The kinetics and mechanism of lead (II) biosorption by powderized Rhizopus oligosporus

Transcription

1 World Journal of Microbiology & Biotechnology 15: 291±298, Ó 1999 Kluwer Academic Publishers. Printed in the Netherlands. The kinetics and mechanism of lead (II) biosorption by powderized Rhizopus oligosporus A.B. Ari *, M. Mel, M.A. Hasan and M.I.A. Karim Department of Biotechnology, Universiti Putra Malaysia, UPM Serdang, Selangor, Malaysia *Author for correspondence: Fax: , Received in revised form 28 April 1998; accepted 16 January 1999 Summary The kinetics and mechanism of lead biosorption by powderized Rhizopus oligosporus were studied using shake ask experiment. The optimum biomass concentration and initial solution ph for lead sorption at initial lead concentrations ranging from 50±200 mg/l was obtained at 0.5 g/l and ph 5, respectively. In term of the ratio of initial lead concentration to biomass concentration ratio, the highest lead adsorption was obtained at 750 mg/g which gave the maximum lead uptake capacity of 126 mg/g. The experimental data of lead sorption by R. oligosporus tted well to the Langmuir sorption isotherm model, indicating that the sorption was similar to that for an ion-exchange resin. This means that the sorption is a single layer metal adsorption that occurred as a molecular surface coverage. This assumption was con rmed by the examination of lead sorption using transmission electron microscope and energy dispersive X-ray analysis, which showed that during sorption most of the lead was adsorbed on the surface of cell. Introduction Biosorption, the metabolism-independent binding of heavy metals to non-living cells, has been explored for its potential in removing toxic metals from aqueous solution. It is a potential alternative to conventional processes for the removal of metals such as chemical precipitation and ion exchange processes. Lead and cadmium for instance, have been e ectively removed from very dilute solutions by dried fungal biomass (Fourest & Roux 1992). Filamentous fungi are widely used in fermentation industries to produce various metabolites such as enzymes, avourings or antibiotics. The fungal biomass contains poorly biodegradable biopolymers (cellulose, chitin and glucans) and make poor fertilizers. Biosorption processes using dead cells can be of great interest, because of the large variety and the low cost of these biosorbent materials (Brierly et al. 1986). A major consideration preceeding the implementation of any sorption operations is the equilibrium distribution of ionic components between the solid and liquid phases for a particular sorption system. An important consideration for the practical utilization of microorganisms for accumulation of metal is the amount of metals that can be accumulated by the cell mass, which varies with the type of microorganism and the biosorption conditions (Volesky 1990). Two key aspects which have to be taken into consideration in conjunction with the metal uptake capacity of the biomass for a speci c metal ions are the characteristics of the solution system and the characteristics of the biomass (Volesky 1992). Biosorption involves physical and chemical reactions between positively charged dissolved species and negatively charged reactive cellular components (Fourest & Roux 1992; Tobin et al. 1990). Cell wall of microorganisms, consisting mainly of polysaccharides, proteins and lipids, o er many functional groups which can bind metal ions such as carboxylate, hydroxyl, sulphate, phosphate and amino groups. Metal sorption performance depends on some

