ARTICLE pubs.acs.org/est
Effect of Light on Iron Uptake by the Freshwater Cyanobacterium Microcystis aeruginosa M. Fujii,† T. C. Dang,† A. L. Rose,‡,† T. Omura,§ and T. D. Waite†,* †
School of Civil and Environmental Engineering, The University of New South Wales, Sydney, NSW 2052, Australia Southern Cross GeoScience, Southern Cross University, Lismore 2480, Australia § Department of Civil and Environmental Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 6-6-06, Sendai, 980-8579, Japan ‡
bS Supporting Information ABSTRACT: Visible light was observed to induce reductive dissociation of organically complexed Fe and dramatically increase the short-term uptake rate of radiolabeled Fe by Microcystis aeruginosa PCC7806 in Fraquil* medium buffered by a single metal chelator, ethylenediaminetetraacetic acid (EDTA). Only wavelengths 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)0 complexation by FZ at this concentration is rapid and should outcompete other reactions involving Fe(II)0 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)0 production would appear reasonable. Because FeIIFZ3 formation followed a first-order relationship with respect to FeIIIEDTA concentration, the rate of photochemical Fe(II)0 generation in our system can be written as: d½FeII FZ3 d½FeðIIÞ0 ¼ ¼ khv ½FeIII EDTA ð1Þ dt dt where khv is a first-order rate constant for photoreductive dissociation of FeIIIEDTA under the conditions examined. Approximating [FeIIIEDTA] ≈ [Fe]T - [FeIIFZ3] and [FZ] ≈ [FZ]T (where
subscript T indicates total concentration) followed by integration of the resulting expression yields a relationship between [FeIIFZ3] and time (part 2 of the Supporting Information and Figure S3 of the Supporting Information for details). As shown in part A of Figure 1, 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 suggests 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)0 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)0 -producing photoredox processes. The effect of light on 55Fe 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, e3.5 106 cell.mL-1) and the use of different concentrations of EDTA and radiolabeled 55Fe(III) (as a replacement for nonradiolabeled Fe). In the absence and presence of light, 55Fe accumulated in cells linearly with time over the 2 h incubations (part B of Figure 1). At 26 μM EDTA and 200 nM Fe, the 55Fe uptake rate (0.33 ( 0.065 amol.cell-1.hr-1, n = 13) under the light was 2 orders of magnitude greater than that under 1393
dx.doi.org/10.1021/es103311h |Environ. Sci. Technol. 2011, 45, 1391–1398
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ARTICLE
Table 1. Kinetic Model for Fe Transformation and Uptake in the Light Chemical Reactions/Diffusion Parameters
Rate Constants/Parameter Values
(a) Chemical reactions FeIIIEDTA þ hv f Fe(II)0 þ EDTAox 0
Fe(II) þ EDTA f Fe EDTA II
0
Fe(II) þ 3FZ f Fe FZ3 II
0
Fe EDTA f Fe(II) þ EDTA II
Fe FZ3 f Fe(II) þ 3FZ 0
0
Fe(II) þ O2 f Fe(III) þ III
0
s-1
kf-EDTA
2.1 10
M-1.s-1
kf-FZ
11 c
3.1 10
M-3.s-1
-3 b
s-1
-5 c
s-1
6b
1.2 10 4.3 10
kd-FZ O2-
Fe EDTA þ O2 f Fe EDTA þ O2 II
6.4 10-6 a
kd-EDTA
0
II
khv
kox -
M-1.s-1
d
8.8
kox-EDTA
31
M-1.s-1
e -9
L.cell-1.s-1
Fe(II) f uptake (b) Diffusion parametersf
kup
3.9 10
diffusion coefficient
D
0.5-1 10-9 -9
m2.s-1
radius of porin
a
0.5-1.5 10
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
-10
1.1 10
As
m
m2
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 4 of the Supporting Information for a detailed discussion of methods and results. c Thompsen and Mottola.23 d Pham and Waite.6 e Fujii et al.22 f Parameters for diffusion and bacterial outer-membrane porin properties are carefully examined in part 6 of the Supporting Information. See also Tables S5 and S6 of the Supporting Information). a
darkness (0.003 ( 0.006 amol.cell-1.hr-1, n = 3), indicating a significant role of light in Fe uptake. Effect of Light Wavelength. Transport of Fe across the (cyto)plasmic membrane is likely an energy-dependent process.24 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 1). Irradiation with light at λ = 500-700 nm, which accounts for the majority (66%) of the total photon flux (Table S1 of the Supporting Information) and which has a substantial impact on cyanobacterial photosynthesis,25 had almost no effect on cellular Fe uptake (part C of Figure 1). Similarly, significant FeIIFZ3 formation only occurred when the sample was exposed to light in the range λ = 400-500 nm (part D of Figure 1). In contrast to the total photon flux density, the FeIIFZ3 formation rate was a linear function of 55Fe uptake rate (Figure S4 of the Supporting Information), supporting the contention that specific light wavelength rather than total intensity is important for the short-term 55Fe 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 17,19 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 FeIIIEDTA rapidly decreases in the visible light region (Figure S5 of theSupporting Information), this complex is still capable of capturing some light at 400-500 nm. Over this
