New Insights into the Role of Chemical Components ...

Water Air Soil Pollut (2015) 225:2006 DOI 10.1007/s11270-014-2006-x

New Insights into the Role of Chemical Components on Metal Ions Sorption by Grape Stalks Waste C. Liu & D. Pujol & N. Fiol & M. À. Olivella & F. de la Torre & J. Poch & I. Villaescusa

Received: 26 March 2014 / Accepted: 14 May 2014 # Springer International Publishing Switzerland 2015

Abstract In this work, metal sorption onto grape stalks waste structural compounds and extractives has been studied for determining their role in Cr(VI), Cu(II) and Ni(II) metal sorption. For this purpose, a sequential extraction of extractives and other compounds from the lignocellulosic material has been carried out. The resulting solid samples obtained in the different extraction processes were used as sorbents of Cr(VI), Cu(II) and Ni(II). Sorption results were discussed taking into account the elemental composition and polarity of the solid extracts. Results indicated that tannins and polyphenols are involved in chromium reduction and sorption. Lignin and celluloses are involved in chromium, Cu(II) and Ni(II) sorption. FTIR analysis confirmed the involvement of lignin moieties in the studied metal ions sorption by grape stalks waste. This study presents a new approach on metal sorption field as the knowledge of the role of the sorbent chemical compounds is essential to determine the key sorbent compounds in the sorption process.

C. Liu : D. Pujol : N. Fiol (*) : M. À. Olivella : F. de la Torre : I. Villaescusa Chemical Engineering Department, Escola Politècnica Superior, Universitat de Girona, Ma Aurèlia Capmany, 61, 17071 Girona, Spain e-mail: [email protected] J. Poch Applied Mathematics Department, Escola Politècnica Superior, Universitat de Girona, Ma Aurèlia Capmany, 61, 17071 Girona, Spain

Keywords Grape stalks waste . Polarity . Chromium . Divalent metals

1 Introduction The use of lignocellulosic materials as sorbent for metal ions removal from aqueous solution has been extensively studied as a low-cost alternative for waste water treatment. Grape stalks (GS) waste, which is generated in large amounts (at about 10,000 t/year) in the Mediterranean areas, is one of these materials investigated as sorbent of metal ions. The ability of this waste to sorb metal ions has been demonstrated in several studies performed (Chubar et al. 2003; Villaescusa et al. 2004; Martinez et al. 2006). A remarkable characteristic of this material is its ability to reduce Cr(VI) to less toxic Cr(III) and sorb chromium in both oxidation state (Fiol et al. 2008; Escudero et al. 2009) and divalent metals by ion exchange between metal ion and the light metals on the sorbent surface (Villaescusa et al. 2004; Miralles et al. 2008). In the above mentioned studies, lignin was reported to be responsible for chromium and divalent metal ions sorption. Apart from lignin, grape stalks being a lignocellulosic material, comprises structural (i.e. cellulose, hemicelluloses) and non-structural components (i.e. extractives) (Kumar et al. 2009). All these chemical components contain a great variety of functional groups (i.e. alcohol, ketone and carboxylic groups as well as alkaline and alkaline earth metals) that are able to undergo complexation reactions and ion exchange processes (Nurchi and

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Villaescusa 2008) thus contributing to metal ion sorption. In a previous work, grape stalk waste was accurately characterised and the chemical composition of this material was previously reported (Pujol et al. 2013). Nevertheless, the role of the different chemical components of grape stalks on metal ions sorption is unknown. Therefore, the aim of this study is to offer some insight into the role of grape stalks chemical components on Cr(VI), Cu(II) and Ni(II) sorption. For this purpose, the grape stalks waste was subjected to successive extractions by solvents of different polarity and to an aqueous alkaline extraction with 1 % NaOH. The treated solid biomass obtained in the different extraction steps were used as sorbents of Cr(VI), Cu(II) and Ni(II). Sorption results were discussed taking into account elemental composition and polarity of the treated samples. Fourier transform infrared (FTIR) analysis of raw GS and its treated biomass before and after metal ion loading were carried out to identify the functional groups involved in metal sorption. The sequential extraction procedure on metal sorption resulted to be really useful to define different features of lignocellulosic materials towards metal ions sorption and to identify the valuable fractions for biosorption purposes.

