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Biosorption Equilibria of Binary Cd(II) and Ni(II)

Systems onto Saccharomyces cerevisiae and
Ralstonia eutropha Cells: Application of
Response Surface Methodology
ARTICLE in JOURNAL OF HAZARDOUS MATERIALS APRIL 2009
Impact Factor: 4.53 DOI: 10.1016/j.jhazmat.2009.03.041 Source: PubMed

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Journal of Hazardous Materials 168 (2009) 14371448

Contents lists available at ScienceDirect

Journal of Hazardous Materials

journal homepage: www.elsevier.com/locate/jhazmat

Biosorption equilibria of binary Cd(II) and Ni(II) systems onto

Saccharomyces cerevisiae and Ralstonia eutropha cells: Application of
response surface methodology
Mohammad Fereidouni, Ali Daneshi, Habibollah Younesi
Department of Environmental Science, Faculty of Natural Resources & Marine Sciences, Tarbiat Modares University, P.O. Box 46414-356, Imam Reza Street, Noor, Iran

a r t i c l e

i n f o

Article history:
Received 9 September 2008
Received in revised form 9 March 2009
Accepted 10 March 2009
Available online 18 March 2009
Keywords:
Biosorption
Ralstonia eutropha
Saccharomyces cerevisiae
RSM
Ni(II)
Cd(II)

a b s t r a c t
Present study investigated the biosorption of Cd(II) and Ni(II) from aqueous solution onto Saccharomyces
cerevisiae and Ralstonia eutropha non-living biomass. Biomass inactivated by heat and pretreated by
ethanol was used in determination of optimum conditions. The important process parameters, such as
initial solution pH (28), initial Ni(II) concentration (1142 mg/l), initial Cd(II) concentration (1142 mg/l),
and biomass dosage (0.24.7 g/l) were optimized using design of experiments (DOE). A central composite
design (CCD) under response surface methodology (RSM) was applied to evaluate and optimize the efciency of removing each adsorbent. Moreover, the two responses were simultaneously studied by using
a numerical optimization methodology. The optimum removal efciency of Cd(II) and Ni(II) onto S. cerevisiae was determined as 43.4 and 65.5% at 7.1 initial solution pH, 4.07 g/l biomass dosage, 16 mg/l initial
Ni(II) concentration and 37 mg/l initial Cd(II) concentration. The optimum removal efciency of Cd(II)
and Ni(II) onto R. eutropha was ascertained as 52.7 and 50.1% at 5.0 initial solution pH, 2.32 g/l biomass
dosage, 28 mg/l initial Ni(II) concentration and 37 mg/l initial Cd(II) concentration. The present analysis
suggests that the predicted values are in good agreement with experimental data. The characteristics of
the possible interactions between biosorbents and metal ions were also evaluated by scanning electron
microscope (SEM) and Fourier transform infrared (FT-IR) spectroscopy analysis.
2009 Published by Elsevier B.V.

1. Introduction
The intensication of industrial activity during recent years is
greatly contributing to the increase of heavy metals in the environment, mainly in the aquatic systems [1]. The main sources of
heavy-metal pollution are mining, milling and surface nishing
industries, discharging a variety of toxic metals such as Cd, Cu, Ni,
Co, Zn and Pb into the environment [2,3]. It is well known that
heavy metals can be extremely toxic as they damage nerves, liver
and bones, and also block functional groups of vital enzymes [2].
Ni(II) is one such heavy metal frequently encountered in raw
wastewater streams from industries such as non-ferrous metal,
mineral processing, paint formulation, electroplating, porcelain
enameling, copper sulphate manufacture and steam-electric power
plants [4,5]. Nickel is also listed as a possible human carcinogen
(group 2B) and associated with reproductive problems and birth
defects. Besides, a range of detrimental effects on fauna and ora are
also well documented [2]. Cadmium has a half-life of 1030 years
[6] and its accumulation in human body affects kidney, bone and

Corresponding author. Tel.: +98 122 625 31013; fax: +98 122 625 3499.
E-mail addresses: hunesi@modares.ac.ir, hunesi@yahoo.com (H. Younesi).
0304-3894/$ see front matter 2009 Published by Elsevier B.V.
doi:10.1016/j.jhazmat.2009.03.041

also causes cancer and its use is increasing in industrial applications

such as electroplating and making pigments and batteries [7].
Since these heavy metals are a valuable resource for different
industrial applications, their recovery and recycling assumes even
greater signicance. Further, strict environmental regulations compel industries to shift to cleaner production methods, demanding
the development of environmentally friendly, low-cost and efcient
treatment techniques for metal rich efuents [2,8]. Biosorption is
an emerging and attractive technology which involves sorption of
dissolved substances by a biomaterial. It is a potential technique
for the removal of heavy metals from solutions and recovery of
precious metals [9,10]. Even though there are many methods for
the removal of metal ions from solutions, such as chemical precipitation [11,12], solvent extraction [1315], membrane processes
[16] and adsorption on activated carbon [17], biosorption processes
show many advantages over these methods. It is selective, effective
and cheap and is able to remove very low levels of heavy metals from
solutions. While the conventional methods have several disadvantages, which include incomplete metal removal, and toxic sludge
generation [18,19]. In present years, different kinds of non-living
biomass including bacteria, fungi, [20] algae, mosses, macrophytes,
higher plants, [21] plant material [22,23] and waste products from
industrial or agricultural operations [24] have been examined as

