International Journal of Biological Macromolecules 79 (2015) 300–308
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An indigenous Halomonas BVR1 strain immobilized in crosslinked chitosan for adsorption of lead and cadmium Manasi a , Vidya Rajesh a , N. Rajesh b,∗ a Department of Biological Sciences, Birla Institute of Technology and Science, Pilani-Hyderabad Campus, Jawahar Nagar, Shameerpet Mandal, R.R. Dist 500 078, India b Department of Chemistry, Birla Institute of Technology and Science, Pilani-Hyderabad Campus, Jawahar Nagar, Shameerpet Mandal, R.R. Dist 500 078, India
a r t i c l e
i n f o
Article history: Received 16 February 2015 Received in revised form 23 April 2015 Accepted 27 April 2015 Available online 6 May 2015 Keywords: Chitosan Halomonas BVR1 Adsorption
a b s t r a c t Biomacromolecules play an important role in the adsorption of metals. In this context, we have studied the potential of an indigenous Halomonas BVR1 strain (isolated from an electronic industry effluent) immobilized in a glutaraldehyde crosslinked chitosan matrix for the adsorption of lead and cadmium. Adequate physico-chemical characterizations and the study of thermodynamic and kinetic parameters authenticated the experimental observations and the interaction mechanism. The adsorption was facile in the pH range 5–7 and pseudo second order kinetic model was favourable for both the metals. The Langmuir adsorption capacities for lead and cadmium were found to be 24.15 mg g−1 and 23.88 mg g−1 respectively. The negative G values confirmed the thermodynamic feasibility and this lucid approach highlights the utility of green methodology for heavy metal adsorption. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The potential toxicity of electronic waste is a global problem that requires efficient remediation strategies [1–3]. The toxic content in electronic waste originates from heavy metals that gain entry into the ecosystem as a result of their bioaccumulation tendency [4]. Lead and cadmium are commonly present in electronic waste. The usage of lead is prevalent in electrical industries, antifouling agents and fungicides. The toxicity of lead leads to intellectual disability in children resulting in developmental delay [5]. High concentrations of lead also cause chronic damage to the nervous system [6]. Cadmium is a typical example of a cumulative toxin that changes the DNA methylation leading to a modified gene expression. It is primarily toxic to kidneys resulting in renal dysfunction. The other long term effects due to excess cadmium include osteoporosis, developmental delay and lung cancer. Hence, removal of these heavy metals is of paramount importance [7,8]. Although, several chemical treatments have been proposed, they have certain intrinsic disadvantages and limitations for practical applications [9]. Methods such as reverse osmosis, precipitation, solvent extraction etc. are associated with high capital costs, sludge generation and residual toxicity. Hence, development of low cost
∗ Corresponding author. Tel.: +91 40 66303503; fax: +91 40 66303998. E-mail address: [email protected] (N. Rajesh). http://dx.doi.org/10.1016/j.ijbiomac.2015.04.071 0141-8130/© 2015 Elsevier B.V. All rights reserved.
and greener biosorbents would be a more viable option [10]. In this context, heavy metal resistant bacterial strains and plant materials have proved their worth [11,12] towards environmental remediation. More recently, multiwalled carbon nanotubes have also been studied for the adsorption of metals [13]. With the increasing hazards due to heavy metal pollution, biosorption is more effective as an eco-friendly option [14,15] for the sequestration of heavy metal ions. Microbial biomasses when used as such for metal remediation possess certain drawbacks such as small particle size, biomass swelling and poor mechanical strength [16,17]. This leads to excessive hydrostatic pressure required to generate suitable flow rates in case of continuous bioreactor or column studies. These drawbacks can be avoided by using suitably immobilized cell systems. Immobilization of microorganisms on a polymeric matrix circumvents these problems by providing controlled particle size, easy regeneration of the biomass, and marginal obstruction under continuous flow processes [18]. Furthermore, porous polymeric adsorbents find more utility for adsorption due to their ability to interact with the metal ions superficially and also through their void volume [19]. Recently, an indigenous heavy metal resistant bacterial strain namely, Halomonas BVR1 was isolated from an electronic industry effluent and the detailed biochemical as well as molecular characterizations (16S-rDNA sequencing) has been reported [20]. Being a halophile, this strain could tolerate high salt concentrations and hence this distinct feature offers advantage over other bacterial strains in the remediation of heavy metals from effluent
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Fig. 1. Graphical illustration of the microbe immobilized in chitosan crosslinked with glutaraldehyde.
samples. Encouraging results have been reported using the indigenously developed strain Halomonas BVR1 as such and after the immobilization in alginate for the adsorption of cadmium [20] and lead [21] respectively. Nevertheless, immobilizing the bacterial strain in other biopolymer matrices such as chitosan offers higher adsorption capacity due to the presence of diverse functional groups required for metal chelation. Hence, chitosan biopolymer was chosen as an effective support for the immobilization of this bacterial strain. Increasing the adsorption capacity coupled with high selectivity for metal ions in solution is attained through chemical crosslinking and grafting of new functional groups on the biosorbent. Chitosan is a green and biodegradable polymer endowed with amino and hydroxyl groups ideally suited for metal chelation. Furthermore, crosslinking with glutaraldehyde enhances the physical and biological stability by imparting mechanical strength to the polymer matrix especially during scale up operations in column studies. The deacetylated form of chitin i.e., chitosan has proved its worth towards metal complexation [22–24]. Chitosan is a renewable biopolymer composed of -2-amino-2-deoxy-d-glucopyranose and residual 2-acetamido2-deoxy-d-glucopyranose units. The free amine and hydroxyl groups present in the chitosan polymer chain interacts with various metal ions through ion exchange and/or chelation mechanism [25]. In the present work, the indigenously isolated bacterial strain was immobilized in chitosan crosslinked with glutaraldehyde. The following sections give details relevant to the preparation, characterization and interaction between the metal ions and the biosorbent. 2. Materials and methods 2.1. Isolation and characterization of the bacterial strain The heavy metal resistant microbial strain Halomonas BVR1 (Gen Bank Accession number: KC178681) was isolated [20] from an electronic industry effluent that discharges lead and cadmium. The selection of this strain was based on its resistance to various metals [20]. This bacterial strain was used for the immobilization in chitosan matrix to study the adsorption of lead and cadmium. 2.2. Preparation of the biosorbent The selected strain [20], being a halophile was grown in a Luria Bertani (LB medium) containing NaCl at an overall concentration of
1.0 mol L−1 . Bacterial cells were harvested at the exponential phase by centrifugation at 7800 rpm for 10 min. The harvested cells were dried at 60 ◦ C and subsequently used for immobilization in chitosan. About 1.0 g of chitosan was dissolved in acetic acid (2%, v/v) and made into slurry. A 0.5 g of the powdered microbial cells was added and the mixture was stirred for 30 min. In order to foster the crosslinking, 5% glutaraldehyde in aqueous medium was added to chitosan in the ratio 40:1 (v/v) and stirred vigorously for 5 min. The resultant mixture was refrigerated at 4 ◦ C for 24 h to promote crosslinking and further washed to obtain neutral pH. The biomass immobilized chitosan (Fig. 1) obtained through the above procedure was dried in an oven at 60 ◦ C [26] and used for biosorption experiments. In order to assess the adsorption efficacy, a comparison of the bacteria immobilized chitosan as well as pure chitosan (without the bacteria) were also studied.
