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Journal of Applied Phycology 14: 267–273, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.
Enhanced sorption of Cu 2+ and Ni 2+ by acid-pretreated Chlorella vulgaris from single and binary metal solutions S.K. Mehta, B.N. Tripathi and J.P. Gaur* Laboratory of Algal Biology, Department of Botany, Banaras Hindu University, Varanasi, 221 005, India; *Author for correspondence (e-mail:
[email protected]) Received 2 November 2001; accepted in revised form 10 March 2002
Key words: Chlorella, Copper, Isotherm model, Metal biosorption, Nickel Abstract The influence of HCl pretreatment (0.1 mM) on sorption of Cu 2+ and Ni 2+ by Chlorella vulgaris was tested using single and binary metal solutions. The optimal initial pH for sorption was 3.5 for Cu 2+ and 5.5 for Ni 2+. Second order rate kinetics described well sorption by untreated and acid-pretreated cells. The kinetic constant q e (metal sorption at equilibrium) for sorption of test metals from single and binary metal solutions was increased after pretreatment of the biomass with HCl. The Langmuir adsorption isotherm was developed for describing the various results for metal sorption. In single metal solution, acid pretreatment enhanced q max for Cu 2+ and Ni 2+ sorption by approximately 70% and 65%, respectively. Cu 2+ and Ni 2+ mutually interfered with sorption of the other metal in the binary system. The combined presence of Cu 2+ and Ni 2+ led to their decreased sorption by untreated biomass by 19% and 88%, respectively. However, acid-pretreated biomass decreased Cu 2+ and Ni 2+ sorption by 15 and 22%, respectively, when both the metals were present in the solution. The results suggest a reduced mutual interference in sorption of Cu 2+ and Ni 2+ from the binary metal system due to the acid pretreatment. Acid-pretreated cells sorbed twice the amount of Cu 2+ and ten times that of Ni 2+ than the untreated biomass from the binary metal system. Acid pretreatment more effectively enhanced the sorption of Ni 2+ form the binary metal solution. The total metal sorption by untreated and acid-pretreated biomass depended on the Cu 2+: Ni 2+ ratio in the binary metal system. Acid pretreatment of C. vulgaris could be an effective and inexpensive strategy for enhancing Cu 2+ and Ni 2+ sorption from single and binary metal solutions. Introduction Since elevated concentrations of heavy metals are well known to affect aquatic biota deleteriously and may eventually the entire food web (De Filippis and Pallaghy 1994), it is necessary to reduce the heavy metal load of industrial wastewaters prior to their disposal. The conventional methods of metal removal like cation exchange, precipitation, solvent extraction, membrane filtration, etc., are inefficient and expensive (Schmiechen et al. 1992). Hence, other methods need to be developed for stripping toxic metals from wastewaters. Research in the last two decades have made clear the high metal sorption capacity of many types of microbial and plant biomass, thereby open-
ing up the possibility of their use in treatment of metal-enriched wastewaters (Aksu 1998). Much information has accumulated on seaweeds (Volesky and Holan 1995), but seaweed biomass may not be readily available for metal sorption away from the coast. In such situations freshwater algae, with their large surface area for metal adsorption, may prove useful metal biosorbents. Earlier studies on metal biosorption by algae were mostly carried out in single metal systems, but industrial effluents often contain high concentrations of more than one metal (Klein et al. 1974), so it is important to characterise the biosorption of metals from multi-metal mixtures. This has, however, received relatively little attention (Mehta and Gaur 2001a Chong and Volesky 1996).
268 In its native form, biomass generally has a low metal sorption ability (Eccles 1999). Therefore, inexpensive pretreatment strategies need to be evolved to enhance its metal sorption capacity. A few researchers have attempted to modify biomass chemically prior to its application for biosorption. Zhou et al. (1994) showed that acid and other kinds of pretreatment increased the metal (Pb 2+, Cu 2+, Zn 2+, Cd 2+, Cr 6+, Mn 2+, Ni 2+, Co 2+) accumulating ability of seaweeds. In contrast, Kuyucak and Volesky (1989) found decreased Co sorption by the marine Ascophyllum nodosum after acid pretreatment. As with most other studies, pretreatment strategies have been developed for seaweeds many in single metal systems (Kuyucak and Volesky 1989). The present study developed from a preliminary report (Mehta and Gaur 2001b) showing 96% enhancement of metal biosorption after acid pretreatment of Chlorella vulgaris. This alga was chosen because it grows abundantly in high rate algal ponds. The aim was to quantify the efficacy of acid (HCl) pretreatment on the ability of C. vulgaris to sorb Cu 2+ and Ni 2+ from single and binary metal solutions.