1438

M. Fereidouni et al. / Journal of Hazardous Materials 168 (2009) 14371448

potential biosorbent for heavy metals. Non-living biomass appears

to present specic advantages in comparison to the use of living microorganisms. Non-living cells may be stored or used for
extended periods at room temperature, they are not subject to
metal toxicity and nutrient supply is not necessary [1].
Response surface methodology (RSM) is a collection of mathematical and statistical techniques useful for analyzing the effects
of several independent variables on the response [25,26]. RSM has
an important application in the process design and optimization
as well as for the improvement of existing design. This methodology has become much more practical with the development of
interactive computer programs compared to traditional method as
it includes interactive effects among the variables while building
on the experimenters prior knowledge and, eventually, it depicts
the overall effects of the parameters on the process [27].
The objective of the present research was to study the effect
of initial Cd(II) and Ni(II) concentrations, pH and biomass concentration on biosorption of Cd(II) and Ni(II) using Saccharomyces
cerevisiae and Ralstonia eutropha non-living biomass. The main aim
of this work was to compare the biosorption characteristics of S.
cerevisiae and R. eutropha for the removal of Cd(II) and Ni(II) in an
aqueous solution. Furthermore, optimum conditions for elimination of Cd(II) and Ni(II) were determined with RSM under design of
experiment (DOE).
2. Materials and methods
2.1. Microorganism and its preparation for biosorption
The yeast S. cerevisiae (PTCC 5010) was provided from Research
and Technology Department of Ministry of Science, Iran (Persian
Type Culture Collection) in the form of freeze-dried, and then cultured in sterilized medium. The composition of growth medium
was (grams per liter): 9; (NH4 )2 SO4 , 40; sugarcane molasses, 2.5;
MgSO4 , 1.00; yeast extract, 1.00; KH2 PO4 , 0.2; K2 HPO4 . The bacterium R. eutropha (DSM 534) was provided by Deutsche Sammlung
von Mikroorganismen und Zellkulturen (DSMZ) in the form of
freeze dry. The composition of growth medium was (grams per
liter): 2.30; KH2 PO4 , 2.90; Na2 HPO4 , 1.00; NH4 Cl, 0.50; MgSO4 ,
0.50; NaHCO3 , 0.01; CaCl2, 0.05; Fe(NH4 ) citrate, 40; sugarcane
molasses and con syrup, and 5 ml trace element solution (grams per
liter): MgSO4 , 2.2 g; FeSO4, 0.1 g; MnSO4 , 0.1 g; K2 SO4, 2.2 g; H3 BO3 ,
0.02 g; CuSO4 , 0.08 g.
The medium was sterilized by autoclaving at a pressure of
1.5 atm and temperature of 121 C for 20 min. Temperature and pH
of growth condition were at ambient temperature (25 C) and 6.8,
respectively, with shaking at 200 rpm. The yeasts and bacteria cells
were collected at the end of the exponential phase, and then centrifuged at 3000 rpm for 15 min, followed thrice by re-suspension
in deionized water and re-centrifugation. Collected cells were oven
dried at 70 C for 20 h. The yeasts and bacteria were ground and
screened through a set of sieve with 230 meshes. The pretreatment of the biosorbent was carried out non-living of cells into 70%
ethanol for 20 min at room temperature. Then, the cells were centrifuged, washed, dried and powdered as mentioned above. Thus
a monotonous powder was produced which was stocked in the
refrigerator at 4 C for future use.
2.2. Scanning electron microscopy (SEM) experiments
Scanning electron microscope (SEM, Phillips XL30, Holland) was
used for the observation of S. cerevisiae and R. eutropha before
and after treatment by 70% ethanol. The morphology of non-living
biomass of S. cerevisiae [28] and R. eutropha was assessed before
and after treatment.

2.3. Fourier transform infrared (FT-IR) spectroscopy analysis

FT-IR spectroscopy was used to detect changes in vibration frequency in the S. cerevisiae and R. eutropha biomass. The spectra
were collected by FTS-135 (Bio-Rad) spectrometer within the range
4004000 cm1 using a KBr window. The background obtained
from the scan of pure KBr was automatically subtracted from the
sample spectra. Spectra were plotted using the same scale on the
absorbance axis.
2.4. Preparation of metal solutions
Cd(II) solution (1000 mg/l) was prepared by dissolving 2.282 g of
Cd(II) sulphate (CdSO4 8/3 H2 O) (Merck) in deionized water. Stock
Ni(II) solution was prepared by dissolving 4.050 g of NiCl2 6H2 O
(Merck) in deionized water. All solutions, their dilutions and
standards were prepared using deionized water (EYELA STILLACE
SA-2100E1). Any pH adjustments were made using 0.1 M H2 SO4 and
0.1 M NaOH.
2.5. Metal biosorption studies
In order to study the effect of initial solution pH, initial Cd(II)
and Ni(II) concentrations and biomass dosage on removal efciency of each ion, thirty batch biosorption experiments designed
by response surface methodology were conducted at the equilibrium time of 240 min, agitation speed of 200 rpm and temperature
of 25 C. Each experiment was carried out in 250 ml Erlenmeyer
asks containing 100 ml Cd(II) and Ni(II) solution by shaking the
asks at 120 rpm for period contact time of 240 min. Samples were
withdrawn at pre-determined time intervals (2, 5, 10, 15, 20, 30, 40,
60, 120 and 240 min) and ltered through 0.25 m lters. Filtered
samples were analyzed for residual Cd(II) and Ni(II) concentration.
Metal removal by both yeast and bacterium were determined as
according to Eq. (1):
R=

C0 Ce
100
C0

(1)

where R is the percentage of metal adsorbed by biomass in percentage, C0 is the initial concentration of metal ion in mg/l and Ce is the
equilibrium concentration of metal ion in mg/l [1,29].
2.6. Experimental and optimization of biosorption
Optimum conditions for the biosorption of Cd(II) and Ni(II) by
S. cerevisiae and R. eutropha were determined by means of central composite design (CCD) under response surface methodology.
The RSM consists of a group of empirical techniques devoted to
the evaluation of relationship existing between a cluster of controlled experimental factors and measured responses according to
one or more selected criteria. Optimization studies were carried out
by studying the effect of four variables including S. cerevisiae and
R. eutropha doses, initial Cd(II) and Ni(II) concentrations and pH
of solutions [3032]. The independent variables used in this study
were coded according to Eq. (2):
xcoded =

XActual (XHi + XLow ) /2

(XHi XLow ) /2

(2)

where x is the coded variables, X is the actual variables. The behavior

of the system is explained by the following empirical second-order
polynomial model Eq. (3):
y = 0 +

k

i=1

i xi +

k

i=1

ii xi2 +

k
k1 

i=1 j=2

ij xi xj +

(3)

M. Fereidouni et al. / Journal of Hazardous Materials 168 (2009) 14371448

1439

Table 1
Experimental ranges and levels of the independent variables.
Independent variables

Range and level

pH (X1 )
Biomass (X2 )
Initial cadmium concentration (X3 )
Initial nickel concentration (X4 )

(1.414)

+1

+ (1.414)

