Chemical and structural characterization of copper adsorbed on mosses (Bryophyta)

Journal of Hazardous Materials 308 (2016) 343–354

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Chemical and structural characterization of copper adsorbed on mosses (Bryophyta) Aridane G. González a,∗ , Felix Jimenez-Villacorta b , Anna K. Beike c,d , Ralf Reski c,e,f , Paola Adamo g , Oleg S. Pokrovsky a,h,i a

GET (Géosciences Environnement Toulouse) UMR 5563CNRS, 14 Avenue Edouard Belin, F-31400 Toulouse, France Instituto de Ciencia de Materiales Madrid, CSIC, Cantoblanco, E-28049 Madrid, Spain c Plant Biotechnology, Faculty of Biology, University of Freiburg, Schaenzlestrasse 1, 79104 Freiburg, Germany d State Museum of Natural History Stuttgart, Rosenstein 1, 70191 Stuttgart, Germany e BIOSS—Centre for Biological Signalling Studies, 79104 Freiburg, Germany f FRIAS—Freiburg Institute for Advanced Studies, 79104 Freiburg, Germany g Department of Agricultural Sciences, University of Naples Federico II, Via Università 100, 80055 Naples, Italy h BIO-GEO-CLIM Laboratory, Tomsk State University, Tomsk, Russia i Institute of Ecological Problems of the North, Russian Academy of Science, Arkhangelsk, Russia b

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

• Cu2+ was adsorbed on four mosses • • • •

used in moss-bag pollution monitoring technique. Thermodynamic approach was used to model Cu speciation based on XAS results. All studied mosses have ∼4.5 O/N atoms at ∼1.95 Å around Cu likely in a pseudo-square geometry. Cu(II)-carboxylates and Cu(II)phosphoryls are the main moss surface binding groups. Moss growing in batch reactor yielded ∼20% of Cu(I) in the form of Cu–S(CN) complexes.

a r t i c l e

i n f o

Article history: Received 7 September 2015 Received in revised form 21 January 2016 Accepted 24 January 2016 Available online 28 January 2016 Keywords: Copper Adsorption XAS XANES Biomonitoring

a b s t r a c t The adsorption of copper on passive biomonitors (devitalized mosses Hypnum sp., Sphagnum denticulatum, Pseudoscleropodium purum and Brachythecium rutabulum) was studied under different experimental conditions such as a function of pH and Cu concentration in solution. Cu assimilation by living Physcomitrella patents was also investigated. Molecular structure of surface adsorbed and incorporated Cu was studied by X-ray Absorption Spectroscopy (XAS). Devitalized mosses exhibited the universal adsorption pattern of Cu as a function of pH, with a total binding sites number 0.05–0.06 mmolgdry −1 and a maximal adsorption capacity of 0.93–1.25 mmolgdry −1 for these devitalized species. The Extended X-ray Absorption Fine Structure (EXAFS) fit of the first neighbor demonstrated that for all studied mosses there are ∼4.5 O/N atoms around Cu at ∼1.95 Å likely in a pseudo-square geometry. The X-ray Absorption Near Edge Structure (XANES) analysis demonstrated that Cu(II)-cellulose (representing carboxylate groups) and Cu(II)-phosphate are the main moss surface binding moieties, and the percentage of these

∗ Corresponding author. E-mail address: [email protected] (A.G. González). http://dx.doi.org/10.1016/j.jhazmat.2016.01.060 0304-3894/© 2016 Elsevier B.V. All rights reserved.

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sites varies as a function of solution pH. P. patens exposed during one month to Cu2+ yielded ∼20% of Cu(I) in the form of Cu–S(CN) complexes, suggesting metabolically-controlled reduction of adsorbed and assimilated Cu2+ . © 2016 Elsevier B.V. All rights reserved.

1. Introduction The study and monitoring of toxic metals in the atmosphere is important due to their impact on the human health and ecosystems. There are many biomaterials that are commonly used for assessing atmospheric deposition of metals and metalloids in rural, urban and industrial areas, but peat moss has been highlighted as one of the most convenient for low costs and high adsorption capacity [1,2]. Naturally-occurring and transplanted mosses have been widely used for pollutant biomonitoring [2–6]. Recently, a standardized protocol, including oven-drying to devitalize the moss, has been proposed to optimize the moss transplant or moss-bag technique and its regular use for environmental monitoring by official institutions [7,8]. However, despite the wide use of mosses as biomonitors, the chemical and structural status of metals adsorbed to the moss surface and incorporated inside the biomass remain poorly understood. In order to enhance the bioaccumulation efficiency of transplanted bryophytes and to assess the contribution of active and passive mechanisms to metal pollutant uptake, different physico-chemical pre-treatments (water-washing, oven drying, acid washing) are applied to mosses before their use as biomonitors [8,9]. The oven-dried moss can be considered the most suitable biomaterial for monitoring trace element deposition from the atmosphere because this treatment does not appear to remarkably change either the chemical composition or the surface morphology of mosses [10,11]. The present work aims to characterize the chemical and structural status of Cu, both an important pollutant and an essential micronutrient, adsorbed on four devitalized mosses (Hypnum sp., Sphagnum denticulatum, Pseudoscleropodium purum and Brachythecium rutabulum) and assimilated by live Physcomitrella patents cultured under laboratory conditions and exposed to Cu during one month. These species are selected in the context of the FP7 European project “MossClone”, aimed at identifying moss species suitable for in vitro-production and moss-bag biomonitorization (http://www.mossclone.eu/). To this end, we combined macroscopic adsorption experiments in a wide range of pH and aqueous copper concentrations with in-situ spectroscopic analyses of adsorbed and assimilated Cu. Specific tasks of the study were to: (i) quantify the adsorption parameters of Cu2+ on naturaldevitalized mosses and develop a surface complexation model thus contributing to the data-base of metal-biosorbents interaction reactions; (ii) suggest the most suitable moss species in order to be used in biomonitoring and (iii) to reveal the chemical identity of surface complexes using X-ray Absorption Spectroscopy (XAS) to characterize the stoichiometry and the structural parameters of Cu binding sites on mosses.