2.3. Biosorption experiments The Halomonas BVR1 strain was selected for this study based on its resistance to heavy metals. Among the various strains isolated, Halomonas BVR1 strain could resist lead and cadmium up to 400 mg L−1 and 250 mg L−1 levels respectively [20,21]. Batch adsorption experiments were conducted at 25 ◦ C using 0.5 g of the immobilized biosorbent and equilibrated individually with 25 mL of varying concentrations of metal ion solutions (Pb and Cd) ranging from 40 mg L−1 to 500 mg L−1 for isotherm studies. Similarly, 0.5 g of pure chitosan (without bacteria) was also equilibrated with varying concentrations of the metal ion solutions. The equilibrations were performed using an orbital incubator shaker (Biotechnics, India) at 120 min and 180 min respectively. The concentrations of metal ions in the solution phase were monitored by atomic absorption spectrometry (Shimadzu AA 7000). The equilibrium adsorption capacities (qe ) for lead and cadmium were obtained from the difference between the initial (Co ) and equilibrium concentrations (Ce ) in the solution phase using the relation [20]: qe =
(Co − Ce )V W
(1)
Based on the optimum concentration at which effective adsorption was observed, 40 mg L−1 lead at pH 7.0 and 80 mg L−1 cadmium at pH 6.8 were chosen as the initial metal ion concentrations for the thermodynamics and kinetic studies respectively.
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Fig. 2. FTIR spectrum of the chitosan polymer and the immobilized strain before and after adsorption of lead and cadmium.
2.4. Analytical characterization The surface characterizations were studied in order to understand the interaction of metal ions with the biosorbent. These studies were performed using FT-IR, SEM and EDAX techniques. The spectral shifts in the functional groups before and after adsorption were ascertained using a Jasco 4200 FTIR spectrometer in the range 400 cm−1 –4000 cm−1 at 4 cm−1 resolution. Morphology of the biosorbent and the involvement of specific groups in metal binding were characterized using SEM-EDAX (Hitachi-S520 and Hitachi SU 1510) studies. Optical imaging was done using an Olympus CH20i microscope. The bacterial pellet (cells harvested after centrifugation) samples were fixed using 4% glutaraldehyde in phosphate buffer (pH 6.9, 0.02 mol L−1 ) for 1 h. The sample was washed with increasing percentage of ethanol ranging from 10 to 100% for 20 min. The pellet was air dried and used for SEMEDAX analysis and optical imaging studies. The optical images were recorded for the microbe immobilized chitosan matrix before and after adsorption of the metal ions. The biosorbent was spread on a glass slide and the images were taken at 4× magnification. All the experiments were carried out in triplicate and chi square test of significance was used to study the applicability of the isotherms.
3. Results and discussion 3.1. FTIR and SEM-EDAX analysis The FTIR spectra of chitosan and microbe immobilized biopolymer before and after adsorption are shown in Fig. 2. The broad band around 3440 cm−1 corresponds to the –NH and –OH stretching vibrations [27]. The C H stretching vibration was observed at 2950 cm−1 . The C H bending vibrations could be identified by a prominent peak at 1424 cm−1 . The peak at 1152 cm−1 could be attributed to C O vibration of the polysaccharide. The peak at 1595 cm−1 corresponds to NH amide band II vibrations. Certain characteristic changes were observed after immobilization with the biomass. The emergence of a peak at 1659 cm−1 indicates the C O in amide group [28]. The biosorbent showed a shift in the amino peak from 1595 cm−1 to 1591 cm−1 [29]. The appearance of peak at 1627 cm−1 is indicative of C N imine group of the glutaraldehyde crosslinked chitosan. After the adsorption of metal ions, the shift in wavenumber from 1659 cm−1 to 1648 cm−1 shows the effectiveness of interaction of lead and cadmium with the C O of the
amide (CONH) group. The amide functionality and the involvement of nitrogen lone pair from the NH2 group towards the binding of cadmium and lead were evident from the shifts in the peak from 1627 cm−1 to 1634 cm−1 respectively [30,31]. The SEM images of the microbe immobilized in crosslinked chitosan taken at 300× magnification showed characteristic coprecipitation of the metal ions in the form of salt like deposition over the biosorbent surface. The biosorbent morphology appeared smooth before the adsorption process (Fig. 3A–C). The optical images (Fig. 3D and E) clearly show that the biosorbent has the potential to accommodate the metal ions in their voids. The images were acquired after adding dithizone as a spot colouring agent [32] to the biosorbent before and after metal adsorption. Accordingly, lead and cadmium which are known to form stable metal chelates with dithizone gave a characteristic pink colour to the biosorbent surface. This implies that the metal ions were adsorbed effectively on the microbe immobilized surface through interaction with the various functional groups. The EDAX spectral analysis (Fig. 3F–H) of the Halomonas BVR1 microbe in chitosan shows the presence of C, N, O and Ca as distinct peaks. In general, the biosorption processes involve an ion exchange mechanism [21,33] wherein, some of the Ca ions in the biosorbent surface are exchanged for the respective metal ions (Pb and Cd) respectively. The decrease in the calcium ion concentration is evident from the disappearance of Ca peak after the adsorption of cadmium. The decrease in intensity of the Ca peaks and emergence of more prominent peaks due to lead adsorption are also evident from the EDAX spectrum. 3.2. Metal adsorption studies and mechanism The influence of pH is a key parameter that needs to be optimized in adsorption processes. The adsorption of metal cations (Pb2+ and Cd2+ ) increases with pH and the maximum adsorption was observed near neutral pH. The protonation of amino group in the biosorbent and its subsequent interaction with the metal ions could be expressed as Microbe-Chitosan–NH3 + + M2+ Microbe-Chitosan–NH2 – M2+ + H+
(2)
With increase in pH of the aqueous solution, the deprotonation of the amino groups are more probable with a corresponding
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Fig. 3. (A) SEM micrographs of the biosorbent after lead adsorption. (B) SEM micrographs of the biosorbent before metal adsorption. (C) SEM micrographs of the biosorbent after cadmium adsorption. (D) Optical images of the biosorbent after heavy metal adsorption. (E) Optical images of the biosorbent before heavy metal adsorption. (F) EDAX spectrum showing the adsorption of Cadmium. (G) EDAX spectrum showing the adsorption of lead. (H) EDAX spectrum of biosorbent.
decrease in the number of H+ ions. This leads to an overall decline in the competition of active binding sites on the biosorbent. However, at an acidic pH, the biosorbent acts as a weak base and hence the amino groups readily get protonated using the protons available
in the aqueous solution. At low pH, the protons would compete with the cationic lead and cadmium for active adsorption sites leading to a reduction in the adsorption. The metal adsorptions were found to be quite effective (Fig. 4) in the pH range 5–7 where the
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parameter relates the effectiveness of Langmuir adsorption for these metal ions and this is given by the expression: RL =
1 1 + bC0
(4)
The RL (in the range 0–1) values were found to be 0.0820 and 0.128 for lead and cadmium signifying a favourable and reversible adsorption process [35]. The Freundlich isotherm is represented as: log qe = log KF +
Fig. 4. Percentage of metal removal at different pH.
