Author’s Accepted Manuscript

Biosorption of low concentration levels of Lead (II)

and Cadmium (II) from aqueous solution by
Saccharomyces cerevisiae: Response surface
methodology

Mohammad Rasoul Hadiani, Kianoush Khosravi

Darani, Nahid Rahimifard, Habibollah Younesi
www.elsevier.com/locate/bab

PII: S1878-8181(18)30283-4
DOI: https://doi.org/10.1016/j.bcab.2018.05.001
Reference: BCAB753
To appear in: Biocatalysis and Agricultural Biotechnology
Received date: 11 April 2018
Revised date: 1 May 2018
Accepted date: 3 May 2018
Cite this article as: Mohammad Rasoul Hadiani, Kianoush Khosravi Darani,
Nahid Rahimifard and Habibollah Younesi, Biosorption of low concentration
levels of Lead (II) and Cadmium (II) from aqueous solution by Saccharomyces
cerevisiae: Response surface methodology, Biocatalysis and Agricultural
Biotechnology, https://doi.org/10.1016/j.bcab.2018.05.001
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Biosorption of low concentration levels of Lead (II) and
Cadmium (II) from aqueous solution by Saccharomyces
cerevisiae: Response surface methodology

Mohammad Rasoul Hadiania,b, Kianoush Khosravi Darania*, Nahid Rahimifardc,

Habibollah Younesid

a
Research Department of Food Technology, National Nutrition and Food
Technology Research Institute, Faculty of Nutrition Sciences and Food
Technology, Shahid Beheshti University of Medical Sciences, P.O.Box: 19395-
4741, Tehran, Iran
b
Food and Drug Reference Laboratories Center, Food and Drug Organization,
Tehran, Iran,
c
Food and Drug Control Laboratories, Ministry of Health and Medical
Education, Tehran, Iran,
d
Department of Environmental Science, Faculty of Natural Resources, Tarbiat
Modares University, Noor, Iran,
hadianmor@sbmu.ac.ir

kiankh@yahoo.com

k.khosravi@sbmu.ac.ir

nahidrahimifard@gmail.com

hunesi@modares.ac.ir

Address for Correspondence: Kianoush Khosravi-Darani (Solution pHD) Full

Prof. of Food Biotechnology Food Technology Research Department, National
Nutrition and Food Technology Research Institute, Shahid Beheshti University of
Medical Sciences, P. O. Box: 19395-4741, Tehran, Iran. Tel: +98-21-22376473;
Fax: +98-21-22376473

1
Abstract

The present study refers to application of Saccharomyces cerevisiae for

bioremoval of very low amounts of heavy metals from aqueous media. The

impact of process variables on biosorption of lead (II) and cadmium (II) by S.

cerevisiae was first evaluated with Taguchi screening design. After determining

of significant variables, optimization of biosorption process was performed by 3

independent parameters (pH, biomass, initial ion concentration) using a central

composite design. So, twenty test runs were done and the experimental data fitted

to the second-order polynomial models. The analysis of variance of the quadratic

models showed that the models are highly significant. The adjusted conditions in

the best set was 5, 52.5 µg/l and 32.5 ×107 CFU for pH, heavy metal

concentration and biomass, respectively. Behavior of biosorption system in a

batch process was analyzed by 3D plots which represented dual simultaneous

interaction effects of variables on metal biosorption yield. The model explained

that heavy metal biosorption in aqueous solution is affected by all the three

factors studied. An optimum lead (II) and cadmium (II) biosorption yield of

70.3% and 76.2% were found at initial ion concentration of 65.0 and 62.6 µg/l,

and S. cerevisiae of 15.0 and 15.2 × 107 CFU, respectively. The results suggest

that S. cerevisiae, as a natural, low-cost and abundant sorbent, has the potential to

be used in biosorption of very low concentrations of lead and cadmium, which is

2
useful to reduce the contaminations of drinking water and foodstuff with green

technologies.

Keywords: biosorption; Saccharomyces cerevisiae; heavy metal; DOE; RSM

1. Introduction

The adverse effects of heavy metals on human health have been studied by

the world developed countries in various organizations and is well documented

(Rajaganapathy et al. 2011). Although heavy metals exist naturally in our

environment, industrial activities can increase their amount in air as well as

human and animals food chains (Yadanaparthi et al., 2009). The toxicity of these

3
elements occurs even in low levels of about 1.0-10 mg/l, however, some of heavy

metals such as lead (Pb) and cadmium (Cd) are very toxic even in lower

concentrations of 1-100 µg/l (Alkorta et al., 2004). Pb and Cd are considered to

be the most toxic pollutants in the environment (Sari and Tuzen, 2008; Barbier et

al., 2000). Pb is applied in various industries such as pigments, paints, batteries,

cables, alloys, glass and plastic industries (Selatnia et al., 2004). Improper

discharge of these industries may cause pollution of the aquatic environment by

heavy metals. On the other hand, the various exposure to Cd may cause renal

dysfunction, hypertension, teratogenic effects, lung damage and hepatic injury

(Sari and Tuzen, 2008; Hajialigol et al., 2006).

For removing heavy metals from aqueous solutions, some of conventional

methods have been studied in detail, such as ion exchange, membrane

technologies, chemical precipitation, adsorption on activated carbon,

electrochemical treatment, etc (Bailey et al., 1999). Chemical precipitation and

electrochemical treatment are -ineffective methods especially when heavy metal

concentration in aqueous solution are less than 100 mg/l, while large amount of

sludge remains is a huge challenge (Wang and Chen, 2006). Other solutions

including activated carbon adsorption, ion exchange and membrane technologies

are expensive procedures, especially for remediation of large amount of water

and wastewater with low level contaminants, and thus are not suitable for use on

a large scale. Along with these methods, various kinds of potentially low-cost

4
sorbents for the removal of metals has been studied (Bailey et al., 1999). Based

on definition, a low-cost sorbent is one which exists in nature plenty, or is a waste

material or by-product of another industry (Low et al., 2000).