2 292 A.B. Ari et al. external factors such as ph, temperature, the presence of other metal ions in solution, biomass and metal concentrations (Friis & Keith 1986; Kuyucak & Volesky 1989). The present study investigated the mechanism and kinetics of lead (II) sorption by powderized Rhizopus oilgosporus at di erent sorption conditions. R. oligosporus is widely used for the production of tempeh, a traditional fermented food originated from Indonesia. Tempeh is a mass of soya beans bound into a cake by a dense cottony growth of Rhizopus sp. white mycelia (Shurtle & Aoyagi 1979). This fungus was chosen as biosorbent material because of the relative lack of information about its sorption abilities. Materials and Methods Microorganism and preparation of biosorbent The fungus, Rhizopus oligosporus, used in this study was obtained from Malaysian Agriculture Research and Development Institute (MARDI), Serdang, Selangor, Malaysia. A 5 ml spore suspension obtained from potato dextrose agar slant was inoculated into a 500 ml shake ask containing 200 ml of liquid medium normally used for fungal fermentation. This medium consisted of; glucose, 40 g/l; yeast extract, 10 g/l; MgSO 4 á 7H 2 O, 1 g/l; NaNO 3, 1 g/l; H 2 KPO 4, 1 g/l. The initial ph of the culture was adjusted to 5.5. The asks were incubated at 30 C in an orbital shaker (Certomat, B. Braun, Germany) agitated at 250 rev/min for 5 days. During the cultivation, the fungus grew in the form of mycelium. The mycelia from the culture broth were separated from the liquid by ltration using Whatman lter paper (No.1) and washed with distilled water. The mycelia were resuspended and rewashed for three times. The wet mycelium biomass was dried for 18 h at 55 C in an oven. Dried material was ground using Waring Blendor and sieved using Endecotts, Octagon 200 Test Sieve Shaker with mesh number 300±600 lm. The powderized R. oligosporus was stored in air tight container prior to use in the sorption experiments. Biosorption equilibrium experiments The lead (II) solutions at a range of the desired concentration (10±700 mg/l) were prepared by dissolving Pb(NO 3 ) 2 stock in double distilled water. The initial ph of the lead solution was adjusted to the required value by using either 0.1 M HNO 3 or 0.1 M NaOH. Sorption equilibrium experiments were carried out using 500 ml shake ask containing 200 mg lead solution/l. After the addition of powderized R. oligosporus, the asks were agitated at 200 rev/min on waterbath shaker and the temperature was controlled at 30 C. Periodically, 1.5 ml samples were collected for analysis. Separation of the eluent solution from the biosorbent was performed by ltration using a membrane lter having 0.45 lm pore size. The concentration of lead was measured using atomic absorption spectrophotometry (Perkin Elmer, Model 3300, Conneticut, USA). All experiments were carried out in triplicate. Lead sorption mechanism experiments The mechanism of lead sorption by powderized R. oligosporus was investigated using transmission electron microscope (TEM) examination and energy dispersive X-ray analysis (EDX). The cell particles were xed in 4% (v/v) glutaraldehyde for 2 days at 4 C. The samples were then washed in 0.1 M cacodylate bu er for 10 min and then dehydrated with a series of acetone solutions at concentrations ranging 35±100% (v/v) and embedded in Spurr's epoxy resin. A thin section of the cell was cut on a LKB microtome knife maker. The section was mounted on copper grids and observed under the transmission electron microscope (Hitachi, H-7100, Nihon Denshi, Japan) at 30 kv. The EDX spectral analysis was recorded using the computer-linked semiconductor detector (LINK exl) with scanning electron microscope (JEOL 6400, Nihon Denshi, Japan). These spectra were used to identify the ions that had been observed earlier from TEM examination. For X-ray analysis, the cell exposed to lead ion was simply spread onto the stub using double-sided tape and mounted in the sample holder. The microprobe was focused at 15 kv and the magni cation was 500 times.

3 Lead sorption by Rhizopus oligosporus 293 Results and Discussion Time course of lead sorption The rate of lead sorption by powderized R. oligosporus in a shake ask is shown in Figure 1. The binding rate of lead was very rapid during the initial stages of the sorption process (0± 20 min). After a very rapid sorption, lead uptake capacity (q) increased slowly with time and reached equilibrium after about 840 min as shown by an equilibrium value of lead speci c uptake (q eq ). This type of biosorption is typical for sorption of metals involving metabolically inert biomass, where metal removal from solution is due to purely physico-chemical interaction between biomass and metal in solution (Schumate et al. 1995). Two step processes, physical and chemical sorptions, normally occurred during metal sorption by microbial biomass (Akzu & Kutsal 1991; Brown 1991; Brady et al. 1994). The physical sorption is relatively a fast adsorption step. The rapid increase in q eq during the initial stages of lead sorption (0± 20 min) by powderized R. oligosporus may be a physical adsorption which can be categorized as extracellular sorption or surface binding. The second step of metal sorption is chemical sorption or chemisorption. This step of sorption needs relatively high energy to adsorb metals into the cell interior (intracellular) and the rate is very slow. The second phase of lead sorption (from 100 to 840 min), which was very much slower than the Figure 1. The time course of lead biosorption by powderized R. oligosporus. Initial lead concentration = 100 mg/l; initial ph = 5; biosorbent concentration = 0.5 g/l. Mean values standard errors of the means of triplicate ask are shown. rst sorption phase (0±100 min), may be chemical sorption. The chemical sorption or intracellular sorption is a more metabolic process than the physical sorption (Lopez et al. 1995). Adsorption mechanism Figure 2 shows typical TEM micrographs of native and lead-exposed cells of R. oligosporus. The TEM micrograph of cells not exposed (Figure 2A) to lead indicates that there was no de nable lead ion. On the other hand, electron-dense layers (black dots) throughout the fungal cell wall were observed in the micrograph of lead-exposed cells (Figure 2B), indicating that lead ion was present at the cell wall of R. oligosporus. The EDX spectra of native and lead-exposed cells are shown in Figure 3. The resulting spectra of X-ray analysis for native and lead exposed cells did not indicate any crystalline structure, implying that the incorporation of lead within the cells did not cause any changes in the amorphous structure of R. oligosporus biomass. The peaks of Mg 2+, P +, S 2+, and K + were noticed as the elements present on the surface of the native cells. On the other hand, the peak of the elements observed on the surface of lead-exposed cells were Pb 2+, and P + suggesting that lead was adsorbed and replaced Mg 2+, S 2+ and K + on the cell surface. It has been reported that Pb 2+ a ects the permeability of the yeast cell membrane causing a loss of K + and Mg 2+ from the cell (Heldwein et al. 1977). From the TEM and EDX analyses, it can be concluded that the exposure of powderized R. oligosporus to solution containing lead caused the lead ions to adsorb to the surface of the cell wall and very little or none was absorbed into the cells. This means that the biosorption of lead ion by R. oligosporus cells was mainly an adsorption phenomenon. Biosorption of metals by yeast cells, Saccharomyces cerevisae, has been reported to be associated with metal penetration through the cell wall (Peng & Koon 1993). In some cases, adsorption on the external cell surface is a biomass defence system against toxic heavy metals, the microorganism producing an external polymeric layer to avoid metal penetration through the cell wall (Scott & Palmer 1990). This is known as microprecipitation mechanism in which ions are removed from solution through the precipitation

