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Determination of the biosorption activation energies of heavy metal ions on Zoogloea ramigera and Rhizopus arrhizus
Process Biochemistry 35 (2000) 801 – 807 www.elsevier.com/locate/procbio
Determination of the biosorption activation energies of heavy metal ions on Zoogloea ramigera and Rhizopus arrhizus Yes¸im Sag˘ *, Tu¨lin Kutsal Department of Chemical Engineering, Faculty of Engineering, Hacettepe Uni6ersity, 06532 Beytepe, Ankara, Turkey Received 2 August 1999; received in revised form 22 October 1999; accepted 6 November 1999
Abstract The activation energies of Fe(III) and Pb(II) ions on Zoogloea ramigera and Fe(III), Cr(VI) and Ni(II) ions on Rhizopus arrhizus were determined using the Arrhenius equation. Batch adsorption kinetics was described by the Langmuir – Hinshelwood model. The applicability of the Langmuir–Hinshelwood model for the metal – microorganism systems was tested at different temperatures in the range 15–45°C. With respect to the magnitude of the activation energy of biosorption, the dominant adsorption mechanism in the whole biosorption process was proposed for each metal – microorganism system. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Biosorption; Z. ramigera; R arrhizus; Heavy metal ions; Temperature effect; Langmuir – Hinshelwood model; Activation energy
1. Introduction Accumulation of metals by microorganisms has been known for a few decades but has received more attention in recent years because of its potential application in environmental protection or recovery of precious or strategic metals. Biological processes for removal of metal ions from solution can be divided into three general categories: (i) biosorption (adsorption) of metal ions onto the surfaces of a microorganism, (ii) intracellular uptake of metal ions, and (iii) chemical transformation of metal ions by microorganisms. The latter two processes require living organisms [1 – 4]. The active mode of metal accumulation by living cells is designated as bioaccumulation. Non-viable microbial biomass frequently exhibits a higher affinity for metal ions compared with viable biomass probably due to the absence of competing protons produced during metabolism. To avoid the problems of toxicity of metals for microbial growth, or inhibition of metal accumulation by nutrient or excreted metabolites, the decoupling of the growth of the biomass from its * Corresponding author. Tel.: +90-312-2977444; fax: + 90-3122992124. E-mail address: [email protected] (Y. Sag˘)
function as a metal-sorbing material is seen as a one of the major advantages of biosorption [5–7]. Biosorption is caused by a number of different physicochemical mechanisms, depending on a number of external environmental factors as well as on the type of a metal, its ionic form in the solution, and on the type of a particular active binding site responsible for sequestering the metal. Temperature, adsorption pH, initial metal ion concentration, biomass concentration, and concentrations of other interfering ions are the environmental influences which are important in the biosorption of heavy metal ions [8–10]. The influence of pH and the metal/biosorbent ratio on heavy metal removal by biosorption have been widely recognized [2,5,11–13], but information on the effect of temperature is still scanty. The binding of most metals to microorganisms by biosorption is observed to enchance as temperature is increased [13,14]. Although the search for new and innovative treatment technologies has focused attention on the metal binding capacities of various microorganisms, the exact interactions between the ligands on the cell walls and the heavy metal ions, the kinetics of the metal uptake process and the description of the thermal properties of the biosorption remain essentially unknown. Although the magnitude of the heat effect for the biosorption
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Y. Sag˘, T. Kutsal / Process Biochemistry 35 (2000) 801–807
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process is the most important criterion to develop a thermodynamic and kinetic relationship for the metal– microorganism interaction process, little attention seems to have been given to the study of the evaluation of the heat change and/or activation energy of biosorption process. The Langmuir model, the most simple used for adsorption phenomena of one component, has a theoretical basis, which relies on a postulated chemical or physical interaction (or both) between solute and vacant sites on the adsorbent surface [15 – 17]. The adsorption rate is ra = kC(1−u)
(1)
the desorption rate is (2)
rd = k%u
where C is the unadsorbed solute concentration in solution, k and k%, respectively the adsorption and desorption rate constants and u, the fraction of surface covered by adsorbed solute. At equilibrium, the equality of these two rates leads to the Langmuir adsorption isotherm: u=
KC 1 + KC
(3)
where the adsorption equilibrium constant is K= k/k%. Combining Eq. (1) and Eq. (3), the Langmuir – Hinshelwood adsorption equation modified for monolayer adsorption is obtained and the rate of adsorption is given as follows [15–17]: r=
kC 1 +KC
(4)
In an experimental data plot of rate versus C, the rate of adsorption is proportional to the first power of the concentration of metal ion at lower bulk metal ion concentrations and can be given using Eq. (5): (5)
r= kC
At higher bulk metal ion concentrations, the rate of adsorption becomes independent of bulk metal ion concentration. Eq. (4) can describe the rate of adsorption very accurately in both of these situations. This kind of rate equation is also defined as ‘saturation type rate’. This rate equation can be linearized by plotting 1/r versus 1/C to determine the rate and equilibrium constants of adsorption from the slope and the intercept, 1/k and K/k, respectively. The rate of adsorption depends on the temperature, through variation of the rate coefficient. According to the Arrhenius equation [15,17]: ln k= −
E1 + ln A0 RT
(6)
where E is activation energy and A0 is a constant called the frequency factor. Consequently, when ln k is plotted versus 1/T, a straight line with slope −E/R is obtained.
2. Materials and methods
2.1. Microorganisms and preparation of the microorganisms for biosorption Zooloea ramigera, an activated sludge bacterium, and Rhizopus arrhizus, a filamentous fungus, were obtained from the US Department of Agriculture Culture Collection. Z. ramigera and R. arrhizus were grown aerobically in batch cultures at 25 and 30°C, respectively as described previously [18,19]. In the stationary phase of growth (120 h), Z. ramigera cells were centrifuged at 5000 rev min − 1 for 5 min, washed twice with distilled water and then dried in an oven at 60°C for 24 h. After the growth period, R. arrhizus was washed twice with distilled water, inactivated using 1% formaldehyde and then dried at 60°C for 24 h. For biosorption studies, a weighed amount of dried cells was suspended in 100 cm3 of distilled water and homogenized for 20 min in a homogenizer (Ultra-Turrax T 25; Janke and Kunkel, IKA-Labortechnik) at 8000 rev min − 1.