Biosorbents are one of the potentially low-cost sorbents with biological origin

such as plants, algae, fungi, yeasts and bacteria which carry out the biosorption

process for pollutants around them with high speed and efficiency. Research has

shown that biosorption methods have great potential to compete with the

conventional technologies for the remediation of heavy metals, and so can be a

suitable candidate for remediation of water sources in high volume and low levels

of heavy metals (Veglio and Beolchini, 1997; Massoud et al., 2017). Waste

biomass resulting from some foods and fermentation industries have shown good

performance as biosorbents for removal of heavy metals (Amirnia and Ray,

2015; Cain et al., 2008; Syed and Chinthala, ; Wang and Chen, 2006). One of

these biosorbents, is Saccharomyces cerevisiae, which is widely used in food and

beverage industries, while it is also a kind of solid waste. Although this

microorganism is an intermediate biosorbent compared to other biosorbents used

for heavy metal removal, it has still a lot of attention due to its unique

characteristics as a worthy biomaterial (Wang and Chen, 2006). Low health

safety concerns , ease of use, easily cultivation at large scale and consequently its

commercialization, are among the benefits of working with S. cerevisiae

(Jianlong and Can, 2006; Soares and Soares, 2012; Vieira and Volesky, 2000).

5
Moreover, its biomass can be easily obtained from food and beverages industries,

and grown on affordable growth media with simple fermentation techniques. On

the other hand, S. cerevisiae is an ideal model organism for research on the

kinetics of bioremediation, especially on molecular-level reactions of metal-

microbe (Wang and Chen, 2006).

The absorption of elemental ions on the functional groups of microorganism cell

wall depends not only on the nature of the microorganism but also on the

chemical properties of absorption media (Ozer and Ozer, 2003). Various factors

can be effective on biosorption of heavy metals such as: biomass dosage,

elemental concentration, solution pH, temperature, viability and presence of other

organic/inorganic compounds (Parvathi and Nagendran, 2007; Mapolelo and

Torto, 2004; Ozer and Ozer, 2003). For accurate optimization studies, it is

necessary to examine the various independent variables simultaneously. But,

when the number of variables increases, the study of all their combined effects

will be very difficult due to time and cost-consuming process, so this problem

shows need to use the fractional factorial design method (Jahadi et al., 2012;

Khosravi-Darani et al., 2008).

The purpose of the present study is evaluating potential of S. cerevisiae for

decontamination of low concentrations of Pb (II) and Cd (II) ions (at ppb levels)

in aqueous solutions. For optimization studies, the impact of some independent

variables proposed in other studies (including biomass dosage, metal ion

6
concentration, solution pH, temperature, contact time, viability of biomass and

shaking rate) was first studied by Taguchi screening design. Then, interactions of

significant variables (biomass dosage, metal ion concentration and solution pH)

were studied at five levels by means of central composite design (CCD) under

response surface methodology in a second-order mathematical model. Since there

is no published data about capability of S. cerevisiae for decontamination of low

levels of lead and cadmium in human health scale, the findings of present study

could open a new step in use of green technology for heavy metal remediation in

food industry and drinking water treatment.

2. Experimental

2.1. Biomass

S. cerevisiae PTCC 5010 was purchased from Research and Technology

Department of Ministry of Sciences (Persian Type Culture collection) in the form

of freeze-dried, and then cultured in a special and sterilized media. The liquid

medium used for the yeast culture, contained (grams per liter): yeast extract, 1;

glucose, 30; KH2PO4, 1; (NH4)2SO4, 9; K2HPO4, 0.2; MgSO4, 2.5. The medium

was then sterilized by autoclaving at a temperature of 121 °C and pressure of 1.4

atm for 20 min. After cooling, the yeast cells were cultured for 16 h (at end of

7
exponential phase) at 27 ºC and 80 rpm and then stored in refrigerator until

biosorption studies.

2.2. Preparation and colony count of biomass seed culture

For each treatment combination, seed culture of S. cerevisiae was daily

activated by inoculation of 5% v/v from master culture (2.1) in the same medium

and shaking for 16 h at 27 ºC and 80 rpm. Then, activated seed culture was

counted by serial dilution method. For this, 1 ml of the seed culture, was diluted

at a ratio of 1:10, and this was repeated 10 times serially. Then, from each

dilution, 1 ml was poured into a Nutrient Agar medium by pour plate method in

duplicate. The plates incubated in 30 ºC for 3 days (Sieuwerts et al., 2008). After

that, counting of yeast colonies was done and showed the mean of 5 × 107

CFU/ml.

2.3. Reagents

8
 For the preparation of yeast culture medium, analytical reagent-grade

chemicals from Merck (Darmstadt, Germany) were used. Also, deionized water

with resistivity of 18.0 MΩ cm (Elga Labwater, Wycombe, Bucks, UK) was used

in all required parts of the test. Standard solutions of Cd (II) and Pb (II) (1000

mg/l in 0.1 M HNO3) were provided by Panreac (Panreac Quimica SA,

Barcelona, Spain). Secondary and working standard solutions were freshly

prepared in 0.1 M HNO3. In order to prevent the transmission of metal

contamination, all glass containers were soaked in 10% (v/v) HNO3 overnight

and rinsed with deionized water prior to analysis. Then, they were sterilized by

autoclaving at a temperature of 121 °C and pressure of 1.4 atm for 20 min for

prevent of microbial contamination.