wavelength range, the average quantum yield for FeIIIEDTA (j = [FeIIFZ3 formation rate]/[number of photons absorbed]) was 0.010 (Table S2 of theSupporting Information), comparable to the published value at similar pH and irradiation wavelength (j = 0.011, when quantum yield data ranging from j = 0.005 at 500 nm to j = 0.02 at 400 nm are averaged 26). 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 photoproduced Fe(II)0 and, potentially, Fe(III)0 . The presence of 1 mM FZ significantly decreased Fe uptake by 27-70% (p < 0.05, part A of Figure S6 of the Supporting Information), supporting the hypothesis that photoproduced Fe(II)0 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. aeruginoasa 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.9 A significant decrease in rate of Fe uptake was also observed with increasing EDTA concentration (part B of Figure S6 of the Supporting Information), indicating that complexation by EDTA at concentrations examined here effectively competes with cellular uptake, as direct FeIIIEDTA acquisition is unlikely. The limited availability of 55Fe-EDTA for dark uptake, even after preliminary photolysis, consistently indicates that light-facilitated Fe uptake is tightly coupled with the availability of photoproduced 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 3 of the Supporting Information and Figure S7 of the Supporting Information). 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 shown in Table 1 and presented schematically in 1394
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Figure 2. Key processes in this model are photoreductive dissociation of FeIIIEDTA to Fe(II)0 , oxygenation of Fe(II)0 to Fe(III)0 , and formation and dissociation of FeIIEDTA. This model is similar to that used by Sunda and Huntsman14 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)0 followed by oxidation of the complex was also considered here. Because inorganic Fe complexes are nonphotoreactive at circumneutral pH,27 photoreduction of Fe(III)0 was ignored. The rate constants for photoreductive dissociation of FeIIIEDTA determined in this work were reasonably similar to the values reported in seawater by Sunda and Huntsman14 (4.4 10-6 s-1 at light intensity of 500 μmol-quanta.m-2.s-1) and Anderson and Morel17 (1.7 10-6 s-1 at 95 μmol-quanta.m-2.s-1). However, in contrast to the Sunda and Huntsman model,14 Fe(III)0 formation via oxidation of photoproduced Fe(II)0 is negligible in the freshwater model presented here because complexation of Fe(II)0 by EDTA or FZ occurs much faster than Fe(II)0 oxygenation (by a factor of at least 104 under the experimental conditions described here, Table 2). Although rates of oxidation and photoproduction of Fe(II)0 are similar in both seawater and freshwater systems, recomplexation of Fe(II)0 by EDTA was ignored in the Sunda model as complexation of Fe0 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.22 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)0 ([Fe(II)0 ]ss) is controlled by photoreductive dissociation of FeIIIEDTA, complexation of photoproduced Fe(II)0 by EDTA and FZ (if present), dissociation of FeIIEDTA and FeIIFZ (if present), oxidation of Fe(II)0 and cellular uptake, then Fe uptake rate (FFe mol.cell-1.s-1) can be described by: 0 FFe ¼ kup ½FeðIIÞ ss ¼
kup ðkhv ½FeIII EDTA þ kd-EDTA ½FeII EDTA þ kd-FZ ½FeII FZ3 Þ kf -EDTA ½EDTA þ kf -FZ ½FZ3 þ kup ½cell þ kox ½O2 ð2Þ
where [cell] is the cell density (cell.L-1) and relevant reaction details are listed in Table 1. As explained in part 5 of the Supporting Information, the thermal dissociation of Fe(II) complexes with EDTA or FZ significantly influences [Fe(II)0 ]ss while Fe(II)0 oxidation has a negligible effect on this parameter. The uptake rate constant kup was estimated by substituting 55Fe uptake rates measured under various conditions together with other known kinetic parameters into eq 2 (part 5 of the Supporting Information for details), yielding an average value ((one standard deviation) of 3.9 ((1.9) 10-9 L.cell-1.s-1 or 35 ((17) L.m-2.s-1 (Table 2). Using this value, Fe uptake rates for a range of cell densities and competitive ligand concentrations were calculated (Figure 3). This model demonstrates that higher cell densities result in a decrease in [Fe(II)0 ]ss and thus a decrease in 55Fe uptake rate (Figure S6 of the Supporting Information). This situation will arise when the rate of uptake is comparable to or higher than the rate of formation of Fe(II) complexes
Figure 2. Fe uptake model by M. aeruginosa in the presence of light. Unchelated Fe(II) (i.e., Fe(II)0 ) is formed from the photoreductive dissociation of ferric EDTA complex (FeIIIEDTA). The photoproduced Fe(II) subsequently passes through nonspecific outer membrane channels (porins) by diffusion. However, cellular Fe uptake competes with Fe(II)0 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 shortterm 55Fe uptake experiment, whereas dotted arrows indicate relatively minor reactions. Rate constants depicted near the arrows correspond to those listed in Table 1.
