Biosorption of lead from aqueous solution by fungal ...scijournal.hcu.ac.th/data/Vol2 Issue 1 Biosorption.pdf · and Rhizopus sp. with NaOH was investigated. Parameters affecting

ว. วิทย. เทคโน. หัวเฉียวเฉลิมพระเกียรติ 15ปีที่ 2 ฉบับที่ 1 มกราคม - มิถุนายน 2559

Biosorption of lead from aqueous solution by fungal biomass of

Aspergillus niger and Rhizopus sp.

วรำภรณ์ พันธุ์ศุภผล1 จิตตินันท์ ยิ่งยงยุทธ1 รุจิรำลัย พูลทวี1 และ มธุรส อ่อนไทย2*

1 สาขาวิชาวิทยาศาสตร์ชีวภาพ คณะวิทยาศาสตร์และเทคโนโลยี มหาวิทยาลัยหัวเฉียวเฉลิมพระเกียรติ

สมุทรปราการ 105402 สาขาวิชาวิทยาศาสตร์กายภาพ คณะวิทยาศาสตร์และเทคโนโลยี มหาวิทยาลัยหัวเฉียวเฉลิมพระเกียรติ

สมุทรปราการ 10540

Waraporn Pansuphaphol1, Jittinan Yingyongyut1, Rujiralai Poontawee1

and Mathuros Ornthai2*

1 Division of Biological Science, Faculty of Science and Technology, Huachiew Chalermprakiet University,

Samutprakarn 105402 Division of Physical Science, Faculty of Science and Technology, Huachiew Chalermprakiet University,

Samutprakarn 10540

AbstractThe biosorption of lead from aqueous solution by pretreated fungal biomass of Aspergillus niger

and Rhizopus sp. with NaOH was investigated. Parameters affecting biosorption such as pH, temperature

and contact time were examined. It was found that the initial pH of the solution strongly affected to the

degree of biosorption. The pH of 5 and 6 were the optimum pH on biosorption of Rhizopus sp. and A. niger, respectively. The wide range of temperature of 30-60°C showed slightly effect on lead adsorption.

The rapid rate of adsorption was found in during the first 60 min and was remained nearly constant

afterwards. The optimum contact time was between 60 and 90 min. Moreover, the lead adsorption by

Rhizopus sp. was higher than that of A. niger in all cases of the study conditions.

Keywords: Fungal biomass, Biosorption, Lead, Aspergillus niger, Rhizopus sp.

Corresponding author: [email protected]

ปีที่ 2 ฉบับที่ 1 มกราคม - มิถุนายน 255916 ว. วิทย. เทคโน. หัวเฉียวเฉลิมพระเกียรติ

บทคัดย่องานวิจัยนี้เป็นการศึกษาการใช้ชีวมวลที่ถูกปรับสภาพของเชื้อรา Aspergillus niger และ Rhizopus sp. ด้วย

โซเดยีมไฮดรอกไซด์ส�าหรบัการดดูซบัทางชวีภาพในการก�าจดัตะกัว่ในสารละลายน�า้ โดยปัจจยัทีศ่กึษาประกอบด้วยอทิธพิล

ของค่าความเป็นกรด-ด่าง อุณหภมู ิและระยะเวลาทีใ่ช้ในการดดูซบั โดยทีค่่าความเป็นกรด-ด่างส่งผลต่อค่าการดดูซบัตะกัว่

อย่างมาก ซึ่งพบว่าค่าความเป็นกรด-ด่างเท่ากับ 5 และ 6 เป็นค่าที่เหมาะสมในการดูดซับตะกั่วของเชื้อรา Rhizopus sp.