2.4. Instrumentation

The concentrations of residual Pb and Cd in the biosorption media were

determined by using a Varian Spectra AA 240FS atomic absorption spectrometer

(Varian Australia, Pty Ltd, Mulgrave, Victoria, Australia), by attaching GTA-120

graphite furnace atomizer and deuterium lamp as a background corrector. Pb and

Cd were measured in wavelengths 283.3 and 228.8 nm, respectively. All

9
measurements were performed using pyrolytic-coated partitioned graphite tubes.

Responses were obtained based on peak height and operating parameters were set

as recommended by the manufacturer. All standards and biosorption solutions

were injected three times

2.5. Analytical quality assurance

In order to ensure even the results obtained from yeast biosorption, method

validation including limit of detection (LOD), limit of quantification (LOQ),

linearity range and accuracy (recovery level) was performed before instrumental

analysis. Based on existing definition, LOD is known as the concentration of

analyte corresponding to three times the standard deviation of 10 reagent blanks

or as 3 σ s–1. In the same way, LOQ is the concentration of analyte corresponding

to 10 times the standard deviation of 10 reagent blanks or as 10 σ s–1, whereas σ

is the standard deviation of the blank signal (n = 20) and s is related to slope of

the calibration curve [Miller mand Miller, 2000/24]. In the present study, LOD of

Pb (II) and Cd (II) ions was obtained 1 and 0.2 ng/ml and LOQ, 3 and 0.6 ng/ml,

respectively. For determine method accuracy, recovery tests were done by

spiking increasing amounts of each metal to culture medium solution, while the

amount of Pb and Cd had been already measured in the culture medium itself.

10
This was performed at three spiking levels within 3 days. The results are reported

in Table 1. Mean recoveries for Pb and Cd, were 95.8% and 95%, respectively.

Table 1. Recovery average of Pb and Cd on spiked solutions within 3 days.

Recovery\ Element Pb Cd
Spike Level (ng/ml) 10 25 50 10 25 50
Mean Obtained Result (ng/ml) 9.4 24.5 47.8 9.1 24.2 48.5
Mean Recovery (%) 94.0 98.0 95.6 91.0 96.8 97.0

2.6. Experimentation and optimization of biosorption

Because according to pre-experience and literature review, different factors

may affect the biosorption yield, it is necessary to examine the interaction effects

of the variables in a biosorption study. The usual approach in experimental

planning is to start with a screening design in which all possible factors are taken

into account and the significant factors are chosen, and then the study continues

with an experimental optimization design, such as central composite design

(Hanrahan and Lu, 2006; Coruh et al., 2012). So, in the present study, design of

experiments (DOE) was applied for these two aims.

11
2.6.1. Taguchi design

Based on the factors that have been studied in preliminary trials and literature

reports for biosorption of heavy metal by S. cerevisiae (El-sayed, 2013; Farhan

and Khadom, 2015; Galedar and Younesi, 2013; Ghorbani et al., 2008), seven

independent variables involved in Pb and Cd biosorption by this microorganism

were considered in the present study; including: S. cerevisiae biomass dosage,

solution pH, temperature, contact time, heavy metal concentration, shaking rate

and biomass viability. Range finding was also based on pre-experience and

literature review. The variables and their experimental levels are shown in Table

2. However, metal concentration was selected in very low levels arbitrarily;

because the purpose of this study is to investigate the ability of S. cerevisiae to

absorb low concentration levels of Pb (II) and Cd (II) ions found in drinking

water and foodstuffs, while no citation information has ever been available. So,

an L-8 orthogonal array was designed by Taguchi for determination of optimum

experimental conditions with the goal of maximum removal of Pb and Cd. The

designed experimental runs are shown in Table 3.

Table 2. Investigated variables and their levels.

12
 Low High
Varia
Description level level
ble
(L1) (L2)

S. cerevisiae biomass dosage ( × 107

A 15 50
CFU)

B solution pH 4 6

C Temperature (°C) 25 35

D Contact time (hr) 24 48

E Metal concentration (µg/l) 25 80

F Shaking rate (rpm) 70 100

Non-
G Biomass viability Live
living

Table 3. Test runs designed by Taguchi.

Biomass
solution Contact Metal Shaking
R dosage Temperature Biomass
pH time concentration rate (rpm)
un (×107 CFU) (°C) (C) viability (G)
(B) (hr) (D) (µg/l) (E) (F)
(A)
1 15 6 35 48 80 70 Live

2 50 6 25 24 80 100 Live

3 15 4 25 48 80 100 Non-living

4 50 4 35 24 80 70 Non-living

5 15 4 25 24 25 70 Live

6 50 6 25 48 25 70 Non-living

13
7 15 6 35 24 25 100 Non-living

8 50 4 35 48 25 100 Live

To conduct batch biosorption studies, 100 ml sterile growth media (section 2.1)

supplemented with Pb (II)/Cd (II) ions in concentration level of 25/50 µg/l were

prepared in 250 mL Erlenmeyer flasks. This media was then inoculated with

activated seed culture (3 or 10 ml in 100 ml growth media equal to 15 × 107 and

50 × 107 CFU) as described in section 2.2. The solution pH (4 and 6) was

adjusted by using 0.1 N NaOH and 0.1 N HCl and then, each flask was incubated

at temperature 25°C and 35°C on the shaker for 24 and 48 h. Also, to carry out

the study on non-living cells, they were killed in Ben-Marie 80°C for 15 min. At

the end of biosorption time, yeast cells were centrifuged at 2000 × g for 15 min.

