Lead

 

Chen et al. / J Zhejiang Zhejiang Univ SCI 2005 6 6B(3):171-174 B(3):171-174 

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Journal of Zhejiang University SCIENCE ISSN 1009-3095 http://www.zju.edu.cn/jzus E-mail: [email protected] 

Bioremediation potential of spirulina: toxicity and biosorption studies of lead  CHEN Hong (陈  红), PAN Shan-shan (潘珊珊) ( Department  Department of Environmental Engineering, Zhejiang University, Hangzhou 310013, 310013, China) China) E-mail: [email protected] [email protected];; [email protected] Received Feb. 9, 2004; 2004; revision accepted June 7, 2004

Abstract:  This study exam examines ines the possibi possibility lity of using live spiruli spirulina na to biologic biologically ally remove aaqueous queous lead of low concentrat concentration ion (below 50 mg/L) from wastewater. The spirulina cells were first immersed for seven days in five wastewater samples containing lead of different concentrations, and the growth rate was determined by light at wavelength of 560 nm. The 72 h-EC 50  (72 h medium effective concentration) was estimated to be 11.46 mg/L (lead). Afterwards, the lead adsorption by live spirulina cells was conducted. It was observed that at the initial stage (0–12 min) the adsorption rate was so rapid that 74% of the metal was biologically adsorbed. The maximum biosorption capacity of live spirulina was estimated to be 0.62 mg lead per 105 alga cells. Key words: Bioadsorption, Bioremediation, Spirulina, Lead doi:10.1631/jzus.2005.B0171 Document code: A

INTRODUCTION

CLC number:  X172 

Environmental contamination by toxic metals is Environmental a serious problem worldwide due to their incremental accumulation in the food chain and continued persistence in the ecosystem. Conventional technologies, such as ion exchange or lime precipitation, are often ineffective and/or expensive, particularly for the removal of heavy metal ions at low concentrations

ment in extracellular capsules, precipitation, com plexation and oxidation-reduction oxidation-reduction reactions. It has  been proved that they are capable capable of adsorbing heavy metals from aqueous solutions, especially for the metal concentration below 50 mg/L (Lu and Wilkins, 1995). The metal-binding capacities of several biological materials have been identified to be very high, including marine algae, fungi and yeasts. It was re ported that these microorganism microorganismss can accumulate accumulate a

(below 50 mg/L). Furthermore, most of these techniques are based on physical displacement or chemical replacement, generating yet another problem in the form of toxic sludge, the disposal of which adds further burden on the techno-economic feasibility of the treatment process. In view of this, the development of new techniques is necessary to meet the environmental standards at affordable costs. Biotechnology has been investigated as an alternative method for treating the metal-containing wastewater of low concentrations. In response to heavy metals, microorganisms have evolved various measures via processes such as transport across the

wide range of metal species. Most of the studies dealing with biological removal of metals used dead biomass. However, recently, it was reported that the live Aspergillus live  Aspergillus niger   cells exhibits higher Ni biosorption capacity than dead biomass, probably due to intracellular Ni uptake (Kapoor et al ., ., 1999). The aim of this study is to examine the possibility of using live spirulina to  biologically remove lead of low concentration (below 50 mg/L) wastewater. The toxicity and biosorption of lead to live spirulina cultures were investigated. Both factors are very important in developing spirulina for the treatment of wastewater containing

cell membrane, biosorption to cell walls and entrap-

heavy metals.

 

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MATERIALS AND METHODS Biosorbent material Spirulina was supplied by the Institute of Botany, Zhejiang University. The alga seed was first centrifugated and then stored in liquid medium (details are in Table 1) for 7 d at 20~26 °C under light generated  by a 40 W white fluorescent lamp. The quantity of

living biomass used for the biosorption studies varied 5 5 from 1×10  to 5×10 cells/ml. Table 1 Composition of growth growth medium Constituents Concentration (g/L) EDTA 0.08 CaCl2·2H2O 0.04  NaCl 1.00  NaNO3  2.50  NaHCO3  16.80 FeSO4·7H2O 0.01 MgSO4·7H2O 0.20 K 2SO4  1.00 K 2HPO4  0.50

Acute toxicity tests Growth inhibition was achieved by adding log  phase cells to five solutions containing aqueous lead of 1, 2, 4, 10 and 20 mg/L. The volume of each solution was 100 ml. The growth rate was determined every 24 h and EC50 was evaluated by probit analysis. Lead sorption experiments experiments The Pb solutions were prepared by diluting standard Pb solution to the desired concentrations. The freshly diluted solutions were used for each  biosorption study. The sorption experiments were conducted in 250 ml flasks containing 100 ml of lead solutions with initial concentrations ranging from 10 to 50 mg/L. During the adsorption process, the flasks were agitated on a shaker for 48 h under ambient temperature (25±2 °C). At the designed period of 5, 15, 20, 30, 60, 100, 120 and 150 min, 5, 12 and 24 h, 10 ml of the solution were collected for analysis. To determine the concentration of the remaining metal ions, the spirulina in the sample solutions was removed by filtration and the filtrate was analysed to measure the lead concentration spectrophotometrically. Data analysis

The Langmuir isotherm model below is com-

monly used to describe the sorption of metals onto microbial surface. q = qmax bc /(1 + bc)  

(1)

where c is the final lead concentration (mg/L), q is the 5 metal uptake (mg/10  cells), b is the sorption binding constant (L/mg), qmax is the saturation capacity (mg/ 105 cells). The linear form of Eq.(1) is: 1 / q = 1 /( qmax bc) + 1 / qmax

(2)

 

from the slope and intercept of a 1/q 1/q vs 1/c 1/c linear plot −1 such that qmax=intercept  and b=intercept/slope. 