2 Experimental 2.1 Materials and Reagents Grape stalks (GS) waste were supplied by a wine producer from the Catalonia region, Spain. Grape stalks were rinsed three times with abundant water and then dried in an oven at 105 °C until constant weight. Then, the waste was sieved for a particle size of 0.25– 0.45 mm. Hexavalent chromium, copper and nickel solutions were prepared by dissolving appropriate amounts of potassium dichromate (K2Cr2O7), copper chloride dihydrate (CuCl2·2H2O) and nickel chloride 6-hydrate (NiCl2·6H2O) in Milli-Q water, respectively. 0.1-M HCl and 0.1-M NaOH solutions were used for initial pH adjustments. Dichloromethane (DCM), ethanol (EtOH) and NaOH solutions were used as extraction solvents. Standard 1,000 mg L−1 solutions of chromium, copper and nickel were used for flame atomic absorption spectroscopy (FAAS) calibrations. All reagents were analytical grade and were purchased from Panreac (Barcelona, Spain).

Water Air Soil Pollut (2015) 225:2006

2.2 Extraction of GS Extractives Grape stalks waste was sequentially extracted by different organic solvents, water and alkaline solution. The flowchart of the extraction process can be seen in Fig. 1. In order to get treated biomass at each of the different extraction level, batches of 16.2 g of original sample were placed inside a glass fibre thimble. In the first batch, GS sample was extracted by DCM to get treated sample GS1. In the second batch, GS sample was extracted by DCM then followed by EtOH to get treated sample GS2. In the third batch, GS sample was extracted sequentially by DCM, EtOH and water to get treated sample GS3. In the last batch, GS sample was treated by DCM, EtOH, water and then by aqueous alkaline extraction with 1 % NaOH (1:50 solid/liquid ratio) obtaining the GS4 sample. The extraction with organic solvents and water was performed in a Soxhlet extractor using the following conditions: 40 °C for 6 h, 78.5 °C for 16 h and 100 °C for 20 h for the extraction with DCM, EtOH and water, respectively. The solvents were recovered afterwards. Each thimble containing treated sample was dried in a fume hood at room temperature if it was needed to be subjected to the next extraction step. Otherwise, the thimble was dried in the fume hood, after that, the free-extractive samples inside were rinsed with Milli-Q water until neutral pH then dried in an oven at 60 °C until constant weight. The alkaline leaching with 1 % NaOH of the free-extractive samples was carried out in a flat bottom flask under reflux at 100 °C for 1 h. Afterwards, the flask was cooled with ice, the solution was filtered by a sand core funnel using a vacuum pump and the solid also rinsed with Milli-Q water until neutral pH. The sand core funnel was put in the oven at 60 °C until constant weight. All the treated samples were washed thoroughly with water to remove the remaining solvents and specially to remove NaOH in the case of GS4 sample. The obtained treated samples (solid) (GS1–GS4) were kept in a desiccator before used for metal sorption and Fourier transform infrared (FTIR) spectroscopy analysis. 2.3 Metal Sorption Procedure Batch experiments were carried out at 20±2 °C in stoppered glass tubes by shaking 0.1 g of either raw material (GS) or treated biomass (GS1–GS4), with


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Raw GS Soxhlet extraction with DCM (40oC, 6 hour)

Aliphatic extractives

GS1 Soxhlet extraction with EtOH (78.5oC, 16 hour)

Polar extractives

GS2 Soxhlet extraction with H20 (100oC, 20 hour)

Polar extractives

GS3 Alkaline reflux with 1% NaOH (100oC, 1 hour)

Tannins, polyphenols and other compounds

GS4

Fig. 1 Flowchart of the extraction process of grape stalks (GS). DCM dichloromethane; EtOH ethanol

15 mL of metal ions solution in a rotary stirrer at 40 rpm (rotator drive STR4, Stuart Scientific). After agitation, the solid was removed by filtration through a 0.45-μm cellulose filter paper (Millipore). The selection of pH and contact time for Cr(VI) removal using raw GS was made on the basis of the results obtained in our previous studies (Fiol et al. 2008). In this study, pH 3.0 and 8 days agitation time were the conditions necessary to achieve maximum Cr(VI) sorption by GS. Therefore, a pH 3.0 and an equilibrium time of 8 days were the conditions selected to sorb Cr(VI) with GS. With the aim to monitor the process of Cr(VI) reduction to Cr(III) two contact times were considered, half-time equilibrium (te½) and equilibrium time (te). For Cu(II) and Ni(II) sorption onto GS, 1-h contact time and pH 5.0 were used (Villaescusa et al. 2004). All sorption experiments were carried out in duplicate and the average results are presented. 2.4 Analysis of Metal Ions The total concentration of chromium, i.e. Cr(VI)+Cr(III) and copper and nickel concentration in the remaining

solution after sorption were determined by flame atomic absorption spectroscopy (FAAS) (Varian Absorption Spectrometer SpectraAA 220FS). Hexavalent chromium was analysed by the standard colorimetric 1,5diphenylcarbazide method (Clesceri et al. 1998) by using a sequential injection system (SIA) recently developed in our laboratory. The concentration of trivalent chromium was determined as the difference between total chromium and hexavalent chromium concentrations, respectively. For comparison’s sake, the Cr(VI) standard used in diphenylcarbazide method was analysed by FAAS. Analytical measurements made by the two techniques were comparable within 5 %.

