Zn2+ biosorption by Oscillatoria anguistissima

Process Biochemistry 34 (1999) 77 – 85

Zn2 + biosorption by Oscillatoria anguistissima Prerna Ahuja, Rani Gupta *, R.K. Saxena Department of Microbiology, Uni6ersity of Delhi, South Campus, Benito Juarez Road, New Delhi-110021, India Received 5 May 1997; received in revised form 22 April 1998; accepted 3 May 1998

Abstract Oscillatoria anguistissima showed a very high capacity for Zn2 + biosorption (641 mg g − 1 dry biomass at a residual concentration of 129·2 ppm) from solution and was comparable to the commmercial ion-exchange resin IRA-400C. Zn2 + biosorption was rapid, pH dependent and temperature independent phenomenon. Zn2 + adsorption followed both Langmuir and Freundlich models. The specific uptake (mg g − 1 dry biomass) of metal decreased with increase in biomass concentration. Pretreatment of biomass did not significantly affect the biosorption capacity of O. anguistissima. The biosorption of zinc by O. anguistissima was an ion-exchange phenomenon as a large concentration of magnesium ions were released during zinc adsorption. The zinc bound to the biomass could be effectively stripped using EDTA (10 mM) and the biomass was effectively used for multiple sorption–desorption cycles with in-between charging of the biomass with tap water washings. The native biomass could also efficiently remove zinc from effluents obtained from Indian mining industries. © 1998 Elsevier Science Ltd. All rights reserved. Keywords: Biosorption; Zinc; Oscillatoria; Biomass; Equilibrium kinetics

1. Introduction Biosorption of metal ions from aquatic systems using microbial biomass, including algae, fungi and bacteria has gained importance in recent years [1,2]. It is a rapid, reversible, economical and ecofriendly technology in contrast to conventional chemical methods of removing metal ions from industrial effluents. According to Volesky [3], it is especially important when the metal ions are dissolved in large volumes in the concentration range 1–100 mg l − 1. Microbes are known to adsorb different metal ions by virtue of the covalent interactions of metal at the cell surface. Various chemical groups including carboxylate, sulphate, hydroxyl, phosphate and amino have been reported to be involved in such an interaction [4,5] Zinc is one of the most important metals often found in effluents discharged from industries involved in galvanization and in the manufacture of alloys. Zinc biosorption by fungi and bacteria is well documented [6,7] but little work has been done using cyanobacteria. We have been engaged in the removal and recovery of * To whom correspondence should be addressed.

heavy metals using cyanobacteria and have reported excellent copper adsorption capacity of a fresh water cyanobacterium O. anguistissima [8] and here we report its biosorption potential for zinc and its application in effluent treatment.

2. Material and methods

2.1. Microorganism Oscillatoria anguistissima culture was obtained from the National Facility for Blue Green Algal Collections, IARI, New Delhi, India. It was cultivated at 25 92°C under 1100 lux light intensity in BGII minimal medium [9] at pH 79 0·2.

2.2. Culture har6est and biomass determination The biomass was harvested in exponential phase after 10 days of growth by centrifugation at 6000 rpm for 10 min in a Remi RC 30 refrigerated centrifuge. Thereafter, the biomass was washed thoroughly with deionised distilled water and was subsequently used for

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P. Ahuja et al. / Process Biochemistry 34 (1998) 77–85

metal biosorption experiments. The dry weight of cells was determined by pelleting a known volume of cell suspension and drying the pellet at 80°C for 48 h until constant weight was obtained.

2.3. Pretreatment of biomass Four milligrams of dry biomass was subjected to various treatments for 10 min: (i) heating at 60/100°C; (ii) treating with 0·01 – 1·0 N NaOH/HCl. Subsequently the biomass was washed thoroughly with deionised distilled water and collected by centrifugation. All the chemicals used in media preparation and the metal salt (ZnSO4·7H2O) except HCl (Merck) were of analytical grade (BDH). The standard solution of Zn2 + (1000 ppm) for Atomic Absorption measurements was obtained from the National Physical Laboratory, New Delhi, India. The resins, IRA 400C and IR 120 (+) H were obtained from ICN chemicals, USA.