2.2. Preparation of biosorption media and biosorption studies Ni(II), Pb(II) and Cr(VI) solutions were prepared by diluting 1.0 g litre − 1 stock solutions of nickel(II), lead(II) and chromium(VI), obtained by dissolving Ni(NO3)2·6H2O, Pb(NO3)2, and K2Cr2O7 in distilled water, respectively. The stock solution of ferric iron (1.0 g litre − 1) from ferrous ammonium sulphate was prepared as described in the literature [20]. The range of concentrations of prepared metal solutions varied from 25 to 200 mg litre − 1. Before mixing with the bacterial and fungal suspensions, the pH of nickel(II) and lead(II) solutions was adjusted to pH 4.5 with 1 mol litre − 1 of HNO3. The pH of biosorption media was adjusted to pH 2.0 for the biosorption of chromium(VI) and iron(III) ions with l mol litre − 1 of H2SO4. In earlier studies, these pH values were determined as the optimum pH values for the biosorption of Ni(II), Pb(II), Cr(VI) and Fe(III) ions on R. arrhizus and Z.ramigera and were held constant in thermodynamic studies [19,21]. Bacterial and fungal suspensions (10 ml) were mixed with 90 ml of the desired metal solutions in an Erlenmeyer flask. Before mixing the fungal suspension and the metal-bearing solution, 3 ml samples were taken from the biosorption media. Subsequently, samples were taken at 1 min intervals at the beginning of adsorption and at 25–30 min intervals after reaching
Y. Sag˘, T. Kutsal / Process Biochemistry 35 (2000) 801–807
803
equilibrium. The samples were centrifuged at 6030× g for 3 min and the supernatant liquid was used to analyse for metal ions.
2.3. Analysis of hea6y metal ions The concentration of unadsorbed Pb(II) ions in the sample supernatant was determined using an atomic absorption spectrophotometer. The concentrations of free Ni(II), Fe(III) and Cr(VI) ions in the solution were determined spectrophotometrically. The coloured complex of Ni(II) ions with sodium diethyl dithiocarbamate, the coloured complex of Fe(III) ions with sodium salicylate and the coloured complex of Cr(VI) ions with diphenly carbazide were read at 400, 530 and 540 nm, respectively [20].
3. Results and discussion Upon contact between the biosorbent and the solution containing the metal species, an equilibrium is established at a given temperature whereby a certain amount of the metal species sequestered by the biosorbent is in equilibrium with its residue left free in the solution containing then the residual, final, or equilibrium concentration of that metal species. Equilibrium considerations of the biosorption process have been extensively investigated [10,21 – 24]. In addition to equilibrium studies, the kinetics of the biosorption has to be determined in order to establish the time course of the metal uptake. Rapid uptake of the metal by the biosorbent is desirable providing for a short solution biosorbent contact time in the actual process. In this paper, the kinetic results are given as the initial adsorption rates, ri (mg g − 1 min − 1). The initial biosorption rate is obtained by calculating the slope of a plot of the adsorbed metal ion quantity q per gram of dried microorganism (mg g − 1) versus time (min) at t =0 (Fig. 1). The initial biosorption rates of Fe(III) and Pb(II) ions by Z. ramigera increased with increasing initial metal ion concentrations, Ci, up to 100 – 300 mg litre − 1 Maximum initial biosorption rates for Fe(III) and Pb(II) ions were determined as 4.15 and 11.60 mg g − 1 min − 1 at 125–150, 150 – 300 mg litre − 1 initial metal ion concentrations, respectively and at 25°C. The initial biosorption rates of Fe(III), Cr(VI) and Ni(II) ions by R. arrhizus increased with increasing metal ion concentrations up to 125– 200 mg litre − 1. Maximum initial biosorption rates for Fe(III), Cr(VI) and Ni(II) ions were 3.90, 8.43 and 10.32 mg g − 1 min − 1 at 100–125, 125 – 150 and 125– 200 mg litre − 1 initial metal ion concentrations, respectively at 25°C. Since a fixed cell biomass offers a finite number of surface binding sites, initial uptake would be expected to show saturation
Fig. 1. The adsorption curve for the biosorption of Fe(III) ions on Z. ramigera at a constant initial metal ion concentration of 100 mg litre − 1 (pH 2.0; temperature: 25°C; biomass concentration: 1.0 g litre − 1; agitation rate: 150 rev min − l).
kinetics at high total metal ion concentrations, and this was observed. The initial biosorption rates of Z. ramigera for Fe(III) and Pb(II) ions also increased with increasing temperatures in the range 35–45°C and were found to be 7.34 and 18.20 mg g − 1 min − 1, respectively. The biosorption of Fe(III) and Cr(VI) ions on R. arrhizus was favoured at higher temperatures, i.e. a maximum initial Cr(VI) biosorption rate of 9.90 mg g − 1 min − 1 was reached at 45°C, although the increase for Ni(II) ions was not very significant. The effect of initial metal ion concentrations on the initial biosorption rates of Fe(III) ions on Z. ramigera and Cr(VI) ions on R. arrhizus was shown at temperatures changing from 15 to 45°C in Figs. 2 and 3, respectively. The linearized Langmuir–Hinshelwood plots for the biosorption of Fe(III)–Pb(II) ions on Z. ramigera and Fe(III)–Cr(VI) ions on R. arrhizus at different tempera-
Fig. 2. The change of initial biosorption rates of Fe(III) ions on Z. ramigera with initial Fe(III) ion concentrations at different temperatures (pH 2.0; biomass concentration: 1.0 g litre − 1 agitation rate: 150 rev min − 1).