2.5 Elemental Analysis Elemental contents (C, H, N and S) of raw materials (GS) and treated biomass (GS1–GS4) were determined using a PerkinElmer EA2400 series II Elemental Analyzer. C, H and N detection limits were 0.72, 0.20 and 1.20 %, respectively. Oxygen content was calculated by difference. The elemental ratios H/C, O/C, C/N, (O+N)/ C were computed for all the samples.

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2.6 FTIR Analysis To give a qualitative and preliminary analysis of the main surface functional groups on raw materials and treated biomass solid fractions, a Fourier transform infrared (FTIR) analysis in solid phase was performed using a Fourier Transform spectrometer (Galaxy Series FTIR 5000, Mattson). Solid-phase FTIR analysis was used to investigate changes in the absorption bands corresponding to the functional groups of the adsorbents as a consequence of the summative extraction and chromium, copper and nickel loading. Spectra of the samples before and after contact with 6-mM Cr(VI), 2-mM Cu(II) and 2-mM Ni(II) solutions were recorded on a Galaxy 5000 FTIR spectrometer (Mattson Instrument Co., Madison, WI). For FTIR analysis, 200-mg KBr disks containing 2 mg of finely ground sample were prepared. Spectra were recorded in the spectral range 3,600 to 600 cm−1 by co-addition of 32 scans with a resolution of 4 cm−1.

3 Results and Discussion 3.1 Isolation and Characterisation of GS and Treated Biomass Samples The mass of each solid (raw materials and treated biomasses) together with the percentage of extractives removed after the successive extractions are presented in Table 1. As seen in this table, the percentage of total extractives removed from GS is very high (75.92 %). The great amount of GS compounds solved in H2O (21.3 %) is remarkable. These results put into evidence that GS2 contains a large amount of high polar watersoluble compounds (polyphenols, condensed tannins). Another distinguishing feature is the effect caused by 1 % NaOH leaching. The quite high loss of GS extractives (45 %) indicates a high content of hydrolysable compounds: tannins and polyphenols insoluble in water and ethanol, lignin moieties and molecules of high molecular weight such as polysaccharides (Frandinho et al. 2002). Miranda et al. (2013) found that the amount of extractives of tree barks solubilised in the alkaline solution are related to the hemicelluloses and suberin content. In a recent work, Pujol et al. (2013) reported a polysaccharides content of 14 % for GS. Compared to other lignocellulosic materials extractives solubilised from GS by the alkaline solution are higher than those


reported for the maritime pine bark from Portugal (11 %) (Frandinho et al. 2002) and within the range of various pine barks (17.2–39.1 %) (Fengel and Wegener 1984). Elemental composition and elemental ratios of raw GS and treated biomasses are given in Table 2. As observed in the table, GS samples present similar polarity index ((O+N)/C) being GS2 treated biomass as the one that showed the highest polarity. 3.2 Sorption Studies Table 3 shows the remaining Cr(VI) and Cr(III) concentration after Cr(VI) sorption onto raw GS and the different GS-treated samples at te1/2 (4 days) and at te (8 days). In the same table, the pH values and the percentage of total chromium removed at equilibrium are also shown. In can be observed that after contact with GS and nonfree extractives samples (GS1–GS3) Cr(III) appears in solution after 4 days contact, indicating the presence of reducing functional groups in all these materials. It must be pointed out that Cr(III) in solution decreases after 8days contact. Thus, part of the formed Cr(III) initially released into the solution is subsequently sorbed onto GS and treated biomass. Note that in the case of the lowest Cr(VI) concentration (i.e. 101 mg L−1), the hexavalent species almost disappeared from the solution after 4-days contact time. When comparing sorption yields showed by raw GS and GS1–GS3 treated biomass, the highest Cr(VI) removal percentage is found for GS3 (99 and 81 % for both respective concentrations). GS3 is the remaining solid obtained as a result of the successive extractions with DCM, EtOH and water after which apolar molecules and most of the polar compounds are eliminated. From the results, it seems Table 1 Grape stalks (GS) mass loss after successive extractions and percentage of extractives removed. The cumulative removal of extractives is indicated in brackets Solvent

