Colloids and Surfaces B: Biointerfaces 46 (2005) 101107

Biosorption of copper(II) and zinc(II) from aqueous solution by Pseudomonas putida CZ1
Xin Cai Chen, Yuan Peng Wang, Qi Lin, Ji Yan Shi , Wei Xiang Wu, Ying Xu Chen
Department of Environmental Engineering, Zhejiang University, Hangzhou 310029, China Received 31 July 2005; received in revised form 10 October 2005; accepted 12 October 2005

Abstract To study Pseudomonas putida CZ1, having high tolerance to copper and zinc on the removal of toxic metals from aqueous solutions, the biosorption of Cu(II) and Zn(II) by living and nonliving P. putida CZ1 were studied as functions of reaction time, initial pH of the solution and metal concentration. It was found that the optimum pH for Zn(II) removal by living and nonliving cells was 5.0, while it was 5.0 and 4.5, respectively, for Cu(II) removal. At the optimal conditions, metal ion biosorption was increased as the initial metal concentration increased. The adsorption data with respect to both metals provide an excellent t to the Langmuir isotherm. The binding capacity of living cells is signicantly higher than that of nonliving cells at tested conditions. It demonstrated that about 4050% of the metals were actively taken up by P. putida CZ1, with the remainder being passively bound to the bacterium. Moreover, desorption efciency of Cu(II) and Zn(II) by living cells was 72.5 and 45.6% under 0.1 M HCl and it was 95.3 and 83.8% by nonliving cells, respectively. It may be due to Cu(II) and Zn(II) uptake by the living cells enhanced by intracellular accumulation. 2005 Elsevier B.V. All rights reserved.
Keywords: Pseudomonas putida CZ1; Copper; Zinc; Biosorption; Bioremediation

1. Introduction Human activities, such as mining operations and the discharge of industrial wastes, have resulted in the accumulation of metals in the environment [1,2]. Some metals (e.g. Ca, Co, Cr, Cu, Zn, Fe, K, Mg, Mn, Na and Ni) are essential micro-nutrients for most, if not all, living organisms. One of the most important functions of micro-nutrients is their role in metalloenzymes. However, when the concentrations of benecial metals in the environment are excessively high, for instance, copper or zinc, can become toxic to these microorganisms and human [3]. Therefore, growing attention is being paid to remove heavy metals from industrial waste water to protect the environment and human health. Although removal of toxic heavy metals from industrial wastewater has been practised for several decades, the conventional physico-chemical removal methods, such as chemical precipitation, electro winning, membrane separation, evaporation or resin ionic exchange, are usually expensive and

Corresponding author. Tel.: +86 571 869 71975; fax: +86 571 869 71898. E-mail address: shijiyan@zju.edu.cn (J.Y. Shi).

sometimes, not effective. Therefore, there is a need for some alternative technique, which is efcient and cost effective. Biosorption, based on living or nonliving microorganisms or plants, could be such an alternative method of treatment. It has distinct advantages over conventional methods of treatments: the process does not produce chemical sludge, hence no secondary pollution, more efcient and easy to operate. High metal binding capacities of several biological materials have already been identied in part. Among the biosorbents, there are marine algae [4], bacteria [5], yeasts [6], fungi and waste mycelia from the fermentation [7] and food industry [8]. Further, the capacities of these microorganisms to accumulate an ample range of metal species have also been described [9,10,4]. Compared to other metals, copper, especially zinc sequestration by bacteria and in relation to Pseudomonas sp. remains little explored. So far some studies have been conducted on P. aeruginosa [1114], Pseudomonas putida [1517] and P. stutzeri [18], but few of them investigated the characteristics of living and nonliving cells of P. putida on the biosorption of Cu(II) and Zn(II) from aqueous solution. Moreover, the performance of any biosorbent also depends on biomass characteristics, physico-chemical properties of the target metals and

