Accepted Manuscript Preparation and characterisation of biodegradable pollen-chitosan microcapsules and its application in heavy metal removal İdris Sargın, Murat Kaya, Gulsin Arslan, Talat Baran, Talip Ceter PII: DOI: Reference:

S0960-8524(14)01669-1 http://dx.doi.org/10.1016/j.biortech.2014.11.067 BITE 14273

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

31 August 2014 14 November 2014 15 November 2014

Please cite this article as: Sargın, İ., Kaya, M., Arslan, G., Baran, T., Ceter, T., Preparation and characterisation of biodegradable pollen-chitosan microcapsules and its application in heavy metal removal, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.11.067

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Preparation and characterisation of biodegradable pollen-chitosan microcapsules and its application in heavy metal removal İdris Sargına*, Murat Kayab, Gulsin Arslanc, Talat Barand, Talip Cetere

a

Selcuk University, Faculty of Science, Department of Chemistry, 42075, Konya, Turkey.

b

Aksaray University, Faculty of Science and Letters, Department of Biotechnology and

Molecular Biology, 68100, Aksaray, Turkey. c

Selcuk University, Faculty of Science, Department of Biochemistry, 42075, Konya, Turkey.

d

e

Aksaray University, Faculty of Science, Department of Chemistry, 68100, Aksaray, Turkey.

Department of Biology, Faculty of Arts and Sciences, Kastamonu University, 37100

Kastamonu, Turkey.

*

Corresponding Author: İdris Sargın, Department of Chemistry, Faculty of Science, Selcuk

University, 42075 Konya, Turkey. Tel: +90-332-2233852 Fax: +90-332-2412499 E-mail address: [email protected] Abstract Biosorbents have been widely used in heavy metal removal. New resources should be exploited to develop more efficient biosorbents. This study reports the preparation of three novel chitosan microcapsules from pollens of three common, wind-pollinated plants (Acer negundo, Cupressus sempervirens and Populus nigra). The microcapsules were characterized (Fourier transform infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy and elemental analysis) and used in removal of heavy metal ions: Cd(II), Cr(III), Cu(II), Ni(II) and Zn(II). Their sorption capacities were compared to those of cross-linked chitosan beads without pollen grains. C. sempervirens-chitosan microcapsules exhibited better performance (Cd(II): 65.98; Cu(II): 67.10 and Zn(II): 49.55 mg g−1)

1

than the other

microcapsules and the cross-linked beads. A. negundo-chitosan microcapsules were more efficient in Cr(III) (70.40 mg g−1) removal. P. nigra-chitosan microcapsules were found to be less efficient. Chitosan-pollen microcapsules (except P. nigra-chitosan microcapsules) can be used in heavy metal removal.

Keywords: pollen; chitosan; microcapsule; biosorbent; heavy metal

Introduction Heavy metal ions are discharged into water bodies via industrial operations such as mining, metal finishing, plating, tannery and fertilizer production. Water bodies that have been contaminated with heavy metal ions are posing risks to the environment (Bilal et al., 2013). Certain heavy metal ions, even in small amounts, are capable of bio-accumulating in the tissues of living organisms and are responsible from developing various diseases and disorders (Abou El-Reash et al., 2011). Many attempts employing various techniques (e.g. coprecipitation, solid phase extraction, ion-exchange separation and adsorption) have been made to remove or recover heavy metal ions from waste waters. However, amongst the techniques mentioned, adsorption manifests itself as an effective and simple method (Sarkar & Majumdar, 2011). Many studies have shown biosorbents are excellent adsorbents (Wan Ngah et al., 2011). Researches on developing biodegradable and eco-friendly biosorbents demanding less chemical treatment during production are on the rise in the last decades. Various sorbents with biological origin, like fungi (Damodaran et al., 2014), algae (He & Chen, 2014), bacteria (Kieu et al., 2011) and yeasts cells (Machado et al., 2010) have been developed for removal and recovery of metal ions from aqueous solutions. Biosorbents have been proved to be effective and, in some cases, superior to the chemical resins in some ways: they are effective, 2