2. Material and methods 2.1. Mosses collection Samples of Hypnum sp., S. denticulatum, P. purum and B. rutabulum moss species were collected in June 2012 in NW Spain (Galicia) in non-urban (rustic) areas of Galicia (Spain) that are far away of any source of pollution [12]. These samples were collected by the group

of Ecotoxicology and Ecophysiology of Vegetal from Universidad de Santiago de Compostela (Spain). The collection was close to 10 g in several sub-samples (20–30) collected from a zone of 30 × 30 cm. Mosses were rinsed three times with Milli-Q water (18 M) and devitalized at 120 ◦ C overnight following the commonly used mossbag protocol [13]. The oven treatment allows the devitalization of the biomass thus eliminating the metabolic influence on the adsorption or uptake of metals [14–16]. In order to approach the moss bag procedure envisaged in the environmental exposure conditions, intact whole mosses without grinding or disaggregation were used. The biomass concentration in the experiments was kept constant at 1 gdry L−1 . 2.2. Adsorption of Cu2+ on mosses The experiments were carried out in 0.01 M NaNO3 electrolyte solutions. Cu(NO3 )2 (Sigma–Aldrich) was used as a source of Cu2+ and it was prepared in Milli-Q water (18 M). The constant pH experiments were buffered with 2.5 mM MES (Merck) at pH 5.5. The pH was measured by a combined electrode (MettlerToledoR ) with a pH-meter analyzer (pHM250-MeterlabTM ) with an uncertainty of ±0.002 units using NIST buffers. Quantitative physico-chemical characterization of copper binding by mosses was studied in adsorption experiments, performed as a function of pH and aqueous metal concentration (adsorption isotherm) following previously elaborated experimental procedures [17]. The aqueous solution was undersaturated with respect to the metal oxide or carbonate as was verified by speciation calculations with the Visual Minteq computer code [18]. The experiments were conducted in polypropylene beakers at room temperature (20 ± 1 ◦ C) and agitated with a suspended Teflon coated magnetic stirrer under continuous nitrogen bubbling. For the pH-edge experiments, the initial Cu2+ concentration was always kept constant at 55 ␮M for a pH range between 1.4 and 6.6 (Table 1). The pH was varied by adding aliquots of NaOH (0.1–0.01 M) or HNO3 (0.1–0.01 M). The adsorption experiments as a function of Cu2+ concentration in solution were carried out from 0.8 to 365 ␮M, at constant pH (5.4 ± 0.2) in the presence of 2.5 mM MES buffer solution. The typical duration of adsorption experiments was 1 h, sufficient to establish reversible adsorption equilibrium [17]. At the end of adsorption, solution in contact with moss was filtered (0.45 ␮m acetate cellulose). Copper concentration was measured in this filtered and acidified (bidistilled HNO3 ) solution using flame atomic adsorption spectroscopy (PerkinElmer AAnalyst 400) with an uncertainty of ±2% and a detection limit of 0.70 ␮mol L−1 . Dissolved Organic Carbon (DOC) was monitored as a function of pH using a Carbon Total Analyzer (Shimadzu TOCVCSN ) with an uncertainty of 2% and a detection limit of 0.1 mg L−1 . The DOC provides the information on the degradability of the samples, and allows evaluating the degree of metal complexation with organic ligands in solution [19]. Two experiments with devitalized S. denticulatum were performed during 1 month of Cu exposure at constant pH in order to assess the effect of long-term assimilation process. Physcomitrella patens was cultured for four weeks in batch reactor (Erlenmeyer flasks) with 180 mL sterile liquid Knop medium [20–22], enriched

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Table 1 List of adsorption experiments and LPM parameters. All metal adsorption experiments were performed in 0.01 M NaNO3 with a constant biomass 1 gdry L−1 . Ks and Km correspond with the equilibrium constant for the reaction between metal in solution and the available sites as a function of pH (pH-edge) and metal aqueous concentration in solution (constant pH-isotherm), respectively. pKs /pKm

Binding sites(mmol gdry −1 )

55

−2.15 −0.75 0.20

0.03 0.01 0.01

1.61–6.50

55

−2.15 −1.20 0.05

0.01 0.01 0.03

pH-edge

1.42–6.38

55

−2.55 −1.20

0.002 0.05

Brachythecium rutabulum

pH-edge

1.54–6.63

55

−3.30 −1.85

0.002 0.05

Hypnum sp.