Optimization of pH for sorption of test metals Solutions of Cu 2+ and Ni 2+ (0.5 mM) were prepared using CuCl 2.2H 2O or NiCl 2.6H 2O. The pH was adjusted to 2.5, 3.5, 4.5, 5.5 or 6.5 with HCl or NaOH. C. vulgaris (100 mg l −1 d. wt) was added to each flask. The flasks were placed on a shaker (Environshaker 3597, Labline Instruments Inc., USA) at 30 rpm for 30 min. The cells were separated from the solution by centrifugation and the metal content in the filtrate was determined with an atomic absorption spectrophotometer (Perkin-Elmer model 2380). Acid pretreatment of biomass The exponentially growing cultures were harvested and pellets washed twice with Milli-Q water. The washed pellets were suspended in 10 ml HCl (0.1 mM) at 25 ± 2 °C and agitated on a shaker (Lab-Line Instrument Inc., USA, Model 3597) for 30 min at 30 rpm. After centrifugation the pellet was washed twice with Milli- Q water. Kinetics of Cu 2+ and Ni 2+ sorption
Materials and methods Test organism, medium and culture conditions C. vulgaris was isolated from a local pond and grown in modified Chu 10 medium (Gerloff et al. 1950) at pH 6.8 ± 0.2. The medium was prepared using Milli-Q water. One litre of medium contained: KNO 3 (0.04 g), K 2HPO 4 (0.019 g, CaCl 2 (0.049 g), MgSO 4 (0.025 g), sodium silicate (0.029 g), ferric citrate (0.003 g), manganous chloride (0.5 mg), sodium molybdate (0.01 mg), boric acid (0.5 mg), CuSO 4 (0.02 mg), CoCl 2 (0.04 mg), ZnSO 4 (0.05 mg). The pH of the medium was adjusted using 0.1 N HCl or NaOH after autoclaving. The cells were harvested by centrifugation at 325 × g and pellets were washed twice with Milli-Q water before adding to metal solution. Three replicates were used in all experiments. Analytical grade chemicals from Loba Chemi, India, were used throughout the study. The glassware were rinsed with dilute HCl and subsequently with Milli-Q water to avoid metal contamination.
The time course of Cu 2+ and Ni 2+ sorption from the binary metal solution by acid-pretreated and untreated biomass was studied. The solution contained a fixed concentration (0.5 mM) of the primary metal (Cu 2+ or Ni 2+) and varying concentrations (0, 0.1, 0.5, 1.0, 1.5 mM) of the secondary (Ni 2+ or Cu 2+, respectively). The pH of the metal solution was adjusted to 3.5 for Cu 2+ and and 5.5 for Ni 2+. These values correspond to the maximum sorption of test metals from their single metal solutions by untreated biomass. Pretreated and untreated washed pellets (100 mg l −1 d. wt) were added to separate flasks containing metal solutions. The cells were harvested at different time periods and the metal content in the filtrate was determined. The data for the time course of metal sorption were fitted to the second order rate equation (Ho et al. 1996): t/q t ⫽
冉
1 2
⫻ k ⫻ q e2
冊
⫹ t/q e
Where k = rate constant for adsorption (g mmol −1 min −1), q e = amount of metal adsorbed at equilibrium (mmol g −1) and
269 q t = the amount of metal adsorbed at a particular time ’t’ (mmol g −1). Kinetic constants were determined using SigmaPlot (version 2.0) curve fitter. Sorption of Cu 2+ and N 2+ from varying concentrations Binary metal solutions were prepared with various concentrations of Cu 2+ and Ni 2+. The concentration of Ni 2+ ranged from 0 to 1.5 mM. To each Ni 2+ concentration, Cu 2+ stock was added to give Cu 2+ concentrations ranging from 0 to 1.5 mM. Acid-pretreated or untreated cells (100 mg l −1 d. wt) were added to each flask and placed for 30 min on a shaker. This period corresponds to the maximum metal sorption obtained from the time-course study of metal sorption from single metal solution. Langmuir isotherm model The Langmuir model was used to describe quantitatively metal sorption. The parameters q max (mass of metal adsorbed when surface site is fully covered, mmol g −1 algal dry weight) and b (constant related to the energy of sorption, L mmol −1) were determined using the equation: q e ⫽ 共q maxbC e兲/共1 ⫹ bC e兲. where q e is metal sorption (mmol g −1 d. wt) and C e is metal concentration at equilibrium.