2.1
0.20
11
11

3.0
0.86
16
16

5.1
2.45
27
27

7.1
4.04
37
37

8.0
4.70
42
42

where y is the predicted response, xi, xj . . . xk are the input variables,

which affect the response y, xi2 , xj2 , . . ., xk2 are the square effects, xixj,
xixk and xjxk are the interaction effects, 0 is the intercept term, i
(i = 1, 2, . . ., k) is the linear effect, ii (i = 1, 2, . . ., k) is the squared
effect, ij (i = 1, 2, . . ., k; j = 1, 2, . . ., k) is the interaction effect and
is a random error [33,34].
The Design-Expert 7.0.0 (Stat-Ease, Inc., Minneapolis, MN, USA)
software was used for regression and graphical analysis of the
obtained data. The most popular response surface methodology
design is the central composite design which is designed to estimate
the coefcients of quadratic Eq. (3). A design of 30 experiments
for the four factor case, i.e. sixteen factorial points, eight axial
(star) points and six replicate points at the central points, were
employed to the quadratic model. The optimum values of the
selected variables were obtained by solving the regression equation at desired values of the process responses as optimization
criteria. Each of the parameters was coded at ve levels of each
factor: , 1, 0, +1 and +. The range and level of the variable
in coded values from RSM studies are given in Table 1. The coded
and actual values of the test variables as well as the experimen-

tal and predicted values of removal efciency of Cd(II) and Ni(II) as

responses for optimization of the process variables are also given in
Table 2.
3. Results and discussion
3.1. Fitting the process models
The results of the statistical analysis for removal efciency of
Cd(II) and Ni(II) onto S. cerevisiae and R. eutropha according to
analysis of variance (ANOVA) are given in Table 3. Model adequacy was tested through lack-of-t (LOF), P-values and F-values
[35]. The lack-of-t term was insignicant as desired (Table 3).
The insignicant value of lack-of-t (more than 0.05) showed that
the models were valid for the present study [36]. The lack-of-t
F-values of 3.60, 0.37, 4.46 and 3.04 for Cd(II) and Ni(II) onto S.
cerevisiae and R. eutropha, respectively, were not statistically signicant as the P-values were greater than 0.05 (Table 3). Adequate
precision is a measure of the range in predicted response relative to its associated error. Its desired value is 4 or more [37].

Table 2
Full factorial central composite design matrix of orthogonal and real values along with observed responses for removal of cadmium(II) and nickel(II).
Run order

Real (coded) values

X1

X2

Removal efciency, %
X3

X4

Saccharomyces cerevisiae
Cadmium(II)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

2.1()
3.0(1)
3.0(1)
3.0(1)
3.0(1)
3.0(1)
3.0(1)
3.0(1)
3.0(1)
5.1(0)
5.1(0)
5.1(0)
5.1(0)
5.1(0)
5.1(0)
5.1(0)
5.1(0)
5.1(0)
5.1(0)
5.1(0)
5.1(0)
7.1(+1)
7.1(+1)
7.1(+1)
7.1(+1)
7.1(+1)
7.1(+1)
7.1(+1)
7.1(+1)
8.0(+)

2.5(0)
4.0(+1)
4.0(+1)
4.0(+1)
0.9(1)
0.9(1)
0.9(1)
4.0(+1)
0.9(1)
0.2()
2.5(0)
2.5(0)
2.5(0)
2.5(0)
2.5(0)
2.5(0)
2.5(0)
4.7(+)
2.5(0)
2.5(0)
2.5(0)
4.0(+1)
4.0(+1)
0.9(1)
0.9(1)
0.9(1)
0.9(1)
4.0(+1)
4.0(+1)
2.5(0)

26.5(0)
37.5(+1)
15.5(1)
37.5(+1)
15.5(1)
37.5(+1)
37.5(+1)
15.5(1)
15.5(1)
26.5(0)
26.5(0)
26.5(0)
42.0(+)
26.5(0)
26.5(0)
26.5(0)
11.0()
26.5(0)
26.5(0)
26.5(0)
26.5(0)
37.5(+1)
15.5(1)
37.5(+1)
15.5(1)
37.5(+1)
15.5(1)
37.5(+1)
15.5(1)
26.5(0)

26.5(0)
37.5(+1)
37.5(+1)
15.5(1)
37.5(+1)
37.5(+1)
15.5(1)
15.5(1)
15.5(1)
26.5(0)
42.0(+)
26.5(0)
26.5(0)
11.0()
26.5(0)
26.5(0)
26.5(0)
26.5(0)
26.5(0)
26.5(0)
26.5(0)
37.5(+1)
37.5(+1)
37.5(+1)
15.5(1)
15.5(1)
37.5(+1)
15.5(1)
15.5(1)
26.5(0)

Ralstonia eutropha
Nickel(II)

Cadmium(II)

Nickel(II)

Experimental

Predicted
value

Experimental

Predicted
value

Experimental

Predicted
value

Experimental

Predicted
value

31.6
26.7
25.6
46.4
42.0
53.0
46.3
47.0
45.9
51.5
50.3
50.5
50.3
62.3
50.4
50.3
34.4
50.9
29.7
61.7
68.6
44.2
43.7
56.1
50.8
62.5
67.8
54.0
54.6
49.7

32.1
26.8
25.6
46.1
41.7
52.7
46.4
46.7
46.0
51.9
50.2
50.2
50.2
62.6
50.2
50.2
34.7
50.2
30.1
62.1
68.9
43.9
43.5
56.3
51.0
62.7
67.9
53.8
54.3
49.9

10.9
12.2
10.1
34.1
36.9
17.1
28.6
23.5
34.9
10.8
26.6
27.3
26.4
37.7
26.4
27.4
49.7
26.6
33.3
35.9
21.0
27.5
19.7
31.7
41.0
42.5
42.5
42.0
40.8
27.2

11.2
11.9
10.1
34.2
36.9
17.1
28.7
23.7
34.4
10.9
26.7
26.7
26.7
37.9
26.7
26.7
50.0
26.7
33.3
36.0
21.2
27.9
19.6
31.6
40.8
42.4
42.3
41.9
41.0
27.3

23.3
14.3
11.3
10.8
10.4
21.2
19.1
22.6
23.0
36.2
18.2
46.2
54.4
30.7
46.6
46.2
45.7
32.9
45.8
45.7
46.0
39.7
24.7
44.8
39.7
52.1
21.1
44.4
40.4
55.0

23.5
13.8
12.0
11.0
10.5
20.5
19.8
22.2
22.7
36.3
18.4
46.2
54.5
30.1
46.2
46.2
45.3
32.4
46.2
46.2
46.2
40.1
24.1
45.2
40.2
51.5
21.0
44.4
41.3
54.5

20.2
22.0
14.3
22.9
10.3
23.5
20.2
36.1
30.9
19.0
34.1
42.1
54.9
37.3
43.6
43.1
50.0
25.0
42.9
42.9
42.8
37.2
20.5
37.9
17.4
18.1
13.3
20.0
24.2
23.3

20.9
22.0
14.4
22.0
10.0
22.8
20.9
36.8
30.4
19.6
34.6
42.9
55.4
36.8
42.9
42.9
49.5
24.4
42.9
42.9
42.9
37.7
19.9
37.3
17.3
18.0
14.2
20.4
24.9
22.7

X1 , pH; X2 , biomass concentration; X3 , initial cadmium concentration; X4 , initial nickel concentration.