low cost, available in large quantities and more importantly have already carry functional groups including amino, carboxyl, hydroxyl and carbonyl (Wang & Chen, 2009). Chitosan is a naturally occurring carbohydrate polymer which can meet the above mentioned traits: it is biocompatible, biodegradable and nontoxic to human and environment and shows antimicrobial and antioxidant activities (Muzzarelli, 2011). These excellent physicochemical features have put chitosan in a unique position amongst the biopolymers: it has found broader applications in a number of fields including pharmaceutics and medicine (Ong et al., 2008), food industry (Aider, 2010), textile (Alonso et al., 2009) and water treatment (Hu et al., 2013). Chitosan also has high affinity towards metal ions due to metal ion binding groups (e.g. –NH2 and –OH) on its polymeric chains (Wu et al., 2010). Chitosan, in raw (Paulino et al., 2007) or functionalized (Wang et al., 2013) form, is one of the widely utilized biosorbents in removal or recovery of heavy metal ions. Among the chitosan-based sorbents, chitosan composites have gained attention and have been considered as an alternative to the conventional biosorbents in recent years. Various chitosan composites with biological materials (e.g. cellulose, cotton, oil palm ash, silk and alginate) have been prepared and used in heavy metal removal (Wan Ngah et al., 2011; Wang & Chen, 2014). However, chitosan composites adsorbents with pollen grains have not been reported in the literature. This is the first study to report the preparation and use of pollen-chitosan composite microcapsules in heavy metal removal. Pollen grains were preferred: (1) since pollens are biomaterial, (2) they are already fine powder and therefore can be easily covered with chitosan and (3) they can be harvested in large quantities when needed. Modification of chitosan composites is easily achieved via crosslinking in glutaraldehyde solution by forming Schiff base. On the other hand, azomethine formation from free amino groups on chitosan

3

polymer reduces metal sorption capacity of the polymer by eliminating coordination sites for metal ions. This study aimed to find out whether pollen grains entrapped in chitosan matrice are capable of counterbalancing this loss and enhancing metal binding capacity of crosslinked chitosan by increasing metal sorption sites, surface area and the porosity of the microcapsules. Chitosan composite microcapsules with pollen grains from three common plant species were prepared: A. negundo, C. sempervirens and P. nigra. Pollen grains from these plants can be harvested easily because these anemophilous plants are wind-pollinated and therefore produce large quantities of pollen grains (Çeter et al., 2011). The present work deals with (1) preparation of three different chitosan-pollen microcapsules, (2) characterisation of the microcapsules employing Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), scanning electron microscopy (SEM) and elemental analysis (EA) and (3) assessment of the heavy metal (Cd(II), Cr(III), Cu(II), Ni(II) and Zn(II)) sorption capacities of the three biosorbents and glutaraldehyde cross-linked chitosan beads without pollen grains. 2. Experimental 2.1. Pollen samples collection and identification The male cones from P. nigra, C. sempervirens and A. negundo were collected in Kastamonu, Turkey and were identified with reference to the guide book by Coden and Cullen (Cooden, 1965). The cones were kept in an oven at 25°C for 1–2 days to release the pollen grains. The pollen grains were sieved to remove any other material and 95% of pollen purity was ensured. Then, the pollen grains were kept at -20°C. The plant samples are kept at the palynology laboratory at Vocational College, Kastamonu University. P. nigra L. (08.04.2013, Voucher: Ceter 56) and A. negundo L. (30.03.2013, Voucher: Ceter 49) cones were collected from the garden of Kastamonu 4

University Vocational College and C. sempervirens L. (30.03.2013, Voucher: Ceter 50) cones were from the garden of Kastamonu İlbank. 2.2. Materials Chitosan powder (medium molecular weight), Cd(NO3)2. 4H2O and NaOH were obtained from Sigma–Aldrich. Glutaraldehyde solution (GA) (25% in water, v:v), the metal salts (Ni(NO3)2. 6H2O, Cr(NO3)3. 9H2O, Zn(NO3)2. 4H2O, Cu(NO3)2. 3H2O), acetic acid and HCl were purchased from Merck. Methanol was obtained from AnalaR Normapur. 2.3. Preparation of chitosan-pollen microcapsules and cross-linked chitosan beads Chitosan (1.000 g) was dissolved and stirred in 50 mL of 2% acetic acid solution. 0.500 g of pollen grains was added to the chitosan solution. To ensure homogeneity, the mixture was stirred for 2 h. Then, the mixture was transferred into a burette. The mixture was dropped into a coagulation solution (100 ml of water, 150 ml of methanol and 30.0 g NaOH) (Pal et al., 2013). The microcapsules were kept in the coagulation solution overnight. Then, they were removed from the solution by filtration. The microcapsules were rinsed with plenty of distilled water to neutrality. Then, the microcapsules in water were recovered by a sieve and transferred into cross-linking reaction solution (mixture of 30 ml of methanol and 0.3 ml of GA) and stirred gently under reflux at 70°C for 6 h. Finally, to remove any unreacted GA molecules, cross-linked microcapsules were rinsed with ethanol and water and finally dried at room temperature. Cross-linked chitosan beads without pollen grains were also prepared following the same method. 2.4. Characterisation of the pollen-chitosan microcapsules 2.4.1. FT-IR spectroscopy

