Chemosphere 103 (2014) 212–219
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Contrasting effects of pyoverdine on the phytoextraction of Cu and Cd in a calcareous soil J.Y. Cornu a,b,⇑, M. Elhabiri c, C. Ferret d, V.A. Geoffroy d, K. Jezequel b, Y. Leva b, M. Lollier b, I.J. Schalk d, T. Lebeau b,e a INRA (Institut National de la Recherche Agronomique), UMR 1220 TCEM (Transfert sol-plante et Cycle des Eléments Minéraux dans les écosystèmes cultivés), CS 20032, 33882 Villenave d’Ornon cedex, France b Université de Haute Alsace, EA 3991 LVBE (Laboratoire Vigne Biotechnologies Environnement), Equipe Dépollution Biologique des Sols, BP 50568, 68008 Colmar cedex, France c CNRS-Université de Strasbourg, UMR 7509 Laboratoire de Chimie Moléculaire, Equipe Chimie Bioorganique et Médicinale, 25 rue Becquerel, 67200 Strasbourg, France d UMR 7242, Université de Strasbourg-CNRS, ESBS, 300 Boulevard Sébastien Brant, F-67412 Illkirch cedex, Strasbourg, France e LUNAM, LPGN UMR 6112 CNRS, Université de Nantes, 2 rue de la Houssinière, BP 92208, 44322 Nantes cedex 3, France
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Pyoverdine (Pvd) coordination
properties towards Cd(II) and Cu(II) are determined. The stability constant of Pvd-Cu is much higher than that of Pvd-Cd. Pyoverdine competes with exogenous chelators for Cu(II) complexation at calcareous pH. Pyoverdine supply improves selectively the phytoextraction of Cu in a calcareous soil. Pyoverdine impact on Cu uptake is restricted to plant roots.
a r t i c l e
i n f o
Article history: Received 1 September 2013 Received in revised form 13 November 2013 Accepted 26 November 2013 Available online 17 December 2013 Keywords: Bacterial siderophore Bioavailability Complexation Coordination properties DGT Metals
Pyoverdine supply
Root ?
Pvd-Cu
Pvd-Cu Absorption
?
Cu
Mobilisation
Cu
?
Convection
Enhancement of Cu phytoextraction
Pvd
Complexation
Cu
L-Cu
Sorption
+ Pvd
Diffusion
+L
L
L-Cu Porewater
Sediment
a b s t r a c t Enhanced metal phytoextraction by the use of siderophore-producing bacteria (SPB) has received a lot of attention in the past decade. Bacterial siderophores are able to bind a wide range of metals other than iron and thus should enhance their phytoavailability in contaminated matrices. However, the impact of bacterial siderophores in the soil–plant transfer of metals is not yet fully elucidated, as underlined by the opposing results reported in the literature regarding the efficiency of coupling phytoextraction with bioaugmentation by SPB. The present study focuses on one bacterial siderophore, the pyoverdine (Pvd), produced by Pseudomonas aeruginosa. The coordination properties of Pvd towards Cd(II) and Cu(II) were determined and the effect of Pvd supply was assessed on (i) the mobility (CaCl2 extractions), (ii) the phytoavailability (DGT measurements) and (iii) the phytoextraction of Cd and Cu, in a calcareous soil. The stability constant 0 0 of Pvd-Cu (KL Cu = 1020.1) was found much higher than that of Pvd-Cd (KL Cd = 108.2). The major finding was the agreement observed between Pvd coordination properties and Pvd impact on metals phytoextraction. Pyoverdine, supplied at 250 lmol kg1 soil, enhanced the mobility, the phytoavailability and the phytoextraction of Cu while the fate of Cd was not affected. All these results were compared to those reported for chelate-assisted phytoextraction. Their relevance in using SPB for phytoremediation is discussed. Ó 2013 Elsevier Ltd. All rights reserved.
⇑ Corresponding author at: INRA (Institut National de la Recherche Agronomique), UMR 1220 TCEM (Transfert sol-plante et Cycle des Eléments Minéraux dans les écosystèmes cultivés), CS 20032, 33882 Villenave d’Ornon cedex, France. Tel.: +33 5 57 12 25 22; fax: +33 5 57 12 25 15. E-mail address: [email protected] (J.Y. Cornu). 0045-6535/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.11.070
J.Y. Cornu et al. / Chemosphere 103 (2014) 212–219
1. Introduction Metal phytoextraction is defined as the use of green plants (either hyperaccumulators or not) to remove metals from contaminated matrices. This technology is environmental friendly, costeffective compared to physico-chemical clean-up process but is generally time-consuming (Glass, 2000). Indeed, the rate of metal phytoextraction is often limited by the low phytoavailability of metals in the contaminated matrix. This is particularly true for Cu, which exhibits a high affinity for the solid phase (high Kd value), especially in calcareous soils (Bravin et al., 2010). One common approach to enhance the rate of metal phytoextraction is to supply the contaminated matrix with an exogenous synthetic chelator (e.g., EDTA) which promotes through efficient complexation the mobilization of metals from the solid phase. Numerous studies have reported a several-fold increase in the removal of metals from contaminated soils when EDTA was supplied (e.g., Luo et al., 2005). This technology called chelate-assisted phytoextraction suffers however from several drawbacks. At the dose of EDTA required to enhance metal phytoextraction (mmol kg1 soil), the leaching of metals to groundwater is unavoidable since plants remove only a small fraction of the metals mobilized from the solid phase (Nowack et al., 2006). Moreover, EDTA is phytotoxic for numerous plants at this dose and affects their biomass (Evangelou et al., 2007). Most of bacteria present in soils and sediments have developed Fe(III) acquisition processes, which involve siderophores. Siderophores are organic chelators with a small molecular weight (150–2000 Da) which are characterized by a very high affinity for Fe(III) (e.g., 1042 M1 for the enterobactin produced by Escherichia coli) (Hider and Kong, 2011). Siderophore-producing bacteria (SPB) such as fluorescent Pseudomonas are commonly isolated in the rhizosphere of crops (Deweger et al., 1995). Siderophore production is regulated by the intracellular concentration of Fe. Under Fe starvation their synthesis is up-regulated (Visca et al., 2007). Recent studies showed that the presence of Cu enhanced the production of the siderophore pyoverdine (Pvd) by