2.4. Analytical methods 2.4.1. Biosorption The desired concentrations of Zn2 + (5 – 200 mg l − 1) were prepared by dissolving ZnSO4·7H2O in deionised distilled water. Short term biosorption trials were performed in 250-ml Erlenmeyer flasks containing 50 ml of ZnSO4·7H2O solution of known initial concentration (pH 2–6) at 25°C for 2 h under shake conditions (100 rpm) unless otherwise mentioned. In all experiments except for the effect of cell density, the biomass concentration was kept constant at 0·08 mg dry weight (DW) ml − 1. Metal free blanks were used to estimate the exact initial concentration of Zn2 + by dilution. Separation of biomass from metal bearing solution was achieved through centrifugation at 6000 rpm for 10 min at room temperature. The supernatant was appropriately diluted and the remaining Zn2 + content esti˚ ) and mated at 213·9 nm wavelength (slit width 3·8 A the magnesium ions released in the metal solution ˚ ). All estimated at 285·2 nm wavelength (slit width 3·8 A experiments were carried out in triplicate and repeated three times. The biosorption capacity of dried biomass of O. anguistissima was compared with the commercial ionexchange resins IRA 400C and IR 120 (+ ) H at different concentrations of zinc (25 – l00 ppm) using 4 mg biomass/resin. Similar experiments were performed at increasing biomass/resin concentration in 50 ppm metal solution. 2.4.2. Desorption Seven desorbing agents, EDTA (0·5 – 10 mM), Na2CO3 (0·75–10 mM), NaHCO3 (0·75 – 10 mM), CaCl2·2H2O (0·5–2 mM), H2SO4 (0·01 – 0·1 N), HCl (0·01 –0·1 N) and citrate buffer (0·2 M, pH 3 – 5) were

used for eluting zinc from the loaded biomass. The loaded biomass was washed once with distilled water and added to 20 ml of different strength desorbing solution in 100 ml of Erlenmeyer flasks. Flasks were incubated at 259 2°C with shaking (100 rpm). Samples were withdrawn after 30, 60 and 90 min and the desorbed zinc was estimated using atomic absorption spectrophotometry.

2.4.3. Resorption Biosorption–desorption procedures were carried out for five cycles to investigate the capacity of O. anguistissima biomass for reusability. At each step, the concentration of zinc in the solution was determined and after each cycle dry weight was measured to estimate the percent loss in biomass. Before each cycle the biomass was charged by washing in tapwater before bisorption. 2.4.4. Effluent analysis Effluent samples were collected from a zinc ore treatment plant, Hindustan Zinc Limited, Udaipur, India. The pH of collected samples was measured before dilution and estimation of zinc content by atomic absorption spectrophotometry. Biosorption of zinc was carried out from effluent samples under the above mentioned conditions and the residual metal was estimated in each case. 2.5. Data analysis The metal uptake capacity in mg g − 1 (q) was calculated from the initial concentration (Ci ) and the final concentration (Cf ) of the metal according to the following equation q= V(Ci− Cf )/M where V is the liquid sample volume and M is the biomass dry weight. The biosorptive metal uptake was evaluated and expressed using Freundlich [10] and Langmuir [11] adsorption models.

3. Results and discussion The non-growing biomass of the fresh water cyanobacterium O. anguistissima adsorbed appreciable quantities of zinc in the initial 15 min from the aqueous solutions. However, equilibria were attained only after 2 h (Fig. 1). These results are in accord with biosorption studies with various groups of microorganisms where fast rates of metal binding have been reported [12–16]. Zinc uptake increased with increasing metal concentration and the highest uptake value of 641·25 mg Zn2 + g − 1 dry weight was observed at a residual/final concen-

P. Ahuja et al. / Process Biochemistry 34 (1998) 77–85

79

Fig. 1. Time course of zinc biosorption in Oscillatoria anguistissima.

tration (Cf ) of 129·2 mg l − 1. The adsorption kinetics of the cells could be explained by Freundlich and Langmuir isotherms. The data fitted the Freundlich model at both pH 5 (Figs. 2 and 3) and 6 (Fig. 4) but the suitability of Langmuir fit was observed only at pH 5 (Fig. 5). The validity of both models was tested at two biomass concentrations — 0·08 and 0·16 mg DW ml − 1. Oscillatoria sp. showed an isotherm that is steep from the origin at low residual concentrations (Fig. 3).