0927-7765/$ see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2005.10.003

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the micro-environment of contact solution, i.e., the initial pH of solution, temperature and interaction with other ions, etc. [19]. Therefore, it was considered that supplementary research in this eld would be useful. In this work, we aimed to study the characteristics of P. putida CZ1 on its removal of toxic metals from aqueous solution. The objectives of the study are to compare the living and nonliving cells of P. putida CZ1 in their removal capacity of Cu(II) and Zn(II) and to model the equilibria of the corresponding adsorption processes. For these purposes, the removal capacity, desorption efciency of living and nonliving cells and various factors affecting the adsorption, such as react time, initial pH of the solution and metal concentration, were investigated with the batch equilibration technique. 2. Materials and methods 2.1. Microorganism and its preparation for biosorption The bacterial strain used in the present study was P. putida CZ1, isolated from metal contaminated soil of mining activities as a copper and zinc tolerant strain described elsewhere. P. putida CZ1 was grown and maintained on nutrient broth medium, which contained 5.0 g beef extract, 10.0 g peptone and 5.0 g sodium chloride per liter with an initial pH of 7.07.2. Cells of P. putida CZ1 were inoculated into 250 ml Erlenmeyer asks containing 100 ml of sterile medium and cultivated aerobically in an orbital rotary shaker (200 rpm) at 30 C for 24 h. After inoculation, cells were harvested by means of centrifugation at 13,000 rpm for 5 min and washed three times with deionized water. Living cells were prepared by resuspending the cell pellet with phosphate buffer solution (PBS) (pH 7.0). Cell concentration in the suspension was determined by drying an aliquot in a pre-weighed aluminum foil container to a constant weight at 60 C [20]. The remaining harvested cells which were freeze dried, autoclaved at 121 C for 30 min, crushed in a blender and resuspended with deionized water, were dened as nonliving cells. 2.2. Preparation of metal solutions Stock solutions (100 mM) of Cu(II) and Zn(II) were prepared by dissolving analytical grade CuSO4 5H2 O and Zn(NO3 )2 6H2 O in distilled water. Before mixing with the biosorbents, the stock solutions were diluted to required concentration. 2.3. Metal biosorption experiments Biosorption experiments were conducted at 30 C in batch with 0.01 g of the living or nonliving cells in a 50 ml plastic tube containing 10 ml of working solution volume. The tubes were then shaked at 200 rpm. Potassium nitrate (0.01 M) was used as a supporting electrolyte for all experiments. Experiments for determining the kinetics of the process were performed at 63.5 and 65.3 mg/l initial metal concentrations for Cu(II) and Zn(II), respectively. Samples were taken at desired

intervals and were subsequently centrifuged at 15,000 g for 5 min. The heavy metal concentration in the resulting supernatant was determined. The impact of the solution pH on the metal biosorption was investigated in the same way except that the initial pH of the solutions was adjusted from 2.0 to 7.0 with the addition of either 0.1 M NaOH or 0.1 M HCl. After 24 h of incubation the metal concentration in supernatants was measured. 2.4. Adsorption isotherms Cu(II) and Zn(II) biosorption isotherms were obtained at constant pH and ionic strength. For each tube, the initial Cu(II) and Zn(II) concentrations were varied from 5.7 to 286.2 and 6.5 to 279.4 mg/l, respectively, and then followed the same procedures for the experiment of pH effect. To test the t of data, the Langmuir and Freundlich isotherm models were applied to this study. The Langmuir isotherm model is valid for monolayer sorption onto surface and nite number of identical sites and given by Eq. (1). qeq = Qmax bCeq 1 + bCeq (1)

where Qmax is the maximum amount of the metal ion per unit weight of cell to form a complete monolayer on the surface bound at high Ceq (mg/g) and b the constant related to the afnity of the binding sites, Qmax represent a practical limiting adsorption capacity when the surface is fully covered with metal ions and assists in the comparison of adsorption performance, particularly in cases where the sorbent did not reach its full saturation in experiments. Qmax and b can be determined from the linear plot of Ceq /qeq versus Ceq . The empirical Freundlich isotherm model based on a heterogeneous surface is given below by Eq. (2). qeq = Kf Ceq
1/n

(2)

where Kf and n are Freundlich constants characteristic of the system. Kf and n are indicators of adsorption capacity and intensity, respectively. The values of Kf and n were evaluated from the intercept and the slope, respectively, of the linear plot of ln qeq versus ln Ceq based on experimental data. The Freundlich isotherm is also more widely used but provides on information on the monolayer adsorption capacity, in contrast to the Langmuir model [2125]. 2.5. Desorption of Cu(II) and Zn(II) Desorption of Cu(II) and Zn(II) from previously loaded living and nonliving P. putida CZ1 was studied by using 0.1 M HCl as eluent. For this purpose, 0.01 g of previously loaded living and nonliving P. putida CZ1 was added to 10 ml of eluent in a 50 ml plastic tube. After 24 h of shaking, supernatants of centrifuged samples were analyzed for the Cu(II) and Zn(II) concentrations. All experiments were conducted in triplicate. Control experiments without biomass were carried out in order to determine the degree of removal of copper and zinc from solution by the