The supernatant solution was then separated to measure the residual metals (Pb &

Cd) using atomic absorption spectrometer. All the experiments were done in

triplicates. The Pb (II)/Cd (II) ions removed by S. cerevisiae are calculated by the

Eq (1):

Eq. 1

while R is the % the metal uptake of Pb (II)/Cd (II) ions adsorbed by yeast

biomass, C0 is the initial concentration of metal ions in the absorption solution in

µg/l, and Ce is the concentration of metal ions in the absorption solution after

14
 biosorption, in µg/l (Goksungur et al., 2005). The data was processed using the

Minitab (version 18.1) statistical software and four variables were identified as

significant variables with ranking 1 to 4, including: solution pH, S. cerevisiae

biomass dosage, heavy metal concentration and biomass viability (Table 4). The

optimization trials was then conducted by fractional factorial design.

Table 4. ANOVA for means and ranking of variables main effect by Minitab.

Heavy

concentration
Temperature
S. cerevisiae

solution pH

Biomass

viability
Shaking
biomass

Contact
dosage

Metal
metal Level
time

rate
removal

1 57.30 26.50 47.17 46.67 40.45 48.40 52.17

2 38.92 69.72 49.05 49.55 55.77 47.83 44.05

Delta 18.38 43.22 1.88 2.88 15.32 0.57 8.12

Pb
Rank 2 1 6 5 3 7 4

Seq SS 675.28 3736.8 7.03 16.53 469.71 0.66 132.03

p-value 0.02 0.008 0.189 0.126 0.024 0.955 0.045

1 55.80 27.60 48.03 47.60 36.58 47.17 51.35

2 40.08 68.28 47.85 48.28 59.30 48.70 44.53

Delta 15.73 40.68 0.17 0.68 22.72 1.53 6.83

Cd
Rank 3 1 7 6 2 5 4

Seq SS 518.42 4032.0 0.08 0.18 684.5 7.22 13.52

p-value 0.075 0.027 0.933 0.900 0.065 0.598 0.402

15
2.6.2. Response surface methodology design (RSM)

The findings from Taguchi design (2.6.1) indicated that three numerical variables

(i.e. solution pH, S. cerevisiae biomass dosage and heavy metal concentration)

and a nominal variable (biomass viability), were significant in biosorption rate of

Pb and Cd ions (Table 4). Accordingly, for more accurate prediction on optimum

conditions of Pb and Cd biosorption by S. cerevisiae and at the same time to

minimize the number of tests, central composite design (CCD) under response

surface methodology was designed. The RSM consists of a group of experimental

methods designed to the evaluation of correlation between a cluster of controlled

experimental factors and obtained responses according to one or more selected

criteria (Kunamneni and Singh, 2005; Preetha and Viruthagiri, 2007). The

experimental design was constructed using Design Expert software (version,

7.1.5, STAT-EASE Inc., Minneapolis, USA). Table 5, shows experimental ranges

of the significant variables for CCD trials. But, because biomass viability is a

nominal variable that showed a greater main effect in its live status on Pb and Cd

removal, so all experiments were performed on live yeast. On the other hand, due

16
 to the fact that the minimum biosorption time was not a priority of the present

study, and also, the shaking rate should be adjusted so that the experimental

solution is neither sediment nor sprinkled around, so contact time, shaking rate

and temperature, were held constant at 24 hours, 70 rpm and 25 °C, respectively.

According to this, the complete design consisted of three factor (S. cerevisiae

biomass dosage, solution pH, initial Pb/Cd ion concentration) and each at five-

levels (-α, -1, 0, +1, + α) (Table 5). Twenty CCD experiments for each metal ions

with their responses are shown in Table 6.

Table 5. The levels of experimental variables for the central composite experimental design.

Range and level

Independent process variable
-α (-1.68179) -1 0 +1 +α (+1.68179)

solution pH (X1) 3.3 4 5 6 6.7

Initial Pb/Cd ion 6.3 25 52.5 80 98.7

concentration, µg/l (X2)
S. cerevisiae biomass dosage, 3.1 15 32.5 50 62
(× 107 CFU) (X3)

17
Table 6. Experimental design showing real and coded process variables and observed responses
for Pb (II) and Cd (II) biosorption by S. cerevisiae.

Real (coded) valuesa) Biosorption rate, %

Pb (II) Cd (II)
Std
no. Observed Predicted Observed Predicted
X1 X2 X3
value value value value