competition from the H+ is relatively low. The availability of lone pair of electrons on the nitrogen atom of the amine functionality also contributes to the interaction with metal cations near neutral pH. 3.3. Isotherm studies The equilibrium data for the adsorption of lead and cadmium at various initial metal ion concentrations were modelled using certain distinct isotherm models. Among them, Langmuir and Freundlich [34] isotherm models were examined in detail to study the adsorption behaviour of metal ions on the biosorbent owing to their best fit to the experimental data. The Langmuir isotherm can be expressed as: 1 Ce Ce = + qe qo b qo
(3)
where qe is the amount of metal ions adsorbed at equilibrium (mg g−1 ), qo is the maximum adsorption capacity (mg g−1 ), Ce is the equilibrium concentration of the adsorbate (mg L−1 ), and b (L mg−1 ) is the Langmuir constant. The maximum adsorption capacity (qo ) for lead and cadmium obtained from the plot of Ce against Ce /qe (Fig. 5A) were found to be 24.15 mg g−1 and 23.88 mg g−1 . In addition, RL a dimensionless
A
1 log Ce , n
(5)
where qe is the amount of metal adsorbed (mg g−1 ), Ce is the equilibrium concentration of the adsorbate (mg L−1 ), and Kf and n are the Freundlich constants which indicate the adsorption capacity and the adsorption intensity respectively. The value of the constants Kf and n from the Freundlich plot of log Ce against log qe (Fig. 5B) were found to be 2.365 and 1.69 mg1−1/n g−1 L1/n for lead and 2.02 and 2.38 mg1−1/n g−1 L1/n for cadmium. The equilibrium adsorption data for cadmium suggests the formation of a monolayer following the Langmuir isotherm and this is also evident from the low chi square (2 ) value (0.222). This feature can be explained by considering the ionic radii of lead and cadmium. Since, the ionic radius of cadmium is less than lead, a minor difference exists between the neighbouring sites in the biosorbent thus favouring Langmuir isotherm [36]. However, the equilibrium data for lead fits well with the Freundlich isotherm model as evident from the low 2 value (0.15). The mean free energies for lead and cadmium adsorption obtained from the Dubinin-Radushkevich (D-R) isotherm model (EDR = 1/(2ˇ)−0.5 ) were found to be 10.20 kJ mol−1 and 1.98 kJ mol−1 respectively. The isotherm parameters obtained from the above three models with and without the immobilized bacteria are summarized in Table 1A and Table 1Brespectively. The Langmuir adsorption capacities were found to be low in the absence of bacteria. This shows that the microorganism immobilized biopolymer has higher uptake efficiency for the metal ions. The D-R model suggests that physisorption phenomenon accounts for the uptake of cadmium and an ion exchange mechanism is more probable in case of lead adsorption. Furthermore, in case of lead, the formation of a metal–ligand complex through an electrostatic or coordination interaction is also in accordance with the physico-chemical adsorption phenomenon. Since in physisorption, the energy of interaction between the cadmium ions and the biosorbent is marginally higher than the energy of condensation of the adsorbate, less activation
B
Fig. 5. Isotherm models. (A) Langmuir plot for both the metals. (B) Freundlich plot of both the metals.
Manasi et al. / International Journal of Biological Macromolecules 79 (2015) 300–308 Table 1A Isotherm parameters obtained from various models for microbe immobilized biopolymer. Sl. no.
Isotherm models
1.
Langmuir
2.
Freundlich
3.
DubininRadushkevich
Parameters
−1
qo (mg g ) b (L mg−1 ) RL r2 2 KF (mg1−1/n g−1 L1/n ) n r2 2 qm (mg g−1 ) ˇ EDR (kJ mol−1 ) r2 2
Values Lead
Cadmium
24.154 0.1149 0.0820 0.820 1.590 2.365 1.691 0.925 0.15 12.590 0.004 10.204 0.518 2.7
23.88 0.136 0.128 0.98 0.22 2.02 2.38 0.81 101.00 14.497 0.127 1.988 0.646 82.35
Table 1B Isotherm parameters obtained from various models for pure chitosan without the bacteria. Sl. no.
Isotherm models
Parameters
1.
Langmuir
2.
Freundlich
3.
DubininRadushkevich
qo (mg g−1 ) b (L mg−1 ) RL r2 KF (mg1−1/n g−1 L1/n ) n r2 qm (mg g−1 ) ˇ EDR (kJ mol−1 ) r2
Values Lead
Cadmium
17.54 0.093 0.017 0.54 2.61 2.032 0.92 16.26 2.817 0.421 0.80
13.848 0.12 0.16 0.78 2.67 2.202 0.87 17.01 5.415 0.303 0.86
energy is needed for adsorption [37]. Hence, the adsorption is favoured at low temperatures. 3.4. Kinetic uptake Kinetics is an important parameter to be evaluated in a batch adsorption experiment. The pseudo first order and pseudo second order models were used to assess the kinetic data (Fig. 6A and B). The first order and second order equations can be expressed as [38,39]: log (qe − qt ) = log qe −
k1 t 2.303
(6)
1 t t = + qt qe k2 q2e
(7)
The approach to adsorption kinetics could be explained through the following modes: a) Film diffusion of metal ions to the external surface of the microbe immobilized chitosan matrix. b) Particle diffusion through the pores of microorganism immobilized biosorbent.
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c) Internal diffusion of Pb2+ and Cd2+ into the biosorbent. The particle diffusion followed by an internal diffusion of the metal ions onto the biosorbent matrix plays a key role in understanding the kinetics of adsorption. The adsorption was quite effective since the equilibrium was reached within 2–3 h duration. However, there were slight differences in the exact equilibrium time attained among the two metals [40]. This could be associated with the difference in the ion exchange affinities of cadmium and lead ions. The probability of lead ions having affinity towards the biosorbent is higher than cadmium. With increase in affinity, the rate of adsorption increases and this leads to a marginal decrease in the pH of the solution. The decrease in pH after the adsorption of lead was more as compared to cadmium. Additionally, lead also has a higher ionic radii and this is evident from the differences between the polarizability of the metal ions. This difference accounts for the higher affinity of lead towards the microbe immobilized biopolymer biosorbent. In accordance with the higher affinities, lead reaches equilibrium faster (2 h) than cadmium (3 h) and the metal adsorption favours pseudo second order kinetics. The experimental and calculated equilibrium adsorption capacity (qe ) values from the pseudo second order model were found to be 4.9 mg g−1 and 5.68 mg g−1 for cadmium and 4.9 mg g−1 and 5.02 mg g−1 for lead respectively. The kinetic parameters (Table 2) show the applicability of second order model with a high regression coefficient. The Weber and Morris intraparticle diffusion model for Pb2+ and Cd2+ were also studied using the expression [41]: √ qt = kint t + C (8) where kint is the intraparticle diffusion constant and qt is the amount of heavy metals adsorbed at time t. The Fig. 6C shows the plot of qt versus t0.5 for a particular concentration of lead (40 mg L−1 ) and cadmium (80 mg L−1 ). The multilinear fit obtained for cadmium shows that the first portion of curve relates to the instantaneous surface adsorption followed by intraparticle diffusion in the second portion. In case of lead, the plot shows a good linear fit with a definite intercept. These plots reflect boundary layer as well as diffusion controlled adsorption processes. 3.5. Adsorption thermodynamics The energetics of biosorption could be described through the study of various thermodynamic parameters involving free energy (G0 ), entropy (S0 ) and enthalpy (H0 ) changes. The above parameters were calculated using the equations [42]: G0 = −RT ln K ln K =
−H 0 RT
+
(9) S 0
(10)
R
The K values were obtained from the ratio of metal ion concentrations on the microbe immobilized chitosan surface to the equilibrium concentration in solution (K = C(biosorbent) /C(solution) ). The free energy changes were accordingly found to be −11.77, −11.76, −10.90 and −7.75 kJ mol−1 for the biosorption of lead at 293, 303, 313 and 323 K respectively. Likewise, the G values for cadmium were also negative at these temperatures (Table 3). The
Table 2 Kinetic and intraparticle rate constant data for the adsorption of metal ions. Metal ion
Second order rate constant k2 [g mg−1 min−1 ]
Regression coefficient
First order rate constant k1 [min−1 ]
Regression coefficient
Intraparticle rate constant kint [mg g−1 min−½ ]
Lead Cadmium
0.07 0.007
0.98 0.99
0.023 0.026
0.974 0.98
20.166 0.069
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Fig. 6. Kinetic plots obtained from the pseudo first-order equation (A), pseudo second-order equation (B), and intraparticle diffusion plot (C).