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plastic tube. Extraneous metal contamination was found to be negligible. 2.6. Analysis of Cu(II) and Zn(II) The concentrations of initial and nal Cu(II) and Zn(II) in the biosorption experiments were determined by using ame atomic absorption spectrophotometry (FAAS, Thermo Element MKII-M6). The results are given as a unit of adsorbed and unadsorbed metal concentrations per gram of living or nonliving biosorbent in solution at equilibrium and calculated by Eq. (3). qe = (C0 Ceq )V X (3)

and Zn(II) to the cells of P. putida CZ1 is mainly due to ionic attraction. Therefore, at low pH values the cell surface becomes more positively charged, reducing the attraction between metal ions and functional groups on the cell wall. In contrast, higher pH results in facilitation of the metal biosorption, since the cell surface is more negatively charged [4,16,28,29]. And copper and zinc will transform into hydroxide complexes at high pH values, however, it could not be considered the biosorption behavior of the cells. However, the inconsistency in literature regarding the inuence of pH on biosorption seems to indicate the way that the pH would alter the adsorption of metal ions to cells varies with the type of adsorbents (cells) and adsorbates (metal ions) [12,16,17]. 3.2. Effect of reaction time on the biosorption Fig. 2 shows the effect of reaction time on the biosorption of Cu(II) and Zn(II) by biosorbent from aqueous solutions. The rate of copper biosorption by the nonliving cells was very rapid, reaching almost 96% of the maximum adsorption capacity within 10 min of contact time. However, it took a longer biosorption time for Zn(II), which reached approximate 90% of the maximum biosorption capacity within 60 min and followed by a nearly constant after 5 h. Noticeably, there was a slight decreasing of Cu(II) sorption by nonliving cells at 120 min and then remained nearly constant. It may be because a small amount of Cu(II) was released back into the solution. Rapid Cu(II) biosorption by nonliving cells of P. putida CZ1 is in agreement with Cu(II) biosorption by lyophilized P. aeruginosa cells [14] and P. cepacia [3], which were completed within 10 min of reaction time. Such rapid biosorption process have been correlated with the characteristics of the biomass, and its physico-chemical interactions with the metal ion [30]. Microbial metal uptake by nonliving cells, which is metabolism-independent passive binding process to cell walls (adsorption), and to other external surfaces, and is generally considered as a rapid process, taking place within a few minutes [31]. The rapid metal sorption is also highly desirable for successful deployment of the biosorbents for practical applications [32].

where X is the biosorbent concentration (g/l), qe the adsorbed metal ion quantity per gram of biosorbent at equilibrium (mg/g), C0 the initial metal concentration (mg/l), Ceq the metal concentration at equilibrium (mg/l) and V is the working solution volume. 3. Results and discussion 3.1. Effect of initial pH on Cu(II) and Zn(II) biosorption Earlier studies on heavy metal biosorption have shown that pH was the single most important parameter affecting the biosorption process [26,27]. In all cases, metal biosorption by the cells increases with increasing pH reaching to a maximum and then showed a rapid decline in biosorption (Fig. 1a and b). Living cells demonstrated the maximum biosorption of 23.9 mg/g Cu(II) at pH 5.0 where as that was 12.8 mg/g at pH 4.5 for nonliving cells. The effect of pH on the biosorption capacity of Zn(II) with living and nonliving cells is shown in Fig. 1b. It indicated the same trends as in the biosorption of Cu(II), but both type of cells achieved its maximum at pH 5.0. The maximum biosorption of Zn(II) by living cells was 26.9 mg/g while that was 12.4 mg/g for nonliving cells. The results demonstrated that copper and zinc biosorption by living and nonliving cells were affected by the initial pH of solution and suggest that the adsorption of Cu(II)

Fig. 1. Effect of initial pH on biosorption capacity of (a) Cu(II) and (b) Zn(II) by living and nonliving Pseudomonas putida CZ1.

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Fig. 2. Effect of reaction time on (a) Cu(II) and (b) Zn(II) by living and nonliving Pseudomonas putida CZ1.

It also can be seen that metals biosorption by living cells consisted of two phases: a primary rapid phase (within 1030 min) and a second slow phase. It indicates that living cells may be not only having surface sorption but also slower and metabolismdependent active uptake of metals. In all cases, the metal adsorption capacity for living cells is apparently higher than that of nonliving cells (Fig. 2), which is consistent with Cu(II) biosorption by resting cells and inactivated cells of Pseudomonas aeruginosa PU21 [12]. This result may be attributed to the intracellular accumulation of metal ions occurring in living cells, resulting in the enhancement in metal uptake capacity. The other possibility is that the autoclave-sterilization step, which may destroy or lose some of metal binding sites, resulting in the decrease in metal uptake capacity of the nonliving cells. 3.3. Effects of initial metal concentrations on the biosorption capacities Biosorption capacity of Cu(II) and Zn(II) by living cells rapidly increased when the initial metal concentration increased up to 50 mg/l and a slight increase thereafter as shown in Fig. 3. Nonliving cells indicated a gradual increase of biosorption of