1440

M. Fereidouni et al. / Journal of Hazardous Materials 168 (2009) 14371448

Table 3
Analysis of variance (ANOVA) for the response surface quadratic model.
Non-living cells

Metal ions

Source of variation

Sum of squares

DF

Mean square

F-value

P-value (Prob > F)

S. cerevisiae

Cadmium(II)

Model
Residual
Lack-of-t
Pure error

3458.54
2.35
2.06
0.29

14
15
10
5

247.04
0.16
0.21
0.057

1579.95

<0.0001

3.60

0.0848

Total

3460.89

29

Model
Residual
Lack-of-t
Pure error

3167.92
1.98
1.04
0.94

14
15
10
5

226.28
0. 13
0.10
0.19

1712.24

<0.0001

0.55

0.8031

Total

3169.90

29

Model
Residual
Lack-of-t
Pure error

5793.47
6.03
5.42
0.61

14
15
10
5

413.82
0.40
0.54
0.12

1029.15

<0.0001

4.46

0.0564

Total

5799.50

29

Model
Residual
Lack-of-t
Pure error

4207.94
8.83
7.58
1.25

14
15
10
5

300.57
0.59
0.76
0.25

510.70

<0.0001

3.04

0.1156

Total

4216.77

29

Nickel(II)

R. eutropha

Cadmium(II)

Nickel(II)

This means that regression models provide an excellent explanation of the relationship between the independent variables (factors)
and the responses (% removal). The model F-value of 1029.15 and
13118.38 for Cd(II) and Ni(II) onto S. cerevisiae and 1029.15, 510.70
for Cd(II) and Ni(II) onto R. eutropha, respectively, and values of
P < 0.0001 indicated that all model terms are signicant. High model
F-values of the models implies that the models are signicant
(Table 3). The associated Prob > F-value for the models lower than
0.0001 (i.e. = 0.05, or 95% condence) indicated that the models
are considered to be statistically signicant (Table 3). The regression equation according to the analysis of variances (ANOVA) gave
the level of Cd(II) and Ni(II) removal as a function of the initial
Cd(II)/Ni(II) concentration, initial solution pH and S. cerevisiae/R.
eutropha biomass dosage. By applying multiple regression analysis on the experimental data, the experimental results of the CCD
design were tted with a quadratic equation. The empirical relationship between Cd(II)/Ni(II) removal (y) and the four variables
(factors) in coded values obtained by the application of RSM are
given in Table 4. Values of Prob > F are less than 0.05 indicate that
the model terms are signicant as presented in Table 4. Accordingly, y is the Cd(II) and Ni(II) removal (responses) in percentage,
x1 , x2 , x3 and x4 are the coded values of the variables, i.e. initial solution pH (x1 ), S. cerevisiae/R. eutropha dosage (x2 ), initial
Cd(II) concentration (x3 ) and initial Ni(II) concentrations (x4 ). The
square of correlation coefcient for each response was computed
as the R-square (R2 ) that is a measure of the amount of variation
around the mean explained by the model. The values of R2 (0.9993
and 0.9999 for Cd(II) and Ni(II) onto R. eutropha and 0.9990 and
0.9979 for Cd(II) and Ni(II) onto S. cerevisiae, respectively) and correspondingly adjusted R2 (0.9980, 0.9987 for Cd(II) and Ni(II) onto
R. eutropha and 0.9980, 0.9960 for Cd(II) and Ni(II) onto S. cerevisiae, respectively) are close to 1.0 that indicate a high correlation
between the observed values and the predicted values. A measure
of the amount of variation in predicted data was also examined by
the model as the predicted R-square. It was observed that the predicted R2 (0.9969 and 0.9970 for Cd(II) and Ni(II) onto R. eutropha
and 0.9937 and 0.9883 for Cd(II) and Ni(II) onto S. cerevisiae, respectively) and the adjusted R2 were also in reasonable agreement with
each other (Table 4). The adequate precision of 98.2 and 83.8 for
Cd(II) and Ni(II) onto R. eutropha and 154.7 and 419.5 for Cd(II) and

Ni(II) onto S. cerevisiae, respectively, indicates an adequate model

discrimination.
The ANOVA was conducted on for quadratic response surface
model and the results are given in Table 5. The signicance of each
coefcient was determined by F-values and P-values as are listed in
Table 5. The larger the magnitude of the F-values and the smaller
P-values, the more signicant is the corresponding coefcients. Values of Prob > F less than 0.05 also indicate high signicant regression
at 95% condence level as presented in Table 5. In this case, the
quadratic model main, square and interaction effects of initial solution pH, biomass dosage, initial Cd(II) and Ni(II) concentration were
signicant model terms. Predicted residual error sum of squares
(PRESS) indicates a measure of how the models t each point in the
design.
3.2. Effect of process variables
In fact, studies of other researchers have shown that the most
critical parameter in the treatment of heavy metal by biosorbents is
initial solution pH of the aqueous solution [10,20,36,38,39]. Therefore, the range and levels of initial solution pH investigated in the
present study have been obtained based on preliminary experiments. From preliminary, it was found that a 100 mg/l of the Cd(II)
and Ni(II) solution started to precipitate when operating the process
at pH more than 8.5. Therefore, the pH range study was between
2 and 8. Furthermore, our previous study on biosorption of Cd(II)
by S. cerevisiae pretreated by ethanol showed that pretreatment
of the biomass by ethanol could signicantly increase the removal
efciency [40]. Therefore, pretreated S. cerevisiae was used in this
study. An increase in removal efciency of heavy metal as a result
of pretreatment with ethanol could be due to an exposure of active
metal binding sites embedded in the cell wall and thus causing
availability of more active anionic sites. The other reason may be
due to removal of surface impurities.
Fig. 1(ad) shows the simultaneous effect of initial solution
pH and biomass dosage on Cd(II) and Ni(II) removal efciency in
an aqueous solution. It can be seen that the 3-dimentional surface of the RSM showed as a function of two variables at a time,
while the other two variables were held at zero level. The 3dimentional surface plot in Fig. 1(a and b) shows that removal

X1 , pH; X2 , biomass concentration; X3 , initial cadmium concentration; X4 , initial nickel concentration; SD, standard deviation; DF, degree of freedom; CV, coefcient of variation; PRESS, predicted residual error sum of squares.