5

The IR spectra of pollen-chitosan microcapsules were recorded with a Perkin Elmer FT-IR Spectrometer over the frequency range of 4000–625 cm−1. 2.4.2. Elemental Analysis Elemental analysis of the pollen-chitosan microcapsules was performed using Thermo Flash 2000. 2.4.3. SEM analysis The pollen-chitosan microcapsules were coated with gold for SEM analysis by Sputter Coater (Cressingto Auto 108). The surface characteristics of the samples were examined by a QUANTA FEG 250 scanning electron microscope. 2.4.4. TGA analysis Thermogravimetric analysis of the microcapsules were conducted using EXSTAR S11 7300 at a heating rate of 10°C min−1. The samples were heated up to 650°C. 2.5. Heavy metal sorption experiments The pollen-chitosan microcapsules or cross-linked chitosan beads (0.1000 g) were added to metal solution (25 mL of 10 mg L−1 at metal solution pH; Cd(II): 5.35, Cr(III): 4.63, Cu(II): 5.18, Ni(II): 5.34, Zn(II): 5.34) and agitated on a shaker (Heidolph Promax 2020) at 200 rpm for 4 h. Then, the sorbent was removed from the solution with a filter paper. The metal ion concentration in the solutions was detected using a flame atomic absorption spectrophotometer (ContrAA 300, Analytikjena). The amount of metal ions recovered per unit mass of the sorbents was determined employing the equation given below: qe = (Ci–Ce)V/W

(1)

6

where q e is the metal sorption capacity of the microcapsules or the cross-linked chitosan beads (mg g−1), Ci and Ce are the initial and equilibrium of metal ion concentrations (mL−1), respectively; V is the volume of metal solution (L), and W is the mass of the sorbent (g). The effects of amount of sorbent (0.0250–0.1250 g), contact time (60–300 min.), metal ion solution pH (3.0, 4.0 and the pH of the metal ion solutions), initial metal ion concentrations (2.5–12.5 mg L−1) and temperature (25, 35 and 45oC) on sorption behaviour of the microcapsules were studied. 3. Results and discussion 3.1. Characterisation of pollen-chitosan microcapsules FT-IR FT-IR spectra can provide an insight into the structure of chitosan polymer chains after the cross-linking reaction has been accomplished. In the FT-IR spectrum of chitosan, an absorption band at 1590 cm−1, which is corresponded to the NH2 groups, appears (Pawlak & Mucha, 2003). This band and any other bands which can be ascribed to the stretching of carbonyl groups (C=O, 1700–1750 cm−1) of glutaraldehyde were not observed in spectra of the three pollen-chitosan microcapsules. Also, the bands appearing at 1650 (A. negundo), 1647 (C. sempervirens) and 1649 cm−1 (P. nigra) can be attributed imine (C=N) groups (Vieira & Beppu, 2006). Furthermore, the bands at 1574 (A. negundo), 1575 (C. sempervirens) and 1572 cm−1 (P. nigra) which can be corresponded to C–N stretching can signify cross-linking of the chitosan polymer. These observations can confirm the condensation of the amino groups of the chitosan with glutaraldehyde (Altun & Cetinus, 2007). EA

7

Nitrogen (N), carbon (C) and hydrogen (H) contents of the pollen-chitosan microcapsules were determined. N, C and H contents of A. negundo-chitosan microcapsules were 5.9, 42.76 and 7.24%; C. sempervirens-chitosan; 5.25, 42.45, 7.18% and P. nigrachitosan; 6.75, 43.77, 7.16% and the cross-linked chitosan beads; 6.91, 42.95, 7.00% respectively. C and H contents of the three microcapsules and the cross-linked chitosan beads were close to each other. However, variations in the N content of the each sorbent was significant and was in the following order: cross-linked chitosan>P. nigra>A. negundo>C. sempervirens. This order may also represent the chemical compositions of the pollens in the same way. However, average metal sorption capacity of the microcapsules followed the opposite order: C. sempervirens>A. negundo>P. nigra (Table 1). It seems that there is probably a negative correlation between the nitrogen containing groups of the pollen grains and the metal binding sites on the biosorbents. Surface morphology SEM images showed that the pollen grains were entrapped in the chitosan matrice, but they were randomly distributed on the surface. Pollen grains from A. negundo and P. nigra were buried in the microcapsules and fewer grains were exposed to the surface. When compared to the others, the microcapsules with C. sempervirens were more spherical and more pollen grains were on the surface; therefore they had larger surface area than the others. This may explain the higher metal sorption capacity of the C. sempervirens-chitosan microcapsules (vide infra). TGA Crystallinity of chitosan determines its thermal stability (Guibal, 2004). Modification of chitosan (i.e., Schiff bases formation) reduces number of pendant amino groups on the polymer chains; this leads to the deformation of intermolecular hydrogen bonds and lowers 8