1 4 (-1) 25 (-1) 15 (-1) 31.5 29.3 34.8 32.5

2 6 (+1) 25 (-1) 15 (-1) 31.0 34.4 36.5 38.0

3 4 (-1) 80 (+1) 15 (-1) 48.5 53.6 50.7 57.1

4 6 (+1) 80 (+1) 15 (-1) 56.4 58.8 60.3 62.5

5 4 (-1) 25 (-1) 50 (+1) 3.5 0.6 6.4 2.5

6 6 (+1) 25 (-1) 50 (+1) 7.0 5.8 9.7 8.1

7 4 (-1) 80 (+1) 50 (+1) 4.1 5.2 7.3 8.2

8 6 (+1) 80 (+1) 50 (+1) 9.5 10.4 10.8 13.7

9 3.3 (-α) 52.5 (0) 32.5 (0) 3.1 3.2 5.7 5.7

10 6.7 (+α) 52.5 (0) 32.5 (0) 14.5 12.0 17.3 15.1

11 5 (0) 6.3 (-α) 32.5 (0) 0.5 3.0 1.7 6.2

12 5 (0) 98.7 (+α) 32.5 (0) 32.4 27.5 38.4 31.7

13 5 (0) 52.5 (0) 3.1 (-α) 91.6 87.2 95.3 91.4

14 5 (0) 52.5 (0) 61.9 (+α) 20.3 22.3 23.4 25.1

15 5 (0) 52.5 (0) 32.5 (0) 44.2 43.7 52.5 51.4

18
16 5 (0) 52.5 (0) 32.5 (0) 43.5 43.7 51.7 51.4

17 5 (0) 52.5 (0) 32.5 (0) 43.2 43.7 51.2 51.3

18 5 (0) 52.5 (0) 32.5 (0) 44.8 43.7 49.7 51.3

19 5 (0) 52.5 (0) 32.5 (0) 42.6 43.7 50.4 51.3

20 5 (0) 52.5 (0) 32.5 (0) 43.6 43.7 52.3 51.4

a) X1: solution pH; X2: initial heavy metal conc.; X3: S. cerevisiae biomass dosage

3. Results and discussion

3.1. RSM approach to achieve maximal the metal uptake of Pb (II) and Cd (II)

ions

The results of Pb/Cd ion biosorption by S. cerevisiae have been represented in

Table 6. These findings showed that S. cerevisiae has high affinity for each two

heavy metal in low level concentrations. The maximum ion biosorption of 91.6%

and 95.3% was obtained for Pb (II) and Cd (II) ions in run 13, respectively. The

application of RSM after analysis of variance (ANOVA) showed the level of Pb

(II) and Cd (II) ion biosorption as the function of three variables, as given in

Table. 7. The values of R-squared for Pb and Cd (R2 = 0.9877 and 0.9845,

respectively) and the adjusted R-squared (adj. R2 = 0.9805 and 0.9755,

respectively) were very close to 1, which indicated high correlation between the

19
observed and predicted values (Grag et al., 2008). The predicted R-squared (pred.

R2) of 0.9428 and 0.9273 for Pb (II) and Cd (II), respectively, are in reasonable

agreement with R2 and adjusted R2.

Table 7. ANOVA results for Pb (II) and Cd (II) biosorption parameters.

Sum of df Mean F p-value

Sourcea)
Squares Square Value

Pbb) biosorption
Model 10075.95 7 1439.42 137.36 < 0.0001
X1 92.14 1 92.14 8.79 0.0118
X2 719.83 1 719.83 68.69 < 0.0001
X3 5072.94 1 5072.94 484.09 < 0.0001
X2 X3 193.06 1 193.06 18.42 0.0010
X12 2349.86 1 2349.86 224.24 < 0.0001
X22 1459.84 1 1459.84 139.31 < 0.0001
X32 219.27 1 219.27 20.92 0.0006
Residual 125.75 12 10.48
Lack of Fit 122.80 7 17.54 29.68 0.0009
Pure Error 2.95 5 0.59
Cor Total 10201.70 19
Cdc) biosorption
Model 11185.11 7 1597.87 109.02 < 0.0001
X1 103.57 1 103.57 7.07 0.0209
X2 783.20 1 783.20 53.44 < 0.0001
X3 5299.34 1 5299.34 361.57 < 0.0001
X2 X3 177.66 1 177.66 12.12 0.0045
X12 3024.04 1 3024.04 206.33 < 0.0001
X22 1893.62 1 1893.62 129.20 < 0.0001
X32 85.22 1 85.22 5.81 0.0328
Residual 175.88 12 14.66
Lack of Fit 169.90 7 24.27 20.29 0.0022
Pure Error 5.98 5 1.20
Cor Total 11360.99 19

a)
X1: solution pH; X2: initial heavy metal concentration; X3: S. cerevisiae biomass dosage
b)
For Pb removal: R2 = 0.9877; R2 adj = 0.9805; R2 pred = 0.9428

20
c)
For Cd removal: R 2= 0.9845; R2 adj = 0.9755; R2 pred = 0.9273

The model summary statistics showed that the regression coefficient was best in

the quadratic model for both Pb and Cd biosorption and it was statistically

significant (p < 0.0001). ANOVA analysis confirmed that both models are

enough to show regression between the variables and responses and it was found

that the second-order polynomial model is the most appropriate to express this

connection. The main effects and square effects of all three variables as well as

interaction effect of initial heavy metal concentration vs. S. cerevisiae biomass

dosage were the significant model terms in Pb (II) and Cd (II) biosorption.

Consequently, the model describing the relation between the independent

variables and response is presented in Eqs. (2) and (3):

Y %Removal of Pb = 43.72 + 2.60 X1 + 7.26 X2 – 19.27 X3 – 4.91 X2X3 – 12.77 X12 –

10.06 X22 + 3.90 X32 (Eq. 2)

Y %Removal of Cd = 51.36 + 2.75 X1 + 7.57 X2 – 19.70 X3 – 4.71 X2X3 – 14.49 X12 –

11.46 X22 + 2.43 X32 (Eq. 3)

21
where Y is the biosorption (removal) rate, and X1, X2 and X3 are independent

process variables i.e., solution pH, initial Pb/Cd ion concentration and S.

cerevisiae biomass dosage, respectively. The models F-value of 137.36 and

109.02 for Pb (II) and Cd (II) respectively, and their values of prob. > F (<

0.0001) indicated that the models are able to satisfactorily predicting the

experimental results.