และ A. niger ตามล�าดับ นอกจากนี้ผลของอุณหภูมิในช่วง 30-60 องศาเซลเซียส ไม่ได้ส่งผลต่อค่าการดูดซับมากนัก โดยที่

อัตราการดูดซับมีค่าสูงมากในช่วงเวลา 60 นาทีแรกของการดูดซับและคงที่หลังจากนั้น ดังนั้นระยะเวลาที่เหมาะสมในการ

ดูดซับคือ ภายใน 60-90 นาที นอกจากนี้ผลของการทดลองบ่งชี้ว่าการดูดซับของ Rhizopus sp. มีค่าการดูดซับตะกั่วที่

มากกว่าของ A. niger ภายใต้เงื่อนไขที่ท�าการศึกษา

ค�ำส�ำคัญ: ชีวมวล การดูดซับทางชีวภาพ ตะกั่ว Aspergillus niger, Rhizopus sp.

Introduction

The contamination of heavy metals in

water streams is a great concern because of their

extreme toxicity for aquatic life and humans. Lead

(Pb) is one of the heavy metals that is extremely

toxic to organisms even at low concentration and

can damage to the nervous system, gastrointestinal

track, encephalopathy with permanent damage,

kidneys and reproductive system, particularly in

children [1]. Many industries including coating,

automotive, aeronautical and steel generate large

quantities of wastewaters containing various

concentrations of lead [2]. Therefore, a removal of

excessive lead from wastewaters before release to

the natural water is necessary.

Generally, the common methods used for

removal heavy metals in wastewaters consist of

chemical precipitation, ion exchange, electrochemical

treatment and reverse osmosis processes [3, 4].

However, the disadvantages of these methods are

complexity, high reagent requirements, generation

of toxic sludges and extremely expensive.

The alternatively low-cost method is biosorption

process by biological materials that offer the

advantages of low operating cost, minimization of

the volume of chemical and/or biological sludge,

high effective removal and easily available in

substantial quantities.

Various biological materials, especially

microorganisms including algae, fungi, bacteria and

yeasts can be prepared as biomass that can be used

for removal of lead in water via biosorption process

[5]. This process results from interactions between

metal ions and the functional groups such as

carboxylate, hydroxyl and amino groups present

on the cell wall surface composed of polysaccharides,

proteins and lipids [6, 7]. The biomass can be utilized

for both living and dead cells. However, dead cells

offer some advantages over the corresponding live

cells such as no limitations for toxicity, no

requirement for growth media and nutrition [8].

Especially, dead cells can be modified the properties

of functional groups on the cell wall surface by

chemically and physically pretreated method that

aims to enhance the metal biosorption capacity.


It was due to that fungi are used in a variety

of industrial fermentation processes. Aspergillus sp.

are used in the production of ferrichrome, kojic acid,

gallic acid, itaconic acid, citric acid and enzymes

like amylases, glucose isomerase, pectinase, lipases

and glucanases [3]. Rhizopus arrhizus are used to

produce various metabolites, such as lipase, fumaric

acid, lactic acid, steroids and celluloses [9].

Therefore, fungi could serve as an economic and

constant supply source of biomass for the removal

of metal ions. Fungi can also be easily cultivated in

substantial amounts using unsophisticated

fermentation techniques and inexpensive growth

media. Research on fungal biosorption was recently

reviewed by Viraraghavan and Srinivasan [10]. This

research focused on fungi in the genera of Aspergillus

and Rhizopus. Although, they have already been

studied as potential biomass for the removal of

heavy metals from aqueous solutions [4, 9-14], the

degree of sorbent affinities and biosorption

capacities were different because of different

operating conditions. The degree of biosorption

capacities of a metal ions on a biomass have been

found to be a function of the biomass dosage, initial

solution pH and initial metal ion concentration,

contact time, temperature and the method of

pretreated biomass.

Herein, the degree of biosorption capacities

of A. niger and Rhizopus sp. which used as pretreated

biomass for removal of lead from aqueous solution

were reported in the study conditions. The effect

of initial pH, contact time and temperature were

investigated. Especially, the operating temperature

was selected above 30°C that was corresponded to

the ambient temperature of Thailand.