diffusion of metal ions [43] from the aqueous solution onto the polymeric interface is more probable near room temperature. The adsorption was thermodynamically feasible and spontaneous as indicated by the negative free energy values. The enthalpy (H0 ) and entropy (S0 ) of biosorption were estimated from the slope and intercept of the plot of ln K against 1/T (Fig. 7). The enthalpy of biosorption (H0 ) for the cadmium and lead systems were found to be −43.6 kJ mol−1 and −50.04 kJ mol−1 respectively. Since, the Gibb’s free energy becomes less negative with temperature, the degree of spontaneity decreases with increase in temperature for both the metal ions. The biosorption process was thermodynamically favourable and exothermic resulting in negative G0 Table 3 Thermodynamic data for the adsorption of metal ions. Metal ion
Temperature/ Kelvin
G0 [kJ mol−1 ]
S0 [J mol−1 K−1 ]
H0 [kJ mol−1 ]
Cadmium
293 303 313 323 293 303 313 323
−7.99 −8.76 −7.057 −4.202 −11.77 −11.76 −10.90 −7.75
−118.80
−43.617
−128.63
−50.043
Lead
Fig. 7. Van’t Hoff isotherm plot obtained for the adsorption of metal ions on the microbe immobilized chitosan surface.
Manasi et al. / International Journal of Biological Macromolecules 79 (2015) 300–308 Table 4 Comparison of adsorption capacity against related biopolymers and bacterial strains. Adsorbent
Heavy metal
Adsorption capacity (mg g−1 )
Chitosan/cotton fibres [44] Chitosan/cellulose [45] Chitosan/sand [46] Alginate chitosan [47] Chitosan immobilized on bentonite [48] Halomonas BVR1 [20] Halomonas BVR1 immobilized in sodium alginate [21] Chitosan (present study)
Cadmium Lead Lead Cadmium Lead
15.74 26.31 12.32 6.63 15.0
Cadmium Lead
12.02 9.68
Lead Cadmium Lead
17.54 13.84 24.15
Cadmium
23.88
Halomonas BVR1 immobilized in chitosan (present study)
and H0 values. The S0 values for adsorption process were also negative (−118.80 J mol−1 K−1 and −128.63 J mol−1 K−1 ) for cadmium and lead respectively suggesting reduced randomness at the solid–solution interface. 3.6. Effect of diverse ions The influence of certain cations and anions that could be commonly associated with the electronic industry effluents were investigated at different concentrations. Ions such as zinc and cobalt did not interfere up to 50 mg L−1 level, while nickel and iron caused a reduction in the adsorption of lead and cadmium beyond 60 mg L−1 concentration. Anions such as chloride and sulphate did not affect the adsorption of lead and cadmium when their concentration levels were in the range 10–60 mg L−1 . Nitrate and phosphate caused a decrease in the percentage adsorption of lead and cadmium beyond 70 mg L−1 . 3.7. Comparison against other biosorbents The proposed method for lead and cadmium were compared in terms of the maximum Langmuir adsorption capacity with other polymeric matrices involving chitosan [44–48]. The results showed that Halomonas BVR1 immobilized chitosan exhibits good adsorption capacity as evident from the comparison shown in Table 4. Evaluation of the potential of this bacterial strain as such [20] and after immobilization in alginate matrix [21] also indicates that Halomonas BVR1–chitosan combination exhibits higher Langmuir adsorption capacity for lead and cadmium. 3.8. Regeneration of biosorbent Preliminary studies for regeneration of the biosorbent showed that 0.5 mM EDTA has the potential to complex both the metal ions and desorb them effectively. Further studies are in progress to check the reusability of the biosorbent. 4. Conclusions The biopolymer strengthens the interaction after immobilization with the bacteria. The biosorption process is spontaneous, exothermic and favours pseudo second order kinetics. The Langmuir adsorption capacities were found to be 24.15 mg g−1 and 23.88 mg g−1 for lead and cadmium respectively. Characterization
307
techniques such as FT-IR, SEM, EDAX and optical imaging techniques ascertained the adsorption of lead and cadmium onto the bacteria immobilized chitosan biosorbent. Electrostatic and ion exchange mechanisms govern the interaction between the metal ions and the biosorbent. As a complexing agent, EDTA showed good potential to desorb lead and cadmium. Hence, the combination of Halomonas BVR1 and chitosan is capable of adsorbing lead and cadmium effectively from aqueous solution. Acknowledgements Thanks to Indian Institute of Chemical Technology, Hyderabad, India and Fore Vision Analytical Instruments, Hyderabad, India for their assistance in the characterization of samples through SEMEDAX analysis. References [1] A. Sepúlveda, M. Schluep, F.G. Renaud, M. Streicher, R. Kuehr, C. Hagelüken, A.C. Gerecke, Environ. Impact Assess. Rev. 30 (2010) 28–41. [2] A.O.W. Leung, N.S. Duzgoren-Aydin, K.C. Cheung, M.H. Wong, Environ. Sci. Technol. 42 (2008) 2674–2680. [3] C.S.C. Wong, S.C. Wu, N.S. Duzgoren-Aydin, A. Aydin, M.H. Wong, Environ. Pollut. 145 (2007) 434–442. [4] D. Singh, A. Tiwari, R. Gupta, J. Agric. Technol. 8 (2012) 1–11. [5] S. Reena, G. Neetu, M. Anurag, G. Rajiv, Indian J. Pharmacol. 43 (2011) 246–253. [6] G. Johannes, S. Franziska, G.S. Christian, E. Vera, B. Paul, R. Andrea, A.G. David, J. Occup. Med. Toxicol. 1 (2006) 1–6. [7] M.E. Romero-Gonzalez, C.J. Williams, P.H. Gardiner, Environ. Sci. Technol. 35 (2001) 25–30. [8] F. Pierron, L. Baillon, M. Sow, S. Gotreau, P. Gonzalez, Environ. Sci. Technol. 48 (2014) 797–803. [9] Z. Hu, L. Lei, Y. Li, Y. Ni, Sep. Purif. Technol. 31 (2003) 13–18. [10] M. Minamisawa, H. Minamisawa, S. Yoshida, N. Takai, J. Agric. Food Chem. 52 (2004) 5606–5611. [11] D.P. Higham, P.J. Sadler, M.D. Scawen, Science 225 (1984) 205–207. [12] A.S. Moffat, Science 285 (1999) 369–370. [13] F.F. Yan, Y.R. He, C. Wu, Y.Y. Cheng, W.W. Li, H.Q. Yu, Environ. Sci. Technol. Lett. 1 (2014) 128–132. [14] N.N.N.A. Rahman, M. Shahadat, C.A. Won, F.M. Omar, RSC Adv. 4 (2014) 58156–58163. [15] N. Gaur, G. Flora, M. Yadav, A. Tiwari, Environ. Sci.: Processes Impacts 16 (2014) 180–193. [16] D. Ivánová, H. Horváthová, J. Kaduková, J.K.C. Ová, Nova Biotechnol. 10 (2010) 45–51. [17] T.A. Davis, B. Volesky, A.A. Mucci, Water Res. 37 (2003) 4311–4330. [18] J. Wang, J,C. Chen, Biotechnol. Adv. 27 (2009) 195–226. [19] M.E. Davis, Nature 417 (2002) 813–821. [20] Manasi, V. Rajesh, A.S.K. Kumar, N. Rajesh, Chem. Eng. J. 235 (2014) 176–185. [21] Manasi, V. Rajesh, A.S.K. Kumar, N. Rajesh, Chem. Eng. J. 248 (2014) 342–351. [22] A. Varma, S. Deshpande, J.F. Kennedy, Carbohydr. Polym. 55 (2004) 77–93. [23] A. Sowmya, S. Meenakshi, Int. J. Biol. Macromol. 69 (2014) 336–343. [24] P. Miretzky, A.F. Cirelli, J. Hazard. Mater. 