RESULTS AND DISCUSSIONS Toxicity of lead on spirulina growth To investigate spirulina’s tolerance of Pb2+, the living cells were cultivated in solution containing 2+ Pb   ions with various concentrations. The growth curves of spirulina are shown in Fig.1. The results indicated that the growth inhibition increased at higher aqueous lead concentration. In the presence of 2+ lowest Pb   concentration (1.0 mg/L), only a slight inhibition of cell growth occurred. By contrast, the highest concentration (20 mg/L) caused a large number of cells to die at first (0–1 d), but the cell growth recovered afterwards. Such responses of live and growing cells to high metal concentration are similar to the pure biological adsorption process using dead/treated biomass. In this study, the EC 50 at 72 h

was estimated to be 11.46 mg/L. 0.16    ) 0.14   m   n 0.12    0    6 0.10    5    (   y    t 0.08    i   s   n 0.06   e    d    l 0.04   a   c    i    t 0.02   p    O

0 mg/L

1 mg/L

2 mg/L

4 mg/L

10 mg/L

20 mg/L

0.00 0

2

4 Time (day)

6

8

Fig.1 Growth of spirulina in solu solutions tions containing a aqueous queous lead of various initial concentrations

 

Chen et al. / J Zhejiang Zhejiang Univ SCI 2005 6B(3):171-174 

Such finding also shows that spirulina has high tolerance to lead with EC50 higher than that of some species previously reported. The EC 50  of  Navicula 6 incerta and  Nitzchia clasterium  clasterium  are 3.01×10   and 6 0.48×10 , respectively. While the EC50  of a green 6 6 alga, Chlorococcum sp. is 2.5×10   to 3.0×10   (Trevors et al ., ., 1986). The resistance of spirulina to lead suggests its suitability for lead treatment.  −

−

−

−

Biological adsorption of lead 1. Time-dependent biosorption The dependence of lead biosorption by living spirulina on time is shown in Fig.2. Generally, it is reported (Ting et al ., ., 1989) that the uptake of metal ions can be divided into two stages: rapid and slow stage. In the ‘rapid’ stage, the metal ions are adsorbed onto the surface of microorganism. In the ‘slow’ stage, the metal ions transport across the cell membrane into the cytoplasm. Swift and Forciniti (1997) investigated different lead uptake mechanisms in various subcellular region of cyanobacteria and  Anabaena cylindrical . They noticed that lead phosphate precipitated on the cell wall and inside the cell. Their results confirmed a very fast uptake in the cell envelope and then a longer uptake period inside the cell. In this study, rapid biosorption was observed at the beginning (0–12 min, with 74% of metal adsorbed), then reached the equilibrium within 24 h with 95% of metal ions adsorbed. Rangsayatorn et al .(2004) .(2004) and Horikoshi et al .(1979) .(1979) reported that cadmium was rapidly adsorbed by S. platensis during platensis during the first 5 min and by C. regularis within regularis within 6 min, respectively. Such rapid uptake of heavy metals by living cells is very significant when the cells are used in bioremediation

 process. 100 90    ) 80    %    ( 70    l 60   a   v   o 50   m   e   r  40    +    2 30    b    P 20 10 0 0

2

4

6

8 10 1 12 2 14 16 18 20 22 24 Time (h)

Fig.2 Time-dependence Time-dependence of lead bio biosorption sorption by spirulina spirulina 2+ (Pb : 30 mg/L)

 

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2. Effect of spirulina concentration on lead biosorption The effect of spirulina concentration on the lead adsorption rate is shown in Fig.3. F ig.3. The living biomass 5 5 concentration varied from 1×10   to 5×10   cells/ml. The results demonstrated that the spirulina concentration considerably affected the metal removal rate. With the increased biomass concentration, the amount of adsorbed lead increased. However, the maximum adsorption rate estimated at equilibrium 2+ 5 stage (mg Pb  removed per 10  alga cells) decreased at higher cell concentration. This is because at higher  biomass concentration, the cells can provide more space for the adsorption process to take place. 100

0.12 0.1

   ) 90    %    (    l   a 80   v   o   m   e   r  70

0.08 0.06 0.04

   +    2

   b    P

60

2+

Pb 2+ removal removal (%) Pb  removal (%)(%) Equii Equilibrium capacity

0.02

50

   )   s   y   l    t    l    i   e   c   a   c   p   a   a   l   c   g   a      5   m    0   u    i   r    1   r    b    i    l    i   e   u   p   q   g   m    E   (