4 294 A.B. Ari et al. Figure 3. Typical EDX spectra of lead-exposed (A) and native (B) R. oligosporus cells. process, enhanced by the compounds produced by the active defence system of the cells. For example, the removal mechanism of cadmium and lead by Citrobacter sp. are by accumulating cadmium phosphate and PbHPO 4 as cell-bound complexes, respectively (Macaskie et al. 1987). Kinetics of lead sorption at di erent initial ph values For a detailed characterization of the adsorption kinetics, the classical adsorption model of Langmuir is often used (de Rome & Gadd 1987; Akzu & Kutsal 1991). The Langmuir isotherm is: q ˆ q max K d C eq Š= 1 K d C eq Š 1 Figure 2. Typical transmission electron micrograph of native (A) and lead-exposed (B) cells of R. oligosporus. (10,000 magni cation). where q is the lead speci c uptake (mg/l), q max is the maximum lead speci c uptake (mg/g), K d is the dissociation constant (1/g) which is related to the energy of adsorption through the Arrhenius equation (Holan et al. 1993), and C eq is the lead equilibrium concentration (mg/l).

5 Lead sorption by Rhizopus oligosporus 295 The lead speci c uptake (q) was determined from the experimental data as follows, q ˆ C 0 C eq Š=x 2 where C 0 is the initial lead concentration (mg/l), C eq is the lead equilibrium concentration (mg/l), and x is the biosorbent concentration in solution (g/l). The sorption isotherms of lead by powderized R. oligosporus at di erent initial ph values follow the typical Langmuir adsorption pattern, which is based on surface adsorption, as shown in Figure 4. This result shows that lead accumulation by powderized R. oligosporus is a chemical, equilibrated and saturatable mechanism. This means that the adsorption increases when the initial lead concentration rises, as long as binding sites are not saturated. The maximum lead speci c uptake (q max ) and dissociation constant (K d ) of lead by R. oligosporus at di erent initial ph values calculated from the linear transformation of the Langmuir equation are given in Table 1. The speci c uptake of a particular biosorbent (q max ) is the amount of metal per g of biomass corresponding to the saturation of adsorption sites. The dissociation constant, which is the metal concentration corresponding to half of the saturation of biosorbent in these experimental conditions, also indicates the a nity (K a = 1/K d ) of lead ion for powderized R. oligosporus. Lead adsorption increased as the initial solution ph increased from 2 to 5 and drastically decreased at initial ph 6. The highest lead speci c uptake was obtained at initial ph 5. The solution ph in uences the active sites of biomass such as chitin amino, proteins, carboxyl and hydroxyl groups thus in uencing the adsorption capacity (Fourest & Roux 1992). It is generally agreed that an optimal ph for heavy metal uptake by microbial biomass is at ph values of around 4 to 5 (Niu et al. 1993; Kuyucak & Volesky 1989; Tsezos & Volesky 1981). The hydroxyl and carboxyl groups may be involved in the adsorption of lead ions. Solutions at high ph values contain more OH ) which is capable of binding with lead ion at a faster rate than solution at low ph value. Thus, the solution becomes less acidic. The tendency to form surface complexes decreases with increasing metal loading of the surfaces because the metal ions bind rst to the surface groups with the highest a nity and subsequently to groups with lower a nity. The binding tendency of cell surfaces for lead ions can be readily interpreted in terms of surface complex formation equilibrium, which is equivalent to Langmuir type adsorption. Kinetics of lead sorption at di erent initial lead and biosorbent concentrations The sorption isotherms of lead by powderized R. oligosporus at di erent initial lead concentrations also follow the typical Langmuir adsorption pattern (Figure 5). The Langmuir model parame- Table 1. Characteristic data for Langmuir isotherm and correlation coe cient (R 2 ) for lead (II) adsorption by powderized R. oligosporus at di erent ph levels. ph K d (l/g) q (mg/g) Correlation coe cient (R 2 ) Figure 4. Langmuir sorption isotherm for lead by powderized R. oligosporus at di erent initial ph values. Biosorbent and initial lead concentrations were 0.5 g/l and 100 mg/l, respectively, (j) ph 2; (e)ph 3; (s) ph 4; (r) ph 5; (d) ph