167 (2009) 10–23. [25] T. Chakrabarty, V.K. Shahi, RSC Adv. 4 (2014) 53245–53252. [26] N. Viswanathan, S. Meenakshi, J. Hazard. Mater. 178 (2010) 226–232. [27] X.F. Sun, S.G. Wang, X.W. Liu, W.X. Gong, N. Bao, Y. Ma, Colloids Surf.: Physicochem. Eng. Asp. 324 (2008) 28–34. [28] G.I. Chen, J. Mi, X. Wu, C. Luo, J. Li, Y. Tang, J. Li, Int. J. Biol. Macromol. 49 (2011) 543–547. [29] N. Kocak, M. Sahin, S. Kucukkolbasi, Z.O. Erdogan, Int. J. Biol. Macromol. 51 (2012) 1159–1166. [30] A.H. Chen, C.Y. Yang, C.Y. Chen, C.Y. Chen, C.W. Chen, J. Hazard. Mater. 163 (2009) 1068–1075. [31] Z.K. George, A.D. Eleni, Molecules 18 (2013) 6193–6214. [32] I. Michalak, K. Chojnacka, A. Witek-Krowiak, Appl. Biochem. Biotechnol. 170 (2013) 1389–1416. [33] C. Gerente, V.K.C. Lee, P. Le Cloirec, G. Mckay, Crit. Rev. Environ. Sci. Technol. 37 (2007) 41–127. [34] K.Y. Foo, B.H. Hameed, Chem. Eng. J. 156 (2010) 2–10. [35] C. Sun, L. Sun, X.A. Sun, Ind. Eng. Chem. Res. 52 (2013) 14251–21426. [36] P. Molyneux, Nature 202 (1964) 368–370. [37] E. Guibal, Sep. Purif. Technol. 38 (2004) 43–74. [38] S. Lagergren, K. Sven, Vetenskapsakad-Akademiens Handlingar 24 (1898) 1–39. [39] Y.S. Ho, J. Hazard. Mater. 136 (2006) 681–689. [40] I.M.N. Vold, K.M. Varum, E. Guibal, O. Smidsrod, Carbohydr. Polym. 54 (2003) 471–477. [41] W.J. Weber, J.C. Morris, J. Sanit. Eng. Div. – American Society of Civil Engineers 89 (1963) 53–61. [42] A. Ramesh, D.J. Lee, J.C. Wong, J. Colloid Interface Sci. 291 (2005) 588–592.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
An indigenous Halomonas BVR1 strain immobilized in crosslinked chitosan for adsorption of lead and cadmium Manasi a , Vidya Rajesh a , N. Rajesh b,∗ a Department of Biological Sciences, Birla Institute of Technology and Science, Pilani-Hyderabad Campus, Jawahar Nagar, Shameerpet Mandal, R.R. Dist 500 078, India b Department of Chemistry, Birla Institute of Technology and Science, Pilani-Hyderabad Campus, Jawahar Nagar, Shameerpet Mandal, R.R. Dist 500 078, India
a r t i c l e
i n f o
Article history: Received 16 February 2015 Received in revised form 23 April 2015 Accepted 27 April 2015 Available online 6 May 2015 Keywords: Chitosan Halomonas BVR1 Adsorption
a b s t r a c t Biomacromolecules play an important role in the adsorption of metals. In this context, we have studied the potential of an indigenous Halomonas BVR1 strain (isolated from an electronic industry effluent) immobilized in a glutaraldehyde crosslinked chitosan matrix for the adsorption of lead and cadmium. Adequate physico-chemical characterizations and the study of thermodynamic and kinetic parameters authenticated the experimental observations and the interaction mechanism. The adsorption was facile in the pH range 5–7 and pseudo second order kinetic model was favourable for both the metals. The Langmuir adsorption capacities for lead and cadmium were found to be 24.15 mg g−1 and 23.88 mg g−1 respectively. The negative G values confirmed the thermodynamic feasibility and this lucid approach highlights the utility of green methodology for heavy metal adsorption. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The potential toxicity of electronic waste is a global problem that requires efficient remediation strategies [1–3]. The toxic content in electronic waste originates from heavy metals that gain entry into the ecosystem as a result of their bioaccumulation tendency [4]. Lead and cadmium are commonly present in electronic waste. The usage of lead is prevalent in electrical industries, antifouling agents and fungicides. The toxicity of lead leads to intellectual disability in children resulting in developmental delay [5]. High concentrations of lead also cause chronic damage to the nervous system [6]. Cadmium is a typical example of a cumulative toxin that changes the DNA methylation leading to a modified gene expression. It is primarily toxic to kidneys resulting in renal dysfunction. The other long term effects due to excess cadmium include osteoporosis, developmental delay and lung cancer. Hence, removal of these heavy metals is of paramount importance [7,8]. Although, several chemical treatments have been proposed, they have certain intrinsic disadvantages and limitations for practical applications [9]. Methods such as reverse osmosis, precipitation, solvent extraction etc. are associated with high capital costs, sludge generation and residual toxicity. Hence, development of low cost
∗ Corresponding author. Tel.: +91 40 66303503; fax: +91 40 66303998. E-mail address: [email protected] (N. Rajesh). http://dx.doi.org/10.1016/j.ijbiomac.2015.04.071 0141-8130/© 2015 Elsevier B.V. All rights reserved.
and greener biosorbents would be a more viable option [10]. In this context, heavy metal resistant bacterial strains and plant materials have proved their worth [11,12] towards environmental remediation. More recently, multiwalled carbon nanotubes have also been studied for the adsorption of metals [13]. With the increasing hazards due to heavy metal pollution, biosorption is more effective as an eco-friendly option [14,15] for the sequestration of heavy metal ions. Microbial biomasses when used as such for metal remediation possess certain drawbacks such as small particle size, biomass swelling and poor mechanical strength [16,17]. This leads to excessive hydrostatic pressure required to generate suitable flow rates in case of continuous bioreactor or column studies. These drawbacks can be avoided by using suitably immobilized cell systems. Immobilization of microorganisms on a polymeric matrix circumvents these problems by providing controlled particle size, easy regeneration of the biomass, and marginal obstruction under continuous flow processes [18]. Furthermore, porous polymeric adsorbents find more utility for adsorption due to their ability to interact with the metal ions superficially and also through their void volume [19]. Recently, an indigenous heavy metal resistant bacterial strain namely, Halomonas BVR1 was isolated from an electronic industry effluent and the detailed biochemical as well as molecular characterizations (16S-rDNA sequencing) has been reported [20]. Being a halophile, this strain could tolerate high salt concentrations and hence this distinct feature offers advantage over other bacterial strains in the remediation of heavy metals from effluent
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Fig. 1. Graphical illustration of the microbe immobilized in chitosan crosslinked with glutaraldehyde.