Constant pH-isotherm

5.4 ± 0.2

0.8–365

1.65 3.75

0.13 4.42

Sphagnum sp.

Constant pH-isotherm

5.4 ± 0.1

0.8–362

1.15 2.85

0.18 0.94

Pseudoscleropodium purum

Constant pH-isotherm

5.4 ± 0.2

0.8–346

2.45

0.99

Brachythecium rutabulum

Constant pH-isotherm

5.4 ± 0.2

0.8–344

1.15 2.45

0.10 1.78

Moss pecies

Type of experiment

pH range

[Cu]aq

Hypnum sp.

pH-edge

1.51–1.64

Sphagnum denticulatum

pH-edge

Pseudoscleropodium purum

in CuSO4 by 10-fold to 0.5 ␮M and by 50-fold to 2.5 ␮M, and maintained at pH 5.8 by KOH addition. The samples enriched by x10 and x50 in Cu relative to usual liquid Knop medium (0.05 ␮M) are abbreviated as P. patens-10 and P. patens-50, respectively. Protonema (the juvenile stage) of P. patens was transferred to the flasks at a concentration of 100 mg L−1 dry weight. The cultures were grown at a light intensity of 70 ␮mol m−2 s−1 (Philips TLD 36 W/33-640) and a photoperiod of 16 h light to 8 h dark at 23 ◦ C. The flasks were shaken continuously on a shaker at 120 rpm. The experimental results were treated with the Linear Programming Model (LPM) in order to compute the apparent equilibrium constants and the site densities for each individual experiment following the approaches elaborated for other biosorbents [23–26]. This model is convenient for describing complex 3-D multi-layer systems having both organic components and rigid cell walls [27,28]. A full description of this model, previously applied to various natural mosses, is available elsewhere [17].

(␮mol L−1 )

were recorded up to a maximum k-value of 14 Å−1 , and the window selected for EXAFS analysis ranged from ∼2.5 Å−1 to ∼11 Å−1 . Multiple scans of 45 min/scan acquisition time were recorded at different spots of the sample to further avoid beam induced reactions. EXAFS data treatment and analysis were performed using the ATHENA and VIPER software, respectively [30,31], following the recommendations of the International X-ray Absorption Society (IXAS, http:// www.ixasportal.net/ixas/). In addition, linear combination fit (LCF) of XANES spectra of all microorganism samples were performed using ATHENA software [30,31], with an average error on the component proportion estimation within ±10%. Fits were performed within an energy range from −20 eV to +100 eV, with respect to the Cu K-edge energy.

3. Results 3.1. Adsorption of copper on devitalized mosses

2.3. Synchrotron XAS analysis X-ray absorption spectroscopy provides quantitative and qualitative information on local environment of Cu in terms of its oxidation state, identity and distance of first and second atomic neighbors [29]. As for any biological matrix, the resolution of integral XAS signal of Cu associated with mosses is within ±10%. XAS spectra, including X-ray Absorption Near Edge Structure (XANES) and Extended X-ray Absorption Fine Structure (EXAFS), of Cu-bearing standard compounds, and freeze dried moss biomass were collected in transmission or fluorescence mode, depending on Cu concentration, at the Cu K-edge over the energy range 8.9–9.1 keV on BM25A-SPLINE beamline at the European Synchrotron Radiation Facility. The energy was selected using an Si(111) double-crystal monochromator constantly calibrated using a 5-␮m-thick copper metal foil; the energy of the first inflection point in the XANES spectrum was set to 8979 eV. The spectra of the mosses were collected in fluorescence mode with a 13-element Si(Li) detector (e2v Instruments). Several samples were tested with and without liquid He (10 K) cryostat and no detectable degradation or variation of the spectra was observed. Reference Cu(I) and Cu(II) organic and inorganic compounds were recorded during the same XAS session to be used as standards of the local atomic environment of Cu in moss samples. Scans

The adsorption of Cu2+ was studied on four devitalized mosses (Hypnum sp., S. denticulatum, P. purum and B. rutabulum) as a function of pH (1.4–6.6). The adsorption of Cu2+ on the studied mosses as a function of pH followed the universal adsorption pH-edge established for various biological surfaces [17,24,32–34], where increasing the solution pH led to deprotonation of the available exchangeable sites on moss surfaces, which thus increases the adsorption capacity with the pH rise (Fig. 1A). In the current experiments, the adsorption of Cu2+ started at pH close to 2 and the maximum adsorption was achieved at pH 5.5–6.0 for all the moss species. An integral parameter pH50 (corresponding to pH at which 50% of initial Cu2+ was adsorbed) was equal to 2.9, 3.0, 3.3 and 4.1 for B. rutabulum, Hypnum sp., P. purum, and S. denticulatum, respectively. The experimental results were fitted with the LPM in order to compute the equilibrium constants (pKs ) and the concentration of binding sites available to complex Cu2+ on the moss surfaces. The concentration of these sites was similar for these four devitalized moss species, ranging from 0.002 to 0.05 mmol gdry −1 (Table 1). However, the pKs , that reflect the strength of the complex between copper ion and functional groups, was the most negative for B. rutabulum (pKs = −3.30), compared to that of the other moss species in this study. According to the binding site numbers and pKs values,