Pseudomonas aeruginosa in a medium supplemented with iron (Braud et al., 2010). Pvd would provide an extracellular protection for bacteria by sequestering Cu and avoiding its diffusion inside the bacteria (Schalk et al., 2011). Although siderophores are generally viewed as biological iron uptake agents, recent evidence has shown that they are able to chelate many other metals (Braud et al., 2009a) and may play significant roles in the biogeochemical cycling and biological uptake of other metals (Kraepiel et al., 2009). Therefore, promoting the colonization of SPB in the rhizosphere could help in the phytoextraction of metals by increasing their phytoavailability. Among bioremediation techniques, bioaugmentation consists in the inoculation of the contaminated matrix with specific competent strains or consortia of microorganisms. Several studies have tested the effect of coupling phytoextraction with soil bioaugmentation (Lebeau et al., 2008). Bioaugmentation by SPB has been observed to both promote and reduce phytoextraction (Sessitsch et al., 2013) depending on the combination of plant, bacterium and metal (Ma et al., 2011). The processes by which SPB may alter the acquisition of metals by plant roots are indeed not elucidated. The effect of metal complexation by bacterial siderophore on metal phytoavailability is of particular interest and is the subject of conflicting theories (Sessitsch et al., 2013). The present study aims to verify that the potential effect of siderophores on metal phytoextraction is based primarily on their ability to complex the metal in soil. This study focuses on one siderophore, i.e., pyoverdine, the major siderophore produced by P. aeruginosa, and two metals: Cd(II) and Cu(II) for which Pvd is assumed to show contrasting binding properties, according to the Irving–Williams series. P. aeruginosa is not the ideal candidate for such environmen-
213
tal cleaning process but it is one of the most studied organism regarding iron acquisition pathways involving siderophores (Schalk et al., 2012). Pyoverdine affinity for Fe(III) reaches 1030.8 M1 (Albrecht-Gary et al., 1994). This siderophore can also chelate several other metals but according to the current knowledge only Fe(III) is assimilated efficiently by the bacterial cell (Braud et al., 2009a). The objectives of this study are (i) to determine the complexation constants of Pvd towards Cd(II) and Cu(II), (ii) to assess the effect of a direct supply of Pvd on the mobility, the phytoavailability and the phytoextraction of Cd and Cu in a calcareous soil so as (iii) to address the relationships between Pvd binding properties and Pvd effect on metal phytoextraction. 2. Materials and methods 2.1. Soil sample The soil sample used in this study was collected in 2009 in the vineyard stormwater basin of Rouffach (Alsace, France). It was sieved to Cu (5.5) > Fe ( Zn > Cd). The concentrations of Pvd and H+ were also assessed in the extracting solution. As expected, Pvd concentration increased with Pvd addition, from 0.1 lM at Pvd25 up to 8.9 lM at Pvd500 (Suppl. data S4), while pH remained unchanged (pH 6.8, Suppl. data S5). Pvd concentration in extracting solution was related (P < 0.001) positively with both Fe (r = 0.87) and Cu (r = 0.98). The percentage of Pvd extracted from soil was however low: more than 90% of the Pvd added to the soil remained sorbed onto the solid phase, even at Pvd500. As the dose of Pvd increased, more Pvd was present in porewater and Cu and Fe solubilization was more complete. The treatment Pvd250 was selected for all the subsequent experiments. It led to a concentration of Pvd in porewater around 10 lM at a realistic soil/water ratio (see Suppl. data T1). This amount of Pvd is much lower than those of EDTA tested in chelate-assisted phytoextraction which often reach several mmol kg1 and led to a porewater concentration of chelate higher than 1 mM (Evangelou et al., 2007).
3.3. Pvd effect on the flux of metals resupply The DGT technique allowed us to characterize the flux of Cu and Cd resupply over a 96 h incubation period. As observed on metal extractability, Pvd induced specifically an increase in the flux of Cu resupply (Fig. 2). The increase in FCu was significant (P < 0.01) for all deployment times and was stronger at shorter times. The values in FCu at t = 4 and 8 h need, however, to be considered
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Table 2 pM values (M = Cd(II), Cu(II) and Fe(III)) for Pvd and environmentally relevant ligands at 25 °C in water. pM values were calculated from the different sets of thermodynamic data using the Hyss 2009 program and for pH values representative of a calcareous (pH 8) and an acidic soil (pH 5). Ligands
pH 5
pH 8
References
pCd
pCu
pFe
pCd
pCu
pFe
Siderophores Pyochelina Pyoverdineb Desferrioxamine Bc,d
– 6.00 6.00
11.46 8.88 6.72
10.79 19.00 19.70
– 7.49 6.17
14.90 17.67 12.46
16.71 28.37 28.52
[1]
Non-humic organic acids Citric acid Oxalic acid Tartaric acid
6.00b 6.00b 6.00g
6.23e 6.23b,e 6.00g
10.58b 9.57f 8.54b
6.02b 6.00b 6.00g
6.24e 6.24b,e 6.00g
11.86b 9.63f 11.64b
[3–5] [6,7] [8–10]
Exogenous ligands EDTAh
10.20
13.23
21.05
14.47
17.50
26.13
[11,12]
[2]
[1] Brandel et al. (2012) Dalton Trans. 41, 2820–2834. [2] Farkas et al. (1999) Polyhedron 18, 2391–2398. [3] Ramamoorthy and Manning (1973) J. Inorg. Nucl. Chem. 35, 1571–1575. [4] Campi et al. (1964) J. Inorg. Nucl. Chem. 26, 553–564. [5] Capone et al. (1986) Talanta 33, 763–767. [6] Sindhu et al. (1991) J. Indian Chem. Soc. 68, 289–290. [7] Deneux et al. (1968) Can. J. Chem. 46, 1383–1388. [8] Ramamoorthy and Manning (1972) J. Inorg. Nucl. Chem. 34, 1977–1989. [9] Stary (1963) Anal. Chim. Acta 28, 132–149. [10] Ramamoorthy and Manning (1975) J. Inorg. Nucl. Chem. 37, 363–367. [11] Bauman (1974) J. Inorg. Nucl. Chem. 36, 1827–1832. [12] Skochdopole and Chaberek (1959) J. Inorg. Nucl. Chem. 11, 222–223. a Solvent: CH3OH/H2O (80/20 w/w); I = 0.1 M ((C2H5)4NClO4); Solvent: H2O. b I = 0.1 M (NaClO4). c I = 0.2 M (KCl). d I = 0.15 M (KNO3), T = 37 °C. e I = 0.1 M (KNO3 or NaNO3). f I = 0.5 M (NaClO4). g I = 1 M (NaClO4). h I = 0.1 M (KClO4).
