Bioresource Technology 96 (2005) 5962

Removal of chlorophenols from aquatic systems using

the dried and dead fungus Pleurotus sajor caju
Adil Denizli
a

a,*

, Nil
ufer Cihangir b, Nalan T
uzmen c, G
uleren Alsancak

Department of Chemistry, Hacettepe University, Biochemistry Division, Beytepe, Ankara, Turkey

b
Department of Biology, Hacettepe University, Beytepe, Ankara, Turkey
c
Department of Chemistry, Suleyman Demirel University, Isparta, Turkey

Received 12 June 2003; received in revised form 15 September 2003; accepted 10 November 2003
Available online 12 April 2004

Abstract
In this study, the potential use of the fungus Pleurotus sajor caju to remove phenols (i.e., phenol, o-chlorophenol, p-chlorophenol
and 2,4,6-trichlorophenol) from aqueous solutions was evaluated. Biosorption of phenol or chlorophenols reached equilibrium in 4
h. The maximum adsorptions of phenol and chlorophenols onto the Pleurotus sajor caju were 0.95 mmol/g for phenol, 1.24 mmol/g
for o-chlorophenol, 1.47 mmol/g for p-chlorophenol and 1.89 mmol/g for 2,4,6-trichlorophenol. The anity order was as follows:
2,4,6-trichlorophenol > p-chlorophenol > o-chlorophenol > phenol. Phenol and chlorophenols bindings onto Pleurotus sajor caju
were clearly pH dependent. The adsorption of phenol and chlorophenols increased with increasing pH. Desorption was achieved
using methanol solution (30%, v/v). Pleurotus sajor caju biomass is suitable for reuse for more than ve cycles without noticeable loss
of adsorption capacity.
2004 Elsevier Ltd. All rights reserved.
Keywords: Chlorophenols; Fungal biomass; Biosorption; Organic pollutants; Pleurotus sajor caju

1. Introduction
Phenolic compounds are very toxic, and many are
known or suspected human carcinogens (EPA, 1988;
Slein and Sansone, 1980; IWRS, 1990). Loss of appetite,
headache, rapid fatigue and severe chronic insomnia are
given as symptoms of chronic phenol intoxication in
humans after long-term intake of excessive phenol concentrations. Phenolic compounds are present in the
wastewater generated from paint, solvent, petroleum and
petrochemical, coal-conversion, pharmaceutical, wood
preserving chemicals, plastic, rubber-proong, pesticide,
iron-steel, phenol-production, paper and pulp industries.
Large-scale coal gasication and carbonization plants
generate huge quantities of high-strength phenolic
wastewater (Dutta et al., 1998). United States Environmental Protection Agency (EPA) regulations call for
lowering phenol content in the wastewater to less than
1 mg/l from the several thousand mg/l often present.

Corresponding author. Tel./fax: +90-312-2992163.

E-mail address: denizli@hacettepe.edu.tr (A. Denizli).

0960-8524/$ - see front matter 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2003.11.029

Traditionally, activated carbon adsorption and solvent extraction are the most widely used methods for the
removal of phenolic compounds. But recently, microorganisms have been considered as one of the most
promising adsorbents (Kennedy et al., 1992; B
ulb
ul and
Aksu, 1997; Brandt et al., 1997; Aksu and Yener, 1998).
In the concept of biosorption, several chemical processes
may be involved, such as adsorption, ion exchange, and
covalent bonding with the biosorptive sites of the
microorganisms including carboxyl, hydroxyl, sulphydryl, amino and phosphate groups (Fourest and Volesky, 1997). Fungal cell walls and their components have
major role in the biosorption (Tsezos et al., 1997; Hafez
et al., 1997; Kapoor and Viraraghavan, 1997). Fungal
biomass can also take up considerable quantities of
pollutants from aqueous solution by adsorption or a
related process, even in the absence of physiological
activity (Gadd and White, 1989).
The purpose of this investigation was to examine the
extent of removal of model phenol or chlorophenol
pollutants.In this study, Pleurotus sajor caju was used as
biosorbent and adsorption/desorption of phenol, ochlorophenol, p-chlorophenol and 2,4,6-trichlorophenol

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A. Denizli et al. / Bioresource Technology 96 (2005) 5962

were investigated because they are some of the most

challenging species of priority pollutants to be removed
from waste streams.