3.2. Effect of solution pH and heavy metal concentration on the metal uptake

pH of solution is one of the environmental factors that greatly affects the

elements speciation and consequently their removal by biosorption methods

(Esposito et al., 2002). The impact of solution pH and heavy metal concentration

on biosorption yield in the present study, can be predicted from the three-

dimensional (3D) surface plots in Fig. 3. The 3D surface plots are graphical

diagrams of regression equations showing two factors, while all other factors are

maintained at fixed levels. These plots can help to better understand both the

main and interaction effects of variables. As shown in the Fig. 3, Pb (II) and Cd

(II) biosorption rate increased with increase of solution pH ranging from 4 to 5 as

well as with metal concentration ranging from 25 to 66 µg/l. But, solution pH

22
values of more and less than 5, reduced the biosorption efficiency; so the uptake

of Pb and Cd ions in aqueous solution by S. cerevisiae at a pH of 5 done better.

23
Design-Expert® Software

Pb removal
91.6

0.5
46

X1 = A: pH
X2 = B: Metal concn. 37.25

Actual Factor
Metal uptake, %

C: Biomass dose = 32.5 28.5

19.75

11

6.00
25.0
5.50
38.8
5.00 52.5

solution pH 4.50 66.3

Pb (II) conc., µg/l
4.00 80.0

Design-Expert® Software

Cd removal
95.3
(b)
1.7
53

X1 = A: pH
X2 = B: Metal concn. 43.5

Actual Factor
Metal uptake, %

C: Biomass dose = 32.5 34

24.5

15

6.00
25.0
5.50
38.8
5.00
52.5

solution pH 4.50 66.3

Cd (II) conc., µg/l
4.00 80.0

24
Fig. 3. 3D plots showing the interactive effect of solution pH and heavy metal ion

concentration on the metal uptake percentage of Pb (a) and Cd (b) ions.

The optimum value of heavy metal concentration for both elements was found

52.5 µg/l and also, their factorial point of S. cerevisiae dosage was 32.5 × 107

CFU. By increasing the concentration of metal ions in the solution, more ions can

be connected to S. cerevisiae receptors at a time, and the process of biosorption

would be done more sufficiently. Therefore, the value of Y will increase as the

metal ions concentration rises (Han et al., 2006).

Some of the previous studies have shown that pH of the biosorption medium is an

important parameter which effects on the absorption of soluble metal ions by

various biosorbents (Saber-Samandari and Gazi, 2013; Farhan and Khadom,

2015; Khakpour et al., 2014). The solution pH is very effective factor in

chemistry of the metal ions, their competition for the binding sites and also the

activity of functional groups on the microorganism cell wall (Liu et al., 2006).

The biosorptive potential of metal cations usually increases with rising pH of the

sorption system, but not in a liner relationship, and at very high pH values is

likely to precipitate of metal complexes (Wang and Chen, 2006). The optimal pH

value is different in various metal biosorption systems. Vianna et al. (2000)

hypothesized that at low pH, the affinity with the proton at the binding sites of

microorganisms is much greater than that of the metal ion (H+≫M2+), compared

with that at higher pH, where M2+≫H+. In accordance with our findings,
25
Goksungur et al. (2005) found that metal biosorption by S. cerevisiae may be

increase with increase in solution pH, and showed a maximum yield at solution

pH of 5 and 6 for Pb and Cd, respectively. The positive effect of increasing the

metal concentration on rising biosorption yield was also seen in their study. In

another study, Han et al. (2006) showed that biosorption yield can be increased

with rising of solution pH from 2 to 6 for Cu and Pb, and the lowest sorption

responses were observed at solution pH less than 2 for both metals. Also, the

increase in biosorption rate was observed with increasing concentrations of metal

ions. In most studies, the solution pH range for maximum elemental biosorption

by S. cerevisiae has been suggested between 5.0 to 9.0, and its optimum values

found between 5 and 6 (Farhan and Khadom, 2015; Parvathi and Nagendran,

2007; Cui et al., 2010, Vasudevan et al., 2003). For instance, Farhan and

Khadom, (2015) were found that maximum biosorption of Pb and Cd ions by S.

cerevisiae may be occurred in pH 6 and 5.5, respectively. But, their findings are

related to the biosorption of these heavy metals in very high concentrations (10-

100 mg/l), whereas our study was conducted at much lower concentrations of Pb

(II) and Cd (II) as food contaminants.

3.3. Effect of biomass dosage and solution pH on the metal uptake

26
The effects of S. cerevisiae biomass dosage and solution pH on Pb (II) and Cd

(II) uptake is shown in Fig. 4. As seen in this figure, the metal removal decreased

with increasing the biomass dosage from 15 to 50 × 107 CFU and its optimum

value was 32.5 × 107 CFU. Although it is reasonable to increase the biosorption

rate by increasing the amount of biomass, and thereby increasing the binding

positions, but there are studies that are contrary to this expectation (Han et al.,

2006/36). Similar to our findings, Vasudevan et al. (2002) found that the Cd

biosorption capacity decreased with increase of biosorbent dosage. Moreover,

similar findings were obtained by Mukhopadhyay et al. (2007) with the study on

Cu biosorption by pretreated Aspergillus niger biomass and also, Al-Asheh and

Duvnjak (1995) in the study of chromium and Cu biosorption by A. carbonarius.