Materials and Methods

Microorganisms and growth conditions

A. niger and Rhizopus sp., the filamentous

fungi, were obtained from the Division of Biological

Science, Faculty of Science and Technology,

Huachiew Chalermprakiet University, Thailand.

These cultures were maintained on potato dextrose

agar (PDA) containing 20 g dextrose, 15 g agar, 200 g

potato, and 1 L water at room temperature (27-30°C)

for 3-5 days. A volume of 20 mL of 0.1% Tween

80 was used for wash spores and mycelium. The

spore suspensions were transferred to 250 mL

Erlenmeyer flasks containing 100 mL growth

medium. This growth medium had the following

composition (g/L): dextrose, 20; peptone, 10; NaCl,

0.2; CaCl2.2H

2O, 0.1; KCl, 0.1; K

2HPO

4, 0.5; NaHCO

3,

0.05; MgSO4, 0.25; Fe (SO

4)2.7H

2O, 0.005. Once

inoculated, flasks were shaken on a rotary shaker at

125 rpm for 3 days at room temperature. After the

fungal growth, the biomass and the culture medium

were separated by vacuum filtration and the

resulting biomass was washed several times

thoroughly with distilled water.

Preparation of biomass

Biomass was pretreated by boiling in 0.5 N

NaOH solution for 15 min in order to enhance the

metal biosorption capacity [4, 8] and then was

separated by vacuum filtration. The biomass was

washed by generous amounts of deionized water

till the pH of the wash solution was in the near-

neutral range (6.7-7.2). After washing, the biomass

was dried at 60°C for 24 h and powdered in a mortar

and pestle. The powdered biomass residue obtained

will be referred to as pretreated biomass in this

paper and was used for biosorption studies.


Lead solutions

A stock solution of Pb(II) was prepared by

dissolving an accurate quantity of 0.1598 g of

anhydrous Pb(NO3)2 in 100 mL of 2% HNO

3 to obtain

concentrations of 1000 mg/L. The Pb(II) working

solution was obtained by dilution of Pb(II) stock

solution to the concentration of 10 mg/L with

deionize water. The pH of the working solutions

was adjusted to a desired value with 0.1 M HCl and

0.1 M NaOH.

Batch biosorption experiments

All batch experiments were carried out

with biomass samples (0.1 g) in Erlenmeyer flasks

(250 mL) on an orbital shaker operating at 125 rpm

to elucidate the optimum conditions (pH,

temperature and contact time). The effect of pH

on the biosorption capacity of Pb(II) on fungal

biomass was determined by equilibrating the

sorption mixture containing pretreated biomass

(0.1 g) and metal solutions (100 mL of 10 mg/L of

Pb(II) solution) at different pH values of 3, 4, 5, 6,

7 and 8. The period of contact time was 240 min

at room temperature (27-30°C). The same protocol

at the optimum pH was used for study the effect

of temperature consisted of 30, 45, 50, 55 and 60°C

on Pb(II) adsorption. The effect of contact time was

studied by sampling of supernatant solution (10 mL/

each sampling) during the biosorption was

proceeding in the same Erlenmeyer flask for 5-240

min at room temperature and optimum pH. All the

experiments were carried out in triplicates and the

arithmetical average values were used.

Metal analysis

The concentrations of unadsorbed Pb(II) in

solutions were determined after the separation of

biomass by centrifugation using flame atomic

absorption spectrometer with the deuterium lamp

background correction (Model iCE™ 3000, Thermo

Fisher Scientific Inc., Waltham, MA, USA). Lead

hallow cathode lamp was used at the wavelengths

of 217 nm. The instrument response was periodically

checked by standard metal solutions. The detection

limit of Pb(II) determination was 0.0174 mg/L.

