAM, A. N., ROSE, A. L. & WAITE, T. D. 2012. Kinetics of Cu(II) reduction by
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SHAKED, Y., KUSTKA, A. B., MOREL, F. M. M. & EREL, Y. 2004. Simultaneous
determination of iron reduction and uptake by phytoplankton. Limnology and
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