Treated samples

Mass loss (g)

Extractives removal (%)

DCM

GS1

0.36

2.22 (2.22)

EtOH

GS2

1.14

7.04 (9.26)

H2O

GS3

3.45

21.30 (30.56)

1 % NaOH

GS4

7.35

45.36 (75.92)

Mass of raw GS=16.2 g DCM dichloromethane, EtOH ethanol


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Table 2 Elemental analysis and elemental ratios H/C, O/C and (O+N)/C of raw grape stalks (GS) and treated samples %C

%H

%N

%Oa

H/C

O/C

(O+N)/C

GS

48.56

5.89

N.D.

45.55

1.46

0.70

0.70

GS1

44.53

6.12

0.51

48.84

1.65

0.82

0.83

GS2

43.36

5.75

1.21

49.68

1.59

0.86

0.88

GS3

46.32

6.24

1.57

45.87

1.62

0.74

0.77

GS4

45.26

6.85

0.34

47.55

1.82

0.79

0.79

a

Oxygen was calculated by mass difference

N.D. not detected

that (i) these two families of compounds do not participate in the chromium reduction/sorption process and (ii) the removal of these compounds favours Cr(VI) interaction with the sorbent active sites where chromium reduction and sorption takes place. When the sorbent is GS4 Cr(VI) reduction rate is very low, trivalent species concentration in solution is almost null, and total chromium removal drops dramatically. As reported, the extraction with 1 % NaOH can dissolve part from lignin moieties, tannins and polyphenols that are insoluble in water and ethanol, as well as molecules of high molecular weight such as polysaccharides (Frandinho et al. 2002). Pujol et al. (2013) who characterised grape stalks waste reported high amounts of tannins and polyphenols (27 %), mainly found in 1 % NaOH extracts which confirm the effect of alkaline leaching on the solution of these two kinds of compounds. Therefore, the loss of tannins and polyphenols which are reported to be involved in the reduction/ sorption process in a great extent (Nakano et al. 2001; Chabaane et al. 2011) together with the loss of lignin moieties could be the cause of the so low sorption yield shown by GS4. Moreover, the lower increase of pH observed when using GS4 in Cr(VI) sorption as compared to the pH increase observed when GS–GS3 treated biomasses are used as Cr(VI) sorbents denotes the lower capacity of this treated biomass to reduce chromium. The results obtained when investigating Cu(II) and Ni(II) sorption onto GS and its treated samples at pH 5.0 are presented in Table 4. In this table, metal concentration and solution pH after 60-min contact time are shown. The final pH values of the different treated samples are also shown in the same table. As can be seen, GS fractions are more efficient to sorb copper than nickel and GS3 provided the best sorption yields. Metal

sorption increases with the increase of extractives removed by the organic solvents and water. However, the alkaline hydrolysis likely provokes the modification of some functional groups that are involved in copper and nickel sorption resulting in a decrease of metal ions sorption. The different extraction steps can provoke changes of the chemical composition, functional groups of the treated samples leading to different physical and chemical characteristics of these solid extracts. All these characteristics may greatly influence on the removal of metal ions (Lee and Rowell 2004). Thus, the sample treatment generates a new surface scenario which might lead to different kinds of interactions between Cu(II) and Ni(II) and the functional groups on the sorbent surface. In this context, the treated sample GS3, once extractives have been fully removed, is mainly composed by lignin and cellulose. The solubilisation of lignin moieties, due to the alkaline solution, would result in a decrease of active sites that would justify the lower sorption yields given by GS4 compared to the ones given by GS3 and the involvement of lignin in metal ions sorption. 3.3 Relations between Metal Ions Sorption and Sorbent Characteristics In an attempt to explain the sorption results presented in Tables 3 and 4, the partition coefficient Kd defined as the ratio of the quantity of the adsorbate adsorbed per mass of solid to the amount of the adsorbate remaining in solution at equilibrium was calculated for each metal adsorbed on each GS sample. In order to find a possible relationship of Kd with polarity and/or sorption affinity, the calculated Kd were related to polarity index (O+N/C) and elemental ratio H/C values. Kd values of Cr(VI) and Cr(III) did not correlate with neither polarity index nor elemental ratios H/C. In the case of the studied divalent metals, Kd values found for Cu(II) showed a low correlation with polarity index values (KdCu = 0.16((O+N)/C)+0.0035 (R2 =0.75, N=5) and a nearzero correlation (R2 = 0.26) for nickel. This result suggests that the oxygen functional groups (i.e. carboxylic COOH and phenolic OH groups) on the sorbents surface are involved in copper binding on the sorbents surface. It was previously found that raw GS contain phenolic groups (0.65 mmol g−1), strong carboxylic groups (0.20 mmol g−1) and weak acidic groups