2. Methods
2.1. Cell line and medium
The strain used was Pleurotus sajor caju which was
kindly supplied by Dr. I.F. Zadrazil (Insitut f
ur
Bodenbiologie, Braunschweig, Germany). Cultures were
maintained on potato-dextrose-agar slaints and subcultured every month, then transferred to storage at 4 C.
Pleurotus sajor caju was grown in a liquid medium
containing (g/l) glucose, 10.0; KH2 PO4 , 2.0;
MgSO4 7H2 O, 0.5; CaCl2 , 0.1; NH4 Cl, 0.12 and thiamine, 0.001 (Blais et al., 1993). The initial pH of the
medium was adjusted to 5.5 before sterilization. Media
were inoculated with Pleurotus sajor caju and incubation
was carried out at 28 C on an orbital shaker incubator
at 150 rpm for 7 days. The biomass in growth media was
detected according to dry weight. The biomass was
separated from the broth by ltration. The growth
media were ltered and preweighed lter paper (Whatmann no. 1) and biomass was washed with deionized
and distilled water then dried in an incubator at 30 C
for 24 h (Presscott et al., 1993). The dried sample was
then ground using a blender and sieved to pass through
a 100-mesh sieve to obtain uniform particle size.
2.2. Adsorption/desorption of phenol or chlorophenols
Adsorption of phenol or chlorophenols was investigated in a batch system. Phenol and chlorinated phenols
were obtained from Sigma (St. Louis, USA). Eects of
the initial concentration of phenol or chlorophenols and
pH of the medium on the adsorption rate and capacity
were studied. 250 ml of aqueous phenol or chlorophenol
solutions with dierent concentrations (in the range of
251000 mg/l) were treated with the biomass (50 mg per
batch) at dierent pH (in the range of 2.08.0, adjusted
with HClNaOH) at room temperature, in the asks
agitated magnetically at 600 rpm. All water used in the
experiments was puried using a Barnstead (Dubuque,
IA) ROpure LP reverse osmosis unit with a high ow
cellulose acetate membrane (Barnstead D2731) followed
by a Barnstead D3804 NANOpure organic/colloid
removal and ion exchange packed-bed system. The
resulting puried water had a specic resistance of 18
M X. The concentration of the phenol or chlorophenols
in the aqueous phase was measured by high performance liquid chromatography. The LC equipment
consisted of a Cecile CE1100 liquid chromatograph
pump and Hewlett Packard HP 3395 integrator. Cecile
CE1220 LC UV variable wavelength monitor was used

as detector. In the chromatographic determination,

Spherisorb ODS1 column containing 5 lm particles was
used. For the detection of the phenols, the wavelength
was set at high absorption wavelength. The amount of
adsorption at equilibrium, q (mmol/g), was obtained as
follows:
q C0
Ce  V =m;

where C0 and Ce are the initial and equilibrium liquid

phase concentrations (mmol/l); V is the volume of the
solution (L); and m is the weight of the dry biomass used
(g).
In order to determine the regeneration of the biomass, consecutive adsorptiondesorption cycles were
repeated ve times by using the same sorbent. Desorption was achieved by using methanol solution (30% v,v).
Phenol and cholorophenol loaded Pleurotus sajor caju
were placed in this desorption medium and stirred at 600
rpm for 2 h at room temperature. After eluting, the
biomass was regenerated by washing with water. The
nal concentration in the aqueous phase was determined
by using a HPLC as described before. The desorption
ratio was calculated from the amount of phenol or
chlorophenols initially loaded on the biomass and the
nal concentration in the desorption medium.