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the adsorption capacity of mosses with respect to Cu2+ follows the order: B. rutabulum ≥ P. purum ≥ S. denticulatum ≥ Hypnum sp. The concentration of DOC released from mosses during Cu2+ adsorption as a function of pH is illustrated in Fig. 1B. We can rank four studied moss species in two different groups, according to the concentration of DOC in solution. P. purum and B. rutabulum yielded significantly higher [DOC] (∼40 mg L−1 ) compared to S. denticulatum and Hypnum sp. (∼15 mg L−1 ) without clear dependence on the pH. The adsorption of Cu2+ on devitalized mosses (Hypnum sp., S. denticulatum, P. purum and B. rutabulum) was also studied as a function of the concentration of Cu2+ in solution (0.8–365 ␮mol L−1 ) at constant pH of 5.5 as illustrated in Fig. 1C. The adsorption capacity of Cu2+ decreased with the increase of the total copper concentration in solution. Parameters of the LPM fit to the experimental data are listed in Table 1. The model provided the equilibrium constant (pKm ) and the concentration of binding sites as a function of Cu2+ concentration in solution. The total concentration of surface sites ranged from 0.10 to 4.42 mmol gdry −1 . The constant-pH adsorption data were also treated by applying a Langmuirian adsorption equation, assuming one-site reaction. The maximum adsorption capacity (qmax ) values were rather similar among 4 studied mosses and equal to 0.93, 0.99, 1.04, 1.25 mmol gdry −1 for Hypnum sp., P. purum, B. rutabulum, and S. denticulatum, respectively.

3.2. XANES analysis of adsorbed and assimilated Cu

Fig. 1. (A) Cu2+ adsorbed (%) onto moss surface as a function of pH, in 0.01 M NaNO3 and 1.0 gdry L−1 biomass. Experimental conditions are listed in Table 1. (B) Dissolved Organic Carbon measured during metal adsorption experiments as a function of pH, in 0.01 M NaNO3 and 1.0 gdry L−1 biomass. (C) Cu2+ adsorbed onto moss surface as a function of Cu2+ concentration in solution (Langmuirian-isotherm), in 0.01 M NaNO3 and 1.0 gdry L−1 biomass at constant pH (5.4 ± 0.2). Experimental conditions are listed in Table 1. Lines represent the Linear Programming Model (LPM) results. Squares represent the samples used for XAS studies.

The samples selected for the XAS analyses are highlighted by squares in Fig. 1, and listed in Table 2 and Table 3. The spectra collected for all the mosses reveal the presence of Cu(II) compounds, consistent with the spectral shape and edge energy position at about 8985 eV. We used standard Cu-compounds representative for the possible functional groups determined by acid–base titrations on the same devitalized moss species [17]. Within the resolution of XANES part of the spectra (±10%), no Cu(I) was detected in all adsorption samples (Figs. 3A, D; 4A, D and Table 3). The contribution of the Cu-reference samples was estimated from Linear Combination Fits (LCF analysis). For this treatment, the Cu(II)-cellulose and Cu(II)-alginate (as a Cu(II)carboxylate group), Cu(II)-phosphate (as a Cu(II)-phosphoryl), Cu2 O, Cu(I)-thiocyanate (as a Cu-thiol) and metal Cu0 were used as reference compounds. The XANES spectra of the references are shown in Fig. 2. The best corresponding standards for moss spectra were chosen after using automated combinatorial fitting implemented in the Athena software from statistical analysis of all possible combinations of two, three or four standards [31]. It was found that four standard compounds were sufficient to reproduce the majority of the experimental spectra. Based on the XANES part of the spectra (Figs. 3A, D; 4A, D and 5A and Table 2), the LCF results demonstrated two marked trends in the chemical composition during the adsorption of Cu2+ on devitalized mosses. When the adsorption of Cu2+ occurred in acidic solution (pH ≤ 3), Cu(II)-cellulose complex dominated the speciation of copper, constituting 97 ± 1% (Hypnum sp.), 87 ± 13% (S. denticulatum), 96 ± 1% (P. purum) and 80 ± 7% (B. rutabulum) of total metal. When the pH increased to pH ≥ 5, Cu2+ become mostly bound to phosphoryl groups (Cu(II)-phosphate) at the expense of Cu(II)-cellulose complexes: Hypnum sp. = 44 to 68%, S. denticulatum = 25–73%, P. purum = 53–71% and B. rutabulum = 49–69%. The presence of metal Cu0 was not excluded within the 10% accuracy of the XAS measurements. It could originate from initially-present Cu0 in devitalized samples, as it is observed in native B. rutabulum which was not subject to Cu2+ exposition in the experiment (Table 2). In this moss sample, the Cu-metal signal contribution was as high as ∼15 ± 5%.