cautiously since the corresponding DGT-labile Cu concentration in bulk solution was not yet stabilized (see Suppl. data T1). One single pool was invoked to describe the time dependence of R at both short and long exposure times. The related kinetics parameters are specified on Fig. S6 (Suppl. data). As expected from F and R values (Suppl. data T2), only the kinetics of Cu resupply was affected by Pvd addition. In Pvd-, Cu was characterized by a large response time (Tc = 3000 s) and a low distribution coefficient (Kdl = 11 cm3 g1). This low Kdl reflects that a substantial part of Cu fixed onto the solid phase was not available for resupply at the time-scale of DGT deployment. One can wonder whether this ‘‘non-available’’ pool of Cu can be mobilized by Pvd. Cu resupply in Pvd+ was characterized by a lower response time (Tc = 10 s) but a fairly similar distribution coefficient (Kdl = 40 cm3 g1) than in Pvd (Suppl. data S6). These fitting parameters suggest that Pvd ensured a faster resupply of Cu from the labile reservoir but did not get access to the nonlabile pool, as underlined for Cd by Stanhope et al. (2000) after EDTA addition. The flux of metal resupply over 24 h is often used as an indicator of metal phytoavailability in soil (Davison et al., 2000). The value of FCu at 24 h in Pvd (1.04 nmol Cu m2 s1) was in total agreement with those reported by Bravin et al. (2010) on calcareous Cu-contaminated soils. The one in Pvd+ was 7.44 nmol Cu m2 s1 which would mean that Pvd addition at 250 lmol kg1 soil improved the phytoavailability of Cu by a factor 7. Regarding our working hypothesis, these results confirm the selective effect of Pvd towards Cu. The increase in FCu after Pvd addition was however not as high as the increase in Cu extractability suggesting that the diffusion rate of Pvd-Cu in porewater was slower than those of the Cu complexes (e.g., with fulvic acids) present in the control.
3.4. Pvd effect on metals phytoextraction A bioassay was performed to assess the impact of Pvd addition on the phytoextraction of metals by a monocotyledonous (strategy II) and a dicotyledonous (strategy I) plant. Pvd addition did not affect significantly (P > 0.05) the plant biomass at harvest (Table 3) and did not induce any visual symptom of toxicity. The concentration of Fe in shoots was over the critical deficiency level reported by Marschner (1995) for higher plants. The synthesis of phytosiderophores by barley roots was thus neglected. Surprisingly, the addition of Pvd did not promote (P > 0.05) the acquisition of Fe by the two plants. This result contrasts with those reported by Shirley et al. (2011) who showed a clear effect of Fe-Pvd supply on the Fe status of both strategy I and strategy II plants. The study of Shirley and co-workers was however performed in liquid medium with Fe-Pvd (100 lM) as single source of iron. Cadmium contents in plant tissues was also not affected (P > 0.05) by Pvd addition (Table 3). The contents in Zn, Ni (Table 3), Mn and Ca (data not shown) were not affected as well (P > 0.05). Conversely, the amount of Cu taken up by the two plants was significantly higher (P < 0.05) when Pvd was supplied (Fig. 3). For barley, it increased from 12.9 lg in the control (3.5 lg for tomato) up to 20.2 lg in the presence of Pvd (4.5 lg for tomato). To our knowledge, this is the first time the direct role of a bacterial siderophore on the phytoextraction of Cu has been underlined in soil. The effect of Pvd was however restricted to the roots. For both plants, root Cu was 2-times higher (P < 0.05) when Pvd was supplied while shoot Cu was not affected (P > 0.05) (Fig. 3). Indeed, plants have evolved mechanisms to limit Cu translocation to the shoots at high exposure levels. Copper transport, chelation, trafficking and sequestration in the root symplasm are highly controlled by homeostatic
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Table 3 Dry weight (DW) and content at harvest in Fe, Cd, Zn and Ni of tomato and barley cropped on the soil supplied (Pvd+) or not (Pvd) with pyoverdine. For each plant and each variable separately, indicates that the value in the presence of Pvd significantly differ (P < 0.05) from the one in the control (absence of Pvd). Roots
Shoots
DW Fe (mg)
Cd Zn Ni DW Fe (lg g1 DW) (mg)
Tomato Pvd 19 Pvd+ 14
1790 169 2117 147
Barley
3725 19 3702 26
Pvd 61 Pvd+ 52
Cd Zn Ni (lg g1 DW)
39 7.6 57 151 44 54 5.2 64 98 39 334 11 137 277 10 145
74 4.5 70 3.0
55 3.2 58 3.3
40 1.3 39 1.3
40
Shoots
Cu content (µg Cu g-1 DW)
20
0 Pvd -
Barley
Tomato
Pvd +
0
100
Roots
200
chelate-assisted phytoextraction like with EDTA, it is widely accepted that metals like Cu are taken up passively under their chelated forms, via the non-selective apoplasmic pathway. The phytoextraction of Cu is thus non-limited by the saturation of transporters like through the symplasm (no Fmax value, see below). Disruption of the Casparian band is however required to achieve the high shoot concentrations needed for phytoextraction. This occurs at the root apex where the Casparian strip is not yet fully developed or might be induced by the addition of EDTA (Nowack et al., 2006). Despite its high molecular weight (4 times higher than EDTA), Pvd is suspected to be internalised by plant roots. Indeed, both strategy I and strategy II plants were found to incorporate Pvd when supplied as Pvd-Fe (Shirley et al., 2011). The uptake of Cu as Pvd-Cu might thus have occurred, presumably via the apoplasm since anionic metal–ligand complexes are supposed non-bioavailable. At the concentration of Pvd used in the bioassay (250 lmol kg1), all dissolved Cu was not assumed to be complexed by Pvd. Indeed, the percentage of Pvd-Cu calculated in DGT bulk solutions was comprised between 25% and 81% (Suppl. data T1). A significant part of Cu was thus presumably taken up via the symplastic pathway through membrane proteins (e.g., from the COPT family) that displayed saturating transport kinetics. The comparison of the maximal uptake flux of Cu by plant roots (Fmax) mentioned in Bravin et al. (2010) for non-hyperaccumulators (0.47 < Fmax < 1.57 nmol Cu m2 s1) with the flux of Cu resupply from the soil measured in Pvd+ (2.53 < FCu < 11.05 nmol Cu m2 s1) suggests that the rate-limiting process of Cu phytoextraction, when Pvd was supplied, was the kinetics of Cu uptake by plant roots. This would explain why Cu phytoextraction was less impacted by Pvd than was Cu phytoavailability assessed by DGT measurements. One way to overcome this issue might be the use of Cu hyperaccumulators whose Fmax value can reach 2.68 nmol Cu m2 s1 according to Bravin et al. (2010). 4. Conclusions