Results Cu 2+ and Ni 2+ sorption by the alga depended on the initial pH of the solution, with maximum sorption at pH 3.5 and 5.5, respectively (Table 1). Figure 1 represents the second order rate kinetics of Cu 2+ and Ni 2+ sorption by untreated and 0.1 mM HCl- pretreated cells from a single metal solution. At each time interval, acid-pretreated C. vulgaris sorbed more metal than the untreated one. Cu 2+ and Ni 2+ were very rapidly sorbed by acid-pretreated and untreated alga. However, the q e for Cu 2+ and Ni 2+ sorption showed an almost 2-fold increase after acid pretreatment (Table 2). The second order rate constant k for Cu 2+ and Ni 2+ sorption from the single metal system decreased by 78 and 23%, respectively, after acid pretreatment.
Figure 1. Time course of Ni 2+ and Cu 2+ sorption from single metal solution (0.5 mM Ni 2+ or Cu 2+) by untreated and acid-pretreated C. vulgaris. Data were fitted to the second order rate kinetics. Symbols represent the data points and curves represent the prediction made by second order rate equation. Vertical bars represent SD; n = 3.
Sorption of test metals by untreated and acid-pretreated cells was also determined at various external concentrations of Cu 2+ and Ni 2+. The equilibrium data of metal sorption by untreated and acid-pretreated biomass fit well (r 2 > 0.9) to the Langmuir adsorption model (Table 3). The Langmuir constant q max for Cu 2+ and Ni 2+ sorption increased by about 70 and 65% after acid-pretreatment of biomass (Table 3). Likewise, acid-pretreatment caused 20 and
270 Table 1. Effect of pH on sorption of Cu 2+ and Ni 2+ by C. vulgaris from solution with 0.5 mM Cu 2+ or Ni 2+. Metal sorption, mmol g −1 dry wt. Cu2+ Ni2+
pH
2.5 3.5 4.5 5.5 6.5
1.12 3.82 2.35 2.28 1.15
± ± ± ± ±
0.09 0.23 0.21 0.21 0.22
1.05 1.12 1.62 2.68 1.85
Metal ± ± ± ± ±
0.07 0.08 0.11 0.14 0.12
Table 2. Second-order rate constants for Cu 2+ and Ni 2+ sorption from single and binary metal solutions by untreated and acid-pretreated C. vulgaris. Inhibitory
Untreated
Acid-pretreated
metal (mM) K (g
q e (mmol
K (g
q e (mmol
mmol −1
g −1)
mmol −1
g −1)
−1
−1
min ) Ni 2+
Cu 2+
0.0 0.1 0.5 1.0 1.5
sorption 0.049 0.061 0.067 0.074 0.079
Cu 2+
Ni 2+
0.0 0.1 0.5 1.0 1.5
sorption 0.066 0.090 0.171 0.345 0.365
Table 3. Langmuir parameters determined for the sorption of Cu 2+ and Ni 2+ by untreated and acid-pretreated C. vulgaris from the single metal system.
min )
4.14 3.81 (7.9)* 2.72 (34.4) 2.05 (50.5) 1.29 (68.8)
0.011 0.011 0.013 0.012 0.010
7.50 7.23 (3.6) 6.39 (14.8) 4.83 (35.6) 4.15 (44.7)
2.82 1.76 (37.6) 0.44 (84.4) 0.19 (93.3) 0.18 (93.6)
0.051 0.069 0.129 0.176 0.276
4.54 4.41 (3.0) 3.56 (21.6) 2.51 (44.7) 0.96 (78.9)
*
Values in parentheses represent per cent decrease in q e for Cu 2+ and Ni 2+ sorption in binary metal system compared to that for the single metal system.