the crystallinity of the chitosan polymer. Maximum decomposition temperature (DTGmax) of raw chitosan has been reported to be 302°C (Kaya et al., 2014), and a decrease in the DTGmax values of the microcapsules was expectable after the cross-linking of the polymeric backbone. As expected, lower DTGmax values were recorded for the pollen-chitosan microcapsules; A. negundo-chitosan: 273, C. sempervirens-chitosan: 258 and P. nigra-chitosan: 269°C. At end of the pyrolysis, 70.5% of A. negundo-chitosan, 84.5% of C. sempervirenschitosan and 75.7% of P. nigra-chitosan microcapsules decomposed.

As mentioned,

microcapsules with A. negundo had the highest DTGmax value. These microcapsules also exhibited the highest thermal stability but the lowest Cd(II) uptake capacity. C. sempervirenschitosan microcapsules, which had the lowest thermal stability and the highest total decomposition rate, showed better performance in metal heavy sorption than the others (vide infra). 3.2. Heavy metal sorption performances of the microcapsules 3.2.1. Adsorbent dosage The minimum amount of the pollen-chitosan microcapsules that was required was determined for each of the metal ions (Fig. 1). The increase in the amount of the sorbents led to an increase in the amount of the metal ions removed until a saturation point. After this point (close to the 0.1 g), the increment in the sorbent dosage did not contribute much to the sorption of the metal ions. Similar trends were observed for the three types of the pollenchitosan microcapsules and the chitosan beads without pollen grains. 3.2.2. Contact time Time that is required for the adsorption of the metal ions onto each of the pollenchitosan microcapsules to attain the equilibrium state was determined (Fig. 2). The studies

9

revealed that the sorption of Cd(II), Cr(III), Cu(II), Ni(II) and Zn(II) on the pollen-chitosan microcapsules or the chitosan beads was slow in nature and an equilibrium state was observed at around 240 min. for each metal ion regardless of the sorbent type. This could be attributed to the diffusion resistance within the polymeric matrice. Additionally; it appears that the incorporation of the pollen grains in chitosan did not significantly change the diffusion characteristics of the microcapsules. 3.2.3. pH Metal solution pH can affect not only metal ion speciation in the solution but physicochemical nature of the sorbent. Chitosan itself exhibits pH depending solubility nature; its structure in the

solution is subject to

changes especially through

protonation/deprotonation of the amino and hydroxyl groups on it. The lower sorption capacities observed for each of the microcapsules at more acidic conditions could be resulted from the decrease in the electron-donating ability of N and O atoms and the repulsion forces upon protonation of the binding sites on the sorbents (Fig. 3). Also, possible competition of hydronium ions with the metal cations for sorption sites could weaken the chelation ability of the sorption sites on the sorbents. 3.2.4. Thermodynamic analysis The effect of the temperature on the uptake of the metal ions was studied and higher sorption capacities for the three microcapsules were accomplished at elevated temperatures. By using the linear van’t Hoff plot of log KC versus 1/T, changes in standard free energy (∆G°), enthalpy (∆H°), and entropy (∆S°) were calculated. ∆G°=−RT lnKC

(2)

∆G°= ∆H°−T∆S°

(3) 10

log KC = (∆S°/2.303R) − (∆H°/2.303RT)