They suggested this comment that higher biosorption by microorganism at lower

biomass dosage, could be due to the ratio of metal ions and biosorbent, which

decrease with increasing in biomass dosage. Some researchers have suggested

this interpretation that adsorption sites on the microorganism cell surface may

remain unsaturated at higher biosorbent dosage (Zou et al., 2006; Vasudevan et

al., 2002). It is also said that higher amounts of biomass dosage may cause

interference between binding sites (DeRome and Gadd, 1987). Accordingly,

biosorption yield can be rise by decreasing in yeast biomass dosage and

increasing in solution pH at the same time. At higher solution pH, availability of

metal binding sites on the cell surface can be increased which facilitated

27
connection of metal ions (Lu et al., 2006). Moreover, Goksungur et al. (2005)

provided the theory that in moderate acidic and basic pH, there is a net negative

charge on the microorganism cell wall which may change ionic state of ligands

such as phosphate, carboxyl and amino groups in order to promote reaction with

metal cations. By reducing the pH, the overall surface charge on the cells goes up

to the more positive, which inhibits the going-on of positively charged cations.

The combination of protons with metal ions for the ligands may also cause

decrease the interaction of metal ions with the cell components (Sag and Kutsal,

1996). In a research on Pseudomonas putida, this result was reaffirmed that Pb

and Cu biosorption yield decreased with increasing the biosorbent concentration

(Uslu and Tanyol, 2006).

28
Design-Expert® Software

Pb removal
91.6
(a)
0.5
68

X1 = A: pH
X2 = C: Biomass dose 54

Actual Factor
Pb (II) uptake, %

B: Metal concn. = 52.5 40

26

12

6.00

15.0
5.50
23.8
5.00
32.5
solution pH 4.50
41.3
Design-Expert® Software 4.00 50.0
Biomass dosage, (× 10^7) CFU

Cd removal
95.3
(b)
1.7 74

X1 = A: pH
X2 = C: Biomass dose 59.5
Cd (II) uptake, %

Actual Factor
45
B: Metal concn. = 52.5

30.5

16

6.00

5.50
15.0

5.00 23.8

32.5
solution pH 4.50
41.3

4.00 50.0 Biomass dosage, (× 10^7) CFU

29
Fig. 4. 3D plots showing the interactive effect of solution pH and S. cerevisiae biomass

dosage on the metal uptake percentage of Pb (a) and Cd (b) ions.

3.4. Effect of biomass dosage and heavy metal concentration on the metal uptake

The interaction effect of metal concentration and biomass dosage is shown in Fig.

5. The solution pH was actual factor that its value determined 5. The optimum

biomass dosage for the maximum removal of Pb (II) and Cd (II) ions by S.

cerevisiae was found to be 15.8 and 15.0 × 107 CFU, respectively. Although the

biosorbent surface area increases with increasing biomass dosage and more

adsorption sites will be available, but in practice, the results showed that the

metal uptake decreases with increasing biosorbent dosage. Perhaps this can be

explained by the fact that notwithstanding by increasing the biomass dosage the

number of sites available for biosorption increases, but these sites may remain

unsaturated during the biosorption process (Vasudevan et al., 2002). From Fig. 5,

it is shown that an optimum Pb (II) and Cd (II) biosorption yield of 70.3% and

76.2% were found at initial ion concentration of 65.0 and 62.6 µg/l, and S.

cerevisiae biomass dosage of 15.0 and 15.2 × 107 CFU, respectively.

30
Fadel et al. (2017) also got the result that biosorption rate increased with

increasing the Mn2+ concentration and decreased with increasing S. cerevisiae

biomass dose. Our results are also supported by literature references, indicate that

reduction in the biomass resulted in the enhancement of the metal removal due to

exhaustion of metal ions in the biosorption media or the interaction between the

metal ion binding sites (Park and Choi, 2002). An increase of electrostatic

interaction at high biomass dosage can prevent metal biosorption. When the

biomass dose is low, metal ions in the solution would not only be adsorbed to the

surface of the biomass, but also can enter into intracellular parts through

facilitating the concentration gradient of metal ion (Wang, 2002). Moreover, at

low biosorbent dosage, inter-cellular distance is higher which can facilitate metal

biosorption process, as this condition ensures optimal electrostatic interaction

between cells (Park and Choi, 2002).

31
Design-Expert® Software

Pb removal
91.6

0.5 (a) 71

X1 = B: Metal concn.
X2 = C: Biomass dose
57

Actual Factor Pb (II) uptake, %

A: pH = 5.00 43

29

15

80.0 15.0
66.3 23.8
52.5 32.5
38.8 41.3
Design-Expert® Software Pb (II) conc., µg/l 25.0 50.0
Biomass dosage, (× 10^7) CFU

Cd removal
95.3

1.7 (b)
77

X1 = B: Metal concn.
X2 = C: Biomass dose
62.5

Actual Factor
Cd (II) uptake, %

A: pH = 5.00 48

33.5

19

80.0 15.0
66.3 23.8
52.5 32.5
38.8 41.3
Cd (II) conc., µg/l Biomass dosage, (× 10^7) CFU
25.0 50.0

Fig. 5. 3D plots showing the interactive effect of S. cerevisiae biomass dosage and heavy metal

ion concentration on the metal uptake of Pb (a) and Cd (b) ions.

32
3.5. Comparison of S. cerevisiae with other biosorbents

As a fungal biosorbent, S. cerevisiae has high resistance to toxic elements and

can also survive in the external unsuitable conditions such as acidic pH.