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Table 3 Cr(VI) sorption onto raw grape stalks (GS) and treated samples. Initial pH 3.00, half-time equilibrium (te1/2) Sample

Cr(VI) t e1/2 (mg L−1)

Cr(III) te1/2 (mg L−1)

pH te1/2

Cr(VI) te (mg L−1)

Cr(III) te (mg L−1)

pH te

Total removal (%)

Initial Cr(VI) 101.26 mg L−1 GS

0.28

20.17

6.32

0.06

17.25

6.31

82.91

GS1

0.01

21.62

6.23

0.00

17.79

6.01

82.43

GS2

0.00

17.06

6.57

0.06

13.44

6.67

86.67

GS3

1.48

2.97

6.84

0.00

0.95

6.55

99.07

GS4

66.95

1.57

5.89

61.90

2.80

5.98

36.11

Initial Cr(VI) 308.90 mg L−1 GS

88.21

28.51

6.76

68.46

35.17

6.99

66.45

GS1

72.34

35.91

6.88

38.18

42.08

7.06

74.02 75.00

GS2

57.25

30.18

7.06

44.94

32.29

7.27

GS3

73.93

1.18

7.30

54.24

3.43

7.46

81.33

GS4

263.37

0.00

5.57

257.94

0.00

5.64

16.50

Contact time=4 days; time at equilibrium (te)=8 days

(0.35 mmol g−1) (Pujol et al. 2013). As seen in Table 4, pH values at equilibrium (pHe) are within the range of 4.28–5.17 and 4.62–6.21 after copper and nickel sorption, respectively. In these pH ranges, strong carboxylic groups are fairly deprotonated (pka =3–5) while phenolic groups remain protonated (pka = 9.5–10.5). The lower pH values range observed after copper sorption suggest that carboxylic acids are deprotonated favouring metal ions and carboxylate anions (−COO − ) binding and thus resulting in higher sorption yields compared with those obtained for nickel. In the literature, some authors using sorbents based on natural organic matter found that copper sorption was strongly influenced by the carboxylic groups content (Sun and Cheng 2002)

while others by that of the phenolic groups (ReyCastro et al. 2009).

3.4 FTIR Analysis The successive removal of GS extractives by the action of the different solvents and NaOH provoke changes in the sorbent structure and consequently in the absorption bands of the spectra. GS and treated biomass spectra are shown in Fig. 2. The differences observed after removal of aliphatic structures (i.e. long-chain n-alkanes and fatty acids) were: peaks at 2,924 and 2,855 cm−1 attributed to C–H vibrations decreased intensity and the peak at 1,735 (GS) corresponding to C=O stretching was shifted to 1,720 cm−1 (GS1–GS4).

Table 4 Cu(II) and Ni(II) in solution at equilibrium and percentage of metal ion removed after contacting with raw GS and treated samples. Initial Cu(II) concentration: 124.66 mg L−1, initial Ni(II) concentration: 124.89 mg L−1, initial pH: 5.00, contact time: 1 h Sample

Cu(II) (mg L−1)

Cu(II) removed (%)

Ni(II) (mg L−1)

Ni(II) removed (%)

pHCu(II)

pHNi(II)

GS

66.46

46.69

96.27

22.92

4.28

4.62

GS1

64.43

48.32

91.72

26.56

4.37

4.84

GS2

59.91

51.94

84.79

32.11

4.54

4.79

GS3

43.59

65.03

70.71

43.38

5.17

6.06

GS4

68.40

45.13

80.06

35.90

5.11

6.21


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Fig. 2 FTIR spectra of GS and samples GS1–GS4