Cu(II) and Zn(II) up to the concentration of 100 mg/l and followed by a slight increase. When the initial Cu(II) concentration was increased from 5.7 to 286.2 mg/l, the biosorption capacity of living cells increased from 3.8 to 28.6 mg/g, but only from 2.0 to 14.4 mg/g for nonliving cells. The biosorption of Zn(II) seemed to the same trends as indicated for Cu(II). When the initial concentration increased from 6.5 to 279.4 mg/l, biosorption increased from 4.5 to 26.1 mg/g for living cells and 2.4 to 15.5 mg/g for nonliving cells. It was also found that the biosorption capacities of living cells to Cu(II) and Zn(II) were signicantly higher than that of the nonliving cells at all metal concentrations. The increase of biosorption capacity of biomass with the increase of metal concentration could be attributed to higher probability of interaction between metal ions and biosorbents [33]. Moreover, higher initial metal concentration provides an increased driving force to overcome all mass transfer resistance of metals between aqueous and solid phases and accelerate the probable collision between metal ion and sorbents which results in higher metal uptake. Only slight difference in Cu(II) and Zn(II) binding capacity of nonliving P. putida CZ1 might be due to the similar properties of ionic size, atomic weight or reduction potential between these two metals [34].

Fig. 3. Effect of initial concentration of (a) Cu(II) and (b) Zn(II) on their biosorption by living and nonliving Pseudomonas putida CZ1.

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Fig. 4. Scatchard plots for (a) Cu(II) and (b) Zn(II) adsorption by living and nonliving Pseudomonas putida CZ1.

3.4. Langmuir and Freundlich adsorption isotherms The isotherm represents the equilibrium relationship between the metal uptake by the sorbent and the nal metal concentration in the aqueous phase, showing the sorption capacity of the sorbent [35]. The pH value of 5.0 was chosen as the experimental condition for the determination of adsorption isotherms except for biosorption of nonliving P. putida CZ1 to Cu(II), which was 4.5. To evaluate and compare the adsorption capacities of Cu(II) and Zn(II) by P. putida CZ1, the adsorption isotherms were analyzed and tted using Scatchard equation. When the Scatchard plot showed a deviation from linearity, greater emphasis was placed on the analysis of the adsorption data in terms of the Freundlich model in order to construct the adsorption isotherms of the ligands at particular concentration in solutions. Fig. 4 presents the adsorption characteristics assessed from the Scatchard plot. In the adsorptions of metals, Scatchard analysis of the equilibrium binding data for Cu(II) and Zn(II) on the cells of P. putida CZ1 nearly gave rise to a linear plot, indicating that the Langmuir model could be applied [31,36].

The linearized Langmuir and Freundlich adsorption isotherms of each metal for living and nonliving P. putida CZ1 were shown in Figs. 5 and 6. The adsorption constants, metal binding constant and correlation coefcients for the metals obtained from Langmuir, Freundlich isotherms and Scatchard analysis are given in Table 1. The adsorption data with respect to both metals provide an excellent t to the Langmuir isotherm. In the experiments of Cu(II) biosorption, the Qmax value of living cells of P. putida CZ1 was 29.9 mg/g, compared to 15.8 mg/g of nonliving cells. It was also found that the Qmax value of Zn(II) biosorption by living cells was higher (27.4 mg/g) than that of nonliving cells (17.7 mg/g). Compared to P. aeruginosa and P. cepacia, copper biosorption capacity value of P. putida CZ1 was lower [3,14]. But its biosorption capacity of living cells can compare well with P. syringae (25.4 mg/g) [37] and P. aeruginosa PU21 (23.0 mg/g) [12]. Moreover, both Cu(II) and Zn(II) biosorption by the cells of P. putida CZ1 are invariably higher than P. putida (6.6 and 6.9 mg/g) [17]. It is known that b is the constant related to the afnity of the binding sites, which allows us to make a comparison of the afnity of the biomass towards the metal ions. As shown in Table 1, the afnity of living cells to Cu(II) and Zn(II) (0.087 and 0.078 l/mg, respectively) was

Fig. 5. Langmuir adsorption isotherms of (a) Cu(II) and (b) Zn(II) on living and nonliving Pseudomonas putida CZ1.

