Redox Transformations of Iron in the Presence of Exudate from the

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Redox transformations of iron in the presence of exudate from the cyanobacterium Microcystis aeruginosa under conditions typical of natural waters Kai Wang, Shikha Garg, and T. David Waite Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b00396 • Publication Date (Web): 24 Feb 2017 Downloaded from http://pubs.acs.org on February 28, 2017

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Environmental Science & Technology

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Redox transformations of iron in the presence of exudate from the cyanobacterium

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Microcystis aeruginosa under conditions typical of natural waters

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Kai Wang, Shikha Garg, and T. David Waite*

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School of Civil and Environmental Engineering, The University of New South Wales, Sydney,

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NSW 2052, Australia

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Revised

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February 2017

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ToC graphic

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2 Environment ACS Paragon Plus

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ABSTRACT

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Interaction of the exudate secreted by a toxic strain of the cyanobacterium Microcystis

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aeruginosa with Fe(II) and Fe(III) was investigated here under both acidic (pH 4) and

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alkaline (pH 8) conditions. At the concentrations of iron and exudate used, iron was present

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as dissolved iron (< 0.025 µm) at pH 4 but principally as small (< 0.45 µm) iron

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oxyhydroxide particles at pH 8 with only ~3-27% present in the dissolved form as a result of

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iron binding by the organic exudate. The formation of strong Fe(III)-exudate and relatively

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weak Fe(II)-exudate complexes alters the reduction potential of the Fe(III)/Fe(II) redox

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couple facilitating more rapid oxidation of Fe(II) at pH 4 and 8 than was the case in the

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absence of exudate. Our results further show that the organic exudate contains Fe(III)

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reducing moieties resulting in production of measureable concentrations of Fe(II). However,

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these reducing moieties are short-lived (with a half-life of 1.9 hours) and easily oxidized in

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air-saturated environments. A kinetic model was developed that adequately describes the

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redox transformation of Fe in the presence of exudate both at pH 4 and pH 8.



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1. INTRODUCTION

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Natural Organic Matter (NOM) is a heterogeneous mixture of various organic moieties and

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occurs ubiquitously in freshwater and marine environments. NOM plays a critical role in a

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variety of important biogeochemical processes in natural waters including generation of

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reactive oxygen species (ROS),1-3 redox cycling of metals,4-8 and degradation of organic

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pollutants.9-11 NOM can be classified into two categories, autochthonous NOM (derived from

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indigenous organisms including algae and bacteria) and allochthonous NOM (derived

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principally from the surrounding catchment). While the characteristics and redox properties

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of allochthonous NOM have been widely investigated,12-15 there has been only a few studies

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in which the nature and redox properties of autochthonous NOM have been investigated.

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to active production by algae and diffusion across the cell membrane. In particular,

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Microcystis aeruginosa, a common alga in cyanobacterial blooms in temperate regions, is

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known to secrete significant quantities of organic exudate20, 21 however the nature and redox

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properties of this organic exudate and the purpose of generation of these moieties are not well

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understood.

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One of the possible reasons for generation and release of this organic exudate is to facilitate

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iron uptake by Microcystis aeruginosa 16 since iron is a critical micronutrient. In oxygenated

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natural surface waters, a majority of the iron is present as either iron(III) oxyhydroxide

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and/or organically complexed ferric iron (Fe(III)L) ; however, both iron(III) oxyhydroxide

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and organically complexed Fe are generally considered to be unavailable for acquisition by

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Microcystis aeruginosa.22 Instead, Microcystis aeruginosa acquires the majority of its Fe

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requirement by uptake of inorganic iron (Fe′) in either the ferric (Fe(III)′) and/or ferrous

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(Fe(II)′) states.22 The concentration of Fe′ is dependent on the rates of Fe(III) reduction and

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Fe(II) oxidation with the kinetics of both of these processes dependent on the nature of the

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Algogenic NOM is believed to be one of the important sources of autochthonous NOM due


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organic moieties present in solution. These organic moieties not only complex Fe but may

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also facilitate Fe(III) reduction via (i) ligand to metal charge transfer (LMCT),

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generation of superoxide resulting in superoxide-mediated Fe reduction (SMIR)

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(iii) reduction by organic compounds containing hydroquinone and/or semiquinone moieties

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oxidation kinetics of Fe(II) are also affected by the presence of NOM with different organic

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groups exhibiting different Fe(II) binding affinity which, in turn, affects the Fe(II) oxidation

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rate.