300
* 400
500
* 1
Fig. 3. Copper concentrations (lg g DM) in shoots and roots of barley and tomato plants grown on the calcareous soil supplied (Pvd+) or not (Pvd) with pyoverdine. For each plant separately, * indicates that the value in root Cu or shoot Cu measured in the presence of Pvd significantly differed (P < 0.05) from the one measured in the control (absence of Pvd). The error bars stand for mean standard deviations.
processes based on the production of chelators (e.g., metallothioneins) and chaperones (Clemens, 2001). The reduced transpiration caused by the low shoot biomass also did not favour the translocation of Cu. As compared to EDTA, the effect of Pvd towards the phytoextraction of Cu was weak. Indeed, several studies showed that the supply of EDTA to the soil can increase the phytoextraction of Cu by non-hyperaccumulators to more than a factor 2 (e.g., Luo et al., 2005). In all these studies, the shoot concentration of Cu was affected. One major reason explaining those differences is the dose of chelator supplied to the cropping matrix. Those of EDTA tested in the literature were often much higher than the one of Pvd used in the bioassay. Adding chelators not only increases the total dissolved metal concentration but may also change the primary route of plant metal-uptake (Nowack et al., 2006). Thereby, in
This study shows a good agreement between the stability constants of the metal complexes with Pvd and its effects on Cu mobility, phytoavailability and phytoextraction in a calcareous soil. Thereby, it validates our working hypothesis assuming that the impact of Pvd on the phytoextraction of one metal is based primarily on its ability to complex this metal. This work underlines several characteristics of the effect of Pvd towards metals phytoextraction. First, the effect of Pvd seems fairly specific to Cu. This specificity would help the phytoextraction of Cu above neutral pH, when Ca is likely to compete for binding with Cu (Nowack et al., 2006) and in metal-polycontaminated matrices. It would however restrict the use of Pvd-producing bacteria to the phytoremediation of Cu-contaminated areas. The high pH-dependence of Pvd coordination properties would also limit the use of Pvd-producing bacteria to alkaline matrices. The rate-limiting uptake and root-to-shoot translocation of Cu in tomato and barley suggests the use of Pvdproducing bacteria together with Cu hyperaccumulating plants to maximize Cu phytoextraction and limit Cu leaching to groundwater. This study suggests that Pvd plays a role in the impact of fluorescent Pseudomonas on the phytoextraction of metals but does not prove that this role is prominent. The discrepancy observed between the effect of purified Pvd and fluorescent Pseudomonas towards, notably, the root-to-shoot translocation of metals (Braud et al., 2009b) suggests that other bacterial process than Pvd production may impact metals phytoextraction. Acknowledgments The authors thank Sylvie Bussière, Cécile Fontaine and Françoise Hoegy for their technical assistance. Financial support for this work
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was provided by the Program Interdisciplinaire CNRS-CEMAGREF ‘‘Ingénierie écologique’’ and the Alsace Region Research Network in Environmental Sciences and Engineering’’ (REALISE). C. Ferret had a fellowship from the Région Alsace and the DGA (Direction Générale de l’Armement). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2013.11.070. References Albrecht-Gary, A.M., Blanc, S., Rochel, N., Ocacktan, A.Z., Abdallah, M.A., 1994. Bacterial iron transport: coordination properties of pyoverdin PaA, a peptidic siderophore of Pseudomonas aeruginosa. Inorg. Chem. 33, 6391–6402. Brandel, J., Humbert, N., Elhabiri, M., Schalk, I.J., Mislin, G.L.A., Albrecht-Gary, A.M., 2012. Role of pyochelin in Pseudomonas aeruginosa: a physico-chemical characterization of the iron(III), copper(II) and zinc(II) complexes. Dalton Trans. 41, 2820–2834. Braud, A., Hoegy, F., Jezequel, K., Lebeau, T., Schalk, I.J., 2009a. New insights into the metal specificity of the Pseudomonas aeruginosa pyoverdine-iron uptake pathway. Environ. Microbiol. 11, 1079–1091. Braud, A., Jezequel, K., Bazot, S., Lebeau, T., 2009b. Enhanced phytoextraction of an agricultural Cr- and Pb-contaminated soil by bioaugmentation with siderophore-producing bacteria. Chemosphere 74, 280–286. Braud, A., Geoffroy, V., Hoegy, F., Mislin, G.L.A., Schalk, I.J., 2010. The siderophores pyoverdine and pyochelin are involved in Pseudomonas aeruginosa resistance against metals: another biological function of these two siderophores. Environ. Microbiol. Rep. 2, 419–425. Bravin, M.N., Le Merrer, B., Denaix, L., Schneider, A., Hinsinger, P., 2010. Copper uptake kinetics in hydroponically-grown durum wheat (Triticum turgidum durum L.) as compared with soil’s ability to supply copper. Plant Soil 331, 91– 104. Chen, Y., Jurkevitch, E., Barness, E., Hadar, Y., 1994. Stability-constants of pseudobactin complexes with transition-metals. Soil Sci. Soc. Am. J. 58, 390– 396. Clemens, S., 2001. Molecular mechanisms of plant metal tolerance and homeostasis. Planta 212, 475–486. Davison, W., Hooda, P.S., Zhang, H., Edwards, A.C., 2000. DGT measured fluxes as surrogates for uptake of metals by plants. Adv. Environ. Res. 3, 550–555. Deweger, L.A., Vanderbij, A.J., Dekkers, L.C., Simons, M., Wijffelman, C.A., Lugtenberg, B.J.J., 1995. Colonization of the rhizosphere of crop plants by plant-beneficial Pseudomonads. Fems Microbiol. Ecol. 17, 221–227. Elhabiri, M., Hamacek, J., Bünzli, J.C.G., Albrecht-Gary, A.M., 2004. Lanthanide homodimetallic triple-stranded helicates: insight into the self-assembly mechanism. Eur. J. Inorg. Chem. 1, 51–62. Ernstberger, H., Davison, W., Zhang, H., Tye, A., Young, S., 2002. Measurement and dynamic modeling of trace metal mobilization in soils using DGT and DIFS. Environ. Sci. Technol. 36, 349–354.
