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in free and alginate immobilized cells. S.Sai Subhashini ... e-mail: [email protected] S.Kaliappan ... exhibited better removal than free cells. Column ...

2011 2nd International Conference on Environmental Science and Technology IPCBEE vol.6 (2011) © (2011) IACSIT Press, Singapore

Removal of heavy metal from aqueous solution using Schizosaccharomyces pombe in free and alginate immobilized cells S.Sai Subhashini


Department of Civil Engineering College of Engineering, Anna University, Chennai Chennai 600 025, India e-mail: [email protected]

Department of Civil Engineering College of Engineering, Anna University, Chennai Chennai 600 025, India e-mail: [email protected]

M. Velan Department of Chemical Engineering A.C. College of Technology, Anna University, Chennai Chennai 600 025, India e-mail: [email protected]

Abstract—The removal of copper ions from aqueous solutions using Shizosaccharomyces pombe (fission yeast) was investigated in batch and column studies. The batch studies were carried out by varying parameters such as pH, temperature and metal concentration. The optimum pH and temperature conditions for metal removal and cell growth were determined to be 4.0 and 25°C respectively. The maximum removal of copper ions was found to be 73% at an initial concentration of 100 ppm with 1% (v/v) of inoculum concentration at 30°C. The cells were subjected to immobilization process using sodium alginate. After the preparation of the immobilized beads, the immobilization studies such as varying the number of beads and initial metal ion concentration were conducted. The immobilized beads exhibited better removal than free cells. Column studies were conducted and that showed better removal at the flow rate of 2 ml and the bed height of 15 cm. Keywords-Bioaccumulation; fissionyeast; Shizosaccharomyces pombe; biosorption;


anions, complexing agents, inhibitors of microbial growth, resistance to stress factors. The mechanisms of metal binding to microbial biomass can be roughly divided into three main types intracellular accumulation, sorption or complex formation on cell surface and extra cellular accumulation or precipitation (Kujan et al. 2005) The metabolically independent biosorption of metals by yeast cells occur with in several minutes (Kratochvil and Volesky 1998; Volesky 2003). Copper contributes markedly to the environmental pollutions, especially of water and soil. It is usually not found in high concentrations but the toxicity of copper is relatively high to that of other heavy metals (Brian et al 2005) these low concentrations can conveniently be removed by low-cost low energy bioaccumulation which is at the same time, a highly efficient process. In this study the removal of copper ions using live and immobilized cells of Schizosaccharomyces pombe was studied. II. MATERIALS ANDMETHODS

I. INTRODUCTION The removal of cations of heavy metals from industrial waste water can be accomplished by biotechnological methods which make use of microorganisms as cation collectors (Babel and kurniawan 2003). Microbial biomass has been derived from various sources, e.g., actinomycetes (Selantia et al. 2004), cyanobacteria, other bacteria, algae (Lodeiro et al. 2005; Akthar et al. 2003), moulds and yeasts (Salinas et al. 2000; Baldrian 2003). The amount of accumulated cations can be very large and depends on many factors ranging from the microbial species and its physiological state to external physio-chemical conditions such as pH, temperature (Xu et al. 2004), initial metal concentration and inoculum concentration etc. Other important factors affecting accumulation capacity are the type of cation, its valence, the presence of other cations or

Microorganisms, media and cultivation conditions: Shizosaccharomyces pombe strain (MTCC2665) was procured from IMTECH Chandigarh and it was cultivated in medium containing (g/L): malt extract 3; yeast extract 3; peptone 5; glucose 10 at room temperature. Subcultures were stored at 4°C. Copper was estimated by neocuprine method (Metcalf and Eddy (1979)) using UV spectrophotometer at 457nm and cell growth was monitored at 600nm A. Immobilization using Calcium Alginate gel: Wet biomass was suspended in the 3% concentration of sodium alginate and this was added drop-wise to a solution of 0.1 M CaCl2. The resulting beads were washed using distilled water and subsequently used for study.


B. Column Study Continuous flow experiments were conducted in a glass column with an internal diameter of 2 cm and 35 cm in length. At the bottom of the column, sieves were attached followed by glass wool. 1 cm high layer of glass beads was placed at the column base in order to provide uniform inlet flow of the solution into the column. The column was then packed densely with immobilized beads and operated in an up flow mode at room temperature. The samples were collected from the effluent outlet at regular intervals of time and it was analyzed for copper concentration using neocuprine method.

different temperatures (15 – 45 °C). The maximum removal of copper occurs at 25°Cshown in Fig 2. The temperature of the accumulation medium could be important for energy dependent mechanisms in metal removal by yeast. Temperature is known to affect the stability of the cell wall, its configurations and can also cause ionization of chemical entities. Energy- independent mechanisms are likely to be affected by temperature since the process responsible for removal is largely physiochemical in nature (Gulay et al 2003).


Figure 2. Effect of Temperature on copper ion removal

Figure 1. Effect of pH on copper ion removal

A. Removal of copper ions at various pH: It is evident from fig.1 that the maximum removal of the copper occurred at pH 4.0 and from pH 5.0 the level of removal started decreasing. Normally yeast cell wall consists of protein coat, which develops a charge by the dissociation of ionizable side groups of the constituent amino acids. The ionic state of ligands such as carboxyl, phosphate, imidazole and amino groups will promote reactions with the positively charged metal ions. At low pH 3.0 cell wall ligands closely associated with the hydronium ions (H3O+) and restricted the accumulation of Cu2+ ions as a result of repulsive forces. Removal of copper and lead by Micrococcus letues (Leung et al 2000) also showed the same trend. The increasing trend from pH 3 to 5 is due to the strong relations of bioaccumulation to the number of surface negative charge, which depends on the dissociation of functional group. At higher alkaline pH values (8 and above), a reduction in the solubility of metals contributes lower uptake rates (Hasan et al 2000) reported the variation of adsorption of nickel at various pH is on the basis of metal chemistry in solution and the surface chemistry of the sorbent..