The amount of adsorbed lead ions (Q) per

gram biomass was calculated using the general

equation as follows [9, 15]:

where Q (adsorbed lead ions, mg/g) is the

amount of metal ions adsorbed on the biomass,

V (L) is the volume of lead containing solution in

contact with the biomass, Ci (mg/L) and C

f (mg/L)

are the initial and final concentration of lead ions

in the solution, respectively and W (g) is the dry

weight of fungal biomass.

Results and Discussion

Biomass used in this work was pretreated

by boiling with 0.5 N NaOH solution for 15 min in

order to enhance the lead biosorption capacity.

This pretreated method has already proved by

Kapoor and Viraraghavan [3, 8] that exhibited higher

lead, cadmium and copper removal capacities than

did live biomass of live A. niger. Pretreatment could

WV)C(CQ fi −=


be exposure of active metal-binding sites embedded

in the cell wall or chemical modifications of the

cell wall components. The alkali pretreatment was

observed to be most effective in increasing the

biosorption capacity of fungal biomass. The alkali

treatment (usually with sodium hydroxide) of fungal

biomass for 4-6 h at 95-100°C deacetylates chitin

presented in the cell wall to form chitosan-glucan

complexes with higher affinity for metal ions [16,

17]. The effect of pH, temperature and contact time

for the degree of lead biosorption capacities of

A. niger and Rhizopus sp. are discussed below.

Effect of pH on biosorption

The pH of the solution strongly affected

to the degree of biosorption of heavy metal ions

on pretreated biomass [4]. The initial pH of

adsorption medium is related to the adsorption

mechanisms onto the surface of biomass from water

and reflects the nature of the physicochemical

interaction of the species in solution and the

adsorptive sites of biomass. Therefore, the batch

equilibrium studies were conducted with different

initial pH values ranging from 3 to 8. Note that

pH 8 did not cause the precipitation of Pb(OH)2 in

the study conditions (checked with Ksp). The results

of various pH on biosorption capacities are

presented in Figure 1.

Figure 1. Effect of initial pH on equilibrium Pb(II) sorption capacity of A. niger and Rhizopus sp. Initial

Pb(II) ion concentration = 10 mg/L, the biomass concentration = 0.1 g/100 mL, temperature = 30°C. The

bars represent the standard error of the mean.

At pH 3, the lowest lead biosorption were

observed in both case of A. niger and Rhizopus sp.

It was due to that at highly acidic pH, the overall

surface charge on the cells became positive and

metal cations and protons compete for binding sites

on cell wall, which results in lower uptake of metal.

It has been suggested that at low pH values, cell

wall ligands would be closely associated with H3O+

that restrict access to ligands by metal ions as a

result of repulsive forces [18]. With an increase in

pH, the negative charge density on the cell surface

increases due to deprotonation of the metal binding


sites and thus increases biosorption [4]. A sudden

increase in sorption with a slight increase in pH is

often referred to as an “adsorption edge” which

were observed at pH 4 and 5 for Rhizopus sp. and

A. niger, respectively. An optimum pH should be

selected above the pH of adsorption edge because

it can avoid the error of pH adjustment. Especially,

when adjusted pH was lower than the pH of

adsorption edge, it would strongly affected to the

efficiency of adsorbed Pb(II). Adsorbed Pb(II) was

not significantly different at the pH above 5 for both

cases of Rhizopus sp. and A. niger. These results

indicated that the pH above 5 would be suitable

for applying to the biosorption processes in this

work. However, when considering in general case

of wastewater which may contain the high

concentration of lead and/or other metal ions, it

may cause the precipitation of lead hydroxide and/

or other metal hydroxides at high pH. Therefore,

the selected optimum pH on biosorption of

Rhizopus sp. and A. niger were 5 and 6, respectively.

An increasing or decreasing the pH from this

optimum pH resulted in a slight reduction of the

biosorption of Pb(II). Moreover, the results indicated

that the lead biosorption capacities of Rhizopus sp.

are higher than that of A. niger in the study

conditions which could be related to the more net

negative charge and appropriate ligands on cell

surface of Rhizopus sp.