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Contrasting effects of pyoverdine on the phytoextraction of Cu and Cd in a calcareous soil J.Y. Cornu a,b,⇑, M. Elhabiri c, C. Ferret d, V.A. Geoffroy d, K. Jezequel b, Y. Leva b, M. Lollier b, I.J. Schalk d, T. Lebeau b,e a INRA (Institut National de la Recherche Agronomique), UMR 1220 TCEM (Transfert sol-plante et Cycle des Eléments Minéraux dans les écosystèmes cultivés), CS 20032, 33882 Villenave d’Ornon cedex, France b Université de Haute Alsace, EA 3991 LVBE (Laboratoire Vigne Biotechnologies Environnement), Equipe Dépollution Biologique des Sols, BP 50568, 68008 Colmar cedex, France c CNRS-Université de Strasbourg, UMR 7509 Laboratoire de Chimie Moléculaire, Equipe Chimie Bioorganique et Médicinale, 25 rue Becquerel, 67200 Strasbourg, France d UMR 7242, Université de Strasbourg-CNRS, ESBS, 300 Boulevard Sébastien Brant, F-67412 Illkirch cedex, Strasbourg, France e LUNAM, LPGN UMR 6112 CNRS, Université de Nantes, 2 rue de la Houssinière, BP 92208, 44322 Nantes cedex 3, France
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Pyoverdine (Pvd) coordination
properties towards Cd(II) and Cu(II) are determined. The stability constant of Pvd-Cu is much higher than that of Pvd-Cd. Pyoverdine competes with exogenous chelators for Cu(II) complexation at calcareous pH. Pyoverdine supply improves selectively the phytoextraction of Cu in a calcareous soil. Pyoverdine impact on Cu uptake is restricted to plant roots.
a r t i c l e
i n f o
Article history: Received 1 September 2013 Received in revised form 13 November 2013 Accepted 26 November 2013 Available online 17 December 2013 Keywords: Bacterial siderophore Bioavailability Complexation Coordination properties DGT Metals
Pyoverdine supply
Root ?
Pvd-Cu
Pvd-Cu Absorption
?
Cu
Mobilisation
Cu
?
Convection
Enhancement of Cu phytoextraction
Pvd
Complexation
Cu
L-Cu
Sorption
+ Pvd
Diffusion
+L
L
L-Cu Porewater
Sediment
a b s t r a c t Enhanced metal phytoextraction by the use of siderophore-producing bacteria (SPB) has received a lot of attention in the past decade. Bacterial siderophores are able to bind a wide range of metals other than iron and thus should enhance their phytoavailability in contaminated matrices. However, the impact of bacterial siderophores in the soil–plant transfer of metals is not yet fully elucidated, as underlined by the opposing results reported in the literature regarding the efficiency of coupling phytoextraction with bioaugmentation by SPB. The present study focuses on one bacterial siderophore, the pyoverdine (Pvd), produced by Pseudomonas aeruginosa. The coordination properties of Pvd towards Cd(II) and Cu(II) were determined and the effect of Pvd supply was assessed on (i) the mobility (CaCl2 extractions), (ii) the phytoavailability (DGT measurements) and (iii) the phytoextraction of Cd and Cu, in a calcareous soil. The stability constant 0 0 of Pvd-Cu (KL Cu = 1020.1) was found much higher than that of Pvd-Cd (KL Cd = 108.2). The major finding was the agreement observed between Pvd coordination properties and Pvd impact on metals phytoextraction. Pyoverdine, supplied at 250 lmol kg1 soil, enhanced the mobility, the phytoavailability and the phytoextraction of Cu while the fate of Cd was not affected. All these results were compared to those reported for chelate-assisted phytoextraction. Their relevance in using SPB for phytoremediation is discussed. Ó 2013 Elsevier Ltd. All rights reserved.