3. Results and discussion

3.1. Adsorption time
The equilibrium adsorption time of phenol or chlorophenols (i.e., o-chlorophenol, p-chlorophenol and 2,4,6trichlorophenol) on the Pleurotus sajor caju biomass was
investigated. The initial concentrations of the phenol or
chlorophenols within the aqueous phase were kept
constant at 500 mg/l. Adsorption amounts of phenol or
chlorophenols were very high at the beginning of
adsorption, and saturation levels were gradually reached
within 4 h for phenol and all chlorophenols. Notice that
2,4,6-trichlorophenol was adsorbed much faster than
phenol and other chlorophenols due to much higher
anity of the interacting groups on the surface of
the biomass. The adsorption rate order is as follows:
2,4,6-trichlorophenol > p-chlorophenol > o-chlorophenol > phenol.
Adsorption of phenolic species was quite fast especially when the initial solution concentration was high.
Data on the adsorption kinetics of phenolic compounds
by various sorbents have shown a wide range of
adsorption rates. For example, Yenkie and Natarajan
(1993) considered 4 h as a equilibrium time in their
phenol adsorption kinetic studies, in which they used
granular activated carbon as sorbent. Ravi et al. (1998)
have investigated phenol and cresol isomers adsorption
on activated carbon and they reported 20 h equilibrium

A. Denizli et al. / Bioresource Technology 96 (2005) 5962

adsorption time. Furuya et al. (1997) investigated the

adsorption of chlorophenols on granular activated carbon and reported that equilibrium adsorption time is 2
weeks. Shu et al. (1997) considered 48 h as a short
equilibrium time in their chlorophenol adsorption kinetic studies, in which they used aluminosilicate-based
sorbent. Streat and Sweetland (1997) studied phenol and
chlorophenols adsorption on a new series of porous
polymeric ion-exchange sorbents and they reported 20
day equilibrium adsorption time. Gupta et al. (1998)
investigated phenol and p-nitrophenol adsorption on a
low cost adsorbent i.e., bagasse y ash and they reported
that the equilibrium adsorption time was 24 h. There are
several parameters which determine the adsorption rate
such as stirring rate (or ow) in the aqueous phase,
structural properties of adsorbent (e.g. porosity, surface
area), amount of adsorbent, adsorbate properties (e.g.
molecular dimensions and solubility), initial concentration of phenolic species and of course existence of other
species which may compete with the phenolic species of
interest for the active adsorption sites. The extent of
biosorption is also inuenced by the surface chemistry
of the biomass.
3.2. Eects of initial concentration of phenol or chlorophenols
The amount of adsorption was increased with the
initial phenol or chlorophenols concentration. The
maximum adsorption capacities of the Pleurotus sajor
caju biomass in the studied range were 0.95 mmol/g for
phenol, 1.24 mmol/g for o-chlorophenol, 1.47 mmol/g
for p-chlorophenol and 1.89 mmol/g for 2,4,6-trichlorophenol at pH 6.0, which are corresponding an initial
concentration of 500 mg/l. The adsorption capacity for
the chlorophenols is higher than that of the phenol,
probably because of the higher solubility of phenol in
water. Aqueous solubilities (mol/l) were 638.4 for phenol, 198.4 for o-chlorophenol; 186 for p-chlorophenol
and 4.3 for 2,4,6-trichlorophenol. The adsorption order
was as follows: 2,4,6-trichlorophenol > p-chlorophenol > o-chlorophenol > phenol.
In literature, dierent sorbents with a wide range of
adsorption capacities for phenolic compounds have
been used. Bouras et al. (2001) studied surfactant
modied iron-pillared bentonite as an adsorbent to remove pentachlorophenol from aqueous stream. They
reported maximum adsorption capacity as 460 lmol/g.
Aksu and Yener (1998) investigated the biosorption of
phenol, o-chlorophenol and p-chlorophenol from aqueous solutions on dried activated sludge. Maximum
adsorption capacity was found to be 1.7 mmol/g. Ravi
et al. (1998) reached adsorption capacity between 3.2
and 4.4 mmol/g with activated carbon for phenol and
cresol isomers. Furuya et al. (1997) used chloro- and
nitrophenols as the test adsorbates, and granular acti-