(4)

where KC is the equilibrium constant, R is universal gas constant (8.314 J mol−1 K−1) and T is the temperature (K). The value of ∆S° and ∆H° were obtained from the slope (∆H◦/2.303R) and the intercept (∆S◦/2.303R) of van’t Hoff plot, log KC versus 1/T of log KC versus 1/T plot. The thermodynamic analysis (Table 2) revealed the exothermic nature of the adsorption of the metal ions onto pollen-chitosan microcapsules. However adsorption process for chitosan beads was found to be endothermic. With the exception of two processes, chitosan beads-Zn(II) and A. negundo-Cr(III), randomness was increased. Cd(II), Ni(II) and Zn(II) sorption on chitosan beads was nonspontaneous, while the sorption of other metal ions was observed to be spontaneous. As for Gibb’s free energy changes of the sorption system of pollen-chitosan microcapsules, it appears that the metal uptake process of the microcapsules was spontaneous. The observed discrepancies in some data have been reported in earlier studies (Liu & Lee, 2014). A recent study addressed this issue and the authors recommended further detailed studies on thermodynamic evaluation of adsorption of heavy metal ions. 3.2.5. Isotherm models Adsorption equilibrium studies of the chitosan-based sorbents have been usually conducted using two isotherms models; the Langmuir and the Freundlich adsorption isotherm models. These mathematical models make it possible to quantify performance of the biosorbent (Wang & Chen, 2014) and their linearized isotherm expressions are employed (Foo & Hameed, 2010): i.

The Freundlich Model:

log qe= log KF+(1/n) logCe

11

(5)

with q e,, amount of solute adsorbed in mmol g−1, Ce, the equilibrium concentration of the adsorbate in mM L−1 and KF and n Freundlich constants denoting adsorption capacity and intensity of adsorption. ii.

The Langmuir model: Ce/qe = Ce/Q0+1/Q0b

(6)

with q e, amount of solute adsorbed in mmol g−1, Ce, the equilibrium concentration of the adsorbate in mmol L−1, Q0(in mmol g-1) and b(in L mmol-1) Langmuir constants related to adsorption capacity and energy of adsorption. Table 3 lists the parameters and the correlation coefficients obtained from the plots of Langmuir (Ce/q e vs. Ce) and Freundlich (log qe vs. log Ce). It appears that adsorbateadsorbent system can be better explained by the Langmuir model for the microcapsules C. sempervirens-chitosan and P. nigra-chitosan and the chitosan beads. On the other hand, the Freundlich model gave higher correlation coefficients (R2) for the adsorption of the metal ions on A. negundo-chitosan microcapsules. This may demonstrate the heterogeneous surface characteristics of the A. negundo-chitosan microcapsules. C. sempervirens-chitosan microcapsules exhibited nearly two times higher affinity for Cd(II) (65.98 mg g−1) than the other sorbents. Also, this sorbent was more efficient in Cu(II) (67.10 mg g−1) and Zn(II) (49.55 mg g−1) removal. In Cr(III) removal, A. negundo-chitosan microcapsules were more effective than all the other adsorbents (70.40 mg g−1). As for Ni(II) removal, pollen microcapsules adsorbed slightly more ions than the chitosan beads. P. nigrachitosan microcapsules were observed to have lower adsorption capacity than the other sorbents including cross-linked chitosan beads (Table 1). Chitosan-pollen microcapsules (except P. nigra-chitosan microcapsules) can be used in heavy metal removal. Affinity of the each

sorbent

for

the

metal

ions

were

in

the

order:

A.

negundo:

Cr(III)>Cu(II)>Cd(II)>Ni(II)>Zn(II); C. sempervirens: Cu(II)>Cr(III)>Cd(II)>Zn(II)>Ni(II) 12

and P. nigra: Cu(II)>Cr(III)>Cd(II)>Zn(II)>Ni(II). W.S. Wan Ngah et al. reported metal adsorption capacity of some chitosan composites in their review paper; chitosan-cotton fibres: Cu(II): 24.78, Ni(II):7.63, Cd(II):15.74 mg g−1, chitosan-cellulose: Cu(II): 26.50, Zn(II): 19.81, Ni: 13.21 and chitosan-alginate; Cu(II): 67.66 mg g−1. In case of Cd(II) ions, all the pollen-chitosan microcapsules were more efficient than the chitosan-cotton fibres adsorbents. In sorption of Cu(II), Zn(II) and Ni(II) ions, C. sempervirens-chitosan microcapsules were more effective than the two adsorbent; chitosan-cotton fibres and chitosan-cellulose composite sorbents. Chitosan-alginate composite showed slightly better performance than C. sempervirens-chitosan sorbent. 4. Conclusions Only C. sempervirens-chitosan microcapsules exhibited higher affinity for Cd(II) than the chitosan beads. C. sempervirens-chitosan microcapsules removed more Cu(II), Cd(II) and Zn(II) ions and can be tested for other metal ions. C. sempervirens-chitosan microcapsules had (1) the lowest thermal stability, (2) the highest pyrolysis rate, (3) the lowest N content and (4) the largest surface. These findings demonstrate that further studies on pollen-chitosan microcapsules can aim to develop more efficient biosorbents using different pollen grains with different surface morphology and chemical composition. Chitosan/pollen ratio, deacetylation degree and molecular weight of chitosan can be manipulated to target various metal ions. Conflict of Interests The authors declare no conflict of interests.