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organically-complexed Fe(II) can be calculated based on Marcus theory using the reduction

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potential of the Fe(III)L/Fe(II)L couple which is, in turn, affected by the stability constant of

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the Fe(III)L and Fe(II)L complexes with the increase in the K Fe( III ) L / K Fe( II ) L value resulting

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in an increase in the Fe(II) oxidation rate constant. Thus, the organic compounds secreted by

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Microcystis aeruginosa may play an important role in the redox transformation of Fe, thereby

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assisting in iron acquisition by Microcystis aeruginosa.

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In this study, we investigate Fe redox transformations in the presence of exudate secreted by

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Microcystis aeruginosa at pH 4 and 8. While the pH 8 studies are representative of both the

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growth medium (pH 8) and natural waters (pH 7-8.5), the acidic conditions have been

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chosen, in part, to avoid complications associated with the fast Fe(II) oxygenation (i.e. Fe(II)

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oxidation due to its interaction with dioxygen) and precipitation of iron oxides and thus assist

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in quantifying the rate and extent of dissolved Fe redox transformations mediated by the

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algal exudate. This work also provides useful insights into the redox properties of the organic

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exudate in comparison with allochthonous NOM. Based on our experimental results, we have

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developed a mathematical model capable of reproducing the Fe redox transformations

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mediated by the organic exudate secreted by Microcystis aeruginosa under particular well-

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defined conditions. While the focus of this study is on the impact of the exudate on Fe(II) and

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(ii)

and/or

which may be intrinsically present in NOM and/or generated on irradiation of NOM. The

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As described by Pham and Waite,

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the second order oxidation rate constant of



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Fe(III) transformations under the conditions of the medium (pH 8) in which the algae has

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been grown, additional attention should be given in future studies to the effects of pH and

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other medium constituents on exudate-mediated iron redox transformations.

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2. EXPERIMENTAL METHODS

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2.1 Reagents

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All reagent solutions were prepared using 18.1 MΩ.cm resistivity Milli-Q water (MQ) unless

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stated otherwise. All glassware and plasticware were pre-soaked in 5% HCl for at least 24 h

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and rinsed three times by MQ prior to use. All experiments were performed at controlled

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room temperature of 22.0°C±0.5°C. All stock solutions were stored at 4°C when not in use

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unless stated otherwise.

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A primary stock solution of 4 mM Fe(II) was prepared in 0.2 M HCl. A 16 µM Fe(II)

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working stock in 0.8 mM HCl was prepared daily by 250-fold dilution of the primary stock

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solution by MQ. A 20 µM Fe(III) working stock in 2 mM HCl was prepared on dilution of 2

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mM Fe(III) stock solution in 0.2 M HCl daily before experiments. The pH of the stock

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solutions of Fe(II) and Fe(III) were sufficiently low to minimize Fe(II) oxygenation and

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Fe(III) precipitation during storage yet sufficiently high to prevent significant pH change

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when they were added to the pH 4 and 8 exudate solutions. A stock solution of 100 µM

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Amplex Red (AR; Invitrogen) mixed with 50 kU.L-1 horseradish peroxidase (HRP; Sigma)

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for H2O2 measurement was prepared as described earlier

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use. Stock solutions of ferrozine (3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,4″-disulfonic

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acid sodium salt; abbreviated as FZ; Sigma), desferrioxamine B (DFB; Sigma) and

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superoxide dismutase (SOD; Sigma) were prepared and stored as reported in our earlier work.