32% increase in the affinity (b) of the cells to sorb Cu 2+ and Ni 2+, respectively. The effect of acid pretreatment on sorption of Cu 2+ and Ni 2+ was also studied from a binary metal solution. The second order rate constants for Cu 2+ and Ni 2+ sorption from binary metal solutions by acidpretreated and untreated biomass are shown in Table 2. The q e for the sorption of the primary metal (Cu 2+ and Ni 2+) decreased with increasing concentration of the secondary (Ni 2+ and Cu 2+, respectively) in the solution. However, the inhibitory effect of the secondary metal on q e for the sorption of the primary
Untreated b q max (mmol
(mM)
r2 −1
(mmol
g −1) Cu 2+ Ni 2+
6.62 4.51
Acid-pretreated b q max (mM)
r2 −1
g −1) 3.33 2.50
0.91 0.89
11.25 7.46
4.00 3.13
0.92 0.90
metal was reduced considerably when the biomass was pretreated with 0.1 mM HCl. The value of k for the sorption of a metal (Cu 2+ or Ni 2+) from the binary metal solution increased with increasing concentration of the inhibitory metal (Ni 2+ or Cu 2+, respectively) in the solution. Acid pretreatment of the biomass greatly decreased k for metal sorption from both single and binary metal systems (Table 2). The efficacy of acid pretreatment on sorption of Cu 2+ and Ni 2+ by C. vulgaris was tested from binary metal solutions containing various concentrations of Cu 2+ and Ni 2+ (Figures 2 and 3). Sorption of primary metals (Cu 2+, Ni 2+) by untreated and acid-pretreated alga decreased with increasing concentration of secondary metals (Ni 2+, Cu 2+, respectively). However, inhibition in Ni 2+ sorption by Cu 2+ was stronger than the inhibition in Cu 2+ sorption by Ni 2+. Acid pretreatment decreased the inhibitory effect of the secondary metal on sorption of the primary metal. Nickel sorption was enhanced by acid pretreatment more than Cu 2+ sorption. Langmuir constants (q max and b) for Cu 2+ and 2+ Ni sorption from single and binary metal systems are shown in Table 3. Acid pretreatment of biomass increased the q max for Cu 2+ and Ni 2+ sorption from single metal solution by 79 and 65%, respectively (Table 3). The constant b for sorption of test metals from single metal solution was also increased after acid pretreatment. The untreated biomass showed 19 and 88% decrease in q max for sorption of Cu 2+ and Ni 2+ when both the metals were concomitantly present in solution; the acid-pretreated biomass showed 15 and 22% decrease, respectively. Acid-pretreatment increased q max for Cu 2+ and Ni 2+ sorption from the binary metal system by about 2- and 10-fold, respectively; however, b increased to about 2-fold for both the metals (Table 4). Figure 4 shows total metal sorption by untreated and acid-pretreated C. vulgaris from the binary metal
271
Figure 2. Langmuir isotherm surfaces for Cu 2+ sorption by (A) untreated and (B) acid-pretreated C. vulgaris from binary metal solutions with various concentrations of Cu 2+ and Ni 2+. n = 3.
solution. Total metal sorption by untreated and acidpretreated alga depended on the molar ratio of Cu 2+ and Ni 2+ in the binary metal solution. When concentration of one metal was kept constant and that of the second increased up to 0.4 mM, the total metal sorption by untreated biomass decreased (Figure 4A). Acid pretreatment of biomass greatly increased the total metal sorption ability of the alga. Low total metal sorption at lower concentrations of test metals by untreated biomass was contrary to the data obtained for the acid-pretreated biomass. The total metal sorption by acid-pretreated biomass continuously increased with rise in concentrations of Cu 2+ and Ni 2+ (Figure 4B).
Figure 3. Langmuir isotherm surfaces for Ni 2+ sorption by untreated (A) and acid-pretreated (B) C. vulgaris from binary metal solutions with various concentrations of Ni 2+ and Cu 2+.
Discussion The second-order rate kinetics described well the sorption of both test metals from single and binary metal systems by acid-pretreated and untreated C. vulgaris. Ho et al. (1996) and Mehta and Gaur (2001a) have also suggested that metal sorption by biological materials can be better described by the second order rate kinetics than the first order rate kinetics. However, Wells and Brown (1987) concluded that first order rate kinetics were suitable to describe Cd uptake by the moss Rhytidiadelphus squarrosus. The present study shows that acid pretreatment caused up to 2-fold increase in q e for the sorption of test metals from a single metal solution. The decrease in k after acid pretreatment enhanced metal sorption.