samples. Encouraging results have been reported using the indigenously developed strain Halomonas BVR1 as such and after the immobilization in alginate for the adsorption of cadmium [20] and lead [21] respectively. Nevertheless, immobilizing the bacterial strain in other biopolymer matrices such as chitosan offers higher adsorption capacity due to the presence of diverse functional groups required for metal chelation. Hence, chitosan biopolymer was chosen as an effective support for the immobilization of this bacterial strain. Increasing the adsorption capacity coupled with high selectivity for metal ions in solution is attained through chemical crosslinking and grafting of new functional groups on the biosorbent. Chitosan is a green and biodegradable polymer endowed with amino and hydroxyl groups ideally suited for metal chelation. Furthermore, crosslinking with glutaraldehyde enhances the physical and biological stability by imparting mechanical strength to the polymer matrix especially during scale up operations in column studies. The deacetylated form of chitin i.e., chitosan has proved its worth towards metal complexation [22–24]. Chitosan is a renewable biopolymer composed of -2-amino-2-deoxy-d-glucopyranose and residual 2-acetamido2-deoxy-d-glucopyranose units. The free amine and hydroxyl groups present in the chitosan polymer chain interacts with various metal ions through ion exchange and/or chelation mechanism [25]. In the present work, the indigenously isolated bacterial strain was immobilized in chitosan crosslinked with glutaraldehyde. The following sections give details relevant to the preparation, characterization and interaction between the metal ions and the biosorbent. 2. Materials and methods 2.1. Isolation and characterization of the bacterial strain The heavy metal resistant microbial strain Halomonas BVR1 (Gen Bank Accession number: KC178681) was isolated [20] from an electronic industry effluent that discharges lead and cadmium. The selection of this strain was based on its resistance to various metals [20]. This bacterial strain was used for the immobilization in chitosan matrix to study the adsorption of lead and cadmium. 2.2. Preparation of the biosorbent The selected strain [20], being a halophile was grown in a Luria Bertani (LB medium) containing NaCl at an overall concentration of
1.0 mol L−1 . Bacterial cells were harvested at the exponential phase by centrifugation at 7800 rpm for 10 min. The harvested cells were dried at 60 ◦ C and subsequently used for immobilization in chitosan. About 1.0 g of chitosan was dissolved in acetic acid (2%, v/v) and made into slurry. A 0.5 g of the powdered microbial cells was added and the mixture was stirred for 30 min. In order to foster the crosslinking, 5% glutaraldehyde in aqueous medium was added to chitosan in the ratio 40:1 (v/v) and stirred vigorously for 5 min. The resultant mixture was refrigerated at 4 ◦ C for 24 h to promote crosslinking and further washed to obtain neutral pH. The biomass immobilized chitosan (Fig. 1) obtained through the above procedure was dried in an oven at 60 ◦ C [26] and used for biosorption experiments. In order to assess the adsorption efficacy, a comparison of the bacteria immobilized chitosan as well as pure chitosan (without the bacteria) were also studied.
2.3. Biosorption experiments The Halomonas BVR1 strain was selected for this study based on its resistance to heavy metals. Among the various strains isolated, Halomonas BVR1 strain could resist lead and cadmium up to 400 mg L−1 and 250 mg L−1 levels respectively [20,21]. Batch adsorption experiments were conducted at 25 ◦ C using 0.5 g of the immobilized biosorbent and equilibrated individually with 25 mL of varying concentrations of metal ion solutions (Pb and Cd) ranging from 40 mg L−1 to 500 mg L−1 for isotherm studies. Similarly, 0.5 g of pure chitosan (without bacteria) was also equilibrated with varying concentrations of the metal ion solutions. The equilibrations were performed using an orbital incubator shaker (Biotechnics, India) at 120 min and 180 min respectively. The concentrations of metal ions in the solution phase were monitored by atomic absorption spectrometry (Shimadzu AA 7000). The equilibrium adsorption capacities (qe ) for lead and cadmium were obtained from the difference between the initial (Co ) and equilibrium concentrations (Ce ) in the solution phase using the relation [20]: qe =
(Co − Ce )V W
(1)
Based on the optimum concentration at which effective adsorption was observed, 40 mg L−1 lead at pH 7.0 and 80 mg L−1 cadmium at pH 6.8 were chosen as the initial metal ion concentrations for the thermodynamics and kinetic studies respectively.
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Fig. 2. FTIR spectrum of the chitosan polymer and the immobilized strain before and after adsorption of lead and cadmium.
2.4. Analytical characterization The surface characterizations were studied in order to understand the interaction of metal ions with the biosorbent. These studies were performed using FT-IR, SEM and EDAX techniques. The spectral shifts in the functional groups before and after adsorption were ascertained using a Jasco 4200 FTIR spectrometer in the range 400 cm−1 –4000 cm−1 at 4 cm−1 resolution. Morphology of the biosorbent and the involvement of specific groups in metal binding were characterized using SEM-EDAX (Hitachi-S520 and Hitachi SU 1510) studies. Optical imaging was done using an Olympus CH20i microscope. The bacterial pellet (cells harvested after centrifugation) samples were fixed using 4% glutaraldehyde in phosphate buffer (pH 6.9, 0.02 mol L−1 ) for 1 h. The sample was washed with increasing percentage of ethanol ranging from 10 to 100% for 20 min. The pellet was air dried and used for SEMEDAX analysis and optical imaging studies. The optical images were recorded for the microbe immobilized chitosan matrix before and after adsorption of the metal ions. The biosorbent was spread on a glass slide and the images were taken at 4× magnification. All the experiments were carried out in triplicate and chi square test of significance was used to study the applicability of the isotherms.
3. Results and discussion 3.1. FTIR and SEM-EDAX analysis The FTIR spectra of chitosan and microbe immobilized biopolymer before and after adsorption are shown in Fig. 2. The broad band around 3440 cm−1 corresponds to the –NH and –OH stretching vibrations [27]. The C H stretching vibration was observed at 2950 cm−1 . The C H bending vibrations could be identified by a prominent peak at 1424 cm−1 . The peak at 1152 cm−1 could be attributed to C O vibration of the polysaccharide. The peak at 1595 cm−1 corresponds to NH amide band II vibrations. Certain characteristic changes were observed after immobilization with the biomass. The emergence of a peak at 1659 cm−1 indicates the C O in amide group [28]. The biosorbent showed a shift in the amino peak from 1595 cm−1 to 1591 cm−1 [29]. The appearance of peak at 1627 cm−1 is indicative of C N imine group of the glutaraldehyde crosslinked chitosan. After the adsorption of metal ions, the shift in wavenumber from 1659 cm−1 to 1648 cm−1 shows the effectiveness of interaction of lead and cadmium with the C O of the
amide (CONH) group. The amide functionality and the involvement of nitrogen lone pair from the NH2 group towards the binding of cadmium and lead were evident from the shifts in the peak from 1627 cm−1 to 1634 cm−1 respectively [30,31]. The SEM images of the microbe immobilized in crosslinked chitosan taken at 300× magnification showed characteristic coprecipitation of the metal ions in the form of salt like deposition over the biosorbent surface. The biosorbent morphology appeared smooth before the adsorption process (Fig. 3A–C). The optical images (Fig. 3D and E) clearly show that the biosorbent has the potential to accommodate the metal ions in their voids. The images were acquired after adding dithizone as a spot colouring agent [32] to the biosorbent before and after metal adsorption. Accordingly, lead and cadmium which are known to form stable metal chelates with dithizone gave a characteristic pink colour to the biosorbent surface. This implies that the metal ions were adsorbed effectively on the microbe immobilized surface through interaction with the various functional groups. The EDAX spectral analysis (Fig. 3F–H) of the Halomonas BVR1 microbe in chitosan shows the presence of C, N, O and Ca as distinct peaks. In general, the biosorption processes involve an ion exchange mechanism [21,33] wherein, some of the Ca ions in the biosorbent surface are exchanged for the respective metal ions (Pb and Cd) respectively. The decrease in the calcium ion concentration is evident from the disappearance of Ca peak after the adsorption of cadmium. The decrease in intensity of the Ca peaks and emergence of more prominent peaks due to lead adsorption are also evident from the EDAX spectrum. 3.2. Metal adsorption studies and mechanism The influence of pH is a key parameter that needs to be optimized in adsorption processes. The adsorption of metal cations (Pb2+ and Cd2+ ) increases with pH and the maximum adsorption was observed near neutral pH. The protonation of amino group in the biosorbent and its subsequent interaction with the metal ions could be expressed as Microbe-Chitosan–NH3 + + M2+ Microbe-Chitosan–NH2 – M2+ + H+
(2)
With increase in pH of the aqueous solution, the deprotonation of the amino groups are more probable with a corresponding
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Fig. 3. (A) SEM micrographs of the biosorbent after lead adsorption. (B) SEM micrographs of the biosorbent before metal adsorption. (C) SEM micrographs of the biosorbent after cadmium adsorption. (D) Optical images of the biosorbent after heavy metal adsorption. (E) Optical images of the biosorbent before heavy metal adsorption. (F) EDAX spectrum showing the adsorption of Cadmium. (G) EDAX spectrum showing the adsorption of lead. (H) EDAX spectrum of biosorbent.