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Fig. 6. Freundlich adsorption isotherms of (a) Cu(II) and (b) Zn(II) on living and nonliving Pseudomonas putida CZ1. Table 1 Adsorption isotherm parameters for Cu(II) and Zn(II) ions on living and nonliving Pseudomonas putida CZ1 Metals Biosorbent Langmuir isotherm Qmax (mg/g) Cu(II) Zn(II) Living Nonliving Living Nonliving 29.9 15.8 27.4 17.7 b (l/mg) 0.087 0.036 0.078 0.032 r2 0.9997 0.9994 0.9996 0.9952 Scatchard analysis Kb 12.62 27.03 11.53 30.40 qm (mg/g) 30.5 15.6 26.8 17.5 r2 0.9810 0.9762 0.9668 0.9194 Freundlich isotherm Kf (mg/g) 4.73 1.45 4.60 1.47 n 2.63 2.19 2.75 2.12 r2 0.8500 0.9316 0.8961 0.9575

higher than that of nonliving cells (0.036 and 0.032 l/mg, respectively). But there was no distinct difference between Cu(II) and Zn(II) biosorption by living or nonliving cells. 3.5. Desorption of Cu(II) and Zn(II) The experiment results of Cu(II) and Zn(II) biosorption and desorption are reported in Table 2. It demonstrated that about 4050% of the metals were actively taken up by P. putida CZ1, with the remainder being passively bound to the bacterium. Nearly 83.895.3% of bound Cu(II) and Zn(II) by nonliving P. putida CZ1 could be desorbed with 0.1 M HCl. However, only 45.672.5% of bound Cu(II) and Zn(II) by living cells could be desorbed. It indicates that 0.1 M HCl can effectively desorb the bound Cu(II) and Zn(II) from nonliving cells, but not very effective for living cells. And the desorption of Cu(II) is more effective than that of Zn(II). It also may be due to the living cells uptake a part of Cu(II) and Zn(II) through intracellular accumuTable 2 Desorption of Cu(II) and Zn(II) from living and nonliving Pseudomonas putida CZ1a Metals Cu(II) Zn(II)
a

lation, since it is possible to remove metals from cell surfaces after biosorption but not bioaccumulation [38]. From the desorption efciency of the living cells, it was also found that a higher percentage of Cu(II) (72.5% desorbed) adsorbed on the living cells surfaces than that of Zn(II) (only 45.6% desorbed). 4. Conclusion In this study, adsorption behavior of Cu(II) and Zn(II) by living and nonliving P. putida CZ1 from aqueous solution was investigated by the batch equilibrium technique under various conditions such as biosorption time, initial pH of the solution and metal concentration. It was found that the optimum initial pH for Zn(II) removal by living and nonliving cells was 5.0, while it was 5.0 and 4.5 for Cu(II) removal by living and nonliving cells, respectively. At the optimal conditions, metal ion biosorption was increased as the initial metal concentration. Both metals provide an excellent t to the Langmuir isotherm. Results of this study demonstrated that the binding capacity of living cells is signicantly higher than that of nonliving cells at tested conditions. It was also found that 0.1 M HCl can effectively desorb the bound Cu(II) and Zn(II) from nonliving cells, but not very effective for living cells. The results of biosorption time and desorption experiments suggested that Cu(II) and Zn(II) uptake by the living cells might be enhanced by intracellular accumulation. Moreover, the autoclave may cause destruction of some metal binding sites. The results suggest that P. putida CZ1 is a potential adsorbing media for metals in the treatment of wastewater containing

Biosorbent Living Nonliving Living Nonliving

Sorption (mg/g) 27.6 0.51 13.2 0.68 24.4 0.50 14.4 0.81

Desorption (mg/g) 20.0 0.78 12.5 0.82 11.1 0.11 12.0 0.89

Desorption (%) 72.5 1.8 95.3 2.6 45.6 1.2 83.8 4.3

Each value is an arithmetical average of a triplicate experimental data and the standard deviation.

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Cu(II) and Zn(II). Moreover, this bacterium was isolated from metal contaminated soil; it can also interact with the heavy metals in soil and inuence their potential bioavailability and impact on the environment. So this bacterium may be employed for metal remediation in simple reactors or even in situ. However, many aspects of metalmicrobe interactions remain unexplored and further researches are necessary. Acknowledgements This work was funded by the National Key Natural Science Foundation of China (40432004), the Scientic Research Foundation for the Returned Overseas Chinese Scholars, Zhejiang Province (G50437) and the Natural Science Foundation of Zhejiang Province (Y504109). Special thanks go to Mr. Perera Anton for revising the English. References
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