This is desirable as it indicates a good affinity of biomass for Zn2 + ions [17]. The highest capacity for Zn2 + adsorption in the present study (641 mg g − 1) is comparable to the best reported in the literature for filamentous fungi Aspergillus niger, Cla6iceps paspali (1000 mg Zn2 + g − 1) and Penicillium chrysogenum (500 mg Zn2 + g − 1) by Luef et al. [6]. However, the values obtained for Oscillatoria biomass are comparatively much higher than Bacillus sp. [18,19] and those of the

Fig. 2. Freundlich adsorption isotherm for zinc at 0·08 mg DW ml − 1 biomass concentration (pH 5·0).

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P. Ahuja et al. / Process Biochemistry 34 (1998) 77–85

Fig. 3. Freundlich adsorption isotherm for zinc at 0·16 mg DW ml − 1 biomass concentration (pH 5·0).

marine algae Sargassum fluitans and Ascophyllum nodosum [20]. Biosorption of zinc was pH dependent and the pH optima was 5·0 (Fig. 6). The value declined by about 30% at pH 6. These results are in agreement with the studies of Kuyucak and Volesky [21] in algae and of Panchanadikar and Das [22] in bacteria. It is well known that both the cell surface metal binding sites and the availability of metal in solution are affected by pH. At low pH, the cell surface sites are closely linked to the H + ions, thereby making these unavailable for other

cations. However, with an increase in pH, there is an increase in ligands with negative charges which results in increased binding of cations. Temperature in the range 25–45°C did not produce any significant difference in Zn2 + adsorption at pH 5·0 (Table 1). The temperature independent biosorption of zinc is in accordance with the study of Aksu and Kutsal [14] in Chlorella 6ulgaris for the removal of heavy metals from aqueous solutions, indicating biosorption to be a passive energy independent process. In contrast, deRome and Gadd [23], Shumate et al. [24], Strandberg

Fig. 4. Freundlich adsorption isotherm for zinc at 0·08 mg DW ml − 1 biomass concentration (pH 6·0).

P. Ahuja et al. / Process Biochemistry 34 (1998) 77–85

81

Fig. 5. Langmuir adsorption isotherm for zinc at 0·08 mg DW ml − 1 biomass concentration (pH 5·0).

et al. [25] and Tsezos and Volesky [26] have reported higher uptake capacities in different organisms at increased temperatures, which seems to involve some of the physiological processes in addition to physico– chemical interaction. The effect of increasing biomass concentration revealed that the specific uptake of zinc decreased when the biomass concentration was increased (Fig. 7) as has been reported in algae [27 – 29] and other microbial

systems [23,30]. The dependence of adsorption on cell density reduces cell distances and thus less sites are available for metal binding [31]. It has also been suggested that at higher biomass concentrations aggregates are formed which can reduce the effective biosorption area [14]. Pretreatment of biomass at high temperature and with alkali did not significantly affect the biosorption of zinc by Oscillatoria sp. However, HCl conditioned

Fig. 6. Effect of initial pH on zinc biosorption by O. anguistissima.

P. Ahuja et al. / Process Biochemistry 34 (1998) 77–85

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Table 1 Effect of temperature on biosorption of Zn2+ by Oscillatoria sp. Temperature (°C)

Zn2+ (mg g−1 dry biomass)

25 30 37 45

264·09 17·73 277·09 34·64 267·59 63.63 275·09 30·00

biomass of Oscillatoria showed significant reduction in zinc adsorption compared with the native biomass (Table 2). The loss of adsorption efficiency of the HCl treated cells is in agreement with the results obtained in Phormidium laminosum by Sampedro et al. [32] and this has been suggested to be due to competition between the bound protons and the dissolved metal ions. This is higher than that resulting from the alkali and alkaline earth cations normally present in the untreated biomass [32]. The presence of another solute can interfere with and generally inhibit the biosorption of the desired metal by competing for binding sites such as amine, carboxylate, phosphate, imidazole and other functional groups in the cell surface proteins and sugars [7,16]. In the present study calcium and magnesium ions inhibited zinc biosorption. The inhibitory effect of Mg2 + was more pronounced than that of Ca2 + . Concentrations of 100 and 200 mg l − 1 Mg2 + resulted in about 52·73% and 53·12% decline as against 23·58% and 31·06%, respectively, in the case of calcium. Similar inhibitory effects of divalent cations on biosorption have been reported in Rhizopus arrhizus by Lewis and Kiff [33].