Table 2 Results of XANES modeling of adsorbed Cu spectra with the contribution (in %) of various Cu references necessary to achieve the best fitting. Long-term exposition experiments were carried out for fresh-alive P. patens and devitalized S. denticulatum for 1 month. B. rutabulum was also measured as devitalized moss, unexposed to copper*. Sample

[Cu]ads ␮mol grdry −1

pH

Cu(II)-cellulose

51.61 0.54 2695.99

3.46 0.28 765.85

1.73 5.46 5.01

97 ± 1% 48 ± 5% 19 ± 4%

54.29 41.38 14.00 3381.75 8.03 0.39 2775.77

0.79 13.69 41.07 80.09 0.16 0.42 686.07

1.76 3.04 5.48 1.49 1.61 5.43 5.13

92 ± 5% 95 ± 4% 75 ± 5% 73 ± 9% 100% 52 ± 3% 12 ± 5%

3159.09 277.97 2854.29

302.75 340.49 607.55

1.42 5.49 5.03

96 ± 1% 42 ± 5% 15 ± 5%

– 3420.46 0.52 2744.90

– 41.38 0.30 691.80

5 ± 1a 1.55 5.53 5.06

85 ± 5% 80 ± 7% 43 ± 4% 17 ± 4%

4.14 2639.06 196.64 983.1

43.82 541.20 0.36 0.90

5.16 4.50 – –

89 56 30 29

± ± ± ±

2% 4% 2% 1%

Cu(II)-alginate

Cu(II)-phosphate

Cu (metal)

2

R-factor (10−3 )

3 ± 1% 44 ± 5% 68 ± 4%

8 ± 5% 13 ± 4%

0.006 0.038 0.024

0.21 1.48 0.96 0.98 0.55 1.01 1.02 5.17 1.04 3.06

8 ± 5%

42 ± 3% 73 ± 5%

6 ± 3% 15 ± 5%

0.024 0.015 0.052 0.025 0.140 0.027 0.074

53 ± 5% 71 ± 4%

4 ± 1% 5 ± 5% 14 ± 5%

0.039 0.038 0.028

1.54 1.41 1.12

15 ± 5%

0.226 0.037 0.033 0.026

8.94 1.43 1.24 1.04

0.005 0.025 0.026 0.041

0.19 0.97 0.45 1.65

5 ± 4% 25 ± 5% 13 ± 9%

14 ± 9%

20 ± 7%

51 ± 2% 46 ± 1%

49 ± 4% 69 ± 4%

8 ± 4% 14 ± 4%

6 ± 2% 36 ± 4% 19 ± 2%b Cu(I)-SCN(-thiocyanate) 25 ± 1%b Cu(I)-SCN(-thiocyanate)

5 ± 2% 8 ± 4% 24 ± 1%

A.G. González et al. / Journal of Hazardous Materials 308 (2016) 343–354

Hypnum sp. HE-1 HE-7 HE-12 Sphagnum denticulatum SE-1 SE-2 SE-3 SE-5 SE-6 SE-7 SE-12 Pseudoscleropodium purum PE-5 PE-7 PE-12 Brachythecium rutabulum a B. rutabulum (natural) BE-5 BE-7 BE-12 Long-term exposition Sphagnum-3 (devitalized) Sphagnum-19 (devitalized) Physcomitrella patens-10 Physcomitrella patens −50

[Cu]sol ␮mol L−1

a

pH for natural samples is assumed to be equal to typical pH of liquid aerosol deposits. b No phosphate ligand is identified.

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Table 3 Structural parameters of the first atomic atom shell of copper obtained from fitting Cu K-edge EXAFS. Long-term exposition experiments were carried out for fresh-alive P. patens and devitalized S. denticulatum for 1 month. Sample

[Cu]sol ␮mol L−1

[Cu]ads ␮mol grdry −1

pH

Cu-ligand

R (Å)

N (ligand)

␴2 (Å−2 )

Cu(II)-phosphate Cu(II)-alginate Cu(II)-cellulose B. rutabulum (natural) Hypnum sp. HE-1 HE-7 HE-12 Sphagnum denticulatum SE-1 SE-2 SE-3 SE-5 SE-6 SE-7 SE-12 Pseudoscleropodium purum PE-5 PE-7 PE-12 Brachythecium rutabulum BE-5 BE-7 BE-12 Long-term exposition Sphagnum-3 Sphagnum-19 Physcomitrella patens-10 Physcomitrella patens −50