⇑ Corresponding author at: INRA (Institut National de la Recherche Agronomique), UMR 1220 TCEM (Transfert sol-plante et Cycle des Eléments Minéraux dans les écosystèmes cultivés), CS 20032, 33882 Villenave d’Ornon cedex, France. Tel.: +33 5 57 12 25 22; fax: +33 5 57 12 25 15. E-mail address: [email protected] (J.Y. Cornu). 0045-6535/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.11.070
J.Y. Cornu et al. / Chemosphere 103 (2014) 212–219
1. Introduction Metal phytoextraction is defined as the use of green plants (either hyperaccumulators or not) to remove metals from contaminated matrices. This technology is environmental friendly, costeffective compared to physico-chemical clean-up process but is generally time-consuming (Glass, 2000). Indeed, the rate of metal phytoextraction is often limited by the low phytoavailability of metals in the contaminated matrix. This is particularly true for Cu, which exhibits a high affinity for the solid phase (high Kd value), especially in calcareous soils (Bravin et al., 2010). One common approach to enhance the rate of metal phytoextraction is to supply the contaminated matrix with an exogenous synthetic chelator (e.g., EDTA) which promotes through efficient complexation the mobilization of metals from the solid phase. Numerous studies have reported a several-fold increase in the removal of metals from contaminated soils when EDTA was supplied (e.g., Luo et al., 2005). This technology called chelate-assisted phytoextraction suffers however from several drawbacks. At the dose of EDTA required to enhance metal phytoextraction (mmol kg1 soil), the leaching of metals to groundwater is unavoidable since plants remove only a small fraction of the metals mobilized from the solid phase (Nowack et al., 2006). Moreover, EDTA is phytotoxic for numerous plants at this dose and affects their biomass (Evangelou et al., 2007). Most of bacteria present in soils and sediments have developed Fe(III) acquisition processes, which involve siderophores. Siderophores are organic chelators with a small molecular weight (150–2000 Da) which are characterized by a very high affinity for Fe(III) (e.g., 1042 M1 for the enterobactin produced by Escherichia coli) (Hider and Kong, 2011). Siderophore-producing bacteria (SPB) such as fluorescent Pseudomonas are commonly isolated in the rhizosphere of crops (Deweger et al., 1995). Siderophore production is regulated by the intracellular concentration of Fe. Under Fe starvation their synthesis is up-regulated (Visca et al., 2007). Recent studies showed that the presence of Cu enhanced the production of the siderophore pyoverdine (Pvd) by Pseudomonas aeruginosa in a medium supplemented with iron (Braud et al., 2010). Pvd would provide an extracellular protection for bacteria by sequestering Cu and avoiding its diffusion inside the bacteria (Schalk et al., 2011). Although siderophores are generally viewed as biological iron uptake agents, recent evidence has shown that they are able to chelate many other metals (Braud et al., 2009a) and may play significant roles in the biogeochemical cycling and biological uptake of other metals (Kraepiel et al., 2009). Therefore, promoting the colonization of SPB in the rhizosphere could help in the phytoextraction of metals by increasing their phytoavailability. Among bioremediation techniques, bioaugmentation consists in the inoculation of the contaminated matrix with specific competent strains or consortia of microorganisms. Several studies have tested the effect of coupling phytoextraction with soil bioaugmentation (Lebeau et al., 2008). Bioaugmentation by SPB has been observed to both promote and reduce phytoextraction (Sessitsch et al., 2013) depending on the combination of plant, bacterium and metal (Ma et al., 2011). The processes by which SPB may alter the acquisition of metals by plant roots are indeed not elucidated. The effect of metal complexation by bacterial siderophore on metal phytoavailability is of particular interest and is the subject of conflicting theories (Sessitsch et al., 2013). The present study aims to verify that the potential effect of siderophores on metal phytoextraction is based primarily on their ability to complex the metal in soil. This study focuses on one siderophore, i.e., pyoverdine, the major siderophore produced by P. aeruginosa, and two metals: Cd(II) and Cu(II) for which Pvd is assumed to show contrasting binding properties, according to the Irving–Williams series. P. aeruginosa is not the ideal candidate for such environmen-
213
tal cleaning process but it is one of the most studied organism regarding iron acquisition pathways involving siderophores (Schalk et al., 2012). Pyoverdine affinity for Fe(III) reaches 1030.8 M1 (Albrecht-Gary et al., 1994). This siderophore can also chelate several other metals but according to the current knowledge only Fe(III) is assimilated efficiently by the bacterial cell (Braud et al., 2009a). The objectives of this study are (i) to determine the complexation constants of Pvd towards Cd(II) and Cu(II), (ii) to assess the effect of a direct supply of Pvd on the mobility, the phytoavailability and the phytoextraction of Cd and Cu in a calcareous soil so as (iii) to address the relationships between Pvd binding properties and Pvd effect on metal phytoextraction. 2. Materials and methods 2.1. Soil sample The soil sample used in this study was collected in 2009 in the vineyard stormwater basin of Rouffach (Alsace, France). It was sieved to Cu (5.5) > Fe ( Zn > Cd). The concentrations of Pvd and H+ were also assessed in the extracting solution. As expected, Pvd concentration increased with Pvd addition, from 0.1 lM at Pvd25 up to 8.9 lM at Pvd500 (Suppl. data S4), while pH remained unchanged (pH 6.8, Suppl. data S5). Pvd concentration in extracting solution was related (P < 0.001) positively with both Fe (r = 0.87) and Cu (r = 0.98). The percentage of Pvd extracted from soil was however low: more than 90% of the Pvd added to the soil remained sorbed onto the solid phase, even at Pvd500. As the dose of Pvd increased, more Pvd was present in porewater and Cu and Fe solubilization was more complete. The treatment Pvd250 was selected for all the subsequent experiments. It led to a concentration of Pvd in porewater around 10 lM at a realistic soil/water ratio (see Suppl. data T1). This amount of Pvd is much lower than those of EDTA tested in chelate-assisted phytoextraction which often reach several mmol kg1 and led to a porewater concentration of chelate higher than 1 mM (Evangelou et al., 2007).