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Supplementary materials FT-IR spectra, TGA thermogram and SEM images of the pollen-chitosan microcapsules are given in supplementary materials. References 1. Abou El-Reash, Y.G., Otto, M., Kenawy, I.M., Ouf, A.M. 2011. Adsorption of Cr(VI) and As(V) ions by modified magnetic chitosan chelating resin. Int. J. Biol. Macromol. 49, 513-522. 2. Aider, M. 2010. Chitosan application for active bio-based films production and potential in the food industry: Review. LWT - Food Sci. Technol. 43, 837-842. 3. Alonso, D., Gimeno, M., Olayo, R., Vázquez-Torres, H., Sepúlveda-Sánchez, J.D., Shirai, K. 2009. Cross-linking chitosan into UV-irradiated cellulose fibers for the preparation of antimicrobial-finished textiles. Carbohydr. Polym. 77, 536-543. 4. Altun, G.D., Cetinus, S.A. 2007. Immobilization of pepsin on chitosan beads. Food Chem. 100, 964-971. 5. Bilal, M., Shah, J.A., Ashfaq, T., Gardazi, S.M., Tahir, A.A., Pervez, A., Haroon, H., Mahmood, Q. 2013. Waste biomass adsorbents for copper removal from industrial wastewater--a review. J. Hazard. Mater. 263 Part 2, 322-333. 6. Cooden, M.C., J. 1965. Flora of Turkey and the East Aegean Island. Edinburgh University Press, Edinburgh. 7. Çeter, T., Pinar, N.M., Güney, K., Yildiz, A., Aşcı, B., Smith, M. 2011. A 2-year aeropalynological survey of allergenic pollen in the atmosphere of Kastamonu, Turkey. Aerobiologia 28, 355-366.

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8. Damodaran, D., Vidya Shetty, K., Raj Mohan, B. 2014. Uptake of certain heavy metals from contaminated soil by mushroom--Galerina vittiformis. Ecotoxicol. Environ. Saf. 104, 414-422. 9. Foo, K.Y., Hameed, B.H. 2010. Insights into the modeling of adsorption isotherm systems. Chem. Eng. J. 156, 2-10. 10. Guibal, E. 2004. Interactions of metal ions with chitosan-based sorbents: a review. Sep. Purif. Technol. 38, 43-74. 11. He, J., Chen, J.P. 2014. A comprehensive review on biosorption of heavy metals by algal biomass: materials, performances, chemistry, and modeling simulation tools. Bioresour. Technol. 160, 67-78. 12. Hu, C.-Y., Lo, S.-L., Chang, C.-L., Chen, F.-L., Wu, Y.-D., Ma, J.-l. 2013. Treatment of highly turbid water using chitosan and aluminum salts. Sep. Purif. Technol. 104, 322-326. 13. Kaya, M., Baran, T., Mentes, A., Asaroglu, M., Sezen, G., Tozak, K.O. 2014. Extraction and characterization of alpha-chitin and chitosan from six different aquatic invertebrates. Food Biophys. 9, 145-157. 14. Kieu, H.T., Muller, E., Horn, H. 2011. Heavy metal removal in anaerobic semicontinuous stirred tank reactors by a consortium of sulfate-reducing bacteria. Water Res. 45, 3863-3870. 15. Liu, X., Lee, D.-J. 2014. Thermodynamic parameters for adsorption equilibrium of heavy metals and dyes from wastewaters. Bioresour. Technol. 160, 24-31. 16. Machado, M.D., Soares, E.V., Soares, H.M. 2010. Removal of heavy metals using a brewer's yeast strain of Saccharomyces cerevisiae: chemical speciation as a tool in the prediction and improving of treatment efficiency of real electroplating effluents. J. Hazard. Mater. 180, 347-353.