Advantages like high safety, easy availability, low cost and an ideal model for

genetic manipulation, have made S. cerevisiae as a suitable biosorbent (Zheng et

al, 2017). In addition, this microorganism is a waste product of the fermentation

industry and is therefore a potential adsorbing material (Dhankhar et al., 2011;

Sakamoto et al., 2010). In the present study, with the aim of using S. cerevisiae in

the remediation of very low concentrations of toxic metals in the water and food

industry, the effects of three significant parameters (solution pH, initial ion

concentration and S. cerevisiae biomass dosage) on Pb (II) and Cd (II) ion

biosorption were studied by Design Expert software, and subsequent statistical

analysis was done by response surface methodology. In order to find the optimum

conditions of these critical parameters for the most biosorption yield, central

composite design (CCD) was applied.

In recent years, studies on removal of heavy metals by microorganisms have

received great attention. In addition to S. cerevisiae, other fungi, bacteria,

cyanobacteria, green algae and seaweeds have also been studied for their

elemental biosorption capacity. In this regard, it can be pointed to studies on

33
 Bacillus sp. (Zhao et al., 2016), Chlorella vulgaris (Amini et al., 2012),

filamentous algae (Singh et al, 2007), Scenedesmus obliquus (Monteiro e al.,

2009), and etc. Table 8 shows various experimental conditions for biosorption of

different elements by S. cerevisiae and some of the other microorganisms. The

difference in the removal efficiency of various biosorbents is due to their inherent

characteristics like structure and functional groups as well as adjustable

environmental conditions. As seen in the initial element concentration, all of

these studies were performed in the high concentration levels (mg/l equivalent to

ppm) of heavy metals, while the importance of the present study, is that it was

done in very low levels of Pb (II) and Cd (II) ions, as common pollutants of water

treatment and food industry.

Table 8: Comparison of metal uptake capacity of S. cerevisiae and some of the other
microorganisms with respect to different solution pH, biomass dosage and initial ion
concentration reported in the literature.

Initial element Biomass

solution
Biosorbent Element concentrationa), dosage, Ref.
pH
mg/l g/l
Cd 6.0 250 3 (El-sayed, 2012)
Pb 5.0 300 3 (El-sayed, 2013)
0.05–0.1
Saccharomyces Pb, Cd 6.0, 5.5 10 (Farhan, 2015)
g
cerevisiae
Cd, Ni,
8.0 100 0.008 (Galedar, 2013)
Co
As 5.0 n.a. 5 (Wu et al. 2012)
Pb, Cd, (Domínguez-
Spirulina maxima 6.5 10, 5, 20 n.a.
Ni Bocanegra, 2014)
Aspergillus niger Pb, Cd,
6.01 89.93 5.22 (Amini, 2009)
Ni

34
Spirogyra neglecta,
Pb 5.0 5 1 (Singh, 2007)
Scenedesmus
Obliquus Cd 7.0 1 0.02 (Monteiro, 2009)

Anabaena ssolution
pHaerica Pb, Cd 3.0, 5.5 50-300 1, 2 (Abdel, 2013)

Utricularia aurea
Pb, Cd 4.0 10 2 (Yoonaiwong, 2011)
Gymnogongrus
Pb, Cd, 0.21, 0.45, 0.77,
torulosus 5.5 1 (Areco, 2010)
Cu, Zn 0.9 mM
a)
n.a.: not available

5. Conclusion

The present study has represented the use of response surface methodology for

determining the conditions leading to maximum heavy metal biosorption. In

order to ensure the accuracy of yeast biosorption results, analytical quality

assurance was done before instrumental analysis. The results showed that

biosorption rate of Pb (II) and Cd (II) ions by S. cerevisiae decreases at low and

high pH values. An optimum condition for Pb (II) and Cd (II) uptake of 70.7%

and 76.9% was achieved by Design-Expert software at initial ion concentration of

68.2 µg/l and S. cerevisiae biomass dosage of 15.0 × 107 CFU. Moreover, it was

indicated that metal biosorption increased with rising up Pb and Cd ion

35
concentrations and the optimum ion concentrations was 52.5 µg/l. On the other

hand, with increasing biomass dosage, the metal biosorption decreased. The

points giving the maximum biosorption of Pb and Cd ions were found to be at

32.5 × 107 CFU biomass dosage.

The fit of the models were checked by getting (R2). In these cases, the values of

the multiple correlation coefficient for Pb and Cd biosorption rates (R2 = 0.9877,

0.9845) indicate that 1.23% and 1.55% of the variations were not explained by

the models, respectively. The results of this research showed that by design of

experiments based on some of the significant factors, including solution pH,

metal ion concentration and biomass dosage, biosorption of very low

concentration levels of Pb (II) and Cd (II) ions by S. cerevisiae from aqueous

media can be practical. Other previous studies had confirmed similar results,

however, at much higher concentrations of metals (ppm) commonly found in

wastewater. Accordingly, the present findings indicate that S. cerevisiae as a

green technology, can be used in decontamination of heavy metals from water

treatment and food processing industry.

Acknowledgement

The authors acknowledge the Food and Drug Reference Laboratories Center of

Food and Drug Organization for instrumental supporting of this research.

36
Conflict of interest: There is no conflict of interests to declare.

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uranium (VI) by Saccharomyces cerevisiae- Crystal formation of chernikovite. Chemosph,

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HIGHLIGHTS
 Saccharomyces cerevisiae can be applied for removal of Pb (II) and Cd (II)
ions from aqueous solutions
 S. cerevisiae is an efficient biosorbent in very low concentration (ppb) of
heavy metals
 Three significant variables for lead and cadmium biosorption are: solution
pH, heavy metal concentration and biomass dosage

47
 Graphic Abstract

Bioremediation

Biosorption by S. cerevisiae

Industrial or agricultural processing

increase Heavy metals toxic Foodstuffs
&

Utilizing fertilizers and pesticides

Pb>Cd

48