3.3. Pvd effect on the flux of metals resupply The DGT technique allowed us to characterize the flux of Cu and Cd resupply over a 96 h incubation period. As observed on metal extractability, Pvd induced specifically an increase in the flux of Cu resupply (Fig. 2). The increase in FCu was significant (P < 0.01) for all deployment times and was stronger at shorter times. The values in FCu at t = 4 and 8 h need, however, to be considered
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Table 2 pM values (M = Cd(II), Cu(II) and Fe(III)) for Pvd and environmentally relevant ligands at 25 °C in water. pM values were calculated from the different sets of thermodynamic data using the Hyss 2009 program and for pH values representative of a calcareous (pH 8) and an acidic soil (pH 5). Ligands
pH 5
pH 8
References
pCd
pCu
pFe
pCd
pCu
pFe
Siderophores Pyochelina Pyoverdineb Desferrioxamine Bc,d
– 6.00 6.00
11.46 8.88 6.72
10.79 19.00 19.70
– 7.49 6.17
14.90 17.67 12.46
16.71 28.37 28.52
[1]
Non-humic organic acids Citric acid Oxalic acid Tartaric acid
6.00b 6.00b 6.00g
6.23e 6.23b,e 6.00g
10.58b 9.57f 8.54b
6.02b 6.00b 6.00g
6.24e 6.24b,e 6.00g
11.86b 9.63f 11.64b
[3–5] [6,7] [8–10]
Exogenous ligands EDTAh
10.20
13.23
21.05
14.47
17.50
26.13
[11,12]
[2]
[1] Brandel et al. (2012) Dalton Trans. 41, 2820–2834. [2] Farkas et al. (1999) Polyhedron 18, 2391–2398. [3] Ramamoorthy and Manning (1973) J. Inorg. Nucl. Chem. 35, 1571–1575. [4] Campi et al. (1964) J. Inorg. Nucl. Chem. 26, 553–564. [5] Capone et al. (1986) Talanta 33, 763–767. [6] Sindhu et al. (1991) J. Indian Chem. Soc. 68, 289–290. [7] Deneux et al. (1968) Can. J. Chem. 46, 1383–1388. [8] Ramamoorthy and Manning (1972) J. Inorg. Nucl. Chem. 34, 1977–1989. [9] Stary (1963) Anal. Chim. Acta 28, 132–149. [10] Ramamoorthy and Manning (1975) J. Inorg. Nucl. Chem. 37, 363–367. [11] Bauman (1974) J. Inorg. Nucl. Chem. 36, 1827–1832. [12] Skochdopole and Chaberek (1959) J. Inorg. Nucl. Chem. 11, 222–223. a Solvent: CH3OH/H2O (80/20 w/w); I = 0.1 M ((C2H5)4NClO4); Solvent: H2O. b I = 0.1 M (NaClO4). c I = 0.2 M (KCl). d I = 0.15 M (KNO3), T = 37 °C. e I = 0.1 M (KNO3 or NaNO3). f I = 0.5 M (NaClO4). g I = 1 M (NaClO4). h I = 0.1 M (KClO4).
cautiously since the corresponding DGT-labile Cu concentration in bulk solution was not yet stabilized (see Suppl. data T1). One single pool was invoked to describe the time dependence of R at both short and long exposure times. The related kinetics parameters are specified on Fig. S6 (Suppl. data). As expected from F and R values (Suppl. data T2), only the kinetics of Cu resupply was affected by Pvd addition. In Pvd-, Cu was characterized by a large response time (Tc = 3000 s) and a low distribution coefficient (Kdl = 11 cm3 g1). This low Kdl reflects that a substantial part of Cu fixed onto the solid phase was not available for resupply at the time-scale of DGT deployment. One can wonder whether this ‘‘non-available’’ pool of Cu can be mobilized by Pvd. Cu resupply in Pvd+ was characterized by a lower response time (Tc = 10 s) but a fairly similar distribution coefficient (Kdl = 40 cm3 g1) than in Pvd (Suppl. data S6). These fitting parameters suggest that Pvd ensured a faster resupply of Cu from the labile reservoir but did not get access to the nonlabile pool, as underlined for Cd by Stanhope et al. (2000) after EDTA addition. The flux of metal resupply over 24 h is often used as an indicator of metal phytoavailability in soil (Davison et al., 2000). The value of FCu at 24 h in Pvd (1.04 nmol Cu m2 s1) was in total agreement with those reported by Bravin et al. (2010) on calcareous Cu-contaminated soils. The one in Pvd+ was 7.44 nmol Cu m2 s1 which would mean that Pvd addition at 250 lmol kg1 soil improved the phytoavailability of Cu by a factor 7. Regarding our working hypothesis, these results confirm the selective effect of Pvd towards Cu. The increase in FCu after Pvd addition was however not as high as the increase in Cu extractability suggesting that the diffusion rate of Pvd-Cu in porewater was slower than those of the Cu complexes (e.g., with fulvic acids) present in the control.