6 296 A.B. Ari et al. ters calculated from the tting of experimental data are shown in Table 2. The maximum lead speci c uptake was increased with increasing initial lead concentration up to 200 mg/l and became equilibrium beyond this concentration. A similar pattern of biosorption was also observed for various metals by marine algae (Andreas et al. 1995). Because lead adsorption by powderized R. oligosporus is a chemical, saturatable and equilibrium mechanism, lead uptake reaches equilibrium when all binding sites are saturated with lead ions (Fourest & Roux 1992). For all initial lead concentrations studied, the maximum lead speci c uptake decreased with increasing biosorbent concentration (Table 3). Obviously, this reduction is attributable to the shortage of lead in solution. This result invalidate Figure 5. Langmuir sorption isotherm for lead by powderized R. oligosporus at di erent initial lead concentrations (C 0 ). Biosorbent concentration and initial ph were 0.5 g/l and 5, respectively. (s) 300 mg/l; (r) 200 mg/l; (j) 100 mg/l; (d) 50 mg/l. Table 2. Characteristic data for Langmuir isotherm and correlation coe cient (R 2 ) for lead (II) adsorption by powderized R. oligosporus at di erent initial lead concentrations. Initial lead K d (l/g) q (mg/g) Correlation concentration (mg/l) coe cient (R 2 ) the hypothesis that electrostatic interaction between cells may be a signi cant factor in the biosorbent dependence of metal adsorption (de Rome & Gadd 1987). Therefore, it is not useful to increase the biomass beyond 1.0, 3.0 and 4.0 g/l to purify a 50, 100 or 200 mg lead solution/l respectively. The electrostatic interactions between cells may be a signi cant factor in the biosorbent concentration dependency of metal adsorption, with a larger quantity of cation being adsorbed when the distance between cells is great (de Rome & Gadd 1987; Xue et al. 1988). Reduction in biosorbent concentration in the suspension at a given lead concentration enhanced the lead/biosorbent (C 0 /x) ratio which in turn increased lead uptake per g biosorbent, as long as the latter is not saturated. Illustration of this behavior is given in Figure 6, which shows lead adsorption by R. oligosporus for three initial lead concentrations at di erent C 0 /x ratios. The increase in lead adsorption with increasing C 0 /x has two possible explanations (Fourest & Roux 1992). Firstly, the reduction of biosorbent concentration decreases the electrostatic interaction between cells, which in turn, increases the lead adsorption rate. Secondly, increases in the ratio of initial lead concentration to biosorbent concentration cause an increase in the number of lead ions available around the cells, environment, which are more easily bound to the active sites of cells as long as the active sites are not saturated. The maximum lead uptake capacity (126 mg/g) was achieved for all concentrations with an initial lead to biosorbent ratio of about Table 3. E ect of biosorbent concentration on lead speci c uptake by powderized R. oligosporus at di erent initial lead concentrations (C 0 ). Biosorbent concentration (g/l) Lead speci c uptake at di erent C 0 50 mg/l 100 mg/l 200 mg/l Values are means of three replicates standard errors.