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and stored at -81°C when not in


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M and was used as the Fe(III) reductant in the measurement of total Fe concentration. A 5 M

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stock solution of ammonium acetate (Sigma) was prepared in MQ to increase the pH of

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acidified samples during measurement of total Fe concentration. A stock solution of 5 µM

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speciation measurement at pH 4 using ion pair solvent extraction method. Ion-pair extracting

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solution was prepared by mixing the ion-paring reagent triotylmethylammonium chloride

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(Aliquat 336, Sigma) in toluene at a concentration of 18 g.L-1.

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2.2 Collection of algal exudate

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Batch cultures of Microcystis aeruginosa were grown in sterilized modified Fraquil medium

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(Fraquil* containing total iron concentration of 1 µM; pH 8)22 at 27°C under 90 µmol

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photons m-2.s-1 of light supplied by cool-white fluorescent tubes on a 14 h : 10 h light : dark

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cycle. During the exponential growth phase, algal cells were collected by centrifuging the

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batch cultures for 10 min at 3500 rpm and then washed twice by MQ to remove the absorbed

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iron and organic matter. Washed cells were subsequently resuspended into a fresh iron and

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organic free Fraquil* medium (defined as “starved medium” hereafter; recipe shown in Table

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S1) to a final density of 7.5×106 -9×106 cells.mL-1. As neither iron nor organic compounds

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were added to the medium prior to inoculation, iron and organics should only be present in

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the solution at trace amounts (if at all) and, as such, their impact on iron redox

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transformations can be ignored. The starved culture was grown for 48 h in the incubator

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under the conditions described above. After 48 h, algal cells were removed firstly by

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centrifuging at 3500 rpm for 10 min followed by filtration through a 0.22 µm PVDF filter

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(Millipore). The filtrate obtained was used and is referred to as fresh exudate from here on.

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Note that there was no apparent release of organic material to solution due to cell rupture

Hydroxylamine hydrochloride (HH, Sigma) was prepared in MQ at concentrations of 0.1

Fe-radiolabeled ferric chloride (Perkin Elmer) was prepared in 5 mM HCl for iron



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during the centrifugation and filtration processes since the dissolved organic carbon (DOC)

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content of the algal exudate prepared immediately after suspension of cells in the starved

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medium was negligible (< 0.1 mg.L-1). A period of 48 h was optimal for release of sufficient

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algal exudate with higher incubation times resulting in evident cell rupture (see Figure S1).

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The procedure of collection of algal exudate was similar to that described in earlier studies 36,

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preparation techniques such as freeze drying. Furthermore, since the preparation procedure

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used is relatively brief, the presence and role of any short-lived organic groups in the organic

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exudate (with lifetimes on the order of minutes to hours) can also be investigated. The pH of

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the exudate was adjusted either to 4.00 ± 0.03 by addition of 1 M HCl (high purity 30% w/v,

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Sigma) or to 8.00 ± 0.03 by addition of 1.5 mM NaHCO3 followed by 1 M HCl to trim to the

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target value (if necessary) immediately before the experiments. The DOC content of the

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exudate was measured using TOC analyser (TOC-5000A, Shimadzu) and was always in the

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range 2.0 - 3.0 mg L-1 with the cell status and cell density similar for each experiment.

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2.3 Experimental setup

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For measurement of Fe redox transformations at pH 4, appropriate concentrations of Fe(III)

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or Fe(II) were added to fresh exudate solution in dark bottles to avoid interference from

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ambient light. Samples were withdrawn at regular time intervals to measure the concentration

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of Fe(II) using the FZ method and H2O2 using the AR method.

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For measurement of Fe(III) reduction rates at pH 8, appropriate concentrations of Fe(III)

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were added to fresh exudate solution containing 1 mM FZ in dark bottles. Ferrozine was

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added to trap any Fe(II) generated prior to its oxidation with the Fe(II)FZ3 production rate

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measured using the Ocean Optics spectrometry system described elsewhere.27 For

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measurement of Fe(II) oxidation rates, appropriate concentrations of Fe(II) were added to

and avoids possible alterations in the structure of the exudate that could be induced by


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fresh exudate solution in dark bottles with samples withdrawn at regular time intervals for