61

vated carbon was used as the adsorbent. They achieved

up to 4 mmol/g adsorption capacity. Shu et al. (1997)
used aluminosilicate-based microporous materials (pillared clays, silicalite and zeolite beta) and they reported
selective phenol adsorption capacity up to 1 mmol/g.
Streat and Sweetland (1997) reported up to 1.5 mmol/g
adsorption capacity for phenol and chlorophenols with
a new series of hypercrosslinked porous polymeric
ion-exchange sorbents (Hypersol-Macronete). Gupta
et al. (1998) showed 60 lmol/g adsorption capacity for
phenol and p-nitrophenol with a low cost adsorbent
i.e., bagasse y ash (a waste generated in local sugar
plants) sorbents. Dargaville et al. (1996) found up to
0.1 mmol/g adsorption capacity for multinuclear phenolic compounds by activated carbon. However, notice
that the adsorption capacities that we achieved are
comparable with the values reported in the previous
publications.
3.3. Eects of pH
The medium pH aects the solubility of phenol or
chlorophenols and the ionisation state of the functional groups (carboxyl, phosphate and amino groups)
of the fungal cell wall. The amino groups carry positive charges that allow the fungal cell wall components to be potential scavengers of chlorophenols. The
adsorption capacities increased with increasing pH.
The interaction forces between phenol or chlorophenols
and biomass are rather weak in the acidic solutions. The
highest adsorption of chlorophenols occurred at pH 6.0
for all species. However within the pH range of 6.08.0
there was no change in the equilibrium adsorption
capacity.
3.4. Regeneration
To be useful in separation and removal processes,
phenolic species adsorbed should be easily desorbed
under suitable conditions and sorbents should be used
many times in order to decrease material cost. Desorption experiments were performed with methanol solution (30%, v/v) as the desorption agent. The biomass
were placed within the desorption medium and the
amount of phenol or chlorophenols desorbed in 2 h was
measured. It must be pointed out that biosorption (i.e.,
binding of phenol or chlorophenols onto fungal biomass) is completely reversible. More than 90% of the
adsorbed phenol or chlorophenols was desorbed in all
cases. This means that methanol breaks down the
interaction forces between phenols and binding sites
onto the surface of the fungal biomass. The biomass can
be used repeatedly without losing signicantly their
adsorption capacities for phenol or all chlorophenols
studied here (Table 1).

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A. Denizli et al. / Bioresource Technology 96 (2005) 5962

Table 1
Adsorptiondesorption cycles for phenol or chlorophenols
Cycle no.

1
2
3
4
5

Phenol

o-Chlorophenol

2,4,6-Trichlorophenol

p-Chlorophenol

Adsorption
(mmol/g)

Desorption
(%)

Adsorption
(mmol/g)

Desorption
(%)

Adsorption
(mmol/g)

Desorption
(%)

Adsorption
(mmol/g)

Desorption
(%)

0.95
0.93
0.92
0.89
0.91

92.1
92.6
90.6
92.7
94.5

1.24
1.22
1.20
1.23
1.21

89.5
93.3
91.6
94.0
92.5

1.47
1.46
1.43
1.44
1.42

91.1
90.8
92.0
93.2
91.0

1.89
1.87
1.88
1.85
1.83

89.5
87.3
92.0
90.9
92.7

Initial concentrations of chlorophenols: 500 mg/l; pH: 6.0, T : 20 C.

4. Conclusions
Wastewaters containing phenolic compounds present
a serious problem. Phenol containing wastewater may
not be conducted into open water without treatment
because of the toxicity of phenol. Recently, polymer
based adsorbents were widely employed for the removal
of phenols. But the high cost of polymers has stimulated
interest in examining the feasibility of using cheaper
adsorbents. Cheaper raw material Pleurotus sajor caju
was used for adsorption/desorption of phenol or chlorophenols (i.e., phenol, o-chlorophenol, p-chlorophenol
and 2,4,6-trichlorophenol) from aqueous solutions. The
present studies led to the following conclusions: The
maximum adsorption capacities of the Pleurotus sajor
caju biomass from their single solutions were 0.95 mmol/
g for phenol, 1.24 mmol/g for o-chlorophenol, 1.47
mmol/g for p-chlorophenol and 1.89 mmol/g for
2,4,6-trichlorophenol. The adsorption order was as follows: 2,4,6-trichlorophenol > p-chlorophenol > o-chlorophenol > phenol. The adsorption of phenol or
chlorophenols increased with increasing pH. Consecutive adsorption and desorption cycles showed the feasibility of the Pleurotus sajor caju biomass for phenol or
chlorophenol removal from aqueous solutions.
References
Aksu, Z., Yener, J., 1998. Investigation of the biosorption of phenol
and monochlorinated phenols on the dried activated sludge.
Process Biochem. 33, 649655.
Blais, J.F., Tyagi, R.D., Auclair, J.C., 1993. Bioleaching of metals
from sewage sludge: microorganisms and growth kinetics. Water
Res. 27, 101110.
Bouras, O., Houari, M., Khalaf, H., 2001. Using of surfactant
modied Fe-pillared bentonite for the removal of pentachlorophenol from aqueous stream. Environ. Technol. 22, 6974.
Brandt, S., Zeng, A.P., Deckwer, W.D., 1997. Adsorption and
desorption of pentachlorophenol on cells of Mycobacterium
chlorophenollicum PCP-1. Biotechnol. Bioeng. 55, 480489.
B
ulb
ul, G., Aksu, Z., 1997. Investigation of wastewater treatment
containing phenol using free and Ca-alginate gel immobilized P.
putida in a batch stirred reactor. Turkish J. Eng. Environ. Sci. 21,
175183.
Dargaville, T.R., Guerzoni, F.N., Looney, M.G., Solomon, D.H.,
1996. The adsorption of multinuclear phenolic compounds on
activated carbon. J. Colloid Interface Sci. 182, 1725.