Effect of temperature on biosorption

The effect of temperature on lead

biosorption was carried out with optimum pH of

each biomass. The results are shown in Figure 2.

Figure 2. Effect of temperature on equilibrium Pb(II) sorption capacity of A. niger and Rhizopus sp. Initial

Pb(II) ion concentration = 10 mg/L, the biomass concentration = 0.1 g/100 mL, pH = 5 and 6 for Rhizopus

sp. and A. niger, respectively. The bars represent the standard error of the mean.


It has been reported that the biosorption

of lead on A. niger was endothermic process in the

temperature of 20-35°C [18]. Thus, the extent of

adsorption could be increased with increasing

temperature, however, the small change in

adsorption were obtained in the temperature of

30-60°C for biomass of A. niger and Rhizopus sp. in

our work. This could be related to the different

interval of temperature that affect to the biosorption

process, which may involve not only physical

sorption but also chemical sorption. These results

suggested that biosorption of lead on the studied

biomass was not sensitive to temperature.

Therefore, a wastewater with a temperature above

30°C can be effectively treated with biomass without

any adjustment of temperature. Furthermore, the

lead biosorption capacities of Rhizopus sp. are also

higher than that of A. niger in these study conditions.

Effect of contact time on biosorption

The effect of the contact time on the

biosorption of lead on A. niger and Rhizopus sp.

biomass are shown in Figure 3.

Figure 3. Effect of contact time on Pb(II) sorption capacity and % Pb(II) removal of A. niger and Rhizopus

sp. Initial Pb(II) ion concentration = 10 mg/L, the biomass concentration = 0.1 g/100 mL, temperature =

30°C, pH = 5 and 6 for Rhizopus sp. and A. niger, respectively. The bars represent the standard error of

the mean.

Rapid biosorption of lead were observed

within 60 min. The biosorption capacities at 60 min

were 9.21 and 8.94 mg Pb(II)/g biomass (92.1% and

89.4% of Pb(II) removal) for Rhizopus sp. and A. niger,

respectively. The biosorption rates remained nearly

constant afterwards. These rapid initial sorption are

also similar to the previous reports on the

biosorption of lead by the same [4] and different

biomass [15, 19]. These plots were suggested that

kinetics of lead biosorption consisted of two phases;

an initial rapid phase where biosorption was fast

and contributed significantly to overall sorption and

a slower second phase whose contribution to the

total lead biosorption was relatively small. These


results indicated that the optimum contact time

should be more than 60 min. However, it should

not be more than 90 min because of long time

consumption and constant adsorption.

Conclusions

The ability of pretreated A. niger and Rhizopus sp. with sodium hydroxide to adsorb Pb(II)

ions from aqueous solution were investigated in a

batch system. The initial pH showed strongly

affected to the degree of biosorption. The very low

lead biosorption were obtained in very acidic pH

of 3. However, the lead biosorption capacities were

increased very sharply when increasing the pH to

4-5 and quite consistent with the pH afterwards.

The temperature showed slightly effect on lead

biosorption in the wide range of 30-60°C. This could

be benefited to effectively treated wastewater

without any adjustment of temperature. The

optimum contact time between biomass and

treated solution was between 60 and 90 min for

highly effective removal of lead. The overview of

the efficiency of biomass to adsorb lead ion was

indicated that Rhizopus sp. has a lead biosorption

capacity higher than that of A. niger in the study

conditions. This research also supported that the

alkaline-pretreated biomass of A. niger and Rhizopus

sp. had a potential to be used in the removal of

lead from aqueous solution.

Acknowledgements

This work was supported by Division of

Physical Science and Biological Science, Faculty of

Science and Technology, Huachiew Chalermprakiet

University.

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Biosorption of lead from aqueous solution by fungal ...scijournal.hcu.ac.th/data/Vol2 Issue 1 Biosorption.pdf · and Rhizopus sp. with NaOH was investigated. Parameters affecting