After water extraction, the peak at 1,065 cm−1 attributed to C–OH stretching of secondary alcohols was significantly reduced and the peak at 1,306 cm−1 attributed to phenol OH bending was shifted to 1,320 cm−1 indicating a partial elimination of tannins by water solubilisation (NetzahuatlMuñoz et al. 2012). After alkaline hydrolysis, the big differences in metal sorption were found (see Tables 3 and 4). The absorption bands of the corresponding spectra GS3 and GS4 were compared with the purpose to find out possible explanations for the different sorption behaviour of these two GS solid fractions. Several peaks assigned in GS3 spectrum were found to shift in GS4 spectrum. The peaks at 1,609 cm−1 (C=C stretching from lignin Fig. 3 FTIR spectra of GS and GS loaded with Cr(VI), Cu(II) and Ni(II)

moieties) and 1,439 cm−1 (C–H deformations of aromatic ring of lignin) were shifted to 1,629 and 1,428 cm−1, respectively and the peak at 1,523 cm−1 attributed to aromatic vibrations of C=C from lignin moieties disappears in GS4. Also, the peak at 1,306 cm−1 associated to OH bending of phenolic groups (Netzahuatl-Muñoz et al. 2012) from lignin and tannins was reduced in intensity after alkaline hydrolysis (GS4). Therefore, these results confirm that alkaline hydrolysis destroys in a great deal the lignin structure and tannins. The removal of these structures may be the cause of the drastic decrease of metal sorption yields exhibited by GS4 as compared to GS3 (see Tables 3 and 4).

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FTIR spectra of GS and GS loaded with Cr(VI), Cu(II) and Ni(II) are shown in Fig. 3. In general, when comparing unloaded and chromium loaded spectra, the most important changes observed in all treated biomass except GS4 indicate the clear involvement of lignin moieties on chromium sorption: the peak at 1,609 was shifted to 1,629 cm−1; the peak at 1,523 cm−1 decreased in intensity (both peaks attributed to aromatic ring stretching of lignin moieties); the peak at 1,735 cm−1 (GS) shifted to 1,720 cm−1 in GS1; and the peak at 1,720 cm−1 in GS2 and GS3 attributed to asymmetric C–H deformations from cellulose and lignin reduced in intensity (Escudero et al. 2009). In addition, the shift of the peak at 1,306 to 1,320 cm−1 attributed to phenol OH bending indicates that tannins also are taking part on chromium sorption as stated in section 3.2. From the obtained results, it is evident that lignin and tannins are involved in chromium sorption onto GS and the partial loss of these components as a consequence of NaOH extraction justify the decrease of chromium sorption shown by GS4 for both Cr(VI) concentrations tested (63 and 70 %, respectively) (Table 3). As regards to copper and nickel sorption, again, the main differences were found between GS3 and 4 sorption yields (Table 4). In all spectra, after copper and nickel sorption, the main difference was found to be at the peak attributed to lignin aromatic C–C bond at around 1,600 cm−1. The peak at 1,609 cm−1 (GS) was shifted to 1,619 cm−1 in GS1, GS2 and GS3 spectra and to 1,629 cm−1 in GS4. This confirms the involvement of lignin ring in all treated biomass tested. In addition, the peak at 1,523 cm−1 was reduced in intensity after copper and nickel sorption in GS1–GS3.

4 Conclusions The sequential extraction proved to be useful to relate the treated biomass sorption features to their behaviour as sorbents for metal ions. The removal of apolar and polar extractives from grape stalks wastes seems to favour the interaction between metal ions and the sorbents matrix. Extractives, i.e. tannins and polyphenols, were found to take part in chromium reduction and sorption. The treatment of GS waste with NaOH generally led to lower metal ions sorption yields and lower capacity of the material to reduce hexavalent chromium due to the


destruction of some moieties of structural components involved in Cr(VI), Cu(II) and Ni(II) removal. The determination of elemental ratios of all GS samples evidenced the involvement of oxygen functional groups only found in copper sorption. FTIR analysis confirmed the involvement of lignin moieties and tannins in the studied metal ions sorption by GS. Moreover, FTIR analysis put into evidence the partial loss of these structures in the spectrum of the NaOH-treated sample (GS4). As a final remark, this study demonstrates that the sequential extraction sheds light on the active biomass key compounds taking part in the sorption of each kind of metal ions. The procedure used is a new approach to metal ions biosorption studies. Acknowledgments This research was funded by the Spanish Ministry of Science and Innovation as part of the projects CTM2010-15185 and CTM2012-37215-C02-01. Chang Liu was financially supported by a fellowship from the Chinese Scholarship Council [2011] 3005. David Pujol was financially support by the Spanish Ministry of Education, Culture and Sport (MHE201100258). We thank Dr. Helena Pereira of the Centro de Estudos Florestais (Lisbon) for her advice and help in extraction procedures.

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