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Y. Sag˘, T. Kutsal / Process Biochemistry 35 (2000) 801–807
Fig. 3. The change of initial biosorption rates of Cr(VI) ions on R. arrhizus with initial Cr(VI) ion concentrations at different temperatures (pH 2.0; biomass concentration: 1.0 g litre − 1; agitation rate: 150 rev min − l).
tures are given in Figs. 4–7. Adsorption rate constant, k and equilibrium constant, K, for the biosoption of heavy metal ions on Z. ramigera and R. arrhizus at a constant temperature were calculated from slope and intercept of a double-reciprocal plot of 1/ri versus 1/Ci and are listed in Tables 1 and 2. The adsorption rate constants of Fe(III) ions on Z. ramigera and Fe(III)– Cr(VI) ions on R. arrhizus were sharply affected by temperature rises while the adsorption rate constants for the biosorption of Pb(II) ions on Z. ramigera and Ni(II) ions on R. arrhizus are relatively insensitive to temperature. The maximum adsorption rate and equilibrium constants were obtained for the biosorption of Fe(III) ions on Z. ramigera. Numerical values for activation energy of biosoption process were obtained by plotting experimental data for rate constants at different temperatures. From the log-
Fig. 4. The linearized Langmuir–Hinshelwood plots for the biosorption of Fe(III) ions on Z. ramigera at different temperatures (pH 2.0; biomass concentration: 1.0 g litre − 1; agitation rate: 150 rev min − l).
Fig. 6. The linearized Langmuir – Hinshelwood plots for the biosorption of Cr(VI) ions on R. arrhizus at different temperatures (pH 2.0; biomass concentration: 1.0 g litre − 1; agitation rate: 150 rev min − l).
Fig. 5. The linearized Langmuir–Hinshelwood plots for the biosorption of Fe(III) ions on R. arrhizus at different temperatures (pH 2.0; biomass concentration: 1.0 g litre − 1; agitation rate: 150 rev min − l).
Fig. 7. The linearized Langmuir – Hinshelwood plots for the biosorption of Pb(II) ions on Z. ramigera at different temperatures (pH 4.5; biomass concentration: 1.0 g litre − 1; agitation rate: 150 rev min − l).
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Table 1 The adsorption rate and equilibrium constants for the biosorption of Fe(III) and Pb(II) ions on Z. ramigera T (°C)
15 25 35 45
Iron(III)
Lead(II)
k (l g−1 min−1)
K (l mg−1)
k (l g−1 min−1)
K (l mg−1)
0.0427 0.1552 0.1855 0.3463
0.0103 0.0304 0.0283 0.0385
0.1314 0.1276 0.1571 0.1392
0.0126 0.0050 0.0020 0.0014
arithmic form of the Arrhenius equation, a plot of ln k versus 1/T yields a slope equal to − E/R. Since the biosorption rates of the metal ions examined increased with temperature, the slopes of Arrhenius plots give negative values and the activation energy is normally found to be positive (Figs. 8 – 10). For biological systems, the reported activation energies in the literature generally change over the range 8.4 – 83.7 kJ mol − 1 (2 – 20 kcal mol − 1) [25]. Two main types of adsorption may occur: physical and chemical adsorption. In physical adsorption, equilibrium between the adsorbent surface and the adsorbate is usually rapidly attained and easily reversible, because the energy requirements are small. The energy of activation for physical adsorption is usually no more than 4.184 kJ mol − 1 (1.0 kcal mol − 1) [15], since the forces involved in physical adsorption are weak. Two kinds of chemisorption are encountered: activated and less frequently, nonactivated. Activated chemisorption means that the rate varies with temperature according to a finite activation energy in the Arrhenius equation (high E). However, in some systems chemisorption occurs very rapidly, suggesting the activation energy is near zero. This is termed nonactivated chemisorption [15]. The observed values of the activation energies of biosorption of Fe(III) ions on both microorganisms and Cr(VI) ions on R. arrhizus are of the same magnitude as the activation energy of chemisorption, while the activation energies for the biosorption of Pb(II) ions on Z. ramigera and Ni(II) ions on R arrhizus have
Fig. 8. The linearized Arrhenius plots for the biosorption of Fe(III) ions on Z. ramigera and R. arrhizus (pH 2.0; biomass concentration: 1.0 g litre − 1; agitation rate: 150 rev min − 1).
Fig. 9. The linearized Arrhenius plot for the biosorption of Cr(VI) ions on R. arrhizus (pH 2.0; biomass concentration: 1.0 g litre − 1; agitation rate: 150 rev min − 1).
boundary values of activation energy of biosorption between the physical and chemical adsorption (Table 3). It is difficult to decide which mechanism is effective on the biosorption of heavy metal ions on microorganisms, only taking activation energies into consideration. With respect to the magnitude of the heat of biosorp-
Table 2 The adsorption rate and equilibrium constants for the biosorption of Fe(III), Cr(VI) and Ni(II) ions on R. arrhizus T (°C)
15 25 35 45
Iron(III)
Chromium(VI)
Nickel(II)
k (l g−1 min−1)
K (l mg−1)
k (l g−1 min−1)
K (l mg−1)
k (l g−1 min−1)
K (l mg−1)
0.0499 0.0797 0.1327 0.1183
0.0107 0.0101 0.0182 0.0158
0.0497 0.1301 0.1076 0.1321
0.0009 0.0077 0.0028 0.0061
0.0695 0.0734 0.0645 0.0866
0.0104 0.0002 0.0053 0.0248
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Fig. 10. The linearized Arrhenius plots for the biosorption of Pb(II) ions on Z. ramigera and Ni(II) ions on R. arrhizus (pH 4.5; biomass concentration: 1.0 g litre − 1; agitation rate: 150 rev min − 1). Table 3 The activation energies for the biosorption of heavy metal ions on Z. ramigera and R. arrhizus Microorganism–metal
E (kJ mol−1)