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measurement of the concentration of Fe(II) remaining by addition of ferrozine to trap any

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Fe(II) present.38

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All experiments were performed in air–saturated solutions unless stated otherwise with air

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saturation assured by maintaining sufficient head-space in the reaction vessels. To determine

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the role of dioxygen in the experimental system investigated here, we conducted one set of

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experiments in which the concentration of dioxygen was reduced by sparging the solutions

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with Ar in a sealed reactor for 4 hours prior to experiments (with pure argon used for the pH

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4 studies and argon containing 300 ppm CO2 used for the pH 8 studies). The concentration of

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dioxygen was reduced to 5-10% of the initial dioxygen concentration in our solution

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(complete removal was not achieved). Sparging was continued during experiments to avoid

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intrusion of O2 into our sample. A rough estimation of dioxygen concentration in the sparged

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solution was made by measuring the rate of oxidation of 100 nM inorganic Fe(II) in pH 8

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buffer solution sparged for the same time in the same reactor using the same flow rate. It

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should be noted that the dioxygen concentration, even in the argon-sparged solutions, would

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be expected to change minimally through the course of any particular iron transformation

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study given the very low concentrations of iron present ( 1 h) since

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the protonation of oxyhydroxide functional groups and subsequent polarization and


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destabilization of iron-oxygen bonds in the surface lattice must occur prior to release of

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Fe(III)′.39 Thus, as shown in a previous study,39 the release of Fe(III)′ during a short-

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acidification step (1 min) should result principally from dissociation of organically-

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complexed Fe(III). Note that the iron oxyhydroxide particles formed in the presence of

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exudate may be quite different to the particles formed in the absence of organic moieties.

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However, as confirmed in a separate study, the reactivity varies by two-three fold at most and

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hence will not result in any significant release of Fe(III)′ within 1 min. To measure the

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concentration of acid-labile iron, the solution was acidified to pH 2 and filtered through 0.025

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µm membrane filters after 1 min of addition of acid. The total iron concentration in the

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filtrate (with the large majority of this acid labile iron expected to be derived from

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organically complexed iron) was measured using the method described below.

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2.4.2 Ion pair solvent extraction method

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To determine the form of Fe (i.e. inorganic Fe and/or organically-complexed Fe) present at

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pH 4, appropriate volumes of

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exudate to achieve final Fe(III) concentrations of 1-2 nM. Subsequently, 4 mL of the solution

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was pipetted into a centrifuge tube that contained 4 mL of the extracting reagent and the

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mixture was shaken vigorously for 30 s. Samples were then centrifuged at 4000 g for 5 min to

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eliminate emulsification and the activity of the organic layer (upper layer) that contained 55Fe

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complex was then determined by liquid scintillation counting. The procedure of Fe speciation

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measurement used here is identical to that described previously. 40

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2.5 Measurement of total Fe

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For measurement of total Fe, 1 mM FZ and 1 mM hydroxylamine hydrochloride were added

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to the samples to reduce all Fe(III) to Fe(II) which was then trapped by FZ. After waiting for

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24-48 h to ensure complete Fe(III) reduction had occurred, an appropriate volume of 5 M

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Fe stock were added to either the starved medium or the



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ammonium acetate was added to adjust the pH to 4 and the concentration of Fe(II)FZ3 was

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measured using the Ocean Optics spectrophotometry system described earlier.27

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2.6 Measurement of Fe(II)

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The concentration of Fe(II) at pH 4 was measured using the modified FZ method as described

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in our earlier work with DFB added to the sample to inhibit FZ mediated Fe(III) reduction.27

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For measurement of Fe(II) at pH 8, addition of DFB was omitted since FZ-mediated Fe(III)

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reduction at pH 8 on the time scales investigated here was found to be negligible. The

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equipment setting and calibration procedure used were identical to those described in our

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earlier study. 27A value of 29000 ± 1000 M-1 cm-1 for the molar absorptivity of the Fe(II)FZ3

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complex was obtained which is close to the published value of 27900 M-1 cm-1. 41