C. Removal of copper ions at various initial metal concentrations: Initial metal ion concentration plays a major role in calculating the bioaccumulative capacity of yeast cell. As metal concentration increased the removal of copper is decreased shown in Fig 3. There is a real danger that these metals may poison the cell and stopping biological activity and growth. It has been inferred in several instances that the accumulation of metal results from the lack of specificity in a normal metal transport system and that, at high concentrations, metal may act as competitive substrate in a transport system. Similar results were obtained in the case of Trametes versicolor for Cu, Pb, and Zn removal (Puranik et al 1997)

B. Removal of copper ions at various temperatures: Bioaccumulation of copper by S. pombe appears to be temperature dependent; the experiment was carried out at


Figure 3. Effect of Metal ion concentration

D. Specific growth rate The specific growth rate for each metal concentration was calculated from the growth curves. Fig.4 shows the Monod curve plotted. Determination of maximum growth rate coefficient (µmax), half-saturation coefficient (Ks) is difficult from the non-linear Monod curve(fig 4). Hence the models such line weaver- Burk and Hanes plots were used to determine the above coefficients .The Monod equation is given by

at 96 hours. The maximum uptake of copper (93%) was noted at 96 hours. After which there was no uptake of metal ions (Fig.5).

μ = μ max(S)/Ks+(S) ----- (1)

Figure 5. Effect of contact time on copper removal before copper accumulation 120 100 Transm ittance %

Figure 4. Monod curve

The Line weaver/Burke equation is given by 1 ----μ

Ks+ (S) = ----------- ------- (2) μ (S)

80 60 40 20 0 0

The Hanes plot equation is given by







wave lenth (Cm )

(a) ------ (3) The values of maximum growth rate coefficient (µmax), half-saturation coefficient (Ks) for various models are given in Table 1

Line weaver-Burk model Hanes plot

120 100 Transmittance %


TABLE 1 Kinetic models µmax (hr-1 )

After copper accumulation

Ks (mg/L)





80 60 40 20 0 0







wave lenth (Cm )


E. Effect of Bead Number on Copper Removal The effect of immobilized bead number on copper removal was studied by varying the bead numbers from 50 300. The maximum removal of copper obtained with 300 beads. Thus the removal efficiency increases with increase in bead number. This is because the number of viable cells in the beads is high with increase in beads.

Figure 6. IR Spectra of S.pombe with 100 ppm of copper (a) before (b) after

FTIR analysis of cells was carried to find out the influence of functional groups on removal of copper ions shown in Fig 6.The removal of copper ions was substantiated with SEM analysis, which indicated the accumulation of copper ions on yeast cells shown in Fig 7.

F. Effect of contact time for copper removal The effect of contact time on copper removal was studied at regular time interval of 12 hours. The equilibrium reaches


(a) Figure 8. Effect of bed height


Figure 9. Effect of flow rate

Figure 7. Scanning electron micrograph of S.pombe on copper accumulation (a) before (b) after

G. Effect of Bed Height The removal performance of immobilized S.pombe was observed with various bed heights at a constant flow rate (2ml/min) and with 100 ppm of copper ion concentration (Fig.8) Maximum removal takes place at the bed height of 15 cm. the removal percentage decreased with decrease in bed height from 15 to 5 cm. this might be due to the relatively small amount of biobeads used. Thus the numbers of viable cells also decrease with decrease in bed height, which reduces percentage removal of copper ions. The highest removal takes place at 15 cm bed height. This is due to the number of viable cells in the bead increases with the increase in beads.

I. Effect of initial metal concentration The effect of initial metal concentration was studied with 100 to 300 ppm of copper solution at constant flow rate of 2 ml/min and 15 cm height. The maximum removal took place at 100 ppm of initial metal concentration. The time taken for the maximum removal of copper at 100 ppm was less (24hrs) compared to other concentrations (36hrs and 42 hrs) of copper ions. Thus the increased initial concentrations increased the time for maximum removal of copper ions Fig.10.

H. Effect of flow rate When the concentration of copper ion (100 ppm) and bed height (15cm) were kept constant, flow rates were changed from 2 - 6 ml/min and the results are furnished in Fig.9. The decrease in the flow rate of the column resulted in the increased removal of copper ions as the slow flow rate provided more contact time. Thus increase in contact time increases the removal percentage of copper ions.


Figure 10. Effect of initial metal concentration

IV. CONCLUSION Growing yeast cells are capable of accumulation of heavy metals. The aim of this work was to study the growth and bioaccumulation characteristics of S.pombe for the removal of Copper (II). Based on the experiment carried out and analysis of the data obtained, the following conclusions are drawn. In copper (II)-containing wastewaters at lower pH (4) values, yeast biomass could provide an effective bioaccumulator for removal of the copper (II) ions. This study showed that, because of its high tolerance to copper (II) ions, S.pombe may be of value as a living biosorbent for treatment processes devoted to wastewater containing copper (II) ions. In 100 ppm of copper ion concentration the removal was 74.85% for free cells, 93% for immobilized cells in pH 4 at 25ºC temperature .The column study conducted with immobilized beads by varying bed heights (5cm to 15Cm), flow rates (2ml/min to 6ml/min) and initial copper ion concentration (100ppm to 300 ppm) shows the optimum bed height at 15cm with 2ml/min flow rate was maximum. These studies show that immobilized beads are more potential in removal of Cu (II) from contaminated water, which is environmentally friendly.




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