49.21
2.59
0.77
83.758
0.9960
0.9979

0.9883

36.50
1.88
0.63
98.202
0.9980
R. eutropha

0.9990

0.9937

7.50
1.25
0.36
154.969
0.9988
0.9994

0.9976

10.73
0.81
0.40
154.763
0.9993

YCd = 50.23 + 6.32X1 + 5.98X2 + 0.20X3 + 1.61X4 4.62X12 + 5.08X22 + 6.05X32 8.92X42 0.47X1 X2 0.21X1 X3
1.93X1 X4 + 1.20X2 X3 5.29X2 X4 1.42X3 X4
YNi = 26.67 + 5.67X1 + 3.66X2 + 065X3 + 5.90X4 3.71X12 5.31X22 + 5.51X32 7.48X42 + 2.31X1 X2 + 1.64X1 X3
3.02X1 X4 3.34X2 X3 + 0.23X2 X4 2.13X3 X4
YCd = 46.19 + 10.96X1 1.39X2 + 3.26X3 4.12X4 3.60X12 5.93X22 + 1.84X32 10.98X42 + 0.40X1 X2 + 3.56X1 X3
1.75X1 X4 2.05X2 X3 + 0.52X2 X4 3.24X3 X4
YNi = 42.90 + 0.64X1 + 1.70X2 + 2.08X3 0.77X4 10.56X12 10.45X22 + 4.77X32 3.61X42 + 0.31X1 X2 + 2.56X1 X3 +
4.34X1 X4 1.30X2 X3 0.48X2 X4 + 5.60X3 X4
S. cerevisiae

0.9969
0.9987

R2
Modied equations with signicant terms
Response

Table 4
ANOVA results for response parameters.

Adjusted
R2

Predicted
R2

Adequate
precision

SD

CV

PRESS

M. Fereidouni et al. / Journal of Hazardous Materials 168 (2009) 14371448

1441

efciency of both metals was sensitive to changes in the solution pH. Fig. 1(a) shows the effect of removal efciency of Ni(II)
onto S. cerevisiae dosage and initial solution pH in 3-dimentional
response surface of the RSM when the other two variables were
held at zero level. Interestingly, the Ni(II) removal efciency of S.
cerevisiae was signicantly increased with an increase in initial
solution pH at 37 when biomass dosage was decreased from 4.04
to 0.86 g/l. In this case, a maximum removal efciency of 30.3%
was obtained at initial solution pH of 7 and biomass dosage of
3.32 g/l. Fig. 1(b) shows the effect of S. cerevisiae dosage and initial solution pH in 3-dimentional response surface plot for removal
efciency of Cd(II) when the other two variables are held at zero
level. The Cd(II) removal efciency of S. cerevisiae increased moderately when biomass dosage and initial pH of the solution was
increased. From Fig. 1(b), a maximum Cd(II) removal efciency
of 63% was achieved at initial solution pH of 6.3 and biomass
dosage of 4.4 g/l. Fig. 1(c) shows the changes in the removal efciency of Ni(II) using R. eutropha with varying initial pH solution
and biomass dosage when other two variables were held at zero
level. A maximum removal efciency of Ni(II) at 43% was reached
when initial solution pH and biomass dosage were 5 and 2.58 g/l,
respectively. Fig. 1(d) shows results of the contribution of the interaction between initial pH and biomass dosage for Cd(II) removal
efciency using R. eutropha. It can be seen from the surface that
the removal efciency of Cd(II) onto R. eutropha increased with
an increase in initial solution pH at 37 when biomass dosage
was decreased from 4.04 to 0.86 g/l. A maximum removal efciency of Cd(II) at 62.9% was achieved when initial solution pH and
biomass dosage were 6.7 and 4.04 g/l, respectively. On the other
hand, increases in pH from 2 to 7 caused more Cd(II) removal by R.
eutropha. The most effective and optimum pH for Cd(II) removal was
observed at 57 for both organisms. At pH less than 3, the amount
of Cd(II) and Ni(II) removal efciency onto both dried biomass was
small. This indicates that initial solution pH inuences not only
metal binding sites on cell surface of S. cerevisiae and R. eutropha,
but also the solution chemistry of the heavy metals in water [40].
Optimum pH by Marques et al. [41] for biosorption of three different cations including Cd(II) was obtained between 4.5 and 5.5.
Vasudevan et al. [42] observed that Cd(II) adsorption capacity of
sorbent increased with increasing pH, and was maximum at a pH
of 6.5. Zhai et al. [43] reported pH 6.0 as optimum pH for Cd(II) and
Ni(II) removal. The result showed that maximum removal of Cd(II)
ions was achieved at pH 5.1 and 8.0 with 68.56 and 55.03% for S.
cerevisiae and R. eutropha, respectively.
Fig. 2(ad) shows the simultaneous effect of initial solution pH
and initial concentration of Cd(II) and Ni(II) on Cd(II) and Ni(II)
removal efciency in an aqueous solution. The contour plot in
Fig. 2(a and b) shows the removal efciency of both metals to
be sensitive to initial pH solution and ions concentration. It can
be seen that Cd(II) and Ni(II) removal efciencies of S. cerevisiae
were increased with an increase in initial solution pH at 27 when
concentration of both ions was at the lowest level. At maximum
concentration of both ions (37 mg/l), the Cd(II) and Ni(II) removal
efciencies of S. cerevisiae were 58.4% at initial pH of 5 and 38% at
initial pH of 6.4, respectively, as presented in Fig. 2(a and b). The
contour plot in Fig. 2(c and d) shows results of the contribution
of the interaction between initial pH and initial ions concentration
for Cd(II) and Ni(II) removal efciency using R. eutropha. Fig. 2(c)
shows that a maximum removal efciency of Ni(II) of 43% was
achieved when initial solution pH and Ni(II) concentration were
5 and 25 mg/l. This result is due to the inuence of adsorption
medium pH on the sorption capacity. From Fig. 2(d), it can be seen
from the contour plot that the removal efciency of Cd(II) onto R.
eutropha increased with an increase in initial solution pH at 37
when Cd(II) was increased from 16 to 37 mg/l. In this case, a maximum removal efciency of Cd(II) at 62% was achieved when initial

1442

M. Fereidouni et al. / Journal of Hazardous Materials 168 (2009) 14371448

Table 5
Regresion analysis using 24 factorial central composite design for S. cerevisiae and R. eutropha.
Biomass

Model term

S. cerevisiae

Intercept
X1
X2
X3
X4
X1 X2
X1 X3
X1 X4
X2 X3
X2 X4
X3 X4
X12
X22
X32
X42

R. eutropha

Intercept
X1
X2
X3
X4
X1 X2
X1 X3
X1 X4
X2 X3
X2 X4
X3 X4
X12
X22
X32
X42

Coefcient estimate

Standard error

Cd(II)

Ni(II)

Cd(II)

Ni(II)

Cd(II)

Ni(II)