– – – –

– – – –

– – – 5 ± 1a

Cu–O Cu–O Cu–O Cu–O

Cu–Cu

1.94 1.96 1.93 1.99

2.42

5.0 4.6 4.9 4.3 1.0

0.0050 0.0065 0.0050 0.0056

0.0062

51.61 0.54 2695.99

3.46 0.28 765.85

1.73 5.46 5.01

Cu–O Cu–O Cu–O

Cu–Cu Cu–Cu

1.95 1.93 1.93

2.42 2.49

5.0 3.6 0.2 3.6 1.1

0.0063 0.0044 0.0010

0.0120 0.0125

54.29 41.38 14.00 3381.75 8.03 0.39 2775.77

0.79 13.69 41.07 80.09 0.16 0.42 686.07

1.76 3.04 5.48 1.49 1.61 5.43 5.13

Cu–O Cu–O Cu–O Cu–O Cu–O Cu–O Cu–O

2.56

4.4 4.4 4.6 5.0 0.8 4.0 4.6 3.6 1.0

0.0050 0.0042 0.0050 0.0083 0.0025 0.0046 0.0101

3159.09 277.97 2854.29

302.75 340.49 607.55

1.42 5.49 5.03

0.0097 0.0092 0.0065

0.0120

3420.46 0.52 2744.90

41.38 0.30 691.80

0.0083 0.0071

4.14 2639.06 196.64 983.1

43.82 541.20 0.36 0.90

a

Cu–Cu

1.97 1.94 1.93 1.98 1.96 1.94 1.93

Cu–O Cu–O Cu–O

Cu–Cu

1.98 1.93 1.93

2.49

3.9 5.0 4.0 0.7

1.55 5.53 5.06

Cu–O Cu–O Cu–O

Cu–Cu Cu–Cu

1.98 1.95 1.93

2.41 2.44

5.3 5.1 1.1 3.6 0.6

0.0085 0.0101 0.0075

5.16 4.50 – –

Cu–O Cu–O Cu–O Cu–S Cu–OCu–SCu–Cu

4.9 4.6 2.6 2.4 3.6 1.3 1.1

0.0059 0.0073 0.0022 0.0022 0.00450.00450.0162

Cu–Cu

2.43

1.94 1.95 1.94 2.09 1.942.082.43

0.0131

0.0011

pH for natural samples is assumed to be equal to typical pH of liquid aerosol deposits.

Long-term interaction of Cu2+ with live P. patens grown produced non-negligible amounts of Cu(I) which was not detected on devitalized S. denticulum moss interacting with Cu over long period. This result is remarkable and unambiguously demonstrates that living moss partially reduces Cu(II) to Cu(I). The latter is mainly present as Cu(I)-S (presumably thiol groups), constituting 19–25% of the total bonded groups. The rest of the Cu was in oxidized form and complexed with cellulose (29–30%) and alginate (46–51%). In contrast, long-term Cu interaction with devitalized S. denticulatum demonstrated the same speciation as that of short-time exposition experiments, where the main group was Cu(II)-cellulose (56–89%) and Cu(II)-phosphate (6–36%). 3.3. EXAFS analysis on mosses Modeling of EXAFS spectra of copper and other metals in natural organic and biologic matrixes is complicated by (i) distorted geometries and the presence of different neighbors (O/N/S) in the nearest metal coordination shell, (ii) predominance in the nextnearest coordination shells of light atoms (C/O/N) exhibiting weak and similar EXAFS signals, and (iii) numerous multiple scattering paths in metal–organic ligand complexes. Since the XANES spectra of the specimens were successfully fitted by the selected reference spectra, most of them having Cu O bonds, it can be suggested that mosses present a first coordination shell dominated by oxygen as ligands to the Cu2+ ions. The EXAFS results (Figs. 3, 4 and and Table 3) demonstrated that all analyzed mosses species have between 4 and 5 O atoms around Cu(II) at a distance between 1.93 and 1.98 Å in the first coordination shell, associated to tetrahedral, planar or square pyramidal geometries. It is important to note that there was no reduction of Cu(II) to Cu(I) during the adsorption onto devitalized mosses, as also demonstrated by XANES results. In the case of long-term exposition to Cu2+ , the EXAFS showed detectable differences between devitalized S. denticulatum and live P. patens. The interatomic distances for Cu assimilated by live moss were

equal to 2.09 ± 0.01 Å for 3.1 ± 0.5 of O atoms, compatible with Cu(I) compounds. For devitalized mosses, the time of exposure did not influence the structure of the first shell, which always comprised 4.8 ± 0.2 of O atoms at 1.95 ± 0.01 Å. We could not find evidence of clear contributions from atoms like C or P expected in the next-nearest shells of Cu(II) in carboxyl (cellulose) or phosphoryl complexes typical of bacterial surface binding of most metals [35,36]. Most of the samples exhibited a strong and broad intensity peak at ∼2 Å, similar to that in metal Cu which probably comes from the metal particles that were accumulated or produced during moss growth in the natural environment (see discussion below). Such a peak was clearly visible for native devitalized B. rutabulum sample, not subjected to Cu2+ treatment (Tables 2–3 and 4F). 4. Discussion 4.1. Adsorption of Cu2+ as a function of pH and Cu concentration Copper, like other transition metals, can be bound to the surface layer of cell walls through cation exchange. The solution pH is one of the most important parameters affecting the biosorption of metal ions [37]: increasing the pH in solution leads to the deprotonation of the available exchangeable sites on moss surfaces thus increasing the adsorption capacity. According to the pH-edge adsorption results, all the studied mosses followed the “universal pH-adsorption edge” [17]. This behavior has been also demonstrated for bacteria consortia and individual bacteria species [24–26,32–34,36,38]. The similarity between different biosorbents stems from the dominance of the same main metal-binding surface moieties, typically, carboxylates. The DOC concentration released by mosses was higher for P. purum and B. rutabulum compared to S. denticulatum and Hypnum sp. Considering its adsorption capacity and the low DOC leached from the moss surface, S. denticulatum seems to be the most suitable candidate for biomonitoring.