0 0

2

4

6

Cell concentration (10 5  cells/ml)

Fig.3 The effect of sspirulina pirulina co concentration ncentration o on n Pb2+  re2+ moval and maximum adsorption capacity (Pb : 30 mg/L, temperature: 25 25 C, agitatio agitation n speed: 166 rpm)  2+

3. Effect of initial Pb   concentration on biosorption 2+ The effect of initial Pb  concentration on bio2+ sorption is shown in Fig.4. The initial Pb  concentration remarkably influenced the metal adsorption 2+ rate at equilibrium stage. It was found that as Pb   concentration was reduced to below 10 mg/L, the Pb  biosorption rate reached 95%. However, when the 2+ 2+ Pb  concentration was above 30 mg/L, the Pb   removal rate decreased. Such decline in lead removal rate is probably caused by the saturation of some adsorption sites. The shape of the adsorption isotherm was also important, and a steep isotherm from the origin at a low residual concentration of the sorbate was highly desirable because it indicated the high affinity of the biosorbent (Volesky, 1990). When the equilibrium Pb2+ concentration was increased from 0 to 35 mg/L approximately, the loading capacity in5 creased from 0 to 0.44 mg per 10  alga cells after 24 h of adsorption. The maximum adsorption capacity and

 

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the adsorption constant were calculated to be 0.62 mg 5  per 10  alga cells and 0.135, respectively. The positive correlation between the maximum adsorption 2+ capacities of biosorbents and the Pb   concentration may be due to the higher collision between metal ions and biosorbents.

CONCLUSION This study led to the conclusion that spirulina’s rapid lead adsorption rate and high lead adsorption capacity made them well suited for the removal of lead in wastewater. In addition, living cells of spirulina were found to have high tolerance to lead and can be regarded as an attractive adsorbate option

10 0

for the biosorption of heavy metal contaminant. However, there are still many uncertainties associated with the development of treating wastewater by living algae and more future work is necessary.

80

   )    %    (    l   a 60   v   o   m   e   r  40

   +    2

   b    P

References  

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Horikoshi, T., Nakajima, A., Sakaguchi, T., 1979. Uptake of uranium by Chlorella regularis.  Agricultural and Biological Chemistry, Chemistry, 43:617-623. Kapoor, A., Viraraghavan, T., Roy, D., 1999. Removal of heavy metalsusing the fungus Aspergillus niger.  Biore sour Technol., Technol., 70:95-104. Lu, Y., Wilkins, E., 1995. Heavy metal removal by caustic-treated yeast immobilized in alginate.  Journal of  Hazardous Materials, Materials, 49(2-3):165-179.

0 0

10 20 30 40 2+ 2+ concentration (mg/L) Pb Pb  concentration (mg/L) (mg/L)

50

(a)    )

0 .5   s    )    l    l   s    l   e    l   c 0 . 4 5   e   c   a      5   g 0 .4    l    0   a    1      5   r   e 0 . 3 5    0   p    1   r   g   e 0 .3   m   p    (   g 0 . 2 5   y    t    i   m   c    (   a   y 0 .2   p    t   a    i   c   c 0 . 1 5   a   m   p   u   a 0 .1    i   r   c    b    i    l   m 0 . 0 5    i   u   u    i   q   r 0    b    E    i    l    i   u   q    E

0

5 10 15 20 25 30 35 Pb c2+o concentration EquilEquilibrium Equilibri ibrium um P b 2+ n c e n t r a t iio o n(mg/L) ( m g /L /L )

Rangsayatorn, N.,Cadmium Pokethitiyook, P., Upatham, Lanze, G.R., 2004. biosorption by cells E.S., of Spirulina  platensis TISTR  platensis  TISTR 8217 immobilized in alginate and silica gel.  E nvironmental nvironmental International , 30(1):57-63. Swift, D.T., Forciniti, D., 1997. Accumulation of lead by  Anabaena cylindrica: cylindrica: mathematical modeling and an energy dispersive X-ray study.  Biotechnology and Bioen gineering , 55:408-419. Ting, Y.P, Lawson, F., Prince, I.G., 1989. Uptake of cadmium andzinc by the alga Chlorella vulgaris: part 1. Individual ion species. Biotechnology species. Biotechnology and Bioengineering , 34:990. Trevors, J.T., Stratton, G.W., Gadd, G.M., 1986. Cadmiumtransport, resistance, and toxicity in bacteria, algae, and fungi. Canadian Journal of Microbiology, Microbiology, 32:464-474. Volesky, B., 1990. Removal and Recovery of Heavy Metals by Biosorption. Biosorption of Heavy Metals. CRC Press, Boca Raton, Florida, p.7-43.

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(b) Fig.4 Lead removal removal rate as a function of Pb2+ concentration (a) and the adsorption isotherm of spirulina (b), initial 5 cell concentration: 5×10   cells/ml, temperature: 25 °C, agitation speed: 166 rpm, time: 24 h  

 

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