Cd(II)

Ni(II)

50.2
6.3
6.0
0.2
1.6
0.5
0.2
1.9
1.2
5.3
1.4
4.6
5.1
6.1
8.9

26.7
5.7
3.7
0.7
5.9
2.3
1.6
3.0
2.1
3.3
0.2
3.7
5.3
5.1
7.5

0.14
0.09
0.09
0.09
0.09
0.10
0.10
0.10
0.10
0.10
0.10
0.13
0.13
0.13
0.13

0.13
0.08
0.08
0.08
0.08
0.09
0.09
0.09
0.09
0.09
0.09
0.12
0.12
0.12
0.12

1579.95
5116.05
4581.27
5.01
333.18
22.73
4.59
379.60
147.25
2866.42
206.26
1271.36
1542.00
2187.08
4751.29

1712.24
4860.27
2022.325
63.94344
5271.576
647.3521
324.2327
1104.229
523.711
1350.148
6.466734
971.7111
1992.584
1871.85
3953.771

<0.0001
<0.0001
<0.0001
0.0408
<0.0001
0.0002
0.049
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001

<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
0.0225
<0.0001
<0.0001
<0.0001
<0.0001

46.2
11.0
1.4
3.3
4.1
0.4
3.6
1.8
2.1
0.5
3.2
3.6
5.9
1.8
11.0

42.9
0.6
1.7
2.1
0.8
0.3
2.6
4.3
1.3
0.5
5.6
10.6
10.5
4.8
3.6

0.23
0.14
0.14
0.14
0.14
0.16
0.16
0.16
0.16
0.16
0.16
0.21
0.21
0.21
0.21

0.28
0.17
0.17
0.17
0.17
0.19
0.19
0.19
0.19
0.19
0.19
0.25
0.25
0.25
0.25

1029.15
5976.10
96.71
529.55
845.56
6.21
504.09
121.96
167.70
10.96
416.82
300.51
815.14
78.44
2797.99

510.6983
13.90677
98.57069
147.4737
20.3137
2.559005
177.7734
513.0987
46.04041
6.192064
851.2721
1769.236
1731.798
360.7605
206.4159

<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
0.0249
<0.0001
<0.0001
<0.0001
0.0048
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001

<0.0001
0.002
<0.0001
<0.0001
0.0004
0.1305
<0.0001
<0.0001
<0.0001
0.0251
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001

solution pH and Cd(II) concentration were 7 and 37 mg/l as presented in Fig. 2(d).
The comparison of removal efciency of pretreated dried
biomass in this study and with those obtained in the literature
shows that S. cerevisiae and R. eutropha are effective for Cd(II) and
Ni(II) removal from aqueous solution. Hanif et al. [18] reported the
uptake of Ni(II) increasing with increase in pH from 3.0 to 6.0 and
then decreasing in the range 7.08.0. zer and zer [44] found that
optimal pH value for Pb(II) and Ni(II) uptake is 5.0. Padmavathy et
al. [5] observed that the Ni(II) adsorption capacity of protonated
deactivated yeast increased with increasing pH, and was maximum at pH of 6.75. Panda et al. [45] employed Lathyrus sativus
for removal Cd(II) and Ni(II) from aqueous solution. The range of
optimum pH in their study was reported at 56. For both metals
under acidic conditions little biosorption occurred. At highly acidic
pH, the overall surface charge on the cells became positive and
metal and proton cations competed for binding sites on the cell
walls, which resulted in lower removal efciency [40,4648]. As
pH increases, the amount of removal efciency increases and maximum increases were observed between pH 4 to 6 for Ni(II) and
67 for Cd(II). For Cd(II) onto R. eutropha, a plateau was reached
at around pH 67, while for Ni(II) onto R. eutropha at pH 5. This
is consistent with the assumption that the deprotonated forms of
the functional groups on the cell surface are mainly responsible for
heavy metal binding and electrostatic interaction may be the main
mechanism of biosorption of heavy metals [49]. The trends of pH
dependence showed that highest and lowest of initial Cd(II) concentration with increment in solution pH caused more removal of
Cd(II) onto S. cerevisiae while R. eutropha showed more biosorption
at high Cd(II) concentration. This consequence was due to the fact
that the initial concentration of metal ions provides a driving force
to overcome mass transfer resistance between biosorbents and bulk
aqueous solutions.
The metal removals as a function of biomass loading for the
two non-living biomass were also studied, presented in Fig. 3(ad).

F-value

P-value

The contour plot in Fig. 3(a and b) demonstrated that dosage of S.

cerevisiae has a noticeable effect on Cd(II) and Ni(II) biosorption.
Fig. 3(a) shows that the Ni(II) removal efciency of S. cerevisiae
was signicantly increased with an increase in biomass dosage at
0.857.04 g/l when initial Ni(II) concentration increased from 16
to 37 mg/l. In this case, a maximum removal efciency of 40% was
obtained at initial Ni(II) concentration of 37 and biomass dosage
of 2.48 g/l. The contour plot in Fig. 3(b), Cd(II) shows how removal
efciency increased with an increase in dosage of biomass from
0.85 to 4.04 g/l when Ni(II) concentration was increased from 16
to 37 mg/l. A maximum removal efciency of Cd(II) was obtained
at 68.5% at maximum biomass dosage and Cd(II) concentration as
demonstrated in Fig. 3(b). Fig. 3(c an d) shows a reverse impact
of R. eutropha dosage on the response so that the increase in
R. eutropha dosage brings about an increase in the response up
to a concentration of 2.98 g/l, while at the concentration higher
than 2.98 g/l showed a decreasing effect. This indicates that an
agglomeration of the biomass could be given as reason for the
decrease in the removal efciency at high concentration of biomass
[47,50]. However, a maximum Cd(II) removal efciency of 52%
was observed at 1.95 g/l and Cd(II) concentration of 37 mg/l as
shown in Fig. 3(c), while a maximum Ni(II) removal efciency of
43% was obtained at biomass dosage of 2.57 g/l and Ni(II) concentration of 25 mg/l, as demonstrated in Fig. 3(d). Sekhar et al.
[51] report that high biomass concentrations result in low metal
sorption due to electrostatic interactions between cells, which
protect binding sites from metal occupation. However, higher surface binding sites on the biosorbents could be obtained with high
dosage of biomass in the aqueous solution which may result in
higher removal efciency. The fact is that the removal efciency
of heavy metals could be related to the ratio of the initial heavy
metal ions concentration to the biomass dosage [39]. The removal
efciency of R. eutropha with respect to Cd(II) and Ni(II) was
observed to increase by about 20% compared to that of S. cerevisiae.