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17. Muzzarelli, R.A.A. 2011. Chitosan composites with inorganics, morphogenetic proteins and stem cells, for bone regeneration. Carbohydr. Polym. 83, 1433-1445. 18. Ong, S.Y., Wu, J., Moochhala, S.M., Tan, M.H., Lu, J. 2008. Development of a chitosan-based wound dressing with improved hemostatic and antimicrobial properties. Biomaterials 29, 4323-4332. 19. Pal, A., Pan, S., Saha, S. 2013. Synergistically improved adsorption of anionic surfactant and crystal violet on chitosan hydrogel beads. Chem. Eng. J. 217, 426-434. 20. Paulino, A.T., Guilherme, M.R., Reis, A.V., Tambourgi, E.B., Nozaki, J., Muniz, E.C. 2007. Capacity of adsorption of Pb2+ and Ni2+ from aqueous solutions by chitosan produced from silkworm chrysalides in different degrees of deacetylation. J. Hazard. Mater. 147, 139-147. 21. Pawlak, A., Mucha, M. 2003. Thermogravimetric and FTIR studies of chitosan blends. Thermochim. Acta 396, 153-166. 22. Sarkar, M., Majumdar, P. 2011. Application of response surface methodology for optimization of heavy metal biosorption using surfactant modified chitosan bead. Chem. Eng. J. 175, 376-387. 23. Vieira, R.S., Beppu, M.M. 2006. Interaction of natural and crosslinked chitosan membranes with Hg(II) ions. Colloid. Surface. A 279, 196-207. 24. Wan Ngah, W.S., Teong, L.C., Hanafiah, M.A.K.M. 2011. Adsorption of dyes and heavy metal ions by chitosan composites: A review. Carbohydr. Polym. 83, 14461456. 25. Wang, J., Chen, C. 2009. Biosorbents for heavy metals removal and their future. Biotechnol. Adv. 27, 195-226. 26. Wang, J., Chen, C. 2014. Chitosan-based biosorbents: modification and application for biosorption of heavy metals and radionuclides. Bioresour. Technol. 160, 129-141.

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27. Wang, W.B., Huang, D.J., Kang, Y.R., Wang, A.Q. 2013. One-step in situ fabrication of a granular semi-IPN hydrogel based on chitosan and gelatin for fast and efficient adsorption of Cu2+ ion. Colloid. Surface. B 106, 51-59. 28. Wu, F.C., Tseng, R.L., Juang, R.S. 2010. A review and experimental verification of using chitosan and its derivatives as adsorbents for selected heavy metals. J. Environ. Manage. 91, 798-806.

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Figure Legends Fig 1. Effect of pollen-chitosan microcapsules (a: A. negundo-chitosan, b: C. sempervirenschitosan, c: P. nigra-chitosan) amount on the adsorption of metals on pollen-chitosan microcapsules at initial pH; metal concentration 10 mg L-1; volume of solution 25 mL; temperature 20°C. Fig. 2. Effect of contact time on the sorption of metals by pollen-chitosan microcapsules (a: A. negundo-chitosan, b: C. sempervirens-chitosan, c: P. nigra-chitosan) at initial pH; amount of pollen-chitosan microcapsules 0.1 g; temperature 20°C; initial metal concentration 10 mg L-1; volume of metal solution, 25 mL. Fig. 3. Effect of pH on the sorption of metals by pollen-chitosan microcapsules (a: A. negundo-chitosan, b: C. sempervirens-chitosan, c: P. nigra-chitosan). Amount of pollenchitosan microcapsules 0.1 g; temperature 20°C; initial metal concentration 10 mg L-1; volume of metal solution, 25 mL.

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Table Legends Table 1. Metals sorption capacity of the pollen-chitosan microcapsules. Table 2. Thermodynamic parameters for the adsorption of metals on pollen-chitosan microcapsules. Table 3. Parameters of Freundlich and Langmuir isotherms for sorption of metals on pollenchitosan microcapsules.

19

20

21

22

Table 1

Sorbents

chitosan beads A. negundochitosan C. sempervirenschitosan P. nigrachitosan

Cd (pH:5.35) % mg g-1 26.60 26.64

Metals Cr (pH:4.63) Cu (pH:5.18) % mg g-1 % mg g-1 58.80 58.80 40.80 40.66

Ni (pH:5.34) % mg g-1 16.20 16.20

Zn (pH:5.34) % mg g-1 21.10 21.11

25.13

32.82

63.77

70.40

48.45

50.58

22.81

22.42

22.24

27.78

52.46

65.98

58.46

64.63

64.37

67.10

22.81

22.42

39.91

49.55

25.77

30.91

38.34

41.54

41.07

31.01

17.25

20.43

21.62

28.23

23

Table 2

Isotherms Metal ions

Sorbents chitosan beads

A. negundo-chitosan

Cd(II)