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Fig. 2. (A) Cu K-edge XANES spectra of selected references at the Cu K-edge energies. (B) Cu K-edge oscillations and the fits of the Cu K-edge XANES spectra. (C) Fourier transform magnitudes of the Cu-references samples used in this study and the fits of the Cu K-edge spectra.

Results of Cu2+ adsorption as a function of aqueous copper concentration are in good agreement with previous data on biological surfaces reporting the range of sites between 0.104 and 1.22 mmol gdry −1 (see compilation in González and Pokrovsky [17]). Likewise, the adsorption capacities computed from Langmuirian isotherm in this work (0.93–1.25 mmol gdry −1 ) were in concordance with previous work on devitalized mosses (qm = 0.78–1.36 mmol gdry −1 [17]. At the same time, these values are higher than those for Danish and Heilongjiang peat (0.54 and 0.40 mmol gdry −1 , respectively [39]), aquatic plants Myriophyllum spicatum (0.16 mmol gdry −1 ), Potamogeton lucensi (0.64 mmol gdry −1 ), Salvinia herzogii (0.31 mmol gdry −1 [40]), herbaceous peat (0.076–0.19 mmol gdry −1 [41]), or Sphagnum peat (0.31 mmol gdry −1 [42]). The elevated mass-normalized adsorption on mosses relative to peat and plants may be due to specific sur-

face area of bryophytes exhibiting highly porous structure with hyaloclasts (i.e., Refs. [13], [14], [19]) 4.2. Chemical structure characterization of mosses under Cu2+ exposition The adsorption of Cu2+ on the surface of biosorbents is controlled by the local ligand environments, Cu coordination and its oxidation state [43]. Although EXAFS analysis in this study could not discriminate between O and N atoms, based on the good match between the samples and reference compounds with O as first-shell neighbor we infer that oxygen is the only first neighbor of copper for devitalized mosses, whereas for living P. patens, exhibits likely contribution from S atoms with Cu(I) such as thiocyanate. XANES spectra of all devitalized mosses showed the pre-edge at

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Fig. 3. (A) Cu K-edge XANES spectra at the Cu K-edge energies for the adsorption of Cu2+ on Hypnum sp. (B) Cu K-edge oscillations for the adsorption of Cu2+ on Hypnum sp., and the fits of the Cu-references samples used in this study. (C) Fourier transform magnitudes of the Hypnum sp. samples and the fits of Cu-references samples used in this study and the fits of the Cu K-edge spectra. (D) Cu K-edge XANES spectra at the Cu K-edge energies for the adsorption of Cu2+ on S. denticulatum. (E) Cu K-edge oscillations for the adsorption of Cu2+ on S. denticulatum, and the fits of the Cu-references samples used in this study. (F) Fourier transformed magnitudes of the S. denticulatum samples and the fits of Cu-references samples used in this study and the fits of the Cu K-edge spectra.

8976 eV and the edge at 8994 eV, linked to the 1s →3d transition. This energy shift is also evidenced for Cu(II) oxide and hydroxide, Cu(II)-phosphate, and organic compounds like Cu(II)-cellulose (Fig. 2). The EXAFS results demonstrate that only Cu(II) was present on the spectra of devitalized mosses used for adsorption experiments which allows to conclude that devitalized mosses do not reduce Cu2+ during its adsorption on the cell surfaces. Therefore, devitalized mosses should act as passive bio-accumulators that adsorb metals without altering their oxidation state. Moreover, it is likely that Cu2+ had only O atoms in its first-shell. Specifically, for all the devitalized mosses we identified ∼4.5 O/N atoms at ∼1.95 Å likely in a pseudo-square geometry. These parameters are in a good agreement with Cu2+ -(O/N)4 planar coordination and corresponding Cu-ligand distances. The same coordination was reported for Cu(II)-bearing solids, organic complexes, Cu adsorbed onto mineral surfaces and in aqueous solutions [29,44–47]. In these studies, chemical and thermodynamic arguments suggested that carboxylate and phosphorylate groups dominate Cu binding to organic

surfaces, as followed from surface complexation modeling based on macroscopic adsorption experiments [17,24,25,36]. This also agrees with general knowledge that metal ion sorption by plants is controlled by carboxylate groups [48–51]. In this study, the adsorption mainly occurred on surface carboxylates at pH 1.4 to 3.0. At circum-neutral pH (5.0–5.5), phosphoryl groups become important, since >R-PO4 H◦ at the moss surface deprotonates in more alkaline solutions compared to the carboxylates [17]. 4.3. Cu assimilated by mosses Cu interaction with living P. patens was dramatically different from that with devitalized moss samples. The same Cu(II) → Cu(I) reduction and Cu(I) complexation with sulfhydryl surface sites could occur without inducing toxicity defense mechanisms, but following the metabolic requirements. Cu reduction upon its interaction with living organisms has also been demonstrated for higher plants, like Larrea tridentate [50] and phototrophic and