M. Fereidouni et al. / Journal of Hazardous Materials 168 (2009) 14371448

1443

Fig. 1. The combined effects of initial biomass dosage and solution pH on removal efciency when other variables held at zero level; (a and b) S. cerevisiae and (c and d) R.
eutropha.

3.3. Optimization using the desirability functions

Myers and Montgomery [35] describe a multiple response
method called desirability. The method makes use of an objective
function, D, called the desirability function. It reects the desirable
ranges for each response (di ). The desirable ranges are from zero
to one (from least to most desirable, respectively). The simultaneous objective function is a geometric mean of all transformed
responses:
D = (d1 d2 . . . dn )

1/n

 n 1/n

di

(4)

i=1

where n is the number of responses in the measure. If any of the

responses or factors fall outside their desirability range, the overall
function becomes zero.
In the numerical optimization, we choose the desired goal for
each factor and response from the menu. The possible goals are:
maximize, minimize, target, within range, none (for responses only)
and set to an exact value (factors only). A minimum and a maximum
level must be provided for each parameter included. A weight can
be assigned for each goal to adjust the shape of its particular desirability function. The goals are combined into an overall desirability
function. Desirability is an objective function that ranges from zero

outside of the limits to one at the goal. The program seeks to maximize this function. The goal seeking begins at a random starting
point and proceeds up the steepest slope to a maximum. There may
be two or more maximums because of curvature in the response
surfaces and their combination into an overall desirability function.
For nding the best local maximum, changes improve by starting from several points in the design space. A multiple response
method was applied for optimizing any combination of four goals,
namely the initial solution pH, biomass dosage, initial Cd(II) and
Ni(II) concentrations, and removal of each metal ion. The numerical optimization found points out how to maximize the desirability
function. The optima of removal efciency for Cd(II) and Ni(II) onto
S. cerevisiae and R. eutropha are shown in Table 6. The obtained values of desirability (above 0.861) showed that the estimated function
may represent the experimental model under desired conditions.
The results showed that pH was an important parameter affecting
the biosorption of heavy metals. From a set of solutions generated by the Design-Expert software, optima of removal efciency
of 67.9% for Cd(II) and 42.3% for Ni(II) onto S. cerevisiae and 51.8%
for Cd(II) and 50.4% for Ni(II) onto R. eutropha were achieved at the
optimum values of the process variables: pH, 7.1; biomass dosage,
4.04 g/l; Cd(II) concentration, 37 mg/l; Ni(II) concentration, 16 mg/l
and pH, 5.0; biomass dosage, 2.32 g/l; Cd(II) concentration, 37 mg/l;
Ni(II) concentration, 28 mg/l, respectively.

1444

M. Fereidouni et al. / Journal of Hazardous Materials 168 (2009) 14371448

Fig. 2. The combined effects of initial ions concentration and solution pH on removal efciency when other variables held at zero level; (a and b) S. cerevisiae and (c and d)
R. eutropha.

In order to verify the model developed, four more experiments

were conducted for Cd(II) and Ni(II) removal efciency onto S.
cerevisiae and R. eutropha as presented in Table 6 using numerical optimization of the RSM. The experiments were conducted
under these conditions and comparison between the experimental
results with the predicted results from the model was made. The
results demonstrated that the model prediction for both responses
in Table 4 agreed reasonably well with the experimental data.
3.4. Characteristics of the biosorbent and mechanism of Cd(II)
and Ni(II) biosorption
Previous studies showed that ethanol treated S. cerevisiae had
increased Cd(II) biosorption capacity and the Cd(II) removal was
two times greater than original S. cerevisiae [1,28]. In order to

evaluate the textual structure of cell surface, SEM micrographs of

non-living biomass of S. cerevisiae and R. eutropha before and after
treatment are shown in Fig. 4(ad). For the treated biomass of S.
cerevisiae (Fig. 4(b)) and R. eutropha (Fig. 4(d)), the cell surface morphology is quite clean surface and generates more accessible space
within -glucan-chitin skeleton with new micro-porous structure
which were absent on the irregular surface of both biomass before
treatment. There were also many thin sheets on the cell surface and
some rudimentary pores were present due to the release of the cell
wall materials containing COOH, phosphates groups, amorphous
polysaccharides and other impurities [52,53]. The irregular surface
morphology of S. cerevisiae and R. eutropha exhibited microstructure porosity for pretreated biomass, and this may be attributed
to the fact that the microstructure plays a role in Cd(II) and Ni(II)
biosorption [54]. Actually chemical treatment of the cell walls using

M. Fereidouni et al. / Journal of Hazardous Materials 168 (2009) 14371448

1445

Fig. 3. The combined effects of initial biomass dosage and Ni(II) and Cd(II) ions concentration on removal efciency when other variables held at zero level; (a and b) S.
cerevisiae and (c and d) R. eutropha.

esterication of ethanol with carboxyl groups ( COOH) has not produced any pore structure on the biosorbent surface but increase
the specic surface area for reaction. Microporous active sites distinguished on the surface morphology of non-viable S. cerevisiae
may precede faster action of biosorption, hence allowing more
heavy metal ions chelation at the surface. These S. cerevisiae par-

ticles with clean surface and high porosity may have application
as biosorbent for heavy metal removal from wastewater efuents
[28].
The FT-IR Spectroscopy is an important analytical technique
which detects the vibration characteristics of chemical functional
groups in a molecule. Upon interaction of an infrared light with

Table 6
Desirability option for Cd(II) and Ni(II) removal by S. cerevisiae and R. eutropha.
Microorganism

pH

Biomass dosage, g/l

Cd(II) concentration,
mg/l

Ni(II) concentration,
mg/l

Cd(II) removal, %

Ni(II) removal, %

Desirability

Experimental

Predicted

Experimental

Predicted

S. cerevisiae

7.1
7.1

4.04
3.87

37
37

16
26

66.5
67.3

67.9
68.6

43.4
35.1

42.3
35.3

0.929
0.861

R. eutropha

5.0
5.0

2.32
2.01

37
37

28
30

52.7
50.8

51.8
51.2

50.1
51.8

50.4
50.2

0.914
0.905

1446

M. Fereidouni et al. / Journal of Hazardous Materials 168 (2009) 14371448

Fig. 4. Micrographs of scanning electron microscope (SEM) of (a) surface of S. cerevisiae before treatment, (b) surface of S. cerevisiae after treatment, (c) surface of R. eutropha
before treatment, and (d) surface of R. eutropha after treatment.