However, cadmium uptake by Tolypothrix tenius was not strongly affected by the presence of Mg2 + and Ca2 + ions [34] and this was suggested to be due to different binding sites or a lower adsorption strength of calcium and magnesium ions than cadmium. The effect of three different anions (sulphate, chloride and nitrate, as ammonium salts) showed that sulphate and chloride ions in the range 0·75–10 mM decreased zinc biosorption. However, nitrate ions did not greatly affect the biosorption of zinc (Table 3) to any significant extent. The dried biomass of O. anguistissima proved to be better than both resins (IR-400C and IR 120 ( +) H) at 25 ppm concentration of zinc and better than IR-400C at higher concentration of zinc (Table 4). The specific uptake of zinc decreased with increasing concentration of biomass/resin (Table 5). At low biomass concentrations, O anguistissima proved to be better than IR-400C and comparable to IR 120(+) for the removal of zinc. However, at higher biomass concentrations, its capacity was comparable to IR 400C but less than that of IR-120 for removal of zinc. A similar mechanism of metal binding by IR 400C and the biomass of Oscillatoria sp. is likely as the pH of the metal solution in both cases increased from 5·0 to 6·5 after biosorption. Kuyucak and Volesky [21] have also reported a high capacity of gold binding by Sagassum natans biomass, outperforming the ion exchange resin IRA 400C. Among the seven desorbing agents tried for desorption of zinc from the loaded biomass, 10 mM EDTA (64·72%) resulted in maximum elution of zinc in 90 min, indicating that it is physico–chemically sequestered on the surface (Table 6).

Fig. 7. Effect of biomass concentration on zinc biosorption by O. anguistissima.

P. Ahuja et al. / Process Biochemistry 34 (1998) 77–85 Table 2 Effect of biomass pretreatments on Zn2+ biosorption by Oscillatoria sp.

83

Table 3 Effect of anions on biosorption of zinc by O. anguistissima

suggests a pH dependent ion-exchange mechanism where zinc ions are exchanged for magnesium ions at the cell surface. Crist et al. [35] and Akhtar et al. [16] have also reported a similar release of magnesium ions during the process of biosorption in algae and fungi. This release of magnesium ions led to an evaluation of the feasibility of reusing the desorbed biomass for subsequent cycles by washing with tap water which contains significant concentrations of divalent cations (Mg2 + and Ca2 + ). The biosorption–desorption procedure for zinc, with in between washings of the biomass with tap water for recharging, revealed that the capacity of O. anguistissima biomass for zinc biosorption was retained for five cycles (Table 7). However, there was

Concentration Zinc biosorbed (mg g−1 dry biomass) of anion (mM)

Table 6 Desorption of zinc with time using different eluants

Pretreatment

Zn2+ (mg g−1 dry biomass)

1. 2. 3. 4. 5. 6.

207·59 34·36 267·5956·62 195·0911·45 140·09 10·60 230·0 9 10·60 232·5 9 26·33

Washing with distilled water Drying at 80°C for 48 h 0·01 N NaOH treatment 0·01 N HCl treatment 60°C incubation 100°C incubation

0·00 0·75 1·00 2·00 5·00 10·00

Sulphate

Chloride

Nitrate

184·33 915·01 158·33 910·40 61·66 9 7·63 68·33 911·54 56·66 9 7·21 27·58 9 2·26

184·339 15·01 139·339 31·97 143·339 18·33 120·339 8·08 97·509 5·00 90·3397·02

184·33915·01 182·50 9 15·20 180·4194·73 169·1692·92 160·00 910·89 166·7598·08

Table 4 Comparison of the biosorptive potential of dry biomass of O. anguistissima and ion-exchange resins at varying concentrations of zinc Conc. of metal Zinc biosorbed (mg g−1 adsorbent) (ppm)

25 50 100

Dry biomass

IR-120

IR-400C

45·00 911·20 186·20 917·10 245.70 919·80

27·50 9 10·10 210·009 10·17 272·109 19·20

32·18 9 7·92 109·719 8·72 190·00 918·20

Table 5 Comparison of the biosorptive potential of dry biomass of O. anguistissima and ion-exchange resins at varying concentrations of biomass/ resin Biomass/resin (mg)

4·00 8·00 10·00

Eluant

IR-120

IR-400C

186·20 917·10 72·00 9 6·91 64·00 95·10

210·00910·17 130·009 7·82 110·009 11·20

109·719 8·72 81·9297·20 71·77 9 9·90

The biosorption of zinc by O. anguistissima was accompanied by release of 2240·009125·29 mg per 50 ml of Mg2 + and a simultaneous rise in pH of the metal solution from 5·0 to 6·37 during biosorption. This