3.4. Pvd effect on metals phytoextraction A bioassay was performed to assess the impact of Pvd addition on the phytoextraction of metals by a monocotyledonous (strategy II) and a dicotyledonous (strategy I) plant. Pvd addition did not affect significantly (P > 0.05) the plant biomass at harvest (Table 3) and did not induce any visual symptom of toxicity. The concentration of Fe in shoots was over the critical deficiency level reported by Marschner (1995) for higher plants. The synthesis of phytosiderophores by barley roots was thus neglected. Surprisingly, the addition of Pvd did not promote (P > 0.05) the acquisition of Fe by the two plants. This result contrasts with those reported by Shirley et al. (2011) who showed a clear effect of Fe-Pvd supply on the Fe status of both strategy I and strategy II plants. The study of Shirley and co-workers was however performed in liquid medium with Fe-Pvd (100 lM) as single source of iron. Cadmium contents in plant tissues was also not affected (P > 0.05) by Pvd addition (Table 3). The contents in Zn, Ni (Table 3), Mn and Ca (data not shown) were not affected as well (P > 0.05). Conversely, the amount of Cu taken up by the two plants was significantly higher (P < 0.05) when Pvd was supplied (Fig. 3). For barley, it increased from 12.9 lg in the control (3.5 lg for tomato) up to 20.2 lg in the presence of Pvd (4.5 lg for tomato). To our knowledge, this is the first time the direct role of a bacterial siderophore on the phytoextraction of Cu has been underlined in soil. The effect of Pvd was however restricted to the roots. For both plants, root Cu was 2-times higher (P < 0.05) when Pvd was supplied while shoot Cu was not affected (P > 0.05) (Fig. 3). Indeed, plants have evolved mechanisms to limit Cu translocation to the shoots at high exposure levels. Copper transport, chelation, trafficking and sequestration in the root symplasm are highly controlled by homeostatic
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Table 3 Dry weight (DW) and content at harvest in Fe, Cd, Zn and Ni of tomato and barley cropped on the soil supplied (Pvd+) or not (Pvd) with pyoverdine. For each plant and each variable separately, indicates that the value in the presence of Pvd significantly differ (P < 0.05) from the one in the control (absence of Pvd). Roots
Shoots
DW Fe (mg)
Cd Zn Ni DW Fe (lg g1 DW) (mg)
Tomato Pvd 19 Pvd+ 14
1790 169 2117 147
Barley
3725 19 3702 26
Pvd 61 Pvd+ 52
Cd Zn Ni (lg g1 DW)
39 7.6 57 151 44 54 5.2 64 98 39 334 11 137 277 10 145
74 4.5 70 3.0
55 3.2 58 3.3
40 1.3 39 1.3
40
Shoots
Cu content (µg Cu g-1 DW)
20
0 Pvd -
Barley
Tomato
Pvd +
0
100
Roots
200
chelate-assisted phytoextraction like with EDTA, it is widely accepted that metals like Cu are taken up passively under their chelated forms, via the non-selective apoplasmic pathway. The phytoextraction of Cu is thus non-limited by the saturation of transporters like through the symplasm (no Fmax value, see below). Disruption of the Casparian band is however required to achieve the high shoot concentrations needed for phytoextraction. This occurs at the root apex where the Casparian strip is not yet fully developed or might be induced by the addition of EDTA (Nowack et al., 2006). Despite its high molecular weight (4 times higher than EDTA), Pvd is suspected to be internalised by plant roots. Indeed, both strategy I and strategy II plants were found to incorporate Pvd when supplied as Pvd-Fe (Shirley et al., 2011). The uptake of Cu as Pvd-Cu might thus have occurred, presumably via the apoplasm since anionic metal–ligand complexes are supposed non-bioavailable. At the concentration of Pvd used in the bioassay (250 lmol kg1), all dissolved Cu was not assumed to be complexed by Pvd. Indeed, the percentage of Pvd-Cu calculated in DGT bulk solutions was comprised between 25% and 81% (Suppl. data T1). A significant part of Cu was thus presumably taken up via the symplastic pathway through membrane proteins (e.g., from the COPT family) that displayed saturating transport kinetics. The comparison of the maximal uptake flux of Cu by plant roots (Fmax) mentioned in Bravin et al. (2010) for non-hyperaccumulators (0.47 < Fmax < 1.57 nmol Cu m2 s1) with the flux of Cu resupply from the soil measured in Pvd+ (2.53 < FCu < 11.05 nmol Cu m2 s1) suggests that the rate-limiting process of Cu phytoextraction, when Pvd was supplied, was the kinetics of Cu uptake by plant roots. This would explain why Cu phytoextraction was less impacted by Pvd than was Cu phytoavailability assessed by DGT measurements. One way to overcome this issue might be the use of Cu hyperaccumulators whose Fmax value can reach 2.68 nmol Cu m2 s1 according to Bravin et al. (2010). 4. Conclusions
300
* 400
500
* 1
Fig. 3. Copper concentrations (lg g DM) in shoots and roots of barley and tomato plants grown on the calcareous soil supplied (Pvd+) or not (Pvd) with pyoverdine. For each plant separately, * indicates that the value in root Cu or shoot Cu measured in the presence of Pvd significantly differed (P < 0.05) from the one measured in the control (absence of Pvd). The error bars stand for mean standard deviations.
processes based on the production of chelators (e.g., metallothioneins) and chaperones (Clemens, 2001). The reduced transpiration caused by the low shoot biomass also did not favour the translocation of Cu. As compared to EDTA, the effect of Pvd towards the phytoextraction of Cu was weak. Indeed, several studies showed that the supply of EDTA to the soil can increase the phytoextraction of Cu by non-hyperaccumulators to more than a factor 2 (e.g., Luo et al., 2005). In all these studies, the shoot concentration of Cu was affected. One major reason explaining those differences is the dose of chelator supplied to the cropping matrix. Those of EDTA tested in the literature were often much higher than the one of Pvd used in the bioassay. Adding chelators not only increases the total dissolved metal concentration but may also change the primary route of plant metal-uptake (Nowack et al., 2006). Thereby, in
This study shows a good agreement between the stability constants of the metal complexes with Pvd and its effects on Cu mobility, phytoavailability and phytoextraction in a calcareous soil. Thereby, it validates our working hypothesis assuming that the impact of Pvd on the phytoextraction of one metal is based primarily on its ability to complex this metal. This work underlines several characteristics of the effect of Pvd towards metals phytoextraction. First, the effect of Pvd seems fairly specific to Cu. This specificity would help the phytoextraction of Cu above neutral pH, when Ca is likely to compete for binding with Cu (Nowack et al., 2006) and in metal-polycontaminated matrices. It would however restrict the use of Pvd-producing bacteria to the phytoremediation of Cu-contaminated areas. The high pH-dependence of Pvd coordination properties would also limit the use of Pvd-producing bacteria to alkaline matrices. The rate-limiting uptake and root-to-shoot translocation of Cu in tomato and barley suggests the use of Pvdproducing bacteria together with Cu hyperaccumulating plants to maximize Cu phytoextraction and limit Cu leaching to groundwater. This study suggests that Pvd plays a role in the impact of fluorescent Pseudomonas on the phytoextraction of metals but does not prove that this role is prominent. The discrepancy observed between the effect of purified Pvd and fluorescent Pseudomonas towards, notably, the root-to-shoot translocation of metals (Braud et al., 2009b) suggests that other bacterial process than Pvd production may impact metals phytoextraction. Acknowledgments The authors thank Sylvie Bussière, Cécile Fontaine and Françoise Hoegy for their technical assistance. Financial support for this work
J.Y. Cornu et al. / Chemosphere 103 (2014) 212–219
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