272 Table 4. Langmuir parameters determined for the sorption of Cu 2+ and Ni 2+ by untreated and acid-pretreated C. vulgaris from the binary metal system (Cu 2++Ni 2+). n = 3. Metal
Untreated
Acid- pretreated −1
2+
Cu Ni 2+
q max (mmol g ) 5.34 0.54
b (mM) 2.08 1.38
−1
2
r 0.83 0.81
q max (mmol g −1) 9.58 5.84
b (mM) −1 4.00 3.12
r2 0.84 0.83
The binary metal system contained varying concentrations of the primary metal (0 to 1.5 mM) and a fixed concentration (0.5 mM) of the secondary metal.
Figure 4. Total metal sorption by untreated (A) and acid pretreated (B) C. vulgaris from binary metal solutions with various concentrations of Cu 2+ and Ni 2+.
Maximal sorption of Cu 2+ and Ni 2+ at different pH values (3.5 and 5.5, respectively) suggests the involvement of specific functional groups in sorption of test metals. Some previous studies have also demonstrated the dependence of metal sorption on the pH of the external medium (Volesky and Holan 1995). The higher q max and b for Cu 2+ than for Ni 2+ sorption suggests that the alga had a higher capacity to sorb Cu 2+. It may be due to larger ionic radius of Cu than Ni, as reported for various metal ions and Rhizopus arrhizus (Tobin et al. 1984). The electronic con-
figuration also favours the sorption of Cu 2+ over Ni 2+. Cu 2+ remains unstable and shows paramagnetic property due to the presence of one electron pair (Purcell and Kotz 1980). Thus Cu 2+ is attracted by a magnetic field possibly originating from biosorbents (Chong and Volesky 1996). In contrast, Ni 2+ remains stable because it has no unpaired electron and is therefore slightly repelled by magnetic field. Higher sorption of Cu 2+ may also be due to greater affinity of Cu 2+ to bind with -SH groups of the cell surface. The study showed that sorption of test metals by both pretreated and untreated cells fitted well (r 2 > 0.9) to the Langmuir model, thereby suggesting monolayer sorption of the metals on both kinds of biomass. About 70 and 65% enhancement in q max for Cu 2+ and Ni 2+ sorption, respectively, after acid pretreatment of alga has been found in the present study. An increased b value after acid pretreatment suggests an enhanced intensity of metal biosorption. Increase in Langmuir constants, q max and b, after acid pretreatment suggests modification of the algal cell surface. The q max and b for metal sorption by the test alga were much higher than the values earlier reported for higher plants and other microorganisms (Al-Asheh and Duvnjak 1998 Röhricht et al. 1990 Volesky 1994). It has been demonstrated that cation exchange plays an important role in metal sorption by algae and other biosorbents (Crist et al. 1980). The algal cell surface has many bound cations like Ca 2+, Mg 2+, K + and H +, which are released during the sorption of metals. Al-Asheh and Duvnjak (1998) have shown a significant release of Ca 2+, Mg 2+, K + and H + from pine bark during the sorption of Cu 2+, Cd 2+ and Ni 2+. The acid pretreatment of biomass possibly released the adsorbed cations, including metal ions from the biomass, thus freeing the binding sites for metal binding. Similar to the present study, Ting et al. (1995) found a tremendous rise in Au uptake by acid-treated C. vulgaris. Another possible reason for enhanced sorption of test metals after acid- pretreatment may
273 be changed architecture of the cell wall resulting in an increased sorption of metals (Ting and Teo 1992). The study showed that the presence of one metal species in solution inhibited the sorption of the other. Likewise, De Carvalho et al. (1995) found mutual interference in the sorption of metals from Cu-Zn, Cu-Cd and Cd-Zn mixtures. The increased inhibition in sorption of the primary metal with increasing concentration of the secondary metal in solution suggested that metal ions are competing for binding sites. The present study showed that the inhibitory effect of Ni 2+ on Cu 2+ sorption was not as strong as was of Cu 2+ on Ni 2+ sorption. The study has shown that acid pretreatment can markedly enhance the metal sorption capacity of C. vulgaris. This effect was shown in both single and binary metal systems, but was greater in the latter, which makes acid pretreatment particularly attractive from a practical point of view. Being inexpensive, acid pretreatment holds great potential for the commercial application of C. vulgaris as a metal biosorbent.
Acknowledgements We thank the Head, Department of Botany, and the Co-ordinator, Centre of Advanced Study in Botany, Banaras Hindu University, for facilities. SKM thanks the Council of Scientific and Industrial Research, New Delhi, for financial support.
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