with EDTA and/or FZ (i.e., kup[cell] g kf-EDTA[EDTA] þ kf-FZ[FZ]3). The apparent high affinity of the cell for Fe(II) was examined by considering the possibility of passive diffusion of photoproduced Fe(II)0 to the intracellular space. In cyanobacteria, nutrients must pass through the outer-membrane into the periplasmic space prior to active translocation across the innermembrane 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 milieu into the periplasmic space most likely occurs via movement through nonselective transmembrane channels called porins, which are ubiquitous in almost all Gram-negative bacteria investigated so far including cyanobacteria.28 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: FFe ¼ Jporin Nporin AS 0 2 1, 000Δ½Fe ¼ - Dπa ð3Þ Nporin AS l 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), Δ[Fe0 ] (= [Fe0 ]in [Fe0 ]out) is the difference between Fe0 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 micrometers29 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 1395
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Table 2. Measured and Calculated Fe Uptake Parameters for Various Culture Conditions Calculated Steady-State or Average Concentrationsd)
Measured Data cellular density kf-EDTA kf-FZ cell.mL-1 [EDTA]s-1 [FZ]3 s-1 kox[O2]a s-1
competitive ligands
FFeb) amol cell -1 hr-1
kupc) L cell-1 s-1
averaged kupc) L cell-1 s-1
Fe(II)0 fM
FeII EDTA nM
FeIIFZ3 nM 0
EDTA 26 μM
2.6 105
55
0.0021
0.28 ((0.034)
2.9 10-9
27
0.17
EDTA 26 μM
1.0 10
6
55
0.0021
0.27 ((0.004)
2.9 10-9
25
0.16
0
EDTA 26 μM
2.7 107
55
0.0021
0.095 ((0.001)
2.2 10-9
8.4
0.053
0
FZ 1 mM
2.6 105
55
0.0021
0.085 ((0.034)
5.8 10-9
e)
310
3.9 10-9
4.1
0.026
4.5
((1.9 10-9)
4.1
0.026
4.5
7.2 10-9 5.7 10-9
3.2 21
0.020 0.13
4.5 0
0.085 ((0.007)
4.0 10-9
6.6
0.16
0
0.010 ((0.005)
1.2 10-9
2.6
0.17
0
-9
FZ 1 mM
1.0 10
6
55
310
0.0021
0.062 ((0.003)
4.2 10
FZ 1 mM EDTA 26 μMe)
2.7 107 3.5 106
55 55
310
0.0021 0.0021
0.069 ((0.000) 0.39 ((0.025)
EDTA 100 μM
3.5 106
210
0.0021
EDTA 260 μM
3.5 10
550
0.0021
6
The first-order oxygenation rate constant for Fe(II)0 at pH 8 was calculated by multiplying the second-order rate constant of 8.8 M-1.s-1 by a saturated dissolved oxygen concentration of 0.24 mM at 27 °C. b) Averaged 55Fe uptake rates and (standard deviations from triplicate experiments. All experiments were performed under nonsaturating conditions. c) kup value for each experimental condition was determined using eq 2. Average and (standard deviations for all kup were also calculated. d) See part 5 of the Supporting Information for detailed methods employed to calculate the steadystate concentrations for Fe(II)0 and FeIIEDTA and time-averaged concentrations for FeIIFZ3. e) The 55Fe uptake experiments were performed on different dates. Although the 55Fe: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. a
Figure 3. Effect of competitive ligand concentrations and cellular densities on calculated Fe uptake rate using eq 2.
M. aeruginosa (cellular radius ∼3 μm). Assuming that unchelated Fe which enters the periplasm is rapidly captured by periplasmic Fe transporters under the nonsaturating conditions investigated here (i.e., [Fe0 ]in ≈ 0), then Δ[Fe0 ] ∼ -[Fe0 ]out = [Fe(II)0 ]ss, leading to an alternative expression for the uptake rate constant kup: 1, 000Dπa2 Nporin AS ð4Þ kup ¼ l 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 1) indicates that diffusion of Fe through such channels is faster than an active transport process by a factor of 101-103. The upper limit for calculated kup (0.06-4.1 10-9 L.cell-1.s-1, Table S6 of the Supporting Information) 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 outermembrane 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 cyanobacterial 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.8 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