Dutta, N.N., Borthakur, S., Baruah, R., 1998. A novel process

for recovery of phenol from alkaline wastewater: laboratory
study and predesign cost estimate. Water Environ. Res. 70,
49.
Fourest, E., Volesky, B., 1997. Alginate properties and heavy metal
biosorption by marine algae. Appl. Biochem. Biotechnol. 67, 215
226.
Furuya, E.G., Chang, H.T., Miura, Y., Noll, K.E., 1997. A fundamental analysis of the isotherm for the adsorption of phenolic
compounds on activated carbon. Sep. Purif. Technol. 11, 69
78.
Gadd, G.M., White, C., 1989. Removal of thorium from simulated
acid process streams by fungal biomass. Biotechnol. Bioeng. 33,
592597.
Gupta, V.K., Sharma, S., Yadav, I.S., Mohan, D., 1998. Utilization of
bagasse y ash generated in the sugar industry for the removal and
recovery of phenol and p-nitrophenol from wastewater. J. Chem.
Technol. Biotechnol. 71, 180186.
Hafez, N., Abdel-Razek, A.S., Hafez, M.B., 1997. Accumulation of
some heavy metals on Aspergillus avus. J. Chem. Technol.
Biotechnol. 68, 1922.
Industrial Wastewater Research Summary, 1990. No. EPA-600/8-10026 (US Environmental Protection Agency, Washington, DC).
Kapoor, A., Viraraghavan, T., 1997. Heavy metal biosorption sites in
Aspergillus niger. Bioresource. Technol. 61, 221227.
Kennedy, K.J., Lu, J., Mohn, W.W., 1992. Biosorption of chlorophenols to anaerobic granular sludge. Water Res. 26, 1085
1092.
Presscott, L.M., Harley, J.P., Klein, D.A., 1993. Microbiology, 2nd ed.
Wm.C. Brown Publishers.
Ravi, V.P., Jasra, R.V., Bhat, T.S.G., 1998. Adsorption of phenol,
cresol isomers and benzyl alcohol from aqueous solution on
activated carbon at 278, 298 and 323 K. J. Chem. Technol.
Biotechnol. 71, 173179.
Shu, H.T., Li, D., Scala, A.S., Ma, Y.H., 1997. Adsorption of
small organic pollutants from aqueous streams by aluminosilicate-based microporous materials. Sep. Purif. Technol. 11, 27
36.
Slein, M.W., Sansone, E.B., 1980. Degradation of Chemical Carcinogens. Van Nostrand Reinhold, New York.
Streat, M., Sweetland, L.A., 1997. Physical and adsorptive properties
of Hypersol-Macronete polymers, 1997. React. Functl. Polym. 35,
99109.
Tsezos, M., Georgousis, Z., Remoudaki, E., 1997. Mechanism of
aluminum interference on uranium biosorption by Rhizopus
arrhizus. Biotechnol. Bioeng. 55, 1627.
US Environmental Protection Agency, 1988. Technology screening
guide for treatment of CERCLA soils and sludges. Publication
EPA/540/2-88/004, Washington, DC.
Yenkie, M.K.N., Natarajan, G.S., 1993. Determination of specic
surface area of granular activated carbon by aqueous phase
adsorption of phenol and from pore size distribution measurements. Sep. Sci. Technol. 28, 11771190.