decrease in the number of H+ ions. This leads to an overall decline in the competition of active binding sites on the biosorbent. However, at an acidic pH, the biosorbent acts as a weak base and hence the amino groups readily get protonated using the protons available
in the aqueous solution. At low pH, the protons would compete with the cationic lead and cadmium for active adsorption sites leading to a reduction in the adsorption. The metal adsorptions were found to be quite effective (Fig. 4) in the pH range 5–7 where the
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parameter relates the effectiveness of Langmuir adsorption for these metal ions and this is given by the expression: RL =
1 1 + bC0
(4)
The RL (in the range 0–1) values were found to be 0.0820 and 0.128 for lead and cadmium signifying a favourable and reversible adsorption process [35]. The Freundlich isotherm is represented as: log qe = log KF +
Fig. 4. Percentage of metal removal at different pH.
competition from the H+ is relatively low. The availability of lone pair of electrons on the nitrogen atom of the amine functionality also contributes to the interaction with metal cations near neutral pH. 3.3. Isotherm studies The equilibrium data for the adsorption of lead and cadmium at various initial metal ion concentrations were modelled using certain distinct isotherm models. Among them, Langmuir and Freundlich [34] isotherm models were examined in detail to study the adsorption behaviour of metal ions on the biosorbent owing to their best fit to the experimental data. The Langmuir isotherm can be expressed as: 1 Ce Ce = + qe qo b qo
(3)
where qe is the amount of metal ions adsorbed at equilibrium (mg g−1 ), qo is the maximum adsorption capacity (mg g−1 ), Ce is the equilibrium concentration of the adsorbate (mg L−1 ), and b (L mg−1 ) is the Langmuir constant. The maximum adsorption capacity (qo ) for lead and cadmium obtained from the plot of Ce against Ce /qe (Fig. 5A) were found to be 24.15 mg g−1 and 23.88 mg g−1 . In addition, RL a dimensionless
A
1 log Ce , n
(5)
where qe is the amount of metal adsorbed (mg g−1 ), Ce is the equilibrium concentration of the adsorbate (mg L−1 ), and Kf and n are the Freundlich constants which indicate the adsorption capacity and the adsorption intensity respectively. The value of the constants Kf and n from the Freundlich plot of log Ce against log qe (Fig. 5B) were found to be 2.365 and 1.69 mg1−1/n g−1 L1/n for lead and 2.02 and 2.38 mg1−1/n g−1 L1/n for cadmium. The equilibrium adsorption data for cadmium suggests the formation of a monolayer following the Langmuir isotherm and this is also evident from the low chi square (2 ) value (0.222). This feature can be explained by considering the ionic radii of lead and cadmium. Since, the ionic radius of cadmium is less than lead, a minor difference exists between the neighbouring sites in the biosorbent thus favouring Langmuir isotherm [36]. However, the equilibrium data for lead fits well with the Freundlich isotherm model as evident from the low 2 value (0.15). The mean free energies for lead and cadmium adsorption obtained from the Dubinin-Radushkevich (D-R) isotherm model (EDR = 1/(2ˇ)−0.5 ) were found to be 10.20 kJ mol−1 and 1.98 kJ mol−1 respectively. The isotherm parameters obtained from the above three models with and without the immobilized bacteria are summarized in Table 1A and Table 1Brespectively. The Langmuir adsorption capacities were found to be low in the absence of bacteria. This shows that the microorganism immobilized biopolymer has higher uptake efficiency for the metal ions. The D-R model suggests that physisorption phenomenon accounts for the uptake of cadmium and an ion exchange mechanism is more probable in case of lead adsorption. Furthermore, in case of lead, the formation of a metal–ligand complex through an electrostatic or coordination interaction is also in accordance with the physico-chemical adsorption phenomenon. Since in physisorption, the energy of interaction between the cadmium ions and the biosorbent is marginally higher than the energy of condensation of the adsorbate, less activation
B
Fig. 5. Isotherm models. (A) Langmuir plot for both the metals. (B) Freundlich plot of both the metals.
Manasi et al. / International Journal of Biological Macromolecules 79 (2015) 300–308 Table 1A Isotherm parameters obtained from various models for microbe immobilized biopolymer. Sl. no.
Isotherm models
1.
Langmuir
2.
Freundlich
3.
DubininRadushkevich
Parameters
−1
qo (mg g ) b (L mg−1 ) RL r2 2 KF (mg1−1/n g−1 L1/n ) n r2 2 qm (mg g−1 ) ˇ EDR (kJ mol−1 ) r2 2
Values Lead
Cadmium
24.154 0.1149 0.0820 0.820 1.590 2.365 1.691 0.925 0.15 12.590 0.004 10.204 0.518 2.7
23.88 0.136 0.128 0.98 0.22 2.02 2.38 0.81 101.00 14.497 0.127 1.988 0.646 82.35
Table 1B Isotherm parameters obtained from various models for pure chitosan without the bacteria. Sl. no.
Isotherm models
Parameters
1.
Langmuir
2.
Freundlich
3.
DubininRadushkevich
qo (mg g−1 ) b (L mg−1 ) RL r2 KF (mg1−1/n g−1 L1/n ) n r2 qm (mg g−1 ) ˇ EDR (kJ mol−1 ) r2
Values Lead
Cadmium
17.54 0.093 0.017 0.54 2.61 2.032 0.92 16.26 2.817 0.421 0.80
13.848 0.12 0.16 0.78 2.67 2.202 0.87 17.01 5.415 0.303 0.86
energy is needed for adsorption [37]. Hence, the adsorption is favoured at low temperatures. 3.4. Kinetic uptake Kinetics is an important parameter to be evaluated in a batch adsorption experiment. The pseudo first order and pseudo second order models were used to assess the kinetic data (Fig. 6A and B). The first order and second order equations can be expressed as [38,39]: log (qe − qt ) = log qe −
k1 t 2.303
(6)
1 t t = + qt qe k2 q2e
(7)
The approach to adsorption kinetics could be explained through the following modes: a) Film diffusion of metal ions to the external surface of the microbe immobilized chitosan matrix. b) Particle diffusion through the pores of microorganism immobilized biosorbent.