Z. Z. R. R. R.
48.8 6.2 23.4 20.7 4.0
ramigera–iron(III) ramigera–lead(II) arrhizus–iron(III) arrhizus–chromium(VI) arrhizus–nickel(II)
tion, the dominant adsorption mechanism in the whole biosorption process can also be proposed for each metal–microorganism system. The enthalpy change for the biosorption process of heavy metal ions on Z. ramigera and R. arrhizus has been evaluated. The heats of biosorption of Fe(III) and Cr(VI) ions on both microorganisms have been determined to be of the same magnitude as the heat of chemisorption, while the biosorption of Pb(II) ions on Z. ramigera and Ni(II) ions on R. arrhizus has shown a heat effect comparable to that of physical adsorption [26]. The pattern of biosorption of the metal ions by Z. ramigera and R. arrhizus also follows this classification [19,21,23,26]. The biosorption of Pb(II) and Ni(II) ions is completely reversible, equilibrium is established very rapidly and cycling of adsorption and desorption, as by raising and lowering the pH using some eluation solutions, can be performed repeatedly. Chemisorption may or may not be reversible. A chemical change in the adsorbate upon desorption is good evidence indeed that chemisorption in fact occurred. Cr(VI) and Fe(III) ions are removed with difficulty from chelating molecules by pH manipulation and the total amount of the bound metal ions may not be eluated from the biomass. Oxidation/reduction reactions are also used to facilitate release of metals from ligands. The classic biological
example of this is hydroxamate siderophore release of iron brought about by reduction of Fe(III) to Fe(II). Fe(II) has a very low affinity for the siderophore and is easily removed. In some cases, the metal ligand complex must undergo hydrolysis in order to release the iron [27]. On the other hand, Cr(VI) in the form of dichromate is anionic and a strong oxidising agent, which properties may account for a number of the observations such as its irreversible binding to the biomass. By treating an alkaline solution, elusion of the bound Cr(VI) can be accomplished. However, if left in contact with microorganisms for extended periods of time or if exposed to very low pH values, the bound Cr(VI) may be reduced to Cr(III) [13]. It should be noted that both physical adsorption and chemisorption can occur together, but any adsorbed layers beyond the first must presumably be physically adsorbed.
4. Conclusions Despite the quite extensive literature available on heavy metal biosorption, little attention seems to have been given to the temperature dependence of the biosoption process. In this study, batch adsorption experiments were performed at different temperatures in the range 15–45°C and the effect of initial metal ion concentration on the initial biosorption rates was investigated. The initial uptake kinetics of heavy metal ions on Z. ramigera and R arrhizus was shown to be represented by Langmuir–Hinshelwood type kinetics or saturation kinetics. From double-reciprocal Langmuir–Hinshelwood plots, the biosorption rate constants were obtained at different temperatures. The biosorption rate constants vary with temperature according to the Arrhenius equation. From the slopes of linearized Arrhenius plots, activation energies were determined and appropriate adsorption mechanism with respect to the magnitude of the activation energy of biosorption has been proposed for each metal–microorganism system. The activation energies for the biosorption of Fe(III) ions on both microorganisms and Cr(VI) ions on R. arrhizus were determined to be of the same order of magnitude as the activation energy of chemisorption.
References [1] Volesky B. Removal and recovery of heavy metals by biosorption. In: Volesky B, editor. Biosorption of Heavy Metals. Boca Raton, FL: CRC Press, 1990:7 – 43. [2] Veglio F, Beolchini F. Removal of metals by biosorption: a review. Hydrometallurgy 1997;44:301 – 16. [3] Corder SL, Reeves M. Biosorption of nickel in complex aqueous waste streams by Cyanobacteria. Appl Biochem Biotechnol 1994;45/46:847– 59.
Y. Sag˘, T. Kutsal / Process Biochemistry 35 (2000) 801–807 [4] Darnall DW, Greene B, Henzl MT, Hosea JM, McPherson RA, Sneddon J, Alexander MD. Selective recovery of gold and other metal ions from an algal biomass. Environ Sci Technol 1986;20:206 – 8. [5] Basnakova G, Macaskie LE. Bioaccumulation of nickel by microbially-enchanced chemisorption into polycrystalline hydrogen uranyl phosphate. Biotechnol Lett 1996;18:257–62. [6] Fourest E, Roux J-C. Heavy metal biosorption by fungal mycelial by-products: mechanisms and influence of pH. Appl Microbiol Biotechnol 1992;37:399–403. [7] Brady JM, Tobin JM, Roux J-C. Continuous fixed bed biosorption of Cu2 + ions: application of a simple two parameter mathematical model. J Chem Technol Biotechnol 1999;74:71 – 7. [8] Wilde EW, Benemann JR. Bioremoval of heavy metals by the use of microalgae. Biotechnol Adv 1993;11:781–812. [9] Panchanadikar VV, Das RP. Biosorption process for removing lead(II) ions from aqueous effluents using Pseudomonas sp. Int J Environ Stud 1994;46:243–50. [10] Veglio F, Beolchini F, Gasbarro A. Biosorption of toxic metals: an equilibrium study using free cells of Arthrobacter sp. Process Biochem 1997;32:99 –105. [11] Zhou JL, Kiff RJ. The uptake of copper from aqueous solution by immobilized fungal biomass. J Chem Technol Biotechnol 1991;52:317 – 30. [12] Geddie JL, Sutherland IW. Uptake of metals by bacterial polysaccharides. J Appl Bacteriol 1993;74:467–72. [13] Bedell GW, Darnall DW. Immobilization of nonviable, biosorbent, algal biomass for the recovery of metal ions. In: Volesky B, editor. Biosorption of Heavy Metals. Boca Raton, FL: CRC Press, 1990:314 – 26. [14] Bedell GW. Propagation of freshwater, algal biosorbents. In: Volesky B, editor. Biosorption of Heavy Metals. Boca Raton, FL: CRC Press, 1990:359–70. [15] Smith JM. Chemical Engineering Kinetics, 3rd edition. New York: McGraw – Hill, 1981.