7 Lead sorption by Rhizopus oligosporus 297 a grant ( UPM/ENV/0010) under IRPA programme. References Figure 6. E ect of initial lead to biosorbent concentration (C 0 /x) ratio on lead adsorption by powderized R. oligosporus. (j) 50 mg/l; (r) 100 mg/l; (d) 200 mg/l. 750 mg/g. These types of data can be used for optimization and scale-up of industrial e uent puri cation. The maximum lead speci c uptake by powderized R. oligosporus obtained in this study (126 mg/ g) at optimal sorption conditions was signi cantly higher than those obtained with other fungi such as R. arrhizus (Fourest & Roux 1992), P. chrysogenum (Niu et al. 1993) and A. niger (Kim et al. 1995) which had q max values of 55 mg/g, 116 mg/g and 30 mg/g, respectively. However, the maximum lead uptake by R. oligosporus was lower than that obtained by Chlorella fusca (Werheim & Wettern 1994) and Arthrobacter sp. (Veglio et al. 1997) which has a q max value of 293 mg/g and 130 mg/g, respectively. The ability of R. oligosporus to biosorp other toxic metals such as cadmium, copper, chromium and zinc is being studied in our laboratory. For an economic biosorption process, the biosorbent should be regenerated for subsequent reuse. The methods of stripping and recovering the bound lead ions that have been biosorbed by R. oligosporus are also being investigated. Acknowledgements The authors are grateful to the Ministry of Science, Technology and Environment, Malaysia for Akzu, Z. & Kutsal, T A bioseparation process for removing lead (II) ions from waste water by using Chlorella vulgaris. Journal of Chemical Technology and Biotechnology 52, 109±118. Andreas, L., Zdenek, R.H. & Volesky, B Biosorption of heavy metals (Cd, Cu, N, Pb, Zn) by chemically reinforced marine algae. Journal of Chemical Technology and Biotechnology 62, 279±288. Brady, D., Stoll, A.D., Starke, L. & Duncan, J.R Chemical and enzymatic extraction of heavy metal binding polymers from isolated cell walls of Saccharomyces cerevisae. Biotechnology and Bioengineering 44, 297±302. Brierly, JA., Goyak, G.M. & Brierly, C.L Consideration for commercial use of natural product for metal recovery. In Immobilisation of Ions by Bio-sorption. eds. H. Eccles & S. Hunt, pp. 105±117, Chichester, England: Ellis Horwood. ISBN Brown, M.J Metal recovery and processing. In Biotechnology, The Science and the Business. eds. V. Moses & R.E. Cape, pp. 567±579. Switzerland: Harvard Academic Publishers. ISBN de Rome, L. & Gadd, G.M Copper adsorption by Rhizopus arrhizus, Cladosporium resinae and Penicillium italicum. Applied Microbiology and Biotechnology 26, 84±90. Fourest, E. & Roux, J Heavy metals biosorption by fungal mycelial by-product: Mechanisms and in uence of ph. Applied Microbiology and Biotechnology 37, 399±403. Friis, N. & Keith, M.P Biosorption of uranium and lead by Streptomyces longwoodensis. Biotechnology and Bioengineering 28, 21±28. Heldwein, R., Tombala, H.W. & Broda, E Aufnahme von cobalt, blei and cadmium durch bakkë erieeèn Zeitschrift fuèr Allgemeine Mikrobiologie 17, 229±234. Holan, Z.R., Volesky, B. & Prasetyo, I Biosorption of cadmium by biomass of marine algae. Biotechnology and Bioengineering 41, 819±825. Kim, Y.H., Yoo, Y.J. & Lee, H.Y Characteristics of lead adsorption by Undaria pinnati da. Biotechnology Letters 17, 345±350. Kuyucak, N. & Volesky, B The mechanism of cobalt biosorption. Biotechnology and Bioengineering 33, 823±831. Lopez, F.A., Perez, C., Sainz, E. & Alonso, M Adsorption of Pb 2+ on blast furnace sludge. Journal of Chemical Technology and Biotechnology 62, 200±206. Macaskie, L.E., Dean, C.R., Cheetham, A.K., Jakeman, J.B. & Skarnulis, A.J Cadmium accumulation by a Citrobacter sp. : The chemical nature of the accumulated metal precipitate and its location on the bacterial cells. Journal of General Microbiology 133, 539±544. Niu H., Xu, X.S. & Wang, J.H Removal of lead from aqueous solutions by Penicillium Biomass. Biotechnology and Bioengineering 42, 785±787.

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