30 min

60 min

90 min

EDTA (mM)

0·00 0·50 1·00 2·00 5·00 10·00

0·05 25·00 27·51 29·91 32·28 34·58

0·12 28·02 27·91 30·40 34·59 34·64

0·12 33·81 28·74 31·17 51·12 64·72

CaCl2·2H2O (mM)

0·00 0·50 0·75 1·00 2·00

1·22 4·29 5·51 6·81 6·74

1·22 4·29 6·13 6·61 6·78

1·22 5·51 6·74 6·92 6·78

Na2CO3 (mM)

0·00 0·75 1·00 2·00 5·00 10·00

0·79 1·00 1·22 1·20 1·23 1·27

0·80 1·12 1·41 1·24 1·23 1·26

0·83 1·12 1·71 1·41 1·32 1·36

NaHCO3 (mM)

0·00 0·75 1·00 2·00 5·00 10·00

2·33 5·33 10·00 12·67 10·66 12·11

2·76 6·66 10·00 12·67 12·90 16·70

2·78 8·00 12·66 14·00 14·98 16·78

HCl (N)

0·00 0·01 0·02 0·05 0·10

1·72 10·17 21·81 34·82 44·71

1·76 10·82 27·92 37·62 47·82

1·79 21·72 32·18 39·48 47·90

H2SO4 (N)

0·00 0·01 0·02 0·05 0·10

1·72 20·17 24·22 37·20 37·40

1·76 23·00 25·00 37·18 37·42

1·79 23·22 25·22 38·00 37·80

Citrate buffer (0·2 M)

Control

2·12

2·23

2·61

pH 3·0 pH 4·0 pH 5·0

25·33 12·00 12·12

28·34 12·46 12·37

31·00 12·62 12·81

Zinc (mg biosorbed per g adsorbent)

Dry biomass

Conc. of eluant % zinc desorption at

P. Ahuja et al. / Process Biochemistry 34 (1998) 77–85

84

Table 7 Biosorption–desorption of zinc by O. anguistissima biomass for five cycles (initial biomass 15 mg) Cycle

Biosorption I Desorption I Biosorption II Desorption II Biosorption III Desorption III Biosorption IV Desorption IV Biosorption V Desorption V

Zn2+ biosorbed Zn2+ (mg) desorbed (mg)

Biomass after each cycle (mg DW)

1750·00 926·45

that cyanobacteria are ideal candidates to be further exploited, as being autotrophs, these are easy to cultivate and harvest.

Acknowledgements The present work was financially supported by the University Grants Commission. Thanks are due to Prof. Daljeet Singh for his valuable help in statistical analysis and in preparation of isotherms.

52·80

14·20

65·20

12·40

78·07

10·00

82·00

9·80

References

81·79

9·70

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1730·009 17·32 1760·66 9 23·09 1668·00 9 16·74 1645·00 9 9·86

Table 8 Removal of zinc from effluents obtained from Hindustan Zinc Limited, Udaipur by O. anguistissima Sample obtained from:

Initial zinc conc. (ppm)

Percent removal after biosorption

1. Balaria mill cyclone overflow (pH 8·05) 2. Balaria zinc rougher 3. Tailings Balaria final waste products (pH 7·96) 4. Mochia CD Tailings (pH 7·95) 5. Balaria lead rougher concentration (pH 7·92)

8·52

93·30

75·0 2·76

91·57 91·41

9·06

95·50

2·22

66·48

about 31% loss of biomass by weight after five cycles of biosorption–desorption. The results are in aggreement with those of Nakajima and Sakaguchi [29] who reported a 50% loss in the dry weight of the free cells of Streptomyces sp. after multiple cycles. The native biomass of O. anguistissima showed excellent removal of zinc from effluent samples (Table 8) and in most cases above 90% removal was observed. The maximum removal (95·5%) was obtained from a sample of Mochia tailings.

4. Conclusions Oscillatoria anguistissima has excellent biosorption capacity for Zn2 + and is comparable to the best known biosorbents. The steep isotherm at low residual concentration, rapid adsorption kinetics and its capacity to remove metal ions from effluent samples suggest that Oscillatoria sp. is an ideal organism for developing a metal biosorbent. The present report also emphasizes

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