305
c) Internal diffusion of Pb2+ and Cd2+ into the biosorbent. The particle diffusion followed by an internal diffusion of the metal ions onto the biosorbent matrix plays a key role in understanding the kinetics of adsorption. The adsorption was quite effective since the equilibrium was reached within 2–3 h duration. However, there were slight differences in the exact equilibrium time attained among the two metals [40]. This could be associated with the difference in the ion exchange affinities of cadmium and lead ions. The probability of lead ions having affinity towards the biosorbent is higher than cadmium. With increase in affinity, the rate of adsorption increases and this leads to a marginal decrease in the pH of the solution. The decrease in pH after the adsorption of lead was more as compared to cadmium. Additionally, lead also has a higher ionic radii and this is evident from the differences between the polarizability of the metal ions. This difference accounts for the higher affinity of lead towards the microbe immobilized biopolymer biosorbent. In accordance with the higher affinities, lead reaches equilibrium faster (2 h) than cadmium (3 h) and the metal adsorption favours pseudo second order kinetics. The experimental and calculated equilibrium adsorption capacity (qe ) values from the pseudo second order model were found to be 4.9 mg g−1 and 5.68 mg g−1 for cadmium and 4.9 mg g−1 and 5.02 mg g−1 for lead respectively. The kinetic parameters (Table 2) show the applicability of second order model with a high regression coefficient. The Weber and Morris intraparticle diffusion model for Pb2+ and Cd2+ were also studied using the expression [41]: √ qt = kint t + C (8) where kint is the intraparticle diffusion constant and qt is the amount of heavy metals adsorbed at time t. The Fig. 6C shows the plot of qt versus t0.5 for a particular concentration of lead (40 mg L−1 ) and cadmium (80 mg L−1 ). The multilinear fit obtained for cadmium shows that the first portion of curve relates to the instantaneous surface adsorption followed by intraparticle diffusion in the second portion. In case of lead, the plot shows a good linear fit with a definite intercept. These plots reflect boundary layer as well as diffusion controlled adsorption processes. 3.5. Adsorption thermodynamics The energetics of biosorption could be described through the study of various thermodynamic parameters involving free energy (G0 ), entropy (S0 ) and enthalpy (H0 ) changes. The above parameters were calculated using the equations [42]: G0 = −RT ln K ln K =
−H 0 RT
+
(9) S 0
(10)
R
The K values were obtained from the ratio of metal ion concentrations on the microbe immobilized chitosan surface to the equilibrium concentration in solution (K = C(biosorbent) /C(solution) ). The free energy changes were accordingly found to be −11.77, −11.76, −10.90 and −7.75 kJ mol−1 for the biosorption of lead at 293, 303, 313 and 323 K respectively. Likewise, the G values for cadmium were also negative at these temperatures (Table 3). The
Table 2 Kinetic and intraparticle rate constant data for the adsorption of metal ions. Metal ion
Second order rate constant k2 [g mg−1 min−1 ]
Regression coefficient
First order rate constant k1 [min−1 ]
Regression coefficient
Intraparticle rate constant kint [mg g−1 min−½ ]
Lead Cadmium
0.07 0.007
0.98 0.99
0.023 0.026
0.974 0.98
20.166 0.069
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Fig. 6. Kinetic plots obtained from the pseudo first-order equation (A), pseudo second-order equation (B), and intraparticle diffusion plot (C).
diffusion of metal ions [43] from the aqueous solution onto the polymeric interface is more probable near room temperature. The adsorption was thermodynamically feasible and spontaneous as indicated by the negative free energy values. The enthalpy (H0 ) and entropy (S0 ) of biosorption were estimated from the slope and intercept of the plot of ln K against 1/T (Fig. 7). The enthalpy of biosorption (H0 ) for the cadmium and lead systems were found to be −43.6 kJ mol−1 and −50.04 kJ mol−1 respectively. Since, the Gibb’s free energy becomes less negative with temperature, the degree of spontaneity decreases with increase in temperature for both the metal ions. The biosorption process was thermodynamically favourable and exothermic resulting in negative G0 Table 3 Thermodynamic data for the adsorption of metal ions. Metal ion
Temperature/ Kelvin
G0 [kJ mol−1 ]
S0 [J mol−1 K−1 ]
H0 [kJ mol−1 ]
Cadmium
293 303 313 323 293 303 313 323
−7.99 −8.76 −7.057 −4.202 −11.77 −11.76 −10.90 −7.75
−118.80
−43.617
−128.63
−50.043
Lead
Fig. 7. Van’t Hoff isotherm plot obtained for the adsorption of metal ions on the microbe immobilized chitosan surface.
Manasi et al. / International Journal of Biological Macromolecules 79 (2015) 300–308 Table 4 Comparison of adsorption capacity against related biopolymers and bacterial strains. Adsorbent
Heavy metal
Adsorption capacity (mg g−1 )
Chitosan/cotton fibres [44] Chitosan/cellulose [45] Chitosan/sand [46] Alginate chitosan [47] Chitosan immobilized on bentonite [48] Halomonas BVR1 [20] Halomonas BVR1 immobilized in sodium alginate [21] Chitosan (present study)
Cadmium Lead Lead Cadmium Lead
15.74 26.31 12.32 6.63 15.0
Cadmium Lead
12.02 9.68
Lead Cadmium Lead
17.54 13.84 24.15
Cadmium
23.88
Halomonas BVR1 immobilized in chitosan (present study)
and H0 values. The S0 values for adsorption process were also negative (−118.80 J mol−1 K−1 and −128.63 J mol−1 K−1 ) for cadmium and lead respectively suggesting reduced randomness at the solid–solution interface. 3.6. Effect of diverse ions The influence of certain cations and anions that could be commonly associated with the electronic industry effluents were investigated at different concentrations. Ions such as zinc and cobalt did not interfere up to 50 mg L−1 level, while nickel and iron caused a reduction in the adsorption of lead and cadmium beyond 60 mg L−1 concentration. Anions such as chloride and sulphate did not affect the adsorption of lead and cadmium when their concentration levels were in the range 10–60 mg L−1 . Nitrate and phosphate caused a decrease in the percentage adsorption of lead and cadmium beyond 70 mg L−1 . 3.7. Comparison against other biosorbents The proposed method for lead and cadmium were compared in terms of the maximum Langmuir adsorption capacity with other polymeric matrices involving chitosan [44–48]. The results showed that Halomonas BVR1 immobilized chitosan exhibits good adsorption capacity as evident from the comparison shown in Table 4. Evaluation of the potential of this bacterial strain as such [20] and after immobilization in alginate matrix [21] also indicates that Halomonas BVR1–chitosan combination exhibits higher Langmuir adsorption capacity for lead and cadmium. 3.8. Regeneration of biosorbent Preliminary studies for regeneration of the biosorbent showed that 0.5 mM EDTA has the potential to complex both the metal ions and desorb them effectively. Further studies are in progress to check the reusability of the biosorbent. 4. Conclusions The biopolymer strengthens the interaction after immobilization with the bacteria. The biosorption process is spontaneous, exothermic and favours pseudo second order kinetics. The Langmuir adsorption capacities were found to be 24.15 mg g−1 and 23.88 mg g−1 for lead and cadmium respectively. Characterization
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