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Fig. 4. (A) Cu K-edge XANES spectra at the Cu K-edge energies for the adsorption of Cu2+ on P. purum. (B) Cu K-edge oscillations for the adsorption of Cu2+ on P. purum, and the fits of the Cu-references samples used in this study. (C) Fourier transform magnitudes of the P. purum samples and the fits of Cu-references samples used in this study and the fits of the Cu K-edge spectra. (D) Cu K-edge XANES spectra at the Cu K-edge energies for the adsorption of Cu2+ on B. rutabulum. (E) Cu K-edge oscillations for the adsorption of Cu2+ on B. rutabulum, and the fits of the Cu-references samples used in this study. (F) Fourier transform magnitudes of the B. rutabulum samples and the fits of Cu-references samples used in this study and the fits of the Cu K-edge spectra.

heterotrophic bacteria [29]. The latter reduced Cu(II) to Cu(I) both at the surface and inside the cells, probably as a detoxification response. A similar mechanism allows plants to tolerate heavy metals via producing Cu-complexing proteins [52] and phytochelatins [53]. Note that, in contrast to Cu(II), Cu(I) complexes are strictly linked to sulfydryl groups [54], as also evidenced by spectra fitting in this study: Cu-SCN yielded the best fit of XANES part of the spectra (Fig. 5). Including Cu2 O compound did not produce any detectable improvement of the spectra fit for Cuassimilated samples. This strongly suggests a general mechanism of Cu2+ interaction with moss surfaces: passive physico-chemical complexation with carboxylates and phosphorylates and active Cu(II)-carboxylate/phosphorylate→Cu(I)-sulfhydryl reduction at the live cell surfaces and inside the cells. The LCF treatment of XANES spectra of all mosses with adsorbed Cu yielded persistent amounts of metal Cu0 (from 5 to 15% with a typical uncertainty of ±5%) regardless of the moss species identity, pH and Cu2+ loading. We verified that this is not an artifact of measurements, spectra collection (i.e., influence of sample holder) or the spectra treatment. In addition, Cu0 contribution was not

increasing with scan number of the same spot, reflecting that Cu0 is not a result of radiation induced reduction (RIR) under the beam. Removal of Cu0 from the list of possible compounds significantly worsened the quality of the spectra fit. We interpret this small, but measurable amount of Cu0 (10 ± 5%) as the native Cu present in mosses prior the experiments. This metal was either metabolically produced during moss life time, or agglutinated (physically assimilated) due to metal atmospheric deposition. The first possibility is more likely as P. patens grown in sterile Cu-bearing medium and not subjected to atmospheric contact also exhibited some metal Cu (Table 2, sample P. patens-10 and 50). Several authors reported Cu0 in natural compounds using XAS analyses. Observations on wetland plants demonstrated that Cu can be transformed into metallic nanoparticles near plant roots as part of a defense mechanism against metal toxicity [47]. The formation of elemental metals has been reported for plants [55], fungi [56], bacteria [57] and algae [58]. Moreover, formation of Cu0 colloids occurred in soils upon changing conditions from oxic to anoxic [59], by the redox-reactive moieties of the organic matter [60].

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Fig. 5. (A) Cu K-edge XANES spectra at the Cu K-edge energies for the long-term exposition of Cu2+ on alive P. patens and devitalized S. denticulatum. (B) Cu K-edge oscillations and the fits of the Cu-references samples used in this study. (C) Fourier transformed magnitudes and the fits of Cu-references samples used in this study and the fits of the Cu K-edge spectra.

5. Conclusions Via a combination of macroscopic adsorption experiments and high-resolution structural analyses, the present study provides important thermodynamic and structural constrains of using devitalized mosses as biomonitors of atmospheric metal pollution. S. denticulatum is the most suitable for biomonitoring as it presented high Cu adsorption capacity being most inert with respect to DOC release to the solution. Physico-chemical experimental approach and linear combination analysis of XAS spectra demonstrated the dominance of carboxylate and phosphorylate group controlling Cu2+ adsorption on moss surfaces. The percentage of bound cop-

per was directly related to the solution pH, and the importance of carboxylate binding relative to phosphorylates decreased with the increase of solution pH from highly acidic to circum-neutral. The 1st shell chemical structure of Cu(II) adsorbed on devitalized mosses remained highly stable over a wide range of pH, duration of exposure and metal loading and comprised ∼4.5 O/N atoms at ∼1.95 Å likely in a planar or square pyramidal geometry. Long-term Cu(II) exposure of live moss P. patens yielded ∼20% of Cu in the form of Cu(I)-S protein bound complexes, presumably reflecting the metabolic responses. Small but detectable amount of native Cu0 (10 ± 5%) assimilated by live moss may reflect a defense mechanism against Cu toxicity.

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Acknowledgements This study was supported by the MOSSclone project by the European Union in the Seventh Framework Program (FP7ENV.2011.3.1.9-1) for Research and Technological Development as well as by BIO-GEO-CLIM grant No 14.B25.31.0001 and RSF grant no. 15-17-10009 (30%). We also thank the Spline staff for the scientific support during the experiments in the ESRF. We would like to express thanks to Katrin Meier for the English proofreading of the manuscript.

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