C. sempervirens-chitosan

P. nigra-chitosan chitosan beads

A. negundo-chitosan

Cr(III)

C. sempervirens-chitosan P. nigra-chitosan

chitosan beads A. negundo-chitosan

Cu(II)

C. sempervirens-chitosan P. nigra-chitosan

chitosan beads A. negundo-chitosan

Ni(II)

C. sempervirens-chitosan

P. nigra-chitosan chitosan beads

A. negundo-chitosan

Zn(II)

C. sempervirens-chitosan P. nigra-chitosan

Freundlich

Langmuir 2

Q0

n

R

0.321

2.119

0.917

0.152

0.827

0.959

19.861

0.654

0.981

0.281

0.466

0.692

1.159

5.181

0.842

0.678

135.593

0.938

2.891

9.709

0.325

0.330

25.404

0.938

2.723

2.198

0.941

1.164

38.805

0.988

179.061

0.618

0.952

1.079

8.111

0.774

10.375

1.484

0.878

1.859

68.842

0.885

1.531

4.464

0.568

0.999

66.600

0.916

5.888

1.706

0.990

1.456

60.650

0.916

111.429

0.549

0.739

0.349

0.802

0.263

1.750

10.989

0.784

1.222

12224.939

0.952

1.365

30.303

0.247

0.750

149.925

0.867

1.153

1.362

0.910

0.813

2.398

0.949

8.974

0.703

0.955

0.561

0.897

0.451

2.848

32.258

0.195

0.541

270.563

0.642

2.163

6.452

0.274

0.398

19.881

0.968

0.552

3.205

0.890

0.246

10.690

0.954

17.620

0.606

0.956

0.382

0.551

0.920

1.151

52.632

0.241

0.867

433.651

0.892

2.124

2.328

0.221

0.471

67.259

0.982

24

b

2

k

R

Table 3

Metals

∆G° (J mol−1)

Sorbents ∆H°(J mol−1)

∆S°(J K−1 mol−1)

T=298.15 (K)

T=308.15 (K)

6933.527

7.927

4569.999

4490.725

4411.452

-6632.900

31.460

-16012.800

-16327.400

-16642.000

-512.595

2.256

-1186.260

-1208.850

-1231.450

-6789.920

31.862

-16289.700

-16608.300

-16927.000

275.924

1.762

-249.304

-266.921

-284.573

A. negundo-chitosan

-4951.500

-11.987

-1377.860

-1257.990

-1138.130

C. sempervirens-chitosan

-1363.730

7.334

-3550.280

-3623.610

-3696.950

-748.308

31.862

-10248.100

-10566.700

-10885.300

2364.790

15.835

-2356.560

-2514.910

-2673.270

-379.132

1.819

-921.488

-939.679

-957.869

-1403.940

0.115

-1438.190

-1439.340

-1440.490

-685.119

0.708

-896.352

-903.437

-910.521

385.834

-8.578

2943.469

3029.252

3115.036

-16704.800

66.597

-36560.700

-37226.700

-37892.700

-3779.830

2.585

-4550.550

-4576.400

-4602.250

-233.798

13.825

-4355.700

-4493.950

-4632.200

2814.771

-2.068

3431.343

3452.023

3472.703

-14144.700

31.460

-23524.600

-23839.200

-24153.800

C. sempervirens-chitosan

-568.890

5.266

-2138.870

-2191.520

-2244.180

P. nigra-chitosan

-995.701

14.016

-5174.690

-5314.860

-5455.620

chitosan beads A. negundo-chitosan

T=318.15 (K)

Cd(II) C. sempervirens-chitosan P. nigra-chitosan chitosan beads

Cr(III)

P. nigra-chitosan chitosan beads A. negundo-chitosan Cu(II) C. sempervirens-chitosan P. nigra-chitosan chitosan beads A. negundo-chitosan Ni(II) C. sempervirens-chitosan P. nigra-chitosan chitosan beads A. negundo-chitosan Zn(II)

25

Highlights Preparation of biodegradable, biocompatible and nontoxic pollen-chitosan microcapsule Characterisation of pollen-chitosan microcapsules by SEM, FTIR, TGA and EA Application in heavy metal removal: Cd(II), Cr(III), Cu(II), Ni(II) and Zn(II) The novel pollen-chitosan biosorbents showed higher performance in Cd(II) removal C. sempervirens pollen-chitosan microcapsules showed the highest removal performance

26