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2.7 H2O2 determination

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The concentration of H2O2 generated on exudate-Fe interaction was quantified

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fluorometrically using a Cary Eclipse fluorescence spectrophotometer (Agent Technologies)

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with 2 µM AR and 1 kU.L-1 HRP as the fluorescence reagent. For H2O2 measurement at pH 4,

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1 mL of sample was mixed with 2 mL of 10 mM phosphate buffer at pH 7 in a quartz cuvette

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followed by the AR-HRP mix. The analysis of samples at pH 8 was undertaken by directly

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mixing the AR-HRP regent solution to 3 mL of sample. The equipment setting and

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calibration procedure used were identical to those described in our earlier study.35 The

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detection limit of the method is determined to be ~ 3.0 nM (defined as 3 times the standard

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deviation of the reagent blank).

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2.8 Kinetic modeling

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The software Kintek Explorer was used for kinetic modelling of our experimental results.

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Kintek Explorer is a kinetic simulation program that allows multiple data sets to be fit


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simultaneously to a single model based on numerical integration of the rate equations

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describing the reaction mechanism.42 The model is developed based on a proposed set of

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chemical reactions and their associated rate equations which are generally either drawn

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from the literature or, if the rate constant is specific to particular experimental

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conditions, deduced from studies of that particular reaction under the conditions of

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interest. As such, good agreement between model output and experimentally determined

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results over a range of conditions confirms that the chosen reaction set is a reasonable

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description of the underlying processes operating under the particular conditions of

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interest. Numerical integration is performed using the Bulirsch–Stoer algorithm with

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adaptive step size, and nonlinear regression to fit data (when required) based on the

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Levenberg–Marquardt method.42

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3. RESULTS and DISCUSSION

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3.1 Chemical speciation of Fe(III) in the presence of Microcystis aeruginosa exudate

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The form of iron present would be expected to exert a huge impact on Fe redox

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transformations and hence, prior to investigating the redox transformations of Fe, we

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determined both the form and acid reactivity of iron present in the exudate solution. The total

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concentration of Fe present after various treatments is shown in Table 1. As shown, 77-95%

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of the total Fe added is present in the filtrate after passage of the solution through 0.025 µm

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filters at pH 4 supporting the conclusion that most of the Fe is present in dissolved form.

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Dynamic light scattering measurement performed on algal exudate containing an even higher

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concentration of Fe(III) (1 µM) at pH 4 shows that no particles are present in this solution

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(Figure S2) further supporting the finding that Fe is present in dissolved form at pH 4 either

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as inorganic Fe(III) and/or Fe(III) complexed by the organic exudate. Since the Fe species



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formed in the presence of algal exudate (DOC ~ 2.5 mg.L-1) at pH 4 can be easily extracted

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using ion pairing solvent (18 g.L-1 of trioctylmethylammonium chloride in toluene; Table S2)

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which is reported to have poor extraction efficiency for inorganic Fe(III) species40, we

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conclude that Fe(III) is principally present as organically-complexed Fe(III) under these

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conditions. No extractable Fe is measured in samples prepared in starved medium containing

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the same total Fe(III) concentration which further supports the conclusion that the speciation

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of Fe in the absence and presence of exudate differs as a result, presumably, of the

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coordinative interaction of the exudate with iron. The complexation of Fe by the algal

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exudate produced by Microcystis aeruginosa is consistent with earlier reports on binding of

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Cu(II) by exudate of the green alga Dunaliella tertiolecta43 and trace metal binding in

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Phaeodactylum tricornutum44 and Emiliania huxleyi.37

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At pH 8, 87-97% of the iron is present as particles smaller than 0.45 µm with a portion (3-

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27%) present in the 0.025 µm filtrate. The large fraction (61-93%) of iron present in the

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colloidal size range (0.025-0.45 µm) is most likely indicative of the presence of exudate-

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coated iron oxyhydroxide particulates (AFO-L). The fraction of Fe present in the 0.025 µm

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filtrate of the acidified samples varies between 14-33% and is slightly higher than that present

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in the

Redox Transformations of Iron in the Presence of Exudate from the

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