matter, chemical bonds will stretch, contract and bend. As a result,

chemical functional groups tend to absorb infrared radiation in
a specic wavelength range regardless of the structure of the
rest of the molecule [55]. Fig. 5 displays a number of absorption
peaks, indicating the complex nature of the examined biomass of
S. cerevisiae and R. eutropha. The broad absorption peak around
32303400 cm1 and 32003520 cm1 are indicative of the existence of the OH groups and the NH groups on S. cerevisiae
and R. eutropha, respectively [56]. Treated S. cerevisiae biomass of
spectrum (b) in comparison with untreated biomass of spectrum
(a) showed more clear peaks as depicted in Fig. 5. Two peaks
were added in the bands 14501510 cm1 and 13801406 cm1
after treatment in spectrum (b), which was taken as a sign of
biomass enrichment. There was a clear disappearance of the
bands 540 cm1 and 1406 cm1 after metal loaded on S. cerevisiae

in spectrum (c). There was also shifting of the bands 822 cm1
(J), 1252 cm1 (H), 1464 cm1 (G), 2879 (C) and 2931 cm1 (B).
The metal loaded of the R. eutropha biomass in spectrum (f) of
Fig. 5 shows that clear disappearance of the bands 700 cm1 and
1464 cm1 and shifting of the bands 550 cm1 (K ), 1103 cm1 (I ),
1258 cm1 (H ), 1562 cm1 (E ), 1688 cm1 (D ) and 2962 cm1 (B )
were observed. These observations could indicate the involvement
of these functional groups in the biosorption process. The FT-IR
wavelengths of each peak and the corresponding functional groups
are depicted in Table 7, which shows that several functional groups
on the surface of the S. cerevisiae and R. eutropha are responsible for
binding of Cd(II) and Ni(II) ions. The FT-IR analysis demonstrates
active functional groups of O H stretching (36003200 cm1 ),
N O stretchalkane C H stretching (28502956 cm1 ),
ing (15001600 cm1 ), amine bending (14501550 cm1 ),

Table 7
FT-IR peaks for S. cerevisiae and R. eutropha.
Biomass

Label

Treated
biomass

Metal loaded
biomass

Change in peak behavior

after metal loaded

IR peak range (cm1 )

Functional group reported corresponding to

the observed peak behavior [55,56]

S. cerevisiae

A
B
C
D
E
F
G
H
I
J

3327.1
2931.8
2879.7
1659.1
1551.2
1464.4
1406.5
1252.3
1082.6
822.3

2966.5
2862.3
1659.1
1551.1
1491.4
Not present
1267.7
1082.6
708.5

Shift
Shift
Reduction
Reduction
Shift
Omit
Shift
Reduction
Shift

36003200
28502956
28502956
17301625
16001500
15501450
14001300
12601000
13001000
7501750

O H stretching vibration
Alkanes CH stretching vibration
Alkanes CH stretching vibration
C O stretching vibration
N O stretching vibration
N H bending vibration
N O stretching vibration
CO stretching vibration, carboxylic acid
C O stretching vibration
Phenyl ring substitution band

R. eutropha

A
B
C
D
E
F
G
H
I
J
K
L

3330.9
2962.6
2875.8
1688.1
1657.2
1562.7
1464.4
1258. 1
1103.8
Not present
700.8
552.3

Not present
2933.6
2875.8
1742.0
1657.2
1551.2
Not present
1385.1
1071.0
841.6
Not present
610.2

Omit
Shift
No change
Shift
No change
Shift
Omit
Shift
Shift
Additional peak
Omit
Shift

32003600
28502956
28502956
17301625

10001260

7501750

O H stretching vibration
Alkanes CH stretching vibration
Alkanes CH stretching vibration
C O stretching vibration

C O stretching vibration, carboxylic acid

Phenyl ring substitution band

M. Fereidouni et al. / Journal of Hazardous Materials 168 (2009) 14371448

1447

onto R. eutropha (52%) was observed at 1.95 g/l and Cd(II) concentration of 37 mg/l, while a maximum Ni(II) removal efciency onto R.
eutropha (43%) was obtained at Ni(II) concentration of 37 mg/l when
initial solution pH and biomass dosage were 5 and 2.58 g/l, respectively. When calculating optimum removal efciency of Cd(II) and
Ni(II) onto S. cerevisiae the points were found to be 43.4 and 65.5%,
respectively, at initial solution pH of 7.1, biomass dosage of 4.07 g/l,
initial Ni(II) concentration of 16 mg/l and initial Cd(II) concentration of 37 mg/l. The points giving optimum removal efciency of
Cd(II) and Ni(II) onto R. eutropha were found to be 52.7 and 50.1%,
respectively, at initial solution pH of 5.0, biomass dosage of 2.32 g/l,
initial Ni(II) concentration of 28 mg/l and initial Cd(II) concentration of 37 mg/l. It was observed that model predictions of Cd(II) and
Ni(II) removal efciencies are in good agreement with experimental
observations.
Acknowledgements
The present research was made possible through a university
grant, sponsored by Ministry of Science, Iran. The authors wish to
thank Mrs. Haghdoust (Technical assistant of Environmental Laboratory) for her assistance, Ellen Vuosalo Tavakoli for English editing,
Tarbiat Modares University and Ministry of Science for their nancial support.
References

Fig. 5. FT-IR spectra of (a) untreated biomass of S. cerevisiae, (b) ethanol pretreated
biomass of S. cerevisiae, (c) Ni(II) and Cd(II) loaded S. cerevisiae biomass, (d) untreated
biomass of R. eutropha, (e) ethanol pretreated biomass of R. eutropha, and (f) Ni(II)
and Cd(II) loaded R. eutropha biomass.

N O
stretching
(13001400 cm1 ),
C O
stretching
1
(16251730 cm ), C O stretching (10001300 cm1 ), carboxylic
acid (10001260 cm1 ), vibrations, and phenyl ring substitution
band (7501750 cm1 ), as shown in Table 7. The above evidence
suggests that the mechanism of the Cd(II) and Ni(II) biosorption
involves hydroxyl ( O H), amine ( NH) and carboxylate ions
( COO ) groups of the polysaccharides on the peptidoglycan layer,
and for the interaction of the metal-biomass at the cell surface and
metal ions can be assumed to be the same.
4. Conclusion
The use of an experimental design allowed the rapid screening of a large experimental domain for optimization of the Cd(II)
and Ni(II) removal efciency of S. cerevisiae and R. eutropha. The
model adequacy, tested through lack-of-t (LOF), P-values and Fvalues, was veried successfully by the validation of experimental
data. A maximum removal efciency of Cd(II) onto S. cerevisiae was
obtained at 68.5% at biomass dosage of 4.04 g/l and Cd(II) concentration of 37 mg/l. However, a maximum Cd(II) removal efciency

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