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[16] Hayward DO, Trapnell BMW. Chemisorption, 2nd edition. London: Butterworth, 1964. [17] Satterfield CN. Heterogeneous Catalysis in Practice. New York: McGraw – Hill, 1980. [18] Aksu Z, Sag˘ Y, Kutsal T. A comparative study of the adsorption of chromium(VI) ions to C. 6ulgaris and Z. ramigera. Environ Technol 1990;11:33 – 40. [19] Sag˘ Y, Kutsal T. The selective biosorption of chromium(VI) and copper(II) ions from binary metal mixtures by R. arrhizus. Process Biochem 1996;31:561 – 72. [20] Snell FD, Snell CT. Colorimetric Methods of Analysis, 3rd edition. New York: Interscience, 1961. [21] Sag˘ Y, Kutsal T. Biosorption of heavy metals by Zoogloea ramigera: use of adsorption isotherms and a comparison of biosorption characteristics. Chem Eng J 1995;60:181 – 8. [22] Tsezos M, Georgousis Z, Remoudaki E. Mechanism of aluminum interference on uranium biosorption by Rhizopus arrhizus. Biotechnol Bioeng 1997;55:16 – 27. [23] Sag˘ Y, Ac¸ıkel U8 , Aksu Z, Kutsal T. A comparative study for the simultaneous biosorption of Cr(VI) and Fe(III) on C. 6ulgaris and R. arrhizus: application of the competitive adsorption models. Process Biochem 1998;33:273 – 81. [24] Chang J-S, Chen C-C. Quantitative analysis and equilibrium models of selective adsorption in multimetal systems using a bacterial biosorbent. Sep Sci Technol 1998;33:611 – 32. [25] Shuler ML, Kargi F. Bioprocess Engineering. Englewood Cliffs, NJ: Prentice Hall, 1992. [26] Sag˘ Y. Selection of the best biosorbent for removal and recovery of the heavy metal ions from waste water and mathematical investigation of the adsorption in the different reactor systems. PhD Thesis, Hacettepe University, Ankara, Turkey, 1993. [27] Huber AL, Holbein BE, Kidby DK. Metal uptake by synthetic and biosynthetic chemicals. In: Volesky B, editor. Biosorption of Heavy Metals. Boca Raton, FL: CRC Press, 1990:249–92.
Determination of the biosorption activation energies of heavy metal ions on Zoogloea ramigera and Rhizopus arrhizus Yes¸im Sag˘ *, Tu¨lin Kutsal Department of Chemical Engineering, Faculty of Engineering, Hacettepe Uni6ersity, 06532 Beytepe, Ankara, Turkey Received 2 August 1999; received in revised form 22 October 1999; accepted 6 November 1999
Abstract The activation energies of Fe(III) and Pb(II) ions on Zoogloea ramigera and Fe(III), Cr(VI) and Ni(II) ions on Rhizopus arrhizus were determined using the Arrhenius equation. Batch adsorption kinetics was described by the Langmuir – Hinshelwood model. The applicability of the Langmuir–Hinshelwood model for the metal – microorganism systems was tested at different temperatures in the range 15–45°C. With respect to the magnitude of the activation energy of biosorption, the dominant adsorption mechanism in the whole biosorption process was proposed for each metal – microorganism system. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Biosorption; Z. ramigera; R arrhizus; Heavy metal ions; Temperature effect; Langmuir – Hinshelwood model; Activation energy
1. Introduction Accumulation of metals by microorganisms has been known for a few decades but has received more attention in recent years because of its potential application in environmental protection or recovery of precious or strategic metals. Biological processes for removal of metal ions from solution can be divided into three general categories: (i) biosorption (adsorption) of metal ions onto the surfaces of a microorganism, (ii) intracellular uptake of metal ions, and (iii) chemical transformation of metal ions by microorganisms. The latter two processes require living organisms [1 – 4]. The active mode of metal accumulation by living cells is designated as bioaccumulation. Non-viable microbial biomass frequently exhibits a higher affinity for metal ions compared with viable biomass probably due to the absence of competing protons produced during metabolism. To avoid the problems of toxicity of metals for microbial growth, or inhibition of metal accumulation by nutrient or excreted metabolites, the decoupling of the growth of the biomass from its * Corresponding author. Tel.: +90-312-2977444; fax: + 90-3122992124. E-mail address: [email protected] (Y. Sag˘)
function as a metal-sorbing material is seen as a one of the major advantages of biosorption [5–7]. Biosorption is caused by a number of different physicochemical mechanisms, depending on a number of external environmental factors as well as on the type of a metal, its ionic form in the solution, and on the type of a particular active binding site responsible for sequestering the metal. Temperature, adsorption pH, initial metal ion concentration, biomass concentration, and concentrations of other interfering ions are the environmental influences which are important in the biosorption of heavy metal ions [8–10]. The influence of pH and the metal/biosorbent ratio on heavy metal removal by biosorption have been widely recognized [2,5,11–13], but information on the effect of temperature is still scanty. The binding of most metals to microorganisms by biosorption is observed to enchance as temperature is increased [13,14]. Although the search for new and innovative treatment technologies has focused attention on the metal binding capacities of various microorganisms, the exact interactions between the ligands on the cell walls and the heavy metal ions, the kinetics of the metal uptake process and the description of the thermal properties of the biosorption remain essentially unknown. Although the magnitude of the heat effect for the biosorption
0032-9592/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 9 9 ) 0 0 1 5 4 - 5
Y. Sag˘, T. Kutsal / Process Biochemistry 35 (2000) 801–807
802
process is the most important criterion to develop a thermodynamic and kinetic relationship for the metal– microorganism interaction process, little attention seems to have been given to the study of the evaluation of the heat change and/or activation energy of biosorption process. The Langmuir model, the most simple used for adsorption phenomena of one component, has a theoretical basis, which relies on a postulated chemical or physical interaction (or both) between solute and vacant sites on the adsorbent surface [15 – 17]. The adsorption rate is ra = kC(1−u)
(1)
the desorption rate is (2)
rd = k%u
where C is the unadsorbed solute concentration in solution, k and k%, respectively the adsorption and desorption rate constants and u, the fraction of surface covered by adsorbed solute. At equilibrium, the equality of these two rates leads to the Langmuir adsorption isotherm: u=
KC 1 + KC
(3)
where the adsorption equilibrium constant is K= k/k%. Combining Eq. (1) and Eq. (3), the Langmuir – Hinshelwood adsorption equation modified for monolayer adsorption is obtained and the rate of adsorption is given as follows [15–17]: r=
kC 1 +KC
(4)
In an experimental data plot of rate versus C, the rate of adsorption is proportional to the first power of the concentration of metal ion at lower bulk metal ion concentrations and can be given using Eq. (5): (5)
r= kC
At higher bulk metal ion concentrations, the rate of adsorption becomes independent of bulk metal ion concentration. Eq. (4) can describe the rate of adsorption very accurately in both of these situations. This kind of rate equation is also defined as ‘saturation type rate’. This rate equation can be linearized by plotting 1/r versus 1/C to determine the rate and equilibrium constants of adsorption from the slope and the intercept, 1/k and K/k, respectively. The rate of adsorption depends on the temperature, through variation of the rate coefficient. According to the Arrhenius equation [15,17]: ln k= −
E1 + ln A0 RT
(6)
where E is activation energy and A0 is a constant called the frequency factor. Consequently, when ln k is plotted versus 1/T, a straight line with slope −E/R is obtained.
2. Materials and methods
2.1. Microorganisms and preparation of the microorganisms for biosorption Zooloea ramigera, an activated sludge bacterium, and Rhizopus arrhizus, a filamentous fungus, were obtained from the US Department of Agriculture Culture Collection. Z. ramigera and R. arrhizus were grown aerobically in batch cultures at 25 and 30°C, respectively as described previously [18,19]. In the stationary phase of growth (120 h), Z. ramigera cells were centrifuged at 5000 rev min − 1 for 5 min, washed twice with distilled water and then dried in an oven at 60°C for 24 h. After the growth period, R. arrhizus was washed twice with distilled water, inactivated using 1% formaldehyde and then dried at 60°C for 24 h. For biosorption studies, a weighed amount of dried cells was suspended in 100 cm3 of distilled water and homogenized for 20 min in a homogenizer (Ultra-Turrax T 25; Janke and Kunkel, IKA-Labortechnik) at 8000 rev min − 1.
2.2. Preparation of biosorption media and biosorption studies Ni(II), Pb(II) and Cr(VI) solutions were prepared by diluting 1.0 g litre − 1 stock solutions of nickel(II), lead(II) and chromium(VI), obtained by dissolving Ni(NO3)2·6H2O, Pb(NO3)2, and K2Cr2O7 in distilled water, respectively. The stock solution of ferric iron (1.0 g litre − 1) from ferrous ammonium sulphate was prepared as described in the literature [20]. The range of concentrations of prepared metal solutions varied from 25 to 200 mg litre − 1. Before mixing with the bacterial and fungal suspensions, the pH of nickel(II) and lead(II) solutions was adjusted to pH 4.5 with 1 mol litre − 1 of HNO3. The pH of biosorption media was adjusted to pH 2.0 for the biosorption of chromium(VI) and iron(III) ions with l mol litre − 1 of H2SO4. In earlier studies, these pH values were determined as the optimum pH values for the biosorption of Ni(II), Pb(II), Cr(VI) and Fe(III) ions on R. arrhizus and Z.ramigera and were held constant in thermodynamic studies [19,21]. Bacterial and fungal suspensions (10 ml) were mixed with 90 ml of the desired metal solutions in an Erlenmeyer flask. Before mixing the fungal suspension and the metal-bearing solution, 3 ml samples were taken from the biosorption media. Subsequently, samples were taken at 1 min intervals at the beginning of adsorption and at 25–30 min intervals after reaching
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equilibrium. The samples were centrifuged at 6030× g for 3 min and the supernatant liquid was used to analyse for metal ions.
2.3. Analysis of hea6y metal ions The concentration of unadsorbed Pb(II) ions in the sample supernatant was determined using an atomic absorption spectrophotometer. The concentrations of free Ni(II), Fe(III) and Cr(VI) ions in the solution were determined spectrophotometrically. The coloured complex of Ni(II) ions with sodium diethyl dithiocarbamate, the coloured complex of Fe(III) ions with sodium salicylate and the coloured complex of Cr(VI) ions with diphenly carbazide were read at 400, 530 and 540 nm, respectively [20].
3. Results and discussion Upon contact between the biosorbent and the solution containing the metal species, an equilibrium is established at a given temperature whereby a certain amount of the metal species sequestered by the biosorbent is in equilibrium with its residue left free in the solution containing then the residual, final, or equilibrium concentration of that metal species. Equilibrium considerations of the biosorption process have been extensively investigated [10,21 – 24]. In addition to equilibrium studies, the kinetics of the biosorption has to be determined in order to establish the time course of the metal uptake. Rapid uptake of the metal by the biosorbent is desirable providing for a short solution biosorbent contact time in the actual process. In this paper, the kinetic results are given as the initial adsorption rates, ri (mg g − 1 min − 1). The initial biosorption rate is obtained by calculating the slope of a plot of the adsorbed metal ion quantity q per gram of dried microorganism (mg g − 1) versus time (min) at t =0 (Fig. 1). The initial biosorption rates of Fe(III) and Pb(II) ions by Z. ramigera increased with increasing initial metal ion concentrations, Ci, up to 100 – 300 mg litre − 1 Maximum initial biosorption rates for Fe(III) and Pb(II) ions were determined as 4.15 and 11.60 mg g − 1 min − 1 at 125–150, 150 – 300 mg litre − 1 initial metal ion concentrations, respectively and at 25°C. The initial biosorption rates of Fe(III), Cr(VI) and Ni(II) ions by R. arrhizus increased with increasing metal ion concentrations up to 125– 200 mg litre − 1. Maximum initial biosorption rates for Fe(III), Cr(VI) and Ni(II) ions were 3.90, 8.43 and 10.32 mg g − 1 min − 1 at 100–125, 125 – 150 and 125– 200 mg litre − 1 initial metal ion concentrations, respectively at 25°C. Since a fixed cell biomass offers a finite number of surface binding sites, initial uptake would be expected to show saturation
Fig. 1. The adsorption curve for the biosorption of Fe(III) ions on Z. ramigera at a constant initial metal ion concentration of 100 mg litre − 1 (pH 2.0; temperature: 25°C; biomass concentration: 1.0 g litre − 1; agitation rate: 150 rev min − l).
kinetics at high total metal ion concentrations, and this was observed. The initial biosorption rates of Z. ramigera for Fe(III) and Pb(II) ions also increased with increasing temperatures in the range 35–45°C and were found to be 7.34 and 18.20 mg g − 1 min − 1, respectively. The biosorption of Fe(III) and Cr(VI) ions on R. arrhizus was favoured at higher temperatures, i.e. a maximum initial Cr(VI) biosorption rate of 9.90 mg g − 1 min − 1 was reached at 45°C, although the increase for Ni(II) ions was not very significant. The effect of initial metal ion concentrations on the initial biosorption rates of Fe(III) ions on Z. ramigera and Cr(VI) ions on R. arrhizus was shown at temperatures changing from 15 to 45°C in Figs. 2 and 3, respectively. The linearized Langmuir–Hinshelwood plots for the biosorption of Fe(III)–Pb(II) ions on Z. ramigera and Fe(III)–Cr(VI) ions on R. arrhizus at different tempera-
Fig. 2. The change of initial biosorption rates of Fe(III) ions on Z. ramigera with initial Fe(III) ion concentrations at different temperatures (pH 2.0; biomass concentration: 1.0 g litre − 1 agitation rate: 150 rev min − 1).
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Fig. 3. The change of initial biosorption rates of Cr(VI) ions on R. arrhizus with initial Cr(VI) ion concentrations at different temperatures (pH 2.0; biomass concentration: 1.0 g litre − 1; agitation rate: 150 rev min − l).
tures are given in Figs. 4–7. Adsorption rate constant, k and equilibrium constant, K, for the biosoption of heavy metal ions on Z. ramigera and R. arrhizus at a constant temperature were calculated from slope and intercept of a double-reciprocal plot of 1/ri versus 1/Ci and are listed in Tables 1 and 2. The adsorption rate constants of Fe(III) ions on Z. ramigera and Fe(III)– Cr(VI) ions on R. arrhizus were sharply affected by temperature rises while the adsorption rate constants for the biosorption of Pb(II) ions on Z. ramigera and Ni(II) ions on R. arrhizus are relatively insensitive to temperature. The maximum adsorption rate and equilibrium constants were obtained for the biosorption of Fe(III) ions on Z. ramigera. Numerical values for activation energy of biosoption process were obtained by plotting experimental data for rate constants at different temperatures. From the log-
Fig. 4. The linearized Langmuir–Hinshelwood plots for the biosorption of Fe(III) ions on Z. ramigera at different temperatures (pH 2.0; biomass concentration: 1.0 g litre − 1; agitation rate: 150 rev min − l).
Fig. 6. The linearized Langmuir – Hinshelwood plots for the biosorption of Cr(VI) ions on R. arrhizus at different temperatures (pH 2.0; biomass concentration: 1.0 g litre − 1; agitation rate: 150 rev min − l).
Fig. 5. The linearized Langmuir–Hinshelwood plots for the biosorption of Fe(III) ions on R. arrhizus at different temperatures (pH 2.0; biomass concentration: 1.0 g litre − 1; agitation rate: 150 rev min − l).
Fig. 7. The linearized Langmuir – Hinshelwood plots for the biosorption of Pb(II) ions on Z. ramigera at different temperatures (pH 4.5; biomass concentration: 1.0 g litre − 1; agitation rate: 150 rev min − l).
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Table 1 The adsorption rate and equilibrium constants for the biosorption of Fe(III) and Pb(II) ions on Z. ramigera T (°C)
15 25 35 45
Iron(III)
Lead(II)
k (l g−1 min−1)
K (l mg−1)
k (l g−1 min−1)
K (l mg−1)
0.0427 0.1552 0.1855 0.3463
0.0103 0.0304 0.0283 0.0385
0.1314 0.1276 0.1571 0.1392
0.0126 0.0050 0.0020 0.0014
arithmic form of the Arrhenius equation, a plot of ln k versus 1/T yields a slope equal to − E/R. Since the biosorption rates of the metal ions examined increased with temperature, the slopes of Arrhenius plots give negative values and the activation energy is normally found to be positive (Figs. 8 – 10). For biological systems, the reported activation energies in the literature generally change over the range 8.4 – 83.7 kJ mol − 1 (2 – 20 kcal mol − 1) [25]. Two main types of adsorption may occur: physical and chemical adsorption. In physical adsorption, equilibrium between the adsorbent surface and the adsorbate is usually rapidly attained and easily reversible, because the energy requirements are small. The energy of activation for physical adsorption is usually no more than 4.184 kJ mol − 1 (1.0 kcal mol − 1) [15], since the forces involved in physical adsorption are weak. Two kinds of chemisorption are encountered: activated and less frequently, nonactivated. Activated chemisorption means that the rate varies with temperature according to a finite activation energy in the Arrhenius equation (high E). However, in some systems chemisorption occurs very rapidly, suggesting the activation energy is near zero. This is termed nonactivated chemisorption [15]. The observed values of the activation energies of biosorption of Fe(III) ions on both microorganisms and Cr(VI) ions on R. arrhizus are of the same magnitude as the activation energy of chemisorption, while the activation energies for the biosorption of Pb(II) ions on Z. ramigera and Ni(II) ions on R arrhizus have
Fig. 8. The linearized Arrhenius plots for the biosorption of Fe(III) ions on Z. ramigera and R. arrhizus (pH 2.0; biomass concentration: 1.0 g litre − 1; agitation rate: 150 rev min − 1).
Fig. 9. The linearized Arrhenius plot for the biosorption of Cr(VI) ions on R. arrhizus (pH 2.0; biomass concentration: 1.0 g litre − 1; agitation rate: 150 rev min − 1).
boundary values of activation energy of biosorption between the physical and chemical adsorption (Table 3). It is difficult to decide which mechanism is effective on the biosorption of heavy metal ions on microorganisms, only taking activation energies into consideration. With respect to the magnitude of the heat of biosorp-
Table 2 The adsorption rate and equilibrium constants for the biosorption of Fe(III), Cr(VI) and Ni(II) ions on R. arrhizus T (°C)
15 25 35 45
Iron(III)
Chromium(VI)
Nickel(II)
k (l g−1 min−1)
K (l mg−1)
k (l g−1 min−1)
K (l mg−1)
k (l g−1 min−1)
K (l mg−1)
0.0499 0.0797 0.1327 0.1183
0.0107 0.0101 0.0182 0.0158
0.0497 0.1301 0.1076 0.1321
0.0009 0.0077 0.0028 0.0061
0.0695 0.0734 0.0645 0.0866
0.0104 0.0002 0.0053 0.0248
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Fig. 10. The linearized Arrhenius plots for the biosorption of Pb(II) ions on Z. ramigera and Ni(II) ions on R. arrhizus (pH 4.5; biomass concentration: 1.0 g litre − 1; agitation rate: 150 rev min − 1). Table 3 The activation energies for the biosorption of heavy metal ions on Z. ramigera and R. arrhizus Microorganism–metal
E (kJ mol−1)
Z. Z. R. R. R.
48.8 6.2 23.4 20.7 4.0
ramigera–iron(III) ramigera–lead(II) arrhizus–iron(III) arrhizus–chromium(VI) arrhizus–nickel(II)
tion, the dominant adsorption mechanism in the whole biosorption process can also be proposed for each metal–microorganism system. The enthalpy change for the biosorption process of heavy metal ions on Z. ramigera and R. arrhizus has been evaluated. The heats of biosorption of Fe(III) and Cr(VI) ions on both microorganisms have been determined to be of the same magnitude as the heat of chemisorption, while the biosorption of Pb(II) ions on Z. ramigera and Ni(II) ions on R. arrhizus has shown a heat effect comparable to that of physical adsorption [26]. The pattern of biosorption of the metal ions by Z. ramigera and R. arrhizus also follows this classification [19,21,23,26]. The biosorption of Pb(II) and Ni(II) ions is completely reversible, equilibrium is established very rapidly and cycling of adsorption and desorption, as by raising and lowering the pH using some eluation solutions, can be performed repeatedly. Chemisorption may or may not be reversible. A chemical change in the adsorbate upon desorption is good evidence indeed that chemisorption in fact occurred. Cr(VI) and Fe(III) ions are removed with difficulty from chelating molecules by pH manipulation and the total amount of the bound metal ions may not be eluated from the biomass. Oxidation/reduction reactions are also used to facilitate release of metals from ligands. The classic biological
example of this is hydroxamate siderophore release of iron brought about by reduction of Fe(III) to Fe(II). Fe(II) has a very low affinity for the siderophore and is easily removed. In some cases, the metal ligand complex must undergo hydrolysis in order to release the iron [27]. On the other hand, Cr(VI) in the form of dichromate is anionic and a strong oxidising agent, which properties may account for a number of the observations such as its irreversible binding to the biomass. By treating an alkaline solution, elusion of the bound Cr(VI) can be accomplished. However, if left in contact with microorganisms for extended periods of time or if exposed to very low pH values, the bound Cr(VI) may be reduced to Cr(III) [13]. It should be noted that both physical adsorption and chemisorption can occur together, but any adsorbed layers beyond the first must presumably be physically adsorbed.
4. Conclusions Despite the quite extensive literature available on heavy metal biosorption, little attention seems to have been given to the temperature dependence of the biosoption process. In this study, batch adsorption experiments were performed at different temperatures in the range 15–45°C and the effect of initial metal ion concentration on the initial biosorption rates was investigated. The initial uptake kinetics of heavy metal ions on Z. ramigera and R arrhizus was shown to be represented by Langmuir–Hinshelwood type kinetics or saturation kinetics. From double-reciprocal Langmuir–Hinshelwood plots, the biosorption rate constants were obtained at different temperatures. The biosorption rate constants vary with temperature according to the Arrhenius equation. From the slopes of linearized Arrhenius plots, activation energies were determined and appropriate adsorption mechanism with respect to the magnitude of the activation energy of biosorption has been proposed for each metal–microorganism system. The activation energies for the biosorption of Fe(III) ions on both microorganisms and Cr(VI) ions on R. arrhizus were determined to be of the same order of